IRAK4 protein degradation agent

Abstract

The invention discloses an IRAK4 protein degradation agent compound as shown in a formula (I), application of the IRAK4 protein degradation agent compound and a pharmaceutical composition containing the IRAK4 protein degradation agent compound. The compound is used for treating hematologic tumors, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an IRAK4 protein degradation agent, use thereof and a pharmaceutical composition containing the same. Background Art

[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 and belongs to the serine / threonine kinase. It 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 myeloid differentiation factor MYD88, leading to the assembly and activation of IRAK4, initiating signal cascade reactions, activating the NF-κB pathway, and producing various inflammatory factors in cells, thereby inducing various immune diseases, such as psoriasis, suppurative hidradenitis, atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus, etc. In addition, as the main regulator of the TLR / IL-1R downstream signaling pathway, IRAK4's overactivation or abnormal activation due to mutations is related to the occurrence and development of inflammatory diseases and tumors. Therefore, in the field of immune diseases and tumor treatment, IRAK4 is an attractive drug development target.

[0004] At present, IRAK4 small molecule inhibitors developed by major pharmaceutical companies such as Pfizer and Bayer have entered clinical trials for the treatment of blood tumors, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus, etc. Recent studies have shown that in addition to the kinase activity of IRAK4 mediating inflammatory signaling pathways, the protein structure can also activate certain inflammatory signaling pathways. Studies have 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. Knocking down IRAK4 expression effectively eliminates inflammatory responses mediated by IL-1, IL-8 and TLR ligands. Therefore, the inflammatory signaling pathway mediated by IRAK4 protein cannot be completely inhibited by ATP-competitive small molecule inhibitors, and IRAK4 small molecule targeted inhibitors have therapeutic limitations.

[0005] PROTAC (Proteolysis 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 the target protein and the E3 ubiquitin ligase 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 the proteolytic enzyme body, thereby achieving the purpose of removing pathogenic proteins. Unlike small molecule inhibitors that can only inhibit the activity of the target protease, PROTAC molecules can not only inhibit the enzymatic activity of the target protein by inducing the degradation of the target protein, but also eliminate the skeleton protein function of the target protein. Summary of the invention

[0006] The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein, L is a connecting group.

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

[0010]

[0011] The present invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable salt in preparing a protein degrader of IRAK4, including the use in treating blood tumors, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus, etc.

[0012] 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.

[0013] 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

[0014] 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.

[0015] Example 1 Synthesis of Compound A1:

[0016]

[0017] At room temperature, compound A1-1 (10 mg, 26 umol) was dissolved in dimethyl sulfoxide (1 mL), and then N, N-diisopropylethylamine (6.64 mg, 51 umol), HATU (11.72 mg, 31 umol) and compound A1-2 (13.51 mg, 26 umol, synthesis reference WO2020173440) were added to the reaction solution in sequence, and the reaction solution was stirred at 25 ° C for 16 hours. Water was added to the reaction mixture, and then ethyl acetate was added for extraction (3x 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 crude product. The crude product was separated and purified by reverse phase column chromatography (C18, 0-32% ACN to water) to obtain a yellow solid, namely the target compound A1 (7.53 mg, 8.74 umol, 33.60% yield). 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, and sodium triacetoxyborohydride (25.4 mg, 0.12 mmol) was gradually added over 10 minutes, and stirred at room temperature. After stirring and reacting for 16 hours, 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, and sodium triacetoxyborohydride (25.4 mg, 0.12 mmol) was gradually added over 10 minutes, and stirred at room temperature. After stirring and reacting for 16 hours, 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 and stirred at 70 degrees for 16 hours. After the reaction was completed, the solvent was removed by vortexing, and the residue was purified by silica gel column 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), nitric acid (0.5 mL) was slowly added at -5 degrees, and the mixture was returned to room temperature and stirred for 2 hours. After the reaction was completed, the mixture 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 spin-dried to obtain A1-c (3 g).

[0036] Step 3:

[0037] A1-c (3 g, 10.5 mmol) obtained in the previous step was dissolved in tetrahydrofuran (100 ml) and water (100 ml), and Boc anhydride (4.61 g, 21.1 mmol) and sodium carbonate (3.36 g, 31.7 mmol) were added in sequence, and then stirred at room temperature overnight. After the reaction was completed, the solvent was removed by vortexing, and the residue was purified by silica gel column 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), potassium carbonate (1.75 g, 12.8 mmol) and R-3-pyrrolidinol (2.5 g, 6.4 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was removed by vortexing, and the residue was purified by silica gel column to obtain A1-e (2.6 g).

[0040] Step 5:

[0041] A1-e (2.6 g, 5.9 mmol) was dissolved in ethanol (50 mL), palladium carbon catalyst (200 mg) was added, and the mixture was stirred overnight at room temperature under hydrogen atmosphere. After the reaction was completed, the palladium carbon was filtered off, and the filtrate was dried to obtain A1-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 in sequence, and then stirred at room temperature overnight. After the reaction was completed, the solvent was removed by vortexing, and the residue was purified by silica gel column to obtain A1-h (1.7 g).

[0044] Step 7:

[0045] A1-h (1.7 g, 2.8 mmol) was dissolved in 4N HCl / dioxane solution and stirred at room temperature for 2 hours. After the reaction was completed, the mixture was directly spin-dried to obtain A1-1 (1.1 g).

[0046] Effect Example 1 Protein Abundance WB Detection Experiment

[0047] 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 cascade amplified 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 show concentration-dose dependence and time-point dependence. DC can be calculated by curve fitting of the protein WB exposure signal. 50 and D max value.

[0048] The experimental operation process is as follows:

[0049] SUDHL2 cells were seeded into 6-well plates (Corning #3506) at 2 × 10 6 2uL RPMI1640 medium (Gibco#A10491) and the compound mixture were added to the 6-well plate, mixed evenly, and placed in a cell culture incubator for 18 hours.

[0050] Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and add 80 uL of pre-cooled cell lysis buffer containing PMSF (Sigma #8553S) and protease inhibitors (Roche #4693124001) to each tube. Place on ice for 60 minutes to fully lyse the cells.

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

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

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

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

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

[0056] The membrane was washed three times with TBST (CST#9997) at room temperature for 10 minutes each time, and the primary antibody (CST#4363S1:1000, CST#97166S) was prepared in protein blocking solution and incubated at 4°C overnight.

[0057] The membrane was washed three times with TBST at room temperature for 10 minutes each time, and the secondary antibody (CST#7074S1:1000, CST#7076S1:1000) was prepared in TBST containing 5% BSA and incubated at room temperature for 1 hour.

[0058] The membrane was washed 3 times with TBST at room temperature for 10 minutes each time. The luminescent substrate solution was prepared in a 1:1 ratio and added to the membrane. The gel imager (BIO-RAD chemiDoc TM MP), and scan the membrane and take pictures. The results are as follows:

[0059]

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 an IRAK4 protein degrader.

5. Use of the compound according to claim 4 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating blood tumors, psoriasis, rheumatoid arthritis, enteritis, systemic lupus erythematosus, etc.

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