PROTAC protein targeted degradation agent and application thereof

Abstract

The invention belongs to the field of pharmacy, and relates to a protein degradation targeting chimera (PROTAC) taking PD-L1 as a target spot and medical application thereof. The protein degradation targeting chimera is a compound composed of a target protein ligand, an E3 ligase ligand and a linker and a pharmaceutically acceptable salt of the compound, the specific structure of the compound is as shown in a formula I, and substituent groups are described in the specification in detail. A Western blot experiment (Western Blot) shows that the compound disclosed by the invention can be used for degrading the PD-L1 protein in a targeted manner.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical science and is a PROTAC protein-targeting degrader and its application. Background Technology

[0002] Cancer immunotherapy, a therapeutic strategy that specifically targets and eliminates tumor cells by activating the host's immune system, has received widespread attention over the past decade. The concept of the immune system's anti-tumor function can be traced back to the 1890s, proposed by William Coley. Current major treatment approaches in this field include tumor-specific T-cell transplantation, immune checkpoint inhibitors (ICIs), and anti-cancer vaccines, all aimed at restoring the body's inherent anti-tumor immune response. In recent years, numerous small-molecule inhibitors have been developed to block the immune escape mechanisms of tumor cells, thereby reactivating the immune system and promoting tumor killing. In particular, small-molecule inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1 have emerged.

[0003] However, small molecule inhibitors targeting PD-L1 often suffer from limitations such as high dosage, easy development of drug resistance, and off-target effects, resulting in less than ideal anti-tumor efficacy. The emergence of protein degradation chimera (PROTAC) technology provides a new approach to overcome these shortcomings. As an emerging drug development paradigm, PROTAC has attracted increasing attention. PROTAC molecules have a heterobifunctional structure, capable of simultaneously binding to the target protein and E3 ubiquitin ligase, inducing ubiquitination modification of the target protein, and degradation via the 26S proteasome pathway. Its structure contains three key parts: one end binds to the target protein, the other end recruits the E3 ligase, and the two are bridged by a linker chain in the middle to form a stable ternary complex. However, different linker structures have different effects. Although PD-L1 biphenyl small molecule inhibitors have been widely studied due to their good inhibitory effects, they are still limited by problems such as high toxicity, drug resistance, and off-target effects. Summary of the Invention

[0004] The purpose of this invention is to design and synthesize PROTAC molecules that target and degrade PD-L1 protein, thereby restoring the body's immune function, killing cancer cells, and ultimately providing a PROTAC protein-targeting degrader and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A PROTAC protein-targeting degrader, wherein the PROTAC protein-targeting degrader is a compound of Formula 1 and its pharmaceutically acceptable salt.

[0007]

[0008] In the formula, part A is selected from structural fragments VHL-A, VHL-B, CRBN, NF, and NC, and their structures are shown below:

[0009]

[0010] In the formula, linker is selected from Furthermore, when the linker is selected When A does not select VHL-A, VHL-B, CRBN, or NC; n is an integer from 3 to 7.

[0011] The PROTAC protein-targeting degrader is

[0012]

[0013]

[0014]

[0015] An application of the PROTAC protein-targeting degrader, wherein the PROTAC protein-targeting degrader compound and its salt are used in the degradation of proteins targeting PD-L1.

[0016] The use of the PROTAC protein-targeting degrader compound and its salt in the preparation of a medicament for treating PD-L1 protein-related diseases.

[0017] The PD-L1 protein-related diseases mentioned above are tumors or other PD-L1 protein-related diseases.

[0018] The tumors mentioned are colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck squamous cell carcinoma, breast cancer, prostate cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, and multiple myeloma.

[0019] Other PD-L1 protein-related diseases mentioned include autoimmune diseases. Attached image description:

[0020] Figure 1 The results show the effects of the compounds in this invention on the degradation of PD-L1 protein in MC-38 cells. Detailed Implementation

[0021] To facilitate understanding of the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below through embodiments. The present invention can be implemented in many different forms, including but not limited to the embodiments described herein.

[0022] Example 1: Synthesis of compound VA-4S-B1

[0023] (1) Synthesis of compound VA-4S-Boc

[0024]

[0025] VHL-A (400 mg, 0.93 mmol), cis-3-(Boc-amino)cyclobutanecarboxylic acid (166.68 mg, 0.77 mmol), N,N-diisopropylethylamine (200.29 mg, 1.55 mmol), EDCI (178.24 mg, 0.93 mmol), and HOBt were administered.

[0026] (125.63 mg, 0.93 mmol) was added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography (dichloromethane:methanol = 8:1) to give VA-4S-Boc (350 mg, 60%) as a white solid. MS m / z: 650.4 [M + Na] + .

[0027] (2) Synthesis of compound VA-4S

[0028]

[0029] VA-4S-Boc (350 mg, 0.56 mmol) was dissolved in 1,4-dioxane hydrochloride solution (4 M, 5 mL). The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum to give VA-4S (285.38 mg, 97%) as a white solid. MS m / z: 528.36 [M+H] + .

[0030] (3) Synthesis of compound VA-4S-B1

[0031]

[0032] VA-4S (50 mg, 0.09 mmol), BMS-1001 (46.96 mg, 0.08 mmol), N-methylmorpholine (20.43 mg, 0.16 mmol), EDCI (18.18 mg, 0.09 mmol), and HOBt (12.81 mg, 0.09 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. VA-4S-B1 (10.46 mg, 10%) was obtained as a white solid by high performance liquid chromatography. HRMS m / z: 1104.4896 [M+H] + . 1 H NMR (600MHz, CD3OD) δ8.87(s,1H),7.93(s,1H),7.80(d,J=7.8Hz,1H),7.71(d,J=7.74Hz,1H ),7.57(t,J=7.74Hz,1H),7.46(d,J=8.22Hz,2H),7.41(d,J=8.28Hz,2H),7.34(d,J=8.22Hz, 1H),7.19(t,J=7.62Hz,1H),7.16(dd,J=1.26,7.68Hz,1H),7.11(s,1H),6.88(d,J=8.16Hz, 1H),6.77(s,1H),6.74-6.71(m,2H),5.26(s,2H),5.12(s,2H),4.60(s,1H),4.55-4.52(m,2H ),4.50-4.49(m,1H),4.35(d,J=15.48Hz,1H),4.27(s,3H),4.17(t,J=8.04Hz,1H),4.12(d, J=13.2Hz,1H),4.06(d,J=13.2Hz,1H),3.89(d,J=11.1Hz,1H),3.82-3.78(m,2H),3.71(q,J= 6.54Hz,1H),3.54(t,J=5.64Hz,1H),2.91-2.85(m,2H),2.46(s,3H),2.40-2.36(m,2H),2.2 3(s,3H),2.16(s,3H),2.12-2.06(m,2H),2.03-1.95(m,2H),1.32-1.28(m,2H),0.99(s,9H). 13C NMR(151MHz,CD3OD)δ175.96,174.46,172.23,157.52,157.31,152.89,1 49.08,144.70,144.27,143.90,140.66,140.25,137.22,136.78,136.54, 135.36,134.51,134.25,133.98,133.46,133.22,132.91,132.25,131.56 ,131.06,130.86,130.41,129.01,128.61,126.58,123.53,123.43,120.7 7,119.66,119.27,119.18,117.96,113.85,113.63,99.18,71.14,71.09,70.77,70.59,70.55,65.77,65.74,60.86,60.77,59.99,59.85,59.71,59 .57,59.16,58.02,57.91,43.73,41.92,39.04,36.76,36.48,34.94,33.89,33.43,33.06,27.08,27.03,16.63,16.47,15.87,15.84,15.76,15.72.

[0033] Example 2: Synthesis of compound VA-4F-B1

[0034]

[0035] It was prepared using the same method as compound VA-4S-B1, and is a white solid with HRMS m / z: 1104.4900 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.86(s,1H),7.88(s,1H),7.80(d,J=7.14Hz,1H),7.71(d,J=7.56Hz,1H),7.59(dd,J=3.48,7.74Hz,1H),7.46-7.44(m,2H),7.41(d,J=8.04Hz,2H),7.36-7.30(m,3H),7.19-7.14(m,4H),7.11(s,1H),6.88(dd,J=3.3,8.1Hz,1H),6.76-6.70(m,3H),5.23(t,J=3.78Hz,2H),5.13(d,J=8.34Hz,2H),4.65-4.62(m,1H),4.59-4.50(m,4H),4.40(t,J=7.74Hz,1H),4.35-4.32(m,2H),4.28(d,J=3.84Hz,5H),4.07-3.98(m,4H),3.93-3.89(m,3H),3.82-3.78(m,2H),3.70-3.67(m,2H),3.59(d,J=12.42Hz,1H),3.51-3.49(m,1H),3.11-3.06(m,2H),2.46(s,1H),2.40(s,1H),2.23(d,J=6Hz,3H),2.16(s,2H),2.11(s,1H),1.02(d,J=24.18Hz,9H). 13C NMR(151MHz,CD3OD)δ174.18,159.73,157.10,152.84,149.07,149.06,144.69 ,144.26,144.25,143.87,140.33,140.24,136.85,136.55,135.27,134.31,13 3.34, 132.84, 132.28, 131.57, 130.98, 130.84, 130.40, 129.02, 128.89, 128.50, 126.54, 123.43, 120.73, 119.69, 119.18, 117.94, 113.76, 99.43, 71.16, 71.1 5,70.92,70.55,70.53,70.51,70.45,65.76,65.74,62.01,61.02,59.85,59.71,59.57,58.09,56.25,43.75,39.04,38.97,38.93,36.76,36.73,36.59,33.12 ,30.88,30.79,30.77,30.70,30.65,30.52,30.50,30.38,30.37,28.81,28.15,27.05,23.79,16.42,15.88,15.85,15.84,15.77,15.74,15.70,15.63,14.49.

[0036] Example 3: Synthesis of compound VA-5S-B1

[0037]

[0038] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1118.5060 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.86(s,1H),7.91(s,1H),7.80(d,J=7.79Hz,1H),7.70(d,J=7.74Hz,1H),7.56(t,J=7.74Hz,1H),7.46(d,J=8.22Hz,2H),7.41(d,J=8.28Hz,2H),7.34(d,J=6.48Hz,1H),7.19(t,J=7.62Hz,1H),7.15(d,J=6.24Hz,1H),7.09(s,1H),6.87(d,J=8.16Hz,1H),6.74-6.71(m,3H),5.23(s,2H),5.11(s,2H),4.48-4.52(m,3H),4.48(t,J=1.68Hz,1H),4.35(d,J=15.42Hz,1H),4.27(s,4H),4.12(t,J=6.36Hz,1H),3.95(s,1H),3.89(d,J=11.04Hz,1H),3.78-3.73(m,2H),3.66(dd,J=6.66,11.22Hz,1H),3.42-3.41(m,1H),2.89(t,J=7.98Hz,1H),2.46(s,2H),2.23(s,3H),2.16(s,3H),2.12-2.05(m,3H),1.85-1.81(m,3H),1.59-1.55(m,3H),1.29-1.28(m,3H),1.00(s,9H). 13C NMR (151MHz, CD3OD) δ178.44,177.66,175.63,174.47,172.47,172.29,159.55,158.80,157.32,157.09 ,152.89,149.08,144.69,144.26,143.88,140.56,140.42,140.32,140.24,137.13,136.91,136.56,13 5.68, 135.33, 134.45, 134.24, 133.79, 133.46, 133.24, 132.86, 132.67, 132.22, 131.57, 131.01, 130.85, 130.41, 129.57, 129.02, 128.58, 126.56, 123.46, 123.43, 120.60, 120.27, 119.68, 119.20, 118.91, 11 7.94, 113.81, 113.67, 99.36, 99.28, 71.18, 71.09, 70.66, 70.55, 70.53, 70.22, 65.76, 65.74, 62.55, 62.53, 60.84, 59.99, 59.85, 59.71, 59.56, 59.43, 59.29, 59.19, 58.00, 57.91, 52.59, 51.65, 51.64, 51.53 ,51.52,47.39,44.13,44.10,43.74,39.09,39.04,38.11,37.67,36.66,33.73,33.47,33.12,30.88,30.78,30.70,30.68,30.52,30.37,29.06,29.05,28.15,27.09,23.79,16.54,16.46,15.85,15.80,14.49.

[0039] Example 4: Synthesis of compound VA-5F-B1

[0040]

[0041] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1118.5055 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.85(s,1H),8.45(s,2H),7.89(s,1H),7.79(d,J=7.68Hz,1H),7.70(d,J=7.62Hz,1H),7.56(t,J=7.74Hz,1H),7.46(d,J=7.98Hz,2H),7.40(d,J=7.68Hz,2H),7.33(d,J=7.5Hz,1H),7.19-7.13(m,4H),7.10(s,1H),6.88-6.85(m,1H),6.75(s,1H),6.73-6.70(m,3H),5.23(s,2H),5.12(s,2H),4.63(s,1H),4.56-4.52(m,3H),4.49(s,1H),4.34(d,J=15.48Hz,1H),4.27(d,J=4.5Hz,4H),4.20(t,J=6.12Hz,1H),4.01(s,2H),3.89(d,J=11.34Hz,1H),3.81(dd,J=3.72,11.04Hz,2H),3.69(q,J=6.54Hz,1H),3.49(t,J=5.22Hz,1H),2.93(t,J=8.1Hz,1H),2.45(s,3H),2.22(s,3H),2.16(s,3H),1.74-1.68(m,2H),1.62-1.57(m,2H),1.51-1.45(m,2H),1.29(s,2H),1.02(s,9H). 13C NMR(151MHz,CD3OD)δ177.83,174.45,172.34,159.68,157.32,157.14,156.99,152.87,149.06,1 44.67,144.25,143.87,140.51,140.31,140.27,137.03,136.85,136.53,135.34,134.49,134.19 ,133.45,133.36,133.29,132.94,132.92,132.86,132.29,132.25,132.21,131.55,131.01,130.94,130.91,130.84,130.50,130.40,129.46,129.01,128.89,128.68,128.59,128.48,126.56,12 3.43,120.70,119.66,119.17,117.94,113.79,99.34,71.13,70.59,70.55,70.22,65.75,65.73,63.12,62.30,61.97,61.03,60.90,60.87,59.99,59.84,59.71,59.56,59.42,58.98,58.09,56.2 6,52.55,51.75,51.51,49.61,47.23,47.20,44.16,43.93,43.73,39.05,38.95,36.95,36.83,33.75,33.11,30.87,30.77,30.64,30.42,30.22,28.15,27.05,23.78,16.44,15.85,15.76,14.49.

[0042] Example 5: Synthesis of compound VA-4L-B1

[0043]

[0044] It was prepared using the same method as compound VA-4S-B1, as a white solid, HRMS m / z: 1145.5251 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.86(s,1H),7.91(s,1H),7.78(t,J=6.84Hz,1H),7.70(d,J=8.7Hz,1H),7.58-7.55(m,1H),7.46-7.45(m,2H),7.41-7.40(m,2H),7.36-7.33(m,1H),7.20-7.14(m,2H),7.10(s,1H),5.23-5.21(m,2H),5.13(s,2H),4.61(s,1H),4.55-4.49(m,3H),4.35(d,J=15.42Hz,1H),4.27(d,J=1.86Hz,4H),4.08-4.04(m,2H),4.01(dd,J=4.14,12.96Hz,1H),3.89(d,J=11.28Hz,1H),3.80-3.77(m,2H),3.68-3.64(m,1H),3.48(t,J=4.74Hz,1H),3.09-3.03(m,1H),2.46(s,3H),2.30-2.19(m,8H),2.17(d,J=4.14Hz,3H),2.11-2.04(m,2H),2.03(s,1H),1.91-1.77(m,3H),1.29-1.28(m,2H),1.01(d,J=6.18Hz,9H). 13C NMR(151MHz,CD3OD)δ176.96,174.47,174.46,172.33,159.82,157.41,152.88,1 49.07,144.69,144.26,143.88,140.32,140.31,136.83,136.52,135.31,134.31 ,133.46,133.22,133.19,132.88,132.18,131.55,131.03,130.86,130.41,129.00,128.53,126.58,123.43,120.69,119.65,119.18,117.94,113.84,99.24,71. 13,70.54,65.73,62.97,62.90,62.22,62.17,60.87,59.99,59.85,59.70,59.56,59.42,58.97,58.06,47.30,47.17,43.84,43.73,43.00,42.91,41.42,40.71,3 9.03, 38.97, 38.28, 38.12, 37.39, 36.77, 35.30, 35.24, 35.00, 34.79, 33.11, 30.88, 30.79, 30.67, 30.50, 30.37, 28.15, 27.02, 23.78, 16.44, 15.85, 15.77, 15.75.

[0045] Example 6: Synthesis of compound VB-4S-B1

[0046]

[0047] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1118.5060 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.87(s,1H),7.93(s,1H),7.80(d,J=7.8Hz,1H),7.71(d,J=7.8Hz,1H),7.57(t,J=7.74Hz,1H),7.44(dd,J=8.4,18.36Hz,4H),7.34(d,J=7.44Hz,1H),7.19(t,J=7.56Hz,1H),7.16(d,J=7.62Hz,1H),7.11(s,1H),6.88(d,J=8.1Hz,1H),6.77(s,1H),6.74-6.71(m,2H),5.26(s,2H),5.12(s,2H),5.01(dd,J=6.9,13.92Hz,1H),4.59(s,1H),4.56(dd,J=8.34,16.5Hz,1H),4.43(t,J=1.8Hz,1H),4.27(s,4H),4.18(t,J=7.74Hz,1H),4.12(d,J=13.14Hz,1H),4.06(d,J=13.02Hz,1H),3.86-3.80(m,2H),3.75-3.68(m,2H),3.54(t,J=4.98Hz,1H),2.92-2.84(m,2H),2.47(s,3H),2.23(s,3H),2.16(s,3H),1.50(d,J=7.02Hz,3H),1.00(s,9H). 13C NMR(151MHz,CD3OD)δ175.98,173.23,172.19,160.03,157.50,152.91,149 .13,145.69,144.70,144.27,143.90,140.26,137.21,136.79,136.54,135. 36, 134.49, 134.27, 133.92, 133.39, 133.22, 132.91, 132.76, 132.24, 131.58, 131.06, 130.86, 130.54, 130.52, 129.44, 128.61, 127.66, 126.58, 123.58 ,123.43,120.77,119.66,119.32,119.18,117.96,117.94,113.85,113.64,99.61,99.19,71.02,70.59,70.55,65.76,65.74,62.68,61.93,60.61,59.2 0,57.99,57.85,50.18,47.18,47.17,41.93,38.89,36.66,36.51,34.91,33.91,33.42,33.11,27.10,27.05,22.42,22.22,16.63,16.47,15.83,15.76.

[0048] Example 7 Synthesis of compound VB-4F-B1

[0049]

[0050] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1118.5055 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.87(s,1H),8.40(s,2H),7.89(s,1H),7.80(d,J=7.74Hz,1H),7.72(d,J=7.62Hz,1H),7.58(t,J=7.8Hz,1H),7.44(dd,J=8.34,17.58Hz,4H),7.34(d,J=7.2Hz,1H),7.19(t,J=7.56Hz,1H),7.16(d,J=6.78Hz,1H),7.11(s,1H),5.24(s,2H),5.13(s,2H),5.01(q,J=6.96Hz,1H),4.63(s,1H),4.56(t,J=8.4Hz,1H),4.44-4.40(m,2H),4.27(s,4H),4.08(q,J=12.48Hz,3H),3.90(d,J=11.16Hz,1H),3.82(dd,J=4.44,11.22Hz,1H),3.77(dd,J=3.78,11.1Hz,1H),3.71(q,J=6.42Hz,1H),3.55-3.51(m,2H),3.11-3.07(m,2H),2.47(s,3H),2.23(s,3H),2.17(s,3H),1.98-1.93(m,2H),1.61(d,J=7.08Hz,1H),1.50(d,J=7.02Hz,2H),1.29-1.28(m,2H),1.03(s,9H). 13C NMR (151MHz, CD3OD) δ177.31,173.25,172.33,172.30,157.44,152.91,149.13,145.68,144.69,144.27,143.90,140.23,136.98,136.8 0,136.54,135.47,135.34,134.56,134.36,133.41,133.29,132.98,132.26,132.21,131.58,131.04,130.93,130.54,128.60,127.66,1 26.58,123.43,120.76,119.64,119.54,119.18,117.95,113.83,99.26,71.04,70.69,70.60,70.55,70.53,65.76,65.74,60.65,59.85,59.71,59.57,59.17,58.06,50.18,44.49,38.83,36.67,36.59,34.81,34.63,34.26,33.60,27.07,22.43,16.54,16.43,15.83,15.73.

[0051] Example 8: Synthesis of compound VB-5S-B1

[0052]

[0053] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1132.5221 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.87(s,1H),7.92(s,1H),7.80(d,J=8.1Hz,1H),7.70(d,J=7.68Hz,1H),7.57(t,J=7.74Hz,1H),7.44(q,J=8.4Hz,4H),7.34(d,J=8.7Hz,1H),7.19(t,J=7.56Hz,1H),7.16(d,J=7.56Hz,1H),7.12(s,1H),6.88(d,J=8.16Hz,1H),6.76(s,1H),6.74-6.71(m,2H),5.25(s,2H),5.13(s,2H),5.01(q,J=6.96Hz,1H),4.56(t,J=3.84Hz,2H),4.41(t,J=1.86Hz,2H),4.27(s,4H),4.15(q,J=6.18Hz,2H),4.03(s,2H),3.86(d,J=11.1Hz,1H),3.80(dd,J=4.74,11.46Hz,1H),3.73-3.67(m,3H),3.51(t,J=5.1Hz,1H),2.91(t,J=7.62Hz,2H),2.47(s,3H),2.23(s,3H),2.16(s,3H),1.97-1.92(m,2H),1.90-1.78(m,5H),1.61-1.53(m,3H),1.50(d,J=7.02Hz,3H),1.01(s,9H). 13C NMR (151MHz, CD3OD) δ178.48,177.69,175.63,173.25,172.44,172.25,158.80,157.41,157.08,1 52.91,149.13,145.70,144.70,144.68,144.25,143.86,140.55,140.33,137.13,136.85,136.60, 136.55, 135.63, 135.34, 134.39, 133.77, 133.39, 133.25, 132.89, 132.65, 132.25, 131.58, 131.05, 131.03, 130.83, 130.54, 128.96, 128.58, 127.68, 126.57, 126.54, 123.43, 120.72, 120.30, 119.6 7,119.21,119.18,117.95,117.94,113.84,113.67,99.39,99.25,71.07,70.97,70.65,70.56,70.55,70.22,66.06,65.76,65.74,60.60,60.59,59.85,59.71,59.56,59.38,59.21,57.98,57.87,52 .69,52.68,51.66,51.51,50.19,50.16,44.12,44.07,40.98,38.91,38.17,37.58,36.60,36.54,33.73,33.48,29.11,29.03,27.11,26.98,22.42,22.37,17.45,17.33,16.54,16.46,15.83,15.79.

[0054] Example 9: Synthesis of compound VB-5F-B1

[0055]

[0056] It was prepared using the same method as compound VA-4S-B1, and was a white solid with HRMS m / z: 1132.5209 [M+H]. + . 1H NMR(600MHz,CD3OD)δ8.87(s,1H),8.42(s,2H),7.90(s,1H),7.80(d,J=7.86Hz,1H),7.70(t,J=7.62Hz,1H),7.58-7.56(m,1H),7.44(dd,J=8.46,17.82Hz,4H),7.35-7.31(m,1H),7.19(dd,J=3.66,7.44Hz,1H),7.16(dd,J=1.32,7.62Hz,1H),7.11(s,1H),6.88(d,J=8.16Hz,1H),6.77(s,1H),6.74-6.71(m,3H),5.24(s,2H),5.14(s,2H),5.01(dd,J=6.96,13.98Hz,1H),4.62(s,1H),4.56(dd,J=8.46,16.62Hz,1H),4.46-4.42(m,1H),4.27(d,J=2.34Hz,5H),4.22(dd,J=5.52,12.96Hz,1H),4.03(s,1H),3.87(d,J=11.1Hz,1H),3.82(q,J=4.62Hz,1H),3.75(dd,J=3.9,10.98Hz,1H),3.70(q,J=6.66Hz,1H),3.52(dd,J=5.76,9.84Hz,1H),2.94(t,J=7.86Hz,1H),2.47(s,2H),2.45(s,1H),2.23(d,J=4.5Hz,3H),2.17(d,J=2.82Hz,3H),2.09-2.01(m,4H),1.97-1.94(m,1H),1.75-1.70(m,1H),1.62-1.55(m,2H),1.48(t,J=7.14Hz,3H),1.29(s,2H),1.03(s,9H). 13C NMR (151MHz, CD3OD) δ177.82,177.78,175.44,173.27,173.22,172.88,172.32,172.27,160.08,159.86,158.80,157.40,157.2 5,157.00,152.91,149.12,145.70,145.65,145.52,144.69,144.26,144.25,143.90,143.89,140.53,140.22,137.04,136.82,1 36.79, 136.59, 136.53, 135.40, 135.35, 135.30, 134.60, 134.50, 134.27, 133.42, 133.30, 133.25, 132.98, 132.93, 132.36, 132.26, 132.25, 131.63, 131.58, 131.56, 131.04, 131.01, 130.94, 130.93, 130.91, 130.54, 130.48, 128.68, 128.60, 128.51, 127.66, 127.63,127.52,126.57,126.55,123.44,123.42,120.91,120.78,119.67,119.66,119.56,119.22,119.18,117.94,113.84,113.83,113.78,99.65,99.45,99.33,71.01,70.80,70.73,70.63,70.56,70.53,65.76,65.74,60.82,60.65,60.63,59.05,59.02, 58.07,58.03,52.59,50.17,49.61,44.15,43.87,43.86,39.05,38.80,38.80,37.31,36.88,36.76,36.68,33.78,33.60,33.12,30.88,30.79,30.68,30.65,30.45,30.38,30.17,28.15,27.06,22.43,22.37,16.50,16.45,16.42,15.83,15.76,15.64,14.49.

[0057] Example 10 Synthesis of compound VB-4L-B1

[0058]

[0059] It was prepared by the same method as compound VA-4S-B1, as a white solid with HRMS m / z: 1159.5415 [M+H]+. 1 H NMR (600MHz, CD3OD) δ8.87(s,1H),7.92(s,1H),7.79(t,J=6.36Hz,1H),7.71(d,J=7.68Hz,1H),7.5 9-7.56(m,1H),7.44-7.40(m,4H),7.35-7.32(m,1H),7.19(t,J=7.62Hz,1H),7.16-7.15(m,1H),7. 11(s,1H),6.88(d,J=8.1Hz,1H),6.76(d,J=2.22Hz,1H),6.74-6.71(m,3H),5.24(d,J=3.84Hz,2H) ,5.13(s,2H),5.01(q,J=6.72Hz,1H),4.60(s,1H),4.57(dd,J=8.22,14.22Hz,2H),4.43(s,1H),4. 27(s,4H),4.08-4.06(m,2H),4.02-4.00(m,2H),3.87(d,J=10.86Hz,1H),3.80-3.77(m,2H),3.75( dd,J=3.96,11.1Hz,1H),3.69-3.65(m,2H),3.49(t,J=4.98Hz,1H),3.10-3.05(m,2H),2.47(s,3H) ,2.46-2.44(m,1H),2.23-2.22(m,5H),2.20-2.19(m,2H),2.17(d,J=3.9Hz,3H),1.51(dd,J=2.88, 6.96Hz,3H),1.29-1.28(m,2H),1.02(d,J=6.12Hz,9H),0.99(d,J=4.08Hz,2H),0.94-0.93(m,1H). 13C NMR (151MHz, CD3OD) δ176.98,173.27,173.24,172.33,159.92,157.45,152.90,149.12,145.71,145.69,144 .70,144.26,143.90,140.54,140.28,136.81,136.53,135.32,134.59,134.37,133.40,133.22,132.91,132. 35,132.21,132.14,131.57,131.05,130.86,130.54,128.58,127.66,127.49,126.62,126.58,123.47,123.43,120.71,119.65,119.18,117.95,113.86,113.81,99.66,99.24,71.02,70.65,70.59,70.55,70.53,65.76 ,65.74,60.63,60.00,59.85,59.71,59.56,59.05,59.02,58.03,50.17,49.61,43.91,43.84,43.76,43.68,43.23,43.00,42.93,41.45,41.18,41.14,40.72,39.02,38.82,38.78,38.30,38.09,37.61,37.39,36.69,36 .63,35.27,35.22,35.04,35.01,34.85,34.81,33.11,30.88,30.81,30.79,30.77,30.68,30.65,30.52,30.38,28.15,27.04,23.79,22.43,16.55,16.52,16.44,16.39,15.85,15.83,15.81,15.76,15.75,15.65,14.49.

[0060] Example 11 Synthesis of compound CL-4S-B1

[0061] (1) Synthesis of compound CL-4S-Boc

[0062]

[0063] 2-(2,6-dioxadiazine-3-yl)-4-fluoroisoindoline-1,3-dione (400 mg, 1.45 mmol), cis-3-amino-1-cyclobutylcarbamate tert-butyl ester (323.46 mg, 1.74 mmol), and N,N-diisopropylethylamine (280.75 mg, 2.17 mmol) were dissolved in N,N-dimethylformamide solution (5 mL) and stirred at 90 °C for 8 h. The mixture was then cooled to room temperature, the solvent was removed, and the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give CL-4S-Boc (246 mg, 38.42%) as a yellow powder. MS m / z: 465.2 [M + Na] + .

[0064] (2) Synthesis of compound CL-4S

[0065]

[0066] A solution of 1,4-dioxane hydrochloride (4 M, 5 mL) was added to CL-4S-Boc (246 mg, 0.56 mmol). The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum to give CL-4S (186.56 mg, 98%) as a yellow powder. MS m / z: 343.19 [M+H] + .

[0067] (3) Synthesis of compound CL-4S-B1

[0068]

[0069] CL-4S (63 mg, 0.18 mmol), BMS-1001 (91.17 mg, 0.15 mmol), N-methylmorpholine (79.32 mg, 0.61 mmol), EDCI (35.29 mg, 0.18 mmol), and HOBt (24.88 mg, 0.18 mmol) were added to N,N-dimethylformamide (3 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. CL-4S-B1 (21.98 mg, 13%) was obtained as a yellow solid by high performance liquid chromatography. HRMS m / z: 919.3672 [M+H] + . 1H NMR (600MHz, CDCl3) δ8.32(s,1H),8.15(s,1H),7.79(s,1H),7.66(d,J=7. 56Hz,1H),7.58(d,J=7.56Hz,1H),7.46(t,J=7.74Hz,1H),7.41(t,J=7.14H z,1H),7.32(t,J=5.16Hz,1H),7.20-7.19(m,2H),7.06-7.04(m,2H),6.91( d,J=8.16Hz,1H),6.80(s,1H),6.76(dd,J=1.62,8.28Hz,1H),6.72(d,J=7. 92Hz,1H),6.50(s,1H),6.28(s,1H),5.13(s,2H),4.97(s,2H),4.90(s,1H) ,4.30(s,4H),4.15(dd,J=7.62,15.24Hz,1H),4.00-3.83(m,4H),3.72-3.6 8(m,1H),3.59(s,1H),2.82(d,J=11.7Hz,2H),2.71(d,J=10.98Hz,2H),2.2 1(s,2H),2.14(s,2H),2.07(s,1H),1.85(s,2H),1.25(s,4H),1.11(s,1H). 13 C NMR (151MHz, CDCl3) δ171.54,169.37,167.50,157.93,157.91,155.49,145.35,143.05,142.66,142.31,138.34,136.18,135 .12,135.11,133.95,133.20,132.41,131.73,131.46,130.75,130.04,129.53,127.13,125.53,122.54,119.83,118.67,118. 21,117.09,116.91,112.78,111.98,110.14,97.71,69.38,64.43,62.34,62.31,59.52,48.90,47.30,47.28,45.44,41.33,41.31,38.90,38.31,38.15,35.96,31.93,31.37,31.24,29.70,29.32,27.21,25.54,22.78,22.70,16.16,15.50,14.13,8.52.

[0070] Example 12 Synthesis of compound CL-4F-B1

[0071]

[0072] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 919.3661 [M+H]. + . 1 HNMR (600MHz, CDCl3) δ8.33-8.27(m,1H),7.78(s,1H),7.67(d,J=7.44Hz,1H),7.6 0(d,J=7.68Hz,1H),7.47(t,J=7.68Hz,1H),7.37(t,J=7.44Hz,1H),7.34(dd,J=3. 3,5.58Hz,1H),7.23-7.20(m,2H),7.08-7.04(m,2H),6.91(t,J=6.42Hz,1H),6.82 -6.80(m,1H),6.76(d,J=8.58Hz,1H),6.62(d,J=8.28Hz,1H),6.51(s,1H),6.28(d, J=3.18Hz,1H),5.13(s,2H),4.99(s,2H),4.90-4.88(m,1H),4.38(d,J=5.88Hz,1H ),4.31(d,J=5.52Hz,4H),4.05(d,J=9.36Hz,1H),3.96-3.84(m,3H),3.68-3.65(m, 1H),2.86(d,J=14.88Hz,1H),2.79-2.67(m,2H),2.39(d,J=5.64Hz,1H),2.35-2.2 2(m,5H),2.19(d,J=8.82Hz,3H),2.09(t,J=6.54Hz,1H),1.25(s,3H),1.11(s,1H). 13C NMR (151MHz, CDCl3) δ171.42,169.34,168.83,167.55,158.04,155.56,145.58,143.06,142.67,142.34,138.24,136.11,135. 09,134.00,133.93,133.18,132.41,131.80,131.55,130.80,130.07,129.58,127.22,127.11,125.55,122.53,119.87,118.6 1,118.21,117.05,116.92,112.83,112.00,110.41,97.78,97.65,69.43,69.36,64.45,62.46,61.57,61.26,59.52,52.58,48.92,47.31,44.13,42.12,42.10,38.15,37.02,36.96,36.90,31.93,31.41,31.24,29.70,22.77,22.70,16.17,15.52,14.13.

[0073] Example 13 Synthesis of compound CL-6JH-B1

[0074]

[0075] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 961.4138 [M+H]. + . 1HNMR(600MHz, CDCl3)δ8.27(s,1H),7.79(s,1H),7.67-7.59(m,3H),7.49-7.44 (m,2H),7.33(t,J=4.08Hz,1H),7.21(d,J=3.84Hz,2H),7.06(d,J=6.24Hz,2H) ,6.91(d,J=8.16Hz,1H),6.83(t,J=8.52Hz,2H),6.76(d,J=8.04Hz,1H),6.50( s,1H),6.30(s,1H),5.14(s,2H),4.99(s,2H),4.91(dd,J=4.86,11.82Hz,1H), 4.30(s,4H),4.09(d,J=12.3Hz,1H),3.98(d,J=12Hz,1H),3.86(s,1H),3.80(s ,1H),3.69(s,1H),3.62(s,1H),3.08(s,2H),2.86(d,J=15.72Hz,1H),2.78-2. 67(m,2H),2.22(s,3H),2.15(s,3H),2.10(t,J=7.26Hz,1H),1.92(d,J=9.06Hz ,1H),1.82(t,J=12.6Hz,2H),1.52(s,1H),1.26(s,3H),1.11(d,J=12.9Hz,4H). 13 C NMR (151MHz, CDCl3) δ171.36,169.55,168.72,167.63,167.61,158.27,155.60,147.03,143.07,142.67,142.34,13 8.19,136.17,135.09,133.93,133.30,132.46,131.79,131.57,130.83,130.08,129.57,127.11,125.55,122.54,1 19.95, 118.61, 118.21, 116.92, 116.67, 113.53, 112.83, 111.45, 109.87, 97.59, 69.45, 69.36, 64.45, 62.34, 61.40, 59.52, 48.88, 48.79, 48.48, 47.07, 38.15, 36.77, 31.95, 31.41, 31.24, 29.70, 29.40, 22.78, 16.17, 15.50, 14.13.

[0076] Example 14 Synthesis of compound CJ-4S-B1

[0077]

[0078] It was prepared using the same method as compound CL-4S-B1, and was a yellow solid. HRMS m / z: 919.3671 [M+H] + . 1 HNMR (600MHz, CDCl3) δ8.40(s,1H),8.20(s,1H),7.77(s,1H),7.62(d,J=7.5Hz,1H),7.55(d,J=7.26Hz,1H),7.43(t ,J=7.38Hz,1H),7.38(s,1H),7.31(s,1H),7.18(s,2H),7.06(s,1H),6.89(d,J=8.1Hz,1H),6.79(s,1H),6.74(d,J=7 .38Hz,2H),6.50(s,2H),5.75(s,1H),5.10(s,2H),4.97(s,2H),4.88(s,1H),4.28(s,4H),4.02-3.84(m,5H),3.61(s ,1H),3.54(s,1H),2.73-2.67(m,4H),2.20(s,3H),2.11(s,2H),2.00(s,2H),1.79(s,2H),1.26(s,2H),1.11(s,1H). 13 C NMR (151MHz, CDCl3) δ172.09,169.72,168.93,168.04,167.48,157.96,155.54,152.66,143.04,142.65,142.29 ,138.34,135.07,134.23,133.91,133.20,131.69,131.50,130.72,130.03,129.50,127.10,125.53,122.52,119 .75,118.72,118.20,117.67,116.91,112.64,97.70,69.34,64.42,62.40,61.46,61.27,59.52,52.33,49.00,47.02,46.98,46.85,41.79,38.62,38.27,38.14,31.92,31.40,31.24,29.69,29.36,22.69,16.15,15.49,14.13.

[0079] Example 15 Synthesis of compound CJ-4F-B1

[0080]

[0081] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 919.3668 [M+H]. + . 1 HNMR (600MHz, CDCl3) δ8.34(s,1H),7.83(s,1H),7.63(d,J=7.62Hz,1H),7.56(d,J=7.5Hz,1H),7.47(dd,J=6.48,13.7 4Hz,2H),7.32(s,1H),7.19(s,2H),7.08(s,1H),6.90(d,J=8.22Hz,1H),6.82-6.79(m,2H),6.75(d,J=8.16Hz,1H),6. 53-6.51(m,2H),5.15-5.09(m,3H),4.98(s,2H),4.91-4.89(m,1H),4.48(s,1H),4.29(s,5H),4.02-3.96(m,2H),3.90 (d,J=12.66Hz,3H),3.64(s,1H),2.80-2.69(m,3H),2.20(s,3H),2.14(s,2H),2.00(s,1H),1.25(s,5H),1.11(s,2H). 13 C NMR (151MHz, CDCl3) δ172.10,172.07,169.50,168.24,168.05,167.46,158.04,155.59,155.35,152.97,143.05,142.66,142.28,138.43,138 .32,135.11,135.09,134.23,133.91,133.29,132.88,131.74,131.50, 130.84,130.77,130.04,130.02,129.52,129.48,127.22,127.09,125. 53,122.53,119.81,118.73,118.21,117.85,116.91,112.86,112.64,97.84,97.67,69.43,69.29,64.45,62.25,61.44,61.33,61.26,59.52,52.49,49.03,46.95,46.84,44.37,42.40,38.15,36.54,36.49,31.93,31.42,31.24,29.66,29.36,27.21,25.54,22.70,16.15,15.50,14.13.

[0082] Example 16 Synthesis of compound CJ-6S-B1

[0083]

[0084] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 947.3982 [M+H]. + . 1 HNMR (600MHz, CDCl3) δ8.33(s,1H),7.81(s,1H),7.65(d,J=7.38Hz,1H),7.61(d,J=7.62Hz,1H),7.50(q,J=6.42Hz,2H),7.29(d,J=6.78Hz, 1H),7.22-7.18(m,2H),7.07(d,J=9.18Hz,1H),6.92-6.90(m,2H),6.82-6.74(m,2H),6.69(d,J=7.98Hz,1H),6.49(s,1H),5.16(d,J=3.78H z,2H),4.97(s,2H),4.91(dd,J=4.8,10.92Hz,1H),4.31(d,J=6.54Hz,3H),4.19(d,J=11.94Hz,1H),3.92-3.80(m,4H),3.45(s,1H),2.86-2 .67(m,3H),2.25-2.23(m,1H),2.21(s,2H),2.14(s,2H),2.10-2.08( m,1H),1.41(s,2H),1.25(s,8H),1.11(s,4H),0.88(t,J=6.78Hz,1H). 13C NMR (151MHz, CDCl3) δ171.43,168.93,168.12,167.45,158.42,155.71,152.98,143.07,142.70,142.37,1 38.12,135.03,134.86,134.61,133.89,131.83,131.41,130.78,130.12,129.59,127.03,125.57,122.53, 120.05,118.59,118.21,117.38,116.94,112.91,97.53,69.48,69.31,64.43,59.53,49.04,38.15,35.96,31.93,31.46,31.24,29.70,29.37,29.33,28.13,27.91,27.51,27.22,25.53,22.70,16.15,15.55,14.13.

[0085] Example 17 Synthesis of compound CJ-6F-B1

[0086]

[0087] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 947.3970 [M+H]. + . 1HNMR (600MHz, CDCl3) δ8.32(s,1H),7.82(s,1H),7.67(d,J=7.92Hz,1H),7.61(d,J=7.62Hz,1H),7.54-7.47(m,2H),7.34(t,J=4.38Hz,1H),7. 22-7.20(m,2H),7.08(d,J=6.24Hz,1H),6.90-6.88(m,1H),6.80(d,J=1.74Hz,1H),6.76(dd,J=1.74,8.16Hz,1H),6.67(d,J=7.74Hz,1H),6.5 2(s,1H),5.14(s,2H),5.00(s,2H),4.92(q,J=4.98Hz,1H),4.29(s,3H ),4.01-3.81(m,3H),3.68-3.63(m,2H),3.22(s,1H),2.84-2.67(m,3H) ,2.22(s,2H),2.17(s,2H),2.08(s,1H),2.01(s,2H),1.92(s,1H),1.82 (s,1H),1.42(s,1H),1.25(s,8H),1.11(s,3H),0.88(t,J=6.84Hz,1H). 13 C NMR (151MHz, CDCl3) δ175.97,171.53,169.15,168.08,167.36,157.98,157.58,155.52,155.38,152.72,143.06,142.67,142.33,13 8.34,135.12,135.09,134.65,133.94,133.19,133.15,132.95,131.75,131.52,130.82,130.06,129.92,129.55,127.22,127.12,1 25.62,125.55,122.53,119.92,118.69,118.21,116.92,112.78,97.70,69.40,64.45,62.40,61.70,61.29,59.52,52.55,50.80,49.09,47.88,47.05,46.84,38.15,35.96,31.93,31.46,31.24,31.01,29.70,29.32,27.21,25.54,22.73,22.70,16.17,15.53,14.13.

[0088] Example 18 Synthesis of compound CJ-6JH-B1

[0089]

[0090] It was prepared using the same method as compound CL-4S-B1, as a yellow solid, HRMS m / z: 961.4138 [M+H]. + . 1 HNMR (600MHz, CDCl3) δ8.29 (s, 1H), 7.79 (s, 1H), 7.66 (d, J = 7.5Hz, 1H), 7.59 (d, J=7.56Hz,1H),7.53(d,J=7.68Hz,1H),7.46(t,J=7.62Hz,1H),7.33(t,J=4.5Hz ,1H),7.21(d,J=3.6Hz,2H),7.07(s,1H),6.91(d,J=7.98Hz,2H),6.80(s,1H),6 .76(d,J=8.28Hz,1H),6.68(d,J=7.98Hz,1H),6.50(s,1H),5.13(s,2H),4.99(s ,2H),4.91(q,J=5.16Hz,1H),4.29(s,4H),4.09(d,J=10.92Hz,1H),3.99(d,J=1 2.66Hz,1H),3.88-3.81(m,2H),3.69(s,1H),3.58(s,1H),3.20(s,1H),2.97(s, 1H),2.82-2.67(m,3H),2.22(s,3H),2.14(s,3H),2.07(s,1H),2.00(s,1H),1.9 0-1.87(m,2H),1.76-1.74(m,3H),1.47(s,1H),1.26(s,1H),1.09-1.03(m,5H). 13C NMR (151MHz, CDCl3) δ171.67,169.18,168.10,167.46,167.38,158.31,155.65,154.00,143.06,142.67, 142.33,138.22,135.07,134.53,133.93,133.35,131.78,131.62,130.85,130.07,129.56,127.09,125.5 5,122.53,119.93,118.69,118.20,117.64,116.93,112.72,97.59,69.45,69.33,64.45,64.42,62.25,61.26,49.36,49.07,48.80,46.92,36.25,31.90,31.46,29.70,29.46,29.42,29.22,22.72,16.17,15.50.

[0091] Example 19 Synthesis of compound CL-3CQ-B1

[0092] (1) Synthesis of compound CL-3CQ-Boc

[0093]

[0094] Cl (400 mg, 1.04 mmol), BMS-1001 (190.78 mg, 1.04 mmol), Pd(dppf)₂Cl₂ (8.5 mg, 0.01 mmol), CuI (1.98 mg, 0.01 mmol), and DIPEA (269.3 mg, 2.08 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at 90 °C for 8 h under a nitrogen atmosphere. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. CL-3CQ-Boc (242.48 mg, 53%) was obtained as a yellow powder by column chromatography. ESI-MS m / z: 462.2 [M + Na] + .

[0095] (2) Synthesis of compound CL-3CQ

[0096]

[0097] A solution of 1,4-dioxane hydrochloride (4 M, 5 mL) was added to CL-3CQ-Boc (242.48 mg, 0.55 mmol). The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum to give CL-3CQ (182.59 mg, 97.5%) as a yellow powder. MS m / z: 340.19 [M+H] + .

[0098] (3) Synthesis of compound CL-3CQ-B1

[0099]

[0100] CL (105 mg, 0.31 mmol), BMS-1001 (153.3 mg, 0.26 mmol), NMM (133.38 mg, 1.03 mmol), EDCI (59.35 mg, 0.31 mmol), and HOBt (41.83 mg, 0.31 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. CL-3CQ-B1 (39.67 mg, 14%) was prepared as a yellow powder by high performance liquid chromatography. HRMS m / z: 916.3561 [M+H] + . 1H NMR(600MHz,CDCl3)δ8.35(s,1H),7.78-7.75(m,2H),7.68-7.62(m,3H),7.58(d,J=7.68Hz,1H),7.46(t,J=7.8Hz,1H),7.33(t,J=4.32Hz,1H),7.21(d,J=3.96Hz,2H),7.06-7.05(m,1H),6.91(d,J=8.22Hz,1H),6.81(d,J=1.98Hz,1H),6.76(dd,J=1.92,8.16Hz,1H),6.48(d,J=7.56Hz,1H),5.14-5.11(m,2H),5.02-4.96(m,3H),4.30(s,4H),4.09(dd,J=12.72,25.74Hz,1H),3.95-3.87(m,2H),3.83-3.77(m,1H),3.61(s,1H),3.50-3.33(m,2H),2.82(d,J=12.36Hz,1H),2.74-2.70(m,2H),2.54-2.43(m,2H),2.22(s,4H),2.09(d,J=11.04Hz,4H),1.82-1.70(m,2H),1.25(s,2H). 13C NMR (151MHz, CDCl3) δ171.53,169.16,169.14,168.79,168.58,167.06,166.95, 166.36,166.31,158.04,158.00,157.37,155.65,155.61,155.31,143.06,142.6 7,142.32,138.50,138.34,135.11,134.19,133.97,133.28,132.80,132.09,131.70,131.57,130.77,130.54,130.50,130.05,129.53,127.22,127.14,125.53,1 22.92,122.88,122.53,121.14,119.75,118.65,118.21,116.92,112.73,98.18,98.12,97.62,97.56,69.45,69.30,64.45,64.43,62.53,62.46,61.84,61.75,61 .56,61.39,52.41,49.45,49.42,47.30,47.24,46.84,38.92,38.81,31.38,31.32,29.69,29.32,27.04,27.00,22.75,22.63,17.68,17.53,16.17,15.49,15.44.

[0101] Example 20 Synthesis of compound NF-C3-B1

[0102] (1) Synthesis of compound NF-C3-Boc

[0103]

[0104] NF (400 mg, 1.11 mmol), Boc-4-aminobutyric acid (147.22 mg, 0.72 mmol), DIPEA (374.49 mg, 2.90 mmol), EDCI (166.63 mg, 0.87 mmol), and HOBt (117.45 mg, 0.87 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography (dichloromethane:methanol = 8:1) to give NF-C3-Boc (502.57 mg, 83%) as a white solid. ESI-MS m / z: 568.3

[0105] [M+Na] + .

[0106] (2) Synthesis of compound NF-C3

[0107]

[0108] NF-C3-Boc (502.57 mg, 0.92 mmol) was dissolved in 1,4-dioxane hydrochloride solution (4 M, 5 mL). The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum to give NF-C3 (402.19 mg, 98%) as a white powder. ESI-MS m / z: 446.24 [M+H] + .

[0109] (3) Synthesis of compound NF-C3-B1

[0110]

[0111] NF-C3 (50 mg, 0.11 mmol), BMS-1001 (55.61 mg, 0.10 mmol), N-methylmorpholine (24.19 mg, 0.19 mmol), EDCI (21.53 mg, 0.11 mmol), and HOBt (15.17 mg, 0.11 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. NF-C3-B1 (22.94 mg, 20%) was obtained as a yellow powder by high performance liquid chromatography. HRMS m / z: 1022.4103 [M+H] + . 1H NMR(600MHz,CDCl3)δ8.26(s,1H),7.80(s,1H),7.75(t,J=6.12Hz,1H),7.68(d,J=7.86Hz,1H),7.63(d,J=7.62Hz,1H),7.52-7.48(m,2H),7.37(d,J=7.68Hz,2H),7.23-7.22(m,2H),7.07(s,1H),6.92(d,J=8.22Hz,1H),6.82(d,J=1.92Hz,1H),6.78(dd,J=1.98,8.22Hz,1H),6.52(s,1H),5.14(s,2H),5.01(s,2H),4.95(q,J=5.28Hz,1H),4.31(s,3H),3.94(dd,J=12.84,30Hz,2H),3.83(s,1H),3.77(s,1H),3.63(s,1H),3.40(s,1H),3.30-3.27(m,1H),3.23(s,1H),3.19(t,J=4.74Hz,2H),2.92-2.88(m,1H),2.82-2.70(m,3H),2.43(q,J=7.14Hz,1H),2.24(s,3H),2.18(s,3H),2.15-2.11(m,1H),2.02-2.00(m,1H),1.86(t,J=6.72Hz,2H),1.26(s,5H),0.89-0.83(m,2H). 13 C NMR(151MHz,CDCl3)δ171.24,170.81,168.00,166.74,166.19,159.09,157.60,157.40,155.33,145.35,143.06,142.67,142.34,138.40,135.20,135.15,133.97,132.86,131.77,131.52,130.83,130.05,129.58,128.97,127.16,125.54,122.55,119.87,118.62,118.22,116.92,113.92,112.88,112.38,112.22,97.86,69.45,64.44,62.33,62.00,49.97,49.73,49.48,47.47,45.20,41.38,39.22,35.95,31.91,31.42,30.65,29.70,29.33,29.24,27.22,25.53,24.46,22.65,16.18,15.52,14.13.

[0112] Example 21 Synthesis of compound NF-C4-B1

[0113]

[0114] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1036.4260 [M+H]. + . 1 H NMR (600MHz, CDCl3) δ8.28(s,1H),7.91(s,1H),7.82(s,1H),7.69(d,J=7.68Hz,1H),7. 60(d,J=7.56Hz,1H),7.50-7.45(m,2H),7.36(dd,J=6.6,9.78Hz,2H),7.21(d,J=4.02H z,2H),7.09(s,1H),6.90(d,J=8.22Hz,1H),6.80(d,J=1.86Hz,1H),6.76(dd,J=1.86,8 .16Hz,1H),6.52(s,1H),5.16(s,2H),5.00(s,2H),4.95(q,J=5.04Hz,1H),4.30(d,J=4. 86Hz,4H),4.13(d,J=12.54Hz,1H),4.04(d,J=12.96Hz,1H),3.95-3.88(m,2H),3.74(s ,2H),3.61(s,2H),3.20(d,J=18.12Hz,6H),2.87-2.84(m,1H),2.79-2.69(m,2H),2.36 (t,J=6.72Hz,2H),2.22(s,3H),2.15(s,3H),2.12-2.10(m,3H),2.01(s,1H),1.62(t,J =6.36Hz,2H),1.50(t,J=6.48Hz,2H),1.26(s,5H),1.11(s,1H),0.88(t,J=6.78Hz,1H). 13C NMR (151MHz, CDCl3) δ171.56,171.35,168.44,168.28,167.07,166.81,166.25,166.24,159.03,158.32,157.33,155.67,145.34,143.06, 142.67,142.33,138.21,135.06,133.96,133.44,131.78,131.64,13 0.87,130.08,129.57,128.93,127.12,125.55,124.77,122.52,119. 90,118.64,118.20,116.91,113.94,112.78,112.33,112.16,97.59,69.46,69.33,64.42,62.05,61.09,59.52,51.61,50.04,49.74,49.47,47.02,45.19,41.33,39.21,38.14,32.28,31.92,31.41,31.24,29.69,29.36,28.66,27.21,22.69,22.64,21.98,16.17,15.47,14.13.

[0115] Example 22 Synthesis of compound NF-C5-B1

[0116]

[0117] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1050.4413 [M+H]. + . 1H NMR(600MHz,CDCl3)δ7.78(s,1H),7.68(d,J=7.92Hz,1H),7.62(d,J=7.68Hz,1H),7.51(dd,J=7.74,15.54Hz,2H),7.36(t,J=7.02Hz,2H),7.22(d,J=3.54Hz,2H),7.06(s,1H),6.91(d,J=8.22Hz,1H),6.81(d,J=1.86Hz,1H),6.77(dd,J=1.92,8.22Hz,1H),6.53(s,1H),5.13(s,2H),5.01(s,2H),4.94(dd,J=5.22,12.36Hz,1H),4.30(s,4H),3.93-3.88(m,2H),3.81-3.76(m,3H),3.64(dd,J=4.62Hz,2H),3.47(s,1H),3.23-3.19(m,5H),2.88-2.85(m,1H),2.80-2.69(m,2H),2.35(t,J=7.2Hz,2H),2.24(s,3H),2.17(s,3H),2.12-2.10(m,1H),1.66-1.61(m,2H),1.51-1.46(m,2H),1.36-1.31(m,2H),1.26(s,3H). 13C NMR (151MHz, CDCl3) δ170.60,170.18,169.53,167.29,166.75,165.76,165.21,158.04,156.60,156.34,154.37,144.35,144.29,142.03,14 1.63,141.28,137.40,134.18,134.10,132.93,131.85,130.72,130.5 4,129.73,129.00,128.55,127.91,126.12,124.51,123.84,123.78,1 21.51,118.75,117.60,117.18,115.88,112.91,112.88,111.77,111.31,111.14,96.80,68.39,63.42,61.51,61.02,49.08,48.77,48.45,46.29,44.23,40.22,37.88,37.12,31.83,30.90,30.38,30.22,28.67,28.33,28.29,28.00,26.18,25.32,23.41,21.61,15.15,14.50,13.10.

[0118] Example 23 Synthesis of compound NF-C7-B1

[0119]

[0120] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1078.4719 [M+H]. + . 1H NMR (600MHz, CDCl3) δ7.77(s,1H),7.69-7.61(m,3H),7.51-7.47(m,2H),7.38-7.35(m,2H),7.22-7.21(m,2H),7.06(s,1H),6.91( d,J=8.22Hz,1H),6.81(d,J=1.86Hz,1H),6.77(dd,J=1.92,8.22Hz,1H),6.52(s,1H),5.13(s,2H),5.01(s,2H),4.94(q,J=5.22Hz, 1H),4.30(s,4H),3.94(q,J=12.9Hz,2H),3.83-3.78(m,4H),3.64(s,2H),3.50(s,1H),3.24-3.15(m,5H),2.92-2.69(m,3H),2.35 (t,J=7.44Hz,2H),2.24(s,3H),2.17(s,3H),2.13-2.11(m,1H),1.62(t,J=6.36Hz,2H),1.44(t,J=6.72Hz,2H),1.31-1.26(m,8H). 13 C NMR (151MHz, CDCl3) δ170.83,170.09,169.26,167.23,166.37,165.76,165.20,158.06,156.66,156.36,154.38,144.37,144 .31,142.04,141.64,141.29,137.38,134.16,134.10,132.92,131.87,130.73,130.53,129.72,129.01,128.56,127.93,126. 12,124.51,123.87,121.51,118.78,117.58,117.19,115.89,112.89,111.79,111.33,111.17,96.78,68.40,63.43,61.51,61.03,49.15,48.84,48.46,46.28,44.28,40.20,38.28,32.05,30.39,28.23,28.11,27.78,25.47,23.96,21.62,15.15,14.49.

[0121] Example 24 Synthesis of compound NF-4S-B1

[0122]

[0123] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1034.4092 [M+H]. + . 1 H NMR (600MHz, CDCl3) δ8.27(s,1H),7.86(d,J=7.98Hz,1H),7.81(s,1H),7.67(d ,J=7.68Hz,1H),7.62(d,J=7.56Hz,1H),7.50(t,J=7.02Hz,2H),7.37(d,J=7.26 Hz,2H),7.22(d,J=5.94Hz,2H),7.05(s,1H),6.91(d,J=8.1Hz,1H),6.80(s,1H) ,6.77(d,J=7.92Hz,1H),6.52(s,1H),5.15(s,2H),5.01(s,2H),4.95(q,J=4.98 Hz,1H),4.35(t,J=8.22Hz,1H),4.30(s,4H),3.93(q,J=12.9Hz,2H),3.80(dd,J =4.5,18.66Hz,4H),3.55(s,2H),3.41(t,J=4.86Hz,1H),3.18(d,J=3.84Hz,3H) ,2.98(t,J=8.58Hz,1H),2.91-2.87(m,1H),2.82-2.71(m,3H),2.56-2.53(m,2H ),2.24(s,3H),2.20-2.18(m,4H),2.14-2.12(m,1H),2.00(s,1H),1.25(s,2H). 13C NMR (151MHz, CDCl3) δ172.21,171.00,170.09,168.14,166.86,166.75,166.20,159.11,157.66,157.41,155.38,145.31,145.25,143.06,142.6 6,142.32,138.47,135.23,135.15,133.96,132.98,131.74,131.52,13 0.83,130.04,129.54,128.95,127.16,125.54,125.02,124.95,122.55, 119.84,118.67,118.22,116.91,113.95,112.82,112.39,112.23,97.86,69.40,64.43,62.21,61.91,61.90,59.52,50.22,49.74,49.49,47.45,44.94,41.63,40.40,38.15,35.96,33.66,33.52,31.93,31.43,31.24,30.32,29.70,29.37,29.32,27.22,25.53,22.65,16.18,15.53,14.13.

[0124] Example 25 Synthesis of compound NF-4F-B1

[0125]

[0126] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1034.4 × 102 [M + H]. + . 1H NMR (600MHz, CDCl3) δ8.34(s,1H),8.07(d,J=6.66Hz,1H),7.79(s,1H),7.68(d,J=7.8Hz,1H),7.62(d,J=7.68Hz,1H),7.51-7.46(m,2H),7 .37-7.35(m,2H),7.23-7.21(m,2H),7.06(s,1H),6.91(d,J=8.16Hz,1H),6.80(d,J=1.98Hz,1H),6.76(dd,J=1.92,8.22Hz,1H),6.52(s,1H ),5.13(s,2H),5.01(s,2H),4.95(q,J=5.22Hz,1H),4.31(d,J=5.58Hz,5H),3.98(d,J=12.9Hz,1H),3.91(d,J=12.72Hz,1H),3.83-3.76(m, 4H),3.51-3.46(m,3H),3.26-3.16(m,5H),2.88-2.85(m,1H),2.80-2 .64(m,5H),2.23(s,3H),2.18(s,3H),2.13-2.11(m,1H),1.26(s,3H). 13 C NMR (151MHz, CDCl3) δ175.92,172.50,171.14,169.80,168.27,167.69,166.77,166.22,159.07,157.80,157.37,155.45,145.37,143.0 5,142.66,142.33,138.33,135.14,135.10,133.94,132.96,131.80,131.58,130.78,130.06,129.61,128.93,127.12,125.55,124.81,1 22.53,119.83,118.63,118.20,116.91,113.92,112.82,112.35,112.18,97.77,69.43,69.39,64.45,64.43,62.47,61.80,50.04,49.79,49.47,47.39,44.84,42.69,41.47,35.96,32.34,31.57,31.42,29.70,29.48,29.32,29.24,27.21,25.54,22.64,16.18,15.54,14.13.

[0127] Example 26 Synthesis of compound NF-5S-B1

[0128]

[0129] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1048.4257 [M+H]. + . 1 H NMR (600MHz, CDCl3) δ8.38(s,1H),8.03(d,J=4.32Hz,1H),7.79(s,1H),7.70(d,J=7.68Hz,1H),7.62(d,J=7.5Hz,1H),7.51- 7.49(m,2H),7.37(d,J=6.54Hz,2H),5.14(s,2H),5.01(s,2H),4.95(q,J=5.1Hz,1H),4.31(s,5H),3.99(d,J=12.96Hz,1H), 3.91(d,J=13.08Hz,1H),3.84-3.77(m,3H),3.71(s,1H),3.39(t,J=4.68Hz,1H),3.28-3.17(m,4H),2.90-2.70(m,3H),2.24 (s,3H),2.19(s,3H),2.14-2.12(m,1H),2.01(s,1H),1.94(d,J=13.32Hz,1H),1.84-1.76(m,2H),1.26(s,5H),1.11(s,2H). 13C NMR (151MHz, CDCl3) δ175.42,171.02,169.64,168.16,168.06,166.72,1 66.18,159.12,157.60,157.42,155.37,145.23,143.05,142.66,142.31 ,138.47,135.24,135.16,133.97,132.96,131.73,131.56,130.82,130. 03,129.56,128.98,127.17,125.53,125.04,122.55,119.81,118.64,11 8.22,116.91,116.33,113.91,112.80,112.40,112.23,97.80,69.48,69.41,64.45,64.43,62.30,62.27,62.03,59.52,51.42,50.20,49.73,49. 50,47.27,45.58,41.79,39.11,38.15,35.47,33.37,31.93,31.42,31.24,29.69,29.36,28.73,27.21,25.54,22.69,22.64,16.17,15.54,14.13.

[0130] Example 27 Synthesis of compound NF-6S-B1

[0131]

[0132] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1062.4415 [M+H]. + . 1H NMR (600MHz, CDCl3) δ8.33(s,1H),7.80(s,1H),7.75-7.69(m,2H),7.61(d,J=7.5Hz,1H),7.51-7.48(m,2H),7.39-7.34(m,2H),7 .22-7.20(m,2H),7.07(s,1H),6.90(d,J=8.22Hz,1H),6.82-6.75(m,2H),6.52(s,1H),5.16(s,2H),5.04-4.94(m,3H),4.31(d,J= 6.66Hz,4H),4.05-3.92(m,3H),3.84-3.77(m,3H),3.68-3.58(m,3H),3.23(d,J=23.16Hz,3H),2.89(t,J=14.88Hz,1H),2.81-2.7 1(m,2H),2.59(s,1H),2.26-2.23(m,3H),2.16(s,3H),2.12(t,J=7.32Hz,1H),1.80-1.68(m,3H),1.61-1.57(m,4H),1.26(s,3H). 13 C NMR (151MHz, CDCl3) δ173.07,170.36,170.34,168.15,167.37,166.56,165.77,165.21,158.04,156.83,156.34,154.55,144.25,1 42.03,141.63,141.27,137.43,134.17,134.08,132.92,132.12,130.69,130.57,129.78,129.00,127.93,126.09,124.50,123.91 ,123.85,121.51,118.73,117.63,117.17,115.87,112.92,111.72,111.34,111.17,96.71,68.44,68.31,63.42,63.40,61.32,61.03,49.33,48.72,48.47,46.27,44.30,43.75,40.34,37.08,30.41,28.67,28.29,28.02,27.89,26.18,23.38,21.62,15.14,14.47.

[0133] Example 28 Synthesis of compound NF-6F-B1

[0134]

[0135] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1062.4413 [M+H]. + . 1 H NMR(600MHz, CDCl3)δ8.31(s,1H),7.79(s,1H),7.71-7.67(m,2H),7.63(d,J=7 .56Hz,1H),7.51-7.48(m,2H),7.39-7.34(m,2H),7.21(s,2H),7.06(s,1H),6. 91(d,J=8.16Hz,1H),6.80(s,1H),6.76(d,J=8.1Hz,1H),6.52(s,1H),5.14(s, 2H),5.00(s,2H),4.95(q,J=4.98Hz,1H),4.30(s,4H),4.04(d,J=12.48Hz,1H), 3.94(d,J=12.78Hz,1H),3.84-3.78(m,4H),3.69-3.63(m,3H),3.24(d,J=20.8 2Hz,3H),2.89(t,J=15.3Hz,1H),2.81-2.70(m,2H),2.48-2.42(m,1H),2.23(s ,3H),2.17(s,3H),2.14-2.12(m,1H),2.01-1.98(m,1H),1.90(d,J=10.08Hz,1 H),1.78(t,J=9.48Hz,2H),1.69(dd,J=12.06,25.32Hz,2H),1.26-1.17(m,4H). 13C NMR (151MHz, CDCl3) δ173.80,171.12,168.24,167.26,166.78,166.22,159.09,158.10,157.39,155.55,145.35,145.29,143.07,142.6 7,142.35,138.24,135.07,133.93,133.10,131.81,131.62,130.82,130.09,129.60,128.96,127.10,125.56,122.52,119.90,118.60, 118.20,116.92,113.92,113.89,112.84,112.38,112.21,97.63,69.47,69.37,64.45,64.42,62.49,61.67,50.37,50.34,49.88,49.48,48.14,47.20,45.17,41.34,39.23,35.97,31.83,31.42,29.77,29.70,29.52,29.32,28.03,27.21,25.53,22.65,16.18,15.53,14.13.

[0136] Example 29 Synthesis of compound NF-4L-B1

[0137]

[0138] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 1074.4415 [M+H]. + . 1H NMR(600MHz,CDCl3)δ8.29(s,1H),7.78(s,1H),7.67(d,J=7.62Hz,1H),7.63(d,J=7.68Hz,1H),7.50(t,J=7.56Hz,2H),7.37(d,J=6.72Hz,2H),7.22(d,J=3.36Hz,2H),7.05(s,1H),6.91(d,J=8.16Hz,1H),6.81(s,1H),6.77(d,J=7.98Hz,1H),6.51(s,1H),5.13(s,2H),5.01(s,2H),4.95(q,J=5.16Hz,1H),4.30(s,4H),4.19-4.17(m,1H),3.96-3.88(m,2H),3.77(s,3H),3.51(s,2H),3.19(s,3H),3.16-3.13(m,1H),2.91-2.88(m,1H),2.82-2.72(m,2H),2.51-2.44(m,1H),2.41-2.34(m,2H),2.28-2.24(m,4H),2.18(s,3H),2.13-2.08(m,2H),1.92-1.79(m,2H),1.26(s,3H),0.89-0.83(m,1H). 13 CNMR(151MHz,CDCl3)δ172.86,171.01,168.15,166.76,166.21,159.11,157.86,157.41,155.43,145.41,145.35,143.07,142.67,142.35,138.33,138.31,135.14,135.10,133.95,132.98,131.80,131.53,130.77,130.07,129.60,128.95,127.14,125.56,124.86,122.54,119.87,118.61,118.21,116.92,113.91,112.87,112.38,112.21,97.75,69.44,69.40,64.45,64.43,62.35,62.29,61.82,61.72,50.21,49.87,49.48,47.33,44.79,43.37,42.37,41.45,40.10,40.08,37.49,36.60,33.51,33.49,32.11,31.42,29.77,29.70,29.32,27.21,25.53,22.65,16.19,15.53,14.13.

[0139] Example 30 Synthesis of compound NC-4F-B1

[0140] (1) Synthesis of compound NC-4F-Boc

[0141]

[0142] NC (300 mg, 1.14 mmol), tert-butyl (trans-3-aminocyclobutyl)carbamate (253.91 mg, 1.36 mmol), N-methylmorpholine (459.65 mg, 4.54 mmol), EDCI (261.34 mg, 1.36 mmol), and HOBt (184.21 mg, 1.36 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. The mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by column chromatography (dichloromethane:methanol = 8:1) to give NC-4F-Boc (378.18 mg, 77%) as a white solid. MS m / z: 455.25 [M + Na] + .

[0143] (2) Synthesis of compound NC-4F

[0144]

[0145] A solution of 1,4-dioxane hydrochloride (4 M, 5 mL) was added to NC-4F-Boc (378.18 mg, 0.87 mmol). The mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum to give NC-4F (305.83 mg, 99%) as a white powder. MS m / z: 340.23 [M+H] + .

[0146] (3) Synthesis of compound NC-4F-B1

[0147]

[0148] NC-4F (100 mg, 0.3 mmol), BMS-1001 (149.01 mg, 0.25 mmol), N-methylmorpholine (129.64 mg, 1 mmol), EDCI (57.69 mg, 0.3 mmol), and HOBt (40.66 mg, 0.3 mmol) were added to N,N-dimethylformamide (5 mL) and stirred at room temperature for 4 h. After removing the solvent, the mixture was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under vacuum. NC-4F-B1 (25.97 mg, 9.50%) was obtained as a white powder by high performance liquid chromatography. HRMS m / z: 909.3821 [M+H] + . 1 H NMR (600MHz, CDCl3) δ8.28(s,1H),7.84(s,1H),7.65(d,J=7.68Hz,1H),7.59(d,J=7.62Hz,1H),7.47(t,J=7.74Hz,1H),7.35 (t,J=4.08Hz,1H),7.20(t,J=8.52Hz,4H),7.08-7.07(m,2H),6.94(d,J=8.7Hz,2H),6.90(d,J=8.22Hz,1H),6.81(d,J=1.86H z,1H),6.76(dd,J=1.86,8.16Hz,1H),6.52(s,1H),5.16-5.12(m,2H),4.99(s,2H),4.46-4.38(m,4H),4.29(s,4H),4.01(dd ,J=12.78,29.46Hz,2H),3.84-3.77(m,4H),3.60(s,1H),2.79(t,J=6.6Hz,2H),2.30-2.22(m,7H),2.16(s,3H),1.25(s,3H). 13C NMR (151MHz, CDCl3) δ169.74,169.13,167.82,167.17,158.06,155.90,155.55,152.26,143.07,142.67,142.31,1 38.31,135.20,135.11,133.94,133.28,131.76,131.50,130.89,130.04,129.50,127.13,126.88,125.54,122.54 ,119.87,118.72,118.21,116.91,115.40,112.77,97.70,69.43,69.33,67.53,64.44,64.42,62.18,61.41,59.52,47.12,45.44,41.90,41.60,38.15,37.11,37.04,31.93,31.35,31.24,29.70,29.36,22.70,16.17,15.52,14.13.

[0149] Example 31 Synthesis of compound NC-6S-B1

[0150]

[0151] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 937.4139 [M+H]. + . 1HNMR(600MHz, CDCl3)δ8.38(s,1H),7.82(s,1H),7.68(d,J=7.8Hz,1H),7.60(d,J =7.68Hz,1H),7.48(t,J=7.74Hz,1H),7.33(t,J=4.56Hz,1H),7.23-7.21(m,4H), 7.04(s,1H),6.99(d,J=8.76Hz,1H),6.92(d,J=8.16Hz,1H),6.81(s,1H),6.77(d d,J=1.8,8.22Hz,1H),6.48(s,1H),6.06(s,1H),5.19(dd,J=12.48,26.46Hz,2H) ,5.00-4.96(m,2H),4.62(s,2H),4.31(s,3H),4.11(d,J=12.66Hz,1H),4.02-3.9 8(m,2H),3.90(s,1H),3.84-3.75(m,3H),3.72-3.67(m,1H),3.63-3.60(m,1H),2 .81(t,J=6.78Hz,2H),2.22(s,3H),2.16(s,3H),1.77(s,2H),1.57-1.51(m,3H), 1.41-1.38(m,1H),1.25(s,3H),1.11(s,1H),0.88(t,J=6.72Hz,1H),0.61(s,2H). 13 C NMR (151MHz, CDCl3) δ169.55,168.29,167.22,166.78,158.45,155.79,153.45,143.09,142.70,142.40,138.13 ,135.26,135.07,134.96,134.01,133.56,131.75,131.56,130.84,130.15,129.53,127.82,127.17,125.58,122 .53,119.68,118.65,118.21,116.94,115.02,112.82,97.47,69.48,69.38,66.88,64.46,62.46,61.76,47.05,45.31,44.32,38.15,31.93,31.36,31.24,29.70,29.37,27.99,27.90,27.47,27.26,22.70,16.18,15.54,14.13.

[0152] Comparative Example 1 Compound NC-6F-B1

[0153]

[0154] As described in Example 30 above, tert-butyl carbamate (trans-3-aminocyclobutyl)carbamate was replaced with tert-butyl carbamate to prepare the product, which is a yellow solid with HRMS m / z: 937.4139 [M+H]. + . 1 HNMR(600MHz, CDCl3)δ8.29(s,1H),7.80(s,1H),7.66(d,J=7.68Hz,1H),7.62(d,J=7.62Hz,1H),7.52-7.48(m,2H),7.36(t,J=3.54Hz,1H),7.25-7 .21(m,4H),7.04(s,1H),6.96(d,J=8.7Hz,2H),6.91(d,J=8.22Hz,1H),6. 81(s,1H),6.77(d,J=8.1Hz,1H),6.51(s,1H),6.43(d,J=8.22Hz,1H),5.1 3(s,2H),5.00(s,2H),4.44(s,2H),4.30(s,4H),3.94-3.89(m,2H),3.81 (t,J=6.66Hz,3H),3.77(d,J=4.74Hz,1H),3.68(d,J=7.2Hz,1H),3.51(s, 1H),2.82(t,J=6.6Hz,2H),2.23(s,3H),2.17(s,3H),1.98(d,J=10.86Hz, 2H),1.92(d,J=11.46Hz,1H),1.87(d,J=11.88Hz,1H),1.31-1.19(m,7H). 13 C NMR (151MHz, CDCl3) δ169.50,167.19,157.77,155.82,155.39,152.11,143.07,142.67,142.3 4,138.35,135.28,135.14,133.96,132.93,131.78,131.51,130.82,130.05,129.55,127.16, 126.89,125.55,122.54,119.88,118.64,118.22,116.92,115.42,97.78,69.42,67.59,64.45,62.50,62.06,47.48,47.34,47.27,45.46,31.39,31.35,31.24,29.70,16.18,15.54,14.13.

[0155] Example 32 Synthesis of compound NC-6JF-B1

[0156]

[0157] It was prepared using the same method as compound NF-C3-B1, as a yellow solid, HRMS m / z: 951.4296 [M+H]. + . 1 HNMR (600MHz, CDCl3) δ8.29 (s, 1H), 7.78 (s, 1H), 7.67 (d, J = 7.62Hz, 1H), 7.61 (d, J=7.62Hz,1H),7.54(s,1H),7.48(t,J=7.68Hz,1H),7.34(t,J=3.96Hz,1H),7.23- 7.21(m,4H),7.04(s,1H),6.95(d,J=8.7Hz,1H),6.91(d,J=8.16Hz,1H),6.81(d,J =1.56Hz,1H),6.77(dd,J=1.68,8.16Hz,1H),6.62(t,J=5.88Hz,1H),6.49(s,1H), 5.13(s,2H),4.99(s,2H),4.49(s,2H),4.30(s,4H),3.99(d,J=12Hz,1H),3.92(d ,J=8.04Hz,1H),3.79(t,J=6.66Hz,4H),3.56(s,2H),3.16(t,J=6.3Hz,2H),2.80( t,J=6.6Hz,2H),2.22(s,3H),2.16(s,3H),2.00(s,1H),1.87(d,J=10.62Hz,1H),1 .78(d,J=10.32Hz,1H),1.67(s,2H),1.38(s,1H),1.25(s,2H),1.08-0.97(m,4H). 13C NMR (151MHz, CDCl3) δ169.67,168.55,168.06,167.20,157.97,155.76,155.49,152.17,143.07,142.67 ,142.34,138.30,135.33,135.11,133.95,133.08,131.78,131.57,130.81,130.07,129.57,127.14,12 6.92,125.55,122.54,119.86,118.62,118.21,116.92,115.27,112.83,97.70,69.45,69.39,67.59,64.45,62.44,61.79,48.51,47.18,45.47,44.52,37.16,31.97,31.37,29.70,29.12,29.09,16.17,15.52.

[0158] Application examples

[0159] Instruments: 96-well plates, low-temperature centrifuge, metal bath, shaker, microplate reader, SDS-PAGE gel electrophoresis apparatus, gel imaging system

[0160] Reagents: RIPA lysis buffer, PMSF protease inhibitor, 1.5ml centrifuge tubes, BCA protein kit, Tris-Mops-SDS Running Buffer Powder, BeyoColor TM Color-stained pre-stained proteins, PVDF membrane, rapid transfer buffer, TBST, protein-free rapid blocking buffer, PD-L1 antibody, GAPDH antibody, horseradish peroxidase-labeled goat anti-mouse IgG (H+L), ECL chemiluminescence solution

[0161] Cell line: MC38 cells

[0162] Western blot analysis was used to detect the degradation activity of the above compounds on PD-L1 protein.

[0163] Mouse colon cancer cells MC38 were grown in RPMI-1640 complete culture medium at 37°C and 5% CO2. Cells were seeded in 6-well plates (2 x 10⁻⁶ cells / well). 5 After incubating for 12 hours, each well was incubated with complete culture medium containing 5 μM of the compound prepared in the above examples and incubated for another 24 hours.

[0164] After washing MC38 cells twice with PBS, they were digested and centrifuged, then placed in pre-chilled homogenization tubes. A mixture containing a protease inhibitor (PMSF) and RAPI lysis buffer (1:100, v / v) was added, and the cells were lysed on ice for 30 min. After lysis, the cells were centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was then collected in pre-chilled centrifuge tubes. The protein concentration was determined using a BCA quantitative assay kit, and the loading volume corresponding to 20 μg of protein was calculated. Proteins were separated by SDS-PAGE electrophoresis, and the gel containing the target protein was cut according to the protein marker position after electrophoresis. Semi-dry transfer was used to transfer the protein bands to a PVDF membrane. The PVDF membrane was placed in an antibody incubation chamber, washed briefly with TBST, and then blocked with protein-free rapid blocking buffer for 30 min on a shaker. After blocking, the PVDF membrane was washed 4–5 times with TBST for 10 min each time. Then, PD-L1 antibody dilution buffer was added, and the bands were incubated overnight at 4°C. The PVDF membrane was washed 4–5 times with TBST for 10 min each time. The bands were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG (H+L) dilution at room temperature for 2 hours. The membrane was developed using ECL chemiluminescence buffer in a gel imaging system, observed, and photographed. Finally, the protein bands were quantified using ImageJ software and GraphPad Prism 10.1.2 software (see [link to image]). Figure 1 ).

[0165] In this study, most of the compounds designed, synthesized, and evaluated based on PD-L1 PROTACs exhibited some degradation ability. Among them, rigid-chain PROTACs using VHL-B as the E3 ligand showed superior degradation activity compared to their counterparts using VHL-A as the ligand. However, the overall degradation activity of the VHL series was not particularly outstanding. Notably, the NF series PD-L1 PROTACs showed significant degradation effects, especially NF-C3-B1, NF-C4-B1, and NF-4L-B1. Furthermore, the three CJ series PD-L1 PROTACs, CJ-4S-B1, CJ-4F-B1, and CJ-6JH-B1, showed significantly better degradation ability than their CL series counterparts with the same linker chain, CL-4S-B1, CL-4F-B1, and CL-6JH-B1.

Claims

1. A PROTAC protein-targeting degrader, characterized in that: PROTAC protein-targeting degradative agents are compounds of Formula 1 and their pharmaceutically acceptable salts. In the formula, part A is selected from structural fragments VHL-A, VHL-B, CRBN, NF, and NC, and their structures are shown below: In the formula, linker is selected from Furthermore, when the linker is selected When A does not select VHL-A, VHL-B, CRBN, or NC; n is an integer from 3 to 7.

2. The PROTAC protein-targeting degrader according to claim 1, characterized in that: The PROTAC protein-targeting degrader is 3. The application of the PROTAC protein-targeting degrader according to claim 1, characterized in that: The PROTAC protein-targeting degradative compound and its salt as described in claim 1 are used in the degradation of proteins targeting PD-L1.

4. The application of the PROTAC protein-targeting degrader according to claim 3, characterized in that: Use of the PROTAC protein-targeting degrader compound and its salt as described in claim 1 in the preparation of a medicament for treating PD-L1 protein-related diseases.

5. The application of the PROTAC protein-targeting degrader according to claim 4, characterized in that: The PD-L1 protein-related diseases mentioned above are tumors or other PD-L1 protein-related diseases.

6. The application of the PROTAC protein-targeting degrader according to claim 5, characterized in that: The tumors mentioned are colorectal cancer, lung cancer, melanoma, bladder cancer, head and neck squamous cell carcinoma, breast cancer, prostate cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, and multiple myeloma.

Images