Maslinic acid PROTACs as well as preparation method and application thereof

Abstract

The invention provides maslinic acid PROTACs as well as a preparation method and application thereof, and belongs to the technical field of drug development. According to the invention, maslinic acid is taken as a POI ligand, lenalidomide and fluoro-thalidomide are taken as E3 ligands, and linker selects a PEG chain or an aliphatic chain, so that the anti-cancer activity of the compound is superior to that of maslinic acid, and the compound has a good potential application prospect in the aspect of anti-tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug development, and particularly relates to a maslinic acid PROTAC and a preparation method and application thereof. BACKGROUND

[0002] Protein degradation targeting chimeras (PROTACs) is a promising new type of drug molecule, which is similar in structure to a dumbbell and consists of three key parts: an E3 ubiquitin ligase ligand (E3 ligand), a target protein ligand (POI ligand), and a linker connecting the two. The mechanism of action of PROTACs relies on its bifunctional nature: one end binds to the target protein, and the other end recruits E3 ubiquitin ligase, forming a POI-PROTAC-E3 ligase ternary complex in the cell.

[0003] In this process, the E3 ubiquitin ligase catalyzes the covalent modification of the target protein by ubiquitin molecules, allowing it to be recognized and degraded by the 26S proteasome. The advantage of PROTACs is that it does not require tight binding to the active site of the target protein, but only requires a short and weak interaction (such as hydrogen bonding, hydrophobic interaction, etc.) to trigger ubiquitin-proteasome system (UPS) mediated protein degradation.

[0004] Due to its unique "event-driven" mode of action (i.e., catalytic degradation rather than inhibition), PROTACs have shown great potential in the field of drug development. Currently, several PROTAC molecules have entered the clinical trial stage, and are expected to provide new treatment strategies for diseases such as cancer and inflammation. And PROTAC drugs have many advantages over traditional small molecule drugs, which are roughly as follows: (1) The design of PROTAC drugs can be free from the Lipinski's rule of five; (2) The pharmacological action of traditional small molecules relies on occupancy-driven binding to the active site of the target protein. The principle is to bind to the active site of the enzyme or receptor to exert its effect. Traditional drugs need to maintain a certain drug concentration in the body, and have high requirements for small molecule binding sites. However, PROTACs can grab the target protein from any corner, and their pharmacological mechanism is to activate the potential target and degrade the target protein to achieve pharmacodynamic effects. Data show that very high drug concentrations are not required to achieve pharmacodynamic effects, and the requirements for binding sites are relatively low, and the pharmacodynamics are better; (3) About 50% of proteins in the human body are found to be associated with 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; (4) Compared with traditional small molecules, PROTACs can ignore drug resistance. In theory, PROTACs can better accommodate mutations in target proteins; (5) PROTACs are more persistent and have stronger inhibitory power than traditional drug molecules.

[0005] Pentacyclic triterpenoids have a wide range of anti-tumor activity and no obvious toxicity, and are a promising leading compound for developing new multi-target anti-tumor drugs. Maslinic acid (MA) is a pentacyclic triterpenoid acid compound, which exists in many natural plants, especially hawthorn, red dates, loquat leaves and olive trees, and has high safety for the human body. It has a wide range of pharmacological benefits, including anti-cancer, anti-inflammatory analgesic effects, anti-platelet aggregation, heart protection and anti-inflammatory, antibacterial, liver protection, anti-diabetic and anti-hyperlipidemic biological activities. Studies have shown that maslinic acid has strong clinical potential. Therefore, it is of great significance to study a maslinic acid PROTAC and its preparation method and application. SUMMARY

[0006] The purpose of the present application is to provide a maslinic acid PROTAC and its preparation method and application, so as to solve the problem of low anti-cancer activity of maslinic acid in the prior art.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a maslinic acid PROTAC, the structure general formula of which is shown in formula I:

[0009]

[0010] Among them, E3 ligase is selected from lenalidomide Or fluorosalidomide

[0011]

[0012] Linker is a first fatty chain, a second fatty chain or a PEG chain;

[0013] The first fatty chain is -(CH2) n1 -CO-, n1 is an integer selected from 1 to 7;

[0014] The second fatty chain is -(CH2) n2 -CH2-, n2 is an integer selected from 1 to 7;

[0015] The PEG chain is -(CH2-CH2-O) n3 -CH2-CH2-, n3 is an integer selected from 1 to 3.

[0016] The present application also provides a preparation method of the above-mentioned maslinic acid PROTAC, comprising the following steps:

[0017] (1) with the first fatty chain corresponding compound and lenalidomide as raw materials, N,N-dimethylformamide as a solvent, activated by N,N-diisopropyl ethylamine and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, an intermediate BS is prepared;

[0018] The first fatty chain corresponding compound is selected from Boc-2-amino acetic acid, Boc-3-amino propionic acid, Boc-4-amino butyric acid, Boc-5-amino pentanoic acid, Boc-6-amino hexanoic acid, Boc-7-amino heptanoic acid or Boc-8-amino octanoic acid;

[0019] (2) an intermediate S is prepared with the intermediate BS and trifluoroacetic acid as raw materials;

[0020] (3) the maslinic acid, N,N-diisopropyl ethylamine and (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) are mixed in a solvent, and then the intermediate S is added for reaction to prepare the maslinic acid PROTACs compound.

[0021] The application further provides a preparation method of the maslinic acid PROTACs, and the preparation method comprises the following steps:

[0022] (1) with the second fatty chain corresponding compound and fluorosaliridomide as raw materials, N,N-dimethylformamide as a solvent, activated by N,N-diisopropyl ethylamine, an intermediate BP is prepared;

[0023] The second fatty chain corresponding compound is selected from N-Boc-1,2-ethylenediamine, N-Boc-1,3-propanediamine, N-Boc-1,4-butanediamine, N-Boc-1,5-pentanediamine, N-Boc-1,6-hexanediamine or N-Boc-1,8-octanediamine;

[0024] (2) an intermediate P is prepared with the intermediate BP and trifluoroacetic acid as raw materials;

[0025] (3) the maslinic acid, N,N-diisopropyl ethylamine and (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) are mixed in a solvent, and then the intermediate P is added for reaction to prepare the maslinic acid PROTACs compound.

[0026] The application further provides a preparation method of the maslinic acid PROTACs, and the preparation method comprises the following steps:

[0027] (1) with the PEG chain corresponding compound and fluorosaliridomide as raw materials, N,N-dimethylformamide as a solvent, activated by N,N-diisopropyl ethylamine, an intermediate BO is prepared;

[0028] The PEG chain corresponds to a compound selected from tert-butyl (2-(2-aminoethoxy)ethyl)carbamate, N-Boc-3,6-dioxa-1,8-octanediamine or N-Boc-1,11-diamino-3,6,9-trioxaundecane;

[0029] (2) preparing intermediate O with intermediate BO and trifluoroacetic acid as raw materials;

[0030] (3) mixing Hawthorn acid, N,N-diisopropylethylamine and (2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate) in a solvent, and then adding intermediate O to react to prepare Hawthorn acid PROTACs compound.

[0031] The application also provides a derivative of the Hawthorn acid PROTACs as described above, wherein the derivative is selected from pharmaceutically acceptable salts of the Hawthorn acid PROTACs compound.

[0032] The application also provides a use of the Hawthorn acid PROTACs as described above in the preparation of an antitumor drug.

[0033] The application has the following beneficial effects:

[0034] The application uses Hawthorn acid as a POI ligand, uses lenalidomide and fluorinated thalidomide as E3 ligands, and selects a PEG chain or a fatty chain as a linker, and the anti-cancer activity is better than that of Hawthorn acid, and the application has a good potential application prospect in the treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Protein bands affected by JAK2, IL-6, P-JAK2, STAT3 and P-STAT3 for immunoblotting analysis of Hawthorn acid MA and Hawthorn acid PROTAC-SZ13;

[0036] Figure 2 Relative abundance column chart of protein bands affected by JAK2, IL-6, P-JAK2, STAT3 and P-STAT3 for immunoblotting analysis of Hawthorn acid MA and Hawthorn acid PROTAC-SZ13;

[0037] Figure 3 Protein bands affected by Bcl-2, Bax, Xiap, Surviving, Caspase-9, Caspase-3, Cleaved caspase-9, Cleaved caspase-3, PARP1 and Cytochrome c for immunoblotting analysis of Hawthorn acid MA and Hawthorn acid PROTAC-SZ13;

[0038] Figure 4Relative abundance column chart of the influence of MA and MA-PROTAC-SZ13 on Bcl-2, Bax, Xiap, Surviving, Caspase-9, Caspase-3, Cleaved caspase-9, Cleaved caspase-3, PARP1, Cytochrome c by Western blot analysis;

[0039] Figure 5 Protein bands of the influence of MA and MA-PROTAC-SZ13 on JNK1, p-JNK1, IRE1, p-IRE1, BIP, CHOP, ATF4, ATF6, Caspase-12, Cleaved caspase-12 by Western blot analysis;

[0040] Figure 6 Relative abundance column chart of the influence of MA and MA-PROTAC-SZ13 on JNK1, p-JNK1, IRE1, p-IRE1, BIP, CHOP, ATF4, ATF6, Caspase-12, Cleaved caspase-12 by Western blot analysis;

[0041] Figure 7 Docking diagram of MA and JAK2 protein, wherein a is a detailed docking diagram of MA and JAK2, b is a docking pocket of MA-JAK2, c is a surface electrostatic force formed by MA and JAK2 protein, d is a two-dimensional visualization of MA-JAK2 docking, and e is a binding energy of MA and JAK2 docking;

[0042] Figure 8 Cell thermal migration experiment was used to verify the binding ability between the anti-tumor target JAK2 and the target protein ligand MA, wherein a is a thermal stability Western blot analysis diagram, and b is a statistical analysis diagram;

[0043] Figure 9 Drug affinity response target stability experiment was used to verify that the target protein ligand MA can improve the anti-enzymatic stability of the anti-tumor target JAK2, wherein a is a DARTS Western blot analysis diagram, and b is a statistical analysis diagram;

[0044] Figure 10 Verification of the dependence of SZ-13 on JAK2 protein degradation, wherein a is a concentration-dependent degradation immunization analysis diagram, and b is a statistical analysis diagram. DETAILED DESCRIPTION

[0045] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0046] The names and structural formulas of the compounds of the hawthorn acid PROTACs prepared in Examples 1-3 are shown in Table 1:

[0047] Table 1 The names and structural formulas of the compounds of the hawthorn acid PROTACs prepared in Examples 1-3

[0048]

[0049]

[0050]

[0051] Example 1

[0052] (1) Preparation of intermediate BS:

[0053] Boc-3-aminopropionic acid (1 g, 4.9 mmol), N, N-diisopropyl ethylamine (DIPEA) (1.71 mL, 9.8 mmol) were dissolved in 1 mL of dry N, N-dimethylformamide (DMF) in turn, stirred at room temperature for 5 min, 2-(7-azabenzotriazol)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU) (1.49 g, 3.92 mmol) was added, and the reaction was monitored by TLC for 0.5 h. Then lenalidomide (826 mg, 3.2 mmol) was added, and the reaction was stirred at room temperature. The reaction was tracked by TLC, and the reaction was complete after 12 h. The reaction was quenched by adding crushed ice to the reaction solution, and extracted with ethyl acetate three times. The organic layers were combined and washed with saturated ammonium chloride solution and saturated brine in turn, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Then PTLC (dichloromethane:methanol = 20:1) was performed to obtain intermediate BS2 (white solid, 1.28 g, 90%). 1 H NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.85 (s, 1H), 7.81 (dd, J = 7.4, 1.6 Hz, 1H), 7.51 (dd, J = 7.5, 1.6 Hz, 1H), 7.50-7.46 (m, 1H), 6.93 (t, J = 5.7 Hz, 1H), 5.15 (dd, J = 13.3, 5.1 Hz, 1H), 4.36 (q, J = 17.6 Hz, 2H), 3.63 (s, 2H), 3.23 (q, J = 6.7 Hz, 2H), 2.97-2.62 (m, 2H), 2.37-1.99 (m, 2H), 1.37 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 172.89, 171.08, 169.57, 167.87, 155.60, 133.82, 133.67, 132.67, 128.62, 125.34, 119.10, 77.66, 54.94, 51.49, 46.44, 36.59, 36.33, 31.22, 28.26, 22.68.

[0054] The reaction conditions for preparing intermediates BS1, BS3 to BS7 and intermediate BS2 are the same, except that the types and amounts of some raw materials are different.

[0055] Raw materials and their amounts for BS1: Boc-4-amino butyric acid (1 g, 5.2 mmol), DIPEA (1.81 mL, 10.4 mmol), HATU (1.6 g, 4.16 mmol), lenalidomide (876 mg, 3.38 mmol).

[0056] Raw materials and their amounts for BS3: Boc-4-amino butyric acid (1 g, 5.2 mmol), DIPEA (1.81 mL, 10.4 mmol), HATU (1.6 g, 4.16 mmol), lenalidomide (876 mg, 3.38 mmol), to obtain intermediate BS3 (white solid, 1.3 g, 90%). 1 H NMR (500 MHz, DMSO-d6) δ 11.29 (s, 1H), 10.02 (s, 1H), 8.06 (dd, J = 7.3, 1.7 Hz, 1H), 7.76 (dd, J = 7.6, 1.7 Hz, 1H), 7.73 (t, J = 7.4 Hz, 1H), 7.05 (t, J = 5.8 Hz, 1H), 5.40 (dd, J = 13.3, 5.1 Hz, 1H), 4.67 - 4.47 (m, 2H), 3.61 (s, 2H), 3.17 - 3.14 (m, 2H), 2.88 - 2.80 (m, 1H), 2.60 (d, J = 7.4 Hz, 2H), 2.27 (ddd, J = 9.7, 5.3, 2.6 Hz, 1H), 1.84 (p, J = 7.6 Hz, 2H), 1.61 (s, 9H), 1.54 (q, J = 8.7, 8.3 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 172.90, 171.33, 171.11, 167.86, 155.60, 133.82, 133.69, 132.68, 128.63, 125.24, 118.98, 77.33, 51.54, 46.49, 35.79, 31.23, 29.34, 28.28, 26.02, 24.87, 22.65.

[0057] Starting materials and their amounts for BS4: Boc-5-amino pentanoic acid (1 g, 4.6 mmol), DIPEA (1.6 mL, 9.2 mmol), HATU (1.4 g, 3.68 mmol), lenalidomide (775 mg, 2.99 mmol) to give intermediate BS4 (white solid, 1.25 g, 91%). 1 H NMR (500 MHz, DMSO-d6) δ 11.18 (s, 1H), 9.92 (s, 1H), 7.96 (dd, J = 7.3, 1.8 Hz, 1H), 7.65 (d, J = 5.7 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 6.96 (q, J = 4.4, 2.9 Hz, 1H), 5.29 (dd, J = 13.3, 5.1 Hz, 1H), 4.62 - 4.40 (m, 2H), 3.08 (t, J = 5.9 Hz, 2H), 2.75 (dt, J = 17.1, 3.7 Hz, 1H), 2.65 (d, J = 2.1 Hz, 2H), 2.17 (dtd, J = 10.5, 5.5, 5.0, 2.5 Hz, 1H), 1.72 (p, J = 7.4 Hz, 2H), 1.57 (q, J = 7.4 Hz, 2H), 1.51 (s, 9H), 1.39 (q, J = 7.2 Hz, 2H). 13 C NMR (126 MHz, DMSO) δ 172.91, 171.30, 171.11, 167.87, 156.36, 133.81, 133.70, 132.69, 128.65, 125.26, 119.01, 77.39, 53.62, 51.55, 46.49, 35.46, 31.24, 29.13, 28.29, 22.67, 22.47.

[0058] Starting materials and their amounts for BS5: Boc-6-amino hexanoic acid (1 g, 4.3 mmol), DIPEA (1.5 mL, 8.6 mmol), HATU (1.31 g, 3.44 mmol), lenalidomide (725 mg, 2.8 mmol) to give intermediate BS5 (white solid, 1.2 g, 92%). 1H NMR (500MHz, DMSO-d6) δ11.03(s,1H),9.79(s,1H),7.82(dd,J=7.4,1.6Hz,1H),7.50(dd,J=7.5,1.6Hz,1H) ,7.48(t,J=7.4Hz,1H),6.79(t,J=5.8Hz,1H),5.14(dd,J=13.3,5.1Hz,1H),4.45–4.29(m,2H),2.90(d,J=6. 8Hz, 2H), 2.61 (dt, J=17.2, 3.6Hz, 1H), 2.35 (d, J=7.5Hz, 2H), 2.02 (ddd, J=9.6, 5.2, 2.6Hz, 1H), 1.59 (p, J= 7.5Hz,2H),1.40(d,J=7.7Hz,2H),1.35(s,9H),1.29(d,J=6.5Hz,2H),1.26–1.14(m,1H),0.93–0.81(m,1H). 13 CNMR(126MHz,DMSO)δ172.91,171.38,171.11,167.90,155.63,133.85,132.69,129.52,128.63 ,125.27,119.00,77.35,54.94,35.81,31.25,29.36,28.30,26.04,24.89,22.67,13.94,10.94.

[0059] The raw materials and dosage of BS6: Boc-7-aminoheptanoic acid (1 g, 4.1 mmol), DIPEA (1.43 mL, 8.2 mmol), HATU (1.25 g, 3.28 mmol), lenalidomide (691 mg, 3.38 mmol) to obtain intermediate BS6 (white solid, 1.53 g, 93%). 1H NMR (500 MHz, DMSO-d6) δ 11.05 (s, 1H), 9.79 (s, 1H), 7.82 (d, J = 6.9 Hz, 1H), 7.51 - 7.39 (m, 1H), 7.14 - 6.46 (m, 1H), 5.40 - 4.92 (m, 1H), 4.37 (q, J = 18.6, 18.0 Hz, 2H), 3.38 (s, 2H), 2.95 - 2.81 (m, 2H), 2.63 (s, 1H), 2.51 (s, 2H), 2.42 - 2.30 (m, 2H), 2.14 - 1.86 (m, 1H), 1.86 - 1.50 (m, 2H), 1.49 (s, 1H), 1.33 (d, J = 34.1 Hz, 9H), 1.26 (d, J = 7.1 Hz, 2H), 1.24 (s, 2H), 1.17 - 0.83 (m, 1H). 13 C NMR (126 MHz, DMSO) δ 173.44, 171.38, 171.11, 168.23, 155.60, 133.82, 133.71, 131.78, 127.73, 125.92, 118.99, 78.07, 51.53, 45.55, 40.02, 36.37, 32.32, 29.39, 28.41, 28.29, 26.10, 25.10, 22.27.

[0060] Starting materials and amounts for BS7: Boc-8-aminooctanoic acid (1 g, 3.8 mmol), DIPEA (1.3 mL, 7.6 mmol), HATU (1.16 g, 3.04 mmol), lenalidomide (640 mg, 2.47 mmol) to give intermediate BS7 (white solid, 1.11 g, 90%). 1H NMR (500 MHz, DMSO-d6) δ 11.02 (s, 1H), 9.78 (s, 1H), 7.81 (dd, J = 7.4, 1.6 Hz, 1H), 7.50 (dd, J = 7.5, 1.7 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H), 6.75 (t, J = 5.6 Hz, 1H), 5.74 (s, 2H), 5.14 (dd, J = 13.3, 5.1 Hz, 1H), 4.41 - 4.31 (m, 2H), 2.88 (t, J = 6.6 Hz, 2H), 2.61 (dt, J = 17.4, 3.8 Hz, 1H), 2.35 (d, J = 7.7 Hz, 2H), 2.02 (dtd, J = 10.5, 5.1, 2.6 Hz, 1H), 1.58 (q, J = 7.2 Hz, 2H), 1.36 (s, 9H), 1.29 (d, J = 3.5 Hz, 2H), 1.27 (s, 2H), 1.26 (d, J = 2.4 Hz, 2H), 1.25 - 1.24 (m, 2H), 1.23 (s, 2H). 13 C NMR (126 MHz, DMSO) δ 172.91, 171.46, 171.12, 167.92, 155.65, 133.87, 133.74, 132.71, 128.65, 125.30, 119.01, 77.35, 54.92, 51.59, 48.64, 46.55, 40.11, 40.02, 39.94, 39.86, 39.78, 39.69, 39.61, 39.52, 39.44, 39.35, 39.19, 39.02, 35.85, 31.25, 29.51, 28.71, 28.54, 28.29, 26.25, 25.10, 22.69.

[0061] (2) Preparation of intermediate S:

[0062] Intermediate BS2 (1 g, 2.25 mmol) was dissolved in 10 mL of a mixed solvent of dichloromethane and methanol (volume ratio of DCM / MeOH was 3:1), 3 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath, after addition, it was slowly moved to room temperature for reaction, TLC was used to monitor the reaction, and the reaction was completed after 12 h. After the reaction was stopped, the solvent was removed by reduced pressure concentration to obtain intermediate S2 (white solid, 713 mg, 96.0%), which was used directly for the next step reaction without purification.

[0063] The intermediates were prepared using intermediates BS1 and BS3 to BS7, respectively, with the same raw material amount and reaction conditions as S2. The mass and yield of S1, S3 to S7 were as follows: S1 was a white solid, with a yield of 690 mg and a yield of 97.0%; S3 was a white solid, with a yield of 675 mg and a yield of 90%; S4 was a white solid, with a yield of 705 mg and a yield of 93.0%; S5 was a white solid, with a yield of 690 mg and a yield of 92.0%; S6 was a white solid, with a yield of 668 mg and a yield of 93.0%; and S7 was a white solid, with a yield of 683 mg and a yield of 91.0%.

[0064] (3) Preparation of final product SZ:

[0065] Specific synthesis route:

[0066]

[0067] Crude ice was added to stop the reaction, and then the reaction solution was extracted with ethyl acetate three times, the organic layers were combined and washed with saturated ammonium chloride solution and saturated brine, respectively. After drying over anhydrous sodium sulfate, the product SZ2 was obtained by filtration, concentration under reduced pressure, and PTLC (dichloromethane:methanol = 20:1).

[0068] SZ1, SZ3 to SZ7 were prepared using intermediates S1, S3 to S7, respectively, with the same preparation method and raw material amount as SZ2. The yield and NMR data were as follows:

[0069] Final product SZ1 (white powder, yield 34.8%) 1HNMR (500 MHz, Chloroform-d) δ 8.68 (dd, J = 4.5, 1.5 Hz, 1H), 8.42 - 8.31 (m, 1H), 7.39 (dd, J = 8.4, 4.5 Hz, 1H), 5.33 (t, J = 3.7 Hz, 1H), 3.68 (ddd, J = 11.3, 9.5, 4.6 Hz, 1H), 3.00 (d, J = 9.5 Hz, 1H), 2.97 - 2.90 (m, 1H), 2.54 (d, J = 66.3 Hz, 2H), 2.29 (dd, J = 13.8, 3.8 Hz, 1H), 2.26 - 2.17 (m, 1H), 2.17 - 2.13 (m, 1H), 2.13 - 2.09 (m, 1H), 2.09 - 2.04 (m, 1H), 2.04 - 2.00 (m, 1H), 2.00 - 1.96 (m, 1H), 1.95 (d, J = 4.4 Hz, 1H), 1.91 - 1.83 (m, 2H), 1.74 (t, J = 13.7 Hz, 1H), 1.70 - 1.57 (m, 2H), 1.55 (d, J = 5.2 Hz, 1H), 1.53 - 1.49 (m, 1H), 1.49 - 1.46 (m, 1H), 1.45 (d, J = 4.5 Hz, 1H), 1.43 (d, J = 8.8 Hz, 1H), 1.39 (h, J = 3.9, 3.2 Hz, 1H), 1.36 (dt, J = 9.7, 2.8 Hz, 1H), 1.32 (t, J = 3.1 Hz, 1H), 1.30 - 1.26 (m, 1H), 1.24 (d, J = 2.1 Hz, 2H), 1.20 (s, 3H), 1.11 (d, J = 38.1 Hz, 1H), 1.03 (s, 3H), 1.00 (d, J = 11.0 Hz, 1H), 0.97 (s, 3H), 0.95 (d, J = 1.6 Hz, 6H), 0.92 - 0.89 (m, 1H), 0.89 - 0.84 (m, 2H), 0.83 (s, 3H), 0.82 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 173.46, 151.69, 142.30, 140.94, 135.12, 129.36, 123.65, 120.79, 83.97, 77.41, 77.36, 77.16, 76.91, 69.00, 55.43, 47.69, 47.59, 46.56, 45.67, 42.07, 41.72, 39.66, 39.32, 38.36, 33.81, 33.05, 32.78, 32.28, 30.74, 28.75, 28.15, 25.87, 23.64, 23.40, 18.48, 17.08, 16.90, 16.80.

[0070] Final product SZ2 (white powder, yield 36.8%): 1 HNMR (500 MHz, Chloroform-d) δ 8.68 (dd, J = 4.4, 1.4 Hz, 1H), 8.38 (dd, J = 8.4, 1.4 Hz, 1H), 7.39 (dd, J = 8.4, 4.5 Hz, 1H), 5.34 (t, J = 3.7 Hz, 1H), 3.75 - 3.60 (m, 1H), 3.00 (d, J = 9.5 Hz, 1H), 2.96 - 2.88 (m, 1H), 2.52 - 2.31 (m, 2H), 2.31 - 2.24 (m, 1H), 2.18 (d, J = 4.6 Hz, 1H), 2.17 - 2.11 (m, 1H), 2.11 - 2.02 (m, 2H), 2.02 - 1.99 (m, 1H), 1.99 - 1.95 (m, 1H), 1.95 - 1.91 (m, 1H), 1.89 (t, J = 4.0 Hz, 1H), 1.75 (t, J = 13.7 Hz, 2H), 1.64 - 1.60 (m, 1H), 1.60 - 1.52 (m, 2H), 1.52 - 1.48 (m, 1H), 1.48 - 1.43 (m, 2H), 1.43 - 1.37 (m, 2H), 1.34 (dt, J = 14.5, 3.3 Hz, 2H), 1.27 (d, J = 3.0 Hz, 1H), 1.26 (s, 1H), 1.25 - 1.23 (m, 2H), 1.21 (s, 3H), 1.16 (s, 1H), 1.04 (s, 3H), 0.98 (s, 3H), 0.96 (d, J = 2.1 Hz, 6H), 0.93 - 0.88 (m, 2H), 0.88 - 0.85 (m, 1H), 0.84 (s, 3H), 0.82 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 174.05, 154.24, 145.64, 141.38, 135.74, 129.96, 122.44, 118.97, 85.21, 67.21, 55.45, 47.71, 47.61, 46.57, 45.69, 42.08, 41.73, 39.68, 39.32, 38.38, 33.83, 33.07, 32.80, 32.30, 29.82, 28.75, 28.16, 25.88, 23.65, 23.41, 18.49, 17.10, 16.89, 16.82.

[0071] Final product SZ3 (white powder, yield 36.8%): 1HNMR (500 MHz, Chloroform-d) δ 8.68 (dd, J = 4.5, 1.4 Hz, 1H), 8.41 - 8.34 (m, 1H), 7.39 (dd, J = 8.4, 4.5 Hz, 1H), 5.34 (t, J = 3.7 Hz, 1H), 3.68 (ddd, J = 11.4, 9.5, 4.5 Hz, 1H), 3.00 (d, J = 9.5 Hz, 1H), 2.98 - 2.89 (m, 1H), 2.46 (d, J = 18.1 Hz, 1H), 2.28 (td, J = 13.8, 3.8 Hz, 1H), 2.21 - 2.12 (m, 1H), 2.12 - 2.03 (m, 2H), 2.01 (d, J = 4.5 Hz, 1H), 1.99 (dd, J = 8.0, 3.4 Hz, 1H), 1.95 (d, J = 4.4 Hz, 1H), 1.90 (dt, J = 11.0, 3.9 Hz, 2H), 1.75 (t, J = 13.7 Hz, 2H), 1.64 - 1.56 (m, 2H), 1.56 - 1.48 (m, 2H), 1.48 - 1.43 (m, 2H), 1.44 - 1.37 (m, 2H), 1.34 (dt, J = 14.2, 3.4 Hz, 2H), 1.27 (dd, J = 5.2, 2.4 Hz, 1H), 1.26 - 1.23 (m, 2H), 1.21 (s, 3H), 1.04 (s, 3H), 0.98 (s, 3H), 0.96 (d, J = 1.9 Hz, 6H), 0.93 - 0.87 (m, 2H), 0.86 (p, J = 3.2, 2.7 Hz, 1H), 0.84 (s, 3H), 0.82 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 173.47, 151.70, 142.32, 140.96, 135.14, 129.38, 123.66, 120.80, 83.04, 70.03, 55.45, 48.24, 47.60, 46.56, 45.69, 42.08, 41.73, 39.68, 39.32, 38.38, 34.36, 33.07, 32.79, 32.29, 30.76, 29.82, 29.44, 28.75, 28.16, 25.88, 23.65, 23.41, 18.09, 17.09, 16.90, 16.81.

[0072] Final product SZ4 (white powder, yield 36.6%): 1HNMR (500 MHz, Chloroform-d) δ 7.86 (dd, J = 4.5, 1.4 Hz, 1H), 7.55 (dd, J = 8.4, 1.4 Hz, 1H), 6.57 (dd, J = 8.4, 4.5 Hz, 1H), 4.51 (t, J = 3.8 Hz, 1H), 2.86 (dd, J = 4.6, 1.9 Hz, 1H), 2.18 (d, J = 9.5 Hz, 1H), 2.14 - 2.08 (m, 1H), 1.44 (dd, J = 13.8, 3.9 Hz, 2H), 1.35 (td, J = 13.6, 4.5 Hz, 2H), 1.30 - 1.21 (m, 2H), 1.18 (ddd, J = 19.4, 6.0, 3.4 Hz, 2H), 1.11 (dd, J = 19.3, 3.9 Hz, 1H), 1.07 (q, J = 5.0, 3.8 Hz, 2H), 0.93 (d, J = 13.7 Hz, 1H), 0.88 (d, J = 11.5 Hz, 1H), 0.87 - 0.85 (m, 1H), 0.84 (s, 1H), 0.81 - 0.74 (m, 2H), 0.74 - 0.65 (m, 2H), 0.65 - 0.62 (m, 2H), 0.62 - 0.56 (m, 2H), 0.56 - 0.50 (m, 2H), 0.49 (dd, J = 5.0, 1.9 Hz, 1H), 0.46 - 0.44 (m, 1H), 0.44 - 0.41 (m, 2H), 0.38 (s, 3H), 0.21 (s, 3H), 0.15 (s, 3H), 0.14 (s, 3H), 0.12 (d, J = 8.2 Hz, 3H), 0.10 (d, J = 2.0 Hz, 1H), 0.08 - 0.07 (m, 1H), 0.06 (d, J = 4.1 Hz, 1H), 0.04 (d, J = 2.3 Hz, 1H), 0.01 (s, 3H), -0.00 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 174.20, 151.71, 142.34, 140.38, 135.16, 129.63, 128.81, 123.67, 119.49, 86.16, 69.07, 66.83, 55.46, 47.73, 47.62, 46.57, 45.71, 42.10, 41.74, 39.70, 39.32, 39.03, 38.40, 34.75, 33.08, 32.81, 32.31, 28.75, 28.18, 25.89, 23.67, 23.42, 19.27, 17.11, 16.89, 16.83, 14.26, 11.23.

[0073] Final product SZ5 (white powder, yield 38.6%):1 HNMR(500MHz,Chloroform-d)δ8.69(dd,J=4.5,1.4Hz,1H),8.38(dd,J=8.4,1.4Hz,1H),7.40 (dd,J=8.4,4.5Hz,1H),5.34(t,J=3.7Hz,1H),3.68(ddd,J=11.2,9.4,4.5Hz,1H),3.00(d,J=9 .5Hz,1H),2.98–2.72(m,2H),2.35(s,1H),2.34–2.20(m,2H),2.20–2.15(m,1H),2.15–2.10(m ,1H),2.10–2.06(m,1H),2.02(dt,J=4.6,2.4Hz,1H),1.99(dd,J=7.3,3.6Hz,1H),1.96(d,J=4 .4Hz,1H),1.90(dt,J=10.9,3.9Hz,2H),1.75(t,J=13.7Hz,2H),1.64–1.56(m,2H),1.54–1.4 7(m,2H),1.45(d,J=3.7Hz,2H),1.43–1.38(m,2H),1.36(q,J=4.2,3.3Hz,1H),1.33(q,J=2.9H z,1H),1.27(q,J=2.3Hz,1H),1.26–1.24(m,2H),1.21(s,3H),1.04(s,3H),0.98(s,3H),0.96( s,3H),0.95(d,J=7.5Hz,3H),0.92–0.88(m,2H),0.88–0.85(m,1H),0.84(s,3H),0.82(s,3H). 13 C NMR (126MHz, CDCl3) δ172.89,150.12,142.32,140.36,135.15,129.62,129 .38,123.67,121.28,89.70,69.04,64.32,55.45,47.71,47.61,46.56,45.1 8,42.08,41.35,39.68,39.31,38.39,33.83,33.07,32.80,32.30,30.76,28.75,28.16,25.88,23.65,23.41,18.49,17.10,16.89,16.82,14.17,11.22.

[0074] Final product SZ6 (white powder, yield 39.4%): 1HNMR (500 MHz, Chloroform-d) δ 8.69 (dd, J = 4.5, 1.4 Hz, 1H), 8.38 (dd, J = 8.4, 1.4 Hz, 1H), 7.40 (dd, J = 8.4, 4.5 Hz, 1H), 5.34 (t, J = 3.7 Hz, 1H), 3.68 (ddd, J = 11.2, 9.4, 4.5 Hz, 1H), 3.00 (d, J = 9.5 Hz, 1H), 2.98 - 2.72 (m, 2H), 2.35 (s, 1H), 2.34 - 2.20 (m, 2H), 2.20 - 2.15 (m, 1H), 2.15 - 2.10 (m, 1H), 2.10 - 2.06 (m, 1H), 2.02 (dt, J = 4.6, 2.4 Hz, 1H), 1.99 (dd, J = 7.3, 3.6 Hz, 1H), 1.96 (d, J = 4.4 Hz, 1H), 1.90 (dt, J = 10.9, 3.9 Hz, 2H), 1.75 (t, J = 13.7 Hz, 2H), 1.64 - 1.56 (m, 2H), 1.54 - 1.47 (m, 2H), 1.45 (d, J = 3.7 Hz, 2H), 1.43 - 1.38 (m, 2H), 1.36 (q, J = 4.2, 3.3 Hz, 1H), 1.33 (q, J = 2.9 Hz, 1H), 1.27 (q, J = 2.3 Hz, 1H), 1.26 - 1.24 (m, 2H), 1.21 (s, 3H), 1.04 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H), 0.95 (d, J = 7.5 Hz, 3H), 0.92 - 0.88 (m, 2H), 0.88 - 0.85 (m, 1H), 0.84 (s, 3H), 0.82 (s, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 140.98, 135.16, 134.37, 129.62, 129.39, 69.07, 67.91, 47.73, 47.62, 41.74, 39.70, 39.31, 39.03, 38.40, 33.84, 33.08, 32.81, 32.31, 30.78, 30.70, 29.83, 29.45, 29.12, 28.75, 28.17, 27.35, 25.89, 24.11, 23.66, 23.42, 23.10, 21.19, 18.50, 17.11, 16.89, 16.83, 14.33, 14.18, 11.23.

[0075] Final product SZ7 (white powder, yield 34.6%): 1HNMR (500 MHz, Chloroform-d) δ 8.71 (dd, J = 4.5, 1.4 Hz, 1H), 8.40 (dd, J = 8.4, 1.4 Hz, 1H), 7.42 (dd, J = 8.4, 4.5 Hz, 1H), 5.36 (t, J = 3.7 Hz, 1H), 4.28 (h, J = 5.6 Hz, 2H), 3.77 - 3.65 (m, 1H), 3.03 (d, J = 9.5 Hz, 1H), 3.00 - 2.93 (m, 1H), 2.40 (d, J = 13.1 Hz, 2H), 2.34 - 2.27 (m, 1H), 2.20 (d, J = 4.6 Hz, 1H), 2.18 - 2.13 (m, 1H), 2.13 - 2.09 (m, 1H), 2.07 - 2.02 (m, 1H), 1.99 (dd, J = 12.4, 4.5 Hz, 1H), 1.93 (dt, J = 11.0, 3.9 Hz, 2H), 1.88 - 1.77 (m, 2H), 1.77 - 1.69 (m, 2H), 1.66 - 1.58 (m, 2H), 1.54 (dd, J = 10.9, 2.8 Hz, 1H), 1.52 - 1.48 (m, 2H), 1.47 (dd, J = 3.8, 2.7 Hz, 2H), 1.46 - 1.43 (m, 2H), 1.42 (dt, J = 6.3, 2.5 Hz, 2H), 1.40 - 1.36 (m, 2H), 1.35 (t, J = 2.5 Hz, 2H), 1.34 - 1.32 (m, 2H), 1.30 (dd, J = 4.8, 2.2 Hz, 1H), 1.27 (d, J = 2.6 Hz, 2H), 1.23 (s, 3H), 1.06 (s, 3H), 1.00 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.97 (s, 2H), 0.95 (s, 1H), 0.94 - 0.92 (m, 2H), 0.92 - 0.90 (m, 2H), 0.89 (q, J = 4.1, 3.7 Hz, 1H), 0.86 (s, 3H), 0.85 (s, 3H).

[0076] Example 2

[0077] (1) Preparation of Intermediate BP

[0078] Fluoro-salidomide (2 g, 7.241 mmol) was dissolved in 20 mL DMF, then DIEA (1.872 mL, 14.481 mmol), N-Boc-1,2-ethanediamine (2 mL, 8.689 mmol) was added, and the reaction was warmed to 90 °C and refluxed for 12 h. After the reaction was completed, it was quenched with ice water, extracted with ethyl acetate, and the combined organic phase was washed with saturated NaHCO3, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain BP1. BP1 (yellow-green powder, yield 41.2%): 1 H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.03 (s, 1H), 7.01 (d, J = 7.3 Hz, 1H), 6.71 (t, J = 6.3 Hz, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 3.37 (d, J = 6.5 Hz, 2H), 3.12 (q, J = 5.9 Hz, 2H), 2.89 (ddd, J = 16.9, 13.8, 5.4 Hz, 1H), 2.59 (dt, J = 16.9, 3.2 Hz, 1H), 2.01 (ddd, J = 10.2, 6.2, 3.7 Hz, 1H), 1.36 (s, 9H), 1.23 (s, 1H). 13 C NMR (126 MHz, DMSO) δ 172.79, 170.03, 168.70, 167.30, 155.89, 146.41, 136.17, 132.21, 117.07, 110.49, 109.24, 77.79, 54.89, 48.52, 41.59, 40.11, 40.02, 39.94, 39.85, 39.78, 39.69, 39.61, 39.52, 39.44, 39.35, 39.19, 39.02, 30.97, 28.19, 22.17.

[0079] Fluoro-salidomide (2 g, 7.241 mmol) was dissolved in 20 mL DMF, then DIEA (1.872 mL, 14.481 mmol), N-Boc-1,3-propanediamine (2 eq) was added, and the reaction was warmed to 90 °C and refluxed for 12 h. After the reaction was completed, it was quenched with ice water, extracted with ethyl acetate, and the combined organic phase was washed with saturated NaHCO3, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to obtain BP2 (yellow-green powder, yield 55.4%). 1H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.03 (s, 1H), 7.01 (d, J = 7.3 Hz, 1H), 6.71 (t, J = 6.3 Hz, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 3.37 (d, J = 6.5 Hz, 2H), 3.12 (q, J = 5.9 Hz, 2H), 2.89 (ddd, J = 16.9, 13.8, 5.4 Hz, 1H), 2.59 (dt, J = 16.9, 3.2 Hz, 1H), 2.01 (ddd, J = 10.2, 6.2, 3.7 Hz, 1H), 1.36 (s, 9H), 1.23 (s, 1H). 13 CNMR (126 MHz, DMSO) δ 172.79, 170.03, 168.70, 167.30, 155.89, 146.41, 136.17, 132.21, 117.07, 110.49, 109.24, 77.79, 54.89, 48.52, 41.59, 40.11, 40.02, 39.94, 39.85, 39.78, 39.69, 39.61, 39.52, 39.44, 39.35, 39.19, 39.02, 30.97, 28.19, 22.17.

[0080] BP3~BP6 were prepared using N-Boc-1,4-butanediamine, N-Boc-1,5-pentanediamine, N-Boc-1,6-hexanediamine, N-Boc-1,8-octanediamine, respectively, in the same manner as the preparation of BP2, and the yields were as follows.

[0081] BP3 (yellow green powder, yield 48.3%): 1 H NMR (500 MHz, Chloroform-d) δ 8.49 (s, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.0 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.23 (t, J = 5.7 Hz, 1H), 4.91 (dd, J = 12.2, 5.4 Hz, 1H), 4.65 (s, 1H), 3.28 (q, J = 6.5 Hz, 2H), 3.16 (q, J = 6.6 Hz, 2H), 2.98 - 2.76 (m, 2H), 2.75 - 2.67 (m, 1H), 2.11 (ddd, J = 12.2, 6.0, 3.5 Hz, 1H), 1.67 (q, J = 7.3 Hz, 2H), 1.59 (p, J = 7.1 Hz, 2H), 1.43 (s, 9H). 13CNMR (126 MHz, CDC13) δ 171.29, 169.64, 168.56, 167.73, 156.15, 146.99, 136.29, 132.60, 116.77, 111.65, 110.07, 79.43, 48.99, 42.37, 40.21, 31.53, 28.54, 27.69, 26.61, 22.92.

[0082] BP4 (yellow-green powder, yield 46.5%): 1 H NMR (500 MHz, Chloroform-d) δ 8.37 (s, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 4.91 (dd, J = 12.2, 5.3 Hz, 1H), 4.59 (t, J = 6.4 Hz, 1H), 3.25 (q, J = 6.6 Hz, 2H), 3.12 (q, J = 9.0, 7.9 Hz, 2H), 2.90 - 2.82 (m, 1H), 2.76 (td, J = 17.2, 16.7, 8.6 Hz, 2H), 2.22 - 2.01 (m, 1H), 1.75 (s, 2H), 1.67 (p, J = 7.3 Hz, 2H), 1.52 (p, J = 7.2 Hz, 2H), 1.43 (s, 9H). 13 CNMR (126 MHz, CDC13) δ 171.29, 169.64, 168.56, 167.73, 156.15, 146.99, 136.29, 132.60, 116.77, 111.65, 110.07, 79.43, 48.99, 42.37, 40.21, 31.53, 28.54, 27.69, 26.61, 22.92.

[0083] BP5 (yellow-green powder, yield 39.1%): 1H NMR(500MHz,Chloroform-d)δ8.35(s,1H),7.48(dd,J=8.6,7.1Hz,1H),7.07(d,J=7.1Hz,1H),6.86(d,J=8. 5Hz,1H),6.22(t,J=5.6Hz,1H),4.91(dd,J=12.3,5.3Hz,1H),4.56(d,J=6.1Hz,1H),3.25(td,J=7.0,5.5Hz ,2H),3.10(d,J=7.6Hz,2H),2.91–2.85(m,1H),2.82–2.70(m,2H),2.11(ddd,J=10.0,5.1,2.3Hz,1H),1.79 –1.71(m,1H),1.65(p,J=7.2Hz,2H),1.52–1.47(m,2H),1.43(s,9H),1.39–1.33(m,2H),1.27–1.23(m,1H). 13 C NMR (126MHz, CDCl3) δ171.28,169.64,168.57,167.76,155.32,147.10,136.25,132.61,116.76 ,111.53,109.98,81.09,48.98,43.50,42.67,31.53,30.12,29.27,28.55,26.74,26.59,22.92.

[0084] BP6 (yellow-green powder, yield 38.4%): 1 H NMR(500MHz,Chloroform-d)δ8.29(s,1H),7.48(dd,J=8.5,7.1Hz,1H),7.08(d,J=7.0Hz,1H),6.87(d ,J=8.5Hz,1H),6.23(t,J=5.5Hz,1H),4.91(dd,J=12.3,5.3Hz,1H),4.54(s,1H),3.25(q,J=6.5Hz,2H ),3.10(q,J=6.8Hz,2H),2.94–2.84(m,1H),2.82–2.67(m,2H),2.12(ddd,J=12.3,6.1,3.3Hz,1H),1. 67–1.63(m,2H),1.43(s,9H),1.35–1.32(m,2H),1.31(s,2H),1.30(s,2H),1.26(s,2H),1.25(s,2H). 13C NMR (126 MHz, CDC13) δ 171.21, 169.65, 168.53, 167.78, 156.14, 147.15, 136.24, 132.63, 116.78, 111.49, 109.97, 79.21, 48.99, 42.74, 40.72, 32.05, 31.55, 30.15, 29.83, 29.29, 29.26, 28.56, 26.94, 26.80, 22.95, 14.25.

[0085] (2) Preparation of intermediate P

[0086] Dissolve 1 g of intermediate BP1 in 10 mL of dichloromethane and place in an ice bath, slowly add 2.5 mL of TFA, after the addition is completed, slowly move the reaction mixture to room temperature to continue the reaction. Monitor the progress of the reaction by thin layer chromatography (TLC), and after 12 h of reaction, the reaction is complete. After the reaction is completed, remove the solvent by concentration under reduced pressure to obtain intermediate P1 (yellow-green powder) with a yield of 97%, which can be directly used in the next step without purification.

[0087] Prepare P2-P6 using BP2-BP6 respectively, and the preparation process is the same as that of P1. The yields are as follows: the yield of P2 (yellow-green powder) is 97%, the yield of P3 (yellow-green powder) is 98%, the yield of P4 (yellow-green powder) is 97%, the yield of P5 (yellow-green powder) is 98%, and the yield of P6 (yellow-green powder) is 98%.

[0088] (3) Preparation of final product SZ

[0089] The specific synthesis route is as follows:

[0090]

[0091] Dissolve the crude product of step 1 (1 eq) and DIPEA (2 eq) in dry DMF. Stir at room temperature for 5 min, then add HATU (1.5 eq), and monitor by TLC. After 30 min, add P1 (1.5 eq), and continue to stir at room temperature. After 12 h of reaction, the reaction is complete. Quench the reaction with ice water, extract with ethyl acetate for 3 times, and then combine the organic layers. Subsequently, wash with saturated ammonium chloride solution and saturated brine solution. Dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify by PTLC (dichloromethane:methanol = 20:1) to obtain the final product SZ8.

[0092] Prepare SZ9-SZ13 using P2-P6 respectively, and the preparation method is similar to that of SZ8. The yields and NMR data of the final products are as follows:

[0093] SZ8 (yellowish green powder, 42.3%): ESI-MS m / z 793.5 [M+Na] + . 1 H NMR (500 MHz, Chloroform-d) δ 8.83 (d, J = 61.7 Hz, 1H), 7.52 (dd, J = 8.5, 7.1 Hz, 1H), 7.13 (d, J = 7.1 Hz, 1H), 7.02 (dd, J = 17.0, 8.6 Hz, 1H), 6.44 - 6.25 (m, 2H), 5.28 - 5.10 (m, 1H), 4.92 (dt, J = 12.2, 5.1 Hz, 1H), 3.76 - 3.61 (m, 2H), 3.52 - 3.38 (m, 2H), 3.30 - 3.13 (m, 1H), 2.98 (dd, J = 9.5, 2.0 Hz, 1H), 2.92 - 2.86 (m, 1H), 2.83 - 2.70 (m, 2H), 2.43 (t, J = 14.0 Hz, 1H), 2.24 - 2.14 (m, 1H), 2.14 - 2.07 (m, 2H), 2.03 (d, J = 9.5 Hz, 2H), 2.01 - 1.96 (m, 2H), 1.95 - 1.90 (m, 2H), 1.82 (dd, J = 18.4, 8.2 Hz, 2H), 1.76 - 1.70 (m, 2H), 1.66 - 1.62 (m, 1H), 1.60 - 1.54 (m, 2H), 1.53 - 1.47 (m, 2H), 1.46 - 1.41 (m, 1H), 1.34 - 1.29 (m, 2H), 1.26 (t, J = 3.5 Hz, 2H), 1.21 (s, 1H), 1.12 (d, J = 1.7 Hz, 3H), 1.00 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.82 (d, J = 18.6 Hz, 3H), 0.79 (d, J = 1.9 Hz, 3H), 0.65 (d, J = 13.8 Hz, 3H). 13C NMR (126 MHz, CDC13) δ 178.11, 171.47, 169.57, 169.01, 167.61, 148.05, 144.73, 136.51, 132.63, 125.09, 117.88, 112.26, 110.40, 83.05, 66.79, 54.36, 49.04, 47.54, 46.77, 46.55, 46.38, 42.34, 42.15, 39.51, 39.29, 38.20, 34.22, 33.07, 32.55, 32.19, 31.57, 30.85, 29.45, 28.71, 27.34, 25.90, 23.98, 23.66, 23.27, 18.77, 17.04, 16.99, 16.87, 16.59, 16.49.

[0094] SZ9 (yellow-green powder, 43.1%): ESI-MS m / z 809.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.39 (d, J = 7.0 Hz, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.10 (d, J = 7.0 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.30 (td, J = 5.9, 2.3 Hz, 1H), 6.06 (t, J = 5.7 Hz, 1H), 5.35 (q, J = 3.3 Hz, 1H), 4.91 (dd, J = 12.2, 5.3 Hz, 1H), 3.68 (ddd, J = 11.3, 9.5, 4.5 Hz, 1H), 3.56 - 3.45 (m, 1H), 3.30 (q, J = 6.6 Hz, 2H), 3.11 (dd, J = 13.1, 6.3 Hz, 1H), 2.99 (d, J = 9.5 Hz, 1H), 2.87 (td, J = 7.8, 6.3, 3.3 Hz, 1H), 2.83 - 2.76 (m, 1H), 2.76 - 2.69 (m, 1H), 2.49 (dd, J = 13.2, 4.3 Hz, 1H), 2.11 (ddd, J = 10.1, 5.1, 2.3 Hz, 1H), 1.96 (t, J = 3.6 Hz, 1H), 1.95 - 1.91 (m, 2H), 1.89 (d, J = 14.5 Hz, 1H), 1.83 (dt, J = 14.2, 7.1 Hz, 2H), 1.79 - 1.72 (m, 2H), 1.72 - 1.69 (m, 1H), 1.66 (s, 1H), 1.63 (d, J = 8.7 Hz, 1H), 1.61 - 1.55 (m, 1H), 1.52 (dd, J = 12.2, 7.2 Hz, 2H), 1.47 (dd, J = 13.2, 3.9 Hz, 1H), 1.44 - 1.38 (m, 1H), 1.38 - 1.30 (m, 2H), 1.30 - 1.27 (m, 1H), 1.26 (d, J = 4.0 Hz, 1H), 1.25 - 1.22 (m, 2H), 1.21 (q, J = 3.4 Hz, 1H), 1.19 - 1.16 (m, 1H), 1.15 (s, 3H), 1.02 (s, 3H), 0.94 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.86 - 0.83 (m, 1H), 0.81 (s, 3H), 0.74 (s, 3H). 13C NMR (126 MHz, CDC13) δ 178.88, 171.30, 169.56, 168.54, 167.69, 146.89, 145.16, 136.33, 132.69, 123.36, 116.67, 111.82, 110.77, 83.95, 67.73, 55.24, 49.03, 47.99, 46.84, 46.48, 46.41, 42.38, 42.23, 40.51, 39.54, 39.31, 38.28, 37.20, 34.23, 33.08, 32.74, 32.30, 31.57, 30.84, 29.82, 28.72, 27.37, 25.89, 23.89, 23.70, 22.92, 18.40, 17.13, 16.87, 16.75, 14.33.

[0095] SZ10 (yellow-green powder, 44.3%): ESI-MS m / z 821.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.84 (d, J = 127.2 Hz, 1H), 7.52 - 7.45 (m, 1H), 7.09 (dd, J = 7.0, 2.4 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.28 - 6.15 (m, 1H), 6.01 (q, J = 5.6 Hz, 1H), 5.38 (dt, J = 12.2, 3.6 Hz, 1H), 4.91 (dd, J = 12.2, 5.4 Hz, 1H), 3.76 - 3.62 (m, 1H), 3.42 (dt, J = 13.3, 6.7 Hz, 1H), 3.28 (q, J = 5.9 Hz, 2H), 3.06 (dt, J = 13.3, 6.3 Hz, 1H), 2.99 (dd, J = 9.6, 4.9 Hz, 1H), 2.91 - 2.84 (m, 1H), 2.81 - 2.69 (m, 2H), 2.52 - 2.43 (m, 1H), 2.21 (t, J = 7.6 Hz, 1H), 2.15 - 2.09 (m, 2H), 2.03 (dd, J = 19.5, 10.1 Hz, 2H), 2.01 - 1.97 (m, 1H), 1.96 - 1.92 (m, 2H), 1.91 (d, J = 5.7 Hz, 1H), 1.77 (dd, J = 13.5, 5.3 Hz, 1H), 1.74 - 1.70 (m, 1H), 1.66 (dd, J = 9.3, 3.8 Hz, 2H), 1.62 (d, J = 4.8 Hz, 2H), 1.60 (d, J = 2.9 Hz, 1H), 1.52 (dd, J = 14.0, 4.1 Hz, 2H), 1.48 - 1.43 (m, 1H), 1.38 - 1.32 (m, 2H), 1.32 (s, 1H), 1.27 (d, J = 8.7 Hz, 2H), 1.25 (d, J = 3.2 Hz, 2H), 1.21 - 1.18 (m, 1H), 1.15 (d, J = 3.6 Hz, 3H), 1.01 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.79 (d, J = 3.0 Hz, 3H), 0.73 (d, J = 6.9 Hz, 3H). 13C NMR (126 MHz, CDC13) δ 180.27, 171.09, 169.61, 169.14, 167.04, 148.83, 145.25, 136.75, 133.36, 125.98, 117.53, 112.83, 108.56, 82.25, 68.93, 56.12, 49.02, 47.56, 46.77, 46.49, 45.56, 42.63, 42.39, 41.80, 39.54, 39.32, 39.08, 38.22, 33.69, 33.10, 31.58, 30.86, 29.45, 28.71, 25.86, 23.84, 23.75, 18.41, 17.18, 17.14, 16.86, 16.84, 16.66, 16.62.

[0096] SZ11 (yellow-green powder, 41.8%): ESI-MS m / z 835.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.46 (d, J = 10.7 Hz, 1H), 7.51 (dd, J = 8.5, 7.1 Hz, 1H), 7.12 (d, J = 7.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.24 (t, J = 5.6 Hz, 1H), 5.99 (t, J = 5.5 Hz, 1H), 5.38 (dd, J = 6.0, 3.3 Hz, 1H), 4.93 (dd, J = 12.2, 5.3 Hz, 1H), 3.69 (dt, J = 10.6, 5.2 Hz, 1H), 3.39 (dd, J = 13.5, 6.7 Hz, 1H), 3.29 (q, J = 6.7 Hz, 2H), 3.03 (dd, J = 16.6, 8.0 Hz, 2H), 2.94 - 2.87 (m, 1H), 2.86 - 2.72 (m, 2H), 2.52 - 2.46 (m, 1H), 2.15 (dd, J = 13.4, 5.2 Hz, 1H), 2.04 (d, J = 28.9 Hz, 2H), 2.00 - 1.96 (m, 2H), 1.95 (d, J = 6.2 Hz, 2H), 1.93 (d, J = 3.5 Hz, 2H), 1.81 - 1.72 (m, 2H), 1.70 (dt, J = 7.4, 3.1 Hz, 2H), 1.69 - 1.63 (m, 2H), 1.56 (d, J = 6.3 Hz, 2H), 1.53 (d, J = 5.1 Hz, 2H), 1.51 - 1.47 (m, 1H), 1.47 - 1.43 (m, 2H), 1.41 (dd, J = 13.5, 2.7 Hz, 1H), 1.36 (tt, J = 8.9, 3.5 Hz, 2H), 1.30 (d, J = 4.5 Hz, 1H), 1.27 (q, J = 3.2 Hz, 2H), 1.22 - 1.20 (m, 1H), 1.17 (d, J = 1.7 Hz, 3H), 1.04 (s, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.82 (d, J = 1.3 Hz, 3H), 0.77 (d, J = 2.1 Hz, 3H). 13C NMR (126 MHz, CDC13) δ 178.43, 171.31, 169.62, 168.56, 167.77, 146.63, 144.89, 136.31, 132.62, 123.17, 117.63, 112.05, 110.06, 85.15, 70.97, 54.83, 48.59, 47.58, 46.85, 46.40, 43.22, 42.47, 42.26, 39.90, 39.39, 39.30, 38.26, 34.93, 33.11, 32.65, 32.34, 31.55, 30.86, 29.29, 29.13, 29.10, 28.71, 27.36, 26.28, 24.68, 24.65, 23.87, 23.71, 22.94, 18.80, 17.17, 16.87, 16.77.

[0097] SZ12 (yellow-green powder, 44.5%): ESI-MS m / z 849.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.57 (d, J = 90.6 Hz, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.19 (q, J = 5.7 Hz, 1H), 5.90 (dt, J = 6.1, 4.0 Hz, 1H), 5.37 (t, J = 3.6 Hz, 1H), 4.91 (ddd, J = 12.4, 5.4, 2.3 Hz, 1H), 3.71 - 3.62 (m, 1H), 3.33 (ddd, J = 13.3, 6.4, 1.8 Hz, 1H), 3.29 - 3.17 (m, 2H), 3.08 - 3.01 (m, 1H), 3.01 - 2.95 (m, 1H), 2.87 (ddd, J = 12.7, 5.3, 2.8 Hz, 1H), 2.81 - 2.66 (m, 2H), 2.48 (d, J = 12.9 Hz, 2H), 2.12 (ddd, J = 11.7, 5.8, 3.2 Hz, 1H), 2.04 (s, 1H), 1.98 - 1.93 (m, 2H), 1.94 - 1.89 (m, 2H), 1.77 (d, J = 13.4 Hz, 1H), 1.73 (d, J = 3.8 Hz, 1H), 1.70 (d, J = 3.8 Hz, 1H), 1.69 - 1.66 (m, 2H), 1.66 - 1.61 (m, 2H), 1.61 - 1.55 (m, 1H), 1.54 (d, J = 4.3 Hz, 1H), 1.51 (dd, J = 7.2, 3.5 Hz, 2H), 1.49 (d, J = 3.1 Hz, 1H), 1.47 - 1.45 (m, 1H), 1.44 - 1.40 (m, 2H), 1.38 (d, J = 3.3 Hz, 1H), 1.37 - 1.34 (m, 2H), 1.32 (t, J = 3.4 Hz, 1H), 1.29 (q, J = 2.9 Hz, 1H), 1.27 (s, 1H), 1.25 (t, J = 3.4 Hz, 2H), 1.24 - 1.17 (m, 2H), 1.15 (s, 3H), 1.11 - 1.03 (m, 1H), 1.02 (d, J = 2.8 Hz, 3H), 0.95 (d, J = 11.0 Hz, 3H), 0.90 (s, 3H), 0.90 (s, 3H), 0.88 - 0.85 (m, 1H), 0.85 - 0.82 (m, 1H), 0.80 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 1.9 Hz, 3H). 13C NMR (126 MHz, CDC13) δ 178.36, 171.75, 169.60, 168.89, 167.42, 147.06, 145.40, 136.27, 133.50, 122.63, 117.53, 111.03, 108.78, 84.04, 67.22, 55.62, 48.98, 47.56, 46.82, 46.41, 43.20, 41.87, 40.03, 39.48, 39.31, 37.73, 33.91, 33.11, 32.64, 32.39, 31.55, 30.87, 29.56, 29.43, 29.18, 29.07, 28.36, 27.39, 27.22, 27.04, 26.70, 25.86, 23.73, 22.21, 19.16, 17.24, 16.88, 16.77, 14.33.

[0098] SZ13 (yellow-green powder, 45.2%): ESI-MS m / z 863.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.49 (d, J = 70.4 Hz, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.09 (d, J = 7.1 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.20 (t, J = 5.6 Hz, 1H), 5.90 (t, J = 5.3 Hz, 1H), 5.37 (t, J = 3.6 Hz, 1H), 4.91 (ddd, J = 12.2, 5.5, 2.6 Hz, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.68 (dddd, J = 13.5, 9.5, 4.2, 1.8 Hz, 1H), 3.31 (ddd, J = 13.5, 6.4, 2.4 Hz, 1H), 3.25 (td, J = 7.1, 5.5 Hz, 2H), 3.09 - 2.96 (m, 2H), 2.91 - 2.85 (m, 1H), 2.74 (tq, J = 14.9, 4.9, 4.0 Hz, 2H), 2.48 (d, J = 13.1 Hz, 1H), 2.17 - 2.09 (m, 1H), 2.03 - 1.98 (m, 1H), 1.97 - 1.94 (m, 2H), 1.93 (d, J = 3.6 Hz, 1H), 1.80 - 1.73 (m, 1H), 1.71 (td, J = 7.6, 3.9 Hz, 1H), 1.67 (s, 1H), 1.65 (d, J = 6.6 Hz, 2H), 1.63 (s, 2H), 1.59 (dd, J = 17.4, 3.2 Hz, 1H), 1.55 (d, J = 4.2 Hz, 1H), 1.53 (d, J = 4.0 Hz, 1H), 1.51 - 1.45 (m, 2H), 1.45 - 1.41 (m, 2H), 1.38 (d, J = 13.9 Hz, 2H), 1.35 (s, 2H), 1.33 (s, 2H), 1.32 (s, 2H), 1.30 (d, J = 3.1 Hz, 2H), 1.29 (s, 1H), 1.27 (d, J = 2.9 Hz, 2H), 1.26 (s, 2H), 1.25 (s, 1H), 1.24 (s, 1H), 1.16 (s, 3H), 1.02 (d, J = 2.9 Hz, 3H), 0.98 (d, J = 1.8 Hz, 3H), 0.90 (s, 6H), 0.81 (d, J = 6.6 Hz, 3H), 0.76 (s, 3H). 13C NMR (126MHz, CDCl3) δ178.67,171.26,170.17,168.80,167.75,147.25,145.45,136.85,132.65,123 .97,116.27,112.82,110.48,86.31,68.39,61.21,55.72,49.84,47.62,46.89,46.47,46.01,42.79 ,42.60,41.93,39.58,39.33,38.32,34.27,33.12,32.64,32.40,31.52,30.40,29.84,29.46,29.18,28.73,27.39,27.15,26.93,25.87,23.86,23.72,23.01,21.19,18.44,17.16,16.86,16.76,14.34.

[0099] Example 3:

[0100] (1) Preparation of intermediate BO

[0101] Fluorothalidamide (2 g, 7.241 mmol, 1 eq) was dissolved in 20 mL of DMF, followed by the addition of DIEA (1.872 ml, 14.481 mmol, 2 eq) and tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (1.2 eq). 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 sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to yield intermediate BO1 (yellow-green powder, 32.6%).

[0102] BO2 and BO3 were prepared using N-Boc-3,6-dioxa-1,8-octanediamine (1.2 eq) and N-Boc-1,11-diamino-3,6,9-trioxaundecane (1.2 eq), respectively, in the same manner as the intermediate BO1. BO1 (yellow-green powder, 32.6%): 1HNMR(500MHz,Chloroform-d)δ8.38(s,1H),7.49(dd,J=8.6,7.1Hz,1H),7.10(d,J=7.0Hz, 1H),6.91(d,J=8.5Hz,1H),6.50(s,1H),5.01(s,1H),4.92(dd,J=12.1,5.3Hz,1H),3.68(t, J=5.3Hz,2H),3.55(t,J=5.2Hz,2H),3.45(t,J=5.3Hz,2H),3.32(q,J=5.4Hz,2H),2.93–2. 83(m,1H),2.82–2.59(m,2H),2.11(ddd,J=13.2,6.1,3.7Hz,1H),1.76(s,1H),1.41(s,9H). 13 C NMR (126MHz, CDCl3) δ171.29,169.52,168.54,167.72,156.12,146.96,136.23,132.59, 116.92,111.91,110.46,79.40,70.39,69.37,49.01,42.35,40.50,31.55,28.50,22.86.

[0103] The yields of BO2~BO3 and their NMR data are as follows.

[0104] BO2 (yellow-green powder, 29.3%): 1 H NMR(500MHz,Chloroform-d)δ8.40(s,1H),7.49(dd,J=8.6,7.1Hz,1H),7.10(d,J=7.1Hz,1H),6.91(d,J= 8.5Hz,1H),6.50(t,J=5.7Hz,1H),5.01(t,J=5.6Hz,1H),4.92(dd,J=12.2,5.3Hz,1H),3.68(t,J=5.3Hz, 2H),3.55(t,J=5.2Hz,2H),3.45(q,J=5.0Hz,2H),3.32(q,J=5.5Hz,2H),2.87(ddd,J=16.0,6.2,2.7Hz,1 H),2.83–2.65(m,2H),2.15–2.08(m,1H),1.77(s,1H),1.41(s,9H),1.40–1.20(m,2H),1.03–0.77(m,1H). 13C NMR (126 MHz, CDCI3) δ 171.30, 169.53, 168.55, 167.72, 156.13, 146.97, 136.23, 132.61, 116.92, 111.91, 110.49, 79.40, 70.40, 69.39, 49.03, 42.36, 40.52, 31.55, 28.51, 22.87.

[0105] BO3 (yellow-green powder, 34.1 %): 1 H NMR (500 MHz, Chloroform-d) δ 7.48 (dd, J = 8.5, 7.1 Hz, 1 H), 7.09 (d, J = 7.1 Hz, 1 H), 6.92 (d, J = 8.5 Hz, 1 H), 6.47 (t, J = 5.7 Hz, 1 H), 5.01 (s, 1 H), 4.95 - 4.86 (m, 1 H), 3.72 (t, J = 5.5 Hz, 2 H), 3.68 (d, J = 2.3 Hz, 2 H), 3.67 (s, 2 H), 3.65 - 3.62 (m, 2 H), 3.61 - 3.58 (m, 2 H), 3.52 (t, J = 5.2 Hz, 2 H), 3.29 (q, J = 5.5 Hz, 2 H), 3.20 (s, 3 H), 3.00 - 2.92 (m, 1 H), 2.82 - 2.69 (m, 2 H), 2.11 - 2.03 (m, 1 H), 1.74 (s, 1 H), 1.42 (s, 9 H), 1.35 - 1.22 (m, 1 H). 13 C NMR (126 MHz, CDCI3) δ 171.30, 169.53, 168.55, 167.72, 156.13, 146.97, 136.23, 132.61, 116.92, 111.91, 110.49, 79.40, 70.40, 69.39, 49.03, 42.36, 40.52, 31.55, 28.51, 22.87.

[0106] (2) Preparation of intermediate O

[0107] Dissolve 1 g of intermediate BO1 in 10 mL of dichloromethane, slowly add 3 mL of TFA under ice bath condition, after the addition is completed, slowly move the reaction mixture to room temperature and continue to react. Monitor the progress of the reaction by thin layer chromatography (TLC), and it can be completed after 12 h of reaction. After the reaction is completed, the solvent is removed by concentration under reduced pressure to obtain O1 in the form of yellow-green powder with a yield of 32.6%.

[0108] O2~O3 were prepared from BO2~BO3 respectively, in the same way as O1: O2 was yellow-green powder with a yield of 29.3%; O3 was yellow-green powder with a yield of 34.1%.

[0109] (3) Preparation of final product SZ14~SZ16

[0110] Mashu acid (1 eq) and DIPEA (2 eq) were dissolved in dry DMF in turn. After stirring at room temperature for 5 min, HATU (1.5 eq) was added, and after 30 min, O1 (1.5 eq) was added. The reaction was complete after 12 h of continuous stirring at room temperature. The reaction was quenched with ice water, and the organic layer was extracted with ethyl acetate three times. The organic layers were combined and washed with saturated ammonium chloride solution and saturated brine in turn. After drying over anhydrous sodium sulfate, the mixture was filtered and concentrated under reduced pressure. The final product SZ14 was obtained by purification using PTLC (dichloromethane:methanol = 20:1).

[0111] SZ15~SZ16 were prepared from O2~O3 respectively, in the same way as SZ14. The yields and NMR data of SZ14~SZ16 prepared are as follows:

[0112] Specific synthesis route:

[0113]

[0114] SZ14 (yellow-green powder, 41.7%): ESI-MS m / z 837.5 [M+Na] + . 1H NMR(500MHz,Chloroform-d)δ8.70(d,J=36.1Hz,1H),7.51(dd,J=8.5,7.2Hz,1H),7.13(d,J=7.1Hz,1H),6.94(dd,J=12.8,8.5Hz,1H),6.48(td,J=5.7,2.8Hz,1H),6.32(q,J=5.5Hz,1H),5.29(s,1H),4.92(dt,J=12.2,6.3Hz,1H),4.11(d,J=7.1Hz,1H),3.68(q,J=4.9Hz,2H),3.65–3.59(m,1H),3.54(q,J=4.3,3.2Hz,2H),3.50(dd,J=9.7,4.8Hz,1H),3.47(dd,J=5.7,3.7Hz,2H),3.37(ddt,J=21.3,13.3,4.4Hz,1H),2.98(d,J=9.5Hz,1H),2.91–2.81(m,2H),2.78(dd,J=12.0,8.8Hz,1H),2.72(ddt,J=13.1,7.7,3.6Hz,1H),2.55–2.33(m,2H),2.10(dtd,J=10.2,5.2,2.8Hz,1H),2.04(s,2H),2.02–1.86(m,2H),1.86–1.82(m,2H),1.72(q,J=6.3Hz,1H),1.70–1.65(m,1H),1.65–1.57(m,2H),1.55(dd,J=11.9,8.8Hz,2H),1.52–1.43(m,2H),1.43–1.37(m,1H),1.33(td,J=12.2,10.2,5.0Hz,2H),1.26(s,1H),1.25(d,J=2.7Hz,2H),1.23(s,1H),1.12(d,J=3.7Hz,3H),1.00(s,3H),0.89(d,J=2.0Hz,3H),0.87(d,J=3.0Hz,6H),0.78(s,3H),0.75(d,J=2.8Hz,3H). 13C NMR (126 MHz, CDC13) δ 178.26, 171.69, 168.73, 167.43, 147.82, 145.64, 135.59, 131.36, 122.58, 117.68, 112.04, 109.50, 84.14, 70.19, 61.23, 55.28, 49.52, 47.62, 46.89, 46.39, 42.79, 42.60, 42.29, 39.58, 39.33, 38.32, 34.27, 33.12, 32.64, 31.52, 30.88, 29.92, 29.41, 29.35, 29.18, 28.73, 27.39, 27.15, 26.93, 25.13, 23.72, 23.06, 21.19, 18.44, 17.16, 16.76, 14.34.

[0115] SZ15 (yellow-green powder, 45.6%): ESI-MS m / z 881.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 9.12 (d, J = 76.8 Hz, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.10 (d, J = 7.1 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.47 (td, J = 5.5, 3.5 Hz, 1H), 6.45 - 6.36 (m, 1H), 5.36 (t, J = 3.7 Hz, 1H), 4.90 (tdd, J = 7.1, 4.5, 1.9 Hz, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.71 (dt, J = 5.5, 2.8 Hz, 2H), 3.67 (dd, J = 5.8, 3.0 Hz, 2H), 3.63 (q, J = 4.5, 3.3 Hz, 2H), 3.54 (t, J = 5.1 Hz, 2H), 3.46 (dd, J = 8.0, 3.5 Hz, 2H), 3.36 - 3.27 (m, 1H), 2.98 (dd, J = 9.5, 2.7 Hz, 1H), 2.84 (dtd, J = 14.9, 8.0, 3.6 Hz, 1H), 2.76 - 2.70 (m, 2H), 2.50 (d, J = 13.0 Hz, 1H), 2.15 - 2.08 (m, 1H), 2.03 (s, 2H), 1.99 - 1.92 (m, 2H), 1.91 (d, J = 4.7 Hz, 2H), 1.88 (s, 2H), 1.78 - 1.68 (m, 2H), 1.64 (dd, J = 21.1, 7.2 Hz, 2H), 1.60 - 1.53 (m, 2H), 1.51 (td, J = 7.6, 3.9 Hz, 2H), 1.43 (dd, J = 12.3, 5.2 Hz, 1H), 1.40 - 1.32 (m, 2H), 1.32 - 1.28 (m, 1H), 1.27 (d, J = 5.5 Hz, 2H), 1.25 (s, 2H), 1.24 (d, J = 4.4 Hz, 2H), 1.13 (s, 3H), 1.00 (s, 3H), 0.94 (d, J = 1.5 Hz, 3H), 0.88 (d, J = 4.0 Hz, 6H), 0.82 (t, J = 2.3 Hz, 1H), 0.78 (s, 3H), 0.75 (d, J = 6.5 Hz, 3H). 13C NMR (126 MHz, CDC13) δ 180.69, 171.66, 169.45, 168.87, 166.53, 147.81, 144.80, 136.75, 132.34, 122.29, 117.52, 112.32, 110.08, 90.85, 70.88, 70.21, 69.76, 69.48, 68.90, 68.75, 59.96, 54.81, 49.40, 47.58, 46.81, 46.45, 42.51, 42.34, 42.15, 39.57, 39.29, 37.76, 34.24, 33.12, 32.55, 32.38, 31.48, 30.84, 29.34, 28.71, 27.36, 25.86, 23.74, 23.04, 21.65, 18.40, 17.05, 15.61, 14.31.

[0116] SZ16 (yellow-green powder, 47.2%): ESI-MS m / z 925.5 [M+Na] + . 1H NMR (500 MHz, Chloroform-d) δ 8.71 (d, J = 40.2 Hz, 1H), 7.49 (dd, J = 8.5, 7.1 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 6.45 (d, J = 21.2 Hz, 1H), 6.37 (t, J = 5.1 Hz, 1H), 5.39 - 5.33 (m, 1H), 4.91 (ddd, J = 12.3, 5.4, 2.5 Hz, 1H), 3.72 (t, J = 5.3 Hz, 2H), 3.68 (s, 2H), 3.66 - 3.62 (m, 2H), 3.60 (t, J = 4.6 Hz, 2H), 3.55 - 3.51 (m, 2H), 3.47 (t, J = 5.5 Hz, 2H), 3.29 (td, J = 9.0, 4.7 Hz, 1H), 3.00 (dd, J = 9.5, 1.5 Hz, 1H), 2.90 - 2.82 (m, 1H), 2.82 - 2.68 (m, 2H), 2.55 - 2.49 (m, 1H), 2.27 - 2.07 (m, 2H), 2.06 - 1.97 (m, 2H), 1.96 (t, J = 5.3 Hz, 2H), 1.94 - 1.92 (m, 2H), 1.91 (d, J = 4.3 Hz, 2H), 1.76 (d, J = 13.4 Hz, 1H), 1.72 - 1.64 (m, 2H), 1.64 - 1.61 (m, 1H), 1.59 (dd, J = 11.8, 5.5 Hz, 1H), 1.56 - 1.51 (m, 2H), 1.51 - 1.38 (m, 2H), 1.38 - 1.31 (m, 2H), 1.30 - 1.26 (m, 2H), 1.25 (s, 1H), 1.25 (s, 1H), 1.18 (d, J = 10.9 Hz, 2H), 1.14 (s, 3H), 1.02 (s, 3H), 0.96 (s, 3H), 0.91 (d, J = 12.0 Hz, 1H), 0.89 (s, 3H), 0.88 (d, J = 2.7 Hz, 3H), 0.85 (d, J = 10.9 Hz, 1H), 0.80 (d, J = 2.2 Hz, 3H), 0.75 (s, 3H). 13C NMR (126 MHz, CDC13) δ 177.81, 171.96, 169.36, 168.68, 167.76, 147.53, 144.42, 135.17, 132.68, 123.75, 117.53, 113.63, 110.09, 85.00, 70.86, 70.78, 70.72, 70.43, 69.81, 69.65, 68.86, 55.71, 53.57, 49.66, 47.62, 46.83, 45.83, 42.55, 42.27, 41.80, 39.58, 39.31, 38.30, 35.75, 33.58, 32.63, 32.38, 31.57, 30.87, 29.83, 29.13, 28.71, 26.97, 24.70, 22.96, 18.43, 17.06, 16.88, 16.82.

[0117] Performance test:

[0118] (1) In vitro anti-tumor activity test (IC 50 50): The conventional CCK-8 method was used to test the tumor cell proliferation inhibition ability of SZ1-SZ14 compounds, and MCF-7 (human breast cancer cells), A549 (human lung cancer cells), and Hela (human cervical cancer cells) were selected as test tumor strains, and maslinic acid (MA) was used as a control group. When MCF-7 (human breast cancer cells), A549 (human lung cancer cells), and Hela (human cervical cancer cells) grew to 80-90% of the culture dish, the cells were digested from the culture dish, centrifuged, and resuspended with fresh DMEM / 1640 complete culture medium, counted under a microscope, and then the cell suspension was diluted to 50 / μL. The outermost circle of the 96-well plate was paved with sterile PBS, 100 μL per well, and the remaining wells were paved with 100 μL of diluted cell suspension per well, and placed in a 37°C, 5% CO2 incubator for culture. After 24 h, the culture medium in the 96-well plate was aspirated, and the drug was diluted with fresh complete culture medium at a certain concentration gradient (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM), and then sequentially added to the 96-well plate. After 48 h, the culture medium containing the drug was removed, and CCK-8 liquid diluted with culture medium (100 μL of culture medium + 10 μL of CCK-8) was added, and placed in a 37°C, 5% CO2 incubator for culture for 30-40 min. The absorbance was detected at OD 450 on the enzyme marker, and the inhibition rate was calculated based on the absorbance at OD 450. The data was calculated using SPSS software to calculate the half inhibitory concentration IC 50 50). The test results are shown in Table 2.

[0119] Calculation formula: inhibition rate = [(Ac-As) / (Ac-Ab)]x100%

[0120] Wherein: As is the experimental hole absorbance (containing cells, culture medium, CCK-8 solution and drug solution);

[0121] Ac is the control hole absorbance (containing cells, culture medium, CCK-8 solution, not containing drugs);

[0122] Ab is the blank hole absorbance (containing culture medium, CCK-8 solution, not containing cells and drugs).

[0123] Table 2 in vitro anti-tumor activity (IC 50 )

[0124]

[0125] As can be seen from Table 1, the anti-tumor activity of the prepared hatic acid PROTACs compound is better than that of hatic acid. Combined with IC 50 and the structure of the compound, SZ13 is selected as the research object to further study the anti-tumor mechanism of hatic acid PROTAC.

[0126] (2) Anti-tumor mechanism research

[0127] The anti-tumor mechanism of hatic acid and hatic acid PROTAC-SZ13 in A549 lung cancer cells was studied by immunoblotting method. MA (20 μM) and different concentrations of SZ13 (5, 10, 20 μM) were used to treat A549 cells. First, the related proteins of JAK2 signaling pathway (such as Figure 1 and Figure 2 ) were analyzed by immunoblotting. In the treatment group, the expression of IL-6, P-JAK2, STAT3 and P-STAT3 decreased in a concentration-dependent manner. SZ13 inhibited the activation of IL-6 / JAK2 / STAT3 signaling pathway, which could effectively inhibit the proliferation and invasion of tumor cells.

[0128] The inhibition of IL-6 / JAK2 / STAT3 signaling pathway often leads to a series of intracellular changes, one of which is the activation of the mitochondrial apoptosis pathway. Immunoblotting analysis showed that Figure 3 and Figure 4), the expression levels of Bcl-2 protein decreased, Bax protein increased, Xiap and Surviving protein decreased, Caspase-9, Caspase-3 protein expression levels decreased and Cleaved caspase-9, Cleaved caspase-3 increased, and PARP decreased, Cytochrome c increased, indicating that SZ13 inhibited the IL-6 / JAK2 / STAT3 signaling pathway and indirectly activated the mitochondrial apoptosis pathway.

[0129] By immunoblotting ( Figure 5 and Figure 6 ), it was found that SZ13 and MA had different anti-tumor mechanisms, which activated endoplasmic reticulum stress and caused apoptosis, and with the increase of SZ13 concentration, the expression levels of endoplasmic reticulum stress-related proteins (such as JNK1, p-JNK1, IRE1, p-IRE1, BIP, CHOP, ATF4, ATF6) were significantly increased.

[0130] (3) Anti-tumor target verification

[0131] Starting from the key anti-tumor proteins affecting cell apoptosis and invasion, the changes of the proteins under the action of SZ13 and its possible mechanism were studied. According to the molecular docking analysis, JAK2, STAT3, Bcl-2, Mdm2, XIAP and other potential binding targets of the precursor of SZ13, the molecular docking binding energy of the protein and SZ13 was-8.8 kcal / mol, -8.6 kcal / mol, -7.8 kcal / mol, -7.9 kcal / mol, -7.9 kcal / mol, respectively. Among them, JAK2 protein and small molecule receptor binding pocket showed high fitting degree, the binding energy was the best, and the two had good binding activity and could bind under natural conditions. In addition, SZ13 and the amino acid residues LYS-736, TRP-737, ALA-733 in JAK2 protein formed three hydrogen bonds. At the same time, SZ13 and JAK2 protein formed non-bonding contact, forming forces represented by electrostatic potential energy and van der Waals force ( Figure 7 ). In summary, SZ13 and JAK2 protein have the potential to form more interactions. Therefore, SZ13 may significantly promote the apoptosis of lung cancer cells by fully degrading JAK2 protein, resulting in a significant increase in cell death rate. Therefore, JAK2 as a potential anti-tumor target, its degradation may provide a new idea for the treatment of lung cancer.

[0132] The binding ability between the potential anti-tumor target JAK2 and the target protein ligand maslinic acid was verified by using the cellular thermal shift assay (CETSA) technology and the drug affinity responsive target stability (DARTS) experiment technology, and the role of this binding relationship in the process of PROTAC-mediated protein degradation was further explored. The experimental results show that ( Figure 8 ) with the increase of temperature, the addition of maslinic acid significantly enhances the thermal stability of JAK2. Specifically, without the addition of maslinic acid, with the gradual increase of temperature, the degradation degree of JAK2 protein gradually increases, indicating that JAK2 is prone to denaturation and degradation at higher temperatures. While under the condition of co-incubation with maslinic acid, the degradation of JAK2 protein is significantly inhibited, and it still maintains high stability at higher temperatures, suggesting that maslinic acid enhances the thermal stability of JAK2 by binding to it, making it not easy to be degraded. These results show that maslinic acid stabilizes the structure of JAK2 protein by binding to it, thereby improving its tolerance to heat. According to the results of DARTS experiment ( Figure 9 ), when maslinic acid is co-incubated with A549 cell lysate (BCA protein quantification result is 3.624 mg / mL) at a concentration of 200 μM, different concentrations of trypsin (1:2000, 1:1000, 1:500) are used for enzymatic treatment. Compared with the solvent group (only adding DMSO), with the increase of trypsin concentration, JAK2 protein without co-incubation with maslinic acid shows a gradually increasing degree of degradation, suggesting that JAK2 is prone to degradation at higher trypsin concentrations. However, under the condition of co-incubation with maslinic acid, the degradation of JAK2 protein is significantly reduced, indicating that maslinic acid effectively enhances the anti-enzymatic stability of JAK2 by binding to it. Combined with the above experimental results, it is confirmed that there is a certain binding relationship between SZ13 and its potential degradation target JAK2, which may be the key mechanism leading to the degradation of target protein by PROTAC.

[0133] In order to further verify whether the degradation of JAK2 protein by SZ13 depends on this classical degradation mechanism, NEDD8 activating enzyme inhibitor MLN4924 was used to inhibit the protein degradation process to explore the degradation mechanism of P4.

[0134] In the experiment of verifying that SZ13 degrades JAK2 by ubiquitin-proteasome system with MLN4924, MLN4924 was first added to A549 cells for pretreatment to study the dependence of SZ13 on JAK2 protein degradation. The results of Western blotting show that Figure 10), after pretreatment with MLN4924, the degradation of JAK2 protein by SZ13 was significantly inhibited, and single MLN4924 had no effect on the degradation of JAK2 protein. This result shows that SZ13 achieves the degradation of JAK2 protein through the ubiquitin-proteasome system, which provides an important mechanism for further studying the potential of SZ13 in anti-tumor.

[0135] In summary, SZ13 inhibits the IL-6 / JAK2 / STAT3 signaling pathway by possibly targeting JAK2 protein to cause its degradation, activates the mitochondrial apoptosis pathway and induces endoplasmic reticulum stress, thereby playing an anti-tumor role. Compared with the natural product maslinic acid, the introduction of maslinic acid PROTAC through PROTAC technology not only enhances the targeting, but also overcomes the limitations of maslinic acid in anti-tumor therapy, providing new experimental evidence for the anti-tumor potential of maslinic acid PROTAC, studying the mechanism of maslinic acid PROTAC in anti-tumor, and providing a new idea for the future treatment strategy of lung cancer and other tumors.

[0136] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A maslinic acid PROTACs, characterized in that Its general structural formula is shown in Formula I: Wherein, E3 ligase is selected from lenalidomide or fluthalidomide Linker is the first fatty chain, the second fatty chain or the PEG chain; The first fatty chain is -(CH2) n1 -CO-, n1 is selected from an integer between 1 and 7; The second fatty chain is -(CH2) n2 -CH2-, n2 is selected from an integer between 1 and 7; The PEG chain is -(CH2-CH2-O) n3 -CH2-CH2-, n3 is selected from an integer between 1 and 3.

2. The maslinic acid PROTACs according to claim 1, characterized in that One selected from the following SZ1 to SZ16 compounds:

3. The method for preparing maslinic acid PROTACs according to claim 1 or 2, characterized in that: The steps include: (1) The first fatty chain corresponding compound and lenalidomide were used as raw materials, N,N-dimethylformamide was used as solvent, and the intermediate BS was prepared by activation with N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; The compound corresponding to the first fatty chain is selected from Boc-2-aminoacetic acid, Boc-3-aminopropionic acid, Boc-4-aminobutyric acid, Boc-5-aminopentanoic acid, Boc-6-aminohexanoic acid, Boc-7-aminoheptanoic acid or Boc-8-aminooctanoic acid; (2) preparing intermediate S using intermediate BS and trifluoroacetic acid as raw materials; (3) Maslinic acid, N,N-diisopropylethylamine, and (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) are mixed in a solvent, and then the intermediate S is added to react to prepare a maslinic acid PROTACs compound.

4. The method for preparing maslinic acid PROTACs according to claim 1 or 2, characterized in that: The steps include: (1) The intermediate BP was prepared by using the corresponding compound of the second fatty chain and fluorothalidomide as raw materials, N,N-dimethylformamide as solvent, and activation with N,N-diisopropylethylamine; The compound corresponding to the second fatty chain is selected from N-Boc-1,2-ethylenediamine, N-Boc-1,3-propylenediamine, N-Boc-1,4-butylenediamine, N-Boc-1,5-pentanediamine, N-Boc-1,6-hexanediamine or N-Boc-1,8-octanediamine; (2) preparing intermediate P using intermediate BP and trifluoroacetic acid as raw materials; (3) Maslinic acid, N,N-diisopropylethylamine, and (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) are mixed in a solvent, and then the intermediate P is added to react to prepare a maslinic acid PROTACs compound.

5. The method for preparing maslinic acid PROTACs according to claim 1 or 2, characterized in that: The steps include: (1) Using the corresponding compound of the PEG chain and fluorothalidomide as raw materials, N,N-dimethylformamide as solvent, and activated with N,N-diisopropylethylamine, the intermediate BO was prepared; The PEG chain corresponding compound is selected from tert-butyl (2-(2-aminoethoxy)ethyl)carbamate, N-Boc-3,6-dioxa-1,8-octanediamine or N-Boc-1,11-diamino-3,6,9-trioxaundecane; (2) preparing intermediate O using intermediate BO and trifluoroacetic acid as raw materials; (3) Maslinic acid, N,N-diisopropylethylamine, and (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) are mixed in a solvent, and then intermediate O is added to react to prepare a maslinic acid PROTACs compound.

6. The derivative of maslinic acid PROTACs according to claim 1 or 2, characterized in that The derivative is selected from pharmaceutically acceptable salts of maslinic acid PROTACs compounds.

7. Use of the maslinic acid PROTACs according to claim 1 or 2 in the preparation of anti-tumor drugs.

8. The use of maslinic acid PROTACs in the preparation of anti-tumor drugs according to claim 7, characterized in that: The tumor includes breast cancer, lung cancer or cervical cancer.

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