A kind of dehydroabietic acid PROTACs and its preparation method and application

Abstract

The present invention discloses a dehydroabietic acid PROTACs, a preparation method and an application thereof, wherein the structural formula is: The present invention can exert anti-liver fibrosis activity by targeted degradation of pathogenic proteins that cause liver fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and specifically relates to dehydroabietic acid PROTACs and a preparation method and application thereof. Background Art

[0002] Protein degradation targeting chimeras (PROTACs) are a new type of drug with good prospects. Its structure is similar to a dumbbell and is mainly composed of three parts, including E3 ligase ligand (E3 ligand), target protein ligand (POI ligand) and a linker connecting the two. One end of the PROTACs molecule binds to the target protein, and the other end binds to the E3 ubiquitin ligase. The E3 ubiquitin ligase can "stick" a small protein called ubiquitin to the target protein, marking the target protein as a defective or damaged protein, and then use the cell's intrinsic proteolytic enzyme (i.e., 26S proteasome) to specifically recognize and degrade the marked target protein. E3 ligands can recruit E3 ligases, and POI ligands can recognize and bind to target proteins. Therefore, PROTACs molecules form a POI:PROTAC:E3 ligase ternary complex in the cell. In the patient's body, the POI ligand of PROTACs binds to the target protein, and the E3 ligand binds to the substrate binding region of the E3 ubiquitin ligase in the cell. The target protein can be "pulled" to the side of the E3 ubiquitin ligase through the Linker, enabling the ubiquitin-proteasome (UPS) system to degrade the target protein. PROTACs molecules do not need to be highly embedded in the highly active region of the target. They only need to have a short-term adhesion effect on its surface, that is, only through some special intermolecular forces and other low-energy weak binding interactions to achieve the degradation of the target molecule. Currently, because the unique driving model of protein degradation mediated by PROTACs has huge therapeutic potential, this technology is currently mainly used in drug development, and some PROTACs have been clinically tested.

[0003] PROTAC drugs have many advantages over traditional small molecule drugs, which are roughly as follows:

[0004] (1) PROTAC drug design is not limited by Lipinski's five-fold rule;

[0005] (2) The pharmacological effects of traditional small molecules rely on the occupation of key sites on the target protein (i.e., occupancy-driven). The principle is to bind to the active site of the enzyme or receptor to exert its effect, and the project efficacy is poor. Traditional drugs need to maintain a certain drug concentration in the body, and the requirements for small molecule binding sites are high. However, PROTACs can capture the target protein through any corner position. Its pharmacological mechanism is to activate potential targets, thereby degrading the target protein and achieving pharmacodynamic effects. Data show that in the process of drug efficacy, very high drug concentrations are not required to achieve it. The requirements for binding sites are relatively low, and the drug efficacy is better.

[0006] (3) About 50% of proteins in the human body are found to be related to human diseases, such as cancer. However, only about 20% of these proteins can be bound by traditional small molecules, while PROTACs can theoretically bind to any part of the protein.

[0007] (4) PROTACs are more resistant to drug resistance than traditional small molecules. In theory, PROTACs can better tolerate mutations in target proteins.

[0008] (5) PROTACs are more durable and have stronger inhibitory effects than traditional drug molecules; Summary of the Invention

[0009] The present invention aims to provide a dehydroabietic acid PROTACs.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned dehydroabietic acid PROTACs.

[0011] Another object of the present invention is to provide the application of the above-mentioned dehydroabietic acid PROTACs.

[0012] The technical solutions of the present invention are as follows:

[0013] A dehydroabietic acid PROTACs, characterized in that: its structural formula is

[0014] in,

[0015] E3 Ligase is lenalidomide or pomalidomide,

[0016] Linker is a fatty chain or a PEG chain. The fatty chain is -NH-(CH2)-NH-(CH2)n1-CH2-CH2-NH-CO- or -NH-(CH2)n2-CH2-NH-CO-. The PEG chain is -(O-CH2-CH2)n3-CH2 CO-COOH. n1 is a natural number from 2 to 7, n2 is a natural number from 1 to 7, and n3 is a natural number from 1 to 3.

[0017] In a preferred embodiment of the present invention, its structural formula is selected from at least one of the following:

[0018]

[0019]

[0020]

[0021] The preparation method of the above-mentioned dehydroabietic acid PROTACs has the following reaction scheme:

[0022] or

[0023]

[0024] or

[0025]

[0026] or

[0027]

[0028] Use of the above-mentioned dehydroabietic acid PROTACs or pharmacologically or physiologically acceptable salts thereof in preparing a cancer treatment composition.

[0029] In a preferred embodiment of the present invention, the cancer includes breast cancer, uterine cancer and liver cancer.

[0030] A cancer treatment composition, the active ingredient of which includes the above-mentioned dehydroabietic acid PROTACs or a pharmacologically or physiologically acceptable salt thereof.

[0031] In a preferred embodiment of the present invention, the cancer includes breast cancer, uterine cancer and liver cancer.

[0032] The beneficial effect of the present invention is that the present invention can exert the activity of resisting liver fibrosis by targeted degradation of pathogenic proteins that cause liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a distribution diagram of the differentially expressed proteins in cells in Example 5 of the present invention.

[0034] Figure 2 The top 20 downregulated differentially expressed proteins of T-PROTAC VS T in Example 5 of the present invention are shown.

[0035] Figure 3 The top 20 downregulated differentially expressed proteins of T-PROTAC VS con in Example 5 of the present invention are shown.

[0036] Figure 4 The top 20 down-regulated differentially expressed proteins of T VS con in Example 5 of the present invention are shown.

[0037] Figure 5 The molecular docking and binding site prediction of TMBIM6 and dehydroabietic acid in Example 6 of the present invention are shown.

[0038] Figure 6 This shows the effects of dehydroabietic acid and T15 on the expression of TMBIM6 protein in Example 7 of the present invention.

[0039] Figure 7 The quantitative analysis of the expression of TMBIM6 protein by dehydroabietic acid and T15 in Example 7 of the present invention is shown. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0041] The following dehydroabietic acid PROTACs prepared in Examples 1 to 3 are listed as follows:

[0042]

[0043]

[0044]

[0045]

[0046] Example 1

[0047] (1) Preparation of intermediates s1-s12 (preparation of E3 ligand)

[0048] A. The structural formula of intermediate s1(n=1) / s3(n=2) / s5(n=3) / s7(n=4) / s9(n=5) / s11(n=6) is The specific synthesis method is as follows:

[0049] Boc-β-alanine (1 g, 5.2 mmol) and DIPEA (1.81 mL, 10.4 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.6 g, 4.16 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (876 mg, 3.38 mmol) was added and stirred at room temperature. TLC monitoring of the reaction indicated that the reaction was complete after approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s1 (white solid, 1.3 g, 90%).

[0050] Boc-4-aminobutyric acid (1 g, 4.9 mmol) and DIPEA (1.71 mL, 9.8 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.49 g, 3.92 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (826 mg, 3.2 mmol) was added and stirred at room temperature. TLC was used to track the reaction, which was complete in approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s3 (white solid, 1.28 g, 90%).

[0051] Boc-5-aminovaleric acid (1 g, 4.6 mmol) and DIPEA (1.6 mL, 9.2 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.4 g, 3.68 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (775 mg, 2.99 mmol) was added and stirred at room temperature. TLC was used to track the reaction, which was complete after approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s5 (white solid, 1.25 g, 91%).

[0052] Boc-6-aminohexanoic acid (1 g, 4.3 mmol) and DIPEA (1.5 mL, 8.6 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.31 g, 3.44 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (725 mg, 2.8 mmol) was added and stirred at room temperature. TLC monitoring of the reaction indicated that the reaction was complete after approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined and washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s7 (white solid, 1.2 g, 92%).

[0053] Boc-7-aminoheptanoic acid (1 g, 4.1 mmol) and DIPEA (1.43 mL, 8.2 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.25 g, 3.28 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (691 mg, 3.38 mmol) was added and stirred at room temperature. TLC monitoring of the reaction indicated that the reaction was complete after approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s9 (white solid, 1.53 g, 93%).

[0054] Boc-8-aminooctanoic acid (1 g, 3.8 mmol) and DIPEA (1.3 mL, 7.6 mmol) were dissolved in 1 mL of dry DMF solution and stirred at room temperature for 5 min. HATU (1.16 g, 3.04 mmol) was added. After approximately 0.5 h of reaction, lenalidomide (640 mg, 2.47 mmol) was added and stirred at room temperature. TLC was used to track the reaction, which was complete after approximately 12 h. Crushed ice was added to the reaction solution to quench the reaction. The solution was extracted three times with ethyl acetate. The organic layers were combined, washed sequentially with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction was analyzed by PTLC (dichloromethane:methanol = 20:1) to afford intermediate s11 (white solid, 1.11 g, 90%).

[0055] B. The structural formula of intermediate s2(n=1) / s4(n=2) / s6(n=3) / s8(n=4) / s10(n=5) / s12(n=6) is The specific synthesis method is as follows:

[0056] Intermediate s1 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s2 (white solid, 713 mg, 96.0%), which was used directly in the next step without purification.

[0057] Intermediate s3 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s4 (white solid, 675 mg, 91%), which was used directly in the next step without purification.

[0058] Intermediate s5 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s6 (white solid, 705 mg, 95.0%), which was used directly in the next step without purification.

[0059] Intermediate s7 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s8 (white solid, 690 mg, 93.0%), which was used directly in the next step without purification.

[0060] Intermediate s9 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 hours. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s10 (white solid, 668 mg, 90.0%), which was used directly in the next step without purification.

[0061] Intermediate s11 (1 g, 2.25 mmol) was dissolved in a DCM / MeOH (v:v = 3:1) mixture (10 mL). TFA (3 mL) was added dropwise under an ice bath. After addition, the mixture was slowly brought to room temperature. The reaction was monitored by TLC and completed in approximately 12 h. After cessation of the reaction, the solvent was removed by concentration under reduced pressure to afford intermediate s12 (white solid, 683 mg, 92.0%), which was used directly in the next step without purification.

[0062] (2) Preparation of intermediate a1

[0063] Dehydroabietic acid (300 mg, 1 mmol) was added to anhydrous dichloromethane (15 mL) and stirred at 0°C. Oxalyl chloride (254 mg, 2 mmol) was then added dropwise to the mixture, and stirred at room temperature for 6 h. After the reaction, the solvent and excess oxalyl chloride were evaporated under reduced pressure to afford intermediate a1 (reddish-brown solid, 375 mg, 90% yield).

[0064] (3) Preparation of final product T1-6

[0065] Intermediate a1 (1 eq), s2 / s4 / s6 / s8 / s10 / s12 (1.96 eq), EDCl (1.96 eq), and DMAP (1 eq) were dissolved in DCM, sequentially. The reaction was stirred at room temperature for 5-8 h, extracted with DCM, and the organic phase was washed with 5% NaHCO₃ solution and saturated sodium chloride, then dried over anhydrous Na₂SO₄. The solid was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was then purified by column chromatography (PE-EA = 10:1 to 1:1, v / v) to obtain the final products T1 / 2 / 3 / 4 / 5 / 6. Their NMR data are as follows:

[0066] Final product T1 (white powder, 36.7%): ESI-MS m / z 635.32 [M+Na]+. 1H NMR (500 MHz, Chloroform-d) δ 9.47 (s, 1H), 9.41 (d, J = 20.7 Hz, 1H), 7.82 (dd, J = 15.1, 7.9 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.35 (td, J = 7.8, 3.1 Hz, 1H), 7.11 (dd, J = 8.4, 1.5 Hz, 1H),6.97(dt,J=8.3,2.2Hz,1H),6.95–6.82(m,1H),6.77(dd,J=18.2,2.0Hz,1H),5.0 3(dq,J=12.5,6.1,5.6Hz,1H),4.32(s,2H),3.54(dt,J=11.2,6.2Hz,2H),2.82–2.71(m ,2H),2.64(d,J=7.3Hz,2H),2.25(d,J=12.4Hz,1H),2.21–2.11(m,1H),2.10–2.05(m, 1H),2.02–1.94(m,1H),1.91–1.82(m,1H),1.81–1.71(m,2H),1.64–1.60(m,1H),1.48 (d,J=10.5Hz,1H),1.39(dq,J=10.3,6.6,5.4Hz,2H),1.21(d,J=2.5Hz,3H),1.19(s,3 H),1.18(s,3H),1.14(dd,J=8.7,3.2Hz,3H),0.95–0.90(m,1H),0.87–0.79(m,2H).13C NMR (126MHz, CDCl3) δ180.21,179.73,172.44,170.89,169.31,147.09,14 5.05,141.95,135.86,133.44,132.06,130.30,129.15,126.97,124.13,12 3.72,123.67,61.26,47.30,45.49,44.16,37.06,34.38,30.22,29.25,25. 89,25.39,24.08,21.15,19.96,19.24,18.57,16.54,15.97,14.99,14.29.

[0067] Final product T2 (white powder, 32.1%): ESI-MS m / z 649.34 [M+Na]+.1H NMR(500MHz,Chloroform-d)δ8.36(s,1H),7.85(t,J=8.6Hz,1H),7.72–7.61(m,1H),7.45(dd,J=14.5,6.9Hz,1H),7.37–7.27(m,1H),7.18–7 .11(m,1H),7.06–6.94(m,1H),6.88–6.77(m,1H),6.08(s,1H),5.41–5 .09(m,2H),4.47(d,J=14.6Hz,2H),4.26(t,J=5.6Hz,1H),3.83(d,J=16 .1Hz,2H),3.65–3.57(m,2H),3.28–3.19(m,1H),2.84–2.79(m,2H),2. 68(s,1H),2.45(s,2H),2.13(d,J=6.1Hz,1H),2.07–1.99(m,2H),1.41 (d,J=1.7Hz,2H),1.37(d,J=1.6Hz,2H),1.36(s,2H),1.34(s,2H),1.25(s,3H),1.22(d,J=5.6Hz,3H),1.20(d,J=2.7Hz,3H),1.11(s,3H).13C NMR (126MHz, CDCl3) δ181.67,172.07,171.71,170.12,169.46,146.94,146. 03,134.66,134.35,133.80,132.76,132.29,129.20,127.18,124.94,124.3 6,120.56,52.56,49.04,45.56,38.91,38.20,37.50,36.60,34.37,33.11,31.52,30.35,29.44,27.09,25.28,24.06,23.41,21.49,19.96,18.73,16.59.

[0068] Final product T3 (white powder, 32.5%): ESI-MS m / z 663.35 [M+Na]+.1H NMR(500MHz,Chloroform-d)δ9.53–9.18(m,1H),9.05–8.89(m,1H),7.84–7.6 9(m,1H),7.59(q,J=7.2Hz,1H),7.45–7.30(m,1H),7.22–7.03(m,1H),6.98(d t,J=7.0,3.3Hz,1H),6.88–6.62(m,1H),6.23–5.95(m,1H),5.02(ddt,J=10.3 ,7.4,5.1Hz,1H),4.40–4.27(m,2H),3.20(dh,J=28.9,7.3Hz,2H),2.84–2.77 (m,2H),2.66(dtd,J=29.6,17.6,15.3,7.2Hz,2H),2.37(d,J=7.4Hz,2H),2.2 7(d,J=15.2Hz,2H),2.12–1.99(m,2H),1.66(d,J=7.6Hz,2H),1.53(q,J=6.8H z,2H),1.48–1.43(m,2H),1.40(d,J=9.7Hz,1H),1.25–1.21(m,3H),1.20(s,3 H),1.19(s,3H),1.18–1.15(m,3H),0.98–0.89(m,2H),0.86–0.81(m,2H).13C NMR (126MHz, CDCl3) δ179.00,172.36,172.07,170.54,169.35,147.77,145.89, 134.56,133.43,132.51,129.60,129.09,126.99,126.45,124.18,124.10,120. 57,53.12,48.18,45.56,44.16,39.58,38.05,37.29,37.05,36.62,35.72,33.54,31.17,30.05,29.35,28.73,27.15,25.41,24.10,23.20,21.22,18.73,16.63.

[0069] Final product T4 (white powder, 38.7%): ESI-MS m / z 677.37 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.35–9.12(m,1H),8.81(d,J=34.7Hz,1H),7.84–7.69(m,1H),7.61(t,J=7.9Hz,1H),7.38(dt,J=15.7,7.8Hz,1H),7.13(dd,J=8.2,4.4Hz,1H),6.98(dd,J=8.2,2.0Hz,1H),6.84(dd,J=4.5,2.0Hz,1H),6.08–5.95(m,1H),5.03(dt,J=13.6,4.3Hz,1H),4.43–4.30(m,2H),3.18(tq,J=20.0,6.6Hz,2H),2.85–2.78(m,2H),2.73–2.58(m,2H),2.37(t,J=7.4Hz,2H),2.29–2.18(m,2H),2.06(ddd,J=12.8,7.8,2.3Hz,2H),2.00–1.93(m,2H),1.88–1.81(m,1H),1.69(d,J=7.1Hz,2H),1.44(d,J=6.8Hz,2H),1.31–1.28(m,2H),1.27–1.25(m,2H),1.22(d,J=4.5Hz,3H),1.20(d,J=5.1Hz,3H),1.19(d,J=5.3Hz,3H),1.18–1.12(m,3H),0.99–0.93(m,1H),0.86(tq,J=10.5,5.4,4.0Hz,2H). 13 C NMR(126MHz,CDCl3)δ185.75,178.74,173.03,170.93,169.13,147.77,147.06,145.85,134.59,133.43,132.27,132.05,128.36,126.99,124.18,123.73,120.31,60.53,53.56,51.41,47.31,45.87,44.37,39.59,37.29,37.11,33.56,31.39,30.08,29.82,29.32,26.33,25.30,25.14,24.10,21.18,16.60,13.95.

[0070] Final product T5 (white powder, 36.3%): ESI-MS m / z 691.38 [M+Na]+.1H NMR(500MHz,Chloroform-d)δ9.16(d,J=13.2Hz,1H),9.01(d,J=21.3Hz,1H),7. 71(t,J=8.2Hz,1H),7.63(dd,J=7.5,3.9Hz,1H),7.45–7.34(m,1H),7.14(d,J=8. 3Hz,1H),6.99(dd,J=8.2,2.0Hz,1H),6.85(t,J=2.4Hz,1H),6.05–5.83(m,1H), 5.03(dd,J=13.2,5.6Hz,1H),4.46–4.30(m,2H),3.17(p,J=6.7Hz,2H),2.87–2.7 9(m,2H),2.74–2.61(m,2H),2.36(t,J=7.3Hz,2H),2.32–2.17(m,2H),2.13–2.0 7(m,2H),2.06–1.99(m,2H),1.76(dd,J=10.4,8.0Hz,2H),1.69–1.65(m,2H),1.6 3–1.51(m,2H),1.48–1.44(m,2H),1.42(d,J=6.8Hz,2H),1.24(s,3H),1.21(s,3H ),1.20(s,3H),1.17(d,J=17.0Hz,3H),0.99–0.88(m,2H),0.87–0.79(m,2H).13C NMR (126MHz, CDCl3) δ178.83,173.56,171.70,170.23,169.26,149.71,147.59,1 45.81,134.90,133.35,132.55,129.50,128.98,126.90,125.58,124.10,120.20, 67.79,54.78,51.87,47.69,45.49,39.89,37.99,37.31,37.03,35.95,33.45,31.44,29.99,29.52,28.25,26.15,25.50,25.22,23.99,23.17,21.17,18.71,15.87.

[0071] Final product T6 (white powder, 35.9%): ESI-MS m / z 705.40 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ9.24(s,1H),9.02(d,J=3.3Hz,1H),7.70(dd,J=11.8,8.2H z,1H),7.62(t,J=7.3Hz,1H),7.42–7.34(m,1H),7.13(dd,J=8.3,3.5Hz,1H),6.98(dd,J =8.2,2.0Hz,1H),6.84(d,J=2.0Hz,1H),5.99(t,J=5.8Hz,2H),5.02(td,J=9.1,4.6Hz,1 H),4.35(dd,J=20.2,5.4Hz,2H),4.11(q,J=7.1Hz,1H),3.17(dt,J=12.8,5.7Hz,2H),2. 84–2.80(m,2H),2.75–2.62(m,2H),2.38(d,J=7.5Hz,2H),2.29–2.19(m,2H),2.08(dt,J =12.5,2.1Hz,2H),1.73(d,J=4.9Hz,2H),1.65(s,2H),1.55–1.48(m,2H),1.45(dd,J=6. 5,2.4Hz,2H),1.41–1.38(m,2H),1.25(d,J=7.2Hz,3H),1.23–1.23(m,3H),1.21(s,3H), 1.19(d,J=2.0Hz,3H),0.95(dt,J=9.3,2.8Hz,2H),0.94–0.86(m,2H),0.86–0.80(m,2H). 13 CNMR(126MHz, CDCl3)δ178.83,172.92,171.89,170.39,169.87,148.07,145.10,13 5.94,133.47,132.61,129.59,129.07,126.98,125.49,125.02,123.62,121.71,67. 18,61.49,51.90,47.35,45.59,39.73,38.63,38.02,37.36,36.73,33.15,31.52,30.67,29.61,28.98,28.66,25.53,25.31,24.09,21.22,18.79,17.04,14.30,12.40.

[0072] Example 2

[0073] (1) Preparation of intermediates w2-6 and 8

[0074] The structural formula of intermediate w2(n=1) / w3(n=2) / w4(n=3) / w5(n=4) / w6(n=5) / w8(n=7) is The specific synthesis method is as follows:

[0075] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF and stirred to dissolve. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,2-ethylenediamine (1.39 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain w2 (a yellow, transparent oil, 1.3533 g, 60% yield).

[0076] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF and stirred to dissolve. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,3-propylenediamine (1.49 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain w3 (a yellow, transparent oil, 1.4209 g, 63% yield).

[0077] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF and stirred to dissolve. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,4-butanediamine (2 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain w4 (a yellow, transparent oil, 1.53374 g, 68% yield).

[0078] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF and stirred to dissolve. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,5-pentanediamine (1.59 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain w5 (a yellow, transparent oil, 1.5111 g, 67% yield).

[0079] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,6-hexanediamine (1.69 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated NaHCO₃ and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford w6 (a yellow, transparent oil, 1.5788 g, 70% yield).

[0080] Fluorothalidamide (2 g, 7.241 mmol) was dissolved in 20 mL of DMF with stirring. DIEA (1.872 mL, 14.481 mmol) and N-Boc-1,8-octanediamine (1.79 mL, 8.689 mmol) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed sequentially with saturated NaHCO₃ and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford W8 (a yellow, transparent oil, 1.5113 g, 67% yield).

[0081] (2) Preparation of intermediate a1 (same as Example 1)

[0082] (3) Preparation of final product T7-12

[0083] Intermediate a1 (1 eq), w2 / w3 / w4 / w5 / w6 / w8 (2 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (2 eq), and 4-dimethylaminopyridine (DMAP) (1 eq) were sequentially dissolved in DCM (5 mL). The reaction was stirred at room temperature for 5-8 h, extracted with DCM, and the organic phase was washed with 5% NaHCO₃ solution and saturated sodium chloride, and dried over anhydrous Na₂SO₄. The solid was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the crude product, which was then purified by column chromatography (PE-EA = 10:1 to 1:1, v / v) to obtain the final products T7 / 8 / 9 / 10 / 11 / 12, whose NMR data are as follows:

[0084] Final product T7 (fluorescent yellow powder, 44.7%): ESI-MS m / z 621.30 [M+Na]+. 1H NMR (500 MHz, Chloroform-d) δ8.81 (d, J=3.7 Hz, 1H), 7.50–7.47 (m, 1H), 7.13 (d, J=8.2 Hz, 1H), 7.08 (d, J=7.0 Hz, 1H), 7.01 (d, J=8.6 Hz, 1H), 6.97 (d, J=8.1 Hz, 1H), 6.82 (s, 1H), 6.43 (s, 1H), 6.40 (q, J=3.4 Hz, 1H), 4.91 (dd, J=9.5, 2.6 Hz, 1H), 3.49–3.46 (m, 2H),3.43(d,J=9.2Hz,2H),2.78(d,J=6.7Hz,2H),2.77(s,2H),2.70(s,1H),2.28(d,J=13.1Hz,1H),2.11–2.05(m,2H),1.74(d ,J=9.2Hz,2H),1.48(d,J=13.3Hz,2H),1.22(s,3H),1.19(s,3H),1.18(s,3H),0.95(d,J=9.4Hz,1H),0.84(t,J=6.2Hz,2H).13C NMR (126MHz, CDCl3) δ179.49,172.22,170.56,168.84,167.69,147.01,1 46.95,145.79,136.42,134.66,132.50,126.92,124.49,123.96,117.10 ,111.95,110.21,50.80,48.98,47.45,45.48,42.12,39.46,38.01,37.2 8,37.10,33.51,31.45,29.94,25.24,24.07,22.82,21.21,18.77,16.54.

[0085] Final product T8 (fluorescent yellow powder, 45.2%): ESI-MS m / z 635.32 [M+Na] + . 1 H NMR(500MHz,Chloroform-d)δ8.80(d,J=6.9Hz,1H),7.49–7.45(m,1H),7.14(d,J =8.1Hz,1H),7.06(dd,J=7.1,2.8Hz,1H),6.98(dd,J=8.1,2.0Hz,1H),6.86(d,J=8 .5Hz,1H),6.84(d,J=2.0Hz,1H),6.38(q,J=5.8Hz,1H),6.27–6.13(m,1H),4.93– 4.88(m,1H),3.38–3.34(m,2H),3.30(d,J=6.4Hz,2H),2.83(dd,J=8.1,3.5Hz,2H) ,2.80–2.78(m,1H),2.71(q,J=3.8Hz,1H),2.28(dd,J=13.7,3.5Hz,1H),2.10–2. 04(m,2H),1.83(t,J=6.7Hz,2H),1.77–1.73(m,2H),1.67(dd,J=9.3,3.5Hz,1H),1 .52(qd,J=9.1,8.6,3.2Hz,2H),1.46(d,J=7.3Hz,1H),1.23(s,3H),1.20(s,3H), 1.19(d,J=1.4Hz,3H),1.15(s,3H),0.98–0.94(m,1H),0.85(h,J=5.6,4.8Hz,2H). 13 C NMR (126MHz, CDCl3) δ178.98,171.73,169.54,168.81,167.69,147.04,1 46.77,145.78,136.27,134.62,132.97,127.37,124.11,124.00,116.67 ,111.64,110.52,48.95,47.34,45.47,40.18,38.02,37.36,37.13,33.5 0,31.45,30.01,29.53,25.26,24.07,24.05,22.81,21.24,18.80,16.57.

[0086] Final product T9 (fluorescent yellow powder, 43.3%): ESI-MS m / z 649.34 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.71(s,1H),7.48–7.44(m,1H),7.14(d,J=8.2Hz, 1H),7.06(d,J=7.1Hz,1H),6.98(dd,J=8.3,2.0Hz,1H),6.87(d,J=8.6Hz,1H),6. 84(d,J=2.0Hz,1H),6.24(t,J=5.8Hz,1H),5.99(t,J=5.8Hz,1H),4.91(dd,J=12. 1,5.4Hz,1H),3.30(d,J=6.6Hz,2H),3.27(d,J=6.6Hz,2H),2.83(dd,J=8.5,3.6H z,2H),2.81–2.77(m,1H),2.77–2.70(m,2H),2.28(dd,J=13.4,3.4Hz,1H),2.12 –2.08(m,2H),1.76(dd,J=9.1,3.8Hz,2H),1.73–1.72(m,1H),1.69–1.65(m,2H), 1.64(d,J=7.2Hz,2H),1.60(dd,J=8.0,5.3Hz,2H),1.52(d,J=9.9Hz,1H),1.23(s ,3H),1.20(s,3H),1.19(s,3H),1.15(s,3H),0.96(d,J=8.6Hz,1H),0.85(s,1H). 13 C NMR (126MHz, CDCl3) δ178.66,172.68,169.62,168.77,167.70,147.05,146. 90,145.77,136.26,134.65,132.51,126.93,124.12,123.98,116.78,111.5 8,109.97,48.93,47.30,45.52,42.23,39.31,38.02,37.38,37.12,33.50,31.46,30.02,27.24,26.63,25.28,24.06,24.05,22.83,21.20,18.80,16.57.

[0087] Final product T10 (fluorescent yellow powder, 48.2%): ESI-MS m / z 663.35 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.54(s,1H),7.50–7.46(m,1H),7.15(d,J=8.1Hz, 1H),7.07(d,J=7.1Hz,1H),6.98(dd,J=8.1,2.0Hz,1H),6.87(s,1H),6.86–6.84( m,1H),6.23(t,J=5.6Hz,1H),5.87(t,J=5.7Hz,1H),4.90(dd,J=12.3,5.3Hz,1H ),3.27(d,J=6.5Hz,2H),3.24(d,J=6.7Hz,2H),2.88–2.84(m,2H),2.82–2.78(m, 1H),2.77–2.69(m,2H),2.29(dd,J=13.3,3.0Hz,1H),2.10(dq,J=12.2,3.3Hz,2 H),1.75(d,J=5.3Hz,2H),1.73–1.71(m,1H),1.69(d,J=7.3Hz,2H),1.55(d,J=7. 8Hz,2H),1.52(d,J=2.8Hz,2H),1.43(d,J=7.1Hz,2H),1.24(s,3H),1.21(s,3H) ,1.20(d,J=4.7Hz,3H),1.19–1.13(m,3H),0.98–0.94(m,1H),0.87–0.83(m,2H). 13 C NMR (126MHz, CDCl3) δ178.55,171.40,169.61,168.64,167.74,147.08,147.0 1,145.82,136.25,134.67,132.96,128.26,124.16,124.01,116.76,111.55,1 09.98,50.85,48.96,47.32,45.63,42.56,39.64,38.06,37.38,37.15,33.53,31.50,30.08,29.50,28.94,25.30,24.33,24.07,22.88,21.20,18.84,17.34.

[0088] Final product T11 (fluorescent yellow powder, 47.3%): ESI-MS m / z 677.37 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.52(s,1H),7.49–7.45(m,1H),7.15(d,J=8.2Hz, 1H),7.08(s,1H),6.98(dd,J=8.2,2.1Hz,1H),6.87(s,1H),6.86–6.84(m,1H),6. 23(t,J=5.7Hz,1H),5.82(d,J=5.5Hz,1H),4.91(dd,J=8.0,4.3Hz,1H),3.26(dd ,J=7.4,2.5Hz,2H),3.25–3.23(m,2H),2.87(d,J=4.3Hz,1H),2.82–2.76(m,2H), 2.76–2.65(m,2H),2.32–2.26(m,1H),2.12–2.08(m,2H),1.76(d,J=9.8Hz,2H), 1.73(q,J=2.3Hz,1H),1.65(d,J=7.4Hz,2H),1.58–1.53(m,2H),1.50(d,J=3.5Hz ,2H),1.43(t,J=6.7Hz,2H),1.38–1.34(m,2H),1.25(d,J=3.1Hz,3H),1.21(s,3 H),1.20(s,3H),1.19(s,3H),0.97(d,J=8.5Hz,1H),0.85(dd,J=6.9,5.4Hz,2H). 13 C NMR (126MHz, CDCl3) δ178.45,171.40,169.61,168.65,167.73,147.10,14 7.05,145.79,136.22,134.69,132.57,126.96,124.15,123.99,116.74,11 1.49,109.95,48.95,47.30,45.62,39.71,38.07,37.40,37.15,33.53,31. 49,30.07,29.66,29.22,26.69,25.31,24.07,22.88,21.19,18.84,16.62.

[0089] Final product T12 (fluorescent yellow powder, 45.2%): ESI-MS m / z 733.43 [M+Na] + . 11H NMR (500 MHz, Chloroform-d) δ 8.40 (d, J = 5.4 Hz, 1H), 7.48 (dd, J = 8.6, 7.0 Hz, 1H), 7.16 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 7.1 Hz, 1H), 6.99 (dd, J = 8.2, 2.0 Hz, 1H), 6.88 (s, 1H), 6.86–6.84 (m, 1H), 6.23 (t, J = 5.6 Hz, 1H), 5.78 (t, J = 5.7 Hz, 1H), 4.91 (ddd, J = 12.3, 5.3, 1.7 Hz, 1H), 3.26 (d, J = 6.6 Hz, 2H), 3.24–3.21 (m, 2H), 2.89–2.84 (m, 2H), 2.84–2.79 (m, 1H), 2.79–2.67 (m, 2H), 2.33–2.23 (m, 1H), 2.11 (ddd, J = 12.3, 5.5, 2.3 Hz, 2H), 1.89 (ddd, J = 15.7, 5.8, 2.4 Hz, 1H), 1.79–1.75 (m, 2H), 1.75–1.69 (m, 2H), 1.66 (q, J = 7.3 Hz, 2H), 1.59 (d, J = 32.9 Hz, 2H), 1.54 (d, J = 2.4 Hz, 1H), 1.51–1.48 (m, 2H), 1.46 (q, J = 3.0 Hz, 2H), 1.39 (d, J = 6.9 Hz, 2H), 1.35 (s, 1H), 1.32 (d, J = 3.1 Hz, 3H), 1.25 (s, 3H), 1.21 (d, J = 1.2 Hz, 3H), 1.20 (s, 3H), 1.17 (s, 1H), 1.00–0.95 (m, 1H), 0.91–0.84 (m, 2H), 0.84–0.72 (m, 1H). 13 13C NMR (126 MHz, CDCl3) δ 178.38, 171.28, 169.62, 168.56, 167.75, 147.13, 147.11, 145.37, 136.72, 134.74, 132.59, 127.55, 124.18, 122.56, 116.76, 112.06, 109.93, 53.56, 48.96, 47.30, 45.66, 42.15, 40.55, 39.88, 39.82, 38.09, 37.41, 37.17, 33.55, 31.52, 30.10, 29.73, 29.25, 29.21, 26.92, 25.34, 23.69, 22.92, 21.18, 18.87, 16.64.

[0090] Example 3

[0091] (1) Preparation of intermediates p1-p3

[0092] The structural formula of the intermediate p1(n=0) / p2(n=1) / p(n=2)3 is

[0093]

[0094] Synthesis of intermediate p1: Fluorothalidamide (100 mg, 0.362 mmol, 1 eq) was dissolved in 3 mL of DMF and stirred to dissolve. DIEA (120 μL, 0.742 mmol, 2 eq) and N-Boc-2,2'-(ethylenemonooxy)diethylamine (129 μL, 0.543 mmol, 1.5 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate p2 (yellow oil, 440 mg, 30%).

[0095] Synthesis of intermediate p2: Fluorothalidamide (100 mg, 0.362 mmol, 1 eq) was dissolved in 3 mL of DMF and stirred to dissolve. DIEA (120 μL, 0.742 mmol, 2 eq) and N-Boc-2,2'-(ethylenedioxy)diethylamine (129 μL, 0.543 mmol, 1.5 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, and concentrated to yield intermediate p2 (yellow oil, 440 mg, 30%).

[0096] Synthesis of intermediate p3: Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF and stirred to dissolve. DIEA (1.872 mL, 14.481 mmol, 2 eq) and N-Boc-2,2'-(ethylenetrioxy)diethylamine (2 mL, 8.689 mmol, 1.2 eq) were then added and the mixture was heated to 90°C and refluxed for 12 h. After completion, the reaction was quenched with ice water and extracted with ethyl acetate. The combined organic phases were washed with saturated NaHCO3 and then saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain intermediate p3 (a yellow, transparent oil, 228.6 mg, 30% yield).

[0097] (2) Preparation of intermediate a1 (same as Example 1)

[0098] (3) Preparation of final product T13-15

[0099] Intermediate a1 (1 eq) and DIPEA (3 eq) were dissolved in 1 mL of dry DMF solution, stirred at room temperature for 5 min, and HATU (1.5 eq) was added. After about 0.5 h of reaction monitored by TLC, p1 / 2 / 33 (2 eq) was added and stirred at room temperature. TLC was used to track the reaction, and the reaction was complete in about 12 h. Crushed ice was added to the reaction solution to quench the reaction, and the mixture was extracted with ethyl acetate three times. The organic layers were combined and washed with saturated ammonium chloride solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The final product T13 / 14 / 15 was obtained by PTLC (dichloromethane:methanol = 15:1). Its NMR data are as follows:

[0100] Final product T13 (fluorescent yellow powder, 42.1%): ESI-MS m / z 665.33 [M+Na]+. 1H NMR (500 MHz, Chloroform-d) δ8.64 (d, J=5.4 Hz, 1H), 7.51–7.47 (m, 1H), 7.15–7.11 (m, 1H), 7.11–7.08 (m, 1H), 6.97 (dt, J=8.2, 2.0 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 6.82 (d, J=2.4Hz,1H),6.50(t,J=5.1Hz,1H),6.31(dq,J=11.5,5.3,4.6Hz,1H),4.91(qd,J=7 .0,6.3,2.1Hz,1H),3.69(t,J=5.1Hz,2H),3.57(t,J=4.9Hz,2H),3.46(s,2H),3.44(s ,1H),2.85(ddd,J=17.6,9.0,4.1Hz,2H),2.79(dd,J=7.7,4.3Hz,2H),2.26(dd,J=13 .2,3.5Hz,1H),2.14–2.06(m,2H),2.00(d,J=6.2Hz,1H),1.79–1.72(m,2H),1.70(dd ,J=10.8,4.9Hz,2H),1.49–1.44(m,2H),1.20(s,3H),1.19(s,3H),1.18(d,J=1.9Hz, 3H),1.15(d,J=25.7Hz,3H),0.96–0.92(m,1H),0.83(dtd,J=9.0,6.3,3.5Hz,2H).13C NMR (126MHz, CDCl3) δ178.81,171.91,169.51,168.62,166.49,148.13,146. 90,144.88,136.25,134.70,132.56,126.90,124.17,123.96,116.84,112.5 2,110.40,70.09,67.82,50.82,49.00,47.28,45.47,41.83,39.64,38.00,37.12,37.04,33.51,31.18,30.11,25.30,24.06,22.84,21.13,18.81,15.54.

[0101] Final product T14 (fluorescent yellow powder, 46.7%): ESI-MS m / z 709.36 [M+Na]+. 1H NMR (500 MHz, Chloroform-d) δ8.93–8.79 (m, 1H), 7.53–7.49 (m, 1H), 7.20–7.12 (m, 1H), 7.11 (s, 1H), 7.00 (dd, J=8.3, 2.0 Hz, 1H), 6.91 (t, J=9.3 Hz, 1H), 6.86 (s, 1H), 6.51 (t ,J=5.4Hz,1H),6.33(dt,J=17.1,5.6Hz,1H),4.94–4.89(m,1H),3.77–3.70(m,2H),3. 67(s,2H),3.60(q,J=4.8,4.3Hz,2H),3.50–3.47(m,2H),3.44(d,J=5.5Hz,1H),2.86(d t,J=10.5,3.8Hz,2H),2.78–2.68(m,2H),2.29(dd,J=13.2,3.9Hz,1H),2.12(ddt,J=1 3.0,10.4,2.7Hz,2H),2.06–2.00(m,1H),1.83–1.76(m,2H),1.73(ddd,J=19.7,9.0,6 .5Hz,2H),1.69(s,1H),1.57–1.51(m,2H),1.50–1.47(m,1H),1.28–1.24(m,3H),1.23 (s,3H),1.21(s,3H),1.19(d,J=14.8Hz,3H),0.98–0.95(m,1H),0.89–0.82(m,2H).13C NMR (126MHz, CDCl3) δ177.26,173.00,169.96,168.30,167.00,148.13,146. 83,144.74,136.17,134.78,132.64,126.93,124.18,123.20,117.34,112.9 3,110.45,70.80,70.18,70.05,69.36,53.55,48.97,47.29,45.57,42.37,39.56,38.03,37.06,33.52,31.48,30.10,25.32,24.08,22.95,18.82,17.20.

[0102] Final product T15 (fluorescent yellow powder, 45.3%): ESI-MS m / z 753.38 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.90(d,J=6.2Hz,1H),7.46(ddd,J=8.7,7.2,2.6Hz,1H),7 .13(d,J=8.2Hz,1H),7.07(d,J=7.1Hz,1H),6.96(dd,J=8.2,2.0Hz,1H),6.89(dd,J=8.6, 4.0Hz,1H),6.83(d,J=2.0Hz,1H),6.47(t,J=5.6Hz,1H),6.40–6.30(m,1H),4.89(dd,J=1 2.0,5.5Hz,1H),3.70(dd,J=10.0,5.2Hz,2H),3.65(s,2H),3.64(d,J=3.9Hz,2H),3.63(s ,1H),3.61–3.60(m,2H),3.53(dt,J=5.3,2.3Hz,2H),3.45(d,J=5.6Hz,2H),3.43(s,2H), 2.87–2.81(m,2H),2.80–2.72(m,2H),2.30–2.23(m,2H),2.13–2.04(m,2H),1.72(dd,J=1 0.9,3.4Hz,2H),1.56–1.50(m,2H),1.48–1.43(m,2H),1.23(d,J=3.1Hz,3H),1.19(s,3H) ,1.18(s,3H),1.18–1.14(m,3H),0.95(dd,J=6.4,3.8Hz,1H),0.83(q,J=5.0,3.7Hz,2H). 13 C NMR (126MHz, CDCl3) δ178.71,171.57,169.81,168.72,167.20,147.07,146.82, 145.02,136.09,134.73,132.56,126.89,124.13,123.89,116.80,111.71,109. 61,70.79,70.58,70.54,70.19,69.97,69.51,48.51,47.25,44.73,42.40,39.46,37.99,37.10,33.47,31.47,30.06,25.30,23.48,22.82,21.09,18.79,16.49.

[0103] Example 4 In vitro antitumor activity test of dehydroabietic acid PROTACs (IC 50 )

[0104] The dehydroabietic acid PROTACs prepared in Examples 1 to 3 of the present invention were tested for their ability to inhibit tumor cell proliferation using a conventional CCK-8 method. When the tumor cells (MCF-7 cells (human breast cancer cells)) grew to 80% to 90% of the culture dish, the cells were digested from the culture dish, centrifuged and resuspended in fresh DMEM / 1640 complete medium, counted under a microscope, and then the cell suspension was diluted to 50 cells / μL. A circle of sterile PBS was spread on the outermost periphery of the 96-well plate, 100 μL per well, and 100 μL of the diluted cell suspension was spread on each well of the remaining wells and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the medium in the 96-well plate was aspirated, and the drug was diluted with fresh complete medium at a certain concentration gradient and then added to the 96-well plate in sequence. After 48 hours, the medium containing the drug was removed, and CCK-8 liquid diluted with medium was added (100 μL medium + 10 μL CCK-8). The cells were incubated in a 37°C, 5% CO2 incubator for 30-40 minutes. The absorbance was measured at OD 450 on a microplate reader, and the inhibition rate was calculated based on the absorbance at OD 450. The data were used to calculate the half-maximal inhibitory concentration (IC50) using SPSS software.

[0105] The calculation formula is as follows:

[0106] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0107] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution);

[0108] Ac: absorbance of control well (containing cells, culture medium, and CCK-8 solution, but no drug);

[0109] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0110] Experimental Results: To further evaluate the in vitro antitumor activity of the target compound, MCF-7, Hela, and A549 (human lung cancer) cells were used as test tumor lines, with T as the control group. The results are shown in Table 1. Overall, the dehydroabietic acid PROTACs demonstrated superior antitumor activity compared to the dehydroabietic acid derivative itself. Based on the IC50 values ​​and compound structure, B13 was selected as the dosing group for further proteomic analysis.

[0111] Table 1 In vitro antitumor activity (IC50) of T and T1 to T15

[0112]

[0113]

[0114] Example 5 Proteomic Analysis of Dehydroabietic Acid PROTACs

[0115] In this example, a total of 9 cell samples were set up and divided into 3 groups, namely PROTACs group (drug group / T-PROTAC), atNP group (control group / T) and blank group (con), and the biological replicates corresponding to each group were 3 cases. Three groups of data were compared: drug group vs. control group, drug group vs. blank group, and control group vs. blank group. The data were searched and analyzed using Proteome Discoverer software (PD) (version 2.4.0.305, Thermo Fisher Scientific) and the built-in Sequest HT search engine.

[0116] Experimental results: Based on LC-MS quantitative analysis of proteins, a total of 6105 proteins and 41392 peptides were identified in this example. The distribution of differential proteins is as follows Figure 1 A, Con VS T-PROTAC differential protein subcellular localization analysis Figure 1 B, Analysis of differential protein subcellular localization of T-PROTACs VS T Figure 1 C, Analysis of differential subcellular localization of con VS T proteins Figure 1 D. Since the mechanism of action of PROTACs small molecules is to directly degrade proteins, this example focuses on the down-regulated differential proteins among the total differential proteins. The top 20 down-regulated differential proteins of T-PROTAC VS T are as follows: Figure 2 The top 20 down-regulated differentially expressed proteins of T-PROTAC VS con are as follows: Figure 3 , (based on the screening principle) screened out the common differential proteins in the two groups, combined with some differential proteins of T VS con ( Figure 4 ), EIF1, TMBIM6, and FANCA were initially identified as potential target proteins for the anti-tumor effects of dehydroabietic acid. TMBIM6 is an inhibitor of apoptosis

[158] and can perform signal transduction by regulating the unfolded protein response. At the same time, TMBIM6 also regulates calcium homeostasis in the endoplasmic reticulum by acting as a calcium leak channel. In addition, further studies have shown that TMBIM6 has also been used to study several pathways related to apoptosis. First, it can directly inhibit the IRE1α signaling pathway and downstream XBP1 mRNA splicing by competing with BAX / BAK for the binding site of IRE1α. Second, because TMBIM6 is located on the ER membrane, it is involved in the [Ca] 2+ ]. Resting ER Ca in cells overexpressing BI-1 2+ Low levels of ER Ca 2+Leakage is higher, and when TMBIM6 is downregulated, it leads to mitochondrial Ca 2+ Increased Ca uptake 2+ Finally, TMBIM6 can prevent the accumulation of ROS under endoplasmic reticulum stress conditions by interacting with MMO members NPR and CYP2E1. TMBIM6 can also promote antioxidant responses through NRF2 and heme oxygenase 1 (HO-1), thereby offsetting the damage caused by ROS to cells.

[0117] Example 6 Molecular docking results of dehydroabietic acid and potential target TMBIM6

[0118] The docking simulation of dehydroabietic acid and TMBIM6 was performed as follows:

[0119] (1) Prepare the software operating environment: First, create a folder named autodock.vina and copy the vina.exe program to this folder. Next, open AutoDock Tools, find set in File-Preferences, and select the autodock vina folder in the startup dictionary. Click make default to complete the setup. All information will be saved in this folder.

[0120] (2) Protein preparation: Download the protein from the PDB database, remove water molecules and receptor proteins using PyMOL software, and save it in PDB format with the file name **.pdb. Then import it into AutoDock Tools software and select Edit-Hydrogens-Add hydrogen, Edit-ChargesComputeGasteige calculate charge, and Edit-Atoms-AssignAD4type to set the atom type. After completion, export it to the software-specific format .pdbqt with the file name **.pdbqt.

[0121] (3) Prepare the receptor molecule: Use Chemdraw to draw the molecular structure of the small molecule, then open the Chemdraw3D software, import the small molecule into it, and click CalcμLationMM2-minimize energy in the menu bar to optimize the small molecule structure. After completion, click Save As in File to output it in PDB format, and the file name is small molecule.pdb. Then use read molecμLe in File in AutoDockTools software to open the small molecule.pdb, and perform Edit-Hydrogens-Add hydrogenation, Ligand-Torsion Tree-DetectRoot to calculate the maximum number of rotatable bonds, and Ligand-TorsionTree-Choose Torsions-Done to set the rotatable bonds. After completion, set it as a ligand and export it in the small molecule.pdbqt special format.

[0122] (4) Molecular docking: Finally, the target and the active ingredient structure were molecularly docked using VINA within the Pyrx software (https: / / pyrx.sourceforge.io / ). The affinity (kcal / mol) value represents the binding affinity between the two. The lower the binding affinity, the more stable the binding between the ligand and the receptor.

[0123] (5) Result processing: Use AutoDock Tools to open resμLt.pdbqt and export it as a pdb format file, then open it with PyMOL. Import resμLt.pdb into PyMOL, select the optimal model from the nine small molecule conformations, and analyze the interaction between the compound and the protein. It is generally believed that a docking energy value less than -4.25 kcal / mol indicates a certain binding activity between the two, less than -5.0 kcal / mol indicates good binding activity, and less than -7.0 kcal / mol indicates strong binding activity.

[0124] Experimental results: The docking results of TMBIM6 protein and small molecules are as follows Figure 5 As shown, the small molecule exhibited a high degree of fit between the protein and the receptor binding pocket, with a binding energy of -8.0 kcal / mol, indicating potential for native binding. Furthermore, TMBIM6 formed a hydrogen bond with amino acid residue PHE-39 in the protein and the small molecule ligand. In summary, TMBIM6 has the potential to form multiple interactions with molecules.

[0125] Example 7 Degradation efficiency test of dehydroabietic acid PROTACS for potential target protein TMBIM6

[0126] The dehydroabietic acid PROTAC compound T15 prepared in Example 3 of the present invention was tested for its potential target protein degradation efficiency. The test method used a conventional WB method. Based on the existing in vitro anti-tumor test data (IC 50 ), the degradation efficiency of the potential target protein TMBIM6 by the dehydroabietic acid PROTAC was further tested. A549 cells were treated with different concentrations of T15, with T as a control group, to study the protein degradation efficiency at different concentrations and explore whether the degradation of TMBIM6 is dependent on the T15 concentration.

[0127] Experimental results: The degradation of TMBIM6 is dependent on the concentration of T15 ( Figure 6 、 7 ), which showed better degradation efficiency than T at 50 μM.

[0128] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A dehydroabietic acid PROTACs, characterized in that: Its structural formula is selected from at least one of the following:

2. Use of the dehydroabietic acid PROTACs or pharmacologically or physiologically acceptable salts thereof according to claim 1 in preparing a cancer therapeutic composition, characterized in that: The cancer is selected from breast cancer, uterine cancer and liver cancer.

3. A cancer treatment composition, characterized in that: The active ingredient comprises the dehydroabietic acid PROTACs or a pharmacologically or physiologically acceptable salt thereof according to claim 1, and the cancer is selected from breast cancer, uterine cancer and liver cancer.

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