CN115304583A - 5-Pyridine-1H-indazoles targeting CLK2 and their applications - Google Patents

Abstract

The invention discloses5-pyridine-1H-indazole compounds for targeted inhibition of CLK2 and application thereof. The compound is a compound shown in a formula (I), a formula (II) or a formula (III) or a medicinal salt, a prodrug, a hydrate or a solvate thereof. The compound has obvious CLK2 inhibition activity, good selectivity on CLK family members, obvious DYRK1A inhibition activity and good anti-osteoarthritis activity in vitro and in vivo.

Description

5-pyridine-1H-indazole compounds targeting to inhibit CLK2 and application thereof

technical field

The invention relates to the field of drug synthesis, in particular to a 5-pyridine-1H-indazole compound targeted to inhibit CLK2 and application thereof.

Background technique

Osteoarthritis (OA) is a worldwide bone and joint disease affecting the health of 250 million people worldwide, characterized by synovial inflammation, cartilage loss, and subchondral bone remodeling. The synovium of OA patients is rich in stem cells, therefore, the inability of patients to regenerate articular cartilage is not due to insufficient supply of stem cells, but improper differentiation of stem cells trying to restore healthy cartilage, and 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 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. In the process of OA, the degradation of cartilage is irreversible. Among them, MMPs proteins (MMP3, MMP13, ADAMTS5, IHH, etc.) are a group of proteins involved in the degradation of cartilage, which play a role in promoting the pathological process of OA. Cartilage Oligomeric Matrix Protein (COMP) is a newly discovered extracellular matrix protein in recent years. Because it has been reported in the literature that it has significance as a biomarker in the process of arthritic cartilage degeneration, it may become an early The "golden index" for diagnosing joint lesions is receiving more and more attention. In addition, OA is an inflammatory disease, and the content of inflammatory factors (IL-6, IL-1β, TNF-α, etc.) in the body of its patients is also high.

The Wnt signaling pathway is a signaling pathway of a set of multiple downstream channels triggered by the binding of the ligand protein Wnt and membrane protein receptors. Through this pathway, extracellular signals are transmitted into the cell through the intracellular activation of cell surface receptors. In the classic Wnt pathway, if there is no Wm protein on the surface of the cell membrane, its downstream β-Catenin protein will be decomposed by the glycogen synthase 3 (GSK3) complex in the cytoplasm, so that it cannot enter the nucleus to start the transcription of related Wnt genes ; and when there is Wm protein on the surface of the cell membrane, it will inhibit the GSK3 complex, so that the β-Catenin protein will accumulate in the nucleus, and finally start the transcription of Wnt pathway-related genes. There is a delicate balance between the homeostasis of bone and joints and the Wnt pathway, and the disruption of this balance may lead to the occurrence of OA.

The protein kinase family CLK (CDK-likekinase) is a dual-specificity protein kinase that regulates intracellular signal transduction through the phosphorylation of substrate proteins on tyrosine, serine or threonine residues; it can be divided into four sub- Types (CLK1, CLK2, CLK3 and CLK4), the protein C segments encoded by these four subtypes all have a highly conserved gene sequence, and have the same amino acid sequence with a similar structure. Among them, the CLK2 subtype is found in most eukaryotes, and it is involved in the phosphorylation of the SR (serine/arginine) protein domain to regulate the selective cleavage of RNA. Studies have confirmed that 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. Recent studies have shown that CLK2 is a potential therapeutic target for the Wm pathway and osteoarthritis.

Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A, DYRK1A) belongs to the DYRK family, which is highly conserved in evolution. In mammals, the DYRK family has five different subtypes, and only DYRK1A is 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. Examples include neurodevelopment, cell proliferation and differentiation, tumorigenesis, and neurodegenerative diseases.

At present, Lorecivivint, a First-in-class small molecule drug developed by Sumumed, has been applied in Phase III clinical trials to treat osteoarthritis, and its specific mechanism is mainly to induce early cartilage formation by inhibiting CLK2-mediated phosphorylation of SR protein; and Inhibit DYRK1A-mediated phosphorylation of SIRT1 and FOXOA to enhance the function of mature chondrocytes; and reduce inflammation by inhibiting NF-γB, STAT3 and other inflammatory factors. Therefore, researchers believe that CLK2 plays a vital role in the Wnt pathway and the generation and differentiation of stem cells, and its development as a drug for the treatment of osteoarthritis has attracted the attention of many scientific research institutions and drug developers. However, research on CLK2 kinase inhibitors is still in its infancy.

Contents of the invention

Purpose of the invention: the present invention provides 5-pyridine-1H-indazole compounds targeting to inhibit CLK2 and applications thereof. Evaluation of biological activity shows that the compound of the present invention has significant CLK2 inhibitory activity, has better selectivity to CLK family members, and has significant DYRK1A inhibitory activity at the same time. The pharmacodynamic results show that the compound of the present invention has good anti-osteoarthritis activity both in vivo and in vitro.

Technical solution: 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 their pharmaceutically acceptable salts, their predecessors Drugs, their hydrates or their solvates:

Among them, R ₁ , R ₂ or R ₄ are selected from the following groups: C1-C10 alkyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 1-3 members selected from N, O and One of the 5-10 membered heteroaryl groups with S heteroatoms, the substituents on the aryl or heteroaryl groups are selected from halogen, Cl~C6 alkyl, Cl~C6 alkoxy, Cl~C6 alkylthio Base, halogenated Cl～C6 alkyl, halogenated Cl～C6 alkoxy, amino, Cl～C6 alkanoyl, NH-CO-Cl～C6 alkyl, -NH(Cl～C6 alkyl), -N( One or more of Cl~C6 alkyl) (Cl~C6 alkyl) and hydroxyl;

R ₃ is selected from the following groups:

Preferably, said R ₁ is selected from:

Preferably, said R ₂ is selected from:

Preferably, said R ₄ is selected from:

As a specific embodiment of the present invention, the compound is as follows:

Further, the pharmaceutically acceptable salt of the compound of the present invention refers to the salt formed by the compound represented by the general formulas I, II and III 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 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-mentioned compounds.

The content of the present invention also includes the application of the compound or the pharmaceutical composition in the preparation of drugs targeted to inhibit CLK2.

The content of the present invention also includes an application of the compound or the pharmaceutical composition in the preparation of a medicine for treating osteoarthritis.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

(1) The 5-pyridine-1H-indazole compound provided by the present invention has high selectivity and is a small molecule inhibitor of CLK2 with good druggability.

(2) The structure of the compounds described in the present invention introduces an alkynyl group, the structure is more novel, and shows effective inhibitory activity on CLK2, wherein several compounds have an inhibitory rate of more than 80% on CLK2 at a concentration of 20 nM, and at the same time, with Compared with Lorecivivint, a CLK2 inhibitor that has entered Phase III clinical trials, the compound of the present invention shows better CLK2 inhibitory activity and better selectivity than positive drugs.

(3) The compound of the present invention shows good anti-osteoarthritis activity both in vitro and in vivo.

Description of drawings

Fig. 1 is the CLKs kinase inhibitory activity of compound LQ23 of the present invention, (n=2, Mean±SD);

Fig. 2 is the regulation of compound LQ23 of the present invention on cartilage degradation protease and inflammatory factor levels in rat chondrocytes, (n=3, Mean±SD);

Fig. 3 is the regulation of the compound LQ23 of the present invention on the levels of cartilage-degrading proteases and inflammatory factors in rat mesenchymal stem cells, (n=3, Mean±SD);

Fig. 4 is the cartilage degrading protease gene expression level in cartilage treated by compound LQ23 in rat MIA osteoarthritis model. (A) Day 11; (B) Day 28. (n=3, Mean±SD);

Fig. 5 is the gene expression levels of COMP and inflammatory factors in serum during the treatment of compound LQ23 of the present invention in rat MIA osteoarthritis model, (A) on the 11th day; (B) on the 28th day. (n=3, Mean±SD);

Fig. 6 The gene expression level of cartilage degrading protease in the cartilage of the treated cartilage of the compound LQ23 of the present invention in the rat ACLT+pMMx osteoarthritis model. (A) the 5th week; (B) the 13th week; (n=3, Mean±SD);

Fig. 7 is the gene expression level of COMP and inflammatory factor in serum in the treatment of compound LQ23 of the present invention in rat ACLT+pMMx osteoarthritis model, (A) the 5th week; (B) the 13th week; (n= 3, Mean ± SD).

Detailed ways

Embodiment 1: the synthesis of compound LQ01

Synthesis of Intermediate 2:

Add raw material 1 (5-bromoindazole, 5g, 25mmol) into a 250mL three-necked flask, add tetrahydrofuran (50mL), p-toluenesulfonic acid (0.48g, 2.5mmol), and add 3,4- Dihydropyran (4.3g, 50mmol) was heated to reflux for 8h. TLC detected that the reaction was complete, and then the solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography to obtain 7.1 g of a white solid with a yield of 63%. The characterization data of intermediate 2 are as follows HRMS(ESI+): cacld for C ₁₂ H ₁₃ BrN ₂ O(M+H)+, 281.0284; found, 281.0256.

Synthesis of intermediate 3:

Intermediate 2 (1.3g, 4.6mmol) was added to a 100mL single-necked flask, and biboronic acid pinacol ester (1.4g, 5.5mmol), potassium acetate (1.4g, 13.8mmol), 1,1'-bis Diphenylphosphinoferrocenepalladium dichloride (0.17g, 0.23mmol), dioxane (13mL), under nitrogen protection, heated to reflux for 6h, TLC detected that the reaction was complete, after cooling down, suction filtered, and the filter cake was washed with a small amount of After washing with ethyl acetate, the filtrate was evaporated to remove the solvent under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain intermediate 3 as a white solid (1.2 g, yield 81%). The characterization data of Intermediate 3 are as follows: ¹ H NMR (300MHz, CDCl ₃ -d ₆ ): δ=8.25(t, J=1.1Hz, 1H), 8.04(s, 1H), 7.82-7.78(dd, J ₁ = 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, J ₁ =2.9Hz, J ₂ =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 C ₁₈ H ₂₅ BN ₂ O ₃ (M+H) ⁺ , 329.2031; found, 329.1998.

Synthesis of Intermediate 5:

The raw material 4 (3-amino-5-bromopyridine, 2g, 12mmol) was dissolved in dichloromethane and then cooled to 0°C in an ice bath, isovaleric acid chloride (2g, 17mmol) was dissolved in dichloromethane and added dropwise Ethylamine (2.3 g, 23 mmol). Stirred under ice bath for 30min. After the completion of the reaction detected by TLC, the solvent was evaporated under reduced pressure, dissolved in ethyl acetate, extracted, the organic layer was washed with dilute hydrochloric acid, saturated sodium bicarbonate, concentrated brine, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was evaporated under reduced pressure Solvent, beating with a small amount of dichloromethane to obtain 2.2 g of white solid with a yield of 74%. The characterization data of intermediate 5 are as follows: ¹ H NMR (300MHz, DMSO-d ₆ ): 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 C ₁₀ H ₁₃ BrN ₂ O(M+H) ⁺ , 257.0284; found, 257.0292.

Synthesis of Intermediate 6:

Intermediate 5 (0.1g, 0.4mmol), Intermediate 3 (0.13g, 0.4mmol), sodium carbonate (0.12g, 1.17mmol), 1,1'-bisdiphenylphosphinoferrocenedichloropalladium (14mg, 0.02mmol), dioxane (2mL) and water (0.3mL) were added into a 50mL single-necked bottle, the air was replaced with nitrogen, and the temperature was raised to reflux for 8h. After the completion of the reaction detected by TLC, the temperature was lowered and suction filtered, the filter cake was washed with a small amount of ethyl acetate, and the filtrate was spin-dried. The crude product was purified by silica gel column chromatography to obtain intermediate 6, a yellow oil, with a yield of 69%. The characterization data of intermediate 6 are as follows: ¹ H NMR (300MHz, DMSO-d ₆ ): 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 C ₂₂ H ₂₆ N ₄ O ₂ (M+H) ⁺ , 379.2179; found, 379.2134.

Synthesis of Intermediate 7:

Intermediate 6 (0.14g, 0.4mmol) and trifluoroacetic acid (1mL) were sequentially added into dichloromethane (1mL) and reacted at room temperature for 1 hour. After the reaction was complete as detected by TLC, the solvent was distilled off under reduced pressure, extracted with ethyl acetate, the organic layer was washed three times with saturated sodium bicarbonate solution, washed with concentrated brine, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was distilled off the solvent under reduced pressure. Vacuum drying afforded 0.1 g of yellow solid with a yield of 84%. The characterization data of intermediate 7 are as follows: HRMS(ESI+): cacld for C ₁₇ H ₁₈ N ₄ O(M+H)+, 295.1553; found, 295.1555.

Synthesis of Intermediate 8:

Intermediate 7 (0.15g, 0.51mmol), potassium hydroxide (0.14g, 2.5mmol), and N,N-dimethylformamide (2mL) were added to a three-necked flask, protected by nitrogen, and cooled to 0 in an ice bath. °C, iodine (0.16 g, 0.61 mmol) was added to the reaction solution in batches, and after the addition was complete, the reaction was carried out at room temperature for 1 hour. After the completion of the reaction detected by TLC, it was quenched with saturated sodium thiosulfate solution, the solid precipitated out, filtered with suction, and the filter cake was dried to obtain Intermediate 8 as a powdery white solid (0.11 g, yield 52%). The characterization data of intermediate 8 are as follows: ¹ HNMR (300MHz, CDCl ₃ -d ₆ ): 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, J ₁ =8.8Hz, J ₂ =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 C ₁₇ H ₁₇ IN ₄ O(M+H) ⁺ , 421.0520; found, 421.0532.

Synthesis of Intermediate 9:

Intermediate 8 (50mg, 0.12mmol), 4-dimethylaminopyridine (1.5mg, 0.012mmol), triethylamine (36mg, 0.36mmol) were added to dichloromethane (0.5mL) and stirred, and dicarbonic acid Di-tert-butyl ester (39 mg, 0.18 mmol) was added to the reaction mixture, and reacted at room temperature for 2 hours. After the completion of the reaction as detected by TLC, the reaction liquid was evaporated to remove the solvent under reduced pressure, slurried with n-hexane, filtered with suction, and the filter cake was vacuum-dried to obtain intermediate 9 as a pale yellow solid (39 mg, yield 63%). The characterization data of intermediate 9 are as follows: ¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =8.8Hz, J ₂ =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 C ₂₂ H ₂₅ IN ₄ O ₃ (M+H) ⁺ , 521.1044; found, 521.1019.

Synthesis of Intermediate 10a:

Intermediate 9 (0.2g, 0.38mmol), 3-ethynylpyridine (59mg, 0.57mmol), bistriphenylphosphinepalladium dichloride (13mg, 0.02mmol), cuprous iodide (7mg, 0.04mmol) , triethylamine (2mL), and N, N-dimethylformamide (1mL) were added to a 50mL single-necked bottle, and the air was replaced with nitrogen, and heated to reflux for 6 hours. Add water (3mL), extract with ethyl acetate three times, combine the organic layers, wash with concentrated brine, dry over anhydrous sodium sulfate, and filter with suction. The filtrate is evaporated to remove the solvent under reduced pressure, and the crude product is purified by silica gel column chromatography to obtain yellow oil 10a (0.1 g, yield 53%). The characterization data of intermediate 10a are as follows: HRMS (ESI+): cacld for C ₂₉ H ₂₉ N ₅ O ₃ (M+H)+, 496.2343; found, 496.2360.

Synthesis of compound LQ01:

Intermediate 10a (0.1 g, 0.2 mmol) was added to 4N hydrogen chloride-ethyl acetate solution (CHCl = 4 mol/L, 1 _mL ), heated to 40 ° C for 1 hour, and after TLC detected that the reaction was complete, suction filtration, The filter cake was washed with ethyl acetate and dried in vacuo to obtain a yellow solid LQ01 (45mg, yield 57%), mp 224-227°C, the structure and characterization data are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 C ₂₄ H ₂₁ N ₅ O(M+H) ⁺ , 396.1819; found, 396.1827.

Embodiment 2: Compound N-(5-(3-((4-cyanophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ02 )Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 4-cyanophenylacetylene, and LQ02 was synthesized by the method of Example 1. The structure and characterization data of LQ02 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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=6.5 Hz, 6H) ppm. ¹³ C NMR (75MHz, DMSO-d ₆ ): δ=172.69, 140.76, 135.53, 132.90 (J=24.6Hz), 130.90 (J=11.0Hz), 128.48 (J=5.8Hz), ^{found _} _{_ _ _} , 420.1852.

Example 3: Synthesis of compound 3-methyl-N-(5-(3-thiazol-4-ylethynyl)-1H-indazol-5-yl)pyridine-3-methylbutyramide (LQ03)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 4-ethynylthiazole, and LQ03 was synthesized by the method of Example 1. The structure and characterization data of LQ03 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ )：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 C ₂₂ H ₁₉ N ₅ OS(M+H) ⁺ , 402.1383; found, 402.1355.

Embodiment 4: Compound N-(5-(3-((4-formylphenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ04 )Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 4-ethynylbenzaldehyde, and the method of Example 1 was used to synthesize LQ04. The structure and characterization data of LQ04 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 HRMS, pp : cacld for C ₂₆ H ₂₂ N ₄ O ₂ (M+H) ⁺ , 423.1816; found, 423.1819.

Example 5: Synthesis of Compound 3-((5-(5-(3-methylbutyramide) pyridin-3-yl)-1H-indazol-3-yl)ethynyl)benzoic acid (LQ05)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 3-ethynylbenzoic acid, and LQ05 was synthesized by the method of Example 1. The structure and characterization data of LQ05 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ CNMR (75MHz, DMSO-d ₆ ): 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 ⁺ ) for C ₂ (M+H) ⁺ , 439.1765; found, 439.1768.

Example 6: Compound N-(5-(3-((3-aminophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ06) Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 3-ethynylaniline, and LQ06 was synthesized by the method of Example 1. The structure and characterization data of LQ06 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO -d ₆ ): 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, 22.0 pp MS: cacld for C ₂₅ H ₂₃ N ₅ O(M+H) ⁺ , 410.1975; found, 410.1974.

Example 7: Synthesis of Compound N-(5-(3-isoquinolin-6-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl-3-methylbutanamide (LQ07)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 6-ethynylisoquinoline, and LQ07 was synthesized by the method of Example 1. The structure and characterization data of LQ07 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =8.6Hz, J ₂ =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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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, 126.12, 128.16, 1 119.14, 112.93, 92.86, 87.45, 45.72, 25.97, 22.75ppm. HRMS (ESI ⁺ ): cacld for C ₂₈ H ₂₃ N ₅ O(M+H) ⁺ , 446.1975; found, 446.1999.

Example 8: Synthesis of compound 3-methyl-N-(5-(3-(pyridin-4-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)butyramide (LQ08)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 4-ethynylpyridine, and LQ08 was synthesized by the method of Example 1. The structure and characterization data of LQ08 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 C ₂₄ H ₂₁ N ₅ O(M+H) ⁺ , 396.1819; found, 396.1825.

Example 9: Compound N-(5-(3-((3-fluorophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ09) Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 3-fluorophenylacetylene, and LQ09 was synthesized by the method in Example 1. The structure and characterization data of LQ09 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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 C ₂₅ H ₂₁ FN ₄ O(M+H) ⁺ , 413.1722; found, 413.1735.

Example 10: Synthesis of 3-methyl-N-(5-(3-(pyrimidin-2-ylethynyl)-1H-indazol-5-ylpyridin-3-yl)butanamide (LQ10)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 2-alkynylpyrimidine, and LQ10 was synthesized by the method of Example 1. The structure and characterization data of LQ10 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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. +H) ⁺ , 397.1771; found, 397.1789.

Example 11: Synthesis of 5-(3-(2-chlorophenyl)ethynyl)-1H-indazol-5-ylpyridin-3-yl-3-methylbutanamide (LQ11)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 2-chlorophenylacetylene, and LQ11 was synthesized by the method in Example 1. The structure and characterization data of LQ11 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 C ₂₅ H ₂₁ ClN ₄ O(M+H) ⁺ , 429.1477; found, 429.1464.

Example 12: Synthesis of 3-methyl-N-(5-(3-(pyridin-2-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)butanamide (LQ12)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 2-ethynylpyridine, and LQ12 was synthesized by the method of Example 1. The structure and characterization data of LQ12 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ = 5.0Hz, J ₂ = 1.3Hz, 1H), 8.42 (d, J = 1.4Hz, 1H), 8.11 (td, J ₁ = 7.7Hz, J ₂ =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, J ₁ =13.5Hz, J ₂ =6.6Hz, 1H), 0.98(d, J=6.6Hz, 6H)ppm. ¹³ C NMR (75MHz, MeOH-d ₆ ): 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, 126.75, 121.59, 0 90.79, 84.44, 45.79, 25.99, 22.74ppm. HRMS (ESI ⁺ ): ccld for C ₂₄ H ₂₁ N ₅ O(M+H) ⁺ , 396.1819; found, 396.1824.

Example 13: Compound N-(5-(3-((1-hydroxycyclohexyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ13) Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by acetylene cyclohexanol, and LQ13 was synthesized by the method of Example 1. The structure and characterization data of LQ13 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, MeOH-d ₆ )：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 C ₂₅ H ₂₈ N ₄ O ₂ (M+H) ⁺ , 417.2285; found, 417.2293.

Example 14: Compound 3-methyl-N-(5-(3-(quinolin-2-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)butanamide (LQ14) synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by 2-ethynylquinoline, and LQ14 was synthesized by the method of Example 1. The structure and characterization data of LQ14 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =8.1Hz, J ₂ =6.8Hz, J ₃ =1.2Hz, 1H), 2.27(d, J=7.0 Hz, 2H), 2.12 (m, 1H), 0.97 (d, J=6.5Hz, 6H) ppm. HRMS (ESI ⁺ ): caeld for C ₂₈ H ₂₃ N ₅ O(M+H) ⁺ , 446.1975; found , 446.1984.

Example 15: Compound 3-methyl-N-(5-(3-((3-nitrophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)butanamide (LQ15 )Synthesis

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by m-nitrophenylacetylene, and LQ15 was synthesized by the method in Example 1. The structure and characterization data of LQ15 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =8.1Hz, J ₂ =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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 C ₂₅ H O ₂₁ N +H) ⁺ , 440.1717; found, 440.1732.

Example 16: Synthesis of compound N-(5-(3-(cyclopropylethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-methylbutanamide (LQ16)

In the synthesis step of intermediate 10a in Example 1, 3-ethynylpyridine was replaced by cyclopropyne, and LQ16 was synthesized by the method in Example 1. The structure and characterization data of LQ16 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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 . ¹³ C NMR (75MHz, DMSO-d ₆ ): 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, 67.88, 61.401, 2 ppm.HRMS(ESI ⁺ ): cacld for C ₂₂ H ₂₂ N ₄ O(M+H) ⁺ , 359.1866; found, 359.1832.

Embodiment 17: the synthesis of compound LQ20

Synthetic route 2:

Synthesis of Intermediate 11a:

Intermediate 9 (0.15g, 0.29mmol), 2-vinylpyridine (45mg, 0.43mmol), palladium acetate (3mg, 0.01mmol), tri(o-methylphenyl)phosphorus (9mg, 0.03 mmol), N,N-diisopropylethylamine (0.11g, 0.87mmol) were added to N,N-dimethylformamide (2mL), the air was replaced with nitrogen, and heated to reflux for 7 hours. Cool the reaction solution, add water (6 mL), extract with ethyl acetate (3 mL×3), wash the organic layers with concentrated brine, dry over anhydrous sodium sulfate, and filter with suction. The filtrate is evaporated under reduced pressure to remove the solvent, and the crude product is passed through a silica gel column Purification by chromatography afforded 11a as a reddish-brown solid (57 mg, yield 40%). HRMS (ESI ⁺ ): cacld for C ₂₉ H ₃₁ N ₅ O ₃ (M+H) ⁺ , 498.2500; found, 498.2518.

Synthesis of compound LQ20:

Intermediate 11a (90mg, 0.18mmol) was added to 4N hydrogen chloride-ethyl acetate solution ( _CHCl = 4mol/L, 1mL), stirred at room temperature for 2 hours, after the reaction was detected by TLC, suction filtered, and the filtrate was evaporated under reduced pressure. The solvent was removed, and the crude product was purified by silica gel column chromatography to obtain 34 mg of a red solid with a yield of 48%. The structure and characterization data of compound LQ20 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 ⁺ ): ccld for C ₂₄ H ₂₃ N ₅ O(M+H) ⁺ , 398.1975; found, 398.1993.

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)

In the synthesis step of intermediate 11a in Example 17, 2-vinylpyridine is replaced by 1-(3-hydroxypyrrolidin-1-yl) propyl-2-en-1-one, using the method of Example 17 Synthesis of LQ21, the structure and characterization data of compound LQ21 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): δ=13.61 (s, 1H), 10.22 (s, 1H), 8.76-8.67 (m, 2H), 8.43 (dt, J ₁ =8.5Hz, J ₂ = 2.3Hz, 1H), 8.33(s, 1H), 7.82(dd, J ₁ =15.7Hz, J ₂ =2.0Hz, 1H), 7.73(s, 2H), 7.20(dd, J ₁ =21.5Hz, J ₂ =15.7Hz, 1H), 5.03(dd, J ₁ =20.4Hz, J ₂ =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, J ₁ =13.7Hz, J ₂ =6.7Hz, 1H), 2.05-1.74(m, 2H) , 0.97 (d, J=6.5Hz, 6H) ppm. HRMS (ESI ⁺ ): cacld for C ₂₄ H ₂₇ N ₅ O ₃ (M+H) ⁺ , 434.2187; found, 434.2198.

Example 19: Compound 5-(3-(3-(3-morpholinyl-3-oxopropyl-1-en-1-yl)-1H-indazol-5-yl)pyridin-3-yl Synthesis of Butanamide (LQ22)

In the synthesis step of intermediate 11a in Example 17, 2-vinylpyridine was replaced by N-acryloylmorpholine, and LQ22 was synthesized by the method of Example 17. The structure and characterization data of compound LQ22 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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 C ₂₄ H ₂₇ N ₅ O ₃ (M+H) ⁺ , 434.2187; found, 434.2249.

Embodiment 20: the synthesis of compound LQ23

Synthetic route 3:

Synthesis of Intermediate 12:

Add raw material 1 (5-bromoindazole, 1 g, 5.1 mmol), potassium hydroxide (0.9 g, 15.3 mmol) into N, N-dimethylformamide (3 mL), cool to 0 ° C in an ice bath, and divide Iodine (2.6 g, 10.2 mmol) was added in portions. Move to room temperature and react for 2 hours. After TLC detects that the reaction is complete, quench with saturated sodium thiosulfate solution, filter with suction, wash the filter cake with a small amount of water, and dry in vacuo to obtain 1.5 g of a gray solid with a yield of 94%. Data characterization of intermediate 12: ¹ H NMR (300 MHz, DMSO-d ₆ ): δ=7.62 (s, 1H), 7.55 (s, 2H) ppm. HRMS (ESI ⁺ ): cacld for C ₇ H ₄ BrIN ₂ (M+H) ⁺ , 322.8675; found, 322.8662.

Synthesis of intermediate 13:

Intermediate 12 (1 g, 3.1 mmol) and p-toluenesulfonic acid (60 mg, 0.3 mmol) were added to tetrahydrofuran (10 mL), and 3,4-dihydro-2H-pyran (0.6 mL, 6.2 mmol) was added dropwise to The reaction solution was heated to reflux for 5 hours. After the reaction was complete as detected by TLC, the heating was stopped, and the solvent was evaporated from the filtrate under reduced pressure. Intermediate 13 was obtained by silica gel column chromatography, 0.8 g of yellow solid, with a yield of 67%. The data of intermediate 13 were characterized as follows: ¹ H NMR (300 MHz, CDCl ₃ -d ₆ ): δ=7.63 (s, 1H), 7.53-7.45 (m, 2H), 5.69-5.65 (dd, J ₁ =2.8Hz , J ₂ =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.

Synthesis of intermediate 14a:

Intermediate 13 (4.2g, 10mmol), 3-ethynylthiophene (1.7g, 15mmol), bistriphenylphosphinepalladium dichloride (86mg, 0.5mmol), cuprous iodide (48mg, 1mmol), tris Ethylamine (20mL) and tetrahydrofuran (20mL) were added to a 100mL single-necked flask, the air was replaced with nitrogen, and the reaction was carried out overnight at room temperature. The intermediate 14a was obtained as 3.2 g of yellow oil, with a yield of 84%. Characterization data of intermediate 14a: HRMS(ESI ⁺ ): cacld for C ₁₈ H ₁₅ BrN ₂ OS(M+H) ⁺ , 387.0161; found, 387.0159.

Synthesis of Intermediate 15a:

Intermediate 14a (0.9g, 2.3mmol), pinacol diboronate (0.7g, 2.8mmol), potassium acetate (0.7g, 6.9mmol), 1,1'-bisdiphenylphosphinoferrocene Palladium chloride (84mg, 0.1mmol) and dioxane (9mL) were added in a 100mL single-necked bottle, the air was replaced by nitrogen, and heated to reflux for 6 hours. The solvent was evaporated under pressure, and the crude product was purified by silica gel column chromatography to obtain 0.5 g of a yellow solid with a yield of 50%. Characterization data of intermediate 15a: ¹ H NMR (300 MHz, DMSO-d ₆ ): δ=8.12 (m, 2H), 7.88-7.73 (m, 2H), 7.72-7.67 (m, 1H), 7.42 (dd, J ₁ =5.0Hz, J ₂ =1.2Hz, 1H), 5.94(dd, J ₁ =9.4Hz, J ₂ =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 C ₂₄ H ₂₇ BN ₂ O ₃ S(M+H) ⁺ , 435.1908; found, 435.1935.

Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentyl methanesulfonate:

Add (S)-(1-hydroxy-4-methylpentan-2-yl) tert-butyl carbamate (1.0g, 4.6mmol) and dichloromethane (10mL) into a 100mL three-necked flask and cool down To 0°C, add pyridine (0.7mL, 9.2mmol), stir for 5 minutes, then add methanesulfonic anhydride (1.2g, 6.9mmol) in batches, move to room temperature and react for 3 hours, after TLC detects that the reaction is complete, add water, Extracted with dichloromethane, the organic layers were combined, washed with concentrated brine, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was evaporated to remove the solvent under reduced pressure to obtain (S)-2-((tert-butoxycarbonyl)amino)-4- Methylpentyl methanesulfonate, orange solid 1.1 g, yield 81%. The characterization data of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentyl methanesulfonate is as follows: ¹ H NMR (300MHz, CDCl ₃ -d ₆ ): 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, J ₁ =2.7Hz, J ₂ =6.6Hz, 6H) ppm.HRMS(ESI ⁺ ): cacld for C ₁₂ H ₂₅ NO ₅ S(M+H) ⁺ , 296.1526; found, 296.1539.

Synthesis of Intermediate 16a:

(S)-2-((tert-butoxycarbonyl)amino)-4-methylpentyl methanesulfonate (0.5g, 1.69mmol), 3-bromo-5-hydroxypyridine (0.38g, 2.2mmol ), cesium carbonate (1.1g, 3.4mmol), N, N-dimethylformamide (5mL) were added to a 25mL single-necked bottle, heated and reacted at 100°C for 3 hours, after TLC detected that the reaction was complete, add water, and use Extracted with ethyl acetate, combined the organic layers, washed with concentrated brine, dried over anhydrous sodium sulfate, filtered with suction, and evaporated the filtrate to remove the solvent under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 0.2 g of colorless oil 16a with a yield of 35%. Characterization data of intermediate 16a: ¹ H NMR (300 MHz, DMSO-d ₆ ): δ=8.29-8.26 (m, 2H), 7.72-7.70 (t, J=2.0 Hz, 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 C ₁₆ H ₂₅ BrN ₂ O ₃ (M+H) ⁺ , 373.1121; found, 373.1141.

Synthesis of Intermediate 17a:

Intermediate 15a (0.3g, 0.8mmol), Intermediate 16a (0.4g, 0.9mmol), 1,1'-bisdiphenylphosphinoferrocenepalladium dichloride (30mg, 0.04mmol), sodium carbonate ( 0.25g, 2.4mmol), dioxane (4mL) and water (1mL) were added to a 25mL single-necked bottle, nitrogen replaced the air, heated to reflux for 6 hours, after TLC detection of complete reaction, suction filtration, The filter cake was washed with a small amount of ethyl acetate, and the filtrate was evaporated to remove the solvent under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 17a, which was dried in vacuo to obtain a yellow solid (0.25 g, yield 52%). Characterization data of intermediate 17a: HRMS (ESI ⁺ ): cacld for C ₃₄ H ₄₀ N ₄ O ₄ S(M+H) ⁺ , 601.2843; found, 601.2857.

Synthesis of compound LQ23:

Add intermediate 17a (0.25g, 0.4mmol), 4N hydrogen chloride-ethyl acetate solution (CHCl=4mol/ _L , 2mL) into a 25mL single-necked bottle, heat to 40° _C for 1 hour, wait for TLC After the reaction was complete, filter with suction, wash the filter cake with a small amount of ethyl acetate, and evaporate the filtrate to remove the solvent under reduced pressure. The crude product was recrystallized from ethyl acetate/ethanol to obtain LQ23 as a pale yellow solid (70mg, yield 42%), the purity was 99.3% by HPLC, mp 227-230°C. The structure and characterization data of LQ23 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =2.9Hz, J ₂ =1.2Hz, 1H), 7.92 (dd, J ₁ =8.8Hz, J ₂ =1.7Hz, 1H), 7.80 (d, J = 8.7Hz , 1H), 7.72 (dd, J ₁ =5.0Hz, J ₂ =2.9Hz, 1H), 7.43 (dd, J ₁ =5.0Hz, J ₂ =1.2Hz, 1H), 4.57 (dd, J ₁ =10.7 Hz, 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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. +H) ⁺ , 417.1744; found, 417.1744.

Example 21: Synthesis of Compound N-(5-(3-cyclopentylkynyl)-1H-indazol-5-ylpyridin-3-yl)-3-methylbutyramide (LQ17)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by cyclopentylacetylene, and intermediate 15c was synthesized by the method in Example 20. The structure and characterization data of intermediate 15c are as follows:

HRMS(ESI+): cacld for C ₂₅ H ₃₃ BN ₂ O ₃ (M+H) ⁺ , 421.2657; found, 421.2655.

In the synthesis step of intermediate 17a in Example 20, intermediate 16a was replaced by intermediate 5, and LQ17 was synthesized by the method of Example 20. The structure and characterization data of LQ17 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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 ⁺ ): ccld for C ₂₄ H ₂₆ N ₄ O(M+H) ⁺ , 387.2179; found, 387.2249.

Example 22: Compound N-(5-(3-([1,1′-biphenyl]-4-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)-3-meth Synthesis of Butyramide (LQ18)

In the synthesis step of intermediate 15c in Example 21, cyclopentylacetylene was replaced by 4-acetylene biphenyl, and LQ18 was synthesized by the method of Example 21. The structure and characterization data of LQ18 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ CNMR (75MHz, DMSO-d ₆ ): 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 C ₃₁ H ₂₆ N ₄ O(M+H) ⁺ , 471.2719; found, 471.2724.

Example 23: Synthesis of Compound 5-(3-(4-methoxyphenyl)ethynyl)-1H-indazol-5-ylpyridin-3-yl-3-methylbutanamide (LQ19)

In the synthesis step of intermediate 15c in Example 21, cyclopentylacetylene was replaced by 4-ethynyl anisole, and LQ19 was synthesized by the method of Example 21. The structure and characterization data of LQ19 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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 C ₂₆ H ₂₄ N ₄ O ₂ (M +H) ⁺ , 425.1972; found, 425.1971.

Example 24: Synthesis of compound N-(5-(3-(thiophen-3-ylethynyl)-1H-indazol-5-yl)pyridin-3-yl)cyclopropanecarboxamide (LQ24)

Synthesis of Intermediate 16b:

Add 3-bromo-5-hydroxypyridine (1.0 g, 5.8 mmol) and dichloromethane (10 mL) into a 100 mL three-necked flask, and cool down to 0°C. Cyclopropylformyl chloride (0.9 g, 8.7 mmol) was dissolved in dichloromethane and added dropwise to the reaction solution, and triethylamine (1.6 mL, 11.6 mmol) was added dropwise. React at 0°C for 3 hours. After the reaction is complete as detected by TLC, evaporate the solvent under reduced pressure and extract with ethyl acetate. The organic layer is washed with 2N dilute hydrochloric acid, water, saturated sodium bicarbonate solution, and concentrated brine. Dry over sodium sulfate, filter with suction, and distill the filtrate to remove the solvent under reduced pressure to obtain 0.6 g of yellow oil with a yield of 43%. Characterization data of intermediate 16b: HRMS(ESI+): cacld for C9H9BrN2O(M+H)+, 240.9971; found, 240.9939.

In the synthesis step of intermediate 17a in Example 20, intermediate 16a was replaced by intermediate 16b, and LQ24 was synthesized by the method of Example 20. The structural formula and characterization data of LQ24 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =2.9Hz, J ₂ =1.2Hz, 1H), 7.84(s, 2H), 7.72(dd, J ₁ =5.0Hz, J ₂ =2.9Hz, 1H), 7.43(dd, J ₁ =5.0Hz, J ₂ =1.2Hz, 1H), 2.06-1.89(m, 1H), 0.93(m, 4H) ppm. ¹³ C NMR (75MHz ，DMSO-d ₆ )：δ＝173.88，140.78，139.60，139.21，134.76，131.38，131.34，130.30，130.03，129.25，127.91，127.64，126.53，125.10，121.17，119.52，112.77，89.09，81.02，15.23，8.83 ppm.HRMS(ESI ⁺ ): cacld for C ₂₂ H ₁₆ N ₄ OS(M+H) ^- , 385.1118; found, 385.1139.

Example 25: Synthesis of Compound 5-(5-(pyrrolidin-2-ylmethoxy)pyridin-3-yl)-3-(thiophen-3-ylethynyl)-1H-indazole (LQ25)

In the synthesis step of intermediate 16a in Example 20, (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentyl methanesulfonate is replaced by 2-hydroxymethylpyrrolidine -1-butyl carboxylate, adopt the method for embodiment 20 to synthesize LQ25, the structural formula and characterization data of LQ25 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =2.9Hz, J ₂ =1.2Hz, 1H), 7.84(s, 2H), 7.72(dd, J ₁ =5.0Hz, J ₂ =2.9Hz, 1H), 7.43(dd, J ₁ =5.0Hz, J ₂ =1.2Hz, 1H), 2.06-1.89(m, 1H), 0.93(m, 4H) ppm. ¹³ C NMR (75MHz ，DMSO-d ₆ )：δ＝173.88，140.78，139.60，139.21，134.76，131.38，131.34，130.30，130.03，129.25，127.91，127.64，126.53，125.10，121.17，119.52，112.77，89.09，81.02，15.23，8.83 ppm.HRMS(ESI ⁺ ): cacld for C ₂₂ H ₁₆ N ₄ OS(M+H) ⁺ , 385.1118; found, 385.1139.

Example 26: Compound 5-(5-((3,3-difluoropyrrolidin-1-yl)methyl)pyridin-3-yl)-3-(thiophen-3-ylethynyl)-1H-ind Synthesis of azole (LQ26)

Synthesis of Intermediate 16d:

3,3-Difluoropyrrolidine (1.0g, 9.43mmol), 5-bromopyridine-3-carbaldehyde (0.8g, 4.6mmol), triethylamine (0.9g, 9.3mmol), dichloromethane (10ml) Add it to a 100ml single-necked bottle, and stir at room temperature for 1 hour. Sodium triacetoxyborohydride (1.5g, 7.0mmol) was added to the reaction solution, and reacted at room temperature overnight. After the reaction was complete as detected by TLC, saturated sodium bicarbonate solution was added, extracted with dichloromethane, and the organic layers were combined and washed with concentrated Washed with brine, dried over anhydrous sodium sulfate, filtered with suction, evaporated the filtrate to remove the solvent under reduced pressure, and dried in vacuo to obtain a brown oil (1.1 g, yield 87%). HRMS(ESI+): cacld for C10H11BrF2N2(M+H)+, 277.0146; found, 277.0169.

In the synthesis step of intermediate 17a in Example 20, intermediate 16a was replaced by intermediate 16d, and LQ26 was synthesized by the method of Example 20. The structural formula and characterization data of LQ26 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): δ = 8.89 (d, J = 1.7Hz, 1H), 8.56 (d, J = 2.6Hz, 1H), 8.30 (s, 2H), 8.09 (dd, J ₁ = 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, J ₁ = 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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, 5, 1182.90, 81.23, 69.11, 57.89, 45.38, 26.74, 23.92ppm. HRMS (ESI ⁺ ): cacld for C ₂₃ H ₁₈ F ₂ O ₄ S(M+H) ⁺ , 421.1293; found, 421.1325.

Example 27: Compound 1-((5-(3-((3-fluorophenyl)ethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy)-4-methylpentane Synthesis of -2-amine (LQ27)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by m-fluorophenylacetylene, and LQ27 was synthesized by the method of Example 20. The structural formula and characterization data of LQ27 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ = 3.2Hz, J ₂ = 5.8Hz, 6H) ppm. HRMS (ESI ⁺ ): cacld for C ₂₆ H ₂₅ FN ₄ O (M+ H) ⁺ , 429.2085; found, 429.2099.

Example 28: Compound 1-(5-(3-(3-fluorophenyl)ethynyl)-1H-indazol-5-ylpyridin-3-yl)-3-phenylpropane-2-amine (LQ28 )Synthesis

In the synthesis step of intermediate 16a in Example 20, (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentyl methanesulfonate is replaced by N-Boc-L-amphetamine Alcohol, using the method of embodiment 27 to synthesize LQ28, the structural formula and characterization data of LQ28 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₂ =5.7Hz, 1H), 3.86(s, 1H), 3.18(dd, J ₁ =13.6Hz, J ₂ =5.5Hz, 1H), 3.05(dd, J ₁ =13.7Hz, J ₂ =9.2Hz, 1H) ppm. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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, 115.93, 1 113.65, 68.40, 51.72, 35.28ppm. HRMS (ESI ⁺ ): cacld for C ₂₉ H ₂₃ FN ₄ O(M+H) ⁺ , 463.1929; found, 463.1988.

Example 29: Compound (S)-1-((5-(3-(cyclopentylethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy)-4-methylpentane -2-amine (LQ29) synthesis

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by cyclopentylacetylene, and LQ29 was synthesized by the method of Example 20. The structural formula and characterization data of LQ29 are as follows:

¹ H NMR (300 MHz, MeOH-d ₆ ): 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. ¹³ C NMR (75MHz, MeOH-d ₆ ): 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 C ₂₅ H ₃₀ N ₄ O(M+H) ⁺ , 403.2492; found, 403.2526 .

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)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by 4-ethynyl biphenyl, and LQ30 was synthesized by the method of Example 26. The structural formula and characterization data of LQ30 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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. ¹³ C NMR (75MHz, DMSO-d ₆ ): 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} _{. _ _ _} M+H) ⁺ , 491.2042; found, 491.2059.

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)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by 4-ethynylbiphenyl, and LQ31 was synthesized by the method of Example 20. The structural formula and characterization data of LQ31 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =9.6Hz, J ₂ =4.5Hz, 1H), 3.96 (dd, J ₁ =9.6Hz, J ₂ = 6.7Hz, 1H), 3.16(m, 1H), 1.93-1.76(m, 1H), 1.30(m, 4H), 0.90(dd, J ₁ =9.4Hz, J ₂ =6.5Hz, 6H)ppm.HRMS (ESI ⁺ ): ccld for C ₃₂ H ₃₀ N ₄ O(M+H) ⁺ , 487.2492; found, 487.2503.

Example 32: Compound 1-((5-(3-(cyclopropylethynyl)-1H-indazol-5-yl)pyridin-3-yl)oxy)-4-methylpentan-2-amine Synthesis of (LQ32)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by cyclopropyne, and LQ32 was synthesized by the method of Example 20. The structural formula and characterization data of LQ32 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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 ⁺ ): ccld for C ₂₃ H ₂₆ N ₄ O(M+H) ⁺ , 375.2179; found, 375.2168.

Example 33: Synthesis of Compound 5-(2-amino-4-methylpentyloxy)pyridin-3-ylpyridin-3-ylpyridin-3-ylethynyl-1H-indazole (LQ33)

In the synthesis step of intermediate 14a in Example 20, 3-ethynylthiophene was replaced by 3-ethynylpyridine, and LQ33 was synthesized by the method of Example 20. The structural formula and characterization data of LQ33 are as follows:

¹ H NMR (300MHz, DMSO-d ₆ ): 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, J ₁ =3.5 Hz, J ₂ =6.3Hz, 6H)ppm. HRMS (ESI ⁺ ): cacld for C ₂₅ H ₂₅ N ₅ O(M+H) ⁺ , 412.2126; found, 412.2137.

Example 34: Enzyme Inhibitory Activity of Compounds of Examples 1-33

Z'LYTE ^TM kit (ThermoFisher) was used to detect the inhibitory rate of compounds on kinases by using the principle of FRET. The in vitro CLK2 enzyme inhibitory activity of the compound LQ01-33 prepared in Examples 1-33 was studied, and the inhibitory rate of the compound at a concentration of 20 nM to the kinase and the IC ₅₀ of the dominant compound were tested.

Experimental principle: The peptide substrate is labeled with two fluorophores, one at each end, to form a FRET pair. In the primary reaction (kinase reaction), kinases transfer the g-phosphate of ATP to a single serine or threonine residue in a synthetic peptide substrate. In a secondary reaction (development reaction), a site-specific protease (development reagent) recognizes and cleaves the non-phosphorylated peptide. Phosphorylated peptides are inhibited from cleavage by imaging reagents. Cleavage disrupts FRET between the donor (ie, coumarin) and acceptor (ie, fluorescein) fluorophores on the peptide, while the non-cleaved phosphorylated peptide maintains FRET. The ratio method calculates the ratio of the donor emission to the acceptor emission (emission ratio) after the donor fluorophore is excited at 400nm, and quantifies the reaction process:

CLK2 enzyme inhibition experiment results (20nM compound's inhibitory rate on kinase, n=2):

CLKs kinase inhibitory activity (IC ₅₀ ) of dominant compound LQ23:

As described in the above table and Figure 1, the dominant compound LQ23 exhibited low nanomolar inhibitory activity on CLK1/2/4 (CLK1 IC ₅₀ =4nM, CLK2 IC ₅₀ =1.4nM, CLK4 IC ₅₀ =12nM), comparable to Lorecivivint Compared with that, the selectivity of LQ23 to CLK2 (VS CLK3 IC50>100nM) was at least 15 times higher. In addition, LQ23 exhibited stronger DYRK1A enzyme inhibitory activity than Lorecivivint (DYRK1A IC ₅₀ =21.7nM).

Example 35: Regulation of LQ23 on levels of cartilage-degrading proteases and inflammatory factors in vitro

After the rat chondrocytes were induced with TNF-α (20ng/mL) and Oncostatin M (10ng/mL) to produce inflammation, they were administered and treated for 48 hours immediately. The expression levels of MMP3, MMP13 and IL-6 genes were detected by qRT-PCR. The results were as follows: Figure 2 shows.

Rat bone marrow mesenchymal stem cells were treated with TNF-α (20ng/mL) and Oncostatin M (10ng/mL) to induce inflammation, and treated for 72 hours immediately. RT-qPCR detected MMP3, MMP13, ADAMTS5, IL-1β and TNF The expression level of -α gene, the results are shown in Figure 3.

The above results showed that, compared with the blank group, the related genes were significantly up-regulated in the model group, indicating that the modeling was successful. The treatment group of the dominant compound LQ23 has a down-regulation effect on related genes, which is better than the positive drug Lorecivivint.

Example 36: Regulation of LQ23 on levels of cartilage-degrading proteases and inflammatory factors in vivo

Based on the good anti-osteoarthritis activity of compound LQ23 in vitro, we further investigated the regulation level of LQ23 on cartilage degradation protease in vivo. Two rat osteoarthritis models were established: intra-articular injection of sodium iodoacetate (Monosodium iodoacetate, MIA) and partial medial meniscectomy combined with anterior cruciate ligament transection and partial medial meniscectomy model (ACLT+pMMx). After sub-articular injection of LQ23 (1.5 μg/kg) and Lorecivivint (1.5 μg/kg), the MIA model was treated for 28 days, and the ACLT+pMMx model was treated for 13 weeks. After the rat cartilage tissue was taken, RT-qPCR was used to detect the cartilage degradation protease and Gene expression levels of inflammatory factors; serum samples were taken, and ELISA kits were used to detect the contents of COMP and inflammatory factors. The experimental results are shown in Figure 4-7.

The above results show that in these two rat osteoarthritis model experiments, compared with the model group, LQ23 can significantly reduce the expression of cartilage-degrading proteases such as MMP13, MMP3, ADAMTS5 and IHH, and significantly reduce the levels of COMP and various inflammatory factors in serum. Factor content, potency is comparable to Lorecivivint. The above shows that LQ23 can significantly reduce cartilage degradation.

Claims (9)

1. 5-pyridine-1H-indazole compounds are characterized in that they are compounds represented by formula (I), formula (II) or formula (III) or pharmaceutically acceptable salts thereof, prodrugs thereof, hydrates thereof or solvates thereof thing:

Among them, R ₁ , R ₂ or R ₄ are selected from the following groups: C1-C10 alkyl, substituted or unsubstituted 6-10 membered aryl, substituted or unsubstituted 1-3 members selected from N, O and One of the 5-10 membered heteroaryl groups with S heteroatoms, the substituents on the aryl or heteroaryl groups are selected from halogen, Cl~C6 alkyl, Cl~C6 alkoxy, Cl~C6 alkylthio Base, halogenated C1～C6 alkyl, halogenated C1～C6 alkoxy, amino, Cl～C6 alkanoyl, NH-CO-Cl～C6 alkyl, -NH(Cl～C6 alkyl), -N( One or more of Cl~C6 alkyl) (Cl~C6 alkyl) and hydroxyl; Wherein, R ₃ is selected from the following groups:

2. The 5-pyridine-1H-indazole compound according to claim 1, wherein said R1 is selected from:

3. The 5-pyridine-1H-indazole compound according to claim 1, wherein said R2 is selected from:

4. 5-pyridine-1H-indazole compound according to claim 1, is characterized in that, described R4 is selected from:

5. 5-pyridine-1H-indazole compound according to claim 1, is characterized in that, described compound is selected from the compound shown in following structure:

6. The 5-pyridine-1H-indazole compound according to any one of claims 1 to 5, characterized in that, the pharmaceutically acceptable salt of the compound is a pharmaceutically acceptable salt form referring to the formula (I) , a salt formed by a compound represented by formula (II) or formula (III) and a pharmaceutically acceptable acid. 7. A pharmaceutical composition comprising the compound according to any one of claims 1-6. 8. A use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a drug targeted to inhibit CLK2. 9. The use of a compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating osteoarthritis.

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