A protacs molecule and uses thereof

Abstract

The application discloses a PROTAC compound with a structure of formula I and application of the PROTAC compound in preparation of a pharmaceutical preparation for preventing or treating diseases caused by mediation of a T cell receptor signal path related protein (including HPK1, LCK, GLK, PCKθ and ITK proteins), such as a malignant tumor.

Description

Technical Field

[0001] This invention relates to the field of small molecule drugs, and more specifically, to a PROTAC molecule that targets proteins related to the T cell receptor signaling pathway, and the applications of such compounds. Background Technology

[0002] The vertebrate immune system is a powerful weapon against cancer cells and foreign pathogens. To provide protection for the host, the immune system must process all intruders. In this context, T cells play a crucial role. Derived from the bone marrow, T cells mature in the thymic microenvironment under the induction of thymic hormones, forming highly differentiated, immunologically active immune cells. Mature T lymphocytes enter the peripheral immune organs via the bloodstream and can circulate through lymphatic vessels and peripheral blood, performing functions such as cellular immunity and immune regulation.

[0003] T cells have a special receptor on their surface called the T cell receptor (TCR), which can recognize antigens such as bacteria, viruses, and small protein fragments on the surface of infected or cancerous somatic cells. It converts extracellular recognition into signals that can be transmitted into the cell. By inducing the activation of tyrosine kinases near the TCR, it promotes the assembly of signal transduction complexes, activates downstream signaling pathways such as MAPK, PKC, and calcium ions, and ultimately activates corresponding transcription factors, regulates the expression of effector protein molecules, and completes the activation of T cells.

[0004] The initiation and transmission of TCR signaling are finely regulated, including both positive and negative regulatory mechanisms. Disruption of this balance can affect the effectiveness of the immune response or induce autoimmunity. Currently, the most studied negative regulatory mechanisms are mediated by inhibitory receptors, such as CTLA4 and PD-1, which have become highly sought-after targets for anti-tumor therapy in recent years. TCR-related proteins, including HPK1, LCK, GLK, PCKθ, and ITK, have become a focus of attention for pharmaceutical researchers.

[0005] Hematopoietic progenitor kinase 1 (HPK1) is an immunosuppressive regulatory kinase and a negative regulator of the T cell receptor (TCR), disrupting the stability of the TCR signaling complex. HPK1 kinase can mediate T cell dysfunction, inhibit the immune function of various cells, and inactivation of its domain is sufficient to trigger an anti-tumor immune response. Therefore, HPK1 is an extremely important candidate target for tumor immunotherapy, and HPK1 inhibitors hold promise as a rising star in tumor immunotherapy and a new partner for immune checkpoint inhibitors.

[0006] More than a decade ago, researchers discovered that HPK1 might be a potential target for cancer immunotherapy. Several compounds have entered clinical trials, such as CFI-402411, BGB-15025, and PRJ1-3024, but so far no related drugs have been marketed.

[0007] Small molecule ligands targeting TCR-related proteins, especially HPK1 (1), have been disclosed in Chinese invention patent application 202310137749.X, and their synthetic route is as follows:

[0008]

[0009] Reagents and conditions: (a) (Boc)₂O, TEA, THF, 50℃, 5h, 85%; (b) 3,4-dimethoxyphenylboronic acid, Pd(dppf)Cl₂, K₂CO₃, 1,4-Dioxane / H₂O, 110℃, 12h, 45%; (c) 4-methylpiperazinylphenylboronic acid, Pd(dppf)Cl₂, K₂CO₃, 1,4-Dioxane / H₂O, 110℃, 12h, 65%; (d) TFA, DCM, 4h, 85%.

[0010] However, current small-molecule inhibitors still lack sufficient inhibitory activity against TCR-related proteins. For example, in the development of HPK1 modulators, the main challenge lies in the different functions of HPK1 family members, making it difficult to design compounds with high selectivity and high inhibitory activity. Furthermore, small-molecule inhibitors, due to their need to occupy target proteins for extended periods, can easily induce secondary drug resistance. Therefore, novel drugs targeting TCR-related proteins remain an urgent need for public health and pharmaceutical research. Summary of the Invention

[0011] The purpose of this invention is to address the shortcomings of existing technologies by providing a PROTAC compound, a method for preparing such a compound, and its application in pharmaceuticals.

[0012] The objective of this invention is achieved through the following technical solution: a PROTACs compound, characterized in that it has the structure of Formula I:

[0013]

[0014] Wherein, L is the connecting chain, which can be attached to the 4- or 5- position of the right benzene ring. It includes nonlinear chains, aliphatic chains, aromatic chains, and heteroaromatic ring structures, and the left and right parts are connected by covalent bonds.

[0015] Furthermore, in the aforementioned PROTACs compounds, the structure of L is as follows:

[0016]

[0017] Furthermore, the structure of the PROTACs compound is as follows:

[0018]

[0019]

[0020]

[0021] This invention also provides a method for preparing PROTACs compounds, specifically including the following steps:

[0022] Step 1: The method disclosed in Chinese invention patent application 202310137749.X is used to synthesize small molecule ligands targeting TCR-related proteins (1);

[0023]

[0024] Step 2: Key intermediates 3, 6, 9, 12, 18, 21, and their analogues were prepared using the following methods and reaction conditions, where n is an integer from 1 to 10:

[0025]

[0026] As shown in S1. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60℃, 6h, 65℃; (b) TFA, DCM, 85%;

[0027]

[0028] S2. Reagents and conditions as shown below: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60℃, 6h, 65℃; (b) TFA, DCM, 85%;

[0029]

[0030] As shown in S3. Reagents and conditions: (a) tert-butylpiperidine-4-carboxylic acid ester, DIPEA, ACN, 80℃, 8h, 75%; (b) TFA, DCM, 80%;

[0031]

[0032] As shown in S4. Reagents and conditions: (a) DIPEA, DMSO, 150℃, 8h, 60-75%; (b) TFA, DCM, 85%;

[0033]

[0034] As shown in S5. Reagents and conditions: (a) K2CO3, DMF, 120℃, 8h, 65%; (b) TFA, DCM, 80%;

[0035]

[0036] As shown in S6. Reagents and conditions: (a) DIPEA, DMF, 100℃, 8h, 55-75%; (b) TFA, DCM, 80%.

[0037] Step 3: The small molecule ligand (1) targeting TCR-related proteins, especially HPK1, obtained in Step 1 and the key intermediate obtained in Step 2 are condensed in THF at 50°C in the presence of HATU and DIPEA to obtain the target product.

[0038] This invention also provides a pharmaceutical application of PROTACs compounds, specifically their use in the preparation of medicaments for the prevention or treatment of diseases mediated by T-cell receptor signaling pathway-related proteins. The T-cell receptor signaling pathway-related proteins include HPK1, LCK, GLK, PCKθ, and ITK proteins. The diseases mediated by T-cell receptor signaling pathway-related proteins include lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumors, carcinoid tumors, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer, and hematologic malignancies. The hematologic malignancies mentioned include acute T-lymphoblastic leukemia, chronic T-lymphoblastic leukemia, acute B-lymphoblastic leukemia, chronic B-lymphoblastic leukemia, plasma cell tumors, multiple myeloma, macroglobulinemia, Jekin's lymphoma, non-Hodgkin's lymphoma, essential thrombocytosis, and polycythemia vera.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] Based on the rapidly developing PROTAC (Proteolysis-targeting chimaeras) technology, this invention addresses the problems of drug resistance and insufficient activity of small molecule inhibitors through protein degradation. The PROTAC compound provided by this invention does not require long-term occupation of the active site like inhibitors; instead, it only needs a brief binding to the target protein to induce protein degradation. Since the degraded protein needs to be resynthesized to restore its function, this significantly delays the development of drug resistance; it remains effective even against proteins that have already developed resistance. Therefore, the degradative agent targeting proteins related to the T-cell receptor signaling pathway provided by this invention may have applications in the preparation of drugs for the prevention or treatment of diseases mediated by these proteins, such as various malignant tumors. Attached Figure Description

[0041] Figure 1 The degradation effect of the target compound on HPK1 protein at different drug concentrations;

[0042] Figure 2 DCs for the degradation of HPK1 protein by the target compound 50 Data chart. Detailed Implementation

[0043] The structure, preparation method and application of the present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0044] The analytical data of the samples were determined using the following instruments:

[0045] The thermometer was not calibrated; Bruker DRX400 NMR spectrometer; Agilent 5975 mass spectrometer; Bruker Vector 22 infrared spectrometer.

[0046] Example 1: Synthesis of intermediates

[0047] Ligands are commonly used ligands for targeting ubiquitinated E3 ligases in PROTAC technology. Their preparation techniques have been published in many papers. Examples of common ligation sites and preparation methods are as follows:

[0048]

[0049] As shown in S1. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60℃, 6h, 65℃; (b) TFA, DCM, 85%.

[0050]

[0051] As shown in S2. Reagents and conditions: (a) tert-butyl 2-bromoacetate, K2CO3, KI, DMF, 60℃, 6h, 65℃; (b) TFA, DCM, 85%.

[0052]

[0053] As shown in S3. Reagents and conditions: (a) tert-butylpiperidine-4-carboxylic acid ester, DIPEA, ACN, 80℃, 8h, 75%; (b) TFA, DCM, 80%.

[0054]

[0055] As shown in S4. Reagents and conditions: (a) DIPEA, DMSO, 150℃, 8h, 60-75%; (b) TFA, DCM, 85%.

[0056]

[0057] As shown in S5. Reagents and conditions: (a) K2CO3, DMF, 120℃, 8h, 65%; (b) TFA, DCM, 80%.

[0058]

[0059] As shown in S6. Reagents and conditions: (a) DIPEA, DMF, 100℃, 8h, 55-75%; (b) TFA, DCM, 80%.

[0060] Example 2: Synthesis of target product A1

[0061] Step 1: Small molecule ligands targeting TCR-related proteins, especially HPK1 (1), have been published in Chinese invention patent application 202310137749.X, and their synthetic route is as follows:

[0062]

[0063] Reagents and conditions: (a) (Boc)₂O, TEA, THF, 50℃, 5h, 85%; (b) 3,4-dimethoxyphenylboronic acid, Pd(dppf)Cl₂, K₂CO₃, 1,4-Dioxane / H₂O, 110℃, 12h, 45%; (c) 4-methylpiperazinylphenylboronic acid, Pd(dppf)Cl₂, K₂CO₃, 1,4-Dioxane / H₂O, 110℃, 12h, 65%; (d) TFA, DCM, 4h, 85%.

[0064] Step 2: The key intermediate obtained in Example 1 and the formula (1) were reacted in THF at 50°C in the presence of HATU and DIPEA to obtain the target product. The structure of A1 is as follows:

[0065]

[0066] 123 mg of A1 was obtained (yield 70%). HPLC purity 96.89%. LC-MS, ESI + ,m / z729.22[M+H] + . 1 HNMR (400MHz, DMSO) δ11.87(s,1H),11.14(s,1H),8.52(d,J=1.8Hz,1H),8.33(d,J=1.6Hz,1H),7.84(dd,J=8.6 ,5.4Hz,2H),7.65(d,J=8.6Hz,2H),7.49(d,J=1.9Hz,1H),7.38(dd,J=8.3,2.2Hz,1H),7.33-7.25(m,2H),7.10( d,J=8.7Hz,2H),7.05(d,J=8.3Hz,1H),5.21(s,2H),5.14(dd,J=12.9,5.3Hz,1H),3.86(s,3H),3.80(s,3H),3. 70-3.60(m,4H),3.33-3.26(m,2H),3.24-3.16(m,2H),2.96-2.83(m,1H),2.65-2.52(m,2H),2.11-2.00(m,1H). 13 CNMR(101MHz,DMSO)δ173.29,170.44,167.43,167.27,165.62,164.18,150.23,1 49.61,148.65,147.68,141.95,134.18,130.47,128.98,128.36,128.07,125.63, 124.92,124.27,123.68,121.67,119.06,117.89,116.83,115.12,112.90,111.00,109.60,66.59,56.05,56.03,49.44,48.93,48.64,44.22,41.53,31.43,22.52.

[0067] Example 3: Synthesis of target product A2

[0068]

[0069] Similar to Example 2, 85 mg of A2 (yield 48%) was obtained with an HPLC purity of 99.10%. LC-MS, ESI + ,m / z 729.22[M+H] + . 1 H NMR (400MHz, DMSO) δ11.86(d,J=1.9Hz,1H),11.14(s,1H),8.52(d,J=1.9Hz,1H),8.32(d,J=1.7Hz,1H),7.83(d,J=2.5H z,1H),7.82-7.75(m,1H),7.65(d,J=8.6Hz,2H),7.46(d,J=7.2Hz,1H),7.39(d,J=8.6Hz,1H),7.33-7.24(m,2H),7.10( d,J=8.7Hz,2H),7.05(d,J=8.3Hz,1H),5.33-5.21(m,2H),5.12(dd,J=12.9,5.3Hz,1H),3.86(s,3H),3.79(s,3H),3.68 -3.59(m,J=19.3Hz,4H),3.34-3.26(m,2H),3.25-3.18(m,2H),2.97-2.82(m,1H),2.66-2.51(m,2H),2.12-1.99(m,1H). 13 C NMR(101MHz,DMSO)δ173.30,170.45,167.29,165.76,165.55,156.09,150.24,149 .62,148.65,147.68,141.95,137.07,133.56,130.45,128.98,128.36,128.07,124 .92,121.27,120.68,119.06,117.89,116.84,116.60,115.94,115.12,112.91,111.00,66.55,56.06,56.03,54.02,49.22,48.64,41.57,31.42,22.47,18.53,17.18.

[0070] Example 4: Synthesis of target product A3

[0071]

[0072] Similar to Example 2, 95 mg of A3 (yield 50%) was obtained with an HPLC purity of 96.82%. LC-MS, ESI + ,m / z 782.34[M+H] + .1 H NMR (400MHz, DMSO) δ11.85(d,J=1.2Hz,1H),11.09(s,1H),8.52(d,J=1.6Hz,1H),8.33(d,J=1.3Hz,1H),7.82(d,J=2.3Hz,1H),7.65(dd ,J=10.5,8.8Hz,3H),7.40-7.27(m,3H),7.24(d,J=8.6Hz,1H),7.06(dd,J=12.9,8.5Hz,3H),5.08(dd,J=12.8,5.3Hz,1H),4.07(d,J=12 .7Hz,2H),3.86(s,3H),3.80(s,3H),3.76-3.58(m,J=33.3Hz,4H),3.28-3.13(m,J=25.2Hz,4H),3.13-2.99(m,J=22.8,11.3Hz,3H),2.9 7-2.83(m,1H),2.67-2.52(m,J=19.8,15.6Hz,2H),2.10-1.96(m,1H),1.82-1.71(m,J=11.0Hz,2H),1.71-1.59(m,J=21.7,10.6Hz,2H). 13 CNMR(101MHz,DMSO)δ173.28,172.75,170.57,168.08,167.43,155.30,150.29,1 49.67,148.67,147.74,141.95,134.51,130.46,129.00,128.41,128.04,125.47 ,124.90,124.24,119.10,118.11,117.92,116.79,115.14,113.00,111.11,108.30,56.10,56.08,49.23,48.81,47.18,45.04,41.46,37.26,31.46,27.92,22.67.

[0073] Example 5: Synthesis of target product B1

[0074]

[0075] Similar to Example 2, 95 mg of B1 (yield 50%) was obtained with an HPLC purity of 96.58%. LC-MS, ESI + ,m / z 786.19[M+H] + . 1H NMR (400MHz, DMSO) δ11.84(d,J=1.4Hz,1H),11.12(s,1H),8.51(d,J=1.7Hz,1H),8.32(d,J=1.5Hz,1H),7.82(d,J=2.4Hz,1H) ,7.58(dd,J=12.3,8.5Hz,3H),7.36-7.25(m,2H),7.12(d,J=8.6Hz,1H),7.08-6.96(m,J=15.3,8.1Hz,4H),6.58(t,J=5.4Hz, 1H),5.06(dd,J=12.8,5.3Hz,1H),3.87(s,3H),3.80(s,3H),3.72(t,J=6.3Hz,2H),3.67-3.57(m,J=4.5Hz,6H),3.51-3.42(m ,2H),3.22-3.06(m,J=16.3Hz,4H),2.96-2.77(m,1H),2.64(t,J=6.3Hz,2H),2.61-2.51(m,J=19.7Hz,2H),2.09-1.95(m,1H). 13 CNMR(101MHz,DMSO)δ173.25,170.53,169.43,169.26,167.75,150.26,149.67,148.65 ,147.74,146.85,141.94,136.69,132.57,130.33,129.02,128.42,127.99,124.89,12 4.22,119.10,117.91,117.86,116.64,115.14,113.00,111.16,111.10,109.76,69.10,67.25,56.10,56.07,55.37,49.05,48.65,45.30,42.19,41.29,33.26,31.46,22.64.

[0076] Example 6: Synthesis of target product B2

[0077]

[0078] Similar to Example 2, 105 mg of B2 (yield 53%) was obtained with an HPLC purity of 98.63%. LC-MS, ESI + ,m / z 830.06[M+H] + . 1H NMR (400MHz, DMSO) δ11.85(s,1H),11.11(s,1H),8.51(s,1H),8.31(s,1H),7.82(s,1H),7.69-7.49(m,J=21.7,7 .7Hz,3H),7.37-7.24(m,J=12.1Hz,2H),7.12(d,J=8.4Hz,1H),7.09-6.96(m,J=6.7Hz,4H),6.68-6.51(m,1H),5. 16-4.97(m,J=8.1Hz,1H),3.86(s,3H),3.80(s,3H),3.69-3.52(m,J=34.8,12.6Hz,12H),3.49-3.41(m,2H),3.2 2-3.10(m,J=14.6Hz,4H),2.97-2.80(m,J=12.8Hz,1H),2.64-2.58(m,2H),2.11-1.96(m,1H),1.29-1.26(m,2H). 13 C NMR(101MHz,DMSO)δ173.25,170.53,169.42,169.35,167.75,150.27,149.67,148.65,14 7.73,146.87,141.93,136.66,132.55,130.35,129.00,128.41,128.01,124.87,124.23, 119.09,117.90,116.69,115.12,113.00,111.13,109.73,70.20,69.36,67.35,56.10,53.81,49.05,48.67,45.26,42.19,42.07,41.24,33.27,31.45,22.62,18.45,17.17,12.67.

[0079] Example 7: Synthesis of target product B3

[0080]

[0081] Similar to Example 2, 120 mg of B3 (yield 54%) was obtained with an HPLC purity of 97.85%. LC-MS, ESI + ,m / z 874.35[M+H] + . 1H NMR (400MHz, DMSO) δ11.84(s,1H),11.10(s,1H),8.51(d,J=1.5Hz,1H),8.32(s,1H),7.82(d,J=2.2Hz,1H),7.62(d,J=8. 5Hz,2H),7.56(t,J=7.8Hz,1H),7.35-7.25(m,2H),7.11(d,J=8.6Hz,1H),7.08-6.97(m,J=11.0,4.8Hz,4H),6.59(t,J=5 .4Hz,1H),5.06(dd,J=12.8,5.3Hz,1H),3.86(s,3H),3.80(s,3H),3.71-3.58(m,J=18.1,5.5Hz,8H),3.57-3.48(m,8H), 3.48-3.39(m,2H),3.25-3.08(m,J=19.9Hz,4H),2.95-2.82(m,1H),2.67-2.56(m,J=13.4,6.9Hz,3H),2.11-1.89(m,2H). 13 C NMR(101MHz,DMSO)δ173.25,170.52,169.41,169.34,167.75,150.29,149.67,148.66,14 7.73,146.86,141.94,136.65,132.55,130.36,129.00,128.41,128.01,124.87,124.23,1 19.09,117.90,117.85,116.70,115.13,112.99,111.10,109.73,70.32,70.26,70.18,69.34,67.28,56.09,56.07,55.37,49.04,48.65,45.25,42.18,41.25,33.27,31.45,22.62.

[0082] Example 8: Synthesis of target product B4

[0083]

[0084] Similar to Example 2, 85 mg of B4 (yield 38%) was obtained with an HPLC purity of 99.46%. LC-MS, ESI + ,m / z 979.45[M+H] + . 1H NMR (400MHz, DMSO) δ11.84(d,J=1.8Hz,1H),11.12(s,1H),8.50(d,J=1.9Hz,1H),8.31(d,J=1.7Hz,1H),7.81(dd,J=5.4, 2.8Hz,2H),7.62(d,J=8.6Hz,2H),7.43(d,J=2.0Hz,1H),7.38-7.24(m,J=14.2,10.4,3.2Hz,3H),7.12-6.99(m,J=7.8Hz ,3H),5.12(dd,J=12.9,5.3Hz,1H),4.36-4.23(m,2H),3.86(s,3H),3.79(s,3H),3.78-3.73(m,2H),3.70-3.55(m,9H),3 .55-3.46(m,10H),3.25-3.09(m,J=21.7Hz,4H),2.94-2.82(m,1H),2.67-2.57(m,J=15.5,9.3Hz,3H),2.12-1.98(m,1H). 13 C NMR(101MHz,DMSO)δ173.21,170.38,169.34,167.32,167.25,164.38,150.29,149.66,14 8.65,147.72,141.93,134.35,130.36,128.99,128.40,128.01,125.71,124.86,123.51,1 21.30,119.08,117.89,116.71,115.11,112.98,111.08,109.35,70.42,70.24,70.18,69.11,68.88,67.30,56.09,56.06,49.44,49.11,48.66,45.26,41.25,33.27,31.42,22.54.

[0085] Example 9: Synthesis of target product C1

[0086]

[0087] Similar to Example 2, 100 mg of C1 (yield 55%) was obtained with an HPLC purity of 98.36%. LC-MS, ESI + ,m / z 756.32[M+H] + . 1H NMR (400MHz, DMSO) δ11.85(s,1H),11.12(s,1H),8.52(s,1H),8.33(s,1H),7.83(s,1H),7.71-7.50(m, 3H),7.41-7.26(m,2H),7.23-7.13(m,J=7.2Hz,1H),7.13-6.96(m,J=7.0Hz,4H),6.68(s,1H),5.07(d,J =6.8Hz,1H),3.87(s,3H),3.80(s,3H),3.61(d,J=18.0Hz,4H),3.25-3.06(m,4H),3.00-2.80(m,1H),2. 69-2.53(m,J=18.9Hz,2H),2.49-2.37(m,2H),2.11-1.95(m,1H),1.92-1.73(m,2H),1.43-1.15(m,2H). 13 CNMR(101MHz,DMSO)δ173.28,170.72,170.57,169.32,167.80,150.29,149.67,148 .67,147.74,146.88,141.95,136.69,132.71,130.39,129.01,128.41,128.02,124 .90,124.24,119.10,117.92,117.73,116.74,115.15,112.99,111.09,110.88,109.61,56.08,55.37,49.03,48.69,45.05,42.07,41.36,31.47,29.84,24.69,22.65.

[0088] Example 10: Synthesis of target product C2

[0089]

[0090] Similar to Example 2, 110 mg of C2 (yield 58%) was obtained with an HPLC purity of 97.35%. LC-MS, ESI + ,m / z 784.35[M+H] + . 1H NMR(400MHz,DMSO)δ11.85(s,1H),11.11(s,1H),8.52(s,1H),8.32(s,1H),7.82(s,1H),7.71-7.47(m,3H),7 .41-7.22(m,2H),7.18-6.91(m,J=7.1Hz,5H),6.53(s,1H),5.06(d,J=7.1Hz,1H),3.86(s,3H),3.80(s,3H), 3.71-3.50(m,4H),3.33-3.24(m,2H),3.24-3.07(m,4H),2.89(t,J=13.0Hz,1H),2.69-2.53(m,J=16.7Hz,2H ),2.43-2.27(m,2H),2.14-1.94(m,1H),1.60-1.56(m,2H),1.45-1.31(m,2H),1.29-1.10(m,J=13.8Hz,2H). 13 C NMR(101MHz,DMSO)δ173.26,171.05,170.56,169.43,167.77,150.30,149.67,148.67 ,147.73,146.90,141.95,136.73,132.66,130.38,129.01,128.41,128.01,124.89,12 4.24,119.09,117.91,117.61,116.73,115.14,112.99,111.10,110.84,109.49,56.07,55.37,49.03,48.75,45.17,42.24,41.26,32.61,31.46,29.03,26.55,24.99,22.65.

[0091] Example 11: Synthesis of target product C3

[0092]

[0093] Similar to Example 2, 98 mg of C3 (yield 50%) was obtained with an HPLC purity of 98.66%. LC-MS, ESI + ,m / z 812.38[M+H] + . 1H NMR (400MHz, DMSO) δ11.85(s,1H),11.11(s,1H),8.52(s,1H),8.32(s,1H),7.82(s,1H),7.62(d,J=7.8Hz,2H),7.57(t,J=7.7Hz,1H), 7.37-7.25(m,J=13.3Hz,2H),7.15-6.97(m,J=11.3,8.1Hz,5H),6.52(t,J=5.0Hz,1H),5.06(dd,J=12.8,5.0Hz,1H),3.86(s,3H),3.8 0(s,3H),3.69-3.52(m,J=21.3Hz,4H),3.33-3.23(m,J=6.1Hz,2H),3.23-3.07(m,J=15.2Hz,4H),2.97-2.81(m,1H),2.68-2.52(m,2H ),2.33(t,J=6.9Hz,2H),2.10-1.95(m,1H),1.62-1.48(m,J=15.5,6.7Hz,4H),1.34-1.30(m,4H),1.27-1.17(m,J=18.1,11.3Hz,2H). 13 C NMR(101MHz,DMSO)δ173.26,171.14,170.55,169.45,167.77,150.30,149.67,148.67,147.73, 146.90,141.94,136.72,132.66,130.40,129.00,128.42,128.01,124.88,124.22,119.09,117 .91,117.61,116.74,115.14,112.99,111.10,110.84,109.50,56.09,56.07,55.37,49.17,49.03,48.76,45.16,42.30,41.24,32.68,31.46,29.20,29.12,29.07,26.71,25.20,22.93,22.64.

[0094] Example 12: Synthesis of target product C4

[0095]

[0096] Similar to Example 2, 75 mg of C4 (yield 36%) was obtained with an HPLC purity of 97.25%. LC-MS, ESI + ,m / z 868.44[M+H] + . 1H NMR (400MHz, DMSO) δ11.83(d,J=1.7Hz,1H),11.10(s,1H),8.51(d,J=1.8Hz,1H),8.31(d,J=1.6Hz,1H),7.81(d,J=2.4Hz,1H),7.6 6-7.60(m,J=8.6Hz,2H),7.59-7.52(m,1H),7.34-7.25(m,2H),7.10-6.98(m,5H),6.50(t,J=5.7Hz,1H),5.05(dd,J=12.9,5.3Hz, 1H),3.86(s,3H),3.79(s,3H),3.67-3.54(m,4H),3.27(dd,J=13.0,6.6Hz,2H),3.23-3.11(m,J=18.4Hz,4H),2.94-2.79(m,1H),2 .64-2.52(m,J=18.9,10.3Hz,2H),2.33(t,J=7.4Hz,2H),2.08-1.97(m,J=12.2,6.6Hz,1H),1.62-1.44(m,5H),1.29-1.24(m,13H). 13 C NMR (101MHz, DMSO) δ173.24,171.15,170.53,169.43,167.77,167.44,150.31,149.67,148.66,147.73,146.90,141.9 3,136.71,132.65,132.20,132.03,130.41,129.12,128.99,128.42,128.01,124.87,124.22,119.08,117.90,117.60 ,116.74,115.12,112.99,111.10,110.82,109.49,67.87,56.09,56.06,49.02,48.78,45.19,42.31,41.25,38.56,32.71,31.45,30.27,29.41,29.35,29.24,29.13,28.83,26.77,25.28,23.72,22.94,22.86,22.63,14.34,11.70,11.25.

[0097] Application Example 1: Determination of biological activity

[0098] Inhibition assay of HPK1 kinase

[0099] 1. Dilute the compound 3-fold with DMSO in a dilution plate, with an initial concentration of 1-10 μM.

[0100] 2. Dilute the compound 50-fold in 1x kinase reaction buffer and shake on a shaker for 20 minutes.

[0101] 3. Prepare 2x kinase using 1x enzyme reaction buffer.

[0102] 4. Add 2 μL of kinase (prepared in step 3) to each well of the reaction plate.

[0103] 5. Add 1 μL of the compound diluted in buffer to each well, seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and let stand at room temperature for 10 minutes.

[0104] 6. Prepare a 4x MBP Protein and ATP (final ATP concentration 10 μM) mixture using 1x enzyme reaction buffer, and add 1 μL of the 4x MBP Protein / ATP mixture to the reaction plate.

[0105] 7. Seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and react at room temperature for 60 minutes.

[0106] 8. Transfer 4 μL of ADP-Glo ​​to a 384 reaction plate, centrifuge at 1000 rpm / min for 1 min, and incubate at 25 °C for 40 min.

[0107] 9. Transfer 8 μL of detection solution to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 40 min.

[0108] 10. Use a Biotek multi-function plate reader to read the RLU (Relative Luminescence Unit) signal. The signal intensity is used to characterize the activity level of the kinase.

[0109] Data processing

[0110] Calculate the inhibition rate for each well and calculate the IC. 50 value.

[0111] Application Example 2: Western Blotting Experiment

[0112] HPK1-positive MCF-7 cell lines were treated with different concentrations of the test substance. After 12 hours of treatment, the cells were collected and lysed (50 mM NaCl, 5 mM EDTA, 0.5% SDS, 0.1 mM sodium orthovanadate, 50 μg / ml lapatinin, 1 mM phenylmethysulfonyl fluoride, and 10 mM Tris-HCl; pH 7.4), then incubated on ice for 30 min, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was used for protein quantification and SDS-PAGE. After electrophoresis, the samples were transferred to nitrocellulose membranes and then subjected to immunoassay. The membranes were blocked at room temperature for 1 h, incubated overnight at 4°C with TMPRSS2 (or TMPRSS2 downstream effector protein) primary antibody, incubated at room temperature with secondary antibody for 2 h, washed with TBST for 2 h, and then incubated with ECL solution for 1 min. After draining the membrane, it was exposed to the dark for several minutes, developed, and fixed. Histone H3 was used as an internal control for statistical analysis.

[0113] The results are shown in Table 1 and Appendix. Figure 1 Appendix Figure 2 .

[0114] Table 1. Structure and pharmacological activity of the PROTAC compounds of the present invention

[0115]

[0116]

[0117] ND: Not measured.

[0118] It is evident that these PROTAC compounds still possess HPK1 inhibitory activity, with A2 exhibiting the highest inhibitory activity (IC50 = 28.17 nM). Simultaneously, these PROTAC compounds demonstrate HPK1 protein degradation ability. At a concentration of 0.1 μM, B1 achieves 100% degradation of HPK1, B2 94.41%, and C2 74.06%; while at a concentration of 1 μM, B1 achieves 100% degradation, B2 95.80%, B4 93.14%, and C2 86.94%; at higher concentrations, such as 10 μM, most compounds show high degradation ability, but the unique "hook" effect of PROTAC compounds is also evident.

Claims

1. A PROTACs compound, characterized in that, Selected from the following structures: ； Formula I-1; ； Formula I-2; ； Formula I-3; ； Formula I-4; ； Formula I-5; ； Formula I-6; ； Formula I-7; ； Formula I-8; ； Formula I-9; ； Formula I-10; ； Formula I-11.

2. The use of the PROTACs compound as described in claim 1 in the preparation of a medicament for the prevention or treatment of diseases mediated by T-cell receptor signaling pathway-related proteins, characterized in that, The T-cell receptor signaling pathway-related protein is HPK1.

3. The application according to claim 2, characterized in that, The diseases mediated by T-cell receptor signaling pathway-related proteins include lymphoma, medulloblastoma, retinoblastoma, liposarcoma, synovial cell sarcoma, carcinoid tumors, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, peritoneal cancer, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer, or hematologic malignancies.

4. The application according to claim 3, characterized in that, The hematologic malignancies mentioned are acute T-lymphoblastic leukemia, chronic T-lymphoblastic leukemia, acute B-lymphoblastic leukemia, chronic B-lymphoblastic leukemia, multiple myeloma, macroglobulinemia, Jekin's lymphoma, non-Hodgkin's lymphoma, essential thrombocytosis, or polycythemia vera.

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