5-Pyridine-1H-indazole compounds, pharmaceutical compositions and uses

Abstract

The present invention discloses a 5-pyridine-1H-indazole compound, pharmaceutical composition and use. The structure of the compound is as shown in formula I or II, and further includes its isomer, pharma-ceutical acceptable salt, or mixture thereof. The compound and its pharmaceutical composition can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and can be prepared as a drug for treating osteoarthritis, and can exert its efficacy at the molecular level, and the therapeutic effect is better, and the optimal level can reach nanomolar concentration. In addition, the preparation method of the compound is simple and easy to operate. [C72] TIFF2025515926000083.tif25166

Description

[Technical field]

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

[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 due to inappropriate differentiation of stem cells. The Wnt pathway plays a central role in organogenesis, cell differentiation, and tissue remodeling, and either abnormal activation or inhibition of the Wnt signaling pathway leads to the development of the disease. Therefore, the Wnt signaling pathway is a potential target for treating osteoarthritis.

[0003] The Wnt signaling pathway is a group of multiple downstream channel signaling pathways triggered by the binding of ligand protein Wnt and membrane protein receptor. Through this pathway, extracellular signals are transmitted into cells by the intracellular activation process of cell surface receptors. In the classical Wnt pathway, in the absence of Wnt protein on the cell membrane surface, its downstream β-Catenin protein is degraded in the cytoplasm by glycogen synthase kinase 3 (GSK3) complex, which prevents it from entering the cell nucleus and initiating the transcription of related Wnt genes; in contrast, in the presence of Wnt protein on the cell membrane surface, the GSK3 complex is inhibited, leading to the accumulation of β-Catenin protein in the cell nucleus, which ultimately initiates the transcription of Wnt pathway-related genes. There is a delicate balance between bone joint homeostasis and the Wnt pathway, and any disruption of this balance may lead to OA.

[0004] The protein kinase family CLK (CDK-like kinase) is a dual-specificity protein kinase that can control intracellular signal transduction by phosphorylating substrate proteins at tyrosine, serine, or threonine residues. It can be classified into four isoforms (CLK1, CLK2, CLK3, and CLK4), and the C segments of the proteins encoded by these four isoforms all have highly conserved gene sequences and share one structurally similar amino acid sequence. Among them, the CLK2 isoform is present in most eukaryotes and is involved in phosphorylation of the SR (serine / arginine) protein domain to regulate alternative splicing of RNA, playing an important role in gluconeogenesis and fatty acid oxidation in the liver. At the same time, it is also a therapeutic target for liver cancer, breast cancer, and Alzheimer's disease, and is also a potential therapeutic target for the Wnt pathway and osteoarthritis.

[0005] Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) belongs to the evolutionarily highly conserved DYRK family. In mammals, the DYRK family has five different isoforms, 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 one protein kinase domain and other special structures. Many important proteins function as substrates of DYRK1A and are regulated by DYRK1A to participate in various biological functions in cells, such as neurodevelopment, cell proliferation and differentiation, tumorigenesis, and neurodegenerative diseases.

[0006] At present, CLK2 inhibitor drugs used for the treatment of osteoarthritis have not yet been successfully marketed, and only one small molecule drug, SM-04690, has entered clinical phase III, so it cannot meet clinical needs. Although SM04690 has significant CLK2 inhibitory activity, it has insufficient selectivity for the CLK family, which may cause certain side effects, and it also has insufficient inhibitory activity against DYRK1A targets and poor water solubility, so its druggability needs to be improved. Summary of the Invention

[0007] Objective of the present invention: In response to problems such as insufficient selectivity for the CLK family and insufficient inhibitory activity against DYRK1A targets present in existing compounds, the present invention aims to provide 5-pyridine-1H-indazole compounds, pharmaceutical compositions and uses that specifically inhibit the activity of CLK2 and DYRK1A proteins.

[0008] Technical solution: In the first aspect of the present invention, the 5-pyridine-1H-indazole compound of the present invention has the structure of Formula I or II, and further includes its isomer, pharma- ceutically acceptable salt, or mixture thereof. Among them, R 1 is a hydrogen atom, a straight or branched chain C substituted with one or more hydrogen atoms, halogen atoms, methoxy groups, trifluoromethyl groups, nitro groups, hydroxyl groups, amino groups, azide groups, sulfonic acid groups, 3- to 6-membered rings, 1 ~C 10 selected from an alkyl group, a phenyl group, a 4- to 6-membered heterocyclic group, or a 4- to 6-membered ring group; L, M are -CH 2 -, -NH-, -O-, or a chemical bond; R 2 teeth, [ka] Selected from the group: R 3 is hydrogen or C 1 ~C 4 It is selected from alkyl groups.

[0009] The present invention synthesizes a series of derivatives through rational drug design, and biological activity evaluation shows that the designed compounds have significant CLK2 inhibitory activity, relatively good selectivity against CLK family members, and significant DYRK1A inhibitory activity.

[0010] Preferably, in the structure of the compound, R 1 is selected from an isobutyl group, a cyclopropylmethyl group, a cyclopentyl group, an α-aminoisopentyl group, a 3,3-difluorotetrahydropyrrolyl group, hydrogen, a morpholinyl group, a methyl group, a tert-butyl group, an ethylsulfonyl group, or a hydroxyl group; R 2 teeth, [ka] It is selected from the group:

[0011] More preferably, the compound is selected from any of the following compounds: [ka] TIFF2025515926000005.tif244166TIFF2025515926000006.tif243166TIFF2025515926000007.tif81169

[0012] Pharmaceutically acceptable salts of the above compounds are salts formed with the above compounds and 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.

[0013] In a second aspect of the present invention, the above compound and a pharma- ceutically acceptable carrier form a pharmaceutical composition, which can be made into a conventional pharmaceutical preparation, such as a tablet, capsule, syrup, suspension or injection, to which conventional pharmaceutical auxiliaries such as flavorings, sweeteners, liquid / solid fillers, diluents, etc. can be added.

[0014] In a third aspect of the present invention, the above compound or a pharmaceutical composition thereof may be prepared as a CLK2 protein inhibitor drug or a DYRK1A protein inhibitor drug, specifically for use in treating inflammation including osteoarthritis, tendonopathy or rheumatoid arthritis, and has the effect of cartilage protection.

[0015] Beneficial Effects: The present invention has the following significant advantages over the prior art: (1) The compound and its pharmaceutical composition can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and inhibit the enzyme level IC 50 The values ​​of both are less than 100 nM, with the optimum being less than 10 nM. In addition, the expression levels of proteases related to cartilage degradation in vivo in an inflammatory model animal can be significantly down-regulated, thereby exerting a cartilage protective effect. (2) The compound and its pharmaceutical composition are widely used and can be manufactured as a drug for treating osteoarthritis, and can exert its medicinal effects at the molecular level and animal level, and has a better therapeutic effect, the optimum of which can reach the nanomolar concentration level; (3) The method for producing the compound is simple and easy to operate. [Brief description of the drawings]

[0016] [Figure 1] Cartilage RT-qPCR results for ACLT model rats at 5 weeks (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The technical solution of the present invention will be further illustrated below with reference to the following examples. Example 1: Synthesis of LH-001 [ka]

[0018] Synthesis of intermediate 1-2: The raw material 1-1 (5-bromoindole, 0.1 mmol) and acetone were added to a three-neck flask, cooled to below 0°C in an ice-salt bath, and an aqueous solution of sodium nitrite (0.8 mmol) was added dropwise, while maintaining the temperature below 0°C. After the addition was completed, dilute hydrochloric acid (2N HCl) was added dropwise continuously. The temperature was then raised to room temperature and reacted for 4 hours. The mixture was then rotary dried and suction filtered to obtain a crude product. The crude product was slurried in dichloromethane to obtain a reddish brown solid 1-2. 1 H NMR (300MHz, DMSO-d 6 )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 C 8 H 6 BrN 2 O[M+H] + :224.9664,found 224.9657. The yield was 67%.

[0019] Synthesis of intermediates 1-3: Intermediate 1-2 (0.1 mmol) was added to a three-neck flask, tetrahydrofuran (80 mL) and p-toluenesulfonic acid (0.02 mmol) were added, 3,4-dihydropyran (0.2 mmol) was added dropwise at room temperature, and the temperature was then raised to 65° C. for 8 h reaction. After the completion of the reaction was detected by TLC, the solvent was distilled off under reduced pressure, and the crude product of intermediate I-3 was obtained by purifying the product by silica gel column chromatography, and the crude product was slurried with petroleum ether to obtain a white solid I-3. 1 H NMR (300MHz, DMSO-d 6)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 BrN 2 O 2 [M+H] + :309.0239,found 309.0239. The yield was 74%.

[0020] Synthesis of intermediate I-4: Add intermediate 1-3 (0.1 mmol) to a one-neck flask and add bis(pinacolato)diboron (0.12 mmol), potassium acetate (0.3 mmol), and Pd(dppf)Cl 2 The mixture was heated to reflux and reacted for 6 h. After completion of the reaction was detected by TLC, the mixture was cooled, suction filtered, rotary dried, and purified by silica gel column chromatography to obtain a white solid 1-4. 1 H NMR (300MHz, DMSO-d 6 )δ9.99(s,1H),8.07(d,J=1.6Hz,1H),7.46(d,J=7.5Hz,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 BN 2 O 4 [M+H] + :357.1986,found 357.1983. The yield was 89%.

[0021] Synthesis of intermediate I-5: Intermediate 1-4 (0.1 mmol) was added to a one-neck flask and dissolved in a mixed solvent of ethanol: dichloromethane (5:1). Then, 2-aminobenzamide (0.1 mmol) and iodine (0.1 mmol) were added, and the mixture was refluxed at 80°C for 30-60 minutes. After the reaction was completed by TLC, 5% sodium thiosulfate was added to quench the reaction, and the mixture was extracted with dichloromethane and saturated saline. 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 product I-5. 1 H NMR (300MHz, DMSO-d 6 )δ12.35(s,1H),8.13(dd,J=7.6,1.9Hz,2H),7.66(dd,J=7.5,2.1Hz,1H),7.5 6(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 BN 4 O 4 [M+H] + :473.2360,found 473.2357. The yield was 68%.

[0022] Synthesis of intermediate 2-2: Isovaleric acid (0.1 mmol) and HATU (0.3 mmol) were dissolved in 40 mL of tetrahydrofuran, then DIPEA (0.3 mmol) was added and stirred at room temperature for about 15 minutes, then raw material 2-1 (0.1 mmol) was added and stirred at room temperature for 1 to 3 hours. After the completion of the reaction of raw material 2-1 was detected by TLC, the reaction was stopped, and the intermediate 2-2 was obtained after separation and purification by column chromatography.

[0023] Synthesis of intermediate 2-3: Intermediate 2-2 (0.1 mmol), Intermediate 1-5 (0.1 mmol), Sodium carbonate (0.3 mmol), Pd(dppf)Cl 2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-neck flask, and the air was replaced with nitrogen gas four times. The mixture was heated to reflux and reacted for 8 h. After cooling, the mixture was suction filtered, rotary dried, and purified by silica gel column chromatography to obtain intermediate 2-3.

[0024] Synthesis of LH-001: 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 to 24 hours. After the completion of the reaction was detected by TLC, the mixture was concentrated under reduced pressure, extracted with dichloromethane and saturated sodium bicarbonate, and the organic phases were combined, dried and concentrated to obtain the final product LH-001. 1 H NMR (300MHz, DMSO-d 6 )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 N 6 O 2 [M+H]+ : 439.1882, found 439.1875。

[0025] Using the same operation as in Example 1, the following compound was produced.

Chemical formula

[0026]

Chemical formula

[0027]

Chem.

[0028]

Chem.

[0029]

Chem.

[0030]

Chem.

[0031]

Chem.

[0032]

Chem.

[0033]

Chem.

[0034]

Chem.

[0035]

Chem.

[0036]

Chem.

[0037]

Chem.

[0038]

Chem.

[0039]

Chem.

[0040]

Chem.

[0041]

Chem.

[0042]

Chem.

[0043]

Chem.

[0044]

Chem.

[0045] [Chemical formula] 1 H NMR (300 MHz, DMSO-d 6 ) δ 14.05 (s, 1H), 12.53 (s, 1H), 8.78 - 8.71 (m, 1H), 8.59 (d, J = 1.3 Hz, 1H), 8.18 - 8.05 (m, 2H), 7.71 - 7.51 (m, 4H), 7.44 (td, J = 7.5, 2.0 Hz, 1H), 7.35 - 7.22 (m, 2H), 7.22 - 7.07 (m, 4H), 4.26 (dd, J = 12.4, 7.0 Hz, 1H), 4.07 (dd, J = 12.4, 6.9 Hz, 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 N 6 O 2 [M + H] + : 4897.2039, found 489.2029。

[0046] [Chemical formula] 1 H NMR (300 MHz, DMSO-d 6 ) δ 14.16 (s, 1H), 12.38 (s, 1H), 8.79 (d, J = 1.1 Hz, 1H), 8.52 (d, J = 1.3 Hz, 1H), 8.36 (d, J = 1.3 Hz, 1H), 8.13 (dd, J = 7.4, 2.0 Hz, 1H), 7.71 - 7.53 (m, 3H), 7.51 - 7.39 (m, 2H), 7.14 (td, J = 7.5, 2.0 Hz, 1H), 3.50 (s, 2H) ppm. HR-MS (ESI): Calculated for C 20 H 15 N 6 O [M + H] + : 355.1307, found 355.1301。

[0047] [Chemical formula] 1 1H NMR (300 MHz, DMSO-d 6 ) δ 14.42 (s, 1H), 12.27 (s, 1H), 8.83 - 8.73 (m, 2H), 8.48 (d, J = 1.3 Hz, 1H), 8.13 (dd, J = 7.4, 2.0 Hz, 1H), 7.89 (t, J = 1.3 Hz, 1H), 7.66 (dd, J = 7.5, 2.0 Hz, 1H), 7.63 - 7.50 (m, 2H), 7.45 (td, J = 7.5, 2.0 Hz, 1H), 7.14 (td, J = 7.5, 2.0 Hz, 1H), 2.48 (s, 3H) ppm. HR-MS (ESI): Calculated for C 21 H 16 N 5 O [M + H] + : 354.1355, found 354.1350。

[0048]

Chem.

[0049]

Chem.

[0050]

Chem.

[0051] [Chemistry] 1 H NMR (300 MHz, DMSO-d 6 ) δ 14.25 (s, 1H), 12.25 (s, 1H), 9.50 (d, J = 1.3 Hz, 1H), 8.86 (d, J = 1.4 Hz, 1H), 8.71 (d, J = 1.3 Hz, 1H), 8.14 (d, J = 9.8 Hz, 2H), 8.06 (d, J = 1.2 Hz, 1H), 8.02 - 7.90 (m, 2H), 7.71 - 7.40 (m, 6H), 7.31 (ddt, J = 9.6, 7.2, 2.0 Hz, 1H), 7.15 (td, J = 7.5, 2.1 Hz, 1H) ppm. HR-MS (ESI): Calculated for C 27 H 19 N 6 O 2 [M + H] + : 459.1569, found 459.1560。

[0052] [Chemistry] 1 H NMR (300 MHz, DMSO-d 6 ) δ 14.56 (s, 1H), 12.17 (s, 1H), 9.80 (s, 1H), 9.61 (d, J = 1.2 Hz, 1H), 8.71 (dd, J = 14.1, 1.4 Hz, 2H), 8.13 (dd, J = 7.4, 2.0 Hz, 1H), 7.81 (t, J = 1.3 Hz, 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 N 6 O 2 [M + H] + : 473.1726, found 473.1719。

[0053] [Chemistry] 1 H NMR (300 MHz, DMSO-d6 ) δ 14.16 (s, 1H), 12.12 (s, 1H), 12.35 (s, 1H), 9.53 - 9.42 (m, 2H), 8.83 (t, J = 1.0 Hz, 1H), 8.70 (d, J = 1.3 Hz, 1H), 8.19 - 8.02 (m, 2H), 7.71 - 7.55 (m, 3H), 7.45 (td, J = 7.5, 2.0 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.14 (td, J = 7.5, 2.0 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H) ppm. HR - MS(ESI): Calculated for C 24 H 17 N 8 O 2 [M + H] + : 449.1474, found 449.1470。

[0054]

Chem.

[0055]

Chem.

[0056]

Chem.

[0057]

Chem.

[0058]

change

[0059] Example 2: Synthesis of LH-036

change

[0060] The other steps were the same as in Example 1 to obtain compound LH-036. [ka] 1 H NMR (300MHz, DMSO-d 6) δ 14.19 (s, 1H), 12.66 (s, 1H), 9.29 (s, 1H), 8.97 (s, 1H), 8.90 (s, 1H), 8.69 (d, J = 5.1 Hz, 1H), 8.61 (s, 1H), 8.18 (s, 1H), 8.02 (d, J = 5.1 Hz, 1H), 7.88 (q, J = 8.7 Hz, 2H), 3.95 (s, 2H), 3.17 - 3.08 (m, 2H), 2.93 (t, J = 6.9 Hz, 2H), 2.38 (dq, J = 15.3, 7.9, 7.4 Hz, 2H) ppm. HR - MS(ESI): Calculated for C 24 H 20 F 2 N 7 O [M + H] + : 460.1697, found 460.1690。

[0061] Using the same procedure as in Example 2, the following compound was prepared.

Chemical Structure

[0062]

Chemical Structure

[0063] Example 3: Synthesis of LH-039 [ka]

[0064] Synthesis of intermediate 1-2: Intermediate 1-1 (0.1 mmol) was added to a three-neck flask, tetrahydrofuran (50 mL) and p-toluenesulfonic acid (0.02 mmol) were added, 3,4-dihydropyran (0.2 mmol) was added dropwise at room temperature, and the reaction was continued for 8 hours at 65° C. After the reaction was completed by TLC, the solvent was distilled off under reduced pressure, and the mixture was purified by silica gel column chromatography to obtain a white solid I-2. 1 H NMR (300MHz, DMSO-d 6 )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(dt,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 12H 14 BrN 2 O [M+H] + :281.0290,found 281.0292. The yield was 85%.

[0065] Synthesis of intermediates 1-3: Add intermediate 1-2 (0.1 mmol) to a one-neck flask and add bis(pinacolato)diboron (0.12 mmol), potassium acetate (0.3 mmol), and Pd(dppf)Cl 2 The mixture was heated to reflux and reacted for 6 h. After the reaction was completed by TLC, the mixture was cooled, suction filtered, rotary dried, and purified by silica gel column chromatography to obtain a white solid 1-3. 1 H NMR (300MHz, DMSO-d 6 )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=1 ppm. HR-MS(ESI):Calculated for C 18 H 26 BN 2 O 3 [M+H] + :329.2036,found 329.2033. The yield was 89%.

[0066] Synthesis of intermediate 2-2: The raw material 2-1 (0.1 mmol) was dissolved in tetrahydrofuran, triethylamine (0.15 mmol) and isovaleric anhydride (0.15 mmol) were added, and the mixture was heated to reflux and reacted for 6 to 8 hours. After the completion of the reaction was detected by TLC, the mixture was concentrated under reduced pressure to remove the solvent, and the reaction liquid was poured into an ice-saturated sodium hydroxide solution to remove excess acid anhydride. The mixture was extracted three times with ethyl acetate and saturated saline, and the organic phases were combined, dried, and concentrated to obtain a yellow powdery solid 2-2. 1 H NMR (300MHz, DMSO-d 6 )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 BrN 2 O [M+H] + :257.0290,found 257.0286. The yield was 85%.

[0067] Synthesis of intermediate 2-3: Intermediate 2-2 (0.1 mmol), Intermediate 1-3 (0.1 mmol), Sodium carbonate (0.3 mmol), Pd(dppf)Cl 2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-neck flask, and the air was replaced with nitrogen gas four times. The mixture was heated to reflux and reacted for 8 h. After cooling, the mixture was suction filtered, rotary dried, and purified by silica gel column chromatography to obtain intermediate 2-3. 1 H NMR (300MHz, DMSO-d 6)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),8.03(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=6.9Hz,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 N 4 O 2 [M + H] + :379.2134, found 379.2132. The yield was 78%.

[0068] Synthesis of Intermediate 2 - 4: Dissolve Intermediate 2 - 3 (0.1 mmol) in 10 mL of tetrahydrofuran, add p - toluenesulfonic acid hydrate (1 mmol) at room temperature, heat up to reflux and react for 16 - 24 hours. After detecting the completion of the reaction by TLC, concentrate under reduced pressure, extract with dichloromethane and saturated sodium bicarbonate, combine the organic phases, dry and concentrate to obtain Intermediate 2 - 4. 1 H NMR(300MHz, DMSO - d 6 )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 N 4 O [M + H] +:295.1559,found 295.1554. The yield was 74%.

[0069] Synthesis of intermediate 2-5: 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 to 24 hours. After completion of the reaction was detected by TLC, the mixture was extracted three times with ethyl acetate and saturated saline, and the organic phases were combined and then dried and concentrated to obtain intermediate 2-5. 1 H NMR (300MHz, DMSO-d 6 )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 IN 4 O [M+H] + :421.0525,found 421.0525. The yield was 65%.

[0070] Synthesis of LH-039: Intermediate 2-5 (0.1 mmol), 3-pyridineboronic acid (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl 2 (0.005mmol), dioxane (9mL), and water (1.5mL) were added to a single-neck flask, and the air was replaced with nitrogen gas four times. The mixture was heated to reflux and reacted for 8 hours. After cooling, the mixture was filtered by suction, rotary dried, and purified by silica gel column chromatography to obtain LH-039. 1 H NMR (300MHz, DMSO-d 6)δ 13.62 (s, 1H), 10.23 (s, 1H), 9.30 (d, J = 2.2 Hz, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.74 (d, J = 2.1 Hz, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.49 (dt, J = 7.9, 2.0 Hz, 1H), 8.40 - 8.32 (m, 2H), 7.83 - 7.73 (m, 2H), 7.60 (dd, J = 8.0, 4.8 Hz, 1H), 2.29 (d, J = 7.1 Hz, 2H), 2.14 (dq, J = 13.5, 6.6 Hz, 1H), 1.00 (s, 3H), 0.98 (s, 3H) ppm. HR-MS (ESI): Calculated for C 22 H 22 N 5 O [M + H] + : 372.1824, found 372.1818。

[0071] Using the same operations as in Example 3, the following compound was produced.

Chemical formula

[0072] Example 4: Synthesis of LH-041

Chemical formula

[0073] Add intermediate LH-041-2 (0.1 mmol) to a one-neck flask and add bis(pinacolato)diboron (0.12 mmol), potassium acetate (0.3 mmol), and Pd(dppf)Cl 2 (0.005 mmol) and dioxane (25 mL) were added. The temperature was raised to reflux and the reaction was carried out for 6 hours. After the reaction was completed by TLC, the temperature was lowered, the mixture was suction filtered, rotary dried, and purified by silica gel column chromatography to obtain intermediate LH-041-3. 1 H NMR (300MHz, DMSO-d 6 )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 BNO 3 [M+H] + :246.1301,found 246.1281. The yield was 88%.

[0074] The other steps were the same as in Example 3 to obtain compound LH-041. [ka] 1H NMR (300MHz, DMSO-d 6 )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 .14(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 N 5 O 2 [M+H] + :412.1773,found 412.1769.

[0075] Example 5: Synthesis of LH-042 [ka]

[0076] Synthesis of intermediate 3-2: 3-1 (0.1 mmol) was added to a one-neck flask, and then excess concentrated sulfuric acid was added and stirred at room temperature for 2 h. After the reaction was completed, the reaction system was cooled to 0°C, and the mixture was alkalized with 10% aqueous sodium hydroxide solution and then extracted with ethyl acetate. The combined extracts were dried over magnesium sulfate, the solvent was removed in vacuum, and then recrystallized from ethanol to obtain 3-2.

[0077] Synthesis of intermediate 3-3: Intermediate 3-2 (0.1 mmol) was added to a one-neck flask and dissolved in ethanol. Then, intermediate 1-4 (0.1 mmol) from Example 1 and iodine (0.1 mmol) were added, and the mixture was allowed to react for 8 hours under reflux at 80°C. After the reaction was completed by TLC, 5% sodium thiosulfate was added to quench the reaction, and the mixture was extracted with dichloromethane and saturated saline. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain intermediate 3-3.

[0078] Synthesis of intermediate 3-4: Intermediate 2-2 (0.1 mmol) of Example 1, Intermediate 3-3 (0.1 mmol), sodium carbonate (0.3 mmol), Pd(dppf)Cl 2 (0.005 mmol), dioxane (9 mL), and water (1.5 mL) were added to a single-neck flask, and the air was replaced with nitrogen gas four times. The mixture was heated to reflux and reacted for 8 h. After cooling, the mixture was suction filtered, rotary dried, and purified by silica gel column chromatography to obtain intermediate 3-4.

[0079] Synthesis of LH-042: Intermediate 3-4 (0.1 mmol) was added to a 50 mL one-neck flask and dissolved in 10 mL of dichloromethane. Excess trifluoroacetic acid (1 mmol) was added and stirred at room temperature overnight. After the reaction was detected to be complete by TLC, the mixture was concentrated in vacuum to remove the solvent and excess trifluoroacetic acid. After sand filtration, the mixture was purified by column chromatography to obtain LH-042. 1 H NMR (300MHz, DMSO-d 6 )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,1 H),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 H21 N 6 O 3 [M+H] + : 429.1675, found 429.1670。

[0080] Using the same operation as in Example 5, the following compound was produced.

Chemical Structure

[0081]

Chemical Structure

[0082]

Chem.

[0083]

Chem.

[0084]

Chem.

[0085] Example 6: Synthesis of LH-048

Chem.

[0086] The other steps were the same as in Example 1 to obtain compound LH-048. [ka] 1 H NMR (300MHz, DMSO-d 6)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(t d,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 N 6 O [M+H] + :425.2090, found 425.2088.

[0087] In Example 6, the following compounds were produced using the same procedure.

change

[0088]

Chem.

[0089]

Chem.

[0090]

Chem.

[0091]

Chem.

[0092]

Chem.

[0093]

Chem.

[0094]

Chem.

[0095] Example 7: Synthesis of LH-057 [ka] The intermediate 1-4 (0.1 mmol) of Route 1 was added to a one-neck flask and dissolved in 5 mL of dimethylacetamide. Then, propylenediamine (0.15 mmol), p-toluenesulfonic acid hydrate (0.15 mmol), and sodium hydrogen sulfite (0.15 mmol) were added and reacted at 120°C for 6 to 8 hours. After the reaction was completed by TLC, the mixture was extracted three times with ethyl acetate and saturated saline. The organic phases were combined, dried, concentrated, and purified by column chromatography to obtain intermediate 1-5-4. 1 H NMR (300MHz, DMSO-d 6)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=11.4,7.0Hz,1H),3.6 2(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 32 BN 4 O 3 [M+H] + :411.2567,found 411.2566. The yield was 78%.

[0096] The other steps were the same as in Example 1 to obtain compound LH-057. [ka] 1 H NMR (300MHz, DMSO-d 6 )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 N 6 O [M+H] + :377.2090,found 377.1998.

[0097] Using a procedure similar to that of Example 7, the following compounds were prepared: [ka] 1 H NMR(300MHz, DMSO-d 6 ) δ 14.35(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 29 N 6 O [M + H] + : 393.2403, found 393.2403。

[0098]

Chem.

[0099] [ka] 1 H NMR (300MHz, DMSO-d 6 )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 F 2 N 6 [M+H] + :397.1952,found 397.1946.

[0100] Example 8: In vitro inhibitory activity of some compounds of the present invention against CLK family and DYEK1A proteins 1. Experimental Method 1. Preparation of 1X kinase reaction buffer [Table A]

[0101] 2. Enzyme activity experiment (1) Preparation of 2X kinase: [Table B]

[0102] (2) Preparation of 4X substrate mixture: [Table C] (1) Positive drugs were diluted 4-fold gradient using DMSO in a dilution plate, and the final starting concentrations of compounds were 1&0.02&0.1 μM. (2) The compound was diluted 50-fold in 1× kinase reaction buffer and shaken on a shaker for 20 minutes. (3) 2X kinase was prepared using 1X enzyme reaction buffer. (4) 2 μL of kinase was added to each well of the reaction plate. (5) 1 μL of a compound diluted with a buffer solution was added to each well, the plate was sealed with a plate seal, centrifuged at 1000 rpm for 60 seconds, and incubated at 25° C. for 10 minutes. (6) A 4X ATP&sub mixture was prepared using 1X enzyme reaction buffer, and 1 μL of the 4X ATP&sub mixture was added to the reaction plate. (7) The plate was sealed with a plate seal, centrifuged at 1000 rpm for 60 seconds, and incubated at 25°C for 60 minutes. (8) 4 μL of ADP-Glo ​​was transferred to a 384 reaction plate, centrifuged at 1000 rpm for 1 min, and incubated at 25° C. for 40 min. (9) 8 μL of the detection solution was transferred to a 384 reaction plate, centrifuged at 1000 rpm for 1 minute, and incubated at 25° C. for 40 minutes. (10) The relative luminescence unit (RLU) signals were read using a BMG multifunctional plate reader, and the signal intensity was used to characterize the activity degree of the kinase.

[0103] 3. Data analysis (1) The ratio for each well was calculated. (2) The inhibition rate was calculated as follows: Compound inhibition rate (%inh) = 100% - (compound - positive control) / (negative control - positive control) x 100% (3) IC 50 was calculated and the inhibition curve of the compound was plotted. The IC of the compound was calculated using the following nonlinear fitting equation: 50 (half maximal inhibitory concentration) was obtained: Data analysis was performed using Graphpad 7.0 software. Y=Bottom+(Top-Bottom) / (1+10^((LogIC50 -X) × HillSlope) X: Log value of compound concentration, Y: inhibition rate (% inhibition).

[0104] II. Experimental results [Table 1] NOTE: A: <10 nM, B: 10–50 nM, C: 50–100 nM, D: >100 nM.

[0105] As is clear from Table 1, the compounds described in the present invention exhibited effective inhibitory activity against CLK2 and DYRK1A. At the same time, the compounds described in the present invention, such as LH-020, exhibited relatively excellent CLK2 inhibitory activity and CLK3 selectivity (IC values ​​for CLK2, CLK3, and DYRK1A). 50 are 2 nM, 81 nM, and 3 nM, respectively, with a selectivity index of 41.5 against CLK3), which provided a basis for LH-020 to exert its pharmacological activity and avoid possible side effects.

[0106] Example 9: In vivo pharmacodynamic (PD) results of LH-020, a representative compound of the present invention, in osteoarthritic rats 1. Experimental Method 1, 40 rats were equally divided into 4 groups: control (normal rats injected with saline into the joint cavity), model (ACLT model rats injected with saline into the joint cavity), low-dose group (ACLT model rats injected with LH020 (1.5 μg / kg) into the joint cavity), and high-dose group (ACLT model rats injected with LH020 (6 μg / kg) into the joint cavity). 2. Knee osteoarthritis was induced in rats by anterior cruciate ligament resection (ACLT model). 3. One week after surgery, saline or LH-020 (1.5 μg / kg, 6 μg / kg) was injected into the joint cavity. 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.

[0107] II. Experimental results As can be seen from Figure 1, compared with the control group (injected saline into the joint cavity), the expression of cartilage-degrading proteases MMP3, MMP13, ADAMTS5, and IHH was significantly increased in the arthritis model. Furthermore, compared with the model group, both the low-dose and high-dose groups of LH-020 could significantly down-regulate the expression of MMP3, MMP13, ADAMTS5, and IHH, and the effect of the high-dose group was superior to that of the low-dose group, indicating that LH-020 has a significant cartilage-protecting effect, which provided a basis for exerting the pharmacological effect of treating osteoarthritis.

Claims

1. A 5-pyridine-1H-indazole compound having the structure of Formula I or II, said compound further including its isomers, pharma- ceutically acceptable salts, or mixtures thereof; 【Chemistry 68】 In the formula, R １ is a hydrogen atom, a straight or branched chain C substituted with one or more hydrogen atoms, halogen atoms, methoxy groups, trifluoromethyl groups, nitro groups, hydroxyl groups, amino groups, azide groups, sulfonic acid groups, 3- to 6-membered rings, １ ~C １０ is selected from an alkyl group, a phenyl group, a 4- to 6-membered heterocyclic group, or a 4- to 6-membered ring group; L and M are -CH ２ -, -NH-, -O-, or a chemical bond; R ２ teeth, 【Chemistry 69】 Selected from the group: R ３ is hydrogen or C １ ~C ４ selected from alkyl groups, The 5-pyridine-1H-indazole compound is characterized in that

2. In the above structure, R １ is selected from an isobutyl group, a cyclopropylmethyl group, a cyclopentyl group, an α-aminoisopentyl group, a 3,3-difluorotetrahydropyrrolyl group, hydrogen, a morpholinyl group, a methyl group, a tert-butyl group, an ethylsulfonyl group, or a hydroxyl group; R ２ teeth, 【Chemistry 70】 is selected from the group The compound according to claim 1 .

3. Selected from any one of the following compounds: The compound according to claim 1 . 【Chemistry 71】 【change】 【change】 【change】

4. The pharma- ceutically acceptable salts are salts formed by the compounds 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; The compound according to claim 1 .

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising:

6. In the manufacture of a CLK2 protein inhibitor drug, the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5, use.

7. In the manufacture of a DYRK1A protein inhibitor drug of the compound according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5, use.

8. The drug is a drug for treating inflammation. Use according to claim 6 or 7.

9. The inflammation is osteoarthritis, tendinopathy, or rheumatoid arthritis; 9. Use according to claim 8.

10. The compound has a chondroprotective effect.

10. The use according to claim 9.

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