WO2024051720A1 - 靶向抑制clk2的5-吡啶-1h-吲唑类化合物及其应用 - Google Patents

靶向抑制clk2的5-吡啶-1h-吲唑类化合物及其应用 Download PDF

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WO2024051720A1
WO2024051720A1 PCT/CN2023/117144 CN2023117144W WO2024051720A1 WO 2024051720 A1 WO2024051720 A1 WO 2024051720A1 CN 2023117144 W CN2023117144 W CN 2023117144W WO 2024051720 A1 WO2024051720 A1 WO 2024051720A1
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compound
synthesis
nmr
dmso
pyridine
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French (fr)
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李志裕
卞金磊
宋佳星
邱志霞
胡天星
刘迈
秦真
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C07D401/14—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 three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the invention relates to the field of drug synthesis, and in particular to a 5-pyridine-1H-indazole compound that targets CLK2 and its application.
  • Osteoarthritis is a worldwide bone and joint disease that affects the health of 250 million people worldwide. It is characterized by synovial inflammation, cartilage loss, and subchondral bone remodeling. The synovium of OA patients is rich in stem cells. Therefore, patients' inability to regenerate articular cartilage is not due to insufficient supply of stem cells, but improper differentiation of stem cells as they try to restore healthy cartilage.
  • the Wnt pathway plays a role in organogenesis, cell differentiation, and tissue remodeling. plays a central role. According to the latest research, abnormal activation or inhibition of the Wnt signaling pathway can lead to the occurrence of the disease. Therefore, the Wnt signaling pathway is a potential target for the treatment of osteoarthritis.
  • MMPs proteins MMP3, MMP13, ADAMTS5, IHH, etc.
  • Cartilage Oligomeric Matrix Protein is a newly discovered extracellular matrix protein in recent years. Because there are reports in the literature that it has significance as a biomarker in the process of arthritis cartilage degeneration, it may become an early The "gold indicator" for diagnosing joint lesions is receiving more and more attention.
  • OA patients also have high levels of inflammatory factors (IL-6, IL-1 ⁇ , TNF- ⁇ , etc.).
  • the Wnt signaling pathway is a set of multi-downstream signaling pathways triggered by the binding of the ligand protein Wnt and membrane protein receptors. Through this pathway, extracellular signals are transmitted into cells through the intracellular activation process of cell surface receptors.
  • Wnt pathway if there is no Wnt protein on the cell membrane surface, its downstream ⁇ -Catenin protein will be decomposed by the glycogen synthase 3 (GSK3) complex in the cytoplasm, resulting in its inability to enter the nucleus to initiate the transcription of related Wnt genes.
  • GSK3 glycogen synthase 3
  • the protein kinase family CLK (CDK-likekinase) is a dual-specificity protein kinase that can regulate intracellular signal transduction through phosphorylation of substrate proteins on tyrosine, serine or threonine residues; it can be divided into four subgroups.
  • Types (CLK1, CLK2, CLK3 and CLK4), the C segments of the proteins encoded by these four subtypes all have a highly conserved gene sequence and have the same structurally similar amino acid sequence.
  • the CLK2 isoform is found in most eukaryotes and is involved in the phosphorylation of the SR (serine/arginine) protein domain to regulate the selective splicing of RNA.
  • CLK2 kinase plays an important role in gluconeogenesis and fatty acid oxidation in the liver; it is also a therapeutic target for liver cancer, breast cancer and Alzheimer's disease.
  • CLK2 is a potential therapeutic target for the Wnt pathway and osteoarthritis.
  • Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A belongs to the DYRK family, which is highly conserved in evolution. In mammals, the DYRK family has five different subtypes, and only DYRK1A Located in the DSCR region of human chromosome 21. DYRK1A is expressed by the dyrk1a gene, and the encoded mature protein consists of 763 amino acids, including a protein kinase domain and other special structures. Studies have shown that many important proteins can serve as substrates of DYRK1A and are regulated by it to participate in various biological functions in cells. For example, neurodevelopment, cell proliferation and differentiation, tumorigenesis, and neurodegenerative diseases.
  • the present invention provides 5-pyridine-1H-indazole compounds that target CLK2 and their applications.
  • Biological activity evaluation shows that the compound of the present invention has significant CLK2 inhibitory activity, good selectivity for CLK family members, and significant DYRK1A inhibitory activity.
  • the pharmacodynamic results show that the compound of the present invention has good anti-osteoarthritis activity both in vivo and in vitro.
  • the technical problem to be solved by the present invention is to provide 5-pyridine-1H-indazole compounds, which are compounds represented by formula (I), formula (II) or formula (III), or pharmaceutically acceptable salts thereof, or their precursors.
  • R 1 , R 2 or R 4 are selected from the following groups: C1 to C10 alkyl, substituted or unsubstituted 6-10 membered aryl group, substituted or unsubstituted containing 1-3 selected from N, O and One of the 5-10 membered heteroaryl groups of S heteroatoms.
  • the substituents on the aryl or heteroaryl groups are selected from halogen, Cl ⁇ C6 alkyl, Cl ⁇ C6 alkoxy, Cl ⁇ C6 alkylthio.
  • halogenated Cl ⁇ C6 alkyl group halogenated Cl ⁇ C6 alkoxy group, amino group, Cl ⁇ C6 alkanoyl group, NH-CO-Cl ⁇ C6 alkyl group, -NH (Cl ⁇ C6 alkyl group), -N( One or more of Cl ⁇ C6 alkyl) (Cl ⁇ C6 alkyl) and hydroxyl;
  • R 3 is selected from the following groups:
  • said R 1 is selected from:
  • said R 2 is selected from:
  • said R 4 is selected from:
  • the compound is as follows:
  • the pharmaceutically acceptable salt form of the compound of the present invention refers to the salt formed by the compound represented by the general formula I, II and III and a pharmaceutically acceptable acid.
  • 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 acid salts include: malic acid, maleic acid, Tartaric acid, citric acid, methanesulfonic acid, succinic acid, acetic acid, p-toluenesulfonic acid, mandelic acid, isobutyric acid, malonic acid, etc.
  • the present invention further provides pharmaceutical compositions of the above compounds.
  • the present invention also includes the use of the compound or the pharmaceutical composition in preparing drugs that target CLK2 inhibition.
  • the present invention also includes the use of a compound as described or a pharmaceutical composition as described in the preparation of drugs for treating osteoarthritis.
  • the present invention has the following advantages:
  • the 5-pyridine-1H-indazole compound provided by the present invention has high selectivity and is a CLK2 small molecule inhibitor with good pharmaceutical properties.
  • the compound structure of the present invention introduces an alkynyl group, the structure is more novel, and shows effective inhibitory activity on CLK2.
  • Several of the compounds have an inhibitory rate of CLK2 of more than 80% at a concentration of 20nM.
  • the compound of the present invention shows better CLK2 inhibitory activity and at the same time shows better selectivity than the positive drug.
  • the compound of the present invention shows good anti-osteoarthritis activity both in vitro and in vivo.
  • Figure 4 shows the gene expression levels of cartilage-degrading proteases in cartilage during the treatment of compound LQ23 in rat MIA osteoarthritis model.
  • A Day 11;
  • Figure 5 shows the effects of COMP and COMP in serum during the treatment of rat MIA osteoarthritis model by compound LQ23 of the present invention.
  • Dissolve raw material 4 (3-amino-5-bromopyridine, 2g, 12mmol) in dichloromethane and cool it to 0°C in an ice bath.
  • Dissolve isovaleric acid chloride (2g, 17mmol) in dichloromethane and add it dropwise.
  • Ethylamine (2.3g, 23mmol). under ice bath Stir for 30 minutes. After TLC detects that the reaction is complete, the solvent is evaporated under reduced pressure, and ethyl acetate is added to dissolve and extract. The organic layer is washed with dilute hydrochloric acid, saturated sodium bicarbonate, concentrated brine, and dried over anhydrous sodium sulfate. It is suction-filtered and the filtrate is evaporated under reduced pressure.
  • LQ05 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ05 are as follows:
  • LQ07 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ07 are as follows:
  • LQ10 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ10 are as follows:
  • LQ11 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ11 are as follows:
  • LQ12 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ12 are as follows:
  • Example 14 Compound 3-methyl-N-(5-(3-(quinolin-2-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)butanamide (LQ14) synthesis
  • LQ14 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ14 are as follows:
  • LQ15 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ15 are as follows:
  • LQ16 was synthesized using the method of Example 1.
  • the structure and characterization data of LQ16 are as follows:
  • Example 18 Compound 5-(3-(3-(3-hydroxypyrrolidin-1-yl)-3-oxopropyl-1-en-1-yl)-1H-indazol-5-ylpyridine Synthesis of -3-yl-3-methylbutanamide (LQ21)
  • Example 27 Compound 1-((5-(3-((3-fluorophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy)-4-methylpentanyl -Synthesis of 2-amine (LQ27)
  • Example 28 Compound 1-(5-(3-(3-fluorophenyl)ethynyl)-1H-indazol-5-ylpyridin-3-yl)-3-phenylpropan-2-amine (LQ28 )Synthesis
  • Example 29 Compound (S)-1-((5-(3-(cyclopentylethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy)-4-methylpentanyl -Synthesis of 2-amine (LQ29)
  • LQ29 was synthesized using the method of Example 20.
  • the structural formula and characterization data of LQ29 are as follows:
  • Example 30 Compound 3-([1,1'-biphenyl]-4-ylethynyl)-5-(5-((3,3-difluoropyrrolidin-1-yl)methyl)pyridine- Synthesis of 3-yl)-1H-indazole (LQ30)
  • LQ30 was synthesized using the method of Example 26.
  • the structural formula and characterization data of LQ30 are as follows:
  • Example 31 Compound 1-((5-(3-([1,1'-biphenyl]-4-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy) Synthesis of -4-methylpentan-2-amine (LQ31)
  • LQ31 was synthesized using the method of Example 20.
  • the structural formula and characterization data of LQ31 are as follows:
  • LQ32 was synthesized using the method of Example 20.
  • the structural formula and characterization data of LQ32 are as follows:
  • LQ33 was synthesized using the method of Example 20.
  • the structural formula and characterization data of LQ33 are as follows:
  • Example 34 Enzyme inhibitory activity of the compounds of Examples 1-33
  • Z'LYTE TM kit (ThermoFisher) was used to detect the inhibitory rate of compounds on kinases using the FRET principle.
  • the compound LQ01-33 prepared in Examples 1-33 was subjected to an in vitro CLK2 enzyme inhibitory activity study, and the inhibitory rate of the compound at a concentration of 20 nM on the kinase and the IC 50 of the dominant compound were tested.
  • the peptide substrate is labeled with two fluorophores, one at each end, forming a FRET pair.
  • a kinase transfers the g-phosphate of ATP to a single serine or threonine residue in a synthetic peptide substrate.
  • site-specific proteases site-specific proteases (development reagents) recognize and cleave non-phosphorylated peptides. Phosphorylated peptides are inhibited from cleavage by developing reagents. Cleavage destroys FRET between the donor (i.e. coumarin) and acceptor (i.e.
  • the ratio method calculates the ratio of donor emission to acceptor emission (emission ratio) after the donor fluorophore is excited at 400nm, and quantifies the reaction process:
  • the selectivity of LQ23 for CLK2 (VS CLK3 IC50>100nM) is at least 15 times improved.
  • Example 35 LQ23 regulates the levels of cartilage-degrading proteases and inflammatory factors in vitro
  • rat chondrocytes were treated with TNF- ⁇ (20ng/mL) and Oncostatin M (10ng/mL) to induce inflammation, they were immediately administered and treated for 48 hours.
  • TNF- ⁇ (20ng/mL
  • Oncostatin M (10ng/mL)
  • Rat bone marrow mesenchymal stem cells were treated with TNF- ⁇ (20ng/mL) and Oncostatin M (10ng/mL) to induce inflammation, and then treated immediately for 72 hours.
  • RT-qPCR detected MMP3, MMP13, ADAMTS5, IL-1 ⁇ and TNF. - ⁇ gene expression level, the results are shown in Figure 3.
  • Example 36 LQ23 regulates the levels of cartilage-degrading proteases and inflammatory factors in vivo
  • MIA monosodium iodoacetate
  • Rat cartilage tissue was taken, and RT-qPCR was used to detect the gene expression levels of cartilage-degrading proteases and inflammatory factors; serum samples were taken and ELISA was used.
  • the kit detects the contents of COMP and inflammatory factors. The experimental results are shown in Figure 4-7.
  • LQ23 can significantly reduce the expression of cartilage-degrading proteases such as MMP13, MMP3, ADAMTS5 and IHH, and significantly reduce COMP and various inflammation in serum.
  • the content of factors and effectiveness are comparable to Lorecivivint.
  • LQ23 can significantly reduce cartilage degradation.

Abstract

本发明公开了一种靶向抑制CLK2的5-吡啶-1H-吲唑类化合物及其应用。本发明所述的化合物为式(I)、式(II)或式(III)所示化合物或其药用盐、其前药、其水合物或其溶剂合物。本发明的化合物具有显著的CLK2抑制活性，对CLK家族成员具有较好的选择性，同时具有显著的DYRK1A抑制活性，并在体外和体内都显示出良好的抗骨关节炎活性。

Description

靶向抑制CLK2的5-吡啶-1H-吲唑类化合物及其应用 技术领域
本发明涉及药物合成领域，特别涉及一种靶向抑制CLK2的5-吡啶-1H-吲唑类化合物及其应用。
背景技术
骨关节炎(Osteoarthritis,OA)是一种世界范围内的骨关节病变，影响着全球2.5亿人的健康，其以滑膜炎症，软骨损失，软骨下骨重塑特征。OA患者滑膜富含丰富的干细胞，因此，患者不能再生关节软骨并不是由于干细胞供应不足，而是干细胞试图恢复健康软骨时分化不当，而Wnt通路在器官发生，细胞分化和组织重塑中发挥着核心作用。据最新研究显示，Wnt信号通路的异常激活或抑制都会导致该疾病的发生。因此，Wnt信号通路是治疗骨关节炎的潜在靶点。在OA进程中，软骨的降解是不可逆的，其中MMPs蛋白(MMP3,MMP13,ADAMTS5,IHH等)是一组参与软骨降解的蛋白，对OA的病理进程起到推动作用。软骨寡聚基质蛋白(Cartilage Oligomeric Matrix Protein,COMP)是近年来新发现的一种细胞外基质蛋白,因有文献报道其在关节炎软骨退变进程中有作为生物标记物的意义，可能成为早期诊断关节病变的"金指标"而正受到越来越多的关注。除此之外，OA作为一种炎症性疾病，其患者体内炎症因子(IL-6,IL-1β,TNF-α等)的含量亦偏高。
Wnt信号传导途径是由配体蛋白质Wnt和膜蛋白受体结合激发的一组多下游通道的信号传导途径。经此途径，通过细胞表面受体胞内的活化过程将细胞外信号传递到细胞内。在经典的Wnt通路中，若细胞膜表面无Wnt蛋白时，其下游的β-Catenin蛋白在细胞质中会被糖原合酶3(GSK3)复合物分解，导致其不能进入细胞核启动相关Wnt基因的转录；而细胞膜表面有Wnt蛋白时，其会抑制GSK3复合物，从而使β-Catenin蛋白在细胞核内积聚，最终启动Wnt通路相关基因的转录。骨关节的稳态与Wnt通路存在着微妙的平衡，这一平衡被打破有可能导致OA的发生。
蛋白激酶家族CLK(CDK-likekinase)是一种双特异性蛋白激酶，可通过酪氨酸、丝氨酸或苏氨酸残基底物蛋白磷酸化调控细胞内信号转导；其可分为四个亚型(CLK1，CLK2，CLK3和CLK4)，此四个亚型编码的蛋白质C段都有一个高度保守的基因序列，并且具有同样一个结构类似的氨基酸序列。其中CLK2亚型被发现于大部分真核生物中，其参与对SR(丝氨酸/精氨酸)蛋白结构域的磷酸化从而调节RNA的选择性剪切。研究证实CLK2激酶在肝脏中糖异生和脂肪酸的氧化发挥着重要作用；同时是肝癌、乳腺癌及阿尔茨海默病的治疗靶点。最新研究显示，CLK2是Wnt通路和骨关节炎的潜在治疗靶点。
双特异性酪氨酸磷酸化调节激酶1A(Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A,DYRK1A)属于DYRK家族，其在进化上高度保守，在哺乳动物中，DYRK家族有五种不同的亚型，只有DYRK1A位于人21号染色体的DSCR区域。DYRK1A由dyrk1a基因表达，编码的成熟蛋白由763个氨基酸组成，包括一个蛋白激酶结构域以及其他特殊结构。研究表明，许多重要蛋白可作为DYRK1A的底物，并受其调控而参与细胞中的多种生物学功能。例如神经发育、细胞增殖与分化、肿瘤发生以及神经退行性疾病等。
目前，Sumumed公司开发的First-in-class小分子药物Lorecivivint已应用于三期临床来治疗骨关节炎，其具体机制主要是通过抑制CLK2介导的SR蛋白磷酸化，从而诱导早期软骨形成；以及抑制DYRK1A介导的SIRT1和FOXOA磷酸化来增强成熟软骨 细胞功能；并且可通过抑制NF-γB，STAT3等炎症因子从而减轻炎症。因此，研究人员认为：CLK2在Wnt通路以及干细胞的生成与分化中发挥着至关重要的作用，其作为治疗骨关节炎药物开发已经受到很多科研单位和药物开发者的重视。但是，CLK2激酶抑制剂的研究尚处于起步阶段。
发明内容
发明目的：本发明提供了靶向抑制CLK2的5-吡啶-1H-吲唑类化合物及其应用。从生物活性评价显示，本发明的化合物具有显著的CLK2抑制活性，对CLK家族成员具有较好的选择性，同时具有显著的DYRK1A抑制活性。药效结果显示，本发明的化合物在体内和体外都具有良好的抗骨关节炎活性。
技术方案：本发明所要解决的技术问题是提供了5-吡啶-1H-吲唑类化合物，为式(Ⅰ)、式(Ⅱ)或式(Ⅲ)所示化合物或其药用盐、其前药、其水合物或其溶剂合物：
其中，R1、R2或R4选自如下基团：C1～C10烷基、取代或者未取代的6-10元芳基、取代或者未取代的含有1-3个选自N、O和S杂原子的5-10元杂芳基中的一种，所述芳基或者杂芳基上的取代基选自卤素、Cl～C6烷基、Cl～C6烷氧基、Cl～C6烷硫基、卤代Cl～C6烷基、卤代Cl～C6烷氧基、氨基、Cl～C6烷酰基、NH-CO-Cl～C6烷基、－NH(Cl～C6烷基)、－N(Cl～C6烷基)(Cl～C6烷基)和羟基中的一个或者多个；
R3选自以下基团：
优选地，所述R1选自：

优选地，所述R2选自：
优选地，所述R4选自：
作为本发明的一种具体实施方式，所述化合物如下所示：


进一步地，本发明所述化合物的药用盐为药学上可接受的盐型是指通式I、Ⅱ和Ⅲ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；有机酸盐包括：苹果酸、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。
本发明进一步提供了上述的化合物的药物组合物。
本发明内容还包括所述的化合物或所述的药物组合物在制备靶向抑制CLK2的药物中的应用。
本发明内容还包括一种如所述的化合物或所述的药物组合物在制备治疗骨关节炎药物中的应用。
有益效果：与现有技术相比，本发明具备以下优点：
(1)本发明提供的5-吡啶-1H-吲唑类化合物，具备高选择性，是具有良好成药性质的CLK2小分子抑制剂。
(2)本发明所述的化合物结构引入炔基，结构更为新颖，且对CLK2显示出有效的抑制活性，其中几个化合物在20nM浓度下对CLK2的抑制率在80％以上，同时，与进入三期临床的CLK2抑制剂Lorecivivint相比，本发明所述化合物显示更优的CLK2抑制活性，同时显示出比阳性药更优的选择性。
(3)本发明所述的化合物在体外和体内都显示出良好的抗骨关节炎活性。
附图说明
图1为本发明化合物LQ23的CLKs激酶抑制活性，(n＝2,Mean±SD)；
图2为本发明化合物LQ23在大鼠软骨细胞中对软骨降解蛋白酶及炎症因子水平的调控，(n＝3,Mean±SD)；
图3为本发明化合物LQ23在大鼠间充质干细胞中对软骨降解蛋白酶及炎症因子水平的调控，(n＝3,Mean±SD)；
图4为化合物LQ23在大鼠MIA骨关节炎模型中的治疗中软骨中软骨降解蛋白酶基因表达水平。(A)第11天；(B)第28天。(n＝3,Mean±SD)；
图5为本发明化合物LQ23在大鼠MIA骨关节炎模型中的治疗中血清中COMP和 炎症因子的基因表达水平，(A)第11天；(B)第28天。(n＝3,Mean±SD)；
图6本发明化合物LQ23在大鼠ACLT+pMMx骨关节炎模型中的治疗中软骨中软骨降解蛋白酶基因表达水平。(A)第5周；(B)第13周；(n＝3,Mean±SD)；
图7为本发明化合物LQ23在大鼠ACLT+pMMx骨关节炎模型中的治疗中的血清中COMP和炎症因子的基因表达水平，(A)第5周；(B)第13周；(n＝3,Mean±SD)。
具体实施方式
实施例1：化合物LQ01的合成
中间体2的合成：
将原料1(5-溴吲唑，5g，25mmol)加入到250mL三颈瓶中，加入四氢呋喃(50mL)、对甲基苯磺酸(0.48g，2.5mmol)，室温下滴加3,4-二氢吡喃(4.3g，50mmol)，加热至回流反应8h。TLC检测反应完全，随后减压蒸除溶剂，粗品通过硅胶柱层析法得到白色固体7.1g，收率63％。中间体2的表征数据如下HRMS(ESI+):cacld for C12H13BrN2O(M+H)+,281.0284；found,281.0256.
中间体3的合成：
将中间体2(1.3g,4.6mmol)加入到100mL单颈瓶中，加入联硼酸频那醇酯(1.4g，5.5mmol)、乙酸钾(1.4g，13.8mmol)、1,1'-双二苯基膦二茂铁二氯化钯(0.17g，0.23mmol)、二氧六环(13mL)，氮气保护，升温至回流反应6h，TLC检测反应完全，降温后抽滤，滤饼用少量乙酸乙酯洗，滤液减压蒸除溶剂，粗产品通过硅胶柱层析法纯化得到中间体3，白色固体(1.2g，收率81％)。中间体3表征数据如下：1H NMR(300MHz，CDCl3-d6):δ＝8.25(t,J＝1.1Hz,1H),8.04(s,1H),7.82–7.78(dd,J1＝1.1Hz,J2＝8.6Hz,1H),7.56(d,J＝8.5Hz,1H),5.75–5.71(dd,J1＝2.7Hz,J2＝9.5Hz,1H),4.11–4.01(m,1H),3.79–3.71(dt,J1＝2.9Hz,J2＝10.5Hz,1H),2.64–2.52(m,1H),2.18–2.03(m,2H),1.83–1.63(m,3H),1.37(s,12H)ppm.HRMS(ESI+):cacld for C18H25BN2O3(M+H)+,329.2031；found,329.1998.
中间体5的合成：
将原料4(3-氨基-5-溴吡啶，2g，12mmol)溶于二氯甲烷后冰浴降温至0℃，异戊酸酰氯(2g，17mmol)溶于二氯甲烷后滴入后加入三乙胺(2.3g，23mmol)。冰浴下 搅拌30min。TLC检测反应完全后，减压蒸除溶剂，加入乙酸乙酯溶解，萃取，有机层经稀盐酸、饱和碳酸氢钠洗、浓盐水洗、无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂，用少量二氯甲烷打浆得白色固体2.2g，收率74％。中间体5表征数据如下：1H NMR(300MHz，DMSO-d6):δ＝10.32(s,1H),8.64(d,1H),8.42-8.37(d,J＝12.0Hz,1H),2.46-2.22(d,J＝7.1Hz,2H),0.94(d,J＝6.6Hz,6H)ppm.HRMS(ESI+):cacld for C10H13BrN2O(M+H)+,257.0284；found,257.0292.
中间体6的合成：
将中间体5(0.1g，0.4mmol)、中间体3(0.13g，0.4mmol)、碳酸钠(0.12g，1.17mmol)、1,1'-双二苯基膦二茂铁二氯化钯(14mg，0.02mmol)、二氧六环(2mL)和水(0.3mL)加入到50mL单颈瓶中，氮气置换空气，升温至回流下反应8h。TLC检测反应完全后，降温抽滤，滤饼用少量乙酸乙酯洗，滤液旋干，粗品经硅胶柱层析纯化得到中间体6，黄色油状物，收率69％。中间体6表征数据如下：1H NMR(300MHz，DMSO-d6):δ＝10.2(s,1H),8.72(s,1H),8.61(s,1H),8.40(s,1H),8.21(s,1H),8.07(s,1H),7.87(m,1H),7.73(m,1H),5.92–5.88(m,1H),3.93–3.89(m,1H),3.81-3.75(m,1H),2.42(m,1H),2.33(m,2H),2.26-2.05(m,3H),1.76(m,1H),1.60(m,2H),0.98(d,6H)ppm.HRMS(ESI+):cacld for C22H26N4O2(M+H)+,379.2179；found,379.2134.
中间体7的合成：
将中间体6(0.14g,0.4mmol)、三氟乙酸(1mL)依次加入到二氯甲烷(1mL)中，室温反应1小时。TLC检测反应完全后，减压蒸除溶剂，加入乙酸乙酯萃取，有机层用饱和碳酸氢钠溶液洗3次，浓盐水洗，无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂，真空干燥得到0.1g黄色固体，收率84％。中间体7表征数据如下：HRMS(ESI+):cacld for C17H18N4O(M+H)+,295.1553；found,295.1555.
中间体8的合成：
将中间体7(0.15g，0.51mmol)，氢氧化钾(0.14g，2.5mmol)，N,N-二甲基甲酰胺(2mL)加入到三颈瓶中，氮气保护，冰浴降温至0℃，将碘(0.16g，0.61mmol)分批加到反应液中，加毕后室温反应1小时。待TLC检测反应完全后，用饱和硫代硫 酸钠溶液淬灭，固体析出，抽滤，滤饼干燥得到中间体8，粉白色固体(0.11g，收率52％)。中间体8表征数据如下：1H NMR(300MHz，CDCl3-d6):δ＝8.63(d,J＝1.6Hz,1H),8.56(d,J＝2.2Hz,2H),8.20(d,J＝8.7Hz,1H),7.83(dd,J1＝8.8Hz,J2＝1.8Hz,1H),7.70-7.62(m,1H),2.32(m,2H),2.25(m,1H),1.74(s,9H),1.05(d,J＝6.2Hz,6H)ppm.HRMS(ESI+):cacld for C17H17IN4O(M+H)+,421.0520；found,421.0532.
中间体9的合成：
将中间体8(50mg，0.12mmol)、4-二甲氨基吡啶(1.5mg，0.012mmol)、三乙胺(36mg，0.36mmol)加到二氯甲烷(0.5mL)中搅拌，后将二碳酸二叔丁酯(39mg，0.18mmol)加到反应液中，室温反应2小时。TLC检测反应完全后，将反应液减压蒸除溶剂，用正己烷打浆，抽滤，滤饼真空干燥得到中间体9，淡黄色固体(39mg，收率63％)。中间体9表征数据如下：1H NMR(300MHz，DMSO-d6):δ＝8.63(d,J＝1.6Hz,1H),8.56(d,J＝2.2Hz,2H),8.20(d,J＝8.7Hz,1H),7.83(dd,J1＝8.8Hz,J2＝1.8Hz,1H),7.70-7.62(m,2H),2.32(d,2H),2.25(m,1H),1.74(s,9H),1.05(d,J＝6.2Hz,6H)ppm.HRMS(ESI+):cacld for C22H25IN4O3(M+H)+,521.1044；found,521.1019.
中间体10a的合成：
将中间体9(0.2g，0.38mmol)、3-乙炔基吡啶(59mg，0.57mmol)、双三苯基磷二氯化钯(13mg，0.02mmol)、碘化亚铜(7mg，0.04mmol)、三乙胺(2mL)、N,N-二甲基甲酰胺(1mL)加到50mL的单颈瓶中，氮气置换空气，加热至回流反应6小时，TLC检测反应完全后，向反应液中加水(3mL)，用乙酸乙酯萃取3次，有机层合并后用浓盐水洗、无水硫酸钠干燥、抽滤，滤液减压蒸除溶剂，粗品通过硅胶柱层析纯化得到黄色油状物10a(0.1g，收率53％)。中间体10a表征数据如下：HRMS(ESI+):cacld for C29H29N5O3(M+H)+,496.2343；found,496.2360.
化合物LQ01的合成：
将中间体10a(0.1g，0.2mmol)加到4N氯化氢-乙酸乙酯溶液(CHCl＝4mol/L,1mL) 中，加热到40℃反应1小时，待TLC检测反应完全后，抽滤，滤饼用乙酸乙酯洗，真空干燥得到黄色固体LQ01(45mg，收率57％)，mp 224-227℃，结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.48(s,1H),9.28(s,1H),9.15(d,2H),9.00(s,1H),8.80(s,1H),8.48(s,1H),8.42(s,1H),7.88(s,2H),7.81(d,1H),2.37(d,J＝6.8Hz,2H),2.15(m,1H),0.97(d,J＝6.1Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.82,147.97,145.28,144.36,140.81,139.40(J＝14Hz),134.60,131.67,129.83,128.17(J＝4.5Hz),126.69,125.47,119.57,112.98ppm.HRMS(ESI+):cacld for C24H21N5O(M+H)+,396.1819；found,396.1827.
实施例2：化合物N-(5-(3-((4-氰基苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ02)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为4-氰基苯乙炔，采用实施例1的方法合成LQ02，LQ02结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.22(s,1H),9.21(s,1H),9.05(s,1H),8.84(s,1H),8.34(s,1H),7.99-7.92(dd,J1＝8.5Hz,J2＝12.3Hz,5H),7.85(s,2H),2.35(d,2H),2.19-2.10(m,1H),0.98(d,J＝6.5Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.69,140.76,135.53,132.90(J＝24.6Hz),130.90(J＝11.0Hz),128.48(J＝5.8Hz),127.17,126.69,125.34,119.42,118.94,112.86,111.74,92.22,85.74,45.80,25.98,22.74ppm.HRMS(ESI+):cacld for C26H21N5O(M+H)+,420.1819；found,420.1852.
实施例3：化合物3-甲基-N-(5-(3-噻唑-4-基乙炔基)-1H-吲唑-5-基)吡啶-3-甲基丁酰胺(LQ03)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为4-乙炔噻唑，采用实施例1的方法合成LQ03，LQ03的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.90(s,1H),10.48(s,1H),9.28(d,J＝2.0Hz,1H),8.85(d,J＝2.2Hz,1H),8.65(d,J＝2.2Hz,1H),8.42(t,J＝2.2Hz,1H),8.35(d,2.0Hz,1H),8.02(s,1H),7.81-7.74(t,J＝10.1Hz,2H),2.28(d,J＝7.0Hz,2H),2.19-2.05(m,1H),0.96(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.05,155.59,142.67,140.30,139.80,137.05,136.70,136.07,131.54,126.94,125.96,125.23,124.49,117.86,112.54,87.68,81.44,45.92,26.04,22.81ppm.HRMS(ESI+):cacld for C22H19N5OS(M+H)+,402.1383；found,402.1355.
实施例4：化合物N-(5-(3-((4-甲酰基苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ04)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为4-乙炔基苯甲醛，采用实施例1的方法合成LQ04，LQ04的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.08(s,1H),10.08(s,1H),8.79(s,1H),8.32(s,1H),8.06-7.89(m,5H),7.89-7.73(m,3H),2.35(d,J＝7.1Hz,2H),2.22-2.06(m,1H),0.98(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝193.04,172.69,140.78,136.25,132.71,130.77,130.20,128.62,128.14,126.76,125.32,119.42,112.86,92.85,85.33,45.83,25.99,22.75ppm.HRMS(ESI+):cacld for C26H22N4O2(M+H)+,423.1816；found,423.1819.
实施例5：化合物3-((5-(5-(3-甲基丁酰胺基)吡啶-3-基)-1H-吲唑-3-基)乙炔基)苯甲酸(LQ05)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为3-乙炔基苯甲酸，采用实施例1的方法合成LQ05，LQ05的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝10.95(s,1H),8.82(s,1H),8.28(m,3H),8.00(m,3H),7.83(m,2H),7.64(m,1H),2.34(s,2H),2.21-1.81(m,1H),1.02-0.81(m,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.84,167.02,140.79,136.20,132.52,131.93,130.31,129.86,128.92,126.76,125.21,122.73,119.78,112.82,92.39,82.51,45.82,25.98,22.74ppm.HRMS(ESI+):cacld for C26H22N4O3(M+H)+,439.1765；found,439.1768.
实施例6：化合物N-(5-(3-((3-氨基苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ06)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为3-乙炔基苯胺，采用实施例1的方法合成LQ06，LQ06的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.22(d,J＝10.8Hz,1H),8.84(s,1H),8.27(s,1H),7.86(s,3H),7.55(m,4H),7.33(d,J＝8.0Hz,1H),2.35(s,2H),2.14(s,1H),0.97(d,J＝6.4Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.79,140.81,134.64,130.80,130.52,128.75,126.73,125.66,125.19,123.86,123.37,119.47,112.89,92.37,82.77,45.80,26.00,22.75ppm.HRMS(ESI+):cacld for C25H23N5O(M+H)+,410.1975；found,410.1974.
实施例7：化合物N-(5-(3-异喹啉-6-基乙炔基)-1H-吲唑-5-基)吡啶-3-基-3-甲基丁酰胺(LQ07)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为6-乙炔异喹啉，采用实施例1的方法合成LQ07，LQ07的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.55(s,1H),9.92(s,1H),9.19(d,J＝48.5Hz,2H),9.04(s,1H),8.76(m,2H),8.60(d,J＝8.7Hz,1H),8.52(d,J＝6.4Hz,1H),8.39(s,1H),8.28(dd,J1＝8.6Hz,J2＝1.5Hz,1H),7.96–7.82(m,2H),2.40(d,J＝7.1Hz,2H),2.17(m,J＝6.8Hz,1H),0.99(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.78,147.55,140.74,139.17,138.34,135.02,133.51,132.70,131.23,130.97,130.64,130.38,129.42,128.34,128.12,126.61,125.49,125.01,119.14,112.93,92.86,87.45,45.72,25.97,22.75ppm.HRMS(ESI+):cacld for C28H23N5O(M+H)+,446.1975；found,446.1999.
实施例8：化合物3-甲基-N-(5-(3-(吡啶-4-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)丁酰胺(LQ08)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为4-乙炔吡啶，采用实施例1的方法合成LQ08，LQ08的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.42(s,1H),9.24(d,J＝2.0Hz,1H),9.08(d,J＝1.8Hz,1H),9.00-8.88(m,4H),8.44(d,J＝1.5Hz,1H),8.25-8.19(m,2H),7.89(d,J＝1.2Hz,2H),2.37(d,J＝7.2Hz,2H),2.25-2.06(m,1H),0.98(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.12,144.02,140.18,138.39,136.32,135.05,130.52,130.19,128.43,127.66,126.83,126.25,125.13,118.72,112.48,91.05,89.94,45.16,25.38,22.14ppm.HRMS(ESI+):cacld for C24H21N5O(M+H)+,396.1819；found,396.1825.
实施例9：化合物N-(5-(3-((3-氟苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ09)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为3-氟苯乙炔，采用实施例1的方法合成LQ09，LQ09的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.18(s,1H),9.21(s,1H),9.05(s,1H),8.83(d,J＝1.9Hz,1H),8.32(d,J＝1.6Hz,1H),7.84(d,J＝1.3Hz,2H),7.69-7.47(m,3H),7.35(m,1H),2.35(d,J＝7.1Hz,2H),2.15(m,1H),0.98(d,J＝6.6Hz,6H)ppm.HRMS(ESI+):cacld for C25H21FN4O(M+H)+,413.1722；found,413.1735.
实施例10：3-甲基-N-(5-(3-(嘧啶-2-基乙炔基)-1H-吲唑-5-基吡啶-3-基)丁酰胺(LQ10)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为2-炔基嘧啶，采用实施例1的方法合成LQ10，LQ10的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.36(s,1H),9.27(s,1H),9.12(s,1H),8.96(d,J＝4.9Hz,2H),8.86(d,J＝1.9Hz,1H),8.54(s,1H),7.91-7.81(m,2H),7.77(s,1H),7.48(t,J＝4.9Hz,1H),2.36(d,J＝7.1Hz,2H),2.14(m,1H),0.97(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.81,162.74,157.77,142.56,142.34,140.11,139.28,134.71,131.98,131.23,129.64,128.29,126.44,126.17,121.05,120.88,120.22,112.95,45.78,25.97,22.73ppm.HRMS(ESI+):cacld for C23H20N6O(M+H)+,397.1771；found,397.1789.
实施例11：5-(3-(2-氯苯基)乙炔基)-1H-吲唑-5-基吡啶-3-基-3-甲基丁酰胺(LQ11)的合成
将实施例1中中间体10a的合成步骤中，3-乙炔基吡啶替换为2-氯苯乙炔，采用实施例1的方法合成LQ11，LQ11的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.96(s,1H),11.10(s,1H),9.16(s,1H),9.01(s,1H),8.80(s,1H),8.22(d,J＝1.4Hz,1H),7.92-7.78(m,3H),7.66(dd,J1＝7.2Hz,J2＝1.9Hz,1H),7.57-7.41(m,2H),2.34(d,J＝7.1Hz,2H),2.14(m,1H),0.97(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.71,140.78,135.59,135.07,134.22,131.28,131.05,130.93,129.98,128.73,128.58,127.98,126.78,125.32,122.09,119.23,112.87,90.14,86.68,45.82,25.99,22.73ppm.HRMS(ESI+):cacld for C25H21ClN4O(M+H)+,429.1477；found,429.1464.
实施例12：3-甲基-N-(5-(3-(吡啶-2-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)丁酰胺(LQ12)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为2-乙炔基吡啶，采用实施例1的方法合成LQ12，LQ12的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.46(s,1H),9.29(d,J＝2.0Hz,1H),9.15(d,J＝1.8Hz,1H),8.97(t,J＝2.0Hz,1H),8.75(dt,J1＝5.0Hz,J2＝1.3Hz,1H),8.42(d,J＝1.4Hz,1H),8.11(td,J1＝7.7Hz,J2＝1.7Hz,1H),8.01(d,J＝7.8Hz,1H),7.89(d,J＝1.3Hz,2H),7.64(ddd,J1＝7.6Hz,J2＝5.1Hz,J3＝1.3Hz,1H),2.37(d,J＝7.1Hz,2H),2.15(dp,J1＝13.5Hz,J2＝6.6Hz,1H),0.98(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,MeOH-d6):δ＝172.80,148.55,140.85,140.32,139.79,139.59,139.27,134.77,131.77,129.98,129.02,128.41,127.88,126.77,125.57,125.29,119.59,113.08,90.79,84.44,45.79,25.99,22.74ppm.HRMS(ESI+):cacld for C24H21N5O(M+H)+,396.1819；found,396.1824.
实施例13：化合物N-(5-(3-((1-羟基环己基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ13)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为乙炔环己醇，采用实施例1的方法合成LQ13，LQ13的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.49(s,1H),10.22(s,1H),8.73(d,J＝2.3Hz,1H),8.61(d,J＝2.1Hz,1H),8.40(t,J＝2.2Hz,1H),7.89(s,1H),7.72(s,2H),5.69(d,1H),2.27(d,J＝7.1Hz,2H),2.12(m,1H),2.02-1.89(m,2H),1.61(m,7H),1.29(m,1H),0.97(d,J＝6.5Hz,6H)ppm.13C NMR(75MHz,MeOH-d6):δ＝173.22,163.49,142.14,140.17,138.80,137.37,130.90,129.50,126.67,125.59,124.89,118.14,111.25,68.27,45.73,35.61,30.31,26.11,25.05,23.07,21.44ppm.HRMS(ESI+):cacld for C25H28N4O2(M+H)+,417.2285；found,417.2293.
实施例14：化合物3-甲基-N-(5-(3-(喹啉-2-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)丁酰胺(LQ14)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为2-乙炔基喹啉，采用实施例1的方法合成LQ14，LQ14的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.89(s,1H),10.23(s,1H),8.80(d,J＝2.2Hz,1H),8.70(d,J＝1.9Hz,1H),8.49(d,J＝8.5Hz,1H),8.41(t,J＝2.2Hz,1H),8.16(t,J＝1.3Hz,1H),8.12-8.01(m,2H),7.93(d,J＝8.5Hz,1H),7.90-7.76(m,3H),7.68(ddd,J1＝8.1Hz,J2＝6.8Hz,J3＝1.2Hz,1H),2.27(d,J＝7.0Hz,2H),2.12(m,1H),0.97(d,J＝6.5Hz,6H)ppm.HRMS(ESI+):cacld for C28H23N5O(M+H)+,446.1975；found,446.1984.
实施例15：化合物3-甲基-N-(5-(3-((3-硝基苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)丁酰胺(LQ15)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为间硝基苯乙炔，采用实施例1的方法合成LQ15，LQ15的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.39(s,1H),9.21(d,2H),8.92(s,1H),8.53(t,J＝2.0Hz,1H),8.42(s,1H),8.32(dd,J1＝8.1Hz,J2＝2.3Hz,1H),8.23-8.15(m,1H),7.97-7.73(m,3H),2.37(d,J＝7.1Hz,2H),2.15(m,1H),0.98(d,J＝6.5Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.77,148.44,140.79,138.31,134.92,131.62,131.03,130.06,128.48,128.25,126.68,126.37,125.30,124.32,123.88,119.69,112.87,91.24,83.91,45.79,25.98,22.73ppm.HRMS(ESI+):cacld for C25H21N5O3(M+H)+,440.1717；found,440.1732.
实施例16：化合物N-(5-(3-(环丙基乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ16)的合成
将实施例1中的中间体10a的合成步骤中，3-乙炔基吡啶替换为环丙乙炔，采用实施例1的方法合成LQ16，LQ16的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝9.10(d,J＝51.6Hz,1H),8.79(s,1H),8.11-7.95(m,1H),7.79-7.70(m,2H),7.66-7.51(m,1H),2.35(d,J＝7.2Hz,2H),2.15(m,1H),1.69(m,1H),0.98(m,8H),0.88(m,2H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.77,140.68,132.06,129.84,129.36,127.73,126.40,125.76,125.07,119.45,119.05,112.60,98.20,67.84,45.81,26.00,22.74,9.18ppm.HRMS(ESI+):cacld for C22H22N4O(M+H)+,359.1866；found,359.1832.
实施例17：化合物LQ20的合成
合成路线2：
中间体11a的合成：
将实施例1中的中间体9(0.15g，0.29mmol)、2-乙烯吡啶(45mg，0.43mmol)、醋酸钯(3mg，0.01mmol)、三(邻甲基苯基)磷(9mg，0.03mmol)、N,N-二异丙基乙胺(0.11g，0.87mmol)加到N,N-二甲基甲酰胺(2mL)中，氮气置换空气，加热至回流反应7小时。将反应液冷却，加水(6mL)，用乙酸乙酯萃取(3mL×3)，有机层合并后用浓盐水洗、无水硫酸钠干燥、抽滤，滤液减压蒸除溶剂，粗品通过硅胶柱层析法纯化得到红棕色固体11a(57mg，收率40％)。HRMS(ESI+):cacld for C29H31N5O3(M+H)+,498.2500；found,498.2518.
化合物LQ20的合成：
将中间体11a(90mg，0.18mmol)加到4N氯化氢-乙酸乙酯溶液(CHCl＝4mol/L,1mL)中，室温搅拌2小时，待TLC检测反应完全后，抽滤，滤液减压蒸除溶剂，粗品通过硅胶柱层析法纯化得到红色固体34mg，收率48％。化合物LQ20的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.83(s,1H),11.05(s,1H),9.15(d,J＝2.0Hz,1H),8.98(d,J＝1.9Hz,1H),8.74(t,J＝2.1Hz,1H),8.47(d,J＝1.6Hz,1H),8.01(dd,J1＝9.1Hz,J2＝2.2Hz,2H),7.86-7.71(m,6H),2.34(d,J＝7.1Hz,2H),2.21-2.07(m,1H),0.98(d,J＝6.6Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝172.69,148.53,141.21,139.87,139.24,138.89,135.74,131.13,130.69,128.21,128.02,127.99,126.92,126.85,126.46,125.69,124.22,120.27,119.98,112.43,112.28,95.37,45.83,26.00,22.76ppm.HRMS(ESI+):cacld for C24H23N5O(M+H)+,398.1975；found,398.1993.
实施例18：化合物5-(3-(3-(3-羟基吡咯烷-1-基)-3-氧代丙基-1-烯-1-基)-1H-吲唑-5-基吡啶-3-基-3-甲基丁酰胺(LQ21)的合成
将实施例17中的中间体11a的合成步骤中，2-乙烯吡啶替换为1-(3-羟基吡咯烷-1-基)丙基-2-烯-1-酮，采用实施例17的方法合成LQ21，化合物LQ21的结构和表征数据 如下：
1H NMR(300MHz，DMSO-d6):δ＝13.61(s,1H),10.22(s,1H),8.76-8.67(m,2H),8.43(dt,J1＝8.5Hz,J2＝2.3Hz,1H),8.33(s,1H),7.82(dd,J1＝15.7Hz,J2＝2.0Hz,1H),7.73(s,2H),7.20(dd,J1＝21.5Hz,J2＝15.7Hz,1H),5.03(dd,J1＝20.4Hz,J2＝3.4Hz,1H),4.43-4.27(d,1H),3.88-3.77(m,1H),3.63-3.50(m,1H),3.45(d,1H),2.27(d,J＝7.1Hz,2H),2.13(dq,J1＝13.7Hz,J2＝6.7Hz,1H),2.05-1.74(m,2H),0.97(d,J＝6.5Hz,6H)ppm.HRMS(ESI+):cacld for C24H27N5O3(M+H)+,434.2187；found,434.2198.
实施例19：化合物5-(3-(3-(3-吗啉基-3-氧代丙基-1-烯-1-基)-1H-吲唑-5-基)吡啶-3-基丁酰胺(LQ22)的合成
将实施例17中的中间体11a的合成步骤中，2-乙烯吡啶替换为N-丙烯酰吗啉，采用实施例17的方法合成LQ22，化合物LQ22的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝10.22(s,1H),8.71(s,2H),8.40(d,J＝16.2Hz,2H),7.87(d,J＝15.7Hz,1H),7.72(s,2H),7.43(d,J＝15.7Hz,1H),3.63(s,8H),2.26(d,J＝7.3Hz,2H),2.17-2.04(m,1H),0.97(d,J＝6.4Hz,6H)ppm.HRMS(ESI+):cacld for C24H27N5O3(M+H)+,434.2187；found,434.2249.
实施例20：化合物LQ23的合成
合成路线3：
中间体12的合成：
将原料1(5-溴吲唑，1g，5.1mmol)、氢氧化钾(0.9g，15.3mmol)加到N,N-二甲基甲酰胺(3mL)中，冰浴降温至0℃，分批加入碘(2.6g，10.2mmol)。移至室温反应2小时，待TLC检测反应完全后，用饱和硫代硫酸钠溶液淬灭，抽滤，滤饼用少量水洗，真空干燥得到灰色固体1.5g，收率94％。中间体12的数据表征：1H NMR(300MHz，DMSO-d6):δ＝7.62(s,1H),7.55(s,2H)ppm.HRMS(ESI+):cacld for C7H4BrIN2(M+H)+,322.8675；found,322.8662.
中间体13的合成：
将中间体12(1g，3.1mmol)和对甲苯磺酸(60mg，0.3mmol)加到四氢呋喃(10mL)中，滴加3,4-二氢-2H-吡喃(0.6mL，6.2mmol)至反应液中，加热至回流反应5小时。待TLC检测反应完全后，停止加热，抽滤，滤液减压蒸除溶剂，经硅胶柱层析法得到中间体13，黄色固体0.8g，收率67％。中间体13的数据表征如下：1H NMR(300MHz，CDCl3-d6):δ＝7.63(s,1H),7.53-7.45(m,2H),5.69-5.65(dd,J1＝2.8Hz,J2＝9.2Hz,1H),4.00(d,J＝11.5Hz,1H),3,76-3.68(m,1H),2.57-2.45(m,1H),2.16-2.04(m,2H),1.79-1.65(m,4H)ppm.
中间体14a的合成：
将中间体13(4.2g，10mmol)、3-乙炔基噻吩(1.7g，15mmol)、双三苯基磷二氯化钯(86mg，0.5mmol)、碘化亚铜(48mg，1mmol)、三乙胺(20mL)、四氢呋喃(20mL)加到100mL的单颈瓶中，氮气置换空气，室温反应过夜，TLC检测反应完全后，抽滤，滤液减压蒸除溶剂，粗品经过硅胶柱层析法得到中间体14a，黄色油状物3.2g，收率84％。中间体14a的表征数据：HRMS(ESI+):cacld for C18H15BrN2OS(M+H)+,387.0161；found,387.0159.
中间体15a的合成：
将中间体14a(0.9g，2.3mmol)、联硼酸频那醇酯(0.7g，2.8mmol)、乙酸钾(0.7g，6.9mmol)、1,1'-双二苯基膦二茂铁二氯化钯(84mg，0.1mmol)、二氧六环(9mL)加到100mL的单颈瓶中，氮气置换空气，加热至回流反应6个小时，待TLC检测反应完全后，抽滤，滤液减压蒸除溶剂，粗品经过硅胶柱层析法纯化得到0.5g黄色固体，收率50％。中间体15a的表征数据：1H NMR(300MHz，DMSO-d6):δ＝8.12(m,2H),7.88-7.73(m,2H),7.72-7.67(m,1H),7.42(dd,J1＝5.0Hz,J2＝1.2Hz,1H),5.94(dd,J1＝9.4Hz,J2＝2.4Hz,1H),3.89(m,1H),3.76(m,1H),2.45-2.25(m,1H),2.01(d,2H),1.88-1.67(m,1H),1.60(s,2H),1.33(s,12H).HRMS(ESI+):cacld for C24H27BN2O3S(M+H)+,435.1908；found,435.1935.
(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯的合成：
把(S)-(1-羟基-4-甲基戊烷-2-基)氨基甲酸叔丁酯(1.0g，4.6mmol)、二氯甲烷(10mL)加到100mL的三颈瓶中，降温至0℃，加入吡啶(0.7mL，9.2mmol)，搅拌5分钟，然后分批加入甲基磺酸酐(1.2g，6.9mmol)，移至室温反应3小时，待TLC检测反应完全后，加水，用二氯甲烷萃取，有机层合并后用浓盐水洗、无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂得到(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯，橙黄色固体1.1g，收率81％。(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯的表征数据如下：1H NMR(300MHz，CDCl3-d6):δ＝4.53-4.48(m,1H),4.29-4.23(m,1H),4.17-4.12(m,1H),3.98-3.90(m,1H),3.03(s,3H),1.76-1.67(m,1H),1.45(s,9H),0.95-0.92(dd,J1＝2.7Hz,J2＝6.6Hz,6H)ppm.HRMS(ESI+):cacld for C12H25NO5S(M+H)+,296.1526；found,296.1539.
中间体16a的合成：
将(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯(0.5g，1.69mmol)、3-溴-5-羟基吡啶(0.38g，2.2mmol)、碳酸铯(1.1g，3.4mmol)、N,N-二甲基甲酰胺(5mL)加到25mL的单颈瓶中，100℃加热反应3个小时，TLC检测反应完全后，加水，用乙酸乙酯萃取，有机层合并后用浓盐水洗，无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂。粗品通过硅胶柱层析法纯化得到0.2g无色油状物16a，收率35％。中间体16a的表征数据：1H NMR(300MHz，DMSO-d6):δ＝8.29-8.26(m,2H),7.72-7.70(t,J＝2.0Hz,1H),6.84-6.81(d,J＝8.7Hz,1H),3.95(d,J＝5.8Hz,2H),3.86-3.77(m,1H),1.71-1.57(m,1H),1.36-1.23(m,11H),0.91-0.82(m,6H)ppm.HRMS(ESI+):cacld for C16H25BrN2O3(M+H)+,373.1121；found,373.1141.
中间体17a的合成：
将中间体15a(0.3g，0.8mmol)、中间体16a(0.4g，0.9mmol)、1,1'-双二苯基膦二茂铁二氯化钯(30mg，0.04mmol)、碳酸钠(0.25g，2.4mmol)、二氧六环(4mL)和水(1mL)加到25mL的单颈瓶中，氮气置换空气，加热至回流反应6个小时，待TLC检测反应完全后，抽滤，滤饼用少量乙酸乙酯洗，滤液减压蒸除溶剂，粗品通过硅胶柱层析法纯化得到17a，真空干燥得黄色固体(0.25g，收率52％)。中间体17a的表征数据：HRMS(ESI+):cacld for C34H40N4O4S(M+H)+,601.2843；found,601.2857.
化合物LQ23的合成：
将中间体17a(0.25g，0.4mmol)、4N氯化氢-乙酸乙酯溶液(CHCl＝4mol/L,2mL)加到25mL单颈瓶中，加热至40℃反应1个小时，待TLC反应完全后，抽滤，滤饼用少量乙酸乙酯洗，滤液减压蒸除溶剂。粗品用乙酸乙酯/乙醇重结晶，得到LQ23，淡黄色固体(70mg，收率42％)，经HPLC检测纯度为99.3％，mp 227-230℃。LQ23的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.92(d,J＝1.7Hz,1H),8.58(d,J＝2.5Hz,1H),8.47(s,2H),8.40-8.27(m,2H),8.10(dd,J1＝2.9Hz,J2＝1.2Hz,1H),7.92(dd,J1＝8.8Hz,J2＝1.7Hz,1H),7.80(d,J＝8.7Hz,1H),7.72(dd,J1＝5.0Hz,J2＝2.9Hz,1H),7.43(dd,J1＝5.0Hz,J2＝1.2Hz,1H),4.57(dd,J1＝10.7Hz,J2＝3.3Hz,2H),4.38(dd,J1＝10.7,J2＝6.7Hz,2H),3.59(s,1H),1.94-1.76(m,1H),1.60(t,J＝7.2Hz,2H),0.93(d,J＝6.4Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝156.60,140.78,139.94,135.06,131.33,130.48,130.31,129.21,128.33,127.71,127.60,126.98,125.05,121.28,119.79,112.52,88.95,81.21,69.78,48.84,38.38,24.06,23.12,22.61ppm.HRMS(ESI+):cacld for C24H24N4OS(M+H)+,417.1744；found,417.1744.
实施例21：化合物N-(5-(3-环戊基炔基)-1H-吲唑-5-基吡啶-3-基)-3-甲基丁酰胺(LQ17)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为环戊基乙炔，采用实施例20的方法合成中间体15c，中间体15c的结构和表征数据如下：
HRMS(ESI+):cacld for C25H33BN2O3(M+H)+,421.2657；found,421.2655.
将实施例20中的中间体17a的合成步骤中，中间体16a替换为中间体5，采用实施 例20的方法合成LQ17，LQ17的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝10.25(t,1H),8.97-8.28(m,3H),8.02-7.35(m,3H),2.72-2.58(m,1H),2.49(m,1H),2.29(d,J＝6.9Hz,2H),2.16(m,2H),2.06-1.92(m,2H),1.83-1.61(m,2H),1.28(m,2H),0.99(d,J＝6.5Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝171.95,143.17,142.75,139.19,132.72,126.24,125.03,124.91,119.39,118.05,46.02,34.00,33.22,30.65,26.05,25.18,24.75,22.80ppm.HRMS(ESI+):cacld for C24H26N4O(M+H)+,387.2179；found,387.2249.
实施例22：化合物N-(5-(3-([1,1'-联苯]-4-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)-3-甲基丁酰胺(LQ18)的合成
将实施例21中的中间体15c的合成步骤中，环戊基乙炔替换为4-乙炔联苯，采用实施例21的方法合成LQ18，LQ18的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.70(s,1H),10.21(s,1H),8.79(d,J＝2.2Hz,1H),8.69(d,J＝2.0Hz,1H),8.39(t,J＝2.3Hz,1H),8.13(s,1H),7.85-7.71(m,8H),7.51(t,J＝7.4Hz,2H),7.42(m,1H),2.27(d,J＝7.1Hz,2H),2.13(m,1H),0.97(d,J＝6.5Hz,6H)ppm.13C NMR(75MHz,DMSO-d6):δ＝171.92,142.91,141.05,140.31,139.73,136.51,136.26,132.68,131.43,129.59,128.51,127.49,127.23,127.01,125.28,124.65,121.43,118.24,112.36,46.03,26.04,22.82ppm.HRMS(ESI+):cacld for C31H26N4O(M+H)+,471.2719；found,471.2724.
实施例23：化合物5-(3-(4-甲氧基苯基)乙炔基)-1H-吲唑-5-基吡啶-3-基-3-甲基丁酰胺(LQ19)的合成
将实施例21中的中间体15c的合成步骤中，环戊基乙炔替换为4-乙炔基苯甲醚，采用实施例21的方法合成LQ19，LQ19的结构和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.00(s,1H),10.21(s,1H),8.73(d,J＝2.2Hz,1H),8.57(s,1H),8.32(t,J＝2.2Hz,1H),8.14-8.05(m,2H),8.02(s,1H),7.67-7.59(m,2H),7.09-7.03(m,2H),3.84(s,3H),2.25(d,J＝7.1Hz,3H),2.12(m,1H),0.95(d,J＝6.5Hz,6H)ppm.HRMS(ESI+):cacld for C26H24N4O2(M+H)+,425.1972；found,425.1971.
实施例24：化合物N-(5-(3-(噻吩-3-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)环丙烷甲酰胺(LQ24)的合成
中间体16b的合成：
将3-溴-5-羟基吡啶(1.0g，5.8mmol)、二氯甲烷(10mL)加到100mL的三颈瓶中，降温至0℃。将环丙基甲酰氯(0.9g，8.7mmol)溶于二氯甲烷中，滴加到反应液中，滴加三乙胺(1.6mL，11.6mmol)。0℃反应3小时，待TLC检测反应完全后，减压蒸除溶剂，用乙酸乙酯萃取，有机层用2N稀盐酸洗、水洗、饱和碳酸氢钠溶液洗、浓盐水洗，有机层用无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂，得到0.6g黄色油状物，收率43％。中间体16b的表征数据：HRMS(ESI+):cacld for C9H9BrN2O(M+H)+,240.9971；found,240.9939.
将实施例20中的中间体17a的合成步骤中，中间体16a替换为中间体16b，采用实施例20的方法合成LQ24，LQ24的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.69(s,1H),9.19(d,J＝2.0Hz,1H),9.06(d,J＝1.9Hz,1H),8.89(t,J＝2.0Hz,1H),8.26(s,1H),8.10(dd,J1＝2.9Hz,J2＝1.2Hz,1H),7.84(s,2H),7.72(dd,J1＝5.0Hz,J2＝2.9Hz,1H),7.43(dd,J1＝5.0Hz,J2＝1.2Hz,1H),2.06-1.89(m,1H),0.93(m,4H)ppm.13C NMR(75MHz,DMSO-d6):δ＝173.88,140.78,139.60,139.21,134.76,131.38,131.34,130.30,130.03,129.25,127.91,127.64,126.53,125.10,121.17,119.52,112.77,89.09,81.02,15.23,8.83ppm.HRMS(ESI+):cacld for C22H16N4OS(M+H)+,385.1118；found,385.1139.
实施例25：化合物5-(5-(吡咯烷-2-基甲氧基)吡啶-3-基)-3-(噻吩-3-基乙炔基)-1H-吲唑(LQ25)的合成
将实施例20中的中间体16a的合成步骤中，(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯替换为2-羟甲基吡咯烷-1-羧酸丁酯，采用实施例20的方法合成LQ25，LQ25的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝11.69(s,1H),9.19(d,J＝2.0Hz,1H),9.06(d,J＝1.9Hz,1H),8.89(t,J＝2.0Hz,1H),8.26(s,1H),8.10(dd,J1＝2.9Hz,J2＝1.2Hz,1H),7.84(s,2H),7.72(dd,J1＝5.0Hz,J2＝2.9Hz,1H),7.43(dd,J1＝5.0Hz,J2＝1.2Hz,1H),2.06-1.89(m,1H),0.93(m,4H)ppm.13C NMR(75MHz,DMSO-d6):δ＝173.88,140.78,139.60,139.21,134.76,131.38,131.34,130.30,130.03,129.25,127.91,127.64,126.53,125.10,121.17,119.52,112.77,89.09,81.02,15.23,8.83ppm.HRMS(ESI+):cacld for C22H16N4OS(M+H)+,385.1118；found,385.1139.
实施例26：化合物5-(5-((3,3-二氟吡咯烷-1-基)甲基)吡啶-3-基)-3-(噻吩-3-基乙炔基)-1H-吲唑(LQ26)的合成
中间体16d的合成：
将3,3-二氟吡咯烷(1.0g，9.43mmol)、5-溴吡啶-3-甲醛(0.8g，4.6mmol)、三乙胺(0.9g，9.3mmol)、二氯甲烷(10ml)加到100ml的单颈瓶中，室温搅拌1个小时。将三乙酰氧基硼氢化钠(1.5g，7.0mmol)加到反应液中，室温反应过夜，TLC检测反应完全后，加饱和碳酸氢钠溶液，用二氯甲烷萃取，有机层合并后用浓盐水洗、无水硫酸钠干燥，抽滤，滤液减压蒸除溶剂，真空干燥得到棕色油状物(1.1g，收率87％)。HRMS(ESI+):cacld for C10H11BrF2N2(M+H)+,277.0146；found,277.0169.
将实施例20中的中间体17a的合成步骤中，中间体16a替换为中间体16d，采用实施例20的方法合成LQ26，LQ26的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.89(d,J＝1.7Hz,1H),8.56(d,J＝2.6Hz,1H),8.30(s,2H),8.09(dd,J1＝3.0Hz,J2＝1.2Hz,1H),7.92(dd,J1＝8.8Hz,J2＝1.7Hz,1H),7.79(d,J＝8.8Hz,1H),7.72(dd,J1＝5.0Hz,J2＝2.9Hz,1H),7.42(dd,J1＝5.0Hz,J2＝1.2Hz,1H),4.59(dd,J1＝10.9Hz,J2＝3.6Hz,1H),4.50(t,J＝9.4Hz,1H),3.23(d,J＝9.8Hz,3H),2.22-2.09(m,1H),2.08-1.87(m,2H),1.86-1.71(m,1H)ppm.13C NMR(75MHz,DMSO-d6):δ＝156.62,140.77,140.08,134.73,131.34,130.33,130.06,129.21,128.17, 127.64,127.05,125.00,121.27,119.90,112.50,88.95,81.23,69.11,57.89,45.38,26.74,23.92ppm.HRMS(ESI+):cacld for C23H18F2O4S(M+H)+,421.1293；found,421.1325.
实施例27：化合物1-((5-(3-((3-氟苯基)乙炔基)-1H-吲唑-5-基)吡啶-3-基)氧基)-4-甲基戊-2-胺(LQ27)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为间氟苯乙炔，采用实施例20的方法合成LQ27，LQ27的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.65(s,1H),8.35(s,1H),8.22(s,1H),7.86-7.75(m,3H),7.65-7.49(m,3H),7.37-7.31(m,1H),4.35-4.32(d,J＝9Hz,1H),4.18-4.13(m,1H),3.51(s,1H),1.83-1.77(m,1H),1.51(d,J＝4.9Hz,2H),0.93(q,J1＝3.2Hz,J2＝5.8Hz,6H)ppm.HRMS(ESI+):cacld for C26H25FN4O(M+H)+,429.2085；found,429.2099.
实施例28：化合物1-(5-(3-(3-氟苯基)乙炔基)-1H-吲唑-5-基吡啶-3-基)-3-苯基丙烷-2-胺(LQ28)的合成
将实施例20中的中间体16a的合成步骤中，(S)-2-((叔丁氧基羰基)氨基)-4-甲基戊基甲磺酸酯替换为N-Boc-L-苯丙胺醇，采用实施例27的方法合成LQ28，LQ28的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.97(d,J＝1.6Hz,1H),8.72-8.49(m,4H),8.34(d,J＝1.7Hz,1H),8.25-8.16(m,1H),7.71-7.54(m,2H),7.43-7.18(m,6H),4.41(dd,J1＝10.7Hz,J2＝3.1Hz,1H),4.25(dd,J1＝10.7Hz,J2＝5.7Hz,1H),3.86(s,1H),3.18(dd,J1＝13.6Hz,J2＝5.5Hz,1H),3.05(dd,J1＝13.7Hz,J2＝9.2Hz,1H)ppm.13C NMR(75MHz,DMSO-d6):δ＝156.22,140.89,138.40,136.46,136.05,132.29,130.10,129.89,129.22,127.53,125.47,123.07,121.93,115.11,113.65,68.40,51.72,35.28ppm.HRMS(ESI+):cacld for C29H23FN4O(M+H)+,463.1929；found,463.1988.
实施例29：化合物(S)-1-((5-(3-(环戊基乙炔基)-1H-吲唑-5-基)吡啶-3-基)氧基)-4-甲基戊-2-胺(LQ29)合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为环戊基乙炔，采用实施例20的方法合成LQ29，LQ29的结构式和表征数据如下：
1H NMR(300MHz，MeOH-d6):δ＝8.51(m,1H),8.33–8.20(m,1H),8.07–7.93(m,1H),7.79–7.59(m,3H),4.25(m,1H),4.12–3.99(m,1H),3.59–3.38(m,1H),2.80–2.49(m,1H),1.86(m,3H),1.54(m,4H),1.41–1.24(m,4H),1.03(t,J＝6.1Hz,6H)ppm.13C NMR(75MHz,MeOH-d6):δ＝155.61,139.95,135.42,135.21,130.67,130.26,126.74,126.37,120.44,118.37,111.14,71.51,41.10,37.86,33.59,29.40,24.67,24.26,21.97,21.33ppm.HRMS(ESI+):cacld for C25H30N4O(M+H)+,403.2492；found,403.2526.
实施例30：化合物3-([1,1'-联苯]-4-基乙炔基)-5-(5-((3,3-二氟吡咯烷-1-基)甲基)吡啶-3-基)-1H-吲唑(LQ30)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为4-乙炔联苯，采用实施例26的方法合成LQ30，LQ30的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝13.71(s,1H),8.94(s,1H),8.53(s,1H),8.21(s,1H),8.10(d,J＝2.1Hz,1H),7.88-7.71(m,8H),7.51(m,2H),7.46-7.36(m,1H),3.77(s,2H),2.94(t,J＝13.3Hz,2H),2.76(t,J＝6.9Hz,2H),2.28(m,2H)ppm.13C NMR(75MHz,DMSO-d6):δ＝148.86,147.43,141.02,140.30,139.61,135.15,132.67,131.37,129.57,128.92,128.49,127.45,127.21,127.11,125.31,121.47,118.41,112.26,93.18,82.81,61.48,56.21,51.87,36.11,35.79ppm.HRMS(ESI+):cacld for C31H24F2N4(M+H)+,491.2042；found,491.2059.
实施例31：化合物1-((5-(3-([1,1'-联苯]-4-基乙炔基)-1H-吲唑-5-基)吡啶-3-基)氧基)-4-甲基戊-2-胺(LQ31)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为4-乙炔联苯，采用实施例20的方法合成LQ31，LQ31的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.61(d,J＝1.8Hz,1H),8.32(d,J＝2.6Hz,1H),8.21(d,J＝1.6Hz,1H),7.90-7.64(m,9H),7.56-7.35(m,3H),4.09(dd,J1＝9.6Hz,J2＝4.5Hz,1H),3.96(dd,J1＝9.6Hz,J2＝6.7Hz,1H),3.16(m,1H),1.93-1.76(m,1H),1.30(m,4H),0.90(dd,J1＝9.4Hz,J2＝6.5Hz,6H)ppm.HRMS(ESI+):cacld for C32H30N4O(M+H)+,487.2492；found,487.2503.
实施例32：化合物1-((5-(3-(环丙基乙炔基)-1H-吲唑-5-基)吡啶-3-基)氧基)-4-甲基戊-2-胺(LQ32)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为环丙乙炔，采用实施例20的方法合成LQ32，LQ32的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.53(d,J＝1.5Hz,1H),8.29(d,J＝2.5Hz,1H),7.97(s,1H),7.78-7.65(m,3H),4.07-3.89(m,2H),3,15(m,1H),1.89-1.77(m,1H),1.72-1.63(m,1H),1.34-1.23(m,4H),0.97-0.86(m,10H)ppm.HRMS(ESI+):cacld for C23H26N4O(M+H)+,375.2179；found,375.2168.
实施例33：化合物5-(2-氨基-4-甲基戊氧基)吡啶-3-基吡啶-3-基吡啶-3-基乙炔基-1H-吲唑(LQ33)的合成
将实施例20中的中间体14a的合成步骤中，3-乙炔基噻吩替换为3-乙炔基吡啶，采用实施例20的方法合成LQ33，LQ33的结构式和表征数据如下：
1H NMR(300MHz，DMSO-d6):δ＝8.94(s,1H),8.65-8.63(d,J＝4.9Hz,2H),8.36(s,1H),8.24(s,1H),8.17-8.14(d,1H),7.87-7.76(m,3H),7.54-7.46(m,2H),7.12-7.09(d,J＝7.7Hz,1H),5.32(s,2H),4.35-4.33(d,1H),4.20-4.15(t,1H),2.29(s,1H),1.54-1.50(t,2H),1.25-1.21(m,2H),0.94-0.91(dd,J1＝3.5Hz,J2＝6.3Hz,6H)ppm.HRMS(ESI+):cacld for  C25H25N5O(M+H)+,412.2126；found,412.2137.
实施例34：实施例1-33的化合物的酶抑制活性
采用Z’LYTETM试剂盒(ThermoFisher)，利用FRET原理检测化合物对激酶的抑制率。对实施例1-33制备的化合物LQ01-33进行了体外CLK2酶抑制活性研究，测试了浓度为20nM的化合物对激酶的抑制率和优势化合物的IC50。
实验原理：肽底物用两个荧光团标记，在两端各一个，组成一个FRET对。在初级反应(激酶反应)中，激酶将ATP的g-磷酸转移到合成肽底物中的单个丝氨酸或苏氨酸残基。在二次反应(发展反应)中，位点特异性蛋白酶(发展试剂)识别和切割非磷酸化肽。磷酸化肽被显影试剂抑制裂解。裂解破坏肽上供体(即香豆素)和受体(即荧光素)荧光团之间的FRET，而未裂解的磷酸化肽维持FRET。比值法计算供体荧光团在400nm激发后供体发射与受体发射的比值(发射比)，定量反应进程：
CLK2酶抑制实验结果(20nM的化合物对激酶的抑制率，n＝2)：





优势化合物LQ23的CLKs激酶抑制活性(IC50)：
如上表和图1所述，优势化合物LQ23对CLK1/2/4表现出低纳摩尔级的抑制活性(CLK1 IC50＝4nM,CLK2 IC50＝1.4nM,CLK4 IC50＝12nM)，与Lorecivivint相比，LQ23对CLK2的选择性(VS CLK3 IC50>100nM)至少提高了15倍。另外，LQ23表现出比Lorecivivint较强的DYRK1A酶抑制活性(DYRK1A IC50＝21.7nM)。
实施例35：LQ23在体外对软骨降解蛋白酶及炎症因子水平的调控
大鼠软骨细胞用TNF-α(20ng/mL)和Oncostatin M(10ng/mL)诱导产生炎症后，立刻给药治疗48h，qRT-PCR检测MMP3,MMP13和IL-6基因的表达水平，结果如图2所示。
大鼠骨髓间充质干细胞用TNF-α(20ng/mL)和Oncostatin M(10ng/mL)诱导产生炎症后，立刻给药治疗72h，RT-qPCR检测MMP3，MMP13，ADAMTS5，IL-1β和TNF-α基因的表达水平，结果如图3所示。
以上结果显示，模型组与空白组相比，相关基因上调明显，表明造模成功。优势化合物LQ23的治疗组对相关基因有下调的作用，且优于阳性药Lorecivivint。
实施例36：LQ23在体内对软骨降解蛋白酶及炎症因子水平的调控
基于化合物LQ23良好的体外抗骨关节炎活性，我们进一步考察LQ23在体内对软骨降解蛋白酶的调控水平。建立了关节腔注射碘乙酸钠(Monosodium iodoacetate，MIA) 和内侧半月板部分切除联合前交叉韧带切断(Anterior cruciate ligament transection and partial medial meniscectomy model，ACLT+pMMx)两种大鼠骨关节炎模型，分别单次关节腔注射LQ23(1.5μg/kg)和Lorecivivint(1.5μg/kg)，MIA模型治疗28天，ACLT+pMMx模型治疗13周后，取大鼠软骨组织，用RT-qPCR检测软骨降解蛋白酶和炎症因子的基因表达水平；取血清样本，用ELISA试剂盒检测COMP及炎症因子的含量，实验结果如图4-7所示。
以上结果表明，在这两种大鼠骨关节炎模型实验中，相较于模型组，LQ23能显著降低MMP13、MMP3、ADAMTS5及IHH等软骨降解蛋白酶的表达，显著降低血清中COMP及多种炎症因子的含量，效力与Lorecivivint相当。以上说明LQ23能够显著降低软骨降解。

Claims (9)

  1. 5-吡啶-1H-吲唑类化合物，其特征在于，为式(I)、式(II)或式(III)所示化合物或其药用盐：
    其中，R1、R2或R4选自如下基团：C1～C10烷基、取代或者未取代的6-10元芳基、取代或者未取代的含有1-3个选自N、O和S杂原子的5-10元杂芳基中的一种，所述芳基或者杂芳基上的取代基选自卤素、C1～C6烷基、C1～C6烷氧基、C1～C6烷硫基、卤代C1～C6烷基、卤代C1～C6烷氧基、氨基、C1～C6烷酰基、NH-CO-C1～C6烷基、-NH(C1～C6烷基)、-N(C1～C6烷基)(C1～C6烷基)和羟基中的一个或者多个；
    其中，R3选自以下基团：
  2. 根据权利要求1所述的5-吡啶-1H-吲唑类化合物，其特征在于，所述R1选自：
  3. 根据权利要求1所述的5-吡啶-1H-吲唑类化合物，其特征在于，所述R2选自：
  4. 根据权利要求1所述的5-吡啶-1H-吲唑类化合物，其特征在于，所述R4选自：
  5. 根据权利要求1所述的5-吡啶-1H-吲唑类化合物，其特征在于，所述化合物选自如下结构所示的化合物：


  6. 根据权利要求1～5任一项所述的5-吡啶-1H-吲唑类化合物，其特征在于，所述化合物的药用盐为药学上可接受的盐型是指式(I)、式(II)或式(III)所示的化合物和药学上可接受的酸形成的盐。
  7. 一种包含有如权利要求1～6任一项所述的化合物的药物组合物。
  8. 一种如权利要求1～6任一项所述的化合物或权利要求7所述的药物组合物在制备靶向抑制CLK2的药物中的应用。
  9. 一种如权利要求1～6任一项所述的化合物或权利要求7所述的药物组合物在制备治疗骨关节炎药物中的应用。
PCT/CN2023/117144 2022-09-07 2023-09-06 靶向抑制clk2的5-吡啶-1h-吲唑类化合物及其应用 WO2024051720A1 (zh)

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