WO2023005281A1 - 一种具有大环骨架结构的新型cdk9抑制剂的制备及其应用 - Google Patents

一种具有大环骨架结构的新型cdk9抑制剂的制备及其应用 Download PDF

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WO2023005281A1
WO2023005281A1 PCT/CN2022/088343 CN2022088343W WO2023005281A1 WO 2023005281 A1 WO2023005281 A1 WO 2023005281A1 CN 2022088343 W CN2022088343 W CN 2022088343W WO 2023005281 A1 WO2023005281 A1 WO 2023005281A1
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卞金磊
李志裕
吴体智
喻彬
张立翱
杜泽坤
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the field of medicinal chemistry, in particular to a macrocyclic CDK9 inhibitor and its preparation method and application.
  • CDKs Cyclin-dependent kinases
  • CDKs are key protein kinases in cell cycle regulation, which can effectively regulate DNA synthesis and mitosis. Combining with the corresponding chaperones to form a complex is a necessary condition for CDK protein function.
  • CDKs can be divided into periodic CDKs (including CDK1, CDK2, CDK3, CDK4, and CDK6) and transcriptional CDKs (including CDK7, CDK8, CDK9, CDK11, CDK12, CDK13, CDK19, etc.).
  • CDK9 protein is widely involved in the initiation, elongation and termination stages of transcription.
  • CDK9 can effectively promote RNA transcription elongation by phosphorylating the CTD end of RNA polymerase II, so CDK9 protein is the key to regulating RNA transcription Factors, and can effectively regulate the protein level of downstream proteins, including the anti-apoptotic protein MCL-1.
  • a number of studies have shown that CDK9 protein imbalance occurs in the occurrence and development of a variety of tumors, including leukemia and other blood tumors, prostate cancer, lung cancer and other solid tumors.
  • an article published in the journal Cell pointed out that the inhibition of CDK9 protein can activate genes that are suppressed by tumors, and promote the expression of tumor suppressor genes and cell differentiation.
  • the above studies show that finding small molecule drugs that target and inhibit CDK9 may be an effective strategy for the development of anti-tumor drugs.
  • CDK9 inhibitors are in the clinical research stage, but most of them are non-selective, which leads to many unpredictable toxic and side effects. Even the clinical trials of some drugs were terminated because of this. Due to the high homology of CDK family members, it is difficult to obtain highly selective CDK9 inhibitors. However, in view of the current clinical research situation and the need to further study the biological role of CDK9, the development of selective CDK9 inhibitors is very important. Currently, there are only three selective CDK inhibitors reported in Linchuan research, including Bayer's BAY-1143572 and BAY-1251152 and AstraZeneca's AZD4573. Clinical trials of BAY-1143572 have been terminated due to neutropenia, but the cause of this side effect has not been reported yet.
  • the main purpose of the present invention is to find a novel CDK9 small molecule inhibitor with high activity, high selectivity and good druggability.
  • Ar is selected from the following groups:
  • X is selected from hydrogen, deuterium, halogen, cyano, methyl, trifluoromethyl;
  • R 1 is selected from the following groups:
  • L is selected from the following structures:
  • the pharmaceutically acceptable salt form in the present invention refers to the salt formed by the compound represented by the general formula I and a pharmaceutically acceptable acid, including inorganic acid salts and organic acid salts.
  • Inorganic acid salts include: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, bicarbonate, nitric acid, monohydrogen phosphate, dihydrogen phosphate, hydrobromic acid or hydroiodic acid;
  • organic acids include: maleic acid, tartaric acid, citric acid , Methanesulfonic acid, succinic acid, acetic acid, p-toluenesulfonic acid, mandelic acid, isobutyric acid, malonic acid, etc.
  • Ar is selected from
  • X is halogen, such as F, Cl, Br, I; in a more specific example, X is F.
  • L is selected from:
  • the present invention provides any of the following compounds or pharmaceutically acceptable salts thereof:
  • the present invention also discloses a synthetic method, and most of the compounds described in the present invention can be obtained rapidly through the following synthetic methods:
  • Ar, X, R 1 and L are as defined above.
  • the present invention also provides a pharmaceutical composition, comprising the compound of the present invention, or a pharmaceutically acceptable salt or prodrug thereof.
  • the present invention also provides the application of the compound of the present invention or its pharmaceutically acceptable salt form in the preparation of CDK9 inhibitor drugs.
  • the inventors found that the compound of the present invention or its pharmaceutically acceptable salt form can effectively inhibit CDK9 protein Activity or application in the discovery of small molecule drugs targeting CDK9.
  • the present invention also provides the application of the compound of the present invention or a pharmaceutically acceptable salt form thereof in the preparation of antiviral drugs or antitumor drugs.
  • the viruses include: HIV virus, cytomegalovirus, Epstein-Barr virus, adenovirus, herpes, and human T-cell lymphoblastic virus.
  • the tumors include glioma, various types of leukemia, lymphoma, liver cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, and lung cancer.
  • the compound described in the present invention is a CDK9 inhibitor with a new skeleton.
  • the inventors designed and synthesized a series of derivatives through a rational drug design method. Inhibitory activity and selectivity of CDK9.
  • Biological activity evaluation shows that the designed compound has significant CDK9 inhibitory activity, has good selectivity (more than 50 times) to CDK family members, and has good anti-proliferation effect on MV4-11, MCF7, HCT116, MOLM13 and other tumor cells active.
  • Embodiment 2 Compound T-2
  • Embodiment 3 Compound T-3
  • Embodiment 4 Compound T-4
  • Embodiment 5 Compound T-5
  • intermediate 12 takes intermediate 2 and methyl 2-methoxy-4-aminobenzoate (intermediate 11) as reaction raw materials, and the synthesis method is the same as that of intermediate 10, with a yield of 73%.
  • HRMS[ESI] + calcd for C 20 H 17 F 2 N 3 O 4 [M+H] + ,402.1187,found 402.1180.
  • Embodiment 6 Compound T-6
  • Embodiment 7 Compound T-7
  • Embodiment 8 Compound T-8
  • Embodiment 9 Compound T-9
  • the synthesis of intermediates 20, 21 and 22 can refer to the preparation method in the synthetic route of compound T1.
  • the synthesis of compound T-27 can refer to the synthesis method of compound T-26 described in Example 26.
  • the preparation of the compounds described in Examples 30 and 31 can refer to the synthesis method and route of compound T-5, and the starting material 4-fluoro-3 methoxyphenylboronic acid in this route is replaced by 4-fluoro-2 methoxy Phenylboronic acid, the linker chain and the amino side chain in the last step of the reaction can be replaced with specific groups.
  • the preparation of the compounds described in Examples 32-35 can refer to the synthesis method and route of compound T-5, and the starting material 4-fluoro-3 methoxyphenylboronic acid in this route is replaced by 5-methoxypyridine-3 -Boronic acid, the linker chain and the amino side chain in the last step of the reaction can be replaced with specific groups.
  • the preparation of the compound described in Example 36 can refer to the synthesis method and route of compound T-18, and the starting material 4-fluoro-3 methoxyphenylboronic acid in this route is replaced by 5-methoxypyridine-3-boronic acid , the linker connecting chain and the amino side chain in the penultimate step of the reaction can be replaced with specific groups.
  • the preparation of the compound described in Example 37 can refer to the synthesis method and route of compound T-18, and the starting material 4-fluoro-3 methoxyphenylboronic acid in this route is replaced by 5-methoxypyridine-3-boronic acid , the linker connecting chain and the amino side chain in the penultimate step of the reaction can be replaced with specific groups.
  • the synthesis of intermediate 43 can refer to the synthesis method of intermediate 18.
  • the synthesis of intermediate 46 can refer to the synthesis method of intermediate 19.
  • the preparation of the compounds described in Examples 39-40 can refer to the preparation method of compound T-5, wherein, 4-fluoro-3-methoxyphenylboronic acid is replaced by the corresponding boric acid or boric acid ester, and the intermediate 2 Synthetic Methods The required starting materials were synthesized.
  • the preparation of the compounds described in Examples 41-44 can refer to the preparation method of compound T-5, wherein the corresponding dichloropyrimidine derivatives can be used to synthesize the required starting materials according to the synthesis method of intermediate 2.
  • reaction solution was raised to room temperature, it was poured into an appropriate amount of ammonium chloride aqueous solution, extracted three times with dichloromethane, and the organic phases were combined and washed twice with saturated aqueous sodium chloride solution. Dry over sodium sulfate, filter with suction, concentrate, and finally use column chromatography to separate compound T-45 with a yield of 36%.
  • Example 46 The preparation of the compound described in Example 46 can refer to the synthesis method and route of compound T-45 in Example 45.
  • Example 47 The preparation of the compound described in Example 47 can refer to the synthesis method and route of compound T-5 in Example 5.
  • Example 50 For the preparation method of Example 50, refer to the synthesis method and route of compound T-25 described in Example 25.
  • the preparation of the compounds described in Examples 51-54 can refer to the synthesis method and route of compound T-5.
  • the experimental method is as follows:
  • the cytotoxicity of the compound to tumor cells with high expression of CDK9 was determined by MTT method: the cells were seeded in 96-well plate at 2000 cells/well, after the cells adhered to the wall, the culture medium was aspirated and added 200uL of the diluted drug was incubated at 37°C with 5% CO2 for 72h, and then 10uL/well of MTT was added. Incubate at 37°C for 4 hours, remove the supernatant, add 150uL of DMSO to each well, and detect the absorbance at 492nm with a multi-functional microplate reader. IC50 was calculated with GraphPad and the cell growth curve was plotted.
  • the experimental method is as follows:
  • the compound was prepared into a 10 mM stock solution with 100% DMSO and stored at low temperature in the dark.
  • the kinase reaction process is as follows:
  • test compound test concentration is 5nM, 20nM or 100nM, and repeated well detection.
  • Compounds were prepared at 100-fold final concentration in a 384-well plate. Then use Echo550 to transfer 250nl to 384 reaction plate for later use. Add 250 nl of 100% DMSO to negative control wells and positive control wells respectively.
  • Conversion%_sample is the conversion rate reading of the sample
  • Conversion%_min the average value of negative control wells, representing the conversion rate readings of wells without enzyme activity
  • Conversion%_max the average value of positive control wells, representing the conversion rate readings of wells without compound inhibition.
  • the concentration of the compound is indicated in the brackets, and the unit is nM, such as 90 (20) indicates that the inhibition rate of the compound to the enzyme is 90% at a concentration of 20 nM.
  • TG-02 is as follows:
  • the compounds of the present invention exhibit effective inhibitory activity on CDK9, and most of the compounds have an inhibitory rate of more than 90% on CDK9 at a concentration of 20 nM.
  • the compound of the present invention shows remarkable CDK9 selectivity.
  • it has significant anti-proliferation inhibitory activity against various tumor cells such as MV4-11, MCF-7, HCT-116 and MOLM13.
  • the dominant compounds T-9, T-10, T-52 and T-53 are all highly selective CDK9 inhibitors, with a selectivity of more than 50 times for CDK2, and they all have good anti-tumor cell proliferation activity.
  • Compound T-53 has an inhibitory IC 50 value of 7 nM for CDK9, and an IC 50 value of greater than 1000 nM for CDK2, with a selectivity greater than 100 times, which is significantly better than that of compound TG02.

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Abstract

本发明公开了一种具有大环骨架结构（I）的新型CDK9抑制剂的制备及其应用，该类化合物可以有效地抑制CDK9蛋白的活性并具有较好的CDK9选择性；本发明还公开了上述化合物的制备方法及其在预防和/或治疗肿瘤相关疾病中的应用，包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌等。

Description

一种具有大环骨架结构的新型CDK9抑制剂的制备及其应用 技术领域
本发明涉及药物化学领域，具体为一种大环类CDK9抑制剂及其制备方法和应用。
背景技术
细胞周期依赖性蛋白激酶(Cyclin-dependent kinases,CDKs)是细胞周期调控的关键的蛋白激酶，可有效调节DNA合成和有丝分裂。与相应的伴侣蛋白结合形成复合物是发挥CDK蛋白功能的必要条件。根据其主要功能，可将CDK分为周期性CDK(包括CDK1，CDK2，CDK3，CDK4和CDK6)和转录性CDK(包括CDK7，CDK8，CDK9，CDK11，CDK12CDK13，CDK19等)。研究表明很多癌症存在“转录成瘾性”，故转录性CDK近来备受关注。其中，CDK9蛋白广泛参与了转录的起始、延伸和终止阶段，特别的是，CDK9可通过磷酸化RNA聚合酶II的CTD端进而有效地促使RNA转录延伸，故CDK9蛋白是调节RNA转录的关键因子，并可以有效调节下游蛋白，包括抗凋亡蛋白MCL-1在内的蛋白水平。多项研究表明，CDK9蛋白的失调出现在多种肿瘤的发生发展中，包括白血病等多种血液瘤，前列腺癌、肺癌等多种实体瘤。2018年，Cell期刊上发表文章指出，CDK9蛋白的抑制可激活被肿瘤抑制的基因，促进抑癌基因的表达和细胞分化。上述研究表明，寻找靶向抑制CDK9的小分子药物可能是一种有效的研发抗肿瘤药物的策略。
目前已有十余个CDK9抑制剂处于临床研究阶段，但其中大部分为非选择性的，进而导致很多难以预料的毒副作用。甚至有部分药物的临床试验因此被终止。由于CDK家族成员的高度同源性，想获得高选择性的CDK9抑制剂有一定困难。但鉴于目前的临床研究情况和进一步深入研究CDK9生物学作用的需求，选择性CDK9抑制剂的开发至关重要。目前已报道的处于临川研究的选择性CDK抑制剂仅有三个，包括拜耳公司的BAY-1143572和BAY-1251152以及阿斯利康公司的AZD4573。由于嗜中性白血球减少症，BAY-1143572的临床试验已经被终止，但导致这一副作用的原因暂没有相关报道。
发明内容
本发明的主要目的是寻找具有高活性，高选择性，且兼具良好成药性质的新型CDK9小分子抑制剂。
一种如通式I所示结构的化合物，或其药学上可接受的盐型：
Figure PCTCN2022088343-appb-000001
其中，Ar选自如下基团：
Figure PCTCN2022088343-appb-000002
X选自氢，氘，卤素，氰基，甲基，三氟甲基；
R 1选自如下基团：
Figure PCTCN2022088343-appb-000003
L选自如下结构：
Figure PCTCN2022088343-appb-000004
本发明所述的药学上可接受的盐型是指通式I所示的化合物和药学上可接受的酸形成的盐，包括无机酸盐和有机酸盐。其中无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；有机酸包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸、丙二酸等。
在一些具体的实例中，Ar选自
Figure PCTCN2022088343-appb-000005
在一些具体的实例中，X为卤素，例如F、Cl、Br、I；在一种更为具体的实例中，X为F。
在一些具体的实例中，L选自：
Figure PCTCN2022088343-appb-000006
在一些具体的实例中，本发明提供如下任一所述的化合物或其药学上可接受的盐：
Figure PCTCN2022088343-appb-000007
Figure PCTCN2022088343-appb-000008
Figure PCTCN2022088343-appb-000009
Figure PCTCN2022088343-appb-000010
Figure PCTCN2022088343-appb-000011
Figure PCTCN2022088343-appb-000012
Figure PCTCN2022088343-appb-000013
Figure PCTCN2022088343-appb-000014
Figure PCTCN2022088343-appb-000015
本发明还公开一种合成方法，本发明所述的大部分化合物均可通过以下合成方法快速获得：
Figure PCTCN2022088343-appb-000016
其中，Ar、X、R 1、L定义如前所述。
本发明还提供一种药物组合物，包括本发明所述的化合物，或其药学上可接受的盐型或者前药。
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备CDK9抑制剂药物中的应用，发明人发现本发明所述化合物或其药学上可接受的盐型可有效抑制CDK9蛋白活性或应用于以CDK9为靶点的小分子药物的发现。
本发明还提供本发明所述化合物或其药学上可接受的盐型在制备抗病毒药物或者抗肿瘤药物中的应用。
在本发明的一些实施例中，所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒。
本发明的一些实施例中，所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。
本发明所述的化合物为新骨架的CDK9抑制剂，发明人通过合理的药物设计方法，设计合成了一系列衍生物，本发明所述的化合物通过大环固定优势化合物的构象，进一步提升其对CDK9的抑制活性和选择性。生物活性评价显示，所设计化合物具有显著的CDK9抑制活性，对CDK家族成员具有较好的选择性(大于50倍)，同时对MV4-11，MCF7，HCT116，MOLM13等肿瘤细胞具有良好的抗增殖活性。
具体实施方式
下面通过具体的实施例对本发明做进一步的详细说明，但并不将本发明限制在所举的实施例范围之中。
实施例1：化合物T-1
Figure PCTCN2022088343-appb-000017
中间体2的合成：
将化合物1(10mmol)，2,4-二氯-5-氟嘧啶(11mmol)溶解于20ml二氧六环中，然后加入Pd(PPh 3) 2Cl 2(3mmol),碳酸钠水溶液(20ml,2N)，使用氮气置换空气三次后升温至100℃反应，约2小时后，TLC检测原料反应完全，停止反应，待反应液冷却后，加入适量水，使用乙酸乙酯萃取三次，合并有几层并用饱和氯化钠水溶液洗涤1次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离纯化得白色固体(中间体2)，收率约80％。HRMS[ESI] +,calcd for C 11H 7ClF 2N 2O[M+H] +,257.0288,found 257.0278.
中间体3的合成：
将中间体2(5mmol)溶解于30ml的DCM中，降温至-10℃以下，然后缓慢滴加三溴化硼(15mmol)，控制温度在-10℃以下，待滴加完毕后将反应液移至室温搅拌反应，约2小时后，TLC检测原料反应完全，停止反应，将反应液降温至-10℃以下，加入适量甲醇淬灭，析出大量固体，抽滤，滤饼使用少量冷甲醇洗涤两次，干燥后得黄色固体，即中间体3，无需纯化直接进行下一步，收 率约85％。HRMS[ESI] +,calcd for C 10H 5ClF 2N 2O[M+H] +,243.0131,found 243.0127.
中间体4的合成：
将中间体3(10mmol)溶解在30ml的DMF中，降温至0℃以下，加入碳酸钾(15mmol)，然后将溴丙炔(11mmol)缓慢滴加到反应液中，控制温度在0℃以下，滴加完毕后将反应液移至室温搅拌反应，约6小时后，TLC检测原料反应完全，停止反应，反应液抽滤，使用适量DMF洗涤三次滤饼，滤液加入适量水并使用乙酸乙酯萃取三次，合并有机相，使用饱和氯化钠水溶液洗涤3次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体4，收率65％。HRMS[ESI] +,calcd for C 13H 9ClF 2N 2O[M+H] +,283.0444,found 283.0437.
中间体6的合成：
将4-硝基水杨酸(5,10mmol)溶解在30ml的DMF中，降温至0℃以下，然后分三次加入氢化钠(22mmol)，再将溴丙炔(22mmol)缓慢滴加到反应液中，控制温度在0℃以下，滴加完毕后将反应液移至室温搅拌反应，约10小时后，TLC检测原料反应完全，停止反应，将反应液降温至0℃以下，加入适量水淬灭剩余的氢化钠，并使用乙酸乙酯萃取三次，饱和氯化钠水溶液洗涤3次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体6，收率80％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.14(s,1H),7.84(s,1H),7.72(s,1H),6.11-6.02(m,2H),5.31-5.43(m,4H),4.72-4.74(m,4H).
中间体7的合成：
将中间体6(2mmol)溶解在20ml四氢呋喃和水(4:1)的混合溶剂中，然后加入氢氧化钠(10mmol)，室温搅拌反应约6小时，待TLC检测原料反应完全，停止反应，浓缩除去四氢呋喃，然后使用稀盐酸调节pH至酸性，析出大量固体，抽滤，用水洗涤滤饼三次，真空干燥后得中间体7，无需纯化直接进行下一步，收率85％。HRMS[ESI] +,calcd for C 10H 9NO 5[M-H] -,222.0841,found 222.0831.
中间体8的合成：
将中间体7(0.5mmol)，HATU(1.75mmol)溶解在4ml的四氢呋喃中，然后加入DIPEA(1mmol)，室温搅约15分钟，然后加入N,N-二甲基乙二胺(0.5mmol)，室温搅拌反应约2小时，TLC检测原料反应完全，停止反应，柱层析分离纯化后可得中间体8，收率56％。HRMS[ESI] +,calcd for C 14H 19N 3O 4[M+H] +,294.1376,found 294.1362.
中间体9的合成：
将中间体8(3mmol)溶解在15ml的乙醇和水(4:1)的混合溶剂中，然后加入铁粉(15mmol)和5滴浓盐酸，升温至70℃反应约1小时，TLC检测原料反应完全，停止反应，冷却后，反应液抽滤，滤液使用碳酸钠水溶液调节pH至碱性，然后使用乙酸乙酯萃取，合并有机相，使用饱和氯化钠水溶液洗涤有机相3次，无水硫酸钠干燥，抽滤，浓缩得9，收率75％，无需纯化直接进行下一步反应。HRMS[ESI] +,calcd for C 14H 21N 3O 2[M+H] +,264.1634,found 264.1626.
中间体10的合成：
将醋酸钯(0.5mmol)和XPhos(0.6mmol)溶解于60ml二氧六环中，氮气置换空气三次后，于室温搅拌约15分钟备用。将中间体4(10mmol)，中间体9(10mmol)，碳酸铯(30mmol)加入150ml三颈瓶中，然后加入上述新制的催化剂，氮气置换空气三次，然后升温至110-120℃回流反应，反应2-4小时后，TLC检测原料反应完全，停止反应，趁热抽滤，滤液浓缩后加入适量水，使用二氯甲烷萃取三次，合并有机相并使用饱和氯化钠洗涤2次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体10，收率86％。HRMS[ESI] +,calcd for C 27H 29F 2N 5O 3[M+H] +,510.2238,found 510.2231.
化合物T-1的合成：
将中间体10(0.5mmol)溶解在20ml二氯甲烷中，加入0.1ml稀盐酸(4N)，氮气置换空气三次后，加入Grubbs二代催化剂(0.05mmol)，再次使用氮气置换空气三次，然后升温至45℃反应，约2小时后，TLC检测原料反应完全，停止反应，冷却后抽滤，滤液浓缩后使用柱层析分离纯化，得化合物T-1，收率63％。
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.72(s,1H),8.19(s,2H),7.82(t,J＝6.0Hz,2H),7.58(s,1H),7.47(s,J＝9.0Hz,1H),6.90(d,J＝9.0Hz,1H),4,30(s,2H),4.10(d,J＝6Hz,2H),3.75(s,4H),3.62-3.60(m,3H),3.29-3.27(m,2H),3.20(s,4H),1.98(s,2H),1.90(s,2H),1.72(s,4H).
实施例2-4所述化合物的制备方法可参考实施例1中的化合物T-1的合成方法和路线进行合成：
实施例2：化合物T-2
Figure PCTCN2022088343-appb-000018
1H NMR(300MHz,CDCl 3):δ(ppm)＝8.68(s,1H),8.38(s,1H),8.23(d,J＝9.0Hz,1H),8.12(d,J＝9.0Hz,1H),7.97(d,J＝9.0Hz,1H),7.76-7.71(m,1H),7.49(s,1H),7.23(t,J＝9.0Hz,1H),6.65(d,J＝9.0Hz,1H),5.80(q,J＝15.0Hz,1H),5.50(q,J＝15.0Hz,1H),4.90(s,2H),4.80(s,2H),3.76(t,J＝6.0Hz,4H),3.64(q,J＝9.0Hz,2H),2.64(t,J＝6.0Hz,2H),2.54(s,4H).
实施例3：化合物T-3
Figure PCTCN2022088343-appb-000019
1H NMR(300MHz,CDCl 3):δ(ppm)＝8.60(s,1H),8.30(s,1H),8.20(d,J＝9.0Hz,1H),8.06(d,J＝9.0Hz,1H),7.91(d,J＝9.0Hz,1H),7.75-7.69(m,1H),7.45(s,1H),7.25(t,J＝9.0Hz,1H),6.60(d,J＝9.0Hz,1H),5.70(q,J＝15.0Hz,1H),5.40(q,J＝15.0Hz,1H),4.80(s,2H),4.44(t,J＝6.0Hz,2H),3.75(t,J＝6.0Hz,4H),3.70-3.59(m,2H),2.65(t,J＝6.0Hz,2H),2.51-2.40(m,6H).
实施例4：化合物T-4
Figure PCTCN2022088343-appb-000020
1H NMR(300MHz,CDCl 3):δ(ppm)＝8.65(s,1H),8.35(s,1H),8.21(d,J＝9.0Hz,1H),8.12(d,J＝9.0Hz,1H),7.95(d,J＝9.0Hz,1H),7.79-7.70(m,1H),7.50(s,1H),7.30(t,J＝9.0Hz,1H),6.65(d,J＝9.0Hz,1H),5.50(q,J＝15.0Hz,1H),5.35 (q,J＝15.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.72(t,J＝6.0Hz,4H),3.68-3.60(m,2H),2.65(t,J＝6.0Hz,2H),2.55(s,4H),2.21(q,J＝6.0Hz,2H),2.1(q,J＝6.0Hz,2H).
实施列5：化合物T-5
化合物T-5的合成路线如下：
Figure PCTCN2022088343-appb-000021
中间体12的合成：
中间体12的合成以中间体2和2-甲氧基-4-氨基苯甲酸甲酯(中间体11)为反应原料，合成方法同中间体10的合成，收率73％。HRMS[ESI] +,calcd for C 20H 17F 2N 3O 4[M+H] +,402.1187,found 402.1180.
中间体13的合成：
将中间体12(10mmol)溶解于60ml的DCM中，降温至-10℃以下，然后缓慢滴加三溴化硼(45mmol)，控制温度在-10℃以下，待滴加完毕后将反应液移至室温搅拌反应，约1.5小时后，TLC检测原料反应完全，停止反应，将反应液降温至-10℃以下，加入适量甲醇淬灭，析出大量固体，抽滤，滤液使用冷甲醇洗涤两次，干燥后得黄色固体，即中间体13，收率65％，无需纯化直接进行下一步。HRMS[ESI] +,calcd for C 18H 13F 2N 3O 4[M+H] +,374.0874,found 374.0863.
中间体14的合成：
将中间体13(5mmol)溶解在20ml的DMF中，加入碳酸铯(12.5mmol)后搅拌约15分钟，然后加入1,4-二溴丁烷(5mmol)，于室温下搅拌反应约6小时，TLC检测反应完全，停止反应，反应液抽滤，使用适量DMF洗涤滤饼，滤 液中加入适量水，然后使用稀盐酸(1N)调节pH至酸性，析出大量固体，抽滤，使用适量水洗涤滤饼三次，干燥后柱层析分离纯化得中间体14，收率45％。HRMS[ESI] +,calcd for C 22H 19F 2N 3O 4[M+H] +,428.1344,found 428.1338.
中间体15的合成：
将中间体14(2mmol)溶解在20ml的甲醇中，然后加入20％的NaOH水溶液(20ml)，升温至75℃反应约5小时，TLC检测原料反应完全，停止反应，反应液冷却，使用稀盐酸调节pH至酸性，析出大量固体，抽滤，使用适量水洗涤滤饼，干燥后得中间体15，收率85％。HRMS[ESI] +,calcd for C 21H 17F 2N 3O 4[M+H] +,414.1187,found 414.1179.
化合物T-5的合成：
化合物T-5的合成使用中间体15和N,N-二甲基乙二胺为原料，合成方法同中间体8的合成，收率42％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.27(s,1H),8.76(d,J＝3.0Hz,1H),8.64(s,1H),8.38(d,J＝6.0Hz,1H),8.15(d,J＝9.0Hz,1H),7.86(d,J＝9.0Hz,1H),7.69-7.65(m,1H),7.49(t,J＝9.0Hz,1H),7.02(d,J＝6.0Hz,1H),4.45(s,2H),4.25(s,2H),3.48(q,J＝6.0Hz,4H),2.33(s,6H),1.97-1.93(m,4H).
实施例6-17所述化合物的制备方法可参考化合物T-5的合成方法和路线进行合成：
实施例6：化合物T-6
Figure PCTCN2022088343-appb-000022
1H NMR(300MHz,CDCl 3):δ(ppm)＝8.63(d,J＝3.0Hz,1H),8.38(d,J＝3.0Hz,1H),8.29(s,1H),8.22(d,J＝9.0Hz,1H),8.14(d,J＝6.0Hz,1H),7.76-7.72(m,1H),7.48(s,1H),7.24(t,J＝9.0Hz,1H),6.65(dd,J 1＝9.0Hz,J 2＝3.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.76(t,J＝6.0Hz,4H),3.70-3.59(m,2H)2.64(t,J＝6.0Hz,2H),2.54(s,4H),2.16(q,J＝6.0Hz,2H),2.01(q,J＝6.0Hz,2H).
实施例7：化合物T-7
Figure PCTCN2022088343-appb-000023
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.01(s,1H),8.73(d,J＝9,1H),8.34(t,J＝3Hz,1H),8.15(d,J＝9.0Hz,1H),7.92(d,J＝9Hz,1H),7.71-7.68(m,1H),7.49-7.43(m,1H),7.02(d,J＝6Hz,1H),4.46(s,2H),4.25(s,2H),3.71-3.68(m,2H),1.86(s,4H).
实施例8：化合物T-8
Figure PCTCN2022088343-appb-000024
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.27(s,1H),8.57(d,J＝3.0Hz,1H),8.36(d,J＝3.0Hz,1H),8.22(dd,J 1＝9.0Hz,J 2＝3.0Hz,1H),7.82(d,J＝9.0Hz,1H),7.68-7.64(m,1H),7.52(s,1H),7.21(t,J＝9.0Hz,1H),6.54(d,J＝9.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.96(t,J＝6.0Hz,4H),3.75(t,J＝6.0Hz,4H),2.65(t,J＝6.0Hz,4H),2.21(q,J＝6.0Hz,2H),2.11(q,J＝6.0Hz,2H).
实施例9：化合物T-9
Figure PCTCN2022088343-appb-000025
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.30(s,1H),8.56(d,J＝3.0Hz,1H),8.38(d,J＝3.0Hz,1H),8.30(s,1H),8.22(d,J＝9.0Hz,1H),7.80-7.72(m,1H),7.52(s,1H),7.21(t,J＝9.0Hz,1H),6.60(d,J＝9.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.72(t,J＝6.0Hz,4H),2.55(s,4H),2.16(q,J＝6.0Hz,2H),2.01(q,J＝6.0Hz,2H),1.80-1.76(m,4H),1.33-1.29(m,1H).
实施例10：化合物T-10
Figure PCTCN2022088343-appb-000026
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.72(s,1H),8.19(s,2H),7.82(t,J＝6.0Hz,2H),7.58(s,1H),7.47(s,J＝9.0Hz,1H),6.90(d,J＝9.0Hz,1H),4.30(s,2H),4.10(d,J＝6Hz,2H),3.75(s,4H),3.62-3.60(m,3H),3.29-3.27(m,2H),3.20(s,4H),1.98(s,2H),1.90(s,2H),1.72(s,4H).
实施例11：化合物T-11
Figure PCTCN2022088343-appb-000027
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.38(s,1H),8.68(d,J＝3.0Hz,1H),8.38(d,J＝3.0Hz,1H),8.23(dd,J 1＝9.0Hz,J 2＝3.0Hz,1H),8.12(d,J＝9.0Hz,1H),7.97(d,J＝9.0Hz,1H),7.76-7.71(m,1H),7.23(t,J＝9.0Hz,1H),6.65(d,J＝9.0Hz,1H),4.45(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),4.25(q,J＝3.0Hz,1H),4.01(dt,J 1＝12.0Hz,J 2＝3.0Hz,2H),3.61(t,J＝9.0Hz,2H),2.11-1.96(m,4H),1.86-1.79(m,4H),1.64-1.53(m,4H).
实施例12：化合物T-12
Figure PCTCN2022088343-appb-000028
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.78(s,1H),8.15(s,2H),7.92(t,J＝6.0Hz,2H),7.63(s,1H),7.47(s,J＝9.0Hz,1H),6.92(d,J＝9.0Hz,1H),4,23(s,2H),4.09(s,2H),3.59-3.57(m,3H),3.31-3.28(m,2H),2.61-2.57(m,4H),1.96(s,2H),1.92(s,2H),1.83(s,8H).
实施例13：化合物T-13
Figure PCTCN2022088343-appb-000029
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.81(s,1H),8.21(s,2H),7.79(t,J＝6.0Hz,2H),7.61(s,1H),7.50(s,J＝9.0Hz,1H),6.91(d,J＝9.0Hz,1H),4.31(s,2H),4.12(d,J＝6Hz,2H),3.75(s,4H),3.64-3.62(m,1H),2.87-2.84(m,2H),2.63-2.61(m,2H),2.59(s,3H),2.03-1.98(m,4H),1.90-1.87(m,4H),1.78(s,4H).
实施例14：化合物T-14
Figure PCTCN2022088343-appb-000030
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.16(s,1H),8.73(d,J＝3.0Hz,1H),8.25-8.20(m,2H),7.86(d,J＝9.0Hz,2H),7.59(s,1H),7.47(t,J＝12.0Hz,1H),6.92(d,J＝9.0Hz,1H),4.31(s,2H),4.0(s,2H),3.53(d,J＝6.0Hz,2H),2.77(s,2H),2.53(s,3H),2.47(s,3H),1.91(s,2H),1.73(s,2H),1.57(s,4H).
实施例15：化合物T-15
Figure PCTCN2022088343-appb-000031
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.08(s,1H),8.75(s,1H),8.35(s,1H),8.18(s,1H),7.92(d,J＝9Hz,1H),7.73-7.69(m,1H),7.49-7.46(m,1H),7.05(d,J＝6Hz,1H),4.38(s,2H),4.21(s,2H),3.69-3.66(m,2H),1.93-1.81(m,4H),1.73(s,4H).
实施例16：化合物T-16
Figure PCTCN2022088343-appb-000032
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.81(s,1H),8.20(s,2H),7.83(t,J＝6.0Hz,2H),7.57(s,1H),7.49(s,1H),6.93(d,J＝9.0Hz,1H),4.4(s,1H),4.21(s,2H),4.12(s,2H),3.45-3.31(m,2H),1.89-1.73(m,6H),1.68-1.42(m,10H).
实施例17：化合物T-17
Figure PCTCN2022088343-appb-000033
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.20(s,1H),8.75(d,J＝3.0Hz,1H),8.30-8.26(m,2H),7.84(d,J＝9.0Hz,2H),7.60(s,1H),7.45(t,J＝12.0Hz,1H),6.90(d,J＝9.0Hz,1H),4.31(s,2H),4.0(s,2H),3.55(d,J＝6.0Hz,2H),2.75(s,2H),2.55(s,3H),2.41(s,3H),1.89(s,2H),1.74(s,2H),1.57(s,4H),1.04(t,J＝9.0Hz,6H).
实施例18：化合物T-18
化合物T-18的合成：
Figure PCTCN2022088343-appb-000034
(1)T-18-1的合成：LW-012-1的合成可参考化合物T5的合成方法与路线。
(2)将上述得到的T-18-1(0.35mmol)溶解在2ml二氯甲烷中，然后加入三氟乙酸(3ml)，氮气保护，常温下反应约2小时，TLC检测反应完全，停止反应，使用2N的碳酸钠水溶液调节pH至碱性，并加入适量水和二氯甲烷搅拌约15分钟，分离有几层，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化，得约105mg固体(T-19)，收率63％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝ 8.84(s,1H),8.51(s,1H),8.23(s,1H),7.74(s,2H),7.54(m,1H),7.49(s,1H),6.99(s,1H),4.18(s,2H),4.02(s,2H),3.64-3.34(m,2H),3.2(s,4H),2.5-2.34(m,6H),1.90-1.81(m,4H),1.68-1.51(m,4H).
实施例19-22所述的制备方法可参考化合物T-18的合成方法与路线进行合成：
实施例19：化合物T-19
Figure PCTCN2022088343-appb-000035
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.81(s,1H),8.59(s,1H),8.26(s,1H),7.79(s,2H),7.42(m,1H),7.41(s,1H),6.91(s,1H),4.31(s,2H),4.19(s,2H),3.61-3.55(m,1H),2.87-2.60(m,3H),2.13-1.98(m,2H),1.96-1.86(m,6H),1.75-1.51(m,6H).
实施例20：化合物T-20
Figure PCTCN2022088343-appb-000036
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.78(s,1H),8.16(s,2H),7.83(t,J＝6.0Hz,2H),7.65(s,1H),7.49(s,J＝9.0Hz,1H),6.94(d,J＝9.0Hz,1H),4.26(s,2H),4.08(s,2H),3.71(s,4H),3.63-3.60(m,1H),2.86-2.84(m,2H),2.65-2.61(m,3H),2.10-1.99(m,4H),1.93-1.86(m,4H),1.75(s,4H).
实施例21：化合物T-21
Figure PCTCN2022088343-appb-000037
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.79(s,1H),8.50(s,1H),8.25(s,1H),7.76(s,2H),7.53(m,1H),7.46(s,1H),6.91(s,1H),4.19(s,2H),4.04(s,2H),3.66-3.36(m,2H),3.3(s,4H),2.54-2.38(m,6H),1.91-1.83(m,4H),1.66-1.51(m,2H).
实施例22：化合物T-22
Figure PCTCN2022088343-appb-000038
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.82(s,1H),8.53(s,1H),8.26(s,1H),7.78(s,2H),7.55(m,1H),7.48(s,1H),6.90(s,1H),4.19(s,2H),4.08(s,2H),3.63-3.34(m,2H),2.51-2.36(m,4H),1.98-1.85(m,4H),1.69(s,2H),1.61-1.50(m,2H).
实施例23：化合物T-23
化合物T-23的合成：
Figure PCTCN2022088343-appb-000039
(1)化合物T-23-1的合成可参考化合物T-5的合成方法与路线。
(2)将上述获得的T-23-1(0.4mmol)溶解在5ml的甲醇中，然后加入50％的氢氧化钠水溶液(3ml)，室温搅拌2小时，TLC检测原料反应完全，停止反应，使用稀盐酸调节pH至酸性，析出白色固体，抽滤后，滤饼干燥，使用柱层析分离得化合物T-23，收率54％。
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.91(s,1H),8.88(s,1H),8.57(s,1H),8.29(s,1H),7.76(s,2H),7.58(m,1H),7.48(s,1H),6.99(s,1H),4.31(s,2H),4.09(s,2H),3.63-3.43(m,1H),2.18-2.03(m,1H),1.98-1.73(m,8H),1.61-1.43(m,8H).
实施例24：化合物T-24
化合物T-24的合成路线：
Figure PCTCN2022088343-appb-000040
中间体16的合成：
将中间体3(10mmol)，6-氯-2-己酮(11mmol)溶解在30ml的DMF中，加入碳酸钾(15mmol)，然后加入碘化钾(1mmol)，升温至60℃反应，约3小时后，TLC检测原料反应完全，停止反应，反应液抽滤，使用适量乙酸乙酯洗涤滤饼，滤液加入适量水并使用乙酸乙酯萃取三次，合并有机相，使用饱和氯化钠水溶液洗涤3次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体16，收率53％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.58(s,1H),7.54(t,J＝6Hz,1H),7.35-7.29(m,2H),4.21(t,J＝6Hz,2H),2.63(t,J＝6Hz,2H),2.04(s,1H),1.71-1.54(m,4H).
中间体17的合成：
将中间体16(10mmol)溶于40ml甲醇中，降温至0℃以下，然后分批次加入硼氢化钠(15mmol)，反应液升温至室温反应约1h，TLC检测原料反应完全，停止反应，将反应液浓缩后降至0℃，加入适量冰水淬灭，然后使用乙酸乙酯萃取三次，再使用饱和氯化钠水溶液洗涤一次，无水硫酸钠干燥，抽滤，浓缩，使用异丙醚重结晶得到中间体17，收率83％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.61(s,1H),7.51(t,J＝6Hz,1H),7.35-7.30(m,2H),4.17(t,J＝6Hz,2H),3.67-3.61(m,1H),1.76-1.58(m,6H),1.24(d,J＝8Hz,3H).
中间体18的合成：
将中间体17(10mmol)溶解在40ml的二氯甲烷中，然后加入三乙胺(20mmol)，反应液降温至0℃以下，然后分批次加入对甲苯磺酰氯(20mmol)，加完后升温至回流反应约3个小时，TLC检测原料反应完全，停止反应，冷却后加入适量冰水，搅拌15分钟后，分离出有机相并使用饱和氯化钠水溶液洗涤两次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体18，收率74％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.21(s,1H),7.77-7.71(m,2H),7.50(t,J＝8Hz,1H),7.40(m,2H),7.26-7.19(m,2H),4.56-4.49(m,1H),4.23(t,J＝6Hz,2H),2.01(s,3H),1.74-1.61(m,4H),1.39-1.25(m,5H).
中间体19的合成：
将中间体18(12mmol)和2-羟基-4-硝基苯甲酸甲酯(10mmol)溶解在80ml的DMF中，然后加入碳酸钾(15mmol)，反应液升温至80℃反应过夜，待TLC检测原料反应完全，停止反应，反应液冷却后，抽滤，向滤液中加入适量水，并使用二氯甲烷萃取三次，合并有机相，并依次使用水、饱和氯化钠水溶液分别洗涤三次有机相，有机相使用无水硫酸钠干燥，抽滤，浓缩，使用乙酸乙酯和甲醇的混合溶剂重结晶得到中间体19，收率54％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.51(s,1H),8.27(s,1H),7.79-7.70(m,2H),7.53(t,J＝8Hz,1H),7.42(m,2H),7.28-7.21(m,2H),4.13(t,J＝6Hz,2H),3.95(s,3H),3.83-3.75(m,1H),2.01(s,3H),1.69-1.63(m,4H),1.41-1.36(m,5H).
中间体20,21和22的合成可参考化合物T1合成路线中的制备方法。
化合物T-24的合成：
化合物T-24的合成使用中间体22和N-(2-氨基乙基)吗啉为原料，合成方法同中间体8的合成，收率42％。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.79(s,1H),8.53(s,1H),8.25(s,1H),7.79(s,2H),7.51(m,1H),7.49(s,1H),6.98(s,1H),4.31(s,2H),4.20-4.04(m,1H),3.87(s,4H),3.66(s,2H),2.6(s,2H),2.49(s,4H),3.3(s,4H),1.90-1.82(m,4H),1.69-1.50(m,5H).
实施例25：化合物T-25
化合物T-25的合成路线：
Figure PCTCN2022088343-appb-000041
中间体24的合成：
将中间体23(10mmol)，1,5-二溴戊烷(12mmol)溶解在30ml的DMF中，加入碳酸钾(15mmol)，然后加入碘化钾(1mmol)，升温至100℃反应，约8小时后，TLC检测原料反应完全，停止反应，反应液抽滤，使用适量二氯甲烷洗涤滤饼，滤液加入适量水并使用二氯甲烷萃取三次，合并有机相，使用饱和氯化钠水溶液洗涤3次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体23，收率43％。HRMS[ESI] +,calcd for C 13H 17BrN 2O 4[M+H] +,345.0372,found 345.0371.
中间体25的合成：
将中间体24(10mmol)，中间体3(10mmol)溶解在40ml DMF中，然后加入碳酸钾(15mmol)，25℃反应约3小时，TLC检测原料反应完全，停止反应，反应液抽滤，使用适量二氯甲烷洗涤滤饼，滤液加入适量水并使用二氯甲烷萃取三次，合并有机相，使用饱和氯化钠水溶液洗涤3次，无水硫酸钠干燥，抽滤，浓缩，使用柱层析分离纯化得中间体25，收率75％。HRMS[ESI] +,calcd for C 23H 21ClF 2N 4O 5[M+H] +,508.1169,found 508.1173.
中间体26-28的合成可参照实施例1中所述的合成方法制备获得。
化合物T-25的合成已中间体28和N-(2-氨基乙基)吗啉为原料，合成方法参照化合物T-1的合成。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.79(s,1H),8.21(s,2H),7.78(t,J＝6.0Hz,2H),7.63(s,1H),7.45(s,J＝9.0Hz,1H),6.92(d,J＝9.0Hz,1H),4.32(s,2H),3.75(s,4H),3.62-3.60(m,2H),3.29-3.27(m,2H),3.04(s,2H),2.75(s,4H),1.97(s,2H),1.91(s,2H),1.78(s,2H).
实施例26：化合物T-26
Figure PCTCN2022088343-appb-000042
中间体29的合成：
将中间体23(10mmol)溶解于20ml的二氯甲烷中，然后加入三乙胺(15mmol)并将反应液降温至0℃以下，将5-溴戊酰氯(20mmol)缓慢滴加进反应液中，控制温度在0℃以下，滴加完毕后将反应液升温回流反应，约2小时后，TLC检测原料反应完全，停止反应，加入适量冰水，搅拌约15分钟，静置分层，分离出有机相并使用饱和氯化钠水溶液洗涤2次，无水硫酸钠干燥，抽滤，浓缩，然后使用异丙醚重结晶即可获得中间体29，收率65％。HRMS[ESI] +,calcd for C 13H 15BrN 2O 5[M+H] +,359.0164,found 359.0154.
后续的合成步骤可参照实施例25中所述的合成方法与路线，最终可得化合物T-26。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.8(s,1H),8.61(s,1H),8.57(s,1H),8.21(s,1H),7.56(s,2H),7.45(m,2H),7.38(s,1H),6.90(s,1H),4.19(s,2H), 3.97(m,4H),3.62-3.59(m,2H),3.00-2.91(m,2H),2.75(s,4H),2.01(s,2H),1.71(s,4H).
实施例27：化合物T-27
化合物T-27的合成路线：
Figure PCTCN2022088343-appb-000043
化合物T-27的合成可参照实施例26所述的化合物T-26的合成方法。
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.2(s,1H),8.69(s,1H),8.64(s,1H),8.23(s,1H),7.56(s,2H),7.47(m,2H),7.39(s,1H),6.92(s,1H),4.20(s,2H),3.98(m,4H),3.63-3.61(m,2H),3.03-2.91(m,2H),2.79(s,4H),2.08(s,2H),1.76(s,4H).
实施例28：化合物T-28
Figure PCTCN2022088343-appb-000044
化合物T-28的合成可参考实施例26所述的化合物T-26的合成方法。
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.1(s,1H),8.71(s,1H),8.56(s,1H),8.24(s,1H),7.56(s,2H),7.49(m,1H),7.42(s,2H),6.91(s,1H),4.21(s,2H),3.94(m,4H),3.63-3.60(m,2H),3.08-2.95(m,2H),2.83(s,4H),2.00(s,2H),1.93(s,2H),1.73(s,4H).
实施例29：化合物T-29
Figure PCTCN2022088343-appb-000045
化合物T-29的合成可参考实施例27所述的化合物T-27的合成方法。
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.99(s,1H),8.79(s,1H),8.61(s,1H),8.29(s,1H),7.52(s,2H),7.41(m,1H),7.37(s,1H),6.99(s,2H),4.28(s,2H),3.91(s,4H),3.63-3.56(m,2H),3.09-2.92(m,2H),2.74(s,4H),2.12(s,2H),1.95(s,2H),1.81(s,4H).
实施例30和31所述化合物的制备可参考化合物T-5的合成方法和路线，将该路线中的起始原料4-氟-3甲氧基苯硼酸替换为4-氟-2甲氧基苯硼酸，linker连接链和最后一步反应中的氨基侧链替换为特定基团即可。
实施例30：化合物T-30
Figure PCTCN2022088343-appb-000046
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.93(s,1H),8.77(s,1H),8.30(t,J＝6.0Hz,1H),8.10(t,J＝6.0Hz,1H),8.22(d,J＝9.0Hz,1H),7.80-7.72(m,1H),7.53(s,1H),7.16(d,J＝9.0Hz,1H),6.99(td,J 1＝9.0Hz,J 2＝3.0Hz,1H),4.45(t,J＝6.0Hz,2H),4.38(t,J＝6.0Hz,2H),3.75-3.71(m,4H),2.60-2.56(m,4H),2.20(q,J＝6.0Hz,2H),2.11(q,J＝6.0Hz,2H),1.80-1.76(m,4H),1.33-1.29(m,1H).
实施例31：化合物T-31
Figure PCTCN2022088343-appb-000047
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.03(s,1H),8.71(s,1H),8.35(t,J＝6.0Hz,1H),8.22(d,J＝9.0Hz,1H),8.10(d,J＝6.0Hz,1H),7.83-7.79(m,1H),7.45(s, 1H),7.20(t,J＝9.0Hz,1H),6.89(d,J＝9.0Hz,1H),4.55(t,J＝6.0Hz,2H),4.40(t,J＝6.0Hz,2H),3.80(t,J＝6.0Hz,4H),3.72-3.65(m,2H)2.62(t,J＝6.0Hz,2H),2.52(s,4H),2.11(q,J＝6.0Hz,2H),2.01(q,J＝6.0Hz,2H).
实施例32-35所述化合物的制备可参考化合物T-5的合成方法和路线，将该路线中的起始原料4-氟-3甲氧基苯硼酸替换为5-甲氧基吡啶-3-硼酸，linker连接链和最后一步反应中的氨基侧链替换为特定基团即可。
实施例32：化合物T-32
Figure PCTCN2022088343-appb-000048
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.21(s,1H),8.71(s,1H),8.28(s,1H),8.08(s,1H),7.80(s,1H),7.33(m,1H),7.24(s,1H),6.91(s,2H),4.31(s,2H),4.21(s,2H),3.97(s,4H),3.51-3.41(m,2H),2.94-2.81(m,2H),2.78(s,4H),1.95(s,4H),1.74(s,4H).
实施例33：化合物T-33
Figure PCTCN2022088343-appb-000049
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.01(s,1H),8.81(s,1H),8.26(s,1H),8.03(s,1H),7.79(s,2H),7.39(m,1H),7.26(s,1H),6.99(s,1H),4.28(s,2H),4.13(s,2H),3.59-3.41(m,2H),2.91-2.73(m,6H),1.93-1.84(m,4H),1.69-1.58(m,8H).
实施例34：化合物T-34
Figure PCTCN2022088343-appb-000050
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.19(s,1H),8.89(s,1H),8.32(s,1H),8.05(s,1H),7.83(s,2H),7.35(m,1H),7.21(s,1H),6.92(s,1H),4.21(s,2H),4.11(s,2H),3.82-3.70(m,2H),1.95(s,4H),1.74(s,4H).
实施例35：化合物T-35
Figure PCTCN2022088343-appb-000051
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.08(s,1H),8.70(s,1H),8.28(s,1H),8.09(s,1H),7.80(s,1H),7.34(m,2H),7.21(s,1H),6.95(s,1H),4.29(s,2H),4.16(s,2H),3.92(s,4H),3.56-3.42(m,2H),2.93-2.81(m,2H),2.73(s,4H),1.90(s,4H),1.71(s,2H).
实施例36：化合物T-36
实施例36所述化合物的制备可参考化合物T-18的合成方法和路线，将该路线中的起始原料4-氟-3甲氧基苯硼酸替换为5-甲氧基吡啶-3-硼酸，linker连接链和倒数第二步反应中的氨基侧链替换为特定基团即可。
Figure PCTCN2022088343-appb-000052
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.01(s,1H),8.73(s,1H),8.23(s,1H),8.04(s,1H),7.83(s,1H),7.35(m,2H),7.30(s,1H),6.91(s,1H),4.23(s,2H),4.15(s,2H),3.91-9.88(m,4H),3.58-3.44(m,3H),2.91-2.78(m,2H),2.71-2.68(m,4H),1.87(s,4H),1.69(s,4H).
实施例37：化合物T-37
实施例37所述化合物的制备可参考化合物T-18的合成方法和路线，将该路线中的起始原料4-氟-3甲氧基苯硼酸替换为5-甲氧基吡啶-3-硼酸，linker连接链和倒数第二步反应中的氨基侧链替换为特定基团即可。
Figure PCTCN2022088343-appb-000053
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.1(s,1H),9.07(s,1H),8.82(s,1H),8.75(s,1H),8.39(s,1H),8.08(s,1H),7.68-7.54(m,2H),7.38(s,1H),6.98(s,1H),4.20(s,2H),3.87(m,4H),3.61-3.57(m,2H),3.32-3.27(m,2H),2.75(s,4H),2.12(s,2H),1.71(s,4H).
实施例38：化合物T-38
化合物T-38的合成路线：
Figure PCTCN2022088343-appb-000054
中间体43的合成可参照中间体18的合成方法。
中间体45的合成：
将中间体44(30mmol)，2,4-二氯-5-氟嘧啶(20mmol)，碳酸钠(0.6mmol)和1,1'-双二苯基膦二茂铁二氯化钯(0.4mmol)加入到70ml的苯甲醚中，氮气置换空气三次。然后升温至130℃反应过夜，TLC检测原料反应完全，停止反应，冷却后抽滤，使用减压蒸馏(158℃)除去溶剂，然后使用柱层析分离纯化，得到中间体45，收率29％。HRMS[ESI] +,calcd for C 8H 6ClFN 4O[M+H] +,229.0214,found 229.0209.
中间体46的合成可参照中间体19的合成方法。
后续中间体的合成方法可参考实施例25所述的化合物T-25的合成方法，最终获得化合物T-38。 1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.81(s,1H),8.74(s,1H),7.96-9.94(m,2H),7.34(m,2H),7.08(s,1H),4.21(s,2H),4.10(s,2H),3.87(s,4H),3.56-3.41(m,2H),3.35(s,3H),2.91-2.80(m,2H),2.71(s,4H),1.74(s,4H),1.35(s,4H).
实施例39-40所述化合物的制备可参照化合物T-5的制备方法，其中，将4-氟-3-甲氧基苯硼酸替换成相应的硼酸或硼酸酯，并参照中间体2的合成方法合成所需的起始原料。
实施例39：化合物T-39
Figure PCTCN2022088343-appb-000055
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.67(s,1H),9.26(s,1H),8.93(s,1H),8.01(d,J＝3.0Hz,1H),7.91(d,J＝9.0Hz,1H),7.79-7.72(m,1H),7.50(s,1H),7.19(t,J＝9.0Hz,1H),6.85(d,J＝9.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.75(s,4H),2.60(s,4H),2.16(t,J＝6.0Hz,2H),2.01(t,J＝6.0Hz,2H),1.92-1.88(m,4H),1.74-1.68(m,4H).
实施例40：化合物T-40
Figure PCTCN2022088343-appb-000056
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.47(s,1H),8.72(s,1H),8.26(d,J＝3.0Hz,1H),8.19(s,1H),8.09(d,J＝6.0Hz,1H),7.67-7.62(m,1H),7.28(s,1H),6.99(d,J＝6.0Hz,1H),6.55(d,J＝3.0Hz,1H),4.44(s,2H),4.36(s,2H),3.89(s,3H),3.77-3.73(m,4H),2.60-2.55(m,4H),2.11-.2.05(m,4H),1.90-1.85(m,4H),1.72-1.67(m,4H).
实施例41-44所述化合物的制备可参照化合物T-5的制备方法，其中，可使用相应的二氯嘧啶衍生物参照中间体2的合成方法合成所需的起始原料。
实施例41：化合物T-41
Figure PCTCN2022088343-appb-000057
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.36(s,1H),8.53(d,J＝3.0Hz,1H),8.40(d,J＝3.0Hz,1H),8.30(s,1H),8.16(d,J＝9.0Hz,1H),7.80-7.76(m,1H),7.53(s,1H),7.26(t,J＝9.0Hz,1H),6.75(d,J＝9.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.74(t,J＝6.0Hz,4H),2.60(t,J＝6.0Hz,4H),2.15(q,J＝6.0Hz,2H),2.06(q,J＝6.0Hz,2H),1.90(t,J＝6.0Hz,4H),1.73(t,J＝6.0Hz,4H).
实施例42：化合物T-42
Figure PCTCN2022088343-appb-000058
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.46(s,1H),8.89(s,1H),8.45(d,J＝3.0Hz,1H),8.30(s,1H),8.22(d,J＝9.0Hz,1H),7.97-7.92(m,1H),7.48(s,1H),7.22(t,J＝9.0Hz,1H),6.70(d,J＝9.0Hz,1H),4.44(t,J＝6.0Hz,2H),4.36(t,J＝6.0Hz,2H),3.73(t,J＝6.0Hz,4H),2.62-2.58(m,4H),2.15(q,J＝6.0Hz,2H),2.08(q,J＝6.0Hz,2H),1.91-1.86(m,4H),1.72-1.67(m,4H).
实施例43：化合物T-43
Figure PCTCN2022088343-appb-000059
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.21(s,1H),8.44(s,1H),8.19(d,J＝9.0Hz,2H),8.09(d,J＝6.0Hz,1H),7.70-7.66(m,1H),7.25(s,1H),6.93(d,J＝6.0 Hz,1H),6.54(d,J＝3.0Hz,1H),4.40(s,2H),4.29(s,2H),3.70-3.64(m,4H),2.55-2.50(m,4H),2.16(t,J＝6.0Hz,2H),2.12(s,3H),2.09-2.06(m,2H),1.86-1.82(m,4H),1.68-1.64(m,4H).
实施例44：化合物T-44
Figure PCTCN2022088343-appb-000060
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝10.37(s,1H),8.30(s,1H),8.16(s,1H),8.00(d,J＝9.0Hz,2H),7.73-7.70(m,1H),7.50(s,1H),7.19(t,J＝9.0Hz,1H),6.70(d,J＝9.0Hz,1H),4.42(t,J＝6.0Hz,2H),4.33(t,J＝6.0Hz,2H),3.74(t,J＝6.0Hz,4H),2.58(t,J＝6.0Hz,4H),2.11(t,J＝6.0Hz,2H),2.03(t,J＝6.0Hz,2H),1.86(t,J＝6.0Hz,4H),1.70(t,J＝6.0Hz,4H).
实施例45：化合物T-45
Figure PCTCN2022088343-appb-000061
化合物T-45的合成：
(1)T-45-1的合成参考实施例5中所述的合成路线。
(2)a：将中间体T-45-1(1mmol)溶解在4ml的无水四氢呋喃中，降温至-10℃，氮气保护下滴加三乙胺(2mmol)，然后再加入三甲基乙酰氯(1.2mmol)，控制温度在-10℃，并在此温度下反应约1小时备用；b：将乙酰胺(1mmol)溶解在3ml无水四氢呋喃，降温至-78℃，氮气保护下缓慢加入n-Bu-Li(1.2mmol，2.7M in hexane)，控制温度在-70℃以下，搅拌约1小时后，将a部分新制的反应液缓慢滴加进去，控制温度在-70℃以下，反应约2小时，TLC检测原料反应完全，反应液捉奸升至室温后倒入适量的氯化铵水溶液中，使用二氯甲烷萃取三次，合并有机相并使用饱和氯化钠水溶液洗涤2次，无水硫酸钠干燥，抽滤，浓缩，最后使用柱层析分离得化合物T-45，收率36％。 1H NMR(300MHz,DMSO-d 6): δ(ppm)＝8.77(s,1H),8.18(s,2H),7.82(t,J＝6.0Hz,2H),7.61(s,1H),7.44(s,J＝9.0Hz,1H),6.90(d,J＝9.0Hz,1H),4.22(s,2H),4.09(s,2H),2.76(s,3H)1.94(s,4H),1.75(s,4H).
实施例46所述化合物的制备可参考实施例45中化合物T-45的合成方法与路线。
实施例46：化合物T-46
Figure PCTCN2022088343-appb-000062
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.79(s,1H),8.21(s,2H),7.85(t,J＝6.0Hz,2H),7.63(s,1H),7.45(s,J＝9.0Hz,1H),6.93(d,J＝9.0Hz,1H),4.24(s,2H),4.11(s,2H),3.75(t,J＝6Hz,4H),2.56-2.51(m,1H),2.12-1.94(m,8H),1.75-1.68(m,4H).
实施例47所述化合物的制备可参考实施例5中化合物T-5的合成方法与路线。
实施例47
Figure PCTCN2022088343-appb-000063
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.80(s,1H),8.23(s,2H),7.84(t,J＝6.0Hz,2H),7.64(s,1H),7.46(s,J＝9.0Hz,1H),6.95(d,J＝9.0Hz,1H),5.28(s,1H),4.67(s,1H),4.25(s,2H),4.11(s,2H),3.75-3.68(m,3H),3.52-3.49(m,2H),1.97-1.93(m,4H),1.77-1.70(m,4H).
实施例48-49所述化合物的制备可参照实施例18所述的合成方法和路线。
实施例48：化合物T-48
Figure PCTCN2022088343-appb-000064
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.82(s,1H),8.24(s,2H),7.87(t,J＝6.0Hz,2H),7.65(s,1H),7.46(s,J＝9.0Hz,1H),6.96(d,J＝9.0Hz,1H),4.26(s,2H),4.12(s,2H),3.62-3.56(m,2H),3.01-2.89(m,2H),2.14-1.93(m,6H),1.76-1.69(m,4H).
实施例49：化合物T-49
Figure PCTCN2022088343-appb-000065
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.84(s,1H),8.51(s,1H),8.23(s,1H),7.74(s,2H),7.54(m,2H),7.49(s,1H),6.99(s,1H),4.18(s,2H),4.02(s,2H),3.64-3.34(m,2H),3.02-2.94(m,4H),2.5-2.34(m,6H),1.92-1.83(m,4H),1.69-1.47(m,6H).
实施例50：化合物T-50
实施例50的制备方法可参照实施例25所述的化合物T-25的合成方法与路线。
Figure PCTCN2022088343-appb-000066
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝8.81(s,1H),8.24(s,2H),7.79(t,J＝6.0Hz,2H),7.65(s,1H),7.46(s,J＝9.0Hz,1H),6.96(d,J＝9.0Hz,1H),4.33(s,2H),3.77(s,4H),3.69-3.63(m,2H),3.25-3.21(m,2H),3.03(s,2H),2.75(s,4H),1.94(s,4H),1.77(s,4H).
实施例51-54所述化合物的制备可参考化合物T-5的合成方法和路线。
实施例51：化合物T-51
Figure PCTCN2022088343-appb-000067
1H NMR(300MHz,DMSO-d 6):δ(ppm)＝9.03(s,1H),8.56(s,1H),8.49(s,1H),8.39(s,1H),8.34(s,1H),8.23-8.19(d,J＝9Hz,1H),8.16(s,1H),7.58(s,1H),4.41(s,2H),4.25(s,2H),3.91(s,4H),3.75-3.63(m,1H),2.13-1.96(m,6H),1.85-1.74(m,6H),1.54-1.40(m,4H).
实施例52：化合物T-52
Figure PCTCN2022088343-appb-000068
1H NMR(300MHz,CDCL3):δ(ppm)＝8.91(s,1H),8.52(s,1H),8.49(s,1H),8.38(s,1H),8.31(s,1H),8.22-8.19(d,J＝9Hz,1H),8.12(s,1H),7.56(s,1H),4.36(s,2H),4.19(s,2H),3.97(s,4H),3.79-3.68(m,1H),2.15-1.99(m,6H),1.82-1.70(m,6H),1.53-1.41(m,6H).
实施例53：化合物T-53
Figure PCTCN2022088343-appb-000069
1H NMR(300MHz,CDCL3):δ(ppm)＝8.37(s,1H),8.22(s,1H),8.14(d,J＝6.0Hz,1H),8.04(s,1H),7.88(s,1H),7.67-7.62(m,1H),7.56(s,1H),7.23-7.16(m,1H),4.38(s,2H),4.19(s,2H),3.98(s,4H),3.77-3.67(m,1H),2.10-1.98(m,6H),1.80-1.1.71(m,6H),1.51-1.40(m,6H).
实施例54：化合物T-54
Figure PCTCN2022088343-appb-000070
1H NMR(300MHz,CDCL3):δ(ppm)＝8.41(s,1H),8.25(s,1H),8.16(d,J＝6.0Hz,1H),8.07(s,1H),7.91(s,1H),7.69-7.62(m,1H),7.55(s,1H),7.23-7.14(m,1H),4.32(s,2H),4.19(s,2H),3.97(s,4H),3.78-3.67(m,1H),2.05-1.93(m,6H),1.86-1.1.72(m,6H),1.50-1.41(m,4H).
以下为此发明部分化合物的生物活性评价，主要包括细胞水平与酶水平两个方面。
一、本发明部分化合物对多种癌细胞的增殖抑制活性
实验方法如下：
以MTT法测定化合物对CDK9高表达的肿瘤细胞(MV4-11，MCF7以及MOLM13)的细胞毒性：细胞以2000个/孔接种于96孔板中，待细胞贴壁后，吸除培养基，加入200uL稀释好的药物，37度，5％CO2培养72h，之后加入10uL/孔的MTT。37度孵育4个小时，吸去上清，每孔加入150uL的DMSO，多功能酶标仪492nm处检测吸光值。用GraphPad计算IC 50并绘制细胞生长曲线。
二、本发明部分化合物对CDK9和CDK2蛋白的抑制活性
实验方法如下：
首先将化合物用100％DMSO配制成10mM储存液于低温下避光保存。
激酶反应过程如下：
1)配制1×Kinase buffer。
2)化合物浓度梯度的配制：受试化合物测试浓度为5nM,20nM或100nM，复孔检测。在384孔板中配置成100倍终浓度的化合物。然后用Echo550转移250nl到384反应板中备用。阴性对照孔和阳性对照孔中分别加250nl的100％DMSO。
3)用1×Kinase buffer配制2.5倍终浓度的激酶溶液。
4)在化合物孔和阳性对照孔分别加10μl的2.5倍终浓度的激酶溶液；在阴性对照孔中加10μl的1×Kinase buffer。
5)1000rpm离心30秒，振荡混匀后室温孵育10分钟。
6)用1×Kinase buffer配制25/15倍终浓度的ATP和Kinase substrate的混合溶液。
7)加入15μl的25/15倍终浓度的ATP和底物的混合溶液，起始反应。
8)将384孔板1000rpm离心30秒，振荡混匀后室温孵育相应的时间。
9)加入30μl终止检测液停止激酶反应，1000rpm离心30秒，振荡混匀。
10)用Caliper EZ Reader读取转化率。
最后进行数据计算和分析：
1)抑制率计算公式：
Figure PCTCN2022088343-appb-000071
其中：Conversion％_sample是样品的转化率读数；Conversion％_min：阴性对照孔均值，代表没有酶活孔的转化率读数；Conversion％_max：阳性对照孔均值，代表没有化合物抑制孔的转化率读数。
2)拟合量效曲线
以浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 5拟合量效曲线，从而得出各个化合物对酶活性的IC 50值。
公式如下：Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
本发明中部分化合物对CDK9和CDK2的抑制活性和其对MV4-11，MCF-7，HCT116以及MOLM13细胞的抗增殖活性如下表1所示：
表1.本发明中部分化合物的酶活性和细胞活性
Figure PCTCN2022088343-appb-000072
Figure PCTCN2022088343-appb-000073
a：抑制率一栏中，括号内表示化合物浓度，单位为nM，如90(20)表示化合物在20nM浓度时对酶的抑制率为90％。
其中，TG-02的结构如下：
Figure PCTCN2022088343-appb-000074
根据表1所示数据，本发明所述的化合物对CDK9显示出有效的抑制活性，大部分化合物在20nM浓度下对CDK9的抑制率在90％以上。同时，与目前唯一报道的处于临床研究阶段的大环类广谱CDK抑制剂TG02相比，本发明所述化合物显示出显著的CDK9选择性。同时，其对MV4-11，MCF-7，HCT-116以及MOLM13等多种肿瘤细胞具有显著的抗增殖抑制活性。其中，优势化合物T-9，T-10，T-52和T-53均是高选择性的CDK9抑制剂，对CDK2的选择性在50倍以上，同时均具有不错的抗肿瘤细胞增殖活性。化合物T-53对CDK9抑制IC 50值为7nM，对CDK2抑制IC 50值大于1000nM，选择性大于100倍，显著优于化合物TG02。

Claims (10)

  1. 一种如通式I所示结构的化合物，或其药学上可接受的盐型，其特征在于：
    Figure PCTCN2022088343-appb-100001
    其中，Ar选自如下基团：
    Figure PCTCN2022088343-appb-100002
    X选自氢，氘，卤素，氰基，甲基，三氟甲基；
    R 1选自如下基团：
    Figure PCTCN2022088343-appb-100003
    L选自如下结构：
    Figure PCTCN2022088343-appb-100004
  2. 根据权利要求1所述化合物，或其药学上可接受的盐型，其特征在于药学上可接受的盐型是指通式I所示的化合物和药学上可接受的酸形成的盐，所述的药学上可接受的酸为无机酸盐或有机酸盐；优选的，无机酸盐包括：盐酸、硫酸、磷酸、碳酸、碳酸氢根、硝酸、磷酸一氢根、磷酸二氢根，氢溴酸或氢碘酸；优选的，有机酸盐包括：马来酸、酒石酸、柠檬酸、甲磺酸、琥珀酸、乙酸、对甲苯磺酸、扁桃酸、异丁酸或丙二酸。
  3. 根据权利要求1所述化合物，或其药学上可接受的盐型，其特征在于Ar选自
    Figure PCTCN2022088343-appb-100005
  4. 根据权利要求1所述化合物，或其药学上可接受的盐型，其特征在于X为卤素；优选X为F、Cl、Br、I；进一步优选X为F。
  5. 根据权利要求1所述化合物，或其药学上可接受的盐型，其特征在于L选自：
    Figure PCTCN2022088343-appb-100006
  6. 如下任一所述的化合物或其药学上可接受的盐：
    Figure PCTCN2022088343-appb-100007
    Figure PCTCN2022088343-appb-100008
    Figure PCTCN2022088343-appb-100009
    Figure PCTCN2022088343-appb-100010
  7. 一种制备权利要求1所述化合物的制备方法，
    Figure PCTCN2022088343-appb-100011
    其中，Ar、X、R 1、L定义如前所述。
  8. 药物组合物，其特征在于，包括权利权利要求1-6任一项所述的化合物，或其药学上可接受的盐型。
  9. 权利要求1-6任一项所述的化合物，或其药学上可接受的盐型在制备CDK9抑制剂药物中的应用。
  10. 权利要求1-6任一项所述的化合物，或其药学上可接受的盐型在制备抗病毒药物或者抗肿瘤药物中的应用；优选的，
    所述的病毒包括：HIV病毒、巨细胞病毒、EB病毒、腺病毒、疱疹、人T细胞淋巴细胞病毒；优选的，所述的肿瘤包括神经胶质瘤、各类白血病、淋巴癌、肝癌、胃癌、前列腺癌、卵巢癌、乳腺癌、肺癌。
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