CN115785071B - 3-Ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and their applications - Google Patents

Abstract

The invention discloses 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and their applications. This type of compound and its pharmaceutical composition can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and can be prepared as a drug for the treatment of osteoarthritis. It can exert its medicinal effect at the molecular level, and the therapeutic effect is better, and the optimal concentration can reach the nanomolar concentration level. . In addition, the compound preparation method is simple and easy to operate.

Description

3-Ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and their applications

Technical Field

The present invention relates to 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and applications thereof, and in particular to 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds, pharmaceutical compositions and applications thereof, which can be prepared to effectively inhibit the activity of CLK2 or DYRK1A proteins.

Background Art

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 insufficient stem cell supply, but to improper stem cell differentiation. The Wnt pathway plays a core role in organogenesis, cell differentiation, and tissue remodeling. Abnormal activation or inhibition of the Wnt signaling pathway can lead to the occurrence of the disease. Therefore, the Wnt signaling pathway is a potential target for the treatment of osteoarthritis.

The Wnt signaling pathway is a signaling pathway with multiple downstream channels that is triggered by the binding of the ligand protein Wnt and the membrane protein receptor. Through this pathway, the activation process of the cell surface receptor transmits the extracellular signal into the cell. In the classical Wnt pathway, when there is no Wnt protein on the cell membrane surface, the downstream β-Catenin protein will be decomposed by the glycogen synthase 3 (GSK3) complex in the cytoplasm, resulting in its inability to enter the cell nucleus to initiate the transcription of related Wnt genes; when there is Wnt protein on the cell membrane surface, it will inhibit the GSK3 complex, thereby causing the β-Catenin protein to accumulate in the cell nucleus, and finally initiate the transcription of genes related to the Wnt pathway. There is a delicate balance between the homeostasis of bone joints and the Wnt pathway. The disruption of this balance may lead to the occurrence of osteoarthritis.

The protein kinase family CLK (CDC-like kinase) is a dual-specific protein kinase that regulates intracellular signal transduction by phosphorylating substrate proteins on tyrosine, serine or threonine residues; it can be divided into four subtypes (CLK1, CLK2, CLK3 and CLK4), and the C segments of the proteins encoded by these four subtypes all have a highly conserved gene sequence and a similar amino acid sequence. CLKs play an important role in alternative splicing. CLKs act as a temperature sensor, which globally controls alternative splicing and gene expression. The activity of CLKs is indeed highly sensitive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Inhibition of CLK2 kinase is considered a way to improve neuronal function and combat intellectual disability and autism; inhibition of CLK2 kinase can kill MYC-driven breast tumors, triple-negative breast cancer and glioblastoma; inhibition of CLK2 and CLK3 can block the production of HIV-1.

Dual Specificity TyrosinePhosphorylation Regulated Kinase 1A (DYRK1A) belongs to the DYRK family, which is highly conserved in evolution. In mammals, the DYRK family has five different subtypes, and only DYRK1A is located in the DSCR region of human chromosome 21. DYRK1A is expressed by the dyrk1a gene, and the encoded mature protein consists of 763 amino acids, including a protein kinase domain and other special structures. Many important proteins can serve as substrates of DYRK1A and are regulated by it to participate in various biological functions in cells. For example, neural development, cell proliferation and differentiation, tumorigenesis, and neurodegenerative diseases.

At present, the first-in-class small molecule drug SM-04690 developed by Sumumed has been applied to the phase III clinical trial to treat osteoarthritis. Its specific mechanism is mainly to induce early cartilage formation by inhibiting CLK2-mediated SR protein phosphorylation; and to enhance the function of mature chondrocytes by inhibiting DYRK1A-mediated SIRT1 and FOXOA phosphorylation; and to reduce inflammation by inhibiting inflammatory factors such as NF-γB and STAT3. Although SM04690 has significant CLK2 inhibitory activity, it is not selective enough for the CLK family, which may lead to certain side effects; in addition, its inhibitory activity on the DYRK1A target is insufficient and its water solubility is poor, so its drugability needs to be improved.

Summary of the invention

The purpose of the present invention is to provide a 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound, a pharmaceutical composition and an application thereof which have the ability to specifically inhibit the activity of CLK2 and DYRK1A proteins, in order to address the problems of insufficient selectivity of the existing compounds for the CLK family and insufficient inhibitory activity on the DYRK1A target.

The purpose of the present invention can be achieved through the following technical solutions:

As the first aspect of the present invention, the 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound represented by formula I of the present invention, its isomer, pharmaceutically acceptable salt or mixture thereof,

Wherein, R1 is selected from the following groups:

R2 is selected from the following groups:

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

Preferably, the structure of the above compound is:

_R1 is selected from H,

R2 is selected from the following groups:

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

The pharmaceutically acceptable salts of the above compounds are salts formed from 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.

As the second aspect of the present invention, the above-mentioned compounds and pharmaceutically acceptable carriers 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.

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, which is specifically used to treat inflammation, including osteoarthritis, tendinopathy or rheumatoid arthritis, and has a cartilage protection effect.

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

(1) Such compounds and their pharmaceutical compositions can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and can also significantly downregulate the expression level of proteases related to cartilage decomposition in inflammatory model animals, thereby exerting a cartilage protective effect;

(2) Such compounds and their pharmaceutical compositions are widely used and can be prepared as drugs for treating osteoarthritis. They can exert their efficacy at the molecular level and animal level, and have better therapeutic effects, with the best concentration reaching nanomolar levels.

(3) The preparation method of the compound is simple and easy to operate.

DETAILED DESCRIPTION

The technical solution of the present invention is further described below in conjunction with embodiments.

Example 1: Synthesis of LBL-001

Synthesis of intermediate II:

5g 5-bromoindazole (25.38mmol, 1eq) was added to a 100ml single-necked bottle, and 12.88g iodine (50.75mmol, 2eq) and 4.27g potassium hydroxide (76.14mmol, 3eq) were added in sequence after dissolving it with 10mL DMF. After stirring at room temperature for 2 hours, TLC monitoring showed that the reaction was complete. Saturated sodium thiosulfate solution was added to quench the reaction solution, and ethyl acetate was used for extraction. The organic layer was retained and dried over anhydrous sodium sulfate. After spin drying, 7.6g yellow-white powder II was obtained, with a yield of 93%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ7.94 (t, J＝1.5Hz, 1H), 7.57 (d, J＝1.6Hz, 1H) ppm. HR-MS (ESI): Calculated for C ₇ H ₄ BrIN ₂ [M+H] ⁺ ：321.8603，found 321.8605.

Synthesis of intermediate III:

Add 5g of intermediate II (15.5mmol, 1eq) to a 100ml three-necked flask, dissolve it in 50mL of tetrahydrofuran, and then drop 2.6g of 3,4-dihydro-2H-pyran (31.0mmol, 2eq). After the dropwise addition is complete, add 0.59g of p-toluenesulfonic acid hydrate (3.1mmol, 0.2eq) in batches. After all the materials are added, heat to reflux at 75°C for reaction. After 5 hours, TLC monitoring shows that the reaction is complete. After the reaction liquid is spin-dried and column chromatography is performed, 5.4g of white powder III is obtained, with a yield of 86.3%. ¹ HNMR (300MHz, DMSO-d6): δ=7.77~7.74(m, 1H), 7.76~7.61(m, 2H), 5.88(dd, j1=2.3Hz, j2=2.0Hz, 1H), 3.88(m, 1H), 3.76(m, 1H), 2.40(m, 1H), 2.02(m, 2H) ), 1.75(m, 1H), 1.58(br, 2H)ppm. HR-MS(ESI): Calculated for C ₁₂ H ₁₂ BrIN ₂ O[M+H] ⁺ : 405.9178, found 405.9156.

Synthesis of intermediate V:

In a 50mL single-necked bottle, add 1.5g of intermediate III (3.69mmol, 1eq), 0.44g of 3-ethynylpyridine (4.42mmol, 1.2eq), 0.1g of bistriphenylphosphine palladium dichloride (0.18mmol, 0.05eq), 70mg of cuprous iodide (0.37mmol, 0.1eq) and 15mL of triethylamine, and dissolve it completely with 7.5mL of DMF. After the addition, replace with nitrogen and stir at room temperature to react. After 5h of reaction, TLC monitoring shows that the reaction is complete. Filter the reaction solution, retain the filtrate, spin dry the filtrate and column chromatograph to obtain 1.3g of yellow solid V, with a yield of 88%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.79 (dd, J＝2.2, 1.6Hz, 1H), 8.69 (ddd, J＝3.3, 2.3, 1.6Hz, 1H), 8.24 (dd, J＝2.3, 0.8Hz, 1H), 7.84 (dt, J＝6.6, 2.2Hz, 1H), 7.74–7.60 ( m,2H),7.40(dd,J＝6.6,3.2Hz,1H),5.92(t,J＝2.4Hz,1H),3.94–3.72(m,2H),2.30–2.06(m,2H),1.97–1.72(m,2H),1.78–1.58(m,2H)ppm. HR-MS (ESI): Calculated for C ₁₉ H ₁₆ BrN ₃ O[M+H] ⁺ : 381.0477, found 381.0467.

Synthesis of intermediate VI:

In a 50mL single-necked bottle, add 1.1g intermediate V (2.76mmol, 1eq), 0.54g trimethylsilylene (5.51mmol, 2eq), 50mg tetrakistriphenylphosphine palladium (0.14mmol, 0.05eq), 10mg cuprous iodide (0.06mmol, 0.02eq) and 10mL triethylamine, and dissolve it completely with 10mL tetrahydrofuran. After the addition is completed, nitrogen replacement is carried out, and the reaction is heated to reflux at 70°C. After 10h of reaction, TLC monitoring shows that the reaction is complete. The reaction solution is filtered, the filtrate is retained, and the filtrate is spin-dried and column chromatography is performed to obtain 0.6g yellow oil VI, with a yield of 45%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.79(dd,J＝2.3,1.6Hz,1H),8.69(ddd,J＝3.3,2.3,1.6Hz,1H),8.35–8.28(m,1H),7.89–7.74(m,2H),7.56(dd,J＝7.2,2.2Hz,1H),7 .40(dd,J＝6.6,3.2Hz,1H),5.92(t,J＝2.4Hz,1H),3.94–3.72(m,2H),2.30–2.06(m,2H),1.97–1.72(m,2H),1.76–1.58(m,2H),0.25(s,8H)ppm. HR-MS(ESI): Calculated for C ₂₄ H ₂₅ N ₃ OSi[M+H] ⁺ : 399.1767, found 399.1765.

Synthesis of intermediate VII:

0.4 g of intermediate VI was added to a 25 ml single-necked bottle, and 0.4 g of potassium carbonate was added after it was completely dissolved in 10 mL of methanol, and stirred at room temperature. After 30 minutes of reaction, TLC monitoring showed that the reaction was complete. Extraction was performed with dichloromethane and water, and the organic layer was retained and dried with anhydrous sodium sulfate. After spin drying, 0.33 g of white solid VII was obtained, with a yield of 100%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.79 (dd, J＝2.3, 1.6Hz, 1H), 8.69 (ddd, J＝3.3, 2.3, 1.6Hz, 1H), 8.40–8.29 (m, 1H), 7.84 (dt, J＝6.6, 2.2Hz, 1H), 7.73–7.63 (m, 2H) ,7.40(dd,J＝6.6,3.2Hz,1H),5.92(t,J＝2.4Hz,1H),3.94–3.72(m,2H),2.30–2.06(m,2H),1.97–1.72(m,2H),1.78–1.58(m,2H)ppm. HR-MS(ESI): Calculated for C ₂₁ H ₁₇ N ₃ O[M+H] ⁺ : 327.1372, found 327.1365.

Synthesis of intermediate IX:

0.26g of intermediate VII (0.75mmol, 1eq) and 7mg of cuprous iodide (0.04mmol, 0.05eq) were added to a 25ml single-necked bottle, and completely dissolved with 4mL of DMF and 0.5mL of methanol. 130mg of trimethylsilyl azide (1.13mmol, 1.5eq) was added, and the reaction was heated at 100°C after nitrogen replacement. After 24h of reaction, TLC monitoring showed that a small amount of raw materials remained. After spin drying, column chromatography was performed to obtain 0.12g of white solid IX, with a yield of 52%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ8.79(dd,J＝2.3,1.6Hz,1H),8.73–8.63(m,2H),8.45(d,J＝1.9Hz,1H),7.99(dd,J＝8.6,2.4Hz,1H),7.84(dt,J＝6.6,2.2Hz,1H),7.7 2(dd,J＝8.6,0.6Hz,1H),7.40(dd,J＝6.6,3.2Hz,1H),5.92(t,J＝2.4Hz,1H), 3.94–3.72(m,2H),2.30–2.06(m,2H),1.97–1.72(m,2H),1.78–1.58(m,2H)pp m. HR-MS(ESI): Calculated for C ₂₁ H ₁₈ N ₆ O[M+H] ⁺ : 370.1542, found 370.1544.

Synthesis of LBL-001:

The product obtained in the previous step was added to a 25ml single-necked bottle, completely dissolved with 8mL of dichloromethane, and then 100μL of triethylsilane was added. The system was white and turbid. After adding 10mL of trifluoroacetic acid, the system became clear and stirred at room temperature for reaction. After 1h of reaction, TLC monitoring showed that the reaction was complete. The reaction solution was spin-dried and extracted with ethyl acetate and sodium bicarbonate solution. The organic layer was retained and dried with anhydrous sodium sulfate. The organic phase was spin-dried and column chromatography was performed to obtain 75mg of white solid LBL-001, with a yield of 100%. ¹ HNMR (300MHz, DMSO-d ₆ ) δ8.79(dd,J＝2.3,1.6Hz,1H),8.73–8.62(m,2H),8.49(d,J＝1.9Hz,1H),8.04(dd,J＝9.7,2.4Hz,1H),7.84(dt,J＝6.6,2.2Hz,1H),7 .59(dd,J＝9.8,0.6Hz,1H),7.40(dd,J＝6.6,3.2Hz,1H)ppm. HR-MS (ESI): Calculated for C ₁₆ H ₁₀ N ₆ [M+H] ⁺ : 286.0967, found 286.0966.

The following compounds were obtained by similar operation as in Example 1:

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.69–8.63(m,1H),8.55–8.45(m,2H),8.33(ddd,J＝7.7,1.9,1.3Hz,1H),8.04(dd ,J＝9.7,2.4Hz,1H),7.76(ddd,J＝6.8,1.9,1.3Hz,1H),7.71–7.54(m,2H)ppm. Calculated for C ₁₇ H ₁₀ N ₆ O ₂ [M+H] ⁺ :330.0865, found330.0854.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (d, J = 2.3 Hz, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz, 1H ),7.59(dd,J＝9.8,0.6 Hz,1H),7.46(td,J＝6.6,4.9 Hz,1H),7.38–7.27(m,2H),7.27–7.16(m,1H)ppm. Calculated for C ₁₇ H ₁₀ FN ₅ [M+H] ⁺ : 303.0920, found 303.0913.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.69–8.63 (m, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz, 1H), 7.96 (t,J＝1.9 Hz,1H),7.68(ddd,J＝9.6,1.9,1.3 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.50(dd,J＝9.6,6.3 Hz, 1H), 7.40 (ddd, J = 6.4, 1.9, 1.3 Hz, 1H) ppm. Calculated for C ₁₈ H ₁₀ F ₃ N ₅ [M+H] ⁺ : 353.0888 found 353.0866.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ9.28 (s, 1H), 8.69–8.62 (m, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.26–7.12(m,2H),6.90(t,J＝1.7 Hz,1H),6.80(dt,J＝6.9,2.0 Hz,1H)ppm. Calculated forC ₁₇ H ₁₁ N ₅ O[M+H] ⁺ :301.0964 found 301.0962.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.69–8.62 (m, 2H), 8.49 (d, J = 1.9 Hz, 2H), 8.04 (dd, J = 9.7, 2.4 Hz, 2H), 7.59 (dd,J＝9.8,0.6 Hz,2H),7.31(t,J＝6.8 Hz,2H),7.25(dt,J＝6.7,1.5 Hz,2H),7.17(t,J＝1.9 Hz,2H) ,7.05–6.96(m,2H)ppm. Calculated for C ₁₈ H ₁₃ N ₅ O[M+H] ⁺ :315.1120 found 315.1121.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.69–8.62 (m, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),7.25(t,J＝6.9 Hz,1H),7.10(ddd,J＝7.0,1.9,1.2 Hz,1H),6.99(t,J＝1.9 Hz, 1H), 6.65 (ddd, J=6.8, 1.9, 1.2 Hz, 1H), 4.97 (s, 2H) ppm. Calculated for C ₁₇ H ₁₂ N ₆ [M+H] ⁺ :300.1123 found300.1114.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.69–8.62 (m, 2H), 8.49 (d, J = 1.9 Hz, 2H), 8.04 (dd, J = 9.7, 2.4 Hz, 2H), 7.92 (t,J＝1.7 Hz,2H),7.65(dd,J＝5.0,1.7 Hz,2H),7.59(dd,J＝9.8,0.6 Hz,2H),7.34(dd,J＝5.0,1.7 Hz, 2H) ppm. Calculated for C ₁₅ H ₉ N ₅ S[M+H] ⁺ :291.0579 found 291.0578.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.68 (dd, J = 2.4, 0.5 Hz, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz ,1H),7.59(dd,J＝9.8,0.6 Hz,1H),3.04(q,J＝6.2Hz,1H),1.37–1.13(m,5H).ppm. Calculated for C ₁₄ H ₁₁ N ₅ [M+H] ⁺ :249.1014 found 249.1011.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.68 (d, J = 2.4 Hz, 1H), 8.49 (d, J = 1.9 Hz, 1H), 8.04 (dd, J = 9.7, 2.4 Hz, 1H ),7.59(d,J＝9.8 Hz,1H),3.05–2.92(m,1H),1.70-1.55(m,J＝13.4,4.8,4.1,2.4,1.5 Hz,8H)ppm. Calculated for C1 ₆ H ₁₅ N ₅ [M+H] ⁺ :277.1327found277.1325.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.79 (dd, J＝2.2, 1.6 Hz, 1H), 8.73–8.63 (m, 2H), 8.50 (s, 1H), 8.05 (dd, J＝ 9.7,2.4 Hz,1H),7.84(dt,J＝6.6,2.2 Hz,1H),7.59(dd,J＝9.8,0.5Hz,1H),7.45–7.20(m,6H),5.37(t,J =0.9 Hz,2H)ppm. Calculated for C ₂₃ H ₁₆ N ₆ [M+H] ⁺ :376.1436 found 376.1428.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.51 (t, J = 1.9 Hz, 1H), 8.50 (s, 1H), 8.33 (ddd ,J＝7.7,1.9,1.3 Hz,1H),8.05(dd,J＝9.7,2.4 Hz,1H),7.76(ddd,J＝6.8,1.9,1.3 Hz,1H),7.71–7.54(m,2H ),7.39–7.20(m,5H),5.37(t,J＝0.9 Hz,2H)ppm. Calculated for C ₂₄ H ₁₆ N ₆ O ₂ [M+H] ⁺ : 420.1335 found 420.1333.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.5 Hz,1H),7.46(td,J＝6.6,5.0 Hz,1H),7.40–7.28(m,5H),7.33–7.21(m,3H),7.21-7.05(m ,J=6.8,2.0,1.1 Hz,1H),5.37(t,J=0.9Hz,2H)ppm. Calculated for C ₂₄ H ₁₆ FN ₅ [M+H] ⁺ :393.1390 found 393.1388.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.96 (t,J＝1.9 Hz,1H),7.68(ddd,J＝9.6,1.9,1.3 Hz,1H),7.63–7.20(m,8H),5.37(t,J＝0.9 Hz,2H)ppm. Calculated for C ₂₅ H ₁₆ F ₃ N ₅ [M+H] ⁺ : 443.1358 found 443.1344.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ9.28 (s, 1H), 8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.39–7.12(m,7H),6.90(t,J＝1.8 Hz,1H),6.80(dt,J＝6.9,1.9 Hz, 1H), 5.37 (t, J = 0.9Hz, 2H) ppm. Calculated for C ₂₄ H ₁₇ N ₅ O[M+H] ⁺ : 391.1433 found 391.1430.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),7.39–7.20(m,8H),7.17(t,J＝1.9 Hz,1H),7.05–6.96(m,1H),5.37(t,J＝0.9 Hz,2H)ppm. Calculated for C ₂₅ H ₁₉ N ₅ O[M+H] ⁺ :405.1590 found 405.1588.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),7.39–7.20(m,7H),7.10(ddd,J＝7.0,1.9,1.2 Hz,1H),6.99(t,J＝1.9 Hz,1H), 6.65 (ddd, J=6.8, 1.9, 1.2 Hz, 1H), 5.37 (t, J=0.9 Hz, 2H), 4.97 (s, 2H) ppm. Calculated for C ₂₄ H ₁₈ N ₆ [M+H] ⁺ :390.1593 found 390.1588.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.92 (t,J＝1.7 Hz,1H),7.65(dd,J＝5.0,1.7 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.39–7.20(m,6H),5.37( t,J=0.9 Hz,2H)ppm. Calculated for C ₂₂ H ₁₅ N ₅ S[M+H] ⁺ :381.1048 found 381.1047.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.68 (dd, J＝2.4, 0.5Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J＝9.7, 2.4Hz, 1H), 7.59 ( dd,J＝9.8,0.5Hz,1H),7.39–7.20(m,6H),5.37(t,J＝0.9Hz,2H),3.04(q,J＝6.2Hz,1H),1.37–1.13(m ,5H)ppm. Calculated for C ₂₁ H ₁₇ N ₅ [M+H] ⁺ : 339.1484found 339.1479.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.68 (d, J＝2.4Hz, 1H), 8.50 (s, 1H), 8.05 (dd, J＝9.7, 2.4Hz, 1H), 7.59 (d, J＝9.8Hz,1H),7.39–7.20(m,5H),5.37(t,J＝0.9Hz,2H),3.05–2.92(m,1H),1.80–1.61(m,8H)ppm. Calculated for C ₂₃ H ₂₁ N ₅ [M+H] ⁺ :367.1797found 367.1793.

Example 2: Synthesis of LBL-021

Synthesis of intermediate IX-II:

In a 25mL single-necked bottle, 0.2g of intermediate VII (0.58mmol, 1eq), 83mg of ethyl azidoacetate (1.1mmol, 1.1eq), 15mg of copper sulfate pentahydrate (0.06mmol, 0.1eq) and 23mg of sodium ascorbate (0.12mmol, 0.2eq) were added, and finally 5mL of DMF was added and stirred at room temperature. After 2h of reaction, TLC monitoring showed that the reaction was complete. Extraction was performed with ethyl acetate and water, and the organic layer was retained and dried with anhydrous sodium sulfate. The organic phase was spin-dried and then column chromatography was performed to obtain 0.12g of white solid IX-II, with a yield of 58%. ¹ H NMR (300MHz, DMSO-d ₆ )δ8.79(dd,J＝2.3,1.6Hz,1H),8.73–8.64(m,2H),8.39(s,1H),8.00(dd,J＝8.5,2.4Hz,1H),7.84(dt,J＝6.6,2.2Hz,1H),7.71(dd,J＝8.6,0.6Hz,1H),7.40 (dd,J＝6.6,3.2Hz ,1H),5.92(t,J＝2.4Hz,1H),5.16(d,J＝0.4Hz,2H),4.28–4.18(m,2H),3.94–3.72(m,2H),2.30–2.06(m,2H),1.97–1.72(m,2H),1.78–1.58(m,2H),1 .24(t,J＝6.6Hz,3H)ppm. Calculated for C ₂₅ H ₂₄ N ₆ O ₃ [M+H] ⁺ :456.1910found456.1908.

The remaining steps were the same as in Example 1 to obtain compound LBL-021.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.79 (dd, J = 2.2, 1.6 Hz, 1H), 8.73–8.63 (m, 2H), 8.39 (s, 1H), 8.05 (dd, J = 9.7 ,2.4Hz,1H),7.84(dt,J＝6.6,2.2Hz,1H),7.59(dd,J＝9.8,0.6Hz,1H),7.40(dd,J＝6.6,3.2Hz,1H),5.16 (s,2H),4.28–4.18(m,2H),1.24(t,J＝6.6Hz,3H)ppm. Calculated for C ₂₀ H ₁₆ N ₆ O ₂ [M+H] ⁺ :372.1335found372.1330.

Using similar operations as in Example 2, the following compounds were obtained:

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.66 (dd, J＝2.4, 0.5Hz, 1H), 8.51 (t, J＝1.9Hz, 1H), 8.42–8.28 (m, 2H), 8.05 ( dd,J＝9.7,2.4Hz,1H),7.76(ddd,J＝6.8,1.9,1.3Hz,1H),7.71–7.54(m,2H),5.16(s,2H),4.28–4.18(m, 2H), 1.24 (t, J = 6.6Hz, 3H) ppm. Calculated for C ₂₁ H ₁₆ N ₆ O ₄ [M+H] ⁺ : 416.1233 found 416.1238.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.66 (dd, J＝2.4, 0.5Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J＝9.7, 2.4Hz, 1H), 7.59 ( dd,J＝9.8,0.6Hz,1H),7.46(td,J＝6.7,5.0Hz,1H),7.38–7.27(m,2H),7.27–7.16(m,1H),5.16(s,2H) ,4.28–4.18(m,2H),1.24(t,J=6.6Hz,3H)ppm. Calculated for C ₂₁ H ₁₆ FN ₅ O ₂ [M+H] ⁺ : 389.1288 found 389.1287.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.96 (t,J＝1.9 Hz,1H),7.68(ddd,J＝9.6,1.9,1.3 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.50(dd,J＝9.6,6.3 Hz,1H),7.40(ddd,J＝6.4,1.9,1.3 Hz,1H),5.16(s,2H),4.28–4.18(m,2H),1.24(t,J＝6.6 Hz,3H)ppm. Calculated for C ₂₂ H ₁₆ F ₃ N ₅ O ₂ [M+H] ⁺ : 439.1256 found 439.1254.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ9.28 (s, 1H), 8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.26–7.12(m,2H),6.90(t,J＝1.8 Hz,1H),6.80(dt,J＝6.9,2.0 Hz, 1H), 5.16 (s, 2H), 4.28–4.18 (m, 2H), 1.24 (t, J = 6.6 Hz, 3H) ppm. Calculated for C ₂₁ H ₁₇ N ₅ O ₃ [M+H] ⁺ : 387.1331 found 387.1330.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),7.36–7.20(m,2H),7.17(t,J＝1.9 Hz,1H),7.05–6.96(m,1H),5.16(s,2H), 4.22 (q, J = 6.6 Hz, 2H), 1.24 (t, J = 6.6 Hz, 3H) ppm. Calculated for C ₂₂ H ₁₉ N ₅ O ₃ [M+H] ⁺ : 401.1488 found 401.1485.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),7.25(t,J＝6.9 Hz,1H),7.10(ddd,J＝7.0,1.9,1.2 Hz,1H),6.99(t,J＝1.9 Hz, 1H),6.65(ddd,J＝6.8,1.9,1.2 Hz,1H),5.16(s,2H),4.97(s,2H),4.28–4.18(m,2H),1.24(t,J＝6.6Hz ,3H)ppm. Calculated for C ₂₁ H ₁₈ N ₆ O ₂ [M+H] ⁺ : 386.1491 found 386.1490.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.66 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.92 (t,J＝1.7 Hz,1H),7.65(dd,J＝5.0,1.7 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.34(dd,J＝5.0,1.7 Hz, 1H), 5.16 (s, 2H), 4.28–4.18 (m, 2H), 1.24 (t, J = 6.6 Hz, 3H) ppm. Calculated for C ₁₉ H ₁₅ N ₅ O ₂ S[M+H] ⁺ : 377.0946 found 377.0944.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.68 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.5 Hz,1H),5.16(s,2H),4.22(q,J＝6.6 Hz,2H),3.04(q,J＝6.2 Hz,1H),1.37–1.13(m, 7H) ppm. Calculated for C ₁₈ H ₁₇ N ₅ O ₂ [M+H] ⁺ : 335.1382 found 335.1380.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.68 (dd, J = 2.4, 0.5 Hz, 1H), 8.39 (s, 1H), 8.05 (dd, J = 9.7, 2.4 Hz, 1H), 7.59 (dd,J＝9.8,0.6 Hz,1H),5.16(s,2H),4.22(q,J＝6.6 Hz,2H),3.05–2.92(m,1H),1.81–1.62(m,8H), 1.24(t,J=6.6 Hz,3H)ppm. Calculated for C ₂₀ H ₂₁ N ₅ O ₂ [M+H] ⁺ : 363.1695 found 363.1694.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.79 (dd, J＝2.3, 1.6 Hz, 1H), 8.69 (ddd, J＝3.3, 2.3, 1.6 Hz, 1H), 8.64–8.57 (m, 1H),8.20(s,1H),8.04(dd,J＝9.8,2.4 Hz,1H),7.89–7.79(m,3H),7.59(dd,J＝9.8,0.6 Hz,1H),7.40(dd ,J＝6.6,3.2 Hz,1H),7.34-7.20(m,J＝8.8,0.8 Hz,2H),2.39(d,J＝1.5 Hz,1H)ppm. Calculated for C ₂₃ H ₁₆ N ₆ O ₂ S[M+H] ⁺ : 440.1055found 440.1050.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57(m,1H),8.51(t,J＝1.9 Hz,1H),8.33(ddd,J＝7.7,1.9,1.3 Hz,1H) ,8.20(s,1H),8.04(dd,J＝9.8,2.4 Hz,1H),7.89–7.80(m,2H),7.76(ddd,J＝6.8,1.9,1.3 Hz,1H),7.66(dd ,J＝7.7,6.8 Hz,1H),7.59(dd,J＝9.7,0.6Hz,1H),7.40–7.29(m,2H),2.39(d,J＝1.4 Hz,1H)ppm. Calculated for C ₂₄ H ₁₆ N ₆ O ₄ S[M+H] ⁺ : 484.0954 found 484.0950.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J = 9.8, 2.4Hz, 1H), 7.89–7.79 (m, 2H),7.59(dd,J＝9.8,0.6 Hz,1H),7.46(td,J＝6.7,4.9 Hz,1H),7.39–7.16(m,5H),2.39(d,J＝1.4 Hz,1H )ppm. Calculated for C ₂₄ H ₁₆ FN ₅ O ₂ S[M+H] ⁺ : 457.1009 found 457.1007.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J＝9.8, 2.4Hz, 1H), 7.96 (t, J＝ 1.9 Hz,1H),7.89–7.79(m,2H),7.68(ddd,J＝9.6,1.9,1.3 Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.50(dd,J ＝9.6,6.4 Hz,1H),7.40(ddd,J＝6.4,1.9,1.3Hz,1H),7.34-7.19(m,J＝8.8,0.7 Hz,2H),2.39(d,J＝1.4 Hz, 1H) ppm. Calculated forC ₂₅ H ₁₆ F ₃ N ₅ O ₂ S[M+H] ⁺ :507.0977 found 507.0975.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ9.28 (s, 1H), 8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J＝9.8, 2.4 Hz, 1H) ,7.89–7.79(m,2H),7.59(dd,J＝9.8,0.6 Hz,1H),7.40–7.29(m,2H),7.26–7.12(m,2H),6.90(t,J＝1.8 Hz , 1H), 6.80 (dt, J = 6.9, 1.9 Hz, 1H), 2.39 (d, J = 1.4 Hz, 1H) ppm. Calculated for C ₂₄ H ₁₇ N ₅ O ₃ S[M+H] ⁺ : 455.1052 found 455.1050.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J = 9.8, 2.4Hz, 1H), 7.89–7.79 (m, 2H),7.59(dd,J＝9.8,0.6 Hz,1H),7.40–7.26(m,3H),7.25(dt,J＝6.7,1.5 Hz,1H),7.17(t,J＝1.9 Hz,1H ),7.05–6.96(m,1H),2.39(d,J＝1.3 Hz,1H)ppm. Calculated for C ₂₅ H ₁₉ N ₅ O ₃ S[M+H] ⁺ : 469.1209 found 469.1208.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J = 9.8, 2.4Hz, 1H), 7.89–7.79 (m, 2H),7.59(dd,J＝9.8,0.6 Hz,1H),7.34 -7.20(m,J＝8.8,0.7 Hz,2H),7.25(t,J＝6.9 Hz,1H),7.10(ddd,J ＝7.0,1.9,1.2 Hz,1H),6.99(t,J＝1.9 Hz,1H),6.65(ddd,J＝6.8,1.9,1.2 Hz,1H),4.97(s,2H),2.39(d, J＝1.4 Hz,1H)ppm. Calculated for C ₂₄ H ₁₈ N ₆ O ₂ S[M+H] ⁺ : 454.1212 found 454.1210.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ8.64–8.57 (m, 1H), 8.20 (s, 1H), 8.04 (dd, J＝9.8, 2.4Hz, 1H), 7.92 (t, J＝ 1.7 Hz,1H),7.89–7.79(m,2H),7.65(dd,J＝5.0,1.7Hz,1H),7.59(dd,J＝9.8,0.6 Hz,1H),7.40–7.29(m,3H ), 2.39 (d, J = 1.4 Hz, 1H) ppm. Calculated for C ₂₂ H ₁₅ N ₅ O ₂ S ₂ [M+H] ⁺ : 445.0667 found 445.0666.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.66 (dd, J＝2.4, 0.5Hz, 1H), 8.20 (s, 1H), 8.04 (dd, J＝9.8, 2.4Hz, 1H), 7.89– 7.79(m,2H),7.59(dd,J＝9.8,0.6Hz,1H),7.34-7.19(m,J＝8.8,0.7Hz,2H),3.04(q,J＝6.2Hz,1H),2.39 (d,J＝1.4Hz,1H),1.37–1.13(m,5H)ppm. Calculated for C ₂₁ H ₁₇ N ₅ O ₂ S[M+H] ⁺ : 403.1103 found 403.1101.

¹ H NMR (300MHz, DMSO-d ₆ ) δ8.66 (dd, J＝2.4, 0.5Hz, 1H), 8.20 (s, 1H), 8.04 (dd, J＝9.8, 2.4Hz, 1H), 7.89– 7.79(m,2H),7.59(dd,J＝9.8,0.5Hz,1H),7.34-7.19(m,J＝8.8,0.8Hz,2H),3.05–2.92(m,1H),2.42–2.36( m,2H),1.81–1.61(m,8H)ppm. Calculated for C ₂₃ H ₂₁ N ₅ O ₂ S[M+H] ⁺ : 431.1416 found 431.1414.

Example 3: In vitro inhibitory activity of some compounds of the present invention on CLK family and DYRK1A proteins

1. Experimental Methods

1. Prepare 1X kinase reaction buffer

2. Enzyme activity test

(1) 2X kinase preparation:

(2) Preparation of 4X substrate mixture:

(1) The positive drug Lorecivivint (SM-04690) was diluted 4-fold in a dilution plate with DMSO, and the final starting concentrations of the compound were 1, 0.02, and 0.1 μM.

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

(3) Prepare 2X kinase using 1X enzyme reaction buffer.

(4) Add 2 μL of kinase to each well of the reaction plate.

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

(6) Prepare 4X ATP & sub mixed solution with 1X enzyme reaction buffer and add 1 μL of 4X ATP & sub mixed solution to the reaction plate.

(7) Seal the plate with sealing film, centrifuge at 1000 rpm for 60 seconds, and incubate at 25°C for 60 minutes.

(8) Transfer 4 μL of ADP-Glo to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

(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.

(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.

2. Experimental Results

Table 1. Enzyme activities of some compounds in the present invention

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

As can be seen from Table 1, the compounds of the present invention show effective inhibitory activity against CLK2 and DYRK1A. At the same time, the compounds of the present invention, such as LBL-001, show better CLK2 inhibitory activity and CLK3 selectivity (IC ₅₀ for CLK2, CLK3, and DYRK1A are 4nM, 120nM, and 3nM, respectively, and the selectivity index for CLK3 is 30), which provides a basis for LBL-001 to exert its pharmacological activity and avoid possible side effects.

Claims (10)

1. A 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound represented by formula I, a pharmaceutically acceptable salt or a mixture thereof , which is characterized by, Wherein R1 is selected from the following groups: H, R2 is selected from the following groups: 2. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to claim 1, pharmaceutically acceptable Salts or their mixtures, characterized by: R ₁ is selected from H, R2 is selected from the following groups: 3. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to claim 1, pharmaceutically acceptable Salts or mixtures thereof, characterized in that they are selected from any of the following compounds: 4. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to claim 1, pharmaceutically acceptable Salts or their mixtures, characterized in that the pharmaceutically acceptable salt is a salt formed by the compound and the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, hydrobromic acid, hydriodic 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 it contains 3-ethynyl-5-(1H-1,2,3-triazole-4 shown in formula I according to any one of claims 1-4 -1H-indazole compounds, pharmaceutically acceptable salts and pharmaceutically acceptable carriers. 6. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to any one of claims 1-4, and its isotopes The use of the construct, pharmaceutically acceptable salt or pharmaceutical composition according to claim 5 in the preparation of CLK2 protein inhibitor drugs. 7. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to any one of claims 1-4, pharmaceutical Use of an acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of DYRK1A protein inhibitor drugs. 8. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I according to any one of claims 1-4, pharmaceutical Use of the acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of medicines for treating inflammation. 9. Application according to claim 8, characterized in that the inflammation is osteoarthritis, tendinopathy or rheumatoid arthritis. 10. The application according to claim 8, characterized in that the compound has a chondroprotective effect.