A protein degrader of irak4

Abstract

The application discloses a kind of IRAK4 protein degradation agent compounds as shown in formula (I), purposes and pharmaceutical compositions containing it.The compounds are used to treat hematological tumors, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus and the like.

Description

Technical Field

[0001] This invention relates to a protein degrader of IRAK4, its uses, and pharmaceutical compositions containing it. Background Technology

[0002] Interleukin-1 receptor-associated kinase 4 (IRAK4) is a protein encoded by the IRAK4 gene. It is a cytoplasmic protein with a full length of 460 amino acids. It belongs to the serine / threonine kinase family and is a key protein in the interleukin-1 (IL-1) receptor family and Toll-like receptor (TLR) signaling, mediating the innate immune response of initial defense.

[0003] When foreign antigens invade or inflammatory stress occurs, TLR and IL-1 receptors recruit the myeloid differentiation factor MYD88, leading to the assembly and activation of IRAK4. This initiates a signaling cascade, activating the NF-κB pathway and causing the production of various inflammatory factors in cells, thereby inducing various immune diseases such as psoriasis, hidradenitis suppurativa, atopic dermatitis, rheumatoid arthritis, and systemic lupus erythematosus. Furthermore, as a major regulator of the downstream TLR / IL-1R signaling pathway, excessive activation of IRAK4, or abnormal activation due to mutations, is associated with the occurrence and development of inflammatory diseases and tumors. Therefore, IRAK4 is an attractive drug development target in the fields of immune disease and tumor treatment.

[0004] Currently, IRAK4 small molecule inhibitors developed by major pharmaceutical companies such as Pfizer and Bayer have entered clinical trials for the treatment of hematological malignancies, psoriasis, rheumatoid arthritis, enteritis, and systemic lupus erythematosus. Recent studies have shown that in addition to kinase activity mediating inflammatory signaling pathways, the protein structure of IRAK4 can also activate certain inflammatory signaling pathways. Research has found that in human dermal fibroblasts, ATP-competitive small molecule inhibitors of IRAK4 cannot effectively inhibit the release of IL-6 and TNF-α stimulated by IL-1β. Knockdown of IRAK4 expression effectively eliminated IL-1, IL-8, and TLR ligand-mediated inflammatory responses. Therefore, the IRAK4 protein-mediated inflammatory signaling pathway cannot be completely inhibited by ATP-competitive small molecule inhibitors, and IRAK4 small molecule targeted inhibitors have therapeutic limitations.

[0005] 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. The PROTAC molecule 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 by proteolytic zymocytes, achieving the goal of eliminating 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, not only inhibit its enzymatic activity but also eliminate the skeletal protein function of the target protein. Summary of the Invention

[0006] This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0007]

[0008] Where L is a linking group.

[0009] The compound or its pharmaceutically acceptable salt described in this invention may be any of the following compounds:

[0010]

[0011] This invention also provides the use of the compounds shown above or pharmaceutically acceptable salts thereof in the preparation of protein degrading agents for IRAK4. This use includes applications in the treatment of hematologic malignancies, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus, etc.

[0012] The present invention also provides a pharmaceutical composition comprising the compound shown above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0013] 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

[0014] 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.

[0015] Example 1: Synthesis of Compound A1:

[0016]

[0017] At room temperature, compound A1-1 (10 mg, 26 μmol) was dissolved in dimethyl sulfoxide (1 mL). Then, N,N-diisopropylethylamine (6.64 mg, 51 μmol), HATU (11.72 mg, 31 μmol), and compound A1-2 (13.51 mg, 26 μmol, synthesis reference WO2020173440) were added sequentially to the reaction mixture. The reaction mixture was stirred at 25 °C for 16 hours. 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 crude product. The crude product was purified by reversed-phase column chromatography (C18, 0-32% ACN to water) to obtain the target compound A1 (7.53 mg, 8.74 μmol, 33.60% yield) as a yellow solid. LCMS(ESI)m / z=862.10[M+H]+.1H NMR (400MHz, DMSO-d6) δ11.05(s,1H),9.80(s,1H),8.96(s,1H),8.68(d,J=5.2Hz,1H),7.86(s,1H),7.77(d,J=4.8Hz,1H), 7.67(s,1H),7.55(d,J=8.4Hz,1H),7.14(s,1H),7.00(d,J=2.0Hz,1H),6.89(dd,J=8.4Hz,2.0Hz,1H),5.02(dd,J=12.8Hz, 5.6Hz,1H),4.87(d,J=3.6Hz,1H),4.28(s,1H),3.71(t,J=6.4Hz,2H),3.62–3.56(m,12H),3.46–3.40(m,1H),3.38–3.35(m ,2H),3.25–3.23(m,1H),2.93–2.79(m,1H),2.67–2.65(m,2H),2.59(s,3H),2.54(s,2H),2.05–1.85(m,2H),1.77(br,1H).

[0018] Example 2: Synthesis of compound A2:

[0019]

[0020] Step 1:

[0021] At room temperature, A2-1 (46.3 mg, 0.135 mmol, synthesis reference WO2021170109) was added to dichloromethane (2.5 mL). The reaction solution was cooled to 0 °C, and Dess-Martin iodate (171 mg, 0.40 mmol) was added at 0 °C. After 30 minutes, the reaction solution was concentrated and purified by reverse-phase C18 column to obtain A2-2 (40 mg, 0.117 mmol, yield 86.8%). LCMS (ESI) m / z = 342.1 [M+1]+.

[0022] Step 2:

[0023] A2-2 (52.7 mg, 0.10 mmol) was suspended in 2 mL of 1,2-dichloroethane. A1-1 (34.1 mg, 0.10 mmol) and triethylamine (20.2 mg, 0.20 mmol) were added, followed by the gradual addition of sodium triacetoxyborohydride (25.4 mg, 0.12 mmol) over 10 minutes. The mixture was stirred at room temperature. After 16 hours of stirring, the mixture was subjected to silica gel column chromatography to obtain a yellow solid product A2 (30 mg, 0.037 mmol, 36.7% yield). LCMS (ESI) m / z = 816.4 [M+H]+.1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),9.80(s,1H),8.96(s,1H),8.67(d,J=4.8Hz,1H),7.86(s,1H),7.77– 7.76(m,1H),7.65–7.62(m,3H),6.77(d,J=1.6Hz,1H),6.66–6.63(m,1H),5.07–5.03(m,1H),4.86(d,J=3. 2Hz,1H),4.29–4.28(m,1H),4.17–4.13(m,2H),3.73–3.56(m,8H),3.44–3.38(m,2H),3.24–3.22(m,2H),3 .06–3.03(m,1H),2.88–2.83(m,1H),2.69–2.67(m,2H),2.59(s,3H),2.54–2.50(m,4H),2.02–1.75(m,5H).

[0024] Example 3: Synthesis of compound A3:

[0025]

[0026] Step 1:

[0027] At room temperature, A3-1 (50.1 mg, 0.135 mmol, synthesis reference WO2020038415) was added to dichloromethane (2.5 mL). The reaction solution was cooled to 0 °C, and Dess-Martin iodate (171 mg, 0.40 mmol) was added at 0 °C. After 30 minutes, the reaction solution was concentrated and purified by reverse-phase C18 column to obtain A3-2 (42 mg, 0.114 mmol, yield 84.3%). LCMS (ESI) m / z = 370.1 [M+1]+.

[0028] Step 2:

[0029] A3-2 (52.7 mg, 0.10 mmol) was suspended in 2 mL of 1,2-dichloroethane. A1-1 (36.9 mg, 0.10 mmol) and triethylamine (20.2 mg, 0.20 mmol) were added, followed by the gradual addition of sodium triacetoxyborohydride (25.4 mg, 0.12 mmol) over 10 minutes. The mixture was stirred at room temperature. After 16 hours of stirring, the mixture was subjected to silica gel column chromatography to obtain a yellow solid product A3 (16 mg, 0.019 mmol, 18.9% yield). LCMS (ESI) m / z = 844.4 [M+H]+.1H NMR (400MHz, DMSO-d6) δ11.07(s,1H),9.80(s,1H),8.96(s,1H),8.67(d,J=4.8Hz,1H),7.86(s,1H),7. 77–7.76(m,1H),7.65–7.64(m,3H),7.31(d,J=1.6Hz,1H),7.25–7.22(m,1H),5.08–5.04(m,1H),4.85(d ,J=3.2Hz,1H),4.28–4.27(m,1H),4.07–4.04(m,2H),3.62–3.56(m,6H),3.44–3.38(m,1H),3.25–3.21 (m,2H),3.02–2.95(m,3H),2.59–2.50(m,8H),2.22–2.21(m,2H),1.92–1.81(m,6H),1.19–1.16(m,2H).

[0030] Synthesis of intermediate A1-1:

[0031]

[0032] Step 1:

[0033] A1-a (5.39 g, 26.8 mmol) was dissolved in tetrahydrofuran (150 ml), and Boc-piperazine (20 g, 107.3 mmol) was added. The mixture was stirred at 70°C for 16 hours. After the reaction was complete, the solvent was removed by evaporation, and the residue was purified by silica gel column chromatography to obtain A1-b (9.1 g).

[0034] Step 2:

[0035] A1-b (1 g, 2.95 mmol) was dissolved in concentrated sulfuric acid (5 mL), and nitric acid (0.5 mL) was slowly added at -5°C. The mixture was then stirred at room temperature for 2 hours. After the reaction was complete, the solution was poured into ice water, adjusted to alkalinity with saturated sodium hydroxide, extracted with dichloromethane, and the organic phase was dried over sodium sulfate and then evaporated to dryness to obtain A1-c (3 g).

[0036] Step 3:

[0037] The A1-c (3 g, 10.5 mmol) obtained in the previous step was dissolved in tetrahydrofuran (100 ml) and water (100 ml), followed by the addition of Boc anhydride (4.61 g, 21.1 mmol) and sodium carbonate (3.36 g, 31.7 mmol), and then stirred overnight at room temperature. After the reaction was complete, the solvent was removed by evaporation, and the residue was purified by silica gel column chromatography to obtain A1-d (800 mg).

[0038] Step 4:

[0039] A1-d (2.47 g, 6.4 mmol) was dissolved in dichloromethane (40 mL) and DMF (20 mL), followed by the addition of potassium carbonate (1.75 g, 12.8 mmol) and R-3-pyrrolidone (2.5 g, 6.4 mmol) and stirring at room temperature for 1 hour. After the reaction was complete, the solvent was removed by evaporation, and the residue was purified by silica gel column chromatography to obtain A1-e (2.6 g).

[0040] Step 5:

[0041] Al-e (2.6 g, 5.9 mmol) was dissolved in ethanol (50 mL), and after adding palladium on carbon catalyst (200 mg), the mixture was stirred overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the palladium on carbon was filtered off, and the resulting filtrate was evaporated to dryness to obtain Al-f (2.4 g).

[0042] Step 6:

[0043] A1-f (2.58 g, 6.4 mmol) and A1-g (1.3 g, 6.4 mmol) were dissolved in DMF, and HATU (2.91 g, 7.6 mmol) and DIPEA (1.65 g, 12.7 mmol) were added sequentially. The mixture was then stirred overnight at room temperature. After the reaction was complete, the solvent was removed by evaporation, and the residue was purified by silica gel column chromatography to obtain A1-h (1.7 g).

[0044] Step 7:

[0045] Al-h (1.7 g, 2.8 mmol) was dissolved in 4 N HCl / dioxane solution and stirred at room temperature for 2 hours. After the reaction was complete, the solution was directly evaporated to dryness to obtain Al-1 (1.1 g).

[0046] Example 1: Protein Abundance Western Blot Detection Experiment

[0047] Western blot (WB) is a classic biochemical assay used to detect 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, resulting in a cascaded signal amplification. The signal is then read out via chemiluminescence or a fluorescent dye. Intracellular protein content is ultimately characterized by the intensity of the optical signal. Protamine compounds induce target protein degradation, exhibiting concentration-dose dependence and time-dependent degradation. The concentration-displacement ratio (DC) can be calculated by curve fitting of the protein WB exposure signal. 50 and D max value.

[0048] The experimental procedure is as follows:

[0049] SUDHL2 cells were seeded into 6-well plates (Corning #3506), 2 × 10⁶ cells per well. 6 cells / 2mL / well. Add 2uL of RPMI 1640 medium (Gibco#A10491) and the compound mixture to a 6-well plate, mix well, and then incubate in a cell culture incubator for 18 hours.

[0050] Transfer the cell suspension to 15 mL centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 80 μL of pre-chilled cell lysis buffer containing PMSF (Sigma #8553S) and protease inhibitor (Roche #4693124001) to each tube, and incubate on ice for 60 minutes to fully lyse the cells.

[0051] The lysed sample was transferred to a 1 mL centrifuge tube and centrifuged at 14,000 rpm for 15 minutes at 4 °C. The supernatant was then transferred to a new 0.6 mL centrifuge tube.

[0052] Protein concentration was determined using the BCA kit (Invitrogen #23225). Loading buffer (Solarbio #P1043) was added to each sample to adjust the protein concentration to 2.5 μg / μL. The samples were then in a 100°C metal bath for 10 minutes and placed on ice until ready for use.

[0053] Western blot was performed using a 4-12% protein precast gel (Invitrogen #WG0335BOX), with 20 μg of protein sample loaded into each well. Electrophoresis was performed at a constant voltage of 120 V for 120 minutes.

[0054] Transfer for 10 minutes at 2.5A 25V.

[0055] The membrane was sealed at room temperature for 2 hours with a 5% BSA solution (Sigma#V900933).

[0056] Wash the membrane 3 times with TBST (CST#9997) at room temperature for 10 minutes each time. Prepare primary antibody (CST#4363S1:1000, CST#97166S) in protein blocking buffer and incubate overnight at 4°C.

[0057] Wash the membrane three times at room temperature with TBST for 10 minutes each time. Prepare secondary antibodies with TBST containing 5% BSA (CST#7074S1:1000, CST#7076S1:1000) and incubate at room temperature for 1 hour.

[0058] Wash the membrane three times at room temperature with TBST, 10 minutes each time. Prepare the luminescent substrate solution at a 1:1 ratio and add it dropwise onto the membrane. Then, use a gel imaging system (BIO-RAD chemiDoc) to perform the gel imaging. TM MP) was used to scan and photograph the membrane. The results are shown in the table below:

[0059]

Claims

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

2. A pharmaceutical composition, characterized in that, It comprises the compound as described in any one of claims 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 an IRAK4 protein degrader.

4. Use of a compound as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hematologic malignancies, psoriasis, rheumatoid arthritis, enteritis, and systemic lupus erythematosus.

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