CN115785071A - 3-Ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and their applications - Google Patents

Abstract

The invention discloses a 3-ethynyl-5- (1H-1,2,3-triazole-4-yl) -1H-indazole compound and application thereof. The compound and the pharmaceutical composition thereof can effectively inhibit activities of CLK2 protein and DYRK1A protein, can be prepared into a medicament for treating osteoarthritis, can exert medicinal effects at a molecular level, have more excellent treatment effect, and optimally reach a nanomolar concentration level. In addition, the preparation method of the compound is simple and convenient and is easy to operate.

Description

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

technical field

The present invention relates to 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds and applications thereof, in particular to a compound that can be prepared to effectively inhibit CLK2 or DYRK1A Protein active 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound, pharmaceutical composition and application.

Background technique

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 failure of articular cartilage to regenerate is not due to insufficient supply of stem cells, but due to improper differentiation of stem cells. The Wnt pathway plays a central role in organogenesis, cell differentiation, and tissue remodeling, and 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 of a set of multiple downstream channels triggered by the binding of the ligand protein Wnt and membrane protein receptors. Through this pathway, intracellular activation of cell surface receptors transmits extracellular signals into the cell. In the classic Wnt pathway, when there is no Wnt protein on the surface of the cell membrane, its downstream β-Catenin protein will be decomposed by the glycogen synthase 3 (GSK3) complex in the cytoplasm, so that it cannot enter the nucleus to start the transcription of related Wnt genes ; and when there is Wnt protein on the surface of the cell membrane, it will inhibit the GSK3 complex, so that the β-Catenin protein will accumulate in the nucleus, and finally start the transcription of Wnt pathway-related genes. There is a delicate balance between the homeostasis of bone and joints and the Wnt pathway, and the disruption of this balance may lead to the occurrence of osteoarthritis.

The protein kinase family CLK (CDC-like kinase) is a dual-specificity protein kinase that regulates intracellular signal transduction through the phosphorylation of substrate proteins on tyrosine, serine or threonine residues; it can be divided into four Subtypes (CLK1, CLK2, CLK3 and CLK4), the protein C segments encoded by these four subtypes all have a highly conserved gene sequence, and have the same amino acid sequence with a similar structure. CLKs play an important role in alternative splicing. CLKs serve as body temperature sensors that globally control alternative splicing and gene expression. The activity of CLKs is indeed highly sensitive to changes in physiological temperature, conferred by structural rearrangements within the kinase activation segment. Inhibition of CLK2 kinase is considered 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 Production of HIV-1.

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

At present, the First-in-class small molecule drug SM-04690 developed by Sumumed has been applied in phase III clinical trials to treat osteoarthritis. The specific mechanism is mainly to induce early cartilage formation by inhibiting CLK2-mediated SR protein phosphorylation ; and inhibit DYRK1A-mediated SIRT1 and FOXOA phosphorylation to enhance the function of mature chondrocytes; and can reduce inflammation by inhibiting NF-γB, STAT3 and other inflammatory factors. Although SM04690 has significant CLK2 inhibitory activity, it has insufficient selectivity for the CLK family, which may cause it to have certain side effects; in addition, its inhibitory activity against the DYRK1A target is insufficient and its water solubility is poor, so its druggability needs to be improved.

Contents of the invention

The object of the present invention is to provide a 3-ethynyl-5-(1H -1,2,3-triazol-4-yl)-1H-indazole compound, pharmaceutical composition and application.

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 compounds represented by the formula I of the present invention, its iso Constructs, pharmaceutically acceptable salts or mixtures thereof,

Wherein, R1 is selected from the following groups:

R2 is selected from the following groups:

The present invention synthesizes a series of derivatives through rational drug design, and the biological activity evaluation shows that the designed compounds have significant CLK2 inhibitory activity, have better selectivity to CLK family members, and have significant DYRK1A inhibitory activity at the same time.

In the structure of the above-mentioned compound preferably:

R ₁ is selected from H,

R2 is selected from the following groups:

More preferably, the above-mentioned compound is selected from any of the following compounds:

The pharmaceutically acceptable salt of the above-mentioned compound is the salt formed by the above-mentioned compound 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 involved in the present invention, the above-mentioned compounds and pharmaceutically acceptable carriers form a pharmaceutical composition, which is made into common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions or injections. The preparations can be added with spices, sweeteners, Flavoring agents, liquid/solid fillers, diluents and other common pharmaceutical excipients.

As the third aspect involved in 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, has the effect of cartilage protection.

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

(1) The compound and its pharmaceutical composition can effectively inhibit the activity of CLK2 protein and DYRK1A protein, and can also significantly down-regulate the expression level of proteases related to cartilage decomposition in inflammatory model animals, and play a cartilage protective effect;

(2) This type of compound and its pharmaceutical composition are widely used, and can be prepared as a drug for treating osteoarthritis, and can exert drug effects at the molecular level and animal level, and the therapeutic effect is better, and the optimal concentration can reach the nanomolar concentration level;

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

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the embodiments.

Example 1: Synthesis of LBL-001

Synthesis of Intermediate II:

Add 5g of 5-bromoindazole (25.38mmol, 1eq) in a 100ml single-necked bottle, dissolve it with 10mL of DMF, and then add 12.88g of iodine (50.75mmol, 2eq) and 4.27g of potassium hydroxide (76.14mmol, 3eq), room temperature After stirring for 2 hours TLC monitoring showed the reaction was complete. The reaction solution was quenched by adding saturated sodium thiosulfate solution, extracted with ethyl acetate, and the organic layer was retained and dried with anhydrous sodium sulfate. After spin-drying, 7.6 g of yellow-white powder II was obtained, with a yield of 93%. ¹ H NMR (300MHz, DMSO-d ₆ ) δ 7.94 (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) into a 100ml three-necked flask, dissolve it with 50mL of tetrahydrofuran, add dropwise 2.6g of 3,4-dihydro-2H-pyran (31.0mmol, 2eq), dropwise After the completion, 0.59 g of p-toluenesulfonic acid hydrate (3.1 mmol, 0.2 eq) was added in batches, and after all the addition was completed, the reaction was heated under reflux at 75°C. TLC monitoring after 5 hours showed the reaction was complete. The reaction solution was spin-dried and then column chromatographed to obtain 5.4 g of white powder III 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:

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) into a 50mL single-necked flask ), 70mg of cuprous iodide (0.37mmol, 0.1eq) and 15mL of triethylamine were completely dissolved with 7.5mL of DMF. After the addition was completed, nitrogen was replaced, and the reaction was stirred at room temperature. After 5 h of reaction, TLC monitoring showed that the reaction was complete. The reaction solution was filtered with suction, the filtrate was retained, spin-dried and then column chromatographed to obtain 1.3 g 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:

Add 1.1g intermediate V (2.76mmol, 1eq), 0.54g trimethylsilyne (5.51mmol, 2eq), 50mg tetrakistriphenylphosphine palladium (0.14mmol, 0.05eq), 10mg iodine into a 50mL single-necked bottle Cuprous chloride (0.06mmol, 0.02eq) and 10mL of triethylamine were completely dissolved with 10mL of tetrahydrofuran. After the addition was complete, nitrogen replacement was carried out, and the mixture was heated to reflux at 70°C for reaction. After 10 h of reaction, TLC monitoring showed that the reaction was complete. The reaction solution was filtered with suction, the filtrate was retained, and the filtrate was spin-dried, followed by column chromatography to obtain 0.6 g of 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:

Add 0.4g of intermediate VI to a 25ml single-necked bottle, dissolve it completely with 10mL of methanol, add 0.4g of potassium carbonate, and stir at room temperature. After 30 minutes of reaction, TLC monitoring showed that the reaction was complete. Extract with dichloromethane and water, keep the organic layer and dry with anhydrous sodium sulfate, spin dry to obtain 0.33g of white solid VII, yield 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:

Add 0.26g of intermediate VII (0.75mmol, 1eq) and 7mg of cuprous iodide (0.04mmol, 0.05eq) into a 25ml single-necked bottle, dissolve it completely with 4mL of DMF and 0.5mL of methanol, and then add 130mg of trimethyl azide Base silane (1.13mmol, 1.5eq) was replaced by nitrogen and heated at 100°C for reaction. After 24 hours of reaction, TLC monitoring showed that a small amount of raw materials remained. After spin-drying, column chromatography gave 0.12 g 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),7.72(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 )ppm. HR-MS (ESI): Calculated for C ₂₁ H ₁₈ N ₆ O [M+H] ⁺ : 370.1542, found 370.1544.

Synthesis of LBL-001:

Add the product obtained in the previous step into a 25ml single-necked bottle, dissolve it completely with 8mL of dichloromethane, then add 100μL of triethylsilane, the system becomes white and turbid, and the system becomes clear after adding 10mL of trifluoroacetic acid, and stir at room temperature reaction. After 1 h of reaction, TLC monitoring showed that the reaction was complete. After the reaction solution was spin-dried, it was extracted with ethyl acetate and sodium bicarbonate solution, and the organic layer was retained and dried with anhydrous sodium sulfate. The organic phase was spin-dried and then column chromatographed to obtain 75 mg 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.

Using similar operations as in Example 1, the following compounds were obtained:

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, found 330.0854.

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=1.9Hz, 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 for C 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 found 300.1114.

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] ⁺ : 277.1327found 277.1325.

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] ⁺ : 443.1358found 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.

Embodiment 2: Synthesis of LBL-021

Synthesis of Intermediate IX-II:

Add 0.2g intermediate VII (0.58mmol, 1eq), 83mg ethyl azidoacetate (1.1mmol, 1.1eq), 15mg copper sulfate pentahydrate (0.06mmol, 0.1eq) and 23mg sodium ascorbate ( 0.12mmol, 0.2eq), and finally add 5mL DMF and stir at room temperature. After 2 h of reaction, TLC monitoring showed that the reaction was complete. Extract with ethyl acetate and water, retain the organic layer and dry it with anhydrous sodium sulfate, spin dry the organic phase and perform column chromatography to obtain 0.12 g 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, 1.6Hz, 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] ⁺ : 372.1335found 372.1330.

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] ⁺ : 416.1233found 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] ⁺ : 389.1288found 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] ⁺ : 387.1331found 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] ⁺ : 377.0946found 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, 0.6 Hz, 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 for C 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=1.4Hz, 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] ⁺ : 403.1103found 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] ⁺ : 431.1416found 431.1414.

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

1. Experimental method

1. Prepare 1X Kinase Reaction Buffer

2. Enzyme activity experiment

(1) 2X kinase preparation:

(2) 4X substrate mixture preparation:

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

(2) The compound was diluted 50 times into 1X kinase reaction buffer, and shaken on a shaker for 20 minutes.

(3) Prepare 2X kinase with 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 mixture with 1X enzyme reaction buffer, add 1 μL of 4X ATP & sub mixture to the reaction plate.

(7) Seal the plate with a 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 at 1000 rpm, centrifuge for 1 min, and incubate at 25° C. for 40 min.

(9) Transfer 8 μL of Detection solution to the 384 reaction plate at 1000 rpm, centrifuge for 1 min, and incubate at 25°C for 40 min.

(10) Use a BMG multifunctional plate reader to read the RLU (Relative luminescence unit) signal, and the signal intensity is used to characterize the activity of the kinase.

2. Experimental results

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

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

It can be seen from Table 1 that the compounds of the present invention exhibit effective inhibitory activity on 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 (the _IC50 for CLK2, CLK3, and DYRK1A are 4nM, 120nM, and 3nM, respectively, and the selectivity index for CLK3 is 30), This provides a basis for LBL-001 to exert its pharmacological activity and avoid possible side effects.

Claims (10)

1. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in a kind of formula I, its isomer, pharmaceutically acceptable Salt or their mixture, characterized in that,

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 compounds shown in formula I according to claim 1, isomers thereof, Pharmaceutically acceptable salts or their mixtures are characterized in that: 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 compounds shown in formula I according to claim 1, isomers thereof, The pharmaceutically acceptable salt or their mixture is characterized in that it is selected from any of the following compounds:

4. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds shown in formula I according to claim 1, isomers thereof, A pharmaceutically acceptable salt or a mixture thereof, wherein the pharmaceutically acceptable salt is a salt formed between the compound and the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, hydrobromic acid, hydroiodide 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 3-ethynyl-5-(1H-1,2,3-triazole-4 shown in formula I described in any one of claims 1-4 -yl)-1H-indazole compounds, their isomers, 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 described in any one of claims 1-4, its iso Construct, pharmaceutically acceptable salt or the application of the pharmaceutical composition described in claim 5 in the preparation of CLK2 protein inhibitor drug. 7. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compound shown in formula I described in any one of claims 1-4, its iso Construct, pharmaceutically acceptable salt or the application of the pharmaceutical composition described in claim 5 in the preparation of DYRK1A protein inhibitor drug. 8. 3-ethynyl-5-(1H-1,2,3-triazol-4-yl)-1H-indazole compounds shown in formula I described in any one of claims 1-4, its iso Construct, pharmaceutically acceptable salt or the application of the pharmaceutical composition described in claim 5 in the preparation of the medicine for treating inflammation. 9. The application according to claim 8, characterized in that the inflammation is osteoarthritis, tendinopathy or rheumatoid arthritis. 10. The use according to claim 9, characterized in that the compound has cartilage protection effect.