Protein degradation agent of PAK1

Abstract

The invention discloses a PAK1 protein degradation agent compound as shown in a formula (I), application of the PAK1 protein degradation agent compound and a pharmaceutical composition containing the PAK1 protein degradation agent compound. The compounds are used for treating tumors. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a protein degradation agent for PAK1, application thereof and a pharmaceutical composition containing the same. Background Art

[0002] PAK1 is a member of the Pak (p21-activated kinase) serine / cysteine ​​protein kinase family and an interactor of RhoGTPases RAC1 and CDC42. Its catalytic activity and scaffolding function play multiple roles in cell signaling. The signal transduction cascades regulated by PAK1 include MAPK, AKT, Wnt1 / β-catenin, ERα, BAD, and NF-κB pathways, which are involved in regulating cell proliferation and survival. PAK1 is also involved in the regulation of cell motility, transmitting various signals that control cytoskeletal dynamics, cell shape, and adhesion.

[0003] PAK1 has shown enhanced function in a variety of human malignancies, which means that PAK1 may play an important role in the development and progression of tumors. Amplification of the PAK1 gene and increased levels of PAK1 protein are often associated with aggressive tumor phenotypes, chemotherapy resistance, and poor prognosis. In addition to gene amplification and protein overexpression, PAK1 may also be overactivated due to mutations in upstream regulatory factors such as RAC1, RAS, and Merlin, thereby transmitting oncogenic signals and causing cells to transform into cancer cell phenotypes. Therefore, targeted interference with PAK1 may be a promising tumor treatment method. In a study of a mouse model of neurofibromatosis type 2, it was found that PAK1 gene deletion could effectively slow down hearing loss and schwannoma growth in mice without obvious systemic toxicity.

[0004] The signal transduction activity of PAK1 is mediated by its enzymatic activity and scaffold function, so PAK1 degraders theoretically have stronger pharmacodynamic potential than PAK1 inhibitors. PROTAC (Proteolytic Targeting Chimera) is a bifunctional compound whose molecular structure includes a target protein binding ligand and an E3 ubiquitin ligase ligand, and the two ligands are connected by a linker. PROTAC molecules can simultaneously bind to target proteins and E3 ubiquitin ligases to form a ternary complex, and use the E3 ubiquitin ligase system to ubiquitinate the target protein, thereby inducing the degradation of the target protein under the action of proteolytic enzymes, thereby achieving the purpose of clearing pathogenic proteins. Unlike small molecule inhibitors that can only inhibit the activity of target proteases, PROTAC molecules can not only inhibit the enzymatic activity of target proteins by inducing the degradation of target proteins, but also eliminate the skeleton protein function of target proteins. Summary of the invention

[0005] The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Wherein, L is a connecting group.

[0008] The compound or pharmaceutically acceptable salt thereof described in the present invention may be any of the following compounds:

[0009]

[0010] The present invention also provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in preparing a protein degradation agent for PAK1, including the use for treating tumors.

[0011] The present invention also provides a pharmaceutical composition, which comprises the compound shown above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0012] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. DETAILED DESCRIPTION

[0013] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0014] Example 1 Synthesis of Compound A1:

[0015]

[0016] Step 1:

[0017] At room temperature, sodium hydroxide solution (5 mL) was added to compound A1-1 (500 mg, 1.79 mmol) to dissolve it, and then 1,2-dibromoethane (5.65 g, 30.43 mmol) and tetrabutylammonium bromide (11.6 mg, 0.036 mmol) were added, and then the mixture was stirred at 90 degrees Celsius for 2 hours. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (3 x 30 mL). The organic phase obtained by extraction was washed twice with brine, then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain a colorless oily liquid, namely the target compound A1-2 (300 mg, yield 43%). LCMS (ESI) m / z = 330.1 [M+H] + .

[0018] Step 2:

[0019] At room temperature, acetonitrile solution (5 mL) was added to compounds A1-2 (300 mg, 0.0.78 mmol) and A1-3 (162 mg, 0.0.39 mmol, synthesis reference Journal of Medicinal Chemistry (2022), 65 (23), 15627-15641) to dissolve them, and then potassium carbonate (162 mg, 1.17 mmol) and potassium iodide (13 mg, 0.08 mmol) were added to the mixture. The mixed reaction solution was stirred at 70 degrees Celsius for 5 hours under nitrogen protection. Post-treatment: The reaction mixture was filtered through a sand core funnel, and the filtrate was then dried and mixed with silica gel. The yellow oily liquid, i.e., the target compound A1-4 (190 mg, yield 69%), was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1). LCMS (ESI) m / z = 700.2 [M+H] + .

[0020] Step 3:

[0021] At room temperature, add hydrogen chloride-1,4-dioxane (1.5 mL) to compound A1-4 (140 mg, 0.20 mmol) to dissolve it, and stir the mixture solution at room temperature for 1 hour. Post-treatment: The reaction mixture is directly spin-dried, and then 1,4-dioxane (4 mL x 3) is added and spin-dried until it is spin-dried to obtain a yellow solid, which is the target compound A1-5 (120 mg, yield 99%). LCMS (ESI) m / z = 600.2 [M+H] + .

[0022] Step 4:

[0023] At room temperature, 1,4-dioxane (6 mL) was added to compound A1-5 (120 mg, 0.20 mmol) to dissolve it, and then triethylamine (0.2 mL) was added to react for 0.5 hours, and then 1-cyanoacetyl-3,5-dimethylpyrazole (49 mg, 0.30 mmol) was added to the mixture, and then the mixture was reacted at 90 degrees Celsius for 2 hours under nitrogen protection. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (2 x 10 mL). The organic phase obtained by extraction was washed twice with brine, and then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by a large thin layer chromatography plate (petroleum ether / ethyl acetate = 1 / 3) to obtain a yellow solid, namely the target compound A1-6 (90 mg, yield 67%). LCMS (ESI) m / z = 667.2 [M+H] + .

[0024] Step 5:

[0025] At room temperature, tetrahydrofuran (2 mL) was added to compound A1-6 (90 mg, 0.14 mmol) to dissolve it, and then 2-formyl thiazole (38 mg, 0.34 mmol) and hexahydropyridine (6 mg, 0.068 mmol) were added thereto, and the reaction mixture was stirred at 70 degrees Celsius for 16 hours. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (2 x 5 mL). The organic phase obtained by extraction was washed twice with brine, then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by a large thin layer chromatography plate (petroleum ether / ethyl acetate = 1 / 2) to obtain a yellow oily liquid, and then separated and purified by high performance liquid chromatography to obtain a white solid A1 (42 mg, purity 99%). LCMS (ESI) m / z = 762.3. 1 H NMR (400 MHz, DMSO-d 6 )δ10.13(s,1H),8.20-8.17(m,2H),8.09–7.99(m,1H),7.60(dd,J 1 =2.0Hz,J 2 =8.8Hz,1H),7.49(d,J=8.8Hz,1H),7.42(d,J=7.6Hz,1H),7.35(d,J=8.4Hz, 1H),7.19–7.15(m,1H),7.05(t,J=11.2Hz,2H),6.89–6.64(m,1H),6.78(dd,J 1 =2.8Hz,J 2 =10Hz,1H),6.22–5.95(m,1H),5.71–5.57(m,1H),4.39–4.27(m,4H),4.08–4.00(m,2H),3.61(s,2H),3.50(s ,2H),3.32–3.04(m,2H),2.78(s,2H),2.60(s,2H),1.98–1.94(m,2H),1.43–1.24(m,2H),1.00–0.96(m,3H).

[0026] Example 2 Synthesis of Compound A2:

[0027]

[0028] Step 1:

[0029] At room temperature, N,N-dimethylformamide (1 mL) was added to compound A2-1 (350 mg, 1.253 mmol) and 1,4-dibromobutane (0.5 mL) to dissolve them, and then sodium hydride (0.2 mL, 1.810 mmol) was added to the mixture, and then the mixture was stirred at room temperature for 1 hour. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (3x40 mL). The organic phase obtained by extraction was washed twice with brine, then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by a large thin layer chromatography plate (dichloromethane / anhydrous methanol=20 / 1) to obtain a green solid, namely the target compound A2-2 (345 mg, yield 60.50%). LCMS (ESI) m / z=413.2[M+H] + .

[0030] Step 2:

[0031] At room temperature, acetonitrile solution (4 mL) was added to compound A2-2 (345 mg, 0.747 mmol) and A1-3 (300 mg, 0.760 mmol) to dissolve them, and then potassium carbonate (342 mg, 2.475 mmol) and potassium iodide (27 mg, 0.163 mmol) were added to the mixture. The mixed reaction solution was stirred at 70 degrees Celsius for 5 hours. Post-treatment: The reaction mixture was filtered through a sand core funnel, and the filtrate was spin-dried and then mixed with silica gel. The yellow oily liquid, i.e., the target compound A2-3 (350 mg, yield 66.40%), was separated and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1). LCMS (ESI) m / z = 727.3 [M + H] + .

[0032] Step 3:

[0033] At room temperature, sodium chloride-1,4-dioxane (5 mL) was added to compound A2-3 (100 mg, 0.137 mmol) to dissolve it, and the mixture solution was stirred at room temperature for 1 hour. Post-treatment: The reaction mixture was directly spin-dried, and then 1,4-dioxane (4 mL x 3) was added and spin-dried until spin-dried to obtain a yellow solid, which was the target compound A2-4 (85 mg, yield 98.90%). LCMS (ESI) m / z = 627.3 [M+H] + .

[0034] Step 4:

[0035] At room temperature, 1,4-dioxane (2 mL) was added to compound A2-4 (85 mg, 0.135 mmol) and reacted for 0.5 hours, then 1-cyanoacetyl-3,5-dimethylpyrazole (33 mg, 0.203 mmol) was added to the mixture, and then the mixture was reacted at 90 degrees Celsius under nitrogen protection for 5 hours. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (2 x 10 mL). The organic phase obtained by extraction was washed twice with brine, then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by a large thin layer chromatography plate (petroleum ether / ethyl acetate = 1 / 2) to obtain a yellow solid, namely the target compound A2-5 (50 mg, yield 53.70%). LCMS (ESI) m / z = 694.3 [M + H] + .

[0036] Step 5:

[0037] At room temperature, tetrahydrofuran (1 mL) was added to compound A2-5 (50 mg, 0.072 mmol) to dissolve it, and then 2-formyl thiazole (0.02 mL) and hexahydropyridine (0.004 mL) were added thereto, and the reaction mixture was stirred at 70 degrees Celsius for 16 hours. Post-treatment: water was added to the reaction mixture, and then ethyl acetate was added for extraction (2 x 5 mL). The organic phase obtained by extraction was washed twice with brine, then dried over anhydrous sodium sulfate, and then the solution was spin-dried to obtain a secondary product. The secondary product was separated and purified by a large thin layer chromatography plate (petroleum ether / ethyl acetate = 1 / 2) to obtain a yellow oily liquid, and then separated and purified by high performance liquid chromatography to obtain a white solid A2 (8.8 mg, purity 99.48%). LCMS (ESI) m / z = 790.3. 1 H NMR (400 MHz, DMSO-d 6)δ9.74(s,1H),8.18(dd,J=7.5,3.1Hz,1H),8.01–7.91(m,1H),7.59(dd,J=8.8,2.4Hz,1H),7.48(d,J=8.8Hz,1H),7.41( d,J=2.3Hz,1H),7.37(d,J=8.5Hz,1H),7.31(d,J=8.7Hz,2H),7.16(dd,J=8.8,5.7Hz,1H),6.96(t,J=9.4Hz,2H),6.89–6 .83(m,1H),6.77(dd,J=10.0,2.9Hz,1H),6.09(t,J=55.3Hz,1H),5.63(d,J=37.1Hz,1H),4.40–4.23(m,2H),4.03(s,5H) ,3.54(d,J=10.5Hz,3H),3.21(s,4H),2.77(s,2H),1.94(s,2H),1.80(s,4H),1.27(d,J=24.5Hz,2H),1.00–0.88(m,3H).

[0038] Effect Example 1 Protein Abundance WB Detection Experiment

[0039] Western blot (WB) is a classic biochemical experimental method for detecting the abundance of target proteins in cells or biological tissue samples. Its basic principle is to detect protein signals in cells or biological tissue samples treated with gel electrophoresis through specific binding of antigens and antibodies; after the protein antigen epitope binds to the specific primary antibody and then couples to the secondary antibody, the signal is amplified in a cascade, and the signal is read out by chemiluminescence or fluorescent groups. The intracellular protein content is ultimately characterized by the high and low optical signal values. PROTAC compound molecules cause target protein degradation, which will be concentration-dose-dependent and time-point-dependent.

[0040] The experimental operation process is as follows:

[0041] 1. Inoculate the experimental cells such as OVCAR3 into a 12-well plate (Corning, 3513), 5*10 5 cells / 500ul / well.

[0042] 2. Mix the compound with 0.5 ml of culture medium to twice the final required concentration, add it to a 12-well plate, mix well, and place it in a cell culture incubator to treat the time point to be tested, such as 16 hours.

[0043] 3. Use a vacuum pump to remove the culture medium in the 12-well plate, wash it with PBS, add 80ul of pre-cooled cell lysis buffer containing protease and phosphatase inhibitors to each well, and lyse on ice for 20 minutes.

[0044] 4. After lysis, transfer the sample to a 600ul centrifuge tube, centrifuge at 13000rpm, 4℃ for 20 minutes, take the supernatant and immediately mix it with 5X protein loading buffer to prepare the sample. Use the BCA kit to determine the protein concentration. According to the measured concentration, use 1X protein loading buffer to adjust the concentration after sample preparation to a consistent level. Boil the sample at 100℃ for 5 minutes and then cool it. The denatured protein sample is frozen at -20℃

[0045] 5. Load 13ul of protein sample into each well, 4-12% Bis-Tris Midi Gel 26-well (invitrogen, WG1403BOX), electrophoresis at 80 volts for 20 minutes, then switch to 120 volts constant voltage electrophoresis for 60 minutes.

[0046] Transfer proteins onto nitrocellulose membrane at 6.250 mA constant current in an ice bath for 80 minutes.

[0047] 7. Block the membrane with protein blocking solution (LI-COR, 927-50000) at room temperature for 1 hour.

[0048] 8. Wash the membrane three times with TBST (CST#9997S) at room temperature for 5 minutes each time, prepare the primary antibody (Rabbitanti-PAK1 CST#2602S1:1000) in protein blocking solution, and incubate at 4°C overnight.

[0049] 9. Wash the membrane 3 times with TBST (CST#9997S) at room temperature for 5 minutes each time, prepare the secondary antibody in TBST containing 5% BSA, and incubate at room temperature for 1-2 hours.

[0050] 10. After washing the membrane three times with TBST (CST#9997S) at room temperature, the membrane was exposed using a LICOR Odyssey DLx machine.

[0051] The degradation rates of different compounds at different concentrations, the test results are as follows:

[0052] Compound Degradation rate at 3.33uM compound concentration Degradation rate at 10uM compound concentration A1 50％ 64％ A2 32％ 61％

Claims

1. A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof: in, L is a linking group.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is any of the following compounds:

3. A pharmaceutical composition, characterized in that It comprises the compound according to any one of claims 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a PAK1 protein degrader.

5. Use of the compound according to claim 4 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors.

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