5-pyridine-1H-indazole compound, pharmaceutical composition, and use

Abstract

Disclosed in the present invention are a 5-pyridine-1H-indazole compound, a pharmaceutical composition, and a use. The structure of the compound is represented by formula I or II, and also comprised a

Description

5-Pyridine-1H-indazole compounds, pharmaceutical compositions and applications Technical Field

[0001] The present invention relates to a 5-pyridine-1H-indazole compound, a pharmaceutical composition and an application, and in particular to a 5-pyridine-1H-indazole compound, a pharmaceutical composition and an application that can be prepared to effectively inhibit the activity of CLK2 or DYRK1A proteins. Background Art

[0002] Osteoarthritis (OA) is characterized by synovial inflammation, cartilage loss, and subchondral bone remodeling. The synovium of OA patients is rich in stem cells, and the inability to regenerate articular cartilage is not due to an insufficient supply of stem cells, but rather to improper stem cell differentiation. The Wnt pathway plays a central role in organogenesis, cell differentiation, and tissue remodeling. Aberrant activation or inhibition of the Wnt signaling pathway contributes to the development of OA. Therefore, the Wnt signaling pathway is a potential target for the treatment of OA.

[0003] The Wnt signaling pathway is a multi-channel signaling pathway initiated by the binding of the ligand protein Wnt to membrane protein receptors. Through this pathway, the intracellular activation of cell surface receptors transmits extracellular signals into the cell. In the canonical Wnt pathway, when Wnt protein is absent from the cell membrane, the downstream β-Catenin protein is degraded in the cytoplasm by the glycogen synthase 3 (GSK3) complex, preventing it from entering the nucleus and initiating transcription of related Wnt genes. However, when Wnt protein is present on the cell membrane, it inhibits the GSK3 complex, allowing β-Catenin protein to accumulate in the nucleus and ultimately initiating transcription of Wnt pathway-related genes. A delicate balance exists between bone and joint homeostasis and the Wnt pathway; disruption of this balance can lead to the development of osteoarthritis.

[0004] The CLK (CDK-like kinase) family of protein kinases is a dual-specificity protein kinase that regulates intracellular signal transduction by phosphorylating substrate proteins at tyrosine, serine, or threonine residues. It can be divided into four subtypes (CLK1, CLK2, CLK3, and CLK4), all of which encode proteins with a highly conserved C-segment gene sequence and a similar structural amino acid sequence. The CLK2 subtype is present in most eukaryotic organisms and participates in the phosphorylation of the SR (serine / arginine) protein domain, thereby regulating the selective splicing of RNA. It 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, and a potential therapeutic target for the Wnt pathway and osteoarthritis.

[0005] Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) belongs to the DYRK family, which is highly conserved across evolution. In mammals, the DYRK family comprises five distinct isoforms, with only DYRK1A located in the DSCR region of human chromosome 21. DYRK1A is expressed by the dyrk1a gene, encoding a mature protein consisting of 763 amino acids containing a protein kinase domain and other specialized structures. Many important proteins serve as substrates for DYRK1A and are regulated by it to participate in diverse cellular biological functions, including neural development, cell proliferation and differentiation, tumorigenesis, and neurodegenerative diseases.

[0006] Currently, no CLK2 inhibitors have been successfully marketed for the treatment of osteoarthritis. Only one small molecule, SM-04690, has entered Phase III clinical trials, thus failing to meet clinical needs. While SM04690 exhibits significant CLK2 inhibitory activity, its limited selectivity for the CLK family may lead to potential side effects. Furthermore, its inhibitory activity against the DYRK1A target and poor water solubility warrant improvement.

[0007] Summary of the Invention

[0008] Purpose of the invention: To address the problems of insufficient selectivity for the CLK family and insufficient inhibitory activity against the DYRK1A target in existing compounds, the present invention aims to provide a 5-pyridine-1H-indazole compound, pharmaceutical composition and application that has the ability to specifically inhibit the activity of CLK2 and DYRK1A proteins.

[0009] Technical solution: As the first aspect of the present invention, the 5-pyridine-1H-indazole compound of the present invention has a structure of Formula I or II, and also includes isomers, pharmaceutically acceptable salts or mixtures thereof:

[0010] wherein R1 is selected from the following groups substituted by one or more hydrogen, halogen, methoxy, trifluoromethyl, nitro, hydroxyl, amino, azido, sulfonic acid, 3-6 membered rings: hydrogen, straight or branched C1-C 10 Alkyl, phenyl, 4-6 membered heterocyclic ring or 4-6 membered ring;

[0011] L and M are selected from -CH2-, -NH-, -O- or a chemical bond;

[0012] R2 is selected from the following groups:

[0013]

[0014] R3 is selected from hydrogen or C1-C4 alkyl.

[0015] The present invention synthesized a series of derivatives through rational drug design. The biological activity evaluation showed that the designed compounds had significant CLK2 inhibitory activity, good selectivity for CLK family members, and significant DYRK1A inhibitory activity.

[0016] Preferably, in the structure of the above compound:

[0017] R1 is selected from isobutyl, cyclopropylmethyl, cyclopentyl, α-aminoisopentyl, 3,3-difluorotetrahydropyrrolyl, hydrogen, morpholinyl, methyl, tert-butyl, ethylsulfonyl or hydroxyl;

[0018] R2 is selected from the following groups:

[0019]

[0020] More preferably, the compound is selected from any one of the following compounds:

[0021]

[0022]

[0023]

[0024]

[0025] The pharmaceutically acceptable salts of the above compounds are salts formed with the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, hydrobromic acid, hydroiodic acid, maleic acid, fumaric acid, tartaric acid, citric acid, malic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, succinic acid, acetic acid, mandelic acid, isobutyric acid or malonic acid.

[0026] As the second aspect of the present invention, the above-mentioned compound and a pharmaceutically acceptable carrier form a pharmaceutical composition to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions or injections. The preparations can be added with common pharmaceutical excipients such as flavors, sweeteners, liquid / solid fillers, diluents, etc.

[0027] As the third aspect of the present invention, the above-mentioned compound or its pharmaceutical composition can be prepared as a CLK2 protein inhibitor drug, and can also be prepared as a DYRK1A protein inhibitor drug, specifically for the treatment of inflammation, including osteoarthritis, tendinopathy or rheumatoid arthritis, and has a cartilage protection effect.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0029] (1) This type of compound and its pharmaceutical composition can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and inhibit IC at the enzyme level 50 The values ​​are all less than 100nM, and the optimal value is less than 10nM. It can also significantly downregulate the expression level of proteases related to cartilage decomposition in inflammatory model animals, exerting a cartilage protective effect.

[0030] (2) Such compounds and pharmaceutical compositions have a wide range of applications and can be prepared as drugs for treating osteoarthritis. They can exert their efficacy at the molecular and animal levels, and their therapeutic effects are even better, with the optimal concentration reaching nanomolar levels.

[0031] (3) The preparation method of the compound is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the RT-qPCR results of cartilage in ACLT model rats at week 5 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0034] Example 1: Synthesis of LH-001

[0035]

[0036] Synthesis of intermediate 1-2:

[0037] Add raw material 1-1 (5-bromoindole, 0.1 mmol) and acetone to a three-necked flask. Cool to below 0°C in an ice-salt bath. Add sodium nitrite aqueous solution (0.8 mmol) dropwise, maintaining the temperature above 0°C. After the addition is complete, add dilute hydrochloric acid (2N HCl) dropwise. Then, warm to room temperature and react for 4 hours. Spin dry and filter to obtain the crude product. Slurry the crude product with dichloromethane to obtain a brownish-red solid 1-2. 1 H NMR(300MHz, DMSO-d6)δ12.29(s,1H),9.80(s,1H),7.88(d,J=1.2Hz,1H),7.44–7.31(m,2H)ppm.HR-MS(ESI):Calculated for C8H6BrN2O[M+H] + :224.9664, found 224.9657. Yield 67%.

[0038] Synthesis of intermediate 1-3:

[0039] Intermediate 1-2 (0.1 mmol) was added to a three-necked flask, followed by tetrahydrofuran (80 mL) and p-toluenesulfonic acid (0.02 mmol). 3,4-Dihydropyran (0.2 mmol) was then added dropwise at room temperature. The temperature was then raised to 65°C and the reaction was allowed to proceed for 8 h. After completion of the reaction as monitored by TLC, the solvent was evaporated under reduced pressure. The crude intermediate I-3 was purified by silica gel column chromatography. The crude product was then slurried with petroleum ether to obtain I-3 as a white solid. 1 H NMR (300MHz, DMSO-d6) δ9.80(s,1H),7.88(s,1H),7.37(d,J=1.0Hz,2H),6.27(t,J=6.9Hz,1H),3.90(dt,J=11.4,7.0Hz,1H),3.77 (dt,J=11.5,7.0Hz,1H),2.52–2.32(m,1H),2.12–1.91(m,2H),1.83–1.64(m,1H),1.58–1.53(m,2H)ppm.HR-MS(ESI):Calculated for C 13 H 14 BrN2O2[M+H] + :309.0239, found 309.0239. Yield 74%.

[0040] Synthesis of intermediate I-4:

[0041] Intermediate 1-3 (0.1 mmol) was added to a single-necked flask, along with pinacol diboronate (0.12 mmol), potassium acetate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), and dioxane (26 mL). The reaction mixture was heated to reflux for 6 h. After completion of the reaction as monitored by TLC, the reaction mixture was cooled, filtered, dried, and purified by silica gel column chromatography to yield a white solid 1-4. 1 H NMR(300MHz,DMSO-d6)δ9.99(s,1H),8.07(d,J=1.6Hz,1H),7.46(d,J=7.5H z,1H),7.27(dd,J=7.5,1.6Hz,1H),6.25(t,J=6.9Hz,1H),3.90(dt,J=11.4, 7.0Hz,1H),3.77(dt,J=11.4,7.0Hz,1H),2.52–2.32(m,1H),2.12–1.91(m,2H),1.86–1.48(m,3H),1.41(d,J=15.1Hz,12H)ppm.HR-MS(ESI):Calculated for C 19 H 26 BN2O4[M+H] +:357.1986, found 357.1983. Yield 89%.

[0042] Synthesis of intermediate I-5:

[0043] Intermediate 1-4 (0.1 mmol) was added to a single-necked flask and dissolved in a mixed solvent of ethanol: dichloromethane (5:1). Then, o-aminobenzamide (0.1 mmol) and elemental iodine (0.1 mmol) were added and refluxed at 80°C for 30-60 min. After the TLC reaction was complete, 5% sodium thiosulfate was added to quench the reaction. The mixture was then extracted with dichloromethane and saturated brine. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain a crude product. The crude product was slurried with (petroleum ether: ethyl acetate = 1:1) to obtain pure I-5. 1 H NMR (300MHz, DMSO-d6) δ12.35(s,1H),8.13(dd,J=7.6,1.9Hz,2H),7.66(dd,J=7.5,2.1Hz ,1H),7.56(d,J=7.4Hz,1H),7.50–7.32(m,2H),7.13(td,J=7.5,2.0Hz,1H),6.31(t,J=6. 9Hz,1H),3.90(dt,J=11.4,7.0Hz,1H),3.77(dt,J=11.4,7.0Hz,1H),2.52–2.32(m,1H),2 .12–1.91(m,2H),1.86–1.48(m,3H),1.42(d,J=15.1Hz,12H)ppm.HR-MS(ESI):Calculated for C 26 H 30 BN4O4[M+H] + :473.2360, found 473.2357. Yield 68%.

[0044] Synthesis of intermediate 2-2:

[0045] Dissolve isovaleric acid (0.1 mmol) and HATU (0.3 mmol) in 40 ml of tetrahydrofuran, then add DIPEA (0.3 mmol) and stir at room temperature for about 15 minutes. Then add raw material 2-1 (0.1 mmol) and stir at room temperature for 1-3 hours. When TLC detects that the reaction of raw material 2-1 is complete, stop the reaction and separate and purify by column chromatography to obtain intermediate 2-2.

[0046] Synthesis of intermediate 2-3:

[0047] Intermediate 2-2 (0.1 mmol), intermediate 1-5 (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-necked flask. The air was replaced with nitrogen four times, and the temperature was raised to reflux and reacted for 8 h. After cooling, the mixture was filtered, dried, and purified by silica gel column chromatography to obtain intermediate 2-3.

[0048] Synthesis of LH-001:

[0049] Intermediate 2-3 (0.1 mmol) was dissolved in 5 ml of dichloromethane, and trifluoroacetic acid (1 mmol) and triethylsilane (0.1 mmol) were added at room temperature. The mixture was reacted at room temperature for 16-24 hours. After TLC detection, the reaction was completed, and the mixture was concentrated under reduced pressure. The mixture was extracted with dichloromethane and saturated sodium bicarbonate. The organic phases were combined, dried, and concentrated to obtain the final product LH-001. 1 H NMR (300MHz, DMSO-d6) δ14.16(s,1H),10.57(s,1H),8.93(t,J=12.4Hz,3H),8.67(d,J=2.1Hz,1H),8.26–8.17(m,1H),7.98–7.82 (m,4H),7.61–7.51(m,1H),6.30(s,1H),2.34(d,J=7.1Hz,2H),2.17(m,H),1.03(s,3H),1.01(s,3H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O2[M+H] + :439.1882,found 439.1875.

[0050] The following compounds were obtained by similar operations to those in Example 1:

[0051]

[0052] 1H NMR(300MHz,DMSO-d6)δ14.25(s,1H),12.27(s,1H),10.69(s,1H),8.91–8.82(m,2H),8.66(s,1H),8.42(s,1H),8.14(d,J=7.9Hz,1H),7.76(d,J=8.4Hz,3H),7.62(d,J=8.7Hz,1H),7.39(t,J=7.2Hz,1H),1.90(q,J=6.2Hz,1H),0.91(q,J=7.9,5.5Hz,4H)ppm.HR-MS(ESI):Calculated for C 24 H 19 N6O2[M+H] + :423.1569,found 423.1562。

[0053]

[0054] 1 H NMR(300MHz,DMSO-d6)δ14.28(s,1H),12.29(s,1H),9.80(s,1H),9.61(d,J=1.3Hz,1H),8.83(d,J=1.5Hz,1H),8.70(d,J=1.3Hz,1H),8.13(dd,J=7.4,2.0Hz,1H),8.03(t,J=1.3Hz,1H),7.71–7.55(m,3H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),2.10(d,J=7.0Hz,2H),1.15(m,1H),0.62–0.44(m,2H),0.32–0.27(m,2H)ppm.HR-MS(ESI):Calculated for C 25 H 21 N6O2[M+H] + :437.1726,found 437.1722。

[0055]

[0056] 1H NMR(300MHz,DMSO-d6)δ14.16(s,1H),10.56(s,1H),8.93(s,2H),8.67(s,1H),8.20(d,J=7.8Hz,1H),7.95(d,J=8.1Hz,1H),7.88(d,J=10.6Hz,3H),7.56(t,J=7.4Hz,1H),5.78(s,2H),2.91(d,J=7.7Hz,1H),1.99–1.57(m,8H)ppm.HR-MS(ESI):Calculated for C 26 H 23 N6O2[M+H] + :451.1882,found 451.1879。

[0057]

[0058] 1 H NMR(300MHz,DMSO-d6)δ14.43(s,1H),11.66(s,1H),10.31(s,1H),8.90(s,1H),8.81(s,1H),8.70(s,1H),8.53(s,1H),8.21(d,J=7.9Hz,1H),7.88(dd,J=7.7,8.3Hz,4H),7.55(t,J=7.3Hz,1H),2.32(d,J=6.9Hz,2H),1.86–1.66(m,6H),1.25(q,J=11.6,10.8Hz,3H),1.05(q,J=11.3Hz,2H)ppm.HR-MS(ESI):Calculated for C 28 H 27 N6O2[M+H] + :479.2195,found 479.2191。

[0059]

[0060] 1H NMR(300MHz,DMSO-d6)δ14.08(s,1H),12.21(s,1H),8.83(s,1H),8.22(d,J=8.2Hz,2H),8.04(d,J=2.4Hz,1H),7.89(d,J=4.0Hz,2H),7.82(s,2H),7.57(dt,J=8.2,4.1Hz,1H),7.38(s,1H),3.05(d,J=6.7Hz,2H),1.88(d,J=12.6Hz,2H),1.77–1.60(m,5H),1.26(t,J=10.7Hz,3H),1.02(dt,J=12.0,6.0Hz,2H)ppm.HR-MS(ESI):Calculated for C 27 H 27 N6O[M+H] + :451.2246,found 451.2241。

[0061]

[0062] 1 H NMR(300MHz,DMSO-d6)δ14.11(s,1H),12.22(s,1H),8.90(d,J=10.1Hz,2H),8.58(s,1H),8.21(d,J=7.9Hz,1H),8.12(s,1H),7.86(dd,J=14.2,7.9Hz,4H),7.57(d,J=7.2Hz,1H),3.86(s,2H),2.64(s,4H),1.80(d,J=5.3Hz,4H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O[M+H] + :423.1933,found 423.1928。

[0063]

[0064] 1H NMR(300MHz,DMSO-d6)δ14.15(s,1H),12.63(s,1H),8.81(s,1H),8.21(d,J=7.9Hz,1H),8.15(s,1H),8.01(d,J=2.5Hz,1H),7.87(d,J=3.6Hz,2H),7.79(d,J=2.6Hz,2H),7.55(dq,J=8.0,3.8,3.2Hz,1H),7.23(s,1H),5.88(d,J=7.9Hz,1H),3.75(d,J=6.8Hz,1H),1.25(s,3H),1.23(s,3H)ppm.HR-MS(ESI):Calculated for C 23 H 21 N6O[M+H] + :397.1777,found 397.1769。

[0065]

[0066] 1 H NMR(300MHz,DMSO-d6)δ14.14(s,1H),12.21(s,1H),8.96(d,J=2.2Hz,1H),8.89(s,1H),8.60(s,1H),8.24–8.13(m,2H),7.88–7.69(m,4H),7.59–7.52(m,1H),3.92(s,2H),3.11(t,J=13.2Hz,2H),2.94–2.88(m,2H),2.43–2.36(m,2H)ppm.HR-MS(ESI):Calculated for C 25 H 21 F2N6O[M+H] + :459.1745,found 459.1739。

[0067]

[0068] 1H NMR(300MHz,DMSO-d6)δ14.27(s,1H),12.28(s,1H),9.00(d,J=2.3Hz,1H),8.85(s,1H),8.65–8.58(m,1H),8.23–8.12(m,2H),7.83(dd,J=9.9,6.4Hz,3H),7.73(dd,J=8.7,1.8Hz,1H),7.56(dd,J=8.0,4.8Hz,1H),7.49–7.42(m,1H)ppm.HR-MS(ESI):Calculated for C 20 H 14 N5O[M+H] + :340.1198,found 340.1192。

[0069]

[0070] 1 H NMR(300MHz,DMSO-d6)δ14.28(s,1H),12.14(s,1H),8.89(s,2H),8.52(s,1H),8.17(d,J=7.9Hz,1H),8.05(s,1H),7.84–7.69(m,4H),7.45(d,J=3.5Hz,1H),3.58(s,2H),2.25(s,6H)ppm.HR-MS(ESI):Calculated for C 23 H 21 N6O[M+H] + :397.1777,found 397.1771。

[0071]

[0072] 1 H NMR(300MHz,DMSO-d6)δ14.09(s,1H),12.25(s,1H),8.91(d,J=2.2Hz,1H),8.87(s,1H),8.57(d,J=1.9Hz,1H),8.21(d,J=7.9Hz,1H),8.12(d,J=2.2Hz,1H),7.93–7.81(m,6H),7.57–7.51(m,1H),3.72(s,2H),3.64(d,J=4.7Hz,6H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O2[M+H] +:439.1882,found 439.1879。

[0073]

[0074] 1 H NMR(300MHz,DMSO-d6)δ14.11(s,1H),12.22(s,1H),8.61–8.47(m,3H),8.19(d,J=7.9Hz,1H),7.91–7.78(m,3H),7.55(dt,J=11.2,5.3Hz,2H),7.44(d,J=5.0Hz,1H),2.36(s,3H)ppm.HR-MS(ESI):Calculated for C 21 H 16 N5O[M+H] + :354.1355,found 354.1348。

[0075]

[0076] 1 H NMR(300MHz,DMSO-d6)δ14.11(s,1H),12.29(s,1H),8.92(d,J=13.6Hz,2H),8.59(s,1H),8.22(d,J=7.9Hz,2H),7.91(d,J=7.4Hz,4H),7.57(ddd,J=8.2,6.0,2.2Hz,1H),3.77(s,1H),3.65(s,2H),1.81(s,2H),1.53(s,3H),1.25(s,3H)ppm.HR-MS(ESI):Calculated for C 26 H 25 N6O2[M+H] + :453.2039,found 453.2041。

[0077]

[0078] 1H NMR(300MHz,DMSO-d6)δ14.14(s,1H),12.20(s,1H),9.68(s,1H),8.98(d,J=11.2Hz,2H),8.77(s,1H),8.60(s,1H),8.29(t,J=7.7Hz,1H),8.04–7.89(m,4H),7.63(d,J=7.7Hz,1H),1.44–1.36(m,9H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O2[M+H] + :439.1882,found 439.1878。

[0079]

[0080] 1 H NMR(300MHz,DMSO-d6)δ14.21(s,1H),12.29(s,1H),10.85(s,1H),8.89(d,J=8.1Hz,2H),8.73(s,1H),8.56(s,1H),8.21(d,J=7.8Hz,1H),7.91(d,J=7.2Hz,4H),7.63–7.50(m,1H),2.93–2.85(m,3H),1.23(s,2H)ppm.HR-MS(ESI):Calculated for C 25 H 19 F2N6O2[M+H] + :473.1538,found 473.1532。

[0081]

[0082] 1 H NMR(300MHz,DMSO-d6)δ14.14(s,1H),12.31(s,1H),10.34(s,1H),8.92(s,1H),8.79(s,1H),8.52(s,1H),8.22(d,J=7.6Hz,1H),8.05(s,1H),7.90(d,J=12.0Hz,4H),7.58(s,1H),3.31(s,2H),1.25(s,3H)ppm.HR-MS(ESI):Calculated for C 22 H 19 N6O3S[M+H] + :447.1239,found 447.1231。

[0083]

[0084] 1 H NMR(300MHz,DMSO-d6)δ14.73(s,1H),12.25(s,1H),8.84(s,1H),8.39(s,1H),8.14(d,J=8.9Hz,2H),7.74(d,J=4.3Hz,3H),7.63(d,J=8.4Hz,1H),7.52(s,1H),7.39(d,J=8.4Hz,1H)ppm.HR-MS(ESI):Calculated for C 20 H 14 N5O2[M+H] + :356.1147,found 356.1141。

[0085]

[0086] 1 H NMR(300MHz,DMSO-d6)δ14.22(s,1H),12.32(s,1H),9.03–8.92(m,2H),8.65(d,J=1.9Hz,1H),8.30(d,J=7.9Hz,1H),8.18(t,J=2.1Hz,1H),8.02–7.90(m,4H),7.65–7.59(m,1H),3.81(s,2H),2.95(s,4H),2.72(s,4H),2.61(s,3H)ppm.HR-MS(ESI):Calculated for C 26 H 26 N7O[M+H] + :452.2199,found 452.2203。

[0087]

[0088] 1H NMR(300MHz,DMSO-d6)δ14.16(s,1H),12.30(s,1H),8.88(s,1H),8.67(d,J=1.7Hz,1H),8.43(d,J=2.6Hz,1H),8.23–8.18(m,2H),7.93–7.84(m,4H),7.61–7.53(m,1H),4.46(dd,J=10.7,3.2Hz,1H),4.25(dd,J=10.6,6.7Hz,1H),3.66(s,1H),2.91(s,1H),2.75(s,1H),1.83-1.60(m,3H),0.98(d,J=1.9Hz,3H),0.96(d,J=2.0Hz,3H)ppm.HR-MS(ESI):Calculated for C 26 H 27 N6O2[M+H] + :455.2195,found 455.2191。

[0089]

[0090] 1 H NMR(300MHz,DMSO-d6)δ14.36(s,1H),12.18(s,1H),8.57(d,J=1.3Hz,1H),8.22–8.08(m,2H),7.72–7.52(m,4H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),4.33(dd,J=12.4,7.0Hz,1H),4.16(dd,J=12.4,7.0Hz,1H),3.25(d,J=7.0Hz,1H),2.97(dt,J=9.4,6.9Hz,1H),2.79(dt,J=9.3,6.9Hz,1H),1.77–1.65(m,3H),1.41(ddd,J=12.3,7.6,6.3Hz,1H),1.19(s,1H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O2[M+H] + :439.1882,found 439.1878。

[0091]

[0092] 1H NMR(300MHz,DMSO-d6)δ14.05(s,1H),12.53(s,1H),8.78–8.71(m,1H),8.59(d,J=1.3Hz,1H),8.18–8.05(m,2H),7.71–7.51(m,4H),7.44(td,J=7.5,2.0Hz,1H),7.35–7.22(m,2H),7.22–7.07(m,4H),4.26(dd,J=12.4,7.0Hz,1H),4.07(dd,J=12.4,6.9Hz,1H),3.67–3.61(m,1H),3.11–2.98(m,1H),2.81–2.71(m,1H),1.54(s,2H)ppm.HR-MS(ESI):Calculated for C 29 H 25 N6O2[M+H] + :4897.2039,found 489.2029。

[0093]

[0094] 1 H NMR(300MHz,DMSO-d6)δ14.16(s,1H),12.38(s,1H),8.79(d,J=1.1Hz,1H),8.52(d,J=1.3Hz,1H),8.36(d,J=1.3Hz,1H),8.13(dd,J=7.4,2.0Hz,1H),7.71–7.53(m,3H),7.51–7.39(m,2H),7.14(td,J=7.5,2.0Hz,1H),3.50(s,2H)ppm.HR-MS(ESI):Calculated for C 20 H 15 N6O[M+H] + :355.1307,found 355.1301。

[0095]

[0096] 1H NMR(300MHz,DMSO-d6)δ14.42(s,1H),12.27(s,1H),8.83–8.73(m,2H),8.48(d,J=1.3Hz,1H),8.13(dd,J=7.4,2.0Hz,1H),7.89(t,J=1.3Hz,1H),7.66(dd,J=7.5,2.0Hz,1H),7.63–7.50(m,2H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),2.48(s,3H)ppm.HR-MS(ESI):Calculated for C 21 H 16 N5O[M+H] + :354.1355,found 354.1350。

[0097]

[0098] 1 H NMR(300MHz,DMSO-d6)δ14.52(s,1H),12.22(s,1H),9.80(s,1H),9.58(d,J=1.3Hz,1H),8.87–8.79(m,1H),8.70(d,J=1.3Hz,1H),8.13(dd,J=7.5,2.0Hz,1H),8.04(t,J=1.3Hz,1H),7.71–7.55(m,3H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),2.20(s,3H)ppm.HR-MS(ESI):Calculated for C 22 H 17 N6O2[M+H] + :397.1413,found 397.1410。

[0099]

[0100] 1H NMR(300MHz,DMSO-d6)δ14.62(s,1H),12.12(s,1H),9.80(s,1H),9.60(d,J=1.3Hz,1H),8.76(d,J=1.6Hz,1H),8.68(d,J=1.3Hz,1H),8.13(dd,J=7.5,2.1Hz,1H),7.91(t,J=1.3Hz,1H),7.66(dd,J=7.5,2.0Hz,1H),7.65–7.49(m,2H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),2.37(t,J=8.1Hz,2H),1.69–1.55(m,2H),0.98(t,J=8.0Hz,3H)ppm.HR-MS(ESI):Calculated for C 24 H 21 N6O2[M+H] + :425.1726,found 425.1720。

[0101]

[0102] 1 H NMR(300MHz,DMSO-d6)δ14.55(s,1H),12.14(s,1H),9.80(s,1H),9.62(d,J=1.4Hz,1H),8.76(d,J=1.5Hz,1H),8.68(d,J=1.3Hz,1H),8.13(dd,J=7.5,2.0Hz,1H),7.91(t,J=1.3Hz,1H),7.71–7.55(m,2H),7.54(dd,J=7.5,1.5Hz,1H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),3.22(d,J=7.0Hz,1H),2.55–2.37(m,2H),2.17–1.82(m,4H)ppm.HR-MS(ESI):Calculated for C 25 H 21 N6O2[M+H] + :437.1726,found 437.1719。

[0103]

[0104] 1H NMR(300MHz,DMSO-d6)δ14.25(s,1H),12.25(s,1H),9.50(d,J=1.3Hz,1H),8.86(d,J=1.4Hz,1H),8.71(d,J=1.3Hz,1H),8.14(d,J=9.8Hz,2H),8.06(d,J=1.2Hz,1H),8.02–7.90(m,2H),7.71–7.40(m,6H),7.31(ddt,J=9.6,7.2,2.0Hz,1H),7.15(td,J=7.5,2.1Hz,1H)ppm.HR-MS(ESI):Calculated for C 27 H 19 N6O2[M+H] + :459.1569,found 459.1560。

[0105]

[0106] 1 H NMR(300MHz,DMSO-d6)δ14.56(s,1H),12.17(s,1H),9.80(s,1H),9.61(d,J=1.2Hz,1H),8.71(dd,J=14.1,1.4Hz,2H),8.13(dd,J=7.4,2.0Hz,1H),7.81(t,J=1.3Hz,1H),7.71–7.28(m,6H),7.28–7.07(m,4H),3.76(s,2H)ppm.HR-MS(ESI):Calculated for C 28 H 21 N6O2[M+H] + :473.1726,found 473.1719。

[0107]

[0108] 1H NMR(300MHz,DMSO-d6)δ14.16(s,1H),12.12(s,1H),12.35(s,1H),9.53–9.42(m,2H),8.83(t,J=1.0Hz,1H),8.70(d,J=1.3Hz,1H),8.19–8.02(m,2H),7.71–7.55(m,3H),7.45(td,J=7.5,2.0Hz,1H),7.35(d,J=7.5Hz,1H),7.14(td,J=7.5,2.0Hz,1H),6.86(d,J=7.5Hz,1H)ppm.HR-MS(ESI):Calculated for C 24 H 17 N8O2[M+H] + :449.1474,found 449.1470。

[0109]

[0110] 1 H NMR(300MHz,DMSO-d6)δ14.11(s,1H),12.27(s,1H),9.48(d,J=1.3Hz,2H),8.90(s,1H),8.80(d,J=1.5Hz,2H),8.71(d,J=1.3Hz,2H),8.13(dd,J=7.4,2.0Hz,2H),7.96(t,J=1.3Hz,2H),7.67(d,J=2.0Hz,1H),7.45(s,1H)ppm.HR-MS(ESI):Calculated for C 24 H 16 N7O3[M+H] + :450.1315,found 450.1310。

[0111]

[0112] 1H NMR(300MHz,DMSO-d6)δ14.11(s,1H),12.27(s,1H),9.49(d,J=1.3Hz,1H),8.90(s,1H),8.79(d,J=1.5Hz,1H),8.70(d,J=1.3Hz,1H),8.13(dd,J=7.5,2.0Hz,1H),8.06(s,1H),7.95(t,J=1.3Hz,1H),7.71–7.50(m,3H),7.45(td,J=7.5,2.0Hz,1H),7.39(s,1H),7.14(td,J=7.5,2.0Hz,1H)ppm.HR-MS(ESI):Calculated for C 24 H 16 N7O3[M+H] + :450.1315,found 450.1309。

[0113]

[0114] 1 H NMR(300MHz,DMSO-d6)δ14.14(s,1H),12.25(s,1H),8.88–8.74(m,2H),8.71(d,J=1.3Hz,1H),8.23–8.13(m,2H),7.71–7.51(m,3H),7.45(td,J=7.5,2.0Hz,1H),7.14(td,J=7.5,2.0Hz,1H),3.69(s,2H),2.43(t,J=5.3Hz,4H),1.62–1.36(m,6H)ppm.HR-MS(ESI):Calculated for C 26 H 25 N6O[M+H] + :437.2090,found 437.2088。

[0115]

[0116] 1H NMR (300MHz, DMSO-d6) δ14.63(s,1H),12.32(s,1H),8.86–8.70(m,3H),8.18–8.04(m,2H),7.71–7.51(m,3H),7.45(td,J=7.5,2.0Hz, 1H),7.14(td,J=7.5,2.0Hz,1H),3.65(s,2H),2.59(q,J=8.0Hz,2H),1.37(s,1H),1.11(t,J=8.0Hz,3H)ppm.HR-MS(ESI):Calculated for C 23 H 21 N6O[M+H] + :397.1777,found 397.1770.

[0117]

[0118] 1 H NMR (300MHz, DMSO-d6) δ14.51(s,1H),12.31(s,1H),8.84–8.74(m,2H),8.65(d,J=1.3Hz,1H),8.20–8.09(m,2H),7.7 1–7.56(m,3H),7.51–7.20(m,6H),7.14(td,J=7.5,2.0Hz,1H),3.65(s,4H),1.69(s,1H)ppm.HR-MS(ESI):Calculated for C 28 H 23 N6O[M+H] + :459.1933,found 459.1926.

[0119] Example 2: Synthesis of LH-036

[0120]

[0121] Intermediate 1-4 (0.1 mmol) was added to a single-necked flask and dissolved with 5 mL of dimethylacetamide. 3-aminopyridine-4-carboxamide (0.15 mmol), p-toluenesulfonic acid hydrate (0.15 mmol), and sodium bisulfite (0.15 mmol) were then added and reacted at 120 ° C for 6-8 hours. After the reaction was complete by TLC, the mixture was extracted three times with ethyl acetate and saturated brine. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain intermediate 1-5-2. 1H NMR (300MHz, DMSO-d6) δ12.31(s,1H),9.47(s,1H),8.41–8.27(m,2H),8.13(d,J=1. 6Hz,1H),7.56(d,J=7.5Hz,1H),7.37(dd,J=7.5,1.6Hz,1H),6.31(t,J=6.9Hz,1H),3 .90(dt,J=11.4,7.0Hz,1H),3.77(dt,J=11.4,7.0Hz,1H),2.52–2.32(m,1H),2.12–1 .91(m,2H),1.86–1.48(m,3H),1.42(d,J=14.9Hz,12H)ppm.HR-MS(ESI):Calculated for C 25 H 29 BN5O4[M+H] + :474.2313, found 474.2310. Yield 75%.

[0122] The remaining steps were the same as in Example 1 to obtain compound LH-036.

[0123]

[0124] 1 H NMR(300MHz,DMSO-d6)δ14.19(s,1H),12.66(s,1H),9.29(s,1H),8.97 (s,1H),8.90(s,1H),8.69(d,J=5.1Hz,1H),8.61(s,1H),8.18(s,1H),8.02(d,J=5.1Hz,1H),7.88(q,J=8.7Hz,2H),3 .95(s,2H),3.17–3.08(m,2H),2.93(t,J=6.9Hz,2H),2.38(dq,J=15.3,7.9,7.4Hz,2H)ppm.HR-MS(ESI):Calculated for C 24 H 20 F2N7O[M+H] + :460.1697,found 460.1690.

[0125] The following compounds were obtained by similar procedures to those in Example 2:

[0126]

[0127] 1H NMR (300MHz, DMSO-d6) δ14.55(s,1H),12.33(s,1H),9.47(s,1H),8.76(t,J=1.0Hz,1H),8.65(d, J=1.3Hz,1H),8.40(d,J=5.0Hz,1H),8.31(d,J=5.0Hz,1H),8.22(d,J=1.4Hz,1H),7.61(dd,J=16 .8,1.2Hz,3H),4.33(dd,J=12.4,7.0Hz,1H),4.08(dd,J=12.4,7.0Hz,1H),3.23–3.13(m,1H),1. 73–1.58(m,2H),1.58–1.40(m,1H),1.34(s,2H),0.99–0.82(m,6H)ppm.HR-MS(ESI):Calculated for C 25 H 26 N7O2[M+H] + :456.2148,found 456.2140.

[0128]

[0129] 1 H NMR (300MHz, DMSO-d6) δ14.34(s,1H),12.21(s,1H),9.80(s,1H),9.64(d,J=1.4Hz,1H),9.47(s,1H),8.82–8.70(m,2H),8.40(d,J=5. 0Hz,1H),8.31(d,J=5.0Hz,1H),8.00(s,1H),7.61(d,J=1.0Hz,2H),2.22–2.05(m,3H),1.08–0.93(m,6H)ppm.HR-MS(ESI):Calculated for C 24 H 22 N7O2[M+H] + :440.1835,found 440.1835.

[0130] Example 3: Synthesis of LH-039

[0131]

[0132] Synthesis of intermediate 1-2:

[0133] Intermediate 1-1 (0.1 mmol) was added to a three-necked flask, followed by tetrahydrofuran (50 mL) and p-toluenesulfonic acid (0.02 mmol). 3,4-Dihydropyran (0.2 mmol) was added dropwise at room temperature, and the mixture was allowed to react at 65°C for 8 h. After the reaction was complete by TLC, the solvent was evaporated under reduced pressure, and the product was purified by silica gel column chromatography to obtain I-2 as a white solid. 1 H NMR(300 MHz, DMSO-d6)δ7.97(d,J=1.5Hz,1H),7.89(s,1H),7.40–7.27(m,2H),6.27–6.16(m,1H),3.90(dt,J=11.4,7.0Hz,1H),3.77(d t,J=11.5,7.0Hz,1H),2.38–2.16(m,1H),2.04–2.01(m,2H),1.84–1.65(m,1H),1.58–1.32(m,2H)ppm.HR-MS(ESI):Calculated for C 12 H 14 BrN2O[M+H] + :281.0290, found 281.0292. Yield 85%.

[0134] Synthesis of intermediate 1-3:

[0135] Intermediate 1-2 (0.1 mmol) was added to a single-necked flask, followed by pinacol diboronate (0.12 mmol), potassium acetate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), and dioxane (26 mL). The reaction mixture was heated to reflux for 6 h. After the reaction was complete by TLC, the mixture was cooled, filtered, dried, and purified by silica gel column chromatography to obtain 1-3 as a white solid. 1 H NMR(300MHz,DMSO-d6)δ7.97(d,J=1.5Hz,1H),7.82–7.74(m,1H),7.39(d,J=7 .5Hz,1H),7.25(dd,J=7.5,1.5Hz,1H),6.21(t,J=6.9Hz,1H),3.90(dt,J=11. 4,7.0Hz,1H),3.77(dt,J=11.4,7.0Hz,1H),2.40–2.21(m,1H),2.16–1.91(m, 2H),1.85–1.48(m,3H),1.40(d,J=15.1Hz,12H)ppm.HR-MS(ESI):Calculated for C 18 H 26 BN2O3[M+H] + :329.2036, found 329.2033. Yield 89%.

[0136] Synthesis of intermediate 2-2:

[0137] The raw material 2-1 (0.1 mmol) was dissolved in tetrahydrofuran, and triethylamine (0.15 mmol) and isovaleric anhydride (0.15 mmol) were added. The temperature was raised to reflux and the reaction was allowed to react for 6-8 hours. After TLC detection, the reaction was completed, and the solvent was removed by concentration under reduced pressure. The reaction solution was poured into an ice-saturated sodium hydroxide solution to remove excess anhydride, and the mixture was extracted three times with ethyl acetate and saturated brine. The organic phases were combined, dried, and concentrated to obtain a yellow powdery solid 2-2. 1 H NMR (300MHz, DMSO-d6) δ9.80 (s, 1H), 9.58 (d, J = 1.3Hz, 1H), 8.49 (d, J = 1.3Hz, 1H), 7.87 (t, J = 1. 3Hz,1H),2.24(m,1H),2.10(d,J=6.9Hz,2H),0.95(d,J=6.7Hz,6H)ppm.HR-MS(ESI):Calculated for C 10 H 14 BrN2O[M+H] + :257.0290, found 257.0286. Yield 85%.

[0138] Synthesis of intermediate 2-3:

[0139] Intermediate 2-2 (0.1 mmol), intermediate 1-3 (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-necked flask. The air was replaced with nitrogen four times, and the temperature was raised to reflux and reacted for 8 h. After cooling, the mixture was filtered, dried, and purified by silica gel column chromatography to obtain intermediate 2-3. 1H NMR(300MHz, DMSO-d6)δ9.80(s,1H),9.63(d,J=1.3Hz,1H),8.69(d,J=1.3Hz,1H),8.19(t,J=1.6Hz,1H),8.0 3(d,J=1.5Hz,1H),7.90(t,J=1.3Hz,1H),7.68(d,J=7.5Hz,1H),7.51(dd,J=7.5,1.5Hz,1H),6.25(t,J=6.9H z,1H),3.90(dt,J=11.5,7.0Hz,1H),3.77(dt,J=11.5,7.0Hz,1H),2.64–2.57(qd,J=12.4,7.0Hz,2H),2.39– 2.13(m,2H),2.13–1.91(m,2H),1.85–1.48(m,3H),0.95(dd,J=15.0,6.8Hz,6H)ppm.HR-MS(ESI):Calculated for C 22 H 27 N4O2[M+H] + :379.2134, found 379.2132. Yield 78%.

[0140] Synthesis of intermediate 2-4:

[0141] Intermediate 2-3 (0.1 mmol) was dissolved in 10 mL of tetrahydrofuran, and p-toluenesulfonic acid hydrate (1 mmol) was added at room temperature. The temperature was raised to reflux and the reaction was reacted for 16-24 hours. After TLC detection, the reaction was completed, and the mixture was concentrated under reduced pressure and extracted with dichloromethane and saturated sodium bicarbonate. The organic phases were combined, dried, and concentrated to obtain intermediate 2-4. 1 H NMR (300MHz, DMSO-d6) δ12.75(s,1H),9.80(s,1H),9.63(d,J=1.3Hz,1H),8.50(d,J=1.3Hz,1H),8.06(d,J=1.6Hz,1H),7.96(t ,J=1.5Hz,1H),7.61(t,J=1.3Hz,1H),7.43–7.24(m,2H),2.24–2.05(m,3H),0.95(d,J=6.3Hz,6H)ppm.HR-MS(ESI):Calculated for C 17 H 19 N4O[M+H] + :295.1559, found 295.1554. Yield 74%.

[0142] Synthesis of intermediate 2-5:

[0143] Intermediate 2-3 (0.1 mmol) was dissolved in 5 mL of dimethylformamide, potassium hydroxide (0.15 mmol) and iodine (0.15 mmol) were added, and the mixture was stirred at room temperature for 16-24 hours. After TLC detection, the reaction was completed, and the mixture was extracted three times with ethyl acetate and saturated brine. The organic phases were combined, dried, and concentrated to obtain intermediate 2-5. 1 H NMR (300MHz, DMSO-d6) δ12.35(s,1H),9.64(s,1H),9.25(d,J=1.3Hz,1H),8.57(d,J=1.3Hz,1H),7.23(t,J=1.5Hz, 1H),7.4(t,J=1.3Hz,1H),7.40–7.22(m,2H),2.24–2.05(m,3H),0.82(d,J=6.3Hz,6H)ppm.HR-MS(ESI):Calculated for C 17 H 18 IN4O[M+H] + :421.0525, found 421.0525. Yield 65%.

[0144] Synthesis of LH-039:

[0145] Intermediate 2-5 (0.1 mmol), 3-pyridineboronic acid (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-necked flask. The air was replaced with nitrogen four times, and the temperature was raised to reflux and reacted for 8 h. After cooling, the product was filtered, dried, and purified by silica gel column chromatography to obtain LH-039. 1 H NMR (300MHz, DMSO-d6) δ13.62(s,1H),10.23(s,1H),9.30(d,J=2.2Hz,1H),8.82(d,J=2 .3Hz,1H),8.74(d,J=2.1Hz,1H),8.67(dd,J=4.8,1.6Hz,1H),8.49(dt,J=7.9,2.0Hz,1H ),8.40–8.32(m,2H),7.83–7.73(m,2H),7.60(dd,J=8.0,4.8Hz,1H),2.29(d,J=7.1Hz,2 H),2.14(dq,J=13.5,6.6Hz,1H),1.00(s,3H),0.98(s,3H)ppm.HR-MS(ESI):Calculated for C 22 H 22 N5O[M+H] +:372.1824,found 372.1818.

[0146] The following compounds were prepared by similar procedures to those in Example 3:

[0147]

[0148] 1 H NMR (300MHz, DMSO-d6) δ13.74(s,1H),10.22(s,1H),8.79(t,J=3.3Hz,1H),8.70(dd,J=4.9,1.8Hz,3H),8.40–8.30(m,2H),8.11–8.01(m,2 H),7.81–7.66(m,2H),2.25(d,J=7.1Hz,2H),2.11(dt,J=13.7,6.8Hz,1H),0.96(s,3H),0.93(d,J=4.7Hz,3H)ppm.HR-MS(ESI):Calculated for C 22 H 22 N5O[M+H] + :372.1824,found 372.1819.

[0149] Example 4: Synthesis of LH-041

[0150]

[0151] The raw material LH-041-1 (2-amino-4-bromophenol, 0.1 mmol) was dissolved in triethyl orthoformate and refluxed overnight, concentrated under reduced pressure and purified by column chromatography to obtain the intermediate LH-041-2. 1 H NMR(300MHz, DMSO-d6)δ8.25(s,1H),7.58(d,J=1.5Hz,1H),7.49(dd,J=7.5,1.6Hz,1H),7.25(d,J=7.5Hz,1H)ppm.HR-MS(ESI):Calculated for C7H5BrNO[M+H] + :197.9555, found 197.9523. Yield 57%.

[0152] Intermediate LH-041-2 (0.1 mmol) was added to a single-necked flask, along with pinacol diboronate (0.12 mmol), potassium acetate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), and dioxane (25 mL). The mixture was heated to reflux for 6 h. After the reaction was complete by TLC, the mixture was cooled, filtered, dried, and purified by silica gel column chromatography to obtain intermediate LH-041-3. 1 H NMR(300MHz, DMSO-d6)δ8.25(s,1H),7.75(dd,J=7.5,1.5Hz,1H),7.58(d,J=7.4Hz,1H),7.48(d,J=1.5Hz,1H),1.40(s,12H)ppm.HR-MS(ESI):Calculated for C 13 H 17 BNO3[M+H] + :246.1301, found 246.1281. Yield 88%.

[0153] The remaining steps were the same as in Example 3 to obtain compound LH-041.

[0154]

[0155] 1 H NMR (300MHz, DMSO-d6) δ13.44(s,1H),10.19(s,1H),8.83(s,1H),8.78(d,J=2.3Hz,1H),8. 70(d,J=2.1Hz,1H),8.41(d,J=1.6Hz,1H),8.34(t,J=2.2Hz,1H),8.30(d,J=1.4Hz,1H),8.1 4(dd,J=8.5,1.7Hz,1H),7.92(dd,J=8.5,0.6Hz,1H),7.78–7.67(m,1H),6.54(s,1H),2.25 (d,J=7.1Hz,2H),2.18–2.04(m,1H),0.96(s,3H),0.94(s,3H)ppm.HR-MS(ESI):Calculated for C 24 H 22 N5O2[M+H] + :412.1773,found 412.1769.

[0156] Example 5: Synthesis of LH-042

[0157]

[0158] Synthesis of intermediate 3-2:

[0159] 3-1 (0.1 mmol) was added to a single-necked flask, followed by an excess of concentrated sulfuric acid and stirring at room temperature for 2 hours. After the reaction was complete, the reaction system was cooled to 0°C, the mixture was basified with 10% sodium hydroxide solution, and then extracted with ethyl acetate. The combined extracts were dried over magnesium sulfate, the solvent was removed in vacuo, and then recrystallized from ethanol to obtain 3-2.

[0160] Synthesis of intermediate 3-3:

[0161] Intermediate 3-2 (0.1 mmol) was added to a single-necked bottle and dissolved with ethanol. Then, intermediate 1-4 (0.1 mmol) of Example 1 and elemental iodine (0.1 mmol) were added and refluxed at 80°C for 8 h. After the reaction was complete by TLC, 5% sodium thiosulfate was added to quench the reaction. The mixture was then extracted with dichloromethane and saturated brine. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain intermediate 3-3.

[0162] Synthesis of intermediate 3-4:

[0163] Intermediate 2-2 (0.1 mmol) from Example 1, intermediate 3-3 (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-necked flask. The air was replaced with nitrogen four times, and the temperature was raised to reflux and reacted for 8 h. After cooling, the mixture was filtered, dried, and purified by silica gel column chromatography to obtain intermediate 3-4.

[0164] Synthesis of LH-042:

[0165] Intermediate 3-4 (0.1 mmol) was added to a 50 mL single-necked flask, dissolved with 10 mL of dichloromethane, and then excess trifluoroacetic acid (1 mmol) was added. The mixture was stirred at room temperature overnight. After the reaction was completed as monitored by TLC, the solvent and excess trifluoroacetic acid were removed by vacuum concentration. After sand preparation, LH-042 was purified by column chromatography. 1H NMR(300MHz,DMSO-d6)δ14.26(s,1H),12.20(s,1H),10.12(s,1H),9.80(s,1H),9.59( d,J=1.3Hz,1H),8.75(d,J=1.5Hz,1H),8.68(d,J=1.3Hz,1H),8.36(s,1H),7.91(t,J= 1.2Hz,1H),7.64–7.49(m,2H),6.88(q,J=7.5Hz,2H),2.53–2.32(m,1H),2.10(d,J=7.0Hz,2H),0.96(d,J=6.7Hz,6H)ppm.HR-MS(ESI):Calculated for C 23 H 21 N6O3[M+H] + :429.1675,found 429.1670.

[0166] Using a procedure similar to Example 5, the following compounds were obtained:

[0167]

[0168] 1 H NMR (300MHz, DMSO-d6) δ12.64(s,1H),10.34(s,1H),8.83(d,J=1.3Hz,1H),8.74(dd,J=6.1,1.5Hz,2H),8.08(t,J=1.3Hz,1H),7.63–7. 50(m,2H),7.31(d,J=7.5Hz,1H),6.98(d,J=7.5Hz,1H),3.69(s,2H),2.70–2.55(m,4H),1.91–1.87(m,2H)ppm.HR-MS(ESI):Calculated for C 23 H 19 F2N6O2[M+H] + :449.1538,found 449.1528.

[0169]

[0170] 1H NMR(300MHz,DMSO-d6)δ12.12(s,1H),10.15(s,1H),8.79–8.71(m,1H),8.60(d,J=1.3Hz,1H),8.18(d,J=1.4Hz,1H),7.60(dt,J=16.7,1.4Hz,3H),7.31(d,J=7.5Hz,1H),6.98(d,J=7.6Hz,1H),4.32(dd,J=12.4,7.0Hz,1H),4.09(dd,J=12.4,7.0Hz,1H),3.22–3.05(m,1H),1.73–1.41(m,3H),1.54(s,2H),0.99–0.81(m,6H)ppm.HR-MS(ESI):Calculated for C 24 H 25 N6O3[M+H] + :445.1988,found 445.1976。

[0171]

[0172] 1 H NMR(300MHz,DMSO-d6)δ13.24(s,1H),10.31(s,1H),9.80(s,1H),9.60(d,J=1.3Hz,1H),8.76(d,J=1.5Hz,1H),8.68(d,J=1.3Hz,1H),7.91(t,J=1.2Hz,1H),7.65–7.49(m,2H),7.36(d,J=7.6Hz,1H),6.85(d,J=7.6Hz,1H),2.32(dt,J=13.6,6.8Hz,1H),2.10(d,J=7.0Hz,2H),0.96(d,J=6.8Hz,6H)ppm.HR-MS(ESI):Calculated for C 23 H 21 N6O2S[M+H] + :445.1447,found 445.1438。

[0173]

[0174] 11H NMR (300 MHz, DMSO-d6) δ 13.26 (s, 1H), 10.18 (s, 1H), 8.84 (d, J = 1.4 Hz, 1H), 8.81–8.68 (m, 2H), 8.10 (t, J = 1.3 Hz, 1H), 7.64–7.50 (m, 2H), 7.36 (d, J = 7.5 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 3.69 (s, 2H), 2.70–2.55 (m, 4H), 1.91–1.75 (m, 2H) ppm. HR-MS (ESI): Calculated for C 23 H 19 F2N6OS [M+H] + : 465.1309, found 465.1298。

[0175]

[0176] 1 1H NMR (300 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.12 (s, 1H), 8.77 (t, J = 1.0 Hz, 1H), 8.60 (d, J = 1.3 Hz, 1H), 8.18 (d, J = 1.3 Hz, 1H), 7.60 (dt, J = 15.1, 1.4 Hz, 3H), 7.36 (d, J = 7.6 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 4.31 (dd, J = 12.4, 7.0 Hz, 1H), 4.09 (dd, J = 12.4, 7.0 Hz, 1H), 3.13–2.92 (m, 1H), 1.75–1.41 (m, 3H), 1.52 (s, 2H), 0.90 (dd, J = 15.0, 6.5 Hz, 6H) ppm. HR-MS (ESI): Calculated for C 24 H 25 N6O2S [M+H] + : 461.1760, found 461.1756。

[0177] Example 6: Synthesis of LH-048

[0178]

[0179] Intermediate 1-4 (0.1 mmol) from Route 1 was added to a single-necked flask and dissolved in 10 mL of dimethylacetamide. Then, o-aminobenzylamine (0.15 mmol), p-toluenesulfonic acid hydrate (0.15 mmol), and sodium bisulfite (0.15 mmol) were added and reacted at 120°C for 6-8 hours. After the reaction was complete by TLC, the product was extracted three times with ethyl acetate and saturated brine. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain Intermediate 1-5-3. 1 H NMR (300MHz, DMSO-d6) δ12.24(s,1H),7.99(d,J=1.5Hz,1H),7.53(d,J=7.5Hz,1H),7.34(dd,J=7.5,1.5Hz, 1H),7.15–7.10(m,1H),7.04(td,J=7.4,2.0Hz,1H),6.89(td,J=7.5,2.1Hz,1H),6.41(dd,J=7.4,2.1Hz,1H) ,6.26(t,J=6.9Hz,1H),4.94(d,J=1.1Hz,2H),3.90(dt,J=11.4,7.0Hz,1H),3.77(dt,J=11.5,7.0Hz,1H),2. 52–2.32(m,1H),2.12–1.91(m,2H),1.86–1.48(m,3H),1.41(d,J=14.9Hz,12H)ppm.HR-MS(ESI):Calculated for C 26 H 32 BN4O3[M+H] + :459.2567, found 459.2558. Yield 78%.

[0180] The remaining steps were the same as in Example 1 to obtain compound LH-048.

[0181]

[0182] 1H NMR (300MHz, DMSO-d6) δ12.36(s,1H),10.28(s,1H),9.80(s,1H),9.51(d,J=1.3Hz,1H),8.67(d,J=1.3Hz,1H),8. 47(d,J=1.5Hz,1H),7.99(t,J=1.3Hz,1H),7.62–7.47(m,2H),7.15–7.11(m,1H),7.04(td,J=7.5,2.1Hz,1H),6.90 (td,J=7.5,2.1Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),2.60(d,J=1.0Hz,2H),2.29(dd,J=13 .5,6.8Hz,1H),2.10(d,J=6.9Hz,2H),0.96(d,J=6.7Hz,6H)ppm.HR-MS(ESI):Calculated for C 25 H 25 N6O[M+H] + :425.2090,found 425.2088.

[0183] The following compounds were obtained by similar procedures to Example 6:

[0184]

[0185] 1 H NMR (300MHz, DMSO-d6) δ12.11(s,1H),10.22(s,1H),8.82(d,J=1.3Hz,1H),8.71(d,J=1.3 Hz,1H),8.48(d,J=1.4Hz,1H),8.08(t,J=1.3Hz,1H),7.60–7.48(m,2H),7.15–7.05(m,1H) ,7.04(td,J=7.5,2.0Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),3. 69(s,2H),2.70–2.55(m,2H),2.60(s,3H),1.91–1.75(m,2H)ppm.HR-MS(ESI):Calculated for C 25 H 23 F2N6[M+H] + :445.1952,found 445.1949.

[0186]

[0187] 1 H NMR(300MHz,DMSO-d6)δ14.21(s,1H),12.31(s,1H),8.56–8.42(m,2H),8.15(d,J=1.3Hz,1H),7.57(dd,J=13.9,1.2Hz,3H),7.15(ddt,J=7.4,2.1,1.1Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),4.95(d,J=1.2Hz,2H),4.33(dd,J=12.4,7.0Hz,1H),4.08(dd,J=12.4,7.0Hz,1H),3.13–3.07(m,1H),1.74–1.39(m,3H),1.56(s,2H),0.90(dd,J=15.0,6.4Hz,6H)ppm.HR-MS(ESI):Calculated for C 26 H 29 N6O[M+H] + :441.2403,found 441.2395。

[0188]

[0189] 1 H NMR(300MHz,DMSO-d6)δ12.32(s,1H),10.21(s,1H),8.82(d,J=1.2Hz,1H),8.69(d,J=1.3Hz,1H),8.49(t,J=1.0Hz,1H),8.10(t,J=1.3Hz,1H),7.55(s,1H),7.15(ddt,J=7.4,2.1,1.0Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.4,2.1Hz,1H),3.69(s,2H),2.68–2.51(m,3H),2.60(s,4H),1.91–1.74(m,4H)ppm.HR-MS(ESI):Calculated for C 25 H 25 N6[M+H] + :409.2141,found 409.2098。

[0190]

[0191] 1 H NMR(300MHz,DMSO-d6)δ12.30(s,1H),10.29(s,1H),8.82(d,J=1.3Hz,1H),8.70(d,J=1.3Hz,1H),8.49(d,J=1.9Hz,1H),8.09(t,J=1.3Hz,1H),7.60–7.48(m,2H),7.15(ddt,J=7.5,2.1,1.0Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),3.69(s,2H),3.58(t,J=4.7Hz,4H),2.60(d,J=1.0Hz,2H),2.39(t,J=4.7Hz,4H)ppm.HR-MS(ESI):Calculated for C 25 H 25 N6O[M+H] + :425.2090,found 425.2081。

[0192]

[0193] 1 H NMR(300MHz,DMSO-d6)δ12.33(s,1H),10.21(s,1H),8.82(d,J=1.3Hz,1H),8.69(d,J=1.3Hz,1H),8.49(t,J=1.0Hz,1H),8.10(t,J=1.3Hz,1H),7.55(s,2H),7.15(ddt,J=7.5,2.2,1.0Hz,1H),7.04(td,J=7.5,2.0Hz,1H),6.90(td,J=7.5,2.1Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),3.69(s,2H),2.60(d,J=1.0Hz,2H),2.48(t,J=5.1Hz,4H),2.34(t,J=5.2Hz,4H),2.24(s,3H)ppm.HR-MS(ESI):Calculated for C 26 H 28 N7[M+H] + :438.2406,found 438.2395。

[0194]

[0195] 1H NMR(300MHz,DMSO-d6)δ13.26(s,1H),12.22(s,1H),9.80(s,1H),9.58(d,J=1.3Hz,1H),8.67(d,J=1.3Hz,1H),8.47(d,J=1.5Hz,1H),7.91(t,J=1.2Hz,1H),7.61–7.47(m,2H),7.15(ddt,J=7.4,2.1,1.0Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),2.60(d,J=1.0Hz,2H),2.20(s,3H)ppm.HR-MS(ESI):Calculated for C 22 H 19 N6O[M+H] + :383.1620,found 383.1598。

[0196]

[0197] 1 H NMR(300MHz,DMSO-d6)δ13.26(s,1H),12.22(s,1H),9.80(s,1H),9.61(d,J=1.3Hz,1H),8.67(d,J=1.3Hz,1H),8.47(d,J=1.4Hz,1H),7.90(t,J=1.3 Hz,1H),7.61–7.47(m,2H),7.15(ddt,J=7.4,2.1,1.0Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7.4,2.0Hz,1H),6.41(dd,J=7.5,2.1Hz,1H),3.21(d,J=6.9Hz,1H),2.60(d,J=1.0Hz,2H),2.48(dt,J=12.4,6.8Hz,2H),2.17–1.82(m,4H)ppm.HR-MS(ESI):Calculated for C 25 H 23 N6O[M+H] + :423.1933,found 423.1912。

[0198]

[0199] 1H NMR (300MHz, DMSO-d6) δ14.26(s,1H),12.22(s,1H),9.50(d,J=1.2Hz,1H),8.68(d,J=1.2Hz,1 H),8.49(d,J=1.4Hz,1H),8.04(s,1H),8.00–7.88(m,3H),7.62–7.47(m,4H),7.31(ddt,J=9.6 ,7.2,2.0Hz,1H),7.15(ddt,J=7.4,2.2,1.0Hz,1H),7.04(td,J=7.5,2.1Hz,1H),6.90(td,J=7 .5,2.1Hz,1H),6.41(dd,J=7.4,2.1Hz,1H),2.60(d,J=1.0Hz,2H)ppm.HR-MS(ESI):Calculated for C 27 H 21 N6O[M+H] + :445.1777,found 445.1765.

[0200] Example 7: Synthesis of LH-057

[0201]

[0202] The intermediate 1-4 (0.1 mmol) of route 1 was added to a single-necked flask and dissolved with 5 mL of dimethylacetamide. Then, propylene diamine (0.15 mmol), p-toluenesulfonic acid hydrate (0.15 mmol), and sodium bisulfite (0.15 mmol) were added and reacted at 120°C for 6-8 hours. After the reaction was complete by TLC, the mixture was extracted three times with ethyl acetate and saturated brine. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain intermediate 1-5-4. 1 H NMR(300MHz,DMSO-d6)δ12.22(s,1H),7.86(d,J=1.5Hz,1H),7.47–7.33(m,2H),7.25( dd,J=7.5,1.6Hz,1H),6.22(t,J=6.8Hz,1H),3.98–3.83(m,3H),3.77(dt,J=11.4,7.0H z,1H),3.62(ddd,J=11.9,4.4,1.7Hz,2H),2.52–2.32(m,1H),2.12–1.91(m,2H),1.86 –1.52(m,3H),1.40(d,J=15.1Hz,13H),1.23–1.08(m,1H)ppm.HR-MS(ESI):Calculated for C 22 H 32BN4O3[M+H] + :411.2567, found 411.2566. Yield 78%.

[0203] The remaining steps were the same as in Example 1 to obtain compound LH-057.

[0204]

[0205] 1 H NMR (300MHz, DMSO-d6) δ14.32 (s, 1H), 9.80 (s, 1H), 9.58 (d, J = 1.3Hz, 1H), 8. 65(d,J=1.3Hz,1H),8.38(d,J=1.3Hz,1H),7.92(t,J=1.3Hz,1H),7.60–7.43 (m,3H),3.52(dt,J=9.2,5.5Hz,4H),2.30–2.10(m,2H),2.09(d,J=1.1Hz,1H),1.60(d,J=5.6Hz,2H),0.97(d,J=6.6Hz,6H)ppm.HR-MS(ESI):Calculated for C 21 H 25 N6O[M+H] + :377.2090,found 377.1998.

[0206] The following compounds were obtained by similar procedures to Example 7:

[0207]

[0208] 1 H NMR (300MHz, DMSO-d6) δ14.35(s,1H),8.56(d,J=1.3Hz,1H),8.16(d,J=1.3Hz,1H),7.61(t,J=1 .3Hz,1H),7.53(d,J=1.1Hz,2H),7.44(s,1H),4.32(dd,J=12.4,7.0Hz,1H),4.08(dd,J=12.4,7 .0Hz,1H),3.92(td,J=12.2,3.1Hz,2H),3.66(ddd,J=11.9,4.4,1.7Hz,2H),3.20–3.04(m,1H), 1.74–1.33(m,6H),1.25–1.12(m,1H),0.90(dd,J=15.0,6.4Hz,6H)ppm.HR-MS(ESI):Calculated for C 22 H 29N6O[M+H] + :393.2403,found 393.2403.

[0209]

[0210] 1 H NMR (300MHz, DMSO-d6)14.35(s,1H),8.56(d,J=1.2Hz,1H),8.38(t,J=1.0Hz,1H), 8.17(d,J=1.4Hz,1H),7.57(dd,J=15.2,1.2Hz,3H),7.45(s,1H),3.93(td,J=12.2, 3.1Hz,2H),3.63(ddd,J=11.9,4.4,1.7Hz,2H),3.21(d,J=6.9Hz,1H),3.03–2.87( m,1H),2.78(dt,J=9.6,7.0Hz,1H),1.89–1.18(m,7H)ppm.HR-MS(ESI):Calculated for C 21 H 23 N7O[M+H] + :389.1964,found 389.1971.

[0211]

[0212] 1 H NMR (300MHz, DMSO-d6) δ14.36(s,1H),8.79(d,J=1.3Hz,1H),8.71(d,J=1.4Hz,1H),8.37(d,J=1.3Hz,1H),8.07(t,J=1.3Hz,1H),7.59–7.42(m, 3H),3.69(s,2H),3.52(dt,J=9.2,5.5Hz,4H),2.70–2.55(m,4H),1.91(tt,J=15.9,6.3Hz,2H),1.60–1.55(m,2H)ppm.HR-MS(ESI):Calculated for C 21 H 23 F2N6[M+H] + :397.1952,found 397.1946.

[0213] Example 8: In vitro inhibitory activity of some compounds of the present invention against CLK family and DYEK1A proteins

[0214] 1. Experimental Methods

[0215] 1. Prepare 1X kinase reaction buffer

[0216] Name Stock solution concentration Volume Final concentration Tris 1M (25X) 240μL 40mM MgCl 2 1M (50X) 120μL 20mM BSA 7.5% (75X) 80μL 0.1% DTT 1M (500X) 3μL 0.5mM ddH2O 5557μL

[0217] 2. Enzyme activity test

[0218] (1) 2X kinase preparation:

[0219] Name Stock concentration Final concentration

[0220] DYRK1A200ng / μL0.2ng / μLCLK1100ng / μL0.5ng / μLCLK2100ng / μL0.5ng / μLCLK3100ng / μL1ng / μLCLK4100ng / μL0.5ng / μL

[0221] (2) Preparation of 4X substrate mixture:

[0222]

[0223] ① Perform a 4-fold serial dilution of the positive drug with DMSO in a dilution plate, with the final starting concentration of the compound being 1, 0.02, and 0.1 μM.

[0224] ② Dilute the compound 50-fold into 1X kinase reaction buffer and shake on a shaker for 20 minutes.

[0225] ③ Prepare 2X kinase using 1X enzyme reaction buffer.

[0226] ④ Add 2 μL of kinase to each well of the reaction plate.

[0227] ⑤ Add 1 μL of compound diluted in buffer to each well, seal the plate with sealing film, centrifuge at 1000 rpm for 60 seconds, and incubate at 25°C for 10 minutes.

[0228] ⑥ Prepare 4X ATP & sub mixture with 1X enzyme reaction buffer and add 1μL 4X ATP & sub mixture to the reaction plate.

[0229] ⑦ Seal the plate with sealing film, centrifuge at 1000 rpm for 60 seconds, and incubate at 25℃ for 60 minutes.

[0230] ⑧ Transfer 4 μL ADP-Glo ​​to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

[0231] ⑨ Transfer 8 μL of detection solution to a 384-well reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

[0232] ①0 The RLU (Relative luminescence unit) signal was read using a BMG multifunctional plate reader. The signal intensity was used to characterize the activity of the kinase.

[0233] 3. Data Analysis

[0234] (1) Calculate the ratio of each well;

[0235] (2) The inhibition rate is calculated as follows:

[0236] Compound inhibition rate (%inh) = 100% - (compound - positive control) / (negative control - positive control) × 100%

[0237] (3) Calculate IC 50 And draw the inhibition curve of the compound:

[0238] The IC of the compound was obtained using the following nonlinear fitting formula: 50 (50% inhibitory concentration): Data analysis was performed using Graphpad 7.0 software.

[0239] Y=Bottom+(Top-Bottom) / (1+10^((Log IC 50 -X)×Hill Slope));

[0240] X: log value of compound concentration, Y: inhibition rate (% inhibition).

[0241] 2. Experimental Results

[0242] Table 1. Enzyme activities (IC 50 )

[0243] IDCLK1CLK3CLK4CLK2DYRK1ALH-001AACABLH-003AADACLH-004ABCBB

[0244] LH-005CCBBBLH-006BCBBBLH-009AABAALH-010BBDABLH-011CCDACLH-015BBAAALH-016ABBABLH-017ADCAALH-01 8CBAAALH-019ADCABLH-020ACBAALH-037AABBALH-039CACBBLH-040CBAAALH-041BABABLH-048AABAALH-049CBDAA

[0245] Note: A: <10nM, B: 10-50nM, C: 50-100nM, D: >100nM.

[0246] As shown in Table 1, the compounds of the present invention exhibited effective inhibitory activity against CLK2 and DYRK1A. At the same time, the compounds of the present invention, such as LH-020, exhibited superior CLK2 inhibitory activity and CLK3 selectivity (IC values ​​for CLK2, CLK3, and DYRK1A). 50 The selectivity index for CLK3 is 41.5), which provides a basis for LH-020 to exert its pharmacological activity and avoid possible side effects.

[0247] Example 9: Pharmacodynamics (PD) results of the representative compound LH-020 of the present invention in osteoarthritis rats

[0248] 1. Experimental Methods

[0249] 1. Forty rats were equally divided into four groups: Control (normal rats were injected with normal saline into the joint cavity), Model (ACLT model rats were injected with normal saline into the joint cavity), low-dose administration group (ACLT model rats were injected with LH020 (1.5 μg / kg) into the joint cavity), and high-dose administration group (ACLT model rats were injected with LH020 (6 μg / kg) into the joint cavity).

[0250] 2. Knee osteoarthritis was induced by resection of the anterior cruciate ligament in rats (ACLT model).

[0251] 3. In the first week after surgery, normal saline or LH-020 (1.5 μg / kg, 6 μg / kg) was injected into the joint cavity.

[0252] 4. Cartilage was harvested 5 weeks after administration and the expression of cartilage-related proteases (MMP3, MMP13, ADAMTS5, IHH, etc.) was detected by RT-qPCR.

[0253] 2. Experimental Results

[0254] As shown in Figure 1, compared to the control group (intra-articular injection of saline), the expression of cartilage-degrading proteases, MMP3, MMP13, ADAMTS5, and IHH, was significantly increased in the arthritis model. Furthermore, compared to the model group, both the low- and high-dose groups of LH-020 significantly downregulated the expression of MMP3, MMP13, ADAMTS5, and IHH, with the high-dose group showing superior effects compared to the low-dose group. This indicates that LH-020 has a significant cartilage-protective effect, providing evidence for its pharmacological effects in the treatment of osteoarthritis.

Claims

1. A 5-pyridine-1H-indazole compound, characterized in that: The compound has a structure of formula I or II, and also includes its isomers, pharmaceutically acceptable salts or mixtures thereof: wherein R1 is selected from the following groups substituted by one or more hydrogen, halogen, methoxy, trifluoromethyl, nitro, hydroxyl, amino, azido, sulfonic acid, 3-6 membered rings: hydrogen, straight or branched C1-C 10 Alkyl, phenyl, 4-6 membered heterocyclic ring or 4-6 membered ring; L and M are selected from -CH2-, -NH-, -O- or a chemical bond; R2 is selected from the following groups: R3 is selected from hydrogen or C1-C4 alkyl.

2. The compound according to claim 1, characterized in that In the structure: R1 is selected from isobutyl, cyclopropylmethyl, cyclopentyl, α-aminoisopentyl, 3,3-difluorotetrahydropyrrolyl, hydrogen, morpholinyl, methyl, tert-butyl, ethylsulfonyl or hydroxyl; R2 is selected from the following groups:

3. The compound according to claim 1, characterized in that Any one of the following compounds:

4. The compound according to claim 1, characterized in that The pharmaceutically acceptable salt is a salt formed by the compound with the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, hydrobromic acid, hydroiodic acid, maleic acid, fumaric acid, tartaric acid, citric acid, malic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, succinic acid, acetic acid, mandelic acid, isobutyric acid or malonic acid.

5. A pharmaceutical composition, characterized in that Comprising the compound according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. Use of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a CLK2 protein inhibitor drug.

7. Use of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5 in the preparation of a DYRK1A protein inhibitor.

8. The use according to claim 6 or 7, characterized in that The medicine is a medicine for treating inflammation.

9. The use according to claim 8, characterized in that The inflammatory condition is osteoarthritis, tendinopathy or rheumatoid arthritis.

10. The use according to claim 9, characterized in that The compounds have a chondroprotective effect.

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