A protein degrader of pak1

Abstract

The application discloses a PAK1 protein degradation agent compound as shown in formula (I), purposes and a pharmaceutical composition containing the same. The compound is used for treating tumors.

Description

Technical Field

[0001] This invention relates to a protein degrader of PAK1, its uses, and pharmaceutical compositions containing it. Background Technology

[0002] PAK1 is a member of the Pak (p21-activated kinase) serine / cysteine ​​protein kinase family and an interacting factor between the RhoGTPases RAC1 and CDC42. Its catalytic activity and scaffolding role play multiple functions in cell signaling. PAK1-regulated signal transduction cascades include the MAPK, AKT, Wnt1 / β-catenin, ERα, BAD, and NF-κB pathways, participating in the regulation of cell proliferation and survival. PAK1 also participates in the regulation of cell motility, transmitting various signals controlling cytoskeleton dynamics, cell shape, and adhesion.

[0003] PAK1 exhibits enhanced function in various human malignancies, suggesting it may play a crucial role in tumor development and progression. PAK1 gene amplification and elevated PAK1 protein levels are often associated with aggressive tumor phenotypes, chemotherapy resistance, and poor prognosis. Besides gene amplification and protein overexpression, PAK1 may also be overactivated by mutations in upstream regulators such as RAC1, RAS, and Merlin, thereby transmitting oncogenic signals and causing cells to transform into cancerous phenotypes. Therefore, targeting and interfering with PAK1 may be a promising cancer treatment approach. Studies in a mouse model of neurofibromatosis type 2 have shown that PAK1 gene deletion effectively slows hearing loss and schwannoma growth in mice without significant systemic toxicity.

[0004] PAK1 signal transduction activity is mediated by its enzymatic activity and scaffold function; therefore, PAK1 degraders theoretically have greater pharmacological 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, linked by a linker. PROTAC molecules can simultaneously bind to both the target protein and the E3 ubiquitin ligase to form a ternary complex. Utilizing the E3 ubiquitin ligase system, the target protein is ubiquitinated, thereby inducing its degradation under the action of proteolytic zymocytes, achieving the goal of clearing pathogenic proteins. Unlike small molecule inhibitors that only inhibit the activity of the target protease, PROTAC molecules, by inducing the degradation of the target protein, can not only inhibit its enzymatic activity but also eliminate the scaffold protein function of the target protein. Summary of the Invention

[0005] This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0006]

[0007] Where L is a linking group.

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

[0009]

[0010] The present invention also provides the use of the compounds shown above or pharmaceutically acceptable salts thereof in the preparation of protein degrading agents for PAK1. This use includes its use in the treatment of tumors.

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

[0012] As described herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of the present invention. Detailed Implementation

[0013] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0014] Example 1: Synthesis of Compound A1:

[0015]

[0016] Step 1:

[0017] At room temperature, compound A1-1 (500 mg, 1.79 mmol) was dissolved in 5 mL of sodium hydroxide solution, followed by the addition of 1,2-dibromoethane (5.65 g, 30.43 mmol) and tetrabutylammonium bromide (11.6 mg, 0.036 mmol). The mixture was then stirred at 90°C for 2 hours. Post-treatment: Water was added to the reaction mixture, followed by extraction with ethyl acetate (3 x 30 mL). The extracted organic phase was washed twice with brine and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness to obtain the secondary product. The secondary product was 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, compounds A1-2 (300 mg, 0.78 mmol) and A1-3 (162 mg, 0.39 mmol, synthesis reference: Journal of Medicinal Chemistry (2022), 65(23), 15627-15641) were dissolved in acetonitrile solution (5 mL). Potassium carbonate (162 mg, 1.17 mmol) and potassium iodide (13 mg, 0.08 mmol) were then added to the mixture. The reaction mixture was stirred at 70°C for 5 hours under nitrogen protection. Post-treatment: The reaction mixture was filtered through a sintered glass funnel, and the filtrate was evaporated to dryness. The solution was then stirred with silica gel and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain a yellow oily liquid, namely the target compound A1-4 (190 mg, yield 69%). LCMS (ESI) m / z = 700.2 [M+H] + .

[0020] Step 3:

[0021] At room temperature, compound A1-4 (140 mg, 0.20 mmol) was dissolved by adding 1,4-dioxane (1.5 mL) of hydrogen chloride, and the mixture was stirred at room temperature for 1 hour. Post-treatment: The reaction mixture was directly evaporated to dryness, followed by the addition of 1,4-dioxane (4 mL x 3) until evaporated to dryness, yielding the target compound A1-5 (120 mg, 99% yield) as a yellow solid. LCMS (ESI) m / z = 600.2 [M+H] + .

[0022] Step 4:

[0023] At room temperature, compound A1-5 (120 mg, 0.20 mmol) was dissolved in 6 mL of 1,4-dioxane, followed by the addition of 0.2 mL of triethylamine and reaction for 0.5 h. Then, 49 mg, 0.30 mmol of 1-cyanoacetyl-3,5-dimethylpyrazole was added to the mixture, and the mixture was reacted at 90°C under nitrogen protection for 2 h. Post-treatment: Water was added to the reaction mixture, followed by extraction with ethyl acetate (2 x 10 mL). The extracted organic phase was washed twice with brine and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness to obtain the secondary product. The secondary product was purified by thin-layer chromatography (petroleum ether / ethyl acetate = 1 / 3) to obtain a yellow solid, namely compound A1-6 (90 mg, yield 67%). LCMS (ESI) m / z = 667.2 [M+H] + .

[0024] Step 5:

[0025] At room temperature, compound A1-6 (90 mg, 0.14 mmol) was dissolved in tetrahydrofuran (2 mL), followed by the addition of 2-aldehydethiazole (38 mg, 0.34 mmol) and hexahydropyridine (6 mg, 0.068 mmol). The reaction mixture was stirred at 70°C for 16 hours. Post-treatment: Water was added to the reaction mixture, followed by extraction with ethyl acetate (2 x 5 mL). The extracted organic phase was washed twice with brine, dried over anhydrous sodium sulfate, and then evaporated to dryness to obtain the secondary product. The secondary product was purified by thin-layer chromatography (petroleum ether / ethyl acetate = 1 / 2) to obtain a yellow oily liquid, which was then purified by high-performance liquid chromatography (HPLC) to obtain a white solid A1 (42 mg, 99% purity). LCMS (ESI) m / z = 762.3. 1 H NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.20-8.17(m,2H),8.09–7.99(m,1H),7.60(dd,J1=2.0Hz,J2=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,J1=2.8Hz,J2=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, compound A2-1 (350 mg, 1.253 mmol) and 1,4-dibromobutane (0.5 mL) were dissolved in N,N-dimethylformamide (1 mL), and then sodium hydride (0.2 mL, 1.810 mmol) was added to the mixture. The mixture was then stirred at room temperature for 1 hour. Post-treatment: Water was added to the reaction mixture, and then ethyl acetate (3 x 40 mL) was added for extraction. The resulting organic phase was washed twice with brine and dried over anhydrous sodium sulfate. The solution was then evaporated to dryness to obtain the sub-product. The sub-product was purified by thin-layer chromatography (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, compounds A2-2 (345 mg, 0.747 mmol) and A1-3 (300 mg, 0.760 mmol) were dissolved in acetonitrile solution (4 mL). Potassium carbonate (342 mg, 2.475 mmol) and potassium iodide (27 mg, 0.163 mmol) were then added to the mixture. The reaction mixture was stirred at 70°C for 5 hours. Post-treatment: The reaction mixture was filtered through a sintered glass funnel, and the filtrate was evaporated to dryness. The solution was then stirred with silica gel and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain a yellow oily liquid, namely compound A2-3 (350 mg, yield 66.40%). LCMS (ESI) m / z = 727.3 [M+H] + .

[0032] Step 3:

[0033] At room temperature, compound A2-3 (100 mg, 0.137 mmol) was dissolved in 5 mL of sodium chloride-1,4-dioxane, and the mixture was stirred at room temperature for 1 hour. Post-treatment: The reaction mixture was directly evaporated to dryness, followed by the addition of 4 mL x 3 mL of 1,4-dioxane, and the mixture was evaporated to dryness 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 h. Then, 1-cyanoacetyl-3,5-dimethylpyrazole (33 mg, 0.203 mmol) was added to the mixture, and the mixture was reacted at 90°C under nitrogen protection for 5 h. Post-treatment: Water was added to the reaction mixture, followed by extraction with ethyl acetate (2 x 10 mL). The extracted organic phase was washed twice with brine, dried over anhydrous sodium sulfate, and then evaporated to dryness to obtain the secondary product. The secondary product was purified by thin-layer chromatography (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, compound A2-5 (50 mg, 0.072 mmol) was dissolved in tetrahydrofuran (1 mL), followed by the addition of 2-aldehydethiazole (0.02 mL) and hexahydropyridine (0.004 mL). The reaction mixture was stirred at 70°C for 16 hours. Post-treatment: Water was added to the reaction mixture, followed by extraction with ethyl acetate (2 x 5 mL). The extracted organic phase was washed twice with brine, dried over anhydrous sodium sulfate, and then evaporated to dryness to obtain the secondary product. The secondary product was purified by thin-layer chromatography (petroleum ether / ethyl acetate = 1 / 2) to obtain a yellow oily liquid, which was then purified by high-performance liquid chromatography (HPLC) to obtain a white solid A2 (8.8 mg, purity 99.48%). LCMS (ESI) m / z = 790.3. 1H NMR(400MHz, DMSO-d6)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,2 H),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] Example 1: Protein Abundance Western Blot Detection Experiment

[0039] Western blot (WB) is a classic biochemical method for detecting the abundance of target proteins in cellular or biological tissue samples. Its basic principle is based on the specific binding of antigens and antibodies to detect protein signals in gel electrophoresis-treated cellular or biological tissue samples. After the protein antigen epitope binds to a specific primary antibody, a secondary antibody is conjugated, amplifying the signal in a cascade. The signal is then read out via chemiluminescence or a fluorescent dye. Intracellular protein content is ultimately characterized by the level of the optical signal. Protamine compounds induce target protein degradation, exhibiting both concentration-dose dependence and time-dependent degradation.

[0040] The experimental procedure is as follows:

[0041] 1. Seed the experimental cells, such as OVCAR3, into 12-well plates (Corning, 3513), 5*102 5 cells / 500ul / well.

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

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

[0044] 4. Transfer the lysed sample to a 600 μL centrifuge tube, centrifuge at 13,000 rpm for 20 minutes at 4°C, collect the supernatant and immediately mix with 5X protein loading buffer. Determine the protein concentration using a BCA kit. Adjust the post-processing concentration to a consistent level using 1X protein loading buffer based on the determined concentration. Boil the sample at 100°C for 5 minutes, then cool. Store the denatured protein sample at -20°C.

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

[0046] 6.250 mA constant current ice bath transfer protein onto nitrocellulose membrane for 80 minutes.

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

[0048] 8. Wash the membrane 3 times with TBST (CST#9997S) at room temperature, 5 minutes each time. Prepare the primary antibody (Rabbitanti-PAK1 CST#2602S1:1000) in protein blocking buffer and incubate overnight at 4°C.

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

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

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

[0052] compound The concentration of the 3.33 μM compound decreased the hydrolysis rate. The concentration of 10 μM compound decreases the hydrolysis rate. A1 50％ 64％ A2 32％ 61％

Claims

1. A compound or a pharmaceutically acceptable salt thereof, as shown below:

2. A pharmaceutical composition, characterized in that, It comprises the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. Use of a compound as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a PAK1 protein degrading agent.

4. Use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.

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