Kinase inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a kinase inhibitor and a preparation method and application thereof, a compound with mixed lineage kinase 3 (MLK3) inhibitory activity is a compound with a structure as shown in a formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, most of the compounds effectively inhibit the activity of MLK3 kinase, and the compound can be used for preparing a medicine for inhibiting MLK3 kinase. Therefore, the compound or the stereoisomer thereof or the pharmaceutically acceptable salt thereof and the related pharmaceutical composition have potential application value in treatment of MLK3 related diseases. (I)

Description

Technical Field

[0001] This invention relates to the field of small molecule drugs, specifically to a compound with mixed-lineage kinase 3 inhibitory activity, its preparation method, and its application. Background Technology

[0002] Mixed lineage kinase 3 (MLK3) is a 97 kDa serine / threonine protein kinase, a core member of the MAP3K family. Its molecular structure includes an SH3 domain, a kinase domain, a double leucine zipper, and a CDC42 / Rac interaction binding motif (CRIB). By regulating MAPK signaling pathways such as JNK, p38, and ERK, it is deeply involved in key physiological processes such as cell proliferation, migration, invasion, and apoptosis. Aberrant activation or mutation of MLK3 is closely related to the development of various diseases, especially exhibiting a clear oncogenic driving role in the field of oncology, making it an important target for small molecule drug development.

[0003] In tumor pathology, the pro-cancer function of MLK3 has been confirmed by multiple studies: In glioblastoma, EGFR signaling drives tumor cell invasion through the DOCK180-Rac1-MLK3-JNK axis, leading to a persistently high recurrence rate after surgical resection and a patient survival of only 12-15 months; in breast cancer, MLK3 regulates focal adhesion turnover through phosphopelin, promoting CXCL12-mediated tumor metastasis, and its high expression is significantly associated with the risk of lung metastasis; in gastrointestinal tumors, 21% of microsatellite instability (MSI) phenotype tumors have somatic mutations in MLK3, these mutations are concentrated in the kinase domain, possess in vitro transformation ability and in vivo tumorigenic activity, and are associated with KRAS / BRAF wild-type tumors. Furthermore, MLK3 also promotes tumor progression in diseases such as ovarian cancer and acoustic neuroma by aberrantly activating the MAPK pathway, and its inhibitors hold promise as a universal strategy for the treatment of multiple cancers.

[0004] Although the clinical value of MLK3 as a target is clear, significant technical bottlenecks remain in the development of existing inhibitors. Currently reported MLK3 inhibitors are represented by URMC-099, whose in vitro IC50... 50 It has a value of 14 nM and can penetrate the blood-brain barrier, but its kinase selectivity is extremely poor, particularly for FLT3 (IC50). 50 =4nM), ABL1 (IC 50 =3nM), LRRK2 (IC 50Multiple kinases, including MLK3 (11 nM), exhibit strong inhibitory activity, leading to significant off-target effects during in vivo application and easily causing side effects such as bone marrow suppression and cardiovascular toxicity, thus limiting their clinical translation. Early patented technologies, such as the multi-lineage kinase inhibitor disclosed in CN1458979A, primarily target neurodegenerative diseases, failing to focus on MLK3's application in cancer treatment and lacking selectivity for MLK family members. Furthermore, while natural derivatives such as licorice-derived DHGA-D can directly bind to MLK3 and inhibit its activity, they are mainly used in anti-inflammatory fields, exhibiting weak anti-tumor activity and low bioavailability.

[0005] From a clinical perspective, existing treatments for MLK3-related tumors have significant limitations: glioblastomas easily develop resistance to radiotherapy and anti-angiogenic therapies, and tumor invasion often makes complete resection impossible; the 5-year survival rate for metastatic breast cancer patients is less than 30%, and there is a lack of targeted drugs targeting the metastatic mechanism; MSI-type gastrointestinal tumors have low sensitivity to chemotherapy, necessitating novel therapeutic drugs with specific targets. Therefore, developing MLK3 small molecule inhibitors with high selectivity, high activity, and favorable pharmacokinetic properties can effectively overcome the technical shortcomings of existing drugs, such as poor selectivity and high toxicity, providing new treatment options for related diseases and possessing significant clinical value and market potential. Summary of the Invention

[0006] This invention provides a kinase inhibitor, its preparation method, and its application. The kinase inhibitor is a compound with mixed lineage kinase 3 (MLK3) inhibitory activity. This type of compound can inhibit MLK3 activity and can be used to treat a variety of diseases, including cancer.

[0007] The technical solution of the present invention is as follows: A compound with the structure shown in formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: (I) in: R1 is selected from the following structures: R2 is selected from the following structures: More specifically, the compound is selected from the following structures: .

[0008] The present invention also provides a method for preparing a compound with the structure shown in formula (I), comprising the following steps: The method for preparing the compound with mixed-lineage kinase 3 inhibitory activity is characterized by comprising the following steps: (1) Under transition metal catalysis, compound (II) is reacted with compound (III) to generate compound (IV); Equation (II); Equation (III); Equation (IV); (2) Under transition metal catalysis, the compound of formula (Ⅳ) is reacted with the compound of formula (Ⅴ) to generate the compound with the structure shown in formula (I). Formula (V); Among them, R1 in equations (III), (IV) and (I) is the same, and R2 in equations (V) and (I) is the same.

[0009] The MLK3 small molecule inhibitor described in this invention can be used alone, or it can be prepared as a pharmaceutically acceptable salt or used in combination with a pharmaceutically acceptable excipient or carrier.

[0010] This invention also provides a compound with the structure shown in formula (I) and its pharmaceutically acceptable salt for use as an MLK3 inhibitor. Specifically, this application is for preparing drugs to prevent or treat diseases caused by MLK3 overexpression, including breast cancer, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ovarian cancer, cervical cancer, prostate cancer, glioblastoma, etc. Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses novel MLK3 inhibitor structures, most of which effectively inhibit the activity of MLK3 kinase, indicating that such compounds, their stereoisomers, or pharmaceutically acceptable salts, as well as related drug combinations, have potential application value in the treatment of MLK3-related diseases. Attached Figure Description

[0011] Figure 1 For compound 2-1 13 C10 NMR structural characterization diagram; Figure 2 For compound 2-1 1 H NMR structural characterization diagram; Figure 3 For compound 3-1 13 C10 NMR structural characterization diagram; Figure 4 For compound 3-1 1 H NMR structural characterization diagram; Figure 5 For compound 4-1 13 C10 NMR structural characterization diagram; Figure 6 For compound 4-1 1 H NMR structural characterization diagram; Figure 7 For compound 1 13 C10 NMR structural characterization diagram; Figure 8 For compound 1 1 H NMR structural characterization diagram; Figure 9 For compound 2 13 C10 NMR structural characterization diagram; Figure 10 For compound 2 1 H NMR structural characterization diagram; Figure 11 For compound 3 13 C10 NMR structural characterization diagram; Figure 12 For compound 3 1 H NMR structural characterization diagram; Figure 13 For compound 4 13 C10 NMR structural characterization diagram; Figure 14 For compound 4 1 H NMR structural characterization diagram; Figure 15 For compound 5 13 C10 NMR structural characterization diagram; Figure 16 For compound 5 1 H NMR structural characterization diagram; Figure 17 For compound 6 13 C10 NMR structural characterization diagram; Figure 18 For compound 6 1 H NMR structural characterization diagram; Figure 19 For compound 7 13 C10 NMR structural characterization diagram; Figure 20 For compound 7 1 H NMR structural characterization diagram; Figure 21 For compound 8 13 C10 NMR structural characterization diagram; Figure 22 For compound 8 1 H NMR structural characterization diagram; Figure 23 For compound 9 13 C10 NMR structural characterization diagram; Figure 24 For compound 9 1 H NMR structure characterization diagram. Detailed Implementation

[0012] Example 1: 3-(3-fluoro-4-methoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 1) Step 1: 5-Bromo-3-(3-fluoro-4-methoxyphenyl)-1H-pyrrole[2,3-b]pyridine (compound 1-1) 5-Bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid tert-butyl ester (500 mg, 1.0 eq) and 3-fluoro-4-methoxyphenylboronic acid pinacol ester (298 mg, 1.0 eq) were dissolved in a mixture of 1,4-dioxane (50 mL) and water (10 mL), followed by the addition of K₂CO₃ (489 mg, 3.0 eq) and Pd(dppf)Cl₂ (43.2 mg, 0.05 eq). The reaction mixture was protected under nitrogen and stirred at 110 °C for 10 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure, followed by purification by silica gel column chromatography to give compound 1-1 (95 mg, 25%).

[0013] Step 2: 3-(3-fluoro-4-methoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 1) Compound 1-1 (44 mg, 1.0 eq) and pinacol 4-(4-methyl-1-piperazinmethyl)phenylboronic acid (43.3 mg, 1.0 eq) were dissolved in a mixture of 1,4-dioxane (10 mL) and water (5 mL), followed by the addition of K₂CO₃ (56.7 mg, 3.0 eq) and Pd(dppf)Cl₂ (5 mg, 0.05 eq). The reaction mixture was protected with nitrogen and stirred at 110 °C for 10 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure, followed by purification by silica gel column chromatography to give compound 6 (18 mg, 30.5%). LC-MS (M+H) + =431.93, 13C NMR (101 MHz, DMSO-D6) δ 149.01, 145.82,142.48, 138.25, 130.07, 129.18, 128.77, 128.70, 127.51, 125.75, 125.05,123.04, 123.01, 117.69, 114.91, 114.51, 114.33, 113.97, 62.11, 56.58, 54.94,52.62, 45.79. 1 H NMR (400 MHz, DMSO- D 6) δ 11.97 (d, J = 2.7 Hz, 1H), 8.51 (d, J =2.1 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 2.6 Hz, 1H), 7.68 (d, J =8.2 Hz, 2H), 7.60 – 7.51 (m, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.19 (t, J = 8.9Hz, 1H), 3.83 (s, 3H), 3.47 (t, 2H), 2.46 – 2.28 (m, 8H), 2.18 (s, 3H). Example 2: (4-(3-(1-methyl-1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)(4-methylpiperazin-1-yl)methyl ketone (Compound 2) Step 1: 5-Bromo-3-(1-Methyl-1H-indol-5-yl)-1H-pyrrole[2,3-b]pyridine (Compound 2-1) The procedure is the same as step 1 in Example 1, where tert-butyl 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (500 mg, 1.0 eq) and pinacol 1-methylindole-5-boronic acid (303.95 mg, 1.0 eq) are reacted to generate compound 2-1 (100 mg, 25.9%). 13 C NMR (101 MHz, DMSO- D6) δ 147.90, 143.17, 135.84, 130.56,129.76, 129.19, 125.66, 125.08, 121.21, 119.97, 118.60, 116.19, 111.54,110.70, 101.07, 33.07. 1 H NMR (400 MHz, DMSO- D 6) δ 12.10 – 11.88 (m, 1H), 8.38(d, J = 2.2 Hz, 1H), 8.29 (d, J = 2.2 Hz, 1H), 7.81 (d, J = 2.3 Hz, 2H), 7.45(d, J = 1.7 Hz, 2H), 7.30 (d, J = 3.0 Hz, 1H), 6.44 (d, J = 3.0 Hz, 1H), 3.77 (s, 3H). The procedure was the same as step 2 in Example 1, reacting compound 2-1 (100 mg, 1.0 eq) with pinacol 4-(4-methylpiperazine-1-carbonyl)phenylboronic acid (101 mg, 1.0 eq) to generate compound 2 (26 mg, 18.8%). LC-MS (M+H) + =450.90, 1 H NMR (400 MHz, DMSO-) D 6) δ 11.90 (d, J = 2.6 Hz, 1H), 8.55 (d, J = 2.1 Hz, 1H), 8.43 (d, J = 2.1 Hz, 1H), 7.88 (d, J = 1.5 Hz, 1H), 7.81 – 7.77 (m, 3H), 7.53(dd, J = 8.5, 1.7 Hz, 1H), 7.49 – 7.45 (m, 3H), 7.30 (d, J = 3.0 Hz, 1H), 6.44 (d, J = 3.0 Hz, 1H), 3.78 (s, 3H), 3.68 – 3.38 (m, 4H), 2.38 – 2.24 (m,4H), 2.18 (s, 3H).13 C NMR (101 MHz, DMSO- D 6) δ 169.39, 149.31, 142.28, 140.91,135.80, 134.75, 130.49, 129.20, 128.30, 128.22, 127.55, 126.27, 126.11,124.19, 121.41, 118.68, 118.29, 116.87, 110.68, 101.04, 46.05, 33.07. Example 3: (4-(3-(benzo[b]thiophene-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)(4-methylpiperazin-1-yl)methyl ketone (compound 3) Step 1: 3-(benzo[b]thiophen-5-yl)-5-bromo-1H-pyrrole[2,3-b]pyridine (compound 3-1) The procedure was the same as step 1 in Example 1, where 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid tert-butyl ester (500 mg, 1.0 eq) and 2-(1-benzothiophene-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane (307.5 mg, 1.0 eq) were reacted to generate compound 3-1 (114 mg, 29.3%). 13 C NMR (101 MHz, DMSO- D 6) δ 147.98,143.51, 140.85, 137.37, 131.08, 129.96, 128.31, 126.27, 124.81, 123.97,123.54, 121.47, 119.64, 114.72, 111.95. 1 H NMR (400 MHz, DMSO- D 6) δ 8.51 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 2.2 Hz, 1H), 8.21 (d, J = 1.7 Hz, 1H), 8.06 –7.96 (m, 2H), 7.74 (d, J = 5.4 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.50 (dd,J = 5.4, 0.8 Hz, 1H). The procedure was the same as step 2 in Example 1, reacting compound 3-1 (120 mg, 1.0 eq) with pinacol 4-(4-methylpiperazine-1-carbonyl)phenylboronic acid (120.4 mg, 1.0 eq) to generate compound 3 (122 mg, 74%). LC-MS (M+H) + =453.86, 1 H NMR (400 MHz, DMSO-) D 6) δ 12.08 (s, 1H), 8.56 (dd, J = 22.7, 2.1 Hz, 2H), 8.28 (d, J = 1.7 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.81 – 7.69 (m, 2H), 7.54 – 7.42 (m, 3H), 3.74 – 3.37 (m,4H), 2.43 – 2.22 (m, 4H), 2.17 (s, 3H). 13 C NMR (101 MHz, DMSO- D 6) δ 169.38,149.37, 142.59, 140.87, 140.78, 137.21, 134.85, 131.69, 128.57, 128.27,128.23, 127.67, 126.24, 125.40, 124.82, 124.18, 123.52, 121.59, 117.99,115.41, 46.06. Example 4: 3-(1-methyl-1H-indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrole[2,3-b]pyridine (compound 4) The procedure was the same as step 2 in Example 1, reacting compound 2-1 (230 mg, 1.0 eq) with pinacol 4-(4-methyl-1-piperazinmethyl)phenylboronic acid (242 mg, 1.0 eq) to generate compound 4 (200 mg, 65%). LC-MS (M+H) + =435.90, 13C NMR (101 MHz, DMSO-) D 6) δ 149.09, 142.23, 138.38, 137.51, 135.77, 130.47, 130.08,129.21, 128.94, 127.41, 126.36, 125.80, 123.98, 121.40, 118.64, 118.28,116.74, 110.67, 101.05, 62.29, 55.26, 53.09, 46.27, 33.06. 1 H NMR (400 MHz, DMSO- D 6) δ 11.82 (d, J = 2.6 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.37 (d, J =2.2 Hz, 1H), 7.87 (s, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.51 (dd, J = 8.5, 1.6 Hz, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.35 (d, J =8.1 Hz, 2H), 7.29 (d, J = 3.0 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 3.77 (s,3H), 3.44 (s, 2H), 2.41 – 2.14 (m, 8H), 2.10 (s, 3H). Example 5: 3-(benzo[b]thiophen-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrole[2,3-b]pyridine (compound 5) The procedure was the same as step 2 in Example 1, reacting compound 3-1 (192 mg, 1.0 eq) with pinacol 4-(4-methyl-1-piperazinmethyl)phenylboronic acid (184.4 mg, 1.0 eq) to generate compound 5 (140 mg, 55%). LC-MS (M+H) + =439.87, 1H NMR (400 MHz, DMSO) δ 12.03 (d, J = 2.7 Hz, 1H), 8.59 (d, J = 2.1 Hz, 1H), 8.52(d, J = 2.2 Hz, 1H), 8.31 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 2.6Hz, 1H), 7.83 – 7.70 (m, 4H), 7.55 (d, J = 5.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 3.50 (s, 2H), 2.49 – 2.25 (m, 8H), 2.18 (s, 3H). 13 C NMR (101 MHz, DMSO) δ149.12, 142.47, 140.80, 138.24, 137.47, 137.11, 131.73, 129.96, 129.22,128.10, 127.46, 125.83, 125.11, 124.75, 124.11, 123.42, 121.49, 117.92,115.24, 62.17, 55.13, 52.89, 46.06. Example 6: 4-(4-(3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-fluorobenzyl)morpholine (compound 6) Step 1: 5-Bromo-3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridine (compound 4-1) The procedure was the same as step 1 in Example 1, where tert-butyl 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid was reacted with 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid (500 mg, 1.0 eq) and 5-indoleboronic acid pinacol ester (287.2 mg, 1.0 eq) to generate compound 4-1 (86.5 mg, 23.5%). 13 C NMR (101 MHz, DMSO- D6) δ 147.89, 143.13, 135.31, 129.77, 128.81,126.27, 125.52, 124.95, 121.20, 120.01, 118.36, 116.40, 112.43, 111.51,101.84. 1 H NMR (400 MHz, DMSO- D 6) δ 11.98 (d, J = 2.6 Hz, 1H), 11.06 (s, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 2.2 Hz, 1H), 7.81 (d, J = 0.9 Hz, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.38 (dd, J = 8.4, 1.7Hz, 1H), 7.32 (t, J = 2.7 Hz, 1H), 6.45 (d, J = 1.0 Hz, 1H). The procedure was the same as step 2 in Example 1, reacting compound 4-1 (74 mg, 1.0 eq) with pinacol ester of 3-fluoro-4-(4-morpholinomethyl)phenylboronic acid (76.2 mg, 1.0 eq) to generate compound 6 (81 mg, 80%). LC-MS (M+H) + =427.84, 1 H NMR (400 MHz, DMSO-) D 6) δ 11.88 (s, 1H), 11.11 (s, 1H), 8.54 (d, J = 2.1 Hz, 1H), 8.43 (d, J = 2.1 Hz, 1H), 7.89 (s, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.63 – 7.52(m, 2H), 7.46 (t, J = 1.4 Hz, 3H), 7.32 (t, J = 2.7 Hz, 1H), 6.45 (t, J= 2.5Hz, 1H), 3.53 (dd, J = 9.7, 5.0 Hz, 6H), 2.40 – 2.30 (m, 4H). 13 C NMR (101 MHz, DMSO- D 6) δ 163.05, 160.62, 149.34, 142.22, 141.06, 135.27, 132.77, 128.83,127.59, 126.16, 126.12, 124.05, 123.26, 121.40, 118.44, 118.25, 117.11,114.27, 114.04, 112.40, 101.81, 66.70, 55.31, 53.49. Example 7: 3-(3-fluorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7) Step 1: 5-Bromo-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (compound 5-1) The procedure is the same as step 1 in Example 1, where tert-butyl 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid (500 mg, 1.0 eq) and pinacol 3-fluorophenylboronic acid (165.4 mg, 1.0 eq) are reacted to generate compound 5-1 (80 mg, 23.3%).

[0014] The procedure was the same as step 2 in Example 1, reacting compound 5-1 (80 mg, 1.0 eq) with pinacol 4-(4-methyl-1-piperazinmethyl)phenylboronic acid (87.3 mg, 1.0 eq) to generate compound 7 (28 mg, 25%). LC-MS (M+H) + =401.93, 1 H NMR (400 MHz, DMSO-) D 6) δ 12.13 (d, J = 2.8 Hz, 1H), 8.53 (d, J = 2.1 Hz, 1H), 8.41 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 2.5 Hz, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.63 (d,J = 7.8 Hz, 1H), 7.57 (d, J = 10.6 Hz, 1H), 7.47 – 7.41 (m, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.03 (td, J = 8.6, 2.6 Hz, 1H), 3.49 – 3.33 (m,4H), 2.40 – 2.24 (m, 6H), 2.13 (s, 3H). 13 C NMR (101 MHz, DMSO- D 6) δ 162.12,149.12, 142.65, 138.15, 137.56, 131.36, 131.27, 130.02, 129.46, 127.55,126.13, 125.88, 122.89, 117.64, 113.34, 113.12, 112.91, 112.70, 62.21, 55.12,52.88, 46.06. Example 8: 4-(2-fluoro-4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)morpholine (compound 8) The procedure was the same as step 2 in Example 1, reacting compound 5-1 (80 mg, 1.0 eq) with pinacol ester of 3-fluoro-4-(4-morpholinomethyl)phenylboronic acid (88.6 mg, 1.0 eq) to generate compound 8 (52 mg, 46.8%). LC-MS (M+H) + =406.91, 1 HNMR (400 MHz, DMSO- D 6) δ 12.16 (d, J = 2.7 Hz, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.01 (d, J = 2.6 Hz, 1H), 7.67 – 7.56 (m, 4H), 7.48 – 7.41 (m, 2H), 7.20 – 6.77 (m, 1H), 3.57 – 3.51 (m, 6H), 2.42 – 2.33 (m, 4H). 13C NMR (101 MHz, DMSO- D 6) δ 162.12, 160.61, 149.34, 142.67, 132.74,131.35, 131.27, 126.32, 126.23, 123.37, 122.97, 117.58, 114.44, 114.21,114.14, 113.40, 113.18, 112.97, 112.76, 66.70, 55.30, 53.49. Example 9: 3-(1H-indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 9) The procedure was the same as step 2 in Example 1, reacting compound 4-1 (100 mg, 1.0 eq) with 4-(4-methylpiperazine-1-ylsulfonyl)phenylboronic acid pinacol ester (129.1 mg, 1.1 eq) to generate compound 9 (20 mg, 13%). LC-MS (M+H) + =472.18, 13 C NMR (101 MHz, DMSO- D 6) δ 149.55, 144.53, 142.37, 135.30, 133.30,128.80, 128.20, 127.20, 126.55, 126.08, 125.95, 124.15, 121.37, 118.48,118.38, 117.31, 112.40, 101.76, 54.05, 46.27, 45.82. 1 H NMR (400 MHz, DMSO- D 6)δ 11.89 (s, 1H), 11.07 (s, 1H), 8.58 (d, J = 2.1 Hz, 1H), 8.47 (d, J = 2.1Hz, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.86 (s, 1H), 7.78 – 7.71 (m, 3H), 7.44(s, 2H), 7.28 (t, J = 2.7 Hz, 1H), 6.43 (t, J= 2.5 Hz, 1H), 2.93 – 2.84 (m,4H), 2.37 – 2.31 (m, 4H), 2.10 (s, 3H). In vitro kinase inhibition assay (refer to CN 113968860 A) 1. Compound preparation Weigh 1 mg of the above compound and dissolve it in 100% DMSO-d6 to prepare a 10 mM stock solution, which is then stored in a nitrogen cabinet away from light.

[0015] 2. Kinase reaction process (1) Prepare 1×Kinase buffer. (2) Preparation of compound concentration gradients: The initial test concentration of the test compound was 2400 nM, with 7 concentrations and replicate detection. The compound was diluted 100 times to a final concentration of 100% DMSO-d in a 384 source plate. 250 nL of the 100-fold final concentration compound was transferred to the target 3575 plate using an Echo 550 dispenser.

[0016] (3) Prepare a kinase solution with a final concentration of 2.5 times using 1×Kinase buffer.

[0017] (4) Add 10 μL of kinase solution at a final concentration of 2.5 times to the compound wells and the positive control wells, respectively; add 10 μL of kinase solution at a final concentration of 2.5 times to the negative control wells. Add 10 μl of 1×Kinase buffer to the well. (5) Centrifuge at 1000 rpm for 30 seconds, shake the reaction plate to mix, and incubate at room temperature for 10 minutes.

[0018] (6) Prepare a mixed solution of ATP and Kinase substrate 2 at a final concentration of 5 / 3 times using 1×Kinase buffer.

[0019] (7) Add 15 μl of a mixture of ATP and substrate at 5 / 3 times the final concentration to initiate the reaction.

[0020] (8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 30 minutes.

[0021] (9) Add 30 μL of the stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix.

[0022] (10) Read the conversion rate using Caliper EZ Reader.

[0023] The results are shown in Table 1. Table 1. MLK3 inhibition results of compounds 1-9 Note: A≤100nM, 100

Claims

1. A kinase inhibitor, characterized in that, Compounds with the structure shown in formula (I), their stereoisomers, or pharmaceutically acceptable salts thereof: ； （I） in: R1 is selected from the following structures: ； R2 is selected from the following structures: 。 2. The kinase inhibitor according to claim 1, characterized in that, The compound with the structure shown in formula (I) has the following structure: ； ； 。 3. The method for preparing the kinase inhibitor according to claim 1 or 2, characterized in that, Includes the following steps: (1) Under transition metal catalysis, compound (II) is reacted with compound (III) to generate compound (IV); Equation (II); Equation (III); Equation (IV); (2) Under transition metal catalysis, the compound of formula (Ⅳ) is reacted with the compound of formula (Ⅴ) to generate the compound with the structure shown in formula (I); Formula (V); Among them, R1 in equations (III), (IV) and (I) is the same, and R2 in equations (V) and (I) is the same.

4. The use of the kinase inhibitor according to claim 1 or 2 in the preparation of a medicament for the prevention or treatment of diseases caused by MLK3 overexpression.

5. The application according to claim 4, characterized in that, The diseases caused by MLK3 overexpression include breast cancer, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, ovarian cancer, cervical cancer, prostate cancer, or glioblastoma.

6. The application according to claim 4, characterized in that, In the aforementioned medicament, the compound having mixed-lineage kinase 3 inhibitory activity may be used alone, or prepared as a pharmaceutically acceptable salt or in combination with a pharmaceutically acceptable excipient or carrier.

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