Bifunctional molecular compound for inducing SHP2 protein degradation based on DCAF16 ligand as well as preparation method and application of bifunctional molecular compound

Abstract

The invention discloses a bifunctional molecule compound for inducing SHP2 protein degradation based on a DCAF16 ligand as well as a preparation method and application of the bifunctional molecule compound, belongs to the technical field of medicines, and relates to a bifunctional molecule for inducing SHP2 protein degradation based on the DCAF16 ligand as shown in a general formula (I) or pharmaceutically acceptable salt of the bifunctional molecule, and a pharmaceutical composition containing an SHP2 protein targeted degradation agent. The invention also relates to a preparation method of the compound and application of the compound in preparation of medicines for treating SHP2-mediated tumors and / or other diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a bifunctional molecular compound based on DCAF16 ligand-induced degradation of SHP2 protein, its preparation method and application. Background Technology

[0002] SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, which is overexpressed in various cancers. Current research indicates that SHP2 is a key node in multiple oncogenic signaling pathways, such as RAS-ERK, PI3K-AKT, and JAK-STAT. Furthermore, SHP2 is involved in the programmed cell death pathway (PD-1 / PD-L1) and inhibits T cell activation. Therefore, targeting the degradation of SHP2 can inhibit tumor cell growth and restore T cell-mediated anti-tumor immune function (Hof P., et al.). Cell . 1998, 92(4), 441-450; Gao X., et al. FASEB J . 2023, 37(4), e22880; HeL., et al. Onco Targets Ther . 2019, 12, 5897-5906; Aceto N., et al. Nat Med .2012, 18(4), 529-537; Igbe I., et al. Oncotarget . 2017, 8(69), 113734-113748;Mainardi S., et al. Nat Med . 2018, 24(7), 961-967.).

[0003] Proteolytic targeting chimeras (PROTACs) are bifunctional small molecules that simultaneously bind to a target protein (POI) and an E3 ligase, thereby inducing ubiquitination of the target protein and subsequent proteasome degradation. Unlike traditional site-driven inhibitors, event-driven PROTACs provide more potent and sustained pharmacological effects. Furthermore, they can simultaneously eliminate both the enzymatic and scaffold functions of the target protein. The properties of the resulting chemicals vary depending on the ubiquitin ligase E3 ligand, the target protein ligand, and the linker. There are no reports in this field of bifunctional molecular compounds mediating SHP2 protein degradation using KB02-type compounds as the ubiquitin ligase E3 ligand and SHP099 as the target protein ligand. Summary of the Invention

[0004] The purpose of this invention is to develop compounds that can target and degrade the SHP2 protein, specifically providing SHP2PROTACs, their preparation method, and uses. Using KB02-type compounds as DCAF16 ligands and SHP099 as SHP2 protein ligands, bifunctional molecular compounds that can target and degrade the SHP2 protein are obtained through different linkers, as well as pharmaceutical compositions containing these compounds. Furthermore, studies have shown that these drugs can be used to prepare remedies for treating SHP2-mediated tumors and / or other diseases.

[0005] The specific technical solution of the present invention is as follows: This invention provides a bifunctional molecular compound or a pharmaceutically acceptable salt or solvate thereof that induces SHP2 protein degradation based on DCAF16 ligand, as shown in formula (I):

[0006] Where L can be any of the following structures:

[0007] Where n is selected from an integer between 1 and 10.

[0008] R is one or two of hydroxyl, cyano, nitro, amino, halogen, hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, 5-6 aryl or heteroaryl, wherein the aryl or heteroaryl is substituted by one or more substituents, wherein the substituent is one or more of nitro, hydroxyl, halogen, amino; wherein the heteroaryl contains 1-3 heteroatoms of O, N or S.

[0009] X is one or two of halogen, hydroxyl, nitro, amino, and C1-C6 alkyl.

[0010] The structures of some of the SHP2 degrading agent compounds of this invention are as follows:

[0011] This invention further provides a method for preparing the above-mentioned SHP2 degrading agent compound, but is not limited to the following preparation method: Step 1: Starting with 2,3-dichlorophenylboronic acid (1) and 2-amino-3-bromo-6-chloropyrazine (2), intermediate (3) was obtained by Suzuki-Miyaura coupling reaction. Then, intermediate (5) was obtained by nucleophilic substitution reaction with (4-methylpiperidin-4-yl)carbamate tert-butyl 4. Finally, the Boc group was removed to obtain SHP099 (6).

[0012]

[0013] Step 2: 1,2,3,4-tetrahydroquinoline-6-ol (7) was treated with chloroacetyl chloride to obtain KB02 (8). KB02 (8) was alkylated with tert-butyl 2-bromoacetate (9) to obtain intermediate (10), which was then deprotected to obtain key intermediate (11). SHP099 (6) was amidated with acids of different chain lengths (12a-j, 14) to obtain the corresponding intermediates (13a-j) and (15). Subsequently, N-deprotection was performed and condensed with intermediate (11) to obtain SK1-SK11.

[0014]

[0015] Step 3: Compound (22a-d) undergoes nucleophilic substitution of piperazine-1-carboxylic acid tert-butyl ester (21) to generate intermediate (23a-d), which is then hydrolyzed to give intermediate (19a-d). SHP099 (6) is amidated with acids of different chain lengths (16a-d, 19a-d) to give the corresponding intermediates (17a-d) and (20a-d). Subsequently, after N-deprotection, it is condensed with intermediate (11) to give SK12, SK14-SK20. In addition, intermediate (17a) is N-deprotected and reacted with intermediate (12b) (i.e., Boc-β-alanine) to produce intermediate (18), which is then N-deprotected and condensed with intermediate (11) to give SK13.

[0016]

[0017] Step 4: KB02 (8) was etherified with propyne bromide to obtain the key intermediate (24). SHP099 (6) was reacted with azide (25a-d) to generate intermediate (26a-d), which was then subjected to a copper-catalyzed cycloaddition reaction with intermediate (24) to obtain compounds SK21-SK24. Compound (28a-d) was condensed with intermediate (11) to obtain intermediate (29a-b). SHP099 (6) was etherified with propyne bromide to obtain intermediate (27), which was then subjected to a copper-catalyzed cycloaddition reaction with intermediate (29a-b) to generate compounds SK25-SK26.

[0018]

[0019] The pharmaceutically acceptable salt of the bifunctional molecular compound for targeted degradation of SHP2 protein described in this invention is a mixture of the derivative of this invention and the salt, wherein the salt is one of inorganic acid salts, organic acid salts, inorganic base salts, and organic base salts. Specifically, the inorganic acid salt is selected from hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; the organic acid salt is selected from salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, glycolic acid, lactic acid, methanesulfonic acid, malonic acid, lipoic acid, acetic acid, methanesulfonic acid, citric acid, fumaric acid, and maleic acid; the inorganic base salt is an alkali metal compound such as sodium, potassium, barium, calcium, magnesium, or zinc; and the organic base salt is selected from meglumine and / or glucosamine, etc.

[0020] Furthermore, the present invention also includes prodrugs derived from the present invention. These prodrugs may have weak or no activity on their own, but after administration, they are converted into the corresponding biologically active form under physiological conditions (such as through metabolism, solvation, or other means).

[0021] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the above-described compounds or a pharmaceutically acceptable salt thereof, combined with one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0022] When the pharmaceutical compositions of the present invention are applied clinically, they can be prepared into various pharmaceutically acceptable dosage forms according to the route of administration and treatment needs, including oral tablets, injectable preparations, topical preparations, and respiratory preparations; wherein oral preparations include tablets, capsules, granules, powders, oral solutions, suspensions, and pills; injectable preparations include sterile powders for injection, injection solutions, and concentrated solutions for injection; topical preparations include ointments, creams, gels, patches, lotions, and liniments; and respiratory preparations include aerosols, powder inhalers, and sprays.

[0023] The present invention relates to the use of bifunctional molecular compounds that target and degrade SHP2 protein, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions thereof, in the preparation of medicaments for treating or preventing tumors. Further, it can be used to prepare medicaments for treating or preventing SHP2-mediated tumors. These SHP2-mediated tumors include cervical cancer, lung cancer, breast cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, leukemia, melanoma, glioma, etc.

[0024] The beneficial effects of this invention are: This invention reports for the first time bifunctional molecular compounds that recruit the E3 ubiquitin ligase DCAF16 to degrade SHP2 protein. These compounds exhibit potent antiproliferative activity in HeLa cells, with the most promising compound SK8 showing potency at low micromolar levels (IC50). 50= 2.32±0.09 μM). SK8 induces SHP2 degradation in a concentration- and time-dependent manner via the ubiquitin-proteasome system. Notably, SK8 exhibits superior antitumor activity compared to SHP099, demonstrating stronger inhibition of cell proliferation, invasion, and migration, as well as apoptosis induction. Preliminary mechanistic studies suggest that this superior potency stems from deeper inhibition of the RAS / MAPK and PI3K / AKT / mTOR pathways, accompanied by a reversal of IFN-γ / JAK / STAT1 inhibition. The SHP2 protein-targeting degrader of this invention provides a novel therapeutic approach for the treatment of SHP2-mediated tumors and / or other diseases. Attached Figure Description

[0025] Figure 1 Evaluation of the antitumor activity of SHP2 PROTACs prepared in this invention; wherein A: IC50 of different test compounds on HeLa cells. 50 Value distribution (dashed line represents the 5 μM activity threshold); B: 24-h dose-dependent inhibition curves of compounds SHP099 and SK8 on HeLa cells.

[0026] Figure 2 The present invention describes the SK8-induced SHP2 degradation mechanism, wherein: A: SK8 reduces SHP2 protein expression in HeLa cells in a concentration-dependent manner; B: SHP099 treatment of HeLa cells for 24 h does not affect the SHP2 protein expression level; C: KB02 treatment of HeLa cells for 24 h shows no SHP2 degradation activity; D: Dose-dependent curves of SHP2 protein expression and degradation rate after SK8 concentration gradient treatment of HeLa cells for 24 h; E: SK8-mediated SHP2 degradation in HeLa cells is time-dependent (15 μMSK8 treatment); F: The proteasome inhibitor MG132 can reverse SK8-induced SHP2 degradation in HeLa cells; G: SHP099 and KB02 can inhibit SK8-induced SHP2 degradation in HeLa cells through ligand competition.

[0027] Figure 3 The SK8 prepared for this invention inhibits the proliferation and migration of HeLa cells and induces apoptosis; wherein A: SK8 inhibits the colony-forming ability of HeLa cells; B: SK8 induces cell cycle arrest in the S phase of HeLa cells; C: SK8 inhibits the scratch healing ability of HeLa cells; D: SK8 inhibits the migration and invasion of HeLa cells; E: SK8 induces apoptosis in HeLa cells; F: SK8 activates the cleavage of apoptosis-related proteins in HeLa cells.

[0028] Figure 4The effects of SK8 prepared in this invention on SHP2-mediated signaling pathways include: A: SK8 inhibits phosphorylation of MAPK pathway proteins in HeLa cells; B: SK8 inhibits AKT / mTOR pathway activation in HeLa cells; C: SK8 enhances IFN-γ-induced STAT1 phosphorylation; D: SK8 upregulates IFN-γ-induced expression of immune-related genes; E: SK8 enhances IFN-γ-induced CD8+ expression. + T cell activation. Detailed Implementation

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0030] Example 1 Synthesis of SHP099(6):

[0031] Synthesis of Intermediate 3: 2-Amino-3-bromo-6-chloropyrazine (1.0 g, 4.8 mmol, 1.0 eq) was dissolved in dioxane / water (V / V = 9:1, 15 mL), followed by the sequential addition of 2,3-dichlorophenylboronic acid (1.1 g, 5.28 mmol, 1.1 eq), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (175 mg, 0.24 mmol, 0.05 eq), and potassium phosphate (2.0 g, 9.6 mmol, 2.0 eq). The reaction mixture was placed under nitrogen and stirred at 120 °C for 4 hours, then cooled to room temperature. The mixture was filtered, and the filtrate was extracted with 20 mL of water and 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate (Na₂SO₄), concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to give Intermediate 3. A yellow solid was obtained in 89% yield. 1 HNMR (600 MHz, DMSO- d 6) d 7.81 (s, 1H), 7.69 (dd, J = 8.0, 1.5 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.34 (dd, J = 7.6, 1.5 Hz, 1H), 6.66 (s, 2H).

[0032] Synthesis of Intermediate 5: Intermediate 3 (1.0 g, 3.6 mmol, 1.0 eq), (4-methylpiperidin-4-yl)carbamate tert-butyl ester (1.56 g, 7.2 mmol, 2.0 eq), and potassium phosphate (764 mg, 3.6 mmol, 1.0 eq) were dissolved in N-methylpyrrolidone (3 mL). The reaction mixture was stirred at 140 °C for 36 hours and then cooled to room temperature. The mixture was extracted with 20 mL of water and 20 mL of ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give Intermediate 5. A yellow solid was obtained in 51% yield. 1 H NMR (600 MHz, DMSO- d 6) d 7.58(dd, J = 8.0, 1.6 Hz, 1H), 7.45 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J =7.6, 1.6 Hz, 1H), 6.56 (s, 1H), 5.57 (s, 2H), 3.73 (dt, J = 13.4, 4.4 Hz, 2H), 3.16 (t, J = 11.2 Hz, 2H), 2.04 (d, J = 13.2 Hz, 2H), 1.41 (ddd, J = 13.9, 10.3, 4.1 Hz, 2H), 1.35 (s, 9H), 1.22 (s, 3H).

[0033] Synthesis of SHP099(6): Intermediate 5 (1.0 g, 2.2 mmol) was dissolved in ethyl acetate and cooled to 0°C. Ethyl hydrochloride solution was added dropwise, and the reaction mixture was stirred continuously until the starting material was completely consumed by thin-layer chromatography (TLC). The solvent was removed under reduced pressure. The resulting residue was diluted with water (10 mL), and the aqueous layer was adjusted to pH 9 with 2M sodium hydroxide. The product was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. SHP099(6) was obtained without further purification.

[0034] Example 2 Synthesis of intermediate 11:

[0035] Synthesis of KB02(8): 1,2,3,4-Tetrahydroquinoline-6-ol (500 mg, 4.15 mmol, 1.0 eq) was dissolved in 1,4-dioxane (10 mL) and cooled to 0 °C. Then, 0.8 M sodium hydroxide aqueous solution was added, followed by dropwise addition of chloroacetyl chloride (515 mg, 4.56 mmol, 1.1 eq). The reaction mixture was heated to room temperature and stirred for 4 hours. After the reaction was complete, the mixture was diluted with water (10 mL) and adjusted to pH 3 with 1 M hydrochloric acid aqueous solution. The product was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with petroleum ether and ethyl acetate (3:1 v / v) to obtain KB02(8). It was a white solid with a yield of 85%. 1 H NMR (600 MHz, DMSO- d 6) d 7.60 (s, 1H), 7.01 (s,1H), 6.97 (dd, J = 8.8, 2.7 Hz, 1H), 4.64 (s, 2H), 3.69 – 3.65 (m, 2H), 2.70(t, J = 6.7 Hz, 2H), 1.87 (p, J = 6.5 Hz, 2H).

[0036] Synthesis of intermediate 10: KB02(8) (500 mg, 2.2 mmol, 1.0 eq) and potassium carbonate (910 mg, 6.6 mmol, 3.0 eq) were dissolved in... N , N To dimethylformamide (10 mL), tert-butyl 2-bromoacetate (515 mg, 2.6 mmol, 1.2 eq) was added dropwise, and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was extracted with 20 mL of water and 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1) to give intermediate 10. The yield was 49%. 1 H NMR (600 MHz, DMSO- d 6) d 7.27 (s, 1H), 6.69 (d, J = 9.5 Hz, 2H), 4.58 (s, 2H), 3.62 (t, J = 6.3 Hz, 2H), 3.33 (s, 2H), 2.63 (s, 2H), 1.83 (s, 2H), 1.39 (s, 9H).

[0037] Synthesis of intermediate 11: Intermediate 10 (100 mg, 0.29 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid was added dropwise. The reaction mixture was stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure. Intermediate 11 was obtained without further purification.

[0038] Example 3 Synthesis of intermediate 13a-j:

[0039] Synthesis of intermediate 13a: Compound 12a, namely Boc-glycine (60 mg, 0.34 mmol, 1.2 eq) and HATU (129 mg, 0.34 mol, 1.2 eq), was dissolved in... N , N In dimethylformamide (10 mL), the reaction mixture was stirred at 0 °C for 15 minutes, and then SHP099(6) (100 mg, 0.28 mmol, 1.0 eq) and [other ingredients] were added sequentially. N , N -Diisopropylethylamine (DIPEA, 146 µL, 0.84 mmol, 3.0 eq) was stirred at room temperature for 8 hours. The reaction mixture was extracted with 20 mL of water and 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 80:1) to give intermediate 13a. The yield was 80%. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0,1.6 Hz, 1H), 7.46 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz,1H), 7.23 (s, 1H), 6.83 (t, J = 6.1 Hz, 1H), 5.56 (s, 2H), 3.83 (dt, J = 13.5, 4.3 Hz, 2H), 3.48 (d, J = 6.1 Hz, 2H), 3.11 (t, J = 12.0 Hz, 2H), 2.10 (d, J= 13.2Hz, 2H), 1.47 – 1.41 (m, 2H), 1.32 (s, 9H), 1.27 (s, 3H).

[0040] Synthesis of intermediate 13b: Refer to the preparation of intermediate 13a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.58 (dd, J = 8.0, 1.5 Hz, 1H), 7.45 (s, 1H), 7.38 (s, 1H), 7.35 (t, J =7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.66 (t, J = 5.6 Hz, 1H), 5.56 (s,2H), 3.81 (dt, J = 13.8, 4.4 Hz, 2H), 3.12 (d, J = 10.0 Hz, 2H), 3.08 (q, J = 6.7Hz, 2H), 2.22 (t, J = 7.2 Hz, 2H), 2.11 (d, J = 13.9 Hz, 2H), 1.41 (ddd, J = 14.2,10.8, 4.2 Hz, 2H), 1.30 (s, 9H), 1.26 (s, 3H).

[0041] Synthesis of intermediate 13c: Refer to the preparation of intermediate 13a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.37 – 7.34 (m, 2H),7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.73 (t, J = 5.7 Hz, 1H), 5.56 (s, 2H), 3.81 (dt, J = 13.5, 4.3 Hz, 2H), 3.13 (d, J= 4.9 Hz, 2H), 2.87 (q, J = 6.6 Hz, 2H), 2.11(d, J = 13.4 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.55 (p, J = 7.3 Hz, 2H), 1.42(ddd, J = 14.1, 10.7, 4.2 Hz, 2H), 1.33 (s, 9H), 1.26 (s, 3H).

[0042] Synthesis of intermediate 13d: Refer to the preparation of intermediate 13a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.1, 1.5 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 2.8 Hz, 1H), 7.26 (dd, J = 7.7, 1.6 Hz, 1H), 6.73 (t, J = 5.8 Hz, 1H), 5.56 (s, 2H), 3.81 (dt, J = 13.4, 4.4 Hz, 2H), 3.13 (ddd, J = 13.5, 10.7, 3.0 Hz,2H), 2.86 (q, J = 6.6 Hz, 2H), 2.11 (d, J = 13.4 Hz, 2H), 2.04 (t, J = 7.3 Hz, 2H), 1.42 (h, J = 5.4, 4.6 Hz, 5H), 1.32 (s, 9H), 1.30 (d, J = 4.9 Hz, 1H), 1.26 (s, 3H).

[0043] Synthesis of intermediate 13e: Refer to the preparation of intermediate 13a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd,J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H),7.32 (s, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.70 (t, J = 5.7 Hz, 1H), 5.56 (s,2H), 3.80 (dt, J = 13.4, 4.4 Hz, 2H), 3.13 (ddd, J = 13.6, 10.8, 3.1 Hz, 2H),2.85 (q, J = 6.7 Hz, 2H), 2.11 (d, J = 13.3 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.43(dt, J = 13.5, 5.3 Hz, 4H), 1.32 (s, 9H), 1.26 (s, 3H), 1.18 (q, J = 8.1, 7.2 Hz, 4H).

[0044] Synthesis of intermediate 13f: For specific procedures and proportions, refer to the preparation of intermediate 13a. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H),7.32 (s, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.70 (t, J = 5.7 Hz, 1H), 5.57 (s,2H), 3.81 (dt, J = 13.5, 4.4 Hz, 2H), 3.12 (ddd, J = 13.4, 11.3, 3.0 Hz, 2H),2.84 (q, J = 6.6 Hz, 2H), 2.11 (d, J = 13.3 Hz, 2H), 2.04 (t, J= 7.4 Hz, 2H), 1.45– 1.40 (m, 4H), 1.32 (s, 9H), 1.26 (s, 3H), 1.20 (q, J = 4.2 Hz, 6H).

[0045] Synthesis of intermediate 13g: For specific procedures and proportions, refer to the preparation of intermediate 13a. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H),7.33 (s, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.71 (t, J = 5.7 Hz, 1H), 5.57 (s,2H), 3.81 (dt, J = 13.5, 4.3 Hz, 2H), 3.12 (ddd, J = 13.6, 10.5, 3.0 Hz, 2H),2.84 (q, J = 6.7 Hz, 2H), 2.11 (d, J = 13.5 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.46– 1.39 (m, 4H), 1.32 (s, 9H), 1.30 (d, J = 7.1 Hz, 2H), 1.26 (s, 3H), 1.22 –1.15 (m, 6H).

[0046] Synthesis of intermediate 13h: For specific procedures and proportions, refer to the preparation of intermediate 13a. 1 H NMR (600 MHz, DMSO- d 6) d 7.59 – 7.56 (m, 1H), 7.46 (d, J = 1.6 Hz, 1H), 7.34 (dd, J = 5.9, 1.7Hz, 2H), 7.28 – 7.25 (m, 1H), 6.70 (t, J= 5.7 Hz, 1H), 5.58 (s, 2H), 3.81 (dt, J = 13.8, 4.4 Hz, 2H), 3.11 (t, J = 11.9 Hz, 2H), 2.83 (q, J = 6.7 Hz, 2H), 2.12(d, J = 14.2 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.46 – 1.38 (m, 4H), 1.32 (d, J =1.7 Hz, 9H), 1.29 (d, J = 6.8 Hz, 2H), 1.26 (s, 3H), 1.20 (s, 8H).

[0047] Synthesis of intermediate 13i: For specific procedures and proportions, refer to the preparation of intermediate 13a. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.36 – 7.32 (m, 2H),7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.70 (t, J = 5.7 Hz, 1H), 5.57 (s, 2H), 3.82 (dt, J = 13.5, 4.4 Hz, 2H), 3.11 (ddd, J = 13.6, 10.5, 2.9 Hz, 2H), 2.84 (q, J = 6.7Hz, 2H), 2.12 (d, J = 14.0 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.48 – 1.38 (m,4H), 1.32 (s, 9H), 1.29 (d, J = 6.8 Hz, 2H), 1.26 (s, 3H), 1.22 – 1.15 (m, 10H).

[0048] Synthesis of intermediate 13j: For specific procedures and proportions, refer to the preparation of intermediate 13a. 1H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H),7.32 (s, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.68 (t, J = 5.7 Hz, 1H), 5.56 (s,2H), 3.81 (dt, J = 13.5, 4.4 Hz, 2H), 3.11 (ddd, J = 13.5, 10.6, 3.0 Hz, 2H),2.83 (q, J = 6.7 Hz, 2H), 2.12 (d, J = 13.5 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.46– 1.40 (m, 4H), 1.32 (s, 9H), 1.30 – 1.28 (m, 2H), 1.26 (s, 3H), 1.21 – 1.17(m, 12H).

[0049] Example 4 Synthesis of intermediate 15:

[0050] For specific procedures and proportions, refer to the preparation of intermediate 13a, and replace intermediate 12a with 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H),7.46 (s, 1H), 7.40 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.5 Hz, 1H), 6.62 (t, J = 5.7 Hz, 1H), 5.55 (s, 2H), 3.81 (dt, J = 13.8, 4.3 Hz, 2H), 3.54 (t,J = 6.4 Hz, 2H), 3.14 (ddd, J = 13.3, 10.6, 2.9 Hz, 2H), 3.01 (q, J = 6.0Hz, 2H), 2.30 (t, J = 6.4 Hz, 2H), 2.11 (d, J = 13.8 Hz, 2H), 1.42 (ddd, J = 14.1,10.6, 4.1 Hz, 2H), 1.33 (s, 9H), 1.27 (s, 3H), 1.21 (d, J = 14.6 Hz, 2H).

[0051] Example 5 Synthesis of compound SK1-10:

[0052] Synthesis of compound SK1: Intermediate 11 (66 mg, 0.24 mmol, 1.2 eq) was dissolved in dichloromethane, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 56 mg, 0.3 mmol, 1.5 eq) and 1-hydroxybenzotriazole (HOBt, 32 mg, 0.24 mmol, 1.2 eq) were added sequentially. The reaction mixture was stirred at 0 °C for 15 min. Intermediate 13a (100 mg, 0.2 mmol, 1.0 eq) was dissolved in ethyl acetate and cooled to 0 °C. Ethyl hydrochloride solution was added dropwise. After the starting material was completely consumed by TLC, the solvent was removed under reduced pressure. A suitable amount of water was added, and the pH was adjusted to 9 by slow dropwise addition of 2M sodium hydroxide aqueous solution. The product was extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The treated intermediate 13a and DIPEA (137 µL, 0.8 mmol, 4.0 eq) were added sequentially to the above reaction mixture, and the resulting reaction system was stirred at room temperature for 8 hours. The mixture was extracted with 20 mL of water and 20 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to give compound SK1. The yield was 34%. 1 H NMR (600 MHz, DMSO- d 6) d 8.15 (t, J = 5.8 Hz, 1H), 7.62 (dd, J= 8.0,1.6 Hz, 1H), 7.50 (s, 2H), 7.39 (t, J = 7.8 Hz, 1H), 7.31 (dd, J = 7.6, 1.6 Hz, 1H), 6.82 (d, J = 9.5 Hz, 2H), 5.62 (s, 2H), 4.52 (s, 2H), 3.85 (dd, J = 14.1, 4.0 Hz, 2H), 3.80 (d, J = 5.6 Hz, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.62 (t, J = 6.3Hz, 1H), 3.19 (ddd, J = 13.5, 9.9, 2.7 Hz, 3H), 2.69 (s, 2H), 2.14 (d, J = 13.4Hz, 2H), 1.88 (s, 2H), 1.49 (ddd, J = 14.0, 10.4, 4.1 Hz, 2H), 1.32 (s, 3H). 13 CNMR (150 MHz, DMSO- d 6) d 168.62, 168.40, 165.73, 165.25, 153.65, 151.18,139.94, 132.57, 132.21, 132.07, 131.16, 130.23, 128.76, 125.46, 125.31,119.94, 117.16, 114.91, 112.94, 67.61, 55.43, 51.67, 42.58, 40.61, 35.29,26.82, 26.38, 23.76, 23.50. + ): m / z Calculated for C 31 H 34 Cl3N7O4[M + H] + , 674.1816 found 674.1813.

[0053] Synthesis of compound SK2: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO-d 6) d 8.03 (t, J = 5.8 Hz, 1H), 7.61 (dd, J = 8.0, 1.7 Hz, 1H), 7.51 (s, 1H),7.48 (s, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.30 (dd, J = 7.7, 1.6 Hz, 1H), 6.79 (d, J = 10.3 Hz, 2H), 5.60 (s, 2H), 4.44 (s, 2H), 3.84 (d, J = 13.6 Hz, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.61 (t, J = 6.3 Hz, 1H), 3.37 – 3.35 (m, 2H), 3.20 (ddd, J =13.7, 10.4, 3.2 Hz, 3H), 2.67 (s, 2H), 2.36 (t, J = 6.8 Hz, 2H), 2.16 (d, J =13.7 Hz, 2H), 1.87 (s, 2H), 1.47 (ddd, J = 14.2, 10.3, 4.4 Hz, 2H), 1.31 (s,3H). 13 C NMR (150 MHz, DMSO- d 6) d 171.08, 168.04, 165.72, 165.23, 153.61, 151.16,143.29, 139.93, 132.59, 132.07, 131.13, 130.22, 128.74, 125.46, 125.22,119.93, 117.15, 114.91, 112.86, 67.72, 55.42, 51.54, 40.66, 36.42, 35.86,35.27, 26.82, 26.36, 23.75, 23.49. HRMS (ESI + ): m / z calculated for C 32 H 36 Cl3N7O4[M+ H]+ , 688.1973 found 688.1972.

[0054] Synthesis of compound SK3: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.13 (t, J = 5.7 Hz, 1H), 7.62 (dd, J = 8.0, 1.5 Hz, 1H), 7.50 (s, 1H), 7.44 (s, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.31 (dd, J = 7.6, 1.6 Hz, 1H), 6.81 (d, J = 10.2 Hz, 2H), 5.61 (s, 2H), 4.45 (s, 2H), 3.85 (d, J = 13.6 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.62 (t, J = 6.3 Hz, 1H), 3.19 – 3.14 (m, 4H), 2.68 (s, 2H), 2.19 – 2.10 (m, 5H), 1.88 (s, 2H), 1.68 (p, J = 7.1 Hz, 2H), 1.47 (ddd, J = 14.0,10.4, 4.0 Hz, 2H), 1.31 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d 172.55, 168.09,165.73, 165.23, 153.64, 151.17, 143.29, 139.93, 132.58, 132.07, 131.15,130.23, 128.75, 125.47, 125.26, 119.94, 117.16, 114.87, 112.93, 67.70, 55.42,51.38, 40.68, 38.64, 35.31, 34.34, 26.84, 26.37, 26.17, 23.77, 23.50. HRMS(ESI + ): m / z Calculated for C 33 H 38 Cl3N7O4[M + H] + , 702.2129 found 702.2129.

[0055] Synthesis of compound SK4: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.09 (q, J = 5.8 Hz, 1H), 7.61 (dd, J = 8.0, 1.8 Hz, 1H), 7.51 (s, 1H),7.45 (t, J = 7.1 Hz, 1H), 7.39 (td, J = 7.9, 1.9 Hz, 2H), 7.31 (dd, J = 7.6, 1.6Hz, 1H), 6.80 (d, J = 11.6 Hz, 2H), 5.61 (s, 2H), 4.44 (d, J = 7.7 Hz, 2H), 3.85(d, J = 11.8 Hz, 2H), 3.67 (t, J = 6.3 Hz, 1H), 3.63 (t, J = 6.3 Hz, 1H), 3.23 –3.12 (m, 5H), 2.68 (s, 2H), 2.18 – 2.10 (m, 4H), 1.88 (s, 2H), 1.56 – 1.39(m, 7H), 1.31 (d, J = 3.7 Hz, 3H). 13 C NMR (150 MHz, DMSO- d 6) d172.78, 167.96,165.72, 165.23, 153.64, 151.17, 143.30, 139.93, 132.60, 132.08, 131.14,130.22, 128.73, 125.45, 125.26, 119.93, 117.18, 114.86, 112.91, 67.73, 55.41,51.32, 40.71, 38.63, 36.33, 35.32, 29.29, 26.83, 26.37, 23.77, 23.51, 23.44.HRMS (ESI + ): m / z Calculated for C 34 H 40 Cl3N7O4[M + H] + , 716.2286 found 716.2281.

[0056] Synthesis of compound SK5: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.04 (q, J = 7.7, 6.7 Hz, 1H), 7.62 (dd, J = 8.0, 1.6 Hz, 1H), 7.50 (s,1H), 7.40 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 5.1 Hz, 1H), 7.31 (dd, J = 7.6, 1.6 Hz, 1H), 6.80 (d, J = 9.9 Hz, 2H), 5.61 (s, 2H), 4.44 (s, 2H), 3.84 (d, J = 13.6 Hz, 2H), 3.67 (t, J = 6.3 Hz, 1H), 3.62 (t, J = 6.3 Hz, 1H), 3.20 – 3.15 (m, 2H), 3.12 (q, J = 5.4, 3.9 Hz, 2H), 2.68 (s, 2H), 2.15 (d, J = 13.4 Hz, 2H), 2.09 (t, J= 7.4 Hz, 2H), 1.92 (s, 1H), 1.88 (s, 1H), 1.53 – 1.40 (m, 7H), 1.30 (s, 3H), 1.25 (q, J = 8.0 Hz, 3H). 13 C NMR (150 MHz, DMSO- d 6) d 172.82, 167.89, 165.72, 165.23, 153.62, 151.17, 143.29, 139.95, 132.58, 132.08, 131.15, 130.21, 128.73, 125.45, 125.25, 119.94, 117.16, 114.87, 112.88, 67.73, 55.43, 51.30, 40.71, 38.73, 36.65, 35.31, 29.43, 26.84, 26.56, 26.40, 25.78, 23.77, 23.51.HRMS (ESI + ): m / z Calculated for C 35 H 42 Cl3N7O4[M + H] + , 730.2442, found 730.2442.

[0057] Synthesis of compound SK6: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.04 – 8.02 (m, 1H), 7.62 (dd, J = 8.0, 2.1 Hz, 1H), 7.50 (s, 1H),7.46 (t, J = 7.7 Hz, 1H), 7.40 (dd, J = 7.8, 1.7 Hz, 1H), 7.38 (d, J = 2.5 Hz, 1H), 7.31 (d, J = 7.7 Hz, 1H), 6.80 (d, J = 12.9 Hz, 2H), 5.60 (s, 2H), 4.44 (s, 2H), 4.15 (s, 2H), 3.85 (d, J= 14.0 Hz, 2H), 3.67 (t, J = 6.3 Hz, 1H), 3.62 (t, J = 6.3Hz, 1H), 3.18 (s, 4H), 3.14 – 3.10 (m, 2H), 2.68 (s, 1H), 2.16 (d, J = 13.5 Hz, 2H), 2.09 (t, J = 7.5 Hz, 2H), 1.88 (s, 1H), 1.50 – 1.40 (m, 6H), 1.31 (s, 3H), 1.25 (s, 4H). 13 C NMR (150 MHz, DMSO- d 6) d 172.97, 167.96, 165.75, 165.22, 153.65, 151.16, 143.28, 139.89, 132.58, 132.05, 131.13, 130.23, 128.76, 125.48, 125.26, 119.92, 117.17, 114.87, 112.88, 67.69, 55.38, 51.30, 49.13, 40.71, 38.77, 36.68, 35.29, 29.51, 28.89, 26.62, 26.39, 26.00, 23.76, 23.50.HRMS (ESI + ): m / z Calculated for C 36 H 44 Cl3N7O4[M + H] + , 744.2599, found 744.2600.

[0058] Synthesis of compound SK7: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.03 (d, J = 6.6 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.50 (s, 1H), 7.40(d, J = 7.8 Hz, 1H), 7.38 (s, 1H), 7.31 (d, J= 7.6 Hz, 1H), 6.79 (d, J = 9.4 Hz,2H), 5.61 (s, 2H), 4.44 (s, 2H), 3.85 (d, J = 13.3 Hz, 2H), 3.67 (t, J = 6.5 Hz, 2H), 3.16 (t, J = 11.8 Hz, 2H), 3.11 (q, J = 6.7 Hz, 2H), 2.68 (s, 2H), 2.16 (d, J = 13.4 Hz, 2H), 2.09 (t, J = 7.5 Hz, 2H), 1.88 (s, 2H), 1.49 – 1.40 (m, 6H), 1.30 (s, 3H), 1.24 (s, 8H). 13 C NMR (150 MHz, DMSO- d 6) d 172.90, 167.92, 165.70, 165.24, 153.63, 151.18, 143.27, 139.92, 132.55, 132.06, 131.16, 130.23, 128.76, 125.48, 125.26, 119.94, 117.09, 114.82, 112.85, 67.66, 55.45, 51.27, 40.68, 38.75, 36.67, 35.27, 29.60, 29.14, 29.03, 26.81, 26.46, 26.00, 23.77, 23.50. HRMS (ESI) + ): m / z Calculated for C 37 H 46 Cl3N7O4[M + H] + , 758.2755, found758.2755.

[0059] Synthesis of compound SK8: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.03 (t, J = 6.5 Hz, 1H), 7.61 (dd, J= 8.0, 1.9 Hz, 1H), 7.51 (s, 1H),7.45 (t, J = 7.7 Hz, 1H), 7.39 (dd, J = 7.8, 1.6 Hz, 1H), 7.37 (d, J = 2.4 Hz, 1H),7.31 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 11.1 Hz, 2H), 5.60 (s, 2H), 4.44 (d, J = 8.2Hz, 2H), 4.13 (q, J = 5.4 Hz, 1H), 3.86 (d, J = 12.1 Hz, 2H), 3.67 (t, J = 6.4 Hz,1H), 3.63 (t, J = 6.4 Hz, 1H), 3.18 (d, J = 4.4 Hz, 5H), 3.14 – 3.08 (m, 2H),2.68 (s, 1H), 2.17 (d, J = 13.2 Hz, 2H), 2.09 (t, J = 6.0 Hz, 2H), 1.88 (s, 1H),1.51 – 1.39 (m, 6H), 1.31 (s, 3H), 1.23 (s, 8H). 13 C NMR (150 MHz, DMSO- d 6) d 172.98, 167.94, 165.22, 153.66, 151.16, 143.29, 139.90, 132.59, 132.06,131.13, 130.23, 128.76, 125.47, 125.27, 119.93, 117.16, 114.85, 112.91,67.70, 55.40, 51.28, 49.14, 40.70, 38.78, 36.71, 35.29, 29.55, 29.23, 29.20,29.12, 26.82, 26.44, 26.02, 23.77, 23.50. HRMS (ESI + ): m / z calculated forC 38H 48 Cl3N7O4[M + H] + , 772.2912, found 772.2910.

[0060] Synthesis of compound SK9: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.02 (t, J = 5.7 Hz, 1H), 7.62 (dd, J = 8.0, 1.6 Hz, 1H), 7.50 (s, 1H),7.39 (t, J = 7.8 Hz, 1H), 7.36 (s, 1H), 7.31 (dd, J = 7.6, 1.6 Hz, 1H), 6.79 (d, J = 10.0 Hz, 2H), 5.61 (s, 2H), 4.44 (s, 2H), 3.86 (d, J = 13.4 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.16 (ddd, J = 13.6, 10.5, 2.9 Hz, 2H), 3.11 (q, J = 6.7 Hz, 2H),2.68 (s, 2H), 2.16 (d, J = 13.2 Hz, 2H), 2.09 (t, J = 7.3 Hz, 2H), 1.88 (s, 2H), 1.51 – 1.39 (m, 6H), 1.30 (s, 3H), 1.23 (d, J = 9.8 Hz, 12H). 13 C NMR (150 MHz, DMSO- d 6) d172.91, 167.91, 165.71, 165.22, 153.65, 151.17, 143.29, 139.93, 132.57, 132.06, 131.14, 130.23, 128.76, 125.47, 125.27, 119.94, 117.13, 114.83, 112.90, 67.71, 55.43, 51.27, 40.69, 38.78, 36.69, 35.29, 31.81, 29.59, 29.46, 29.25, 29.15, 26.83, 26.47, 26.03, 23.77, 23.51, 22.62. HRMS(ESI + ): m / z Calculated for C 39 H 50 Cl3N7O4[M + H] + , 786.3068, found 786.3065.

[0061] Synthesis of compound SK10: For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 7.98 (t, J = 6.5 Hz, 1H), 7.57 (dd, J = 8.1, 1.7 Hz, 1H), 7.45 (s, 1H),7.41 (t, J = 7.6 Hz, 1H), 7.35 (dd, J = 7.7, 1.4 Hz, 1H), 7.33 (d, J = 5.5 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 11.1 Hz, 2H), 5.56 (s, 2H), 4.39 (s, 2H), 3.81 (d, J = 13.7 Hz, 2H), 3.62 (t, J = 6.3 Hz, 1H), 3.58 (t, J = 6.3 Hz, 1H), 3.11(t, J= 12.0 Hz, 2H), 3.08 – 3.04 (m, 2H), 2.63 (s, 2H), 2.12 (d, J = 12.9 Hz, 2H), 2.04 (td, J = 7.4, 2.7 Hz, 2H), 1.84 (d, J = 7.5 Hz, 2H), 1.46 – 1.34 (m,6H), 1.25 (s, 3H), 1.18 (d, J = 10.7 Hz, 14H). 13 C NMR (150 MHz, DMSO- d 6) d 172.95, 167.92, 165.71, 165.22, 153.65, 151.16, 143.29, 139.92, 132.58, 132.06, 131.13, 130.22, 128.75, 125.47, 125.27, 119.93, 117.14, 114.85, 112.88, 67.70, 55.41, 51.27, 49.04, 40.69, 38.79, 36.69, 35.29, 31.81, 29.58, 29.48, 29.27, 29.14, 26.86, 26.47, 26.04, 23.77, 23.51, 22.62. HRMS (ESI + ): m / z Calculated for C 40 H 52 Cl3N7O4[M + H] + , 800.3225, found 800.3224.

[0062] Example 6 Synthesis of compound SK11:

[0063] For specific procedures and proportions, refer to the preparation of compound SK1. 1 H NMR (600 MHz, DMSO- d 6) d 8.04 (t, J =5.7 Hz, 1H), 7.63 (dd, J= 8.0, 1.6 Hz, 1H), 7.50 (s, 1H), 7.47 (s, 1H), 7.40(t, J = 7.9 Hz, 1H), 7.32 (dd, J = 7.6, 1.6 Hz, 1H), 6.82 (d, J = 9.5 Hz, 2H), 5.61(s, 2H), 4.47 (s, 2H), 3.86 (d, J = 13.6 Hz, 2H), 3.68 (t, J = 6.3 Hz, 2H), 3.62(t, J = 6.4 Hz, 2H), 3.45 (t, J = 6.0 Hz, 2H), 3.30 (q, J = 5.9 Hz, 2H), 3.20 (ddd, J = 13.4, 10.6, 2.9 Hz, 2H), 2.70 (s, 2H), 2.37 (t, J = 6.5 Hz, 2H), 2.16 (d, J =13.7 Hz, 2H), 1.89 (s, 2H), 1.47 (ddd, J = 14.1, 10.6, 4.1 Hz, 2H), 1.32 (s,3H), 1.27 – 1.24 (m, 2H). 13 C NMR (150 MHz, DMSO- d 6) d 170.73, 168.20, 165.73,165.23, 153.64, 151.17, 143.28, 139.88, 132.57, 132.06, 131.15, 130.24,128.76, 125.48, 125.21, 119.94, 117.12, 114.90, 112.88, 68.99, 67.63, 67.35,55.42, 51.48, 40.66, 38.75, 37.37, 35.24, 26.82, 26.39, 23.76. HRMS (ESI + ): m / z calculated for C 34 H 40 Cl3N7O5[M + H] +,732.2235, found 732.2232.

[0064] Example 7 Synthesis of intermediate 17a:

[0065] Compound 16a, namely 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid (89 mg, 0.34 mmol, 1.2 eq), and HATU (129 mg, 0.34 mmol, 1.2 eq) were dissolved in... N , N In dimethylformamide (10 mL), the reaction mixture was stirred at 0 °C for 15 min, followed by the sequential addition of SHP099(6) (100 mg, 0.28 mmol, 1.0 eq) and DIPEA (146 µL, 0.84 mmol, 3.0 eq). The resulting reaction mixture was stirred at room temperature for 6 h. The mixture was extracted with 20 mL of water and 20 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 60:1) to give intermediate 17a. The yield was 68%. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J =8.2, 1.7 Hz, 1H), 7.47 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 7.6, 1.7Hz, 1H), 7.04 (s, 1H), 6.75 (t, J = 5.7 Hz, 1H), 5.58 (s, 2H), 3.82 (d, J = 12.2Hz, 4H), 3.55 (dd, J = 6.0, 3.0 Hz, 2H), 3.51 (d, J = 4.5 Hz, 2H), 3.37 (t, J = 6.1Hz, 2H), 3.14 (ddd, J = 13.5, 10.3, 3.0 Hz, 2H), 3.05 (q, J = 6.1 Hz, 2H), 2.14(d, J = 14.2 Hz, 2H), 1.50 (ddd, J= 14.0, 10.5, 4.0 Hz, 2H), 1.33 (s, 9H), 1.31 (s, 3H).

[0066] Example 8 Synthesis of intermediate 17b-d:

[0067] Synthesis of intermediate 17b: Refer to the preparation of intermediate 17a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.58 (dd, J = 8.0, 1.6 Hz, 1H), 7.47 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 7.03 (s, 1H), 6.70 (t, J = 5.7 Hz, 1H), 5.58 (s,2H), 3.83 – 3.79 (m, 4H), 3.57 – 3.55 (m, 2H), 3.53 (dt, J = 6.0, 1.8 Hz, 2H), 3.50 (dd, J = 6.3, 3.8 Hz, 2H), 3.47 – 3.45 (m, 2H), 3.35 – 3.33 (m, 2H), 3.14(ddd, J = 13.4, 10.4, 3.1 Hz, 2H), 3.03 (q, J = 6.0 Hz, 2H), 2.14 (d, J = 13.2 Hz, 2H), 1.50 (ddd, J = 14.1, 10.4, 4.2 Hz, 2H), 1.32 (s, 9H), 1.31 (s, 3H). Synthesis of intermediate 17c-d: Refer to the preparation of intermediate 17a for specific operations and proportions.

[0068] Example 9 Synthesis of compound SK12:

[0069] Intermediate 17a (100 mg) was dissolved in ethyl acetate and cooled to 0°C. Ethyl hydrochloride solution was added dropwise, and after complete consumption of the starting material was monitored by TLC, the solvent was removed under reduced pressure. A suitable amount of water was added, and the pH was adjusted to 9 by slow dropwise addition of 2M sodium hydroxide aqueous solution. The product was extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. Intermediate 11 (66 mg, 0.24 mmol, 1.2 eq) was dissolved in dichloromethane, and EDCI (56 mg, 0.3 mmol, 1.5 eq) and HOBt (32 mg, 0.24 mmol, 1.2 eq) were added sequentially. The reaction mixture was stirred at 0°C for 15 minutes. The treated intermediate 17a and DIPEA (137 µL, 0.8 mmol, 4.0 eq) were added sequentially to the reaction mixture, and the resulting reaction system was stirred at room temperature for 6 hours. The mixture was extracted with 20 mL of water and 20 mL of dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to give compound SK12. The yield was 33%. 1 HNMR (600 MHz, DMSO- d 6) d 8.07 (t, J = 5.9 Hz, 1H), 7.62 (dd, J = 8.0, 1.7 Hz, 1H),7.50 (s, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.6, 1.5 Hz, 1H), 7.08 (s,1H), 6.79 (d, J = 8.0 Hz, 2H), 5.62 (s, 2H), 4.47 (s, 2H), 3.87 – 3.81 (m, 4H), 3.66 (t, J = 6.4 Hz, 2H), 3.60 (dd, J = 6.0, 2.9 Hz, 2H), 3.56 (dd, J = 5.9, 3.2Hz, 2H), 3.49 (t, J = 5.8 Hz, 2H), 3.32 (t, J = 6.0 Hz, 2H), 3.18 (ddd, J = 13.5,10.5, 3.1 Hz, 2H), 2.67 (s, 2H), 2.17 (d, J= 13.6 Hz, 2H), 1.87 (s, 2H), 1.53(ddd, J = 14.0, 10.1, 4.1 Hz, 2H), 1.34 (s, 3H), 1.23 (s, 2H). 13 C NMR (150 MHz, DMSO- d 6) d 169.57, 168.26, 165.71, 165.23, 153.61, 151.17, 143.29, 139.90,132.57, 132.18, 132.06, 131.14, 130.24, 128.76, 125.42, 119.94, 117.15,114.91, 112.85, 70.82, 70.61, 69.81, 69.37, 67.66, 55.43, 51.54, 40.64,38.64, 35.32, 26.82, 26.15, 23.76. HRMS (ESI + ): m / z Calculated for C 35 H 42 Cl3N7O6[M + H] + , 762.2340, found 762.2354.

[0070] Example 10 Synthesis of intermediate 18:

[0071] Intermediate 12b, namely Boc-β-alanine (38 mg, 0.20 mmol, 1.2 eq), and HATU (76 mg, 0.20 mmol, 1.2 eq) were dissolved in... N , NIn dimethylformamide, the reaction mixture was stirred at 0°C for 15 minutes. Intermediate 17a (100 mg, 0.17 mmol, 1.0 eq) was dissolved in ethyl acetate and cooled to 0°C. Ethyl hydrochloride solution was added dropwise, and after complete consumption of the starting material was monitored by TLC, the solvent was removed under reduced pressure. A suitable amount of water was added, and the pH was adjusted to 9 by slow dropwise addition of 2M sodium hydroxide aqueous solution. The product was extracted with ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The treated intermediate 17a and DIPEA (117 µL, 0.67 mmol, 4.0 eq) were added sequentially to the above reaction mixture, and the resulting reaction system was stirred at room temperature for 6 hours. Extraction was performed with 20 mL of water and 20 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 60:1) to give intermediate 18. The yield was 67%. 1 H NMR (600 MHz, DMSO- d 6) d 7.88 (t, J = 5.7 Hz, 1H), 7.58 (dd, J = 8.0, 1.6Hz, 1H), 7.47 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H),7.05 (s, 1H), 6.66 (t, J = 5.8 Hz, 1H), 5.58 (s, 2H), 3.80 (d, J = 12.9 Hz, 4H), 3.56 (dt, J = 4.3, 2.8 Hz, 2H), 3.52 (dt, J = 4.2, 2.6 Hz, 2H), 3.39 (t, J = 6.0Hz, 2H), 3.16 (dd, J = 10.8, 4.6 Hz, 4H), 3.09 – 3.05 (m, 2H), 2.19 (t, J = 7.4Hz, 2H), 2.14 (d, J = 12.5 Hz, 2H), 1.50 (ddd, J = 14.0, 10.3, 4.2 Hz, 2H), 1.32(s, 9H), 1.31(s, 3H).

[0072] Example 11 Synthesis of compound SK13:

[0073] For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 8.08 (t, J = 5.8 Hz, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.61 (dd, J = 8.0, 1.7 Hz, 1H), 7.51 (s,1H), 7.39 (t, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.6, 1.6 Hz, 1H), 7.09 (s, 1H), 6.78(d, J = 10.3 Hz, 2H), 5.63 (s, 2H), 4.44 (s, 2H), 3.87 – 3.82 (m, 4H), 3.66 (t, J = 6.3 Hz, 2H), 3.60 (dd, J = 6.0, 3.1 Hz, 2H), 3.55 (dd, J = 5.7, 2.9 Hz, 2H), 3.44 (t, J = 6.0 Hz, 2H), 3.33 (d, J = 6.9 Hz, 1H), 3.24 – 3.15 (m, 5H), 2.68 (s,2H), 2.32 (t, J = 7.1 Hz, 2H), 2.17 (d, J = 14.6 Hz, 2H), 1.87 (s, 2H), 1.54(ddd, J = 14.1, 10.4, 4.1 Hz, 2H), 1.35 (s, 3H), 1.23 (s, 2H). 13 C NMR (150 MHz, DMSO- d 6) d171.06, 169.58, 168.03, 165.72, 165.24, 153.62, 151.19, 139.88, 132.58, 132.18, 132.07, 131.14, 130.25, 128.76, 125.39, 119.94, 117.13, 114.88, 112.88, 70.80, 70.60, 69.84, 69.61, 67.67, 55.43, 51.55, 40.65, 38.92, 35.64, 35.55, 35.32, 29.52, 26.81, 26.17, 23.76. HRMS (ESI + ): m / z Calculated for C 38 H 47 Cl3N8O7[M + H] + , 833.2712, found 833.2708.

[0074] Example 12 Synthesis of compound SK14-16:

[0075] Synthesis of compound SK14: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 8.06 (t, J = 5.9 Hz, 1H), 7.61 (dd, J = 8.0, 1.6 Hz, 1H), 7.51 (s, 1H),7.39 (t, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.6, 1.6 Hz, 1H), 7.07 (s, 1H), 6.79 (d, J = 10.5 Hz, 2H), 5.63 (s, 2H), 4.46 (s, 2H), 3.87 – 3.83 (m, 4H), 3.66 (t, J =6.4 Hz, 2H), 3.60 – 3.58 (m, 2H), 3.58 – 3.56 (m, 2H), 3.54 – 3.53 (m, 2H), 3.53 – 3.51 (m, 2H), 3.46 (t,J = 5.9 Hz, 2H), 3.31 (q, J = 5.9 Hz, 3H), 3.18(ddd, J = 13.5, 10.4, 3.1 Hz, 2H), 2.67 (s, 2H), 2.17 (d, J = 13.7 Hz, 2H), 1.87(s, 2H), 1.53 (ddd, J = 14.0, 10.5, 4.2 Hz, 2H), 1.34 (s, 3H), 1.23 (s, 1H). 13 CNMR (150 MHz, DMSO- d 6) d 169.59, 168.23, 165.71, 153.61, 151.19, 139.86,132.57, 132.16, 132.07, 131.15, 130.26, 128.76, 125.36, 117.09, 114.88,112.86, 70.84, 70.58, 70.28, 70.10, 70.06, 69.37, 67.65, 55.44, 51.53, 40.63,38.77, 35.32, 26.82, 26.18, 23.76. HRMS (ESI + ): m / z Calculated for C 37 H 46 Cl3N7O7[M + H] + , 806.2603, found 806.2610.

[0076] Synthesis of compound SK15: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 8.02 (t, J = 5.7 Hz, 1H), 7.57 (dd, J = 8.0, 1.9 Hz, 1H), 7.47 (s, 1H), 7.34 (td, J = 7.8, 1.6 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 6.75 (d, J= 11.0 Hz, 2H), 5.59 (s, 2H), 4.42 (s, 2H), 3.81 (s, 4H), 3.62 (t, J = 6.4 Hz, 2H), 3.55 (d, J = 5.0 Hz, 2H), 3.52 (d, J = 4.7 Hz, 2H), 3.49 (d, J = 4.5 Hz, 4H),3.47 (s, 6H), 3.41 (t, J = 5.9 Hz, 2H), 3.26 (q, J = 5.8 Hz, 2H), 3.14 (t, J = 11.9Hz, 2H), 2.63 (s, 2H), 2.13 (d, J = 13.3 Hz, 2H), 1.83 (s, 2H), 1.50 (q, J =10.1, 6.9 Hz, 2H), 1.31 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d 169.58, 168.20, 165.71, 165.24, 153.61, 151.18, 143.29, 139.89, 132.58, 132.17, 132.07, 131.14, 130.24, 128.75, 125.40, 119.94, 117.12, 114.88, 112.85, 70.85, 70.57, 70.33, 70.27, 70.09, 70.06, 69.36, 67.63, 55.44, 51.52, 40.62, 38.77, 35.32, 26.83, 26.17, 23.77, 23.50. HRMS (ESI + ): m / z Calculated for C 39 H 50 Cl3N7O8[M + H] + , 850.2865, found 850.2872.

[0077] Synthesis of compound SK16: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO-d 6) d 8.02 (t, J = 6.0 Hz, 1H), 7.57 (dd, J = 8.0, 1.7 Hz, 1H), 7.46 (s, 1H),7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.7, 1.6 Hz, 1H), 7.04 (s, 1H), 6.82 –6.69 (m, 2H), 5.60 (s, 2H), 4.42 (s, 2H), 3.84 – 3.79 (m, 4H), 3.62 (t, J = 6.4Hz, 2H), 3.58 (t, J = 6.3 Hz, 1H), 3.56 – 3.54 (m, 2H), 3.52 (dd, J = 5.9, 3.3Hz, 2H), 3.49 (d, J = 5.1 Hz, 2H), 3.48 (d, J = 2.0 Hz, 1H), 3.46 (t, J = 5.3 Hz,10H), 3.40 (q, J = 5.5 Hz, 2H), 3.25 (q, J = 4.9, 4.1 Hz, 2H), 3.17 – 3.11 (m,2H), 2.64 (s, 2H), 2.13 (d, J = 13.2 Hz, 2H), 1.87 (s, 1H), 1.83 (s, 1H), 1.50(ddd, J = 14.2, 10.6, 4.0 Hz, 2H), 1.31 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d169.59, 168.20, 165.71, 165.24, 153.61, 151.18, 143.28, 139.89, 132.57, 132.06, 131.14, 130.25, 128.76, 125.47, 125.39, 119.94, 117.11, 114.86, 112.86, 70.85, 70.56, 70.32, 70.26, 70.09, 70.04, 69.35, 67.62, 55.44, 51.52, 40.62, 38.77, 35.31, 26.83, 26.17, 23.76, 21.58. HRMS (ESI + ): m / z calculated for C 41 H 54 Cl3N7O9[M + H] + , 894.3127, found 894.3125.

[0078] Example 13 Synthesis of intermediate 19a-d:

[0079] Synthesis of intermediate 19a: A mixture of N-Boc-piperazine (1.86 g, 10.0 mmol, 1.0 eq), sodium iodide (1.49 g, 10.0 mmol, 1.0 eq), potassium carbonate (4.14 g, 12.0 mmol, 1.2 eq), and methyl 4-bromobutyrate (2.17 g, 12.0 mmol, 1.2 eq) was placed in acetonitrile (60 mL) and stirred at 60 °C for 16 hours. The solvent was removed under reduced pressure to obtain the crude ester, which was used directly in the next reaction. The crude ester was dissolved in tetrahydrofuran / methanol / water (15 mL / 9 mL / 6 mL), and lithium hydroxide hydrate (1.68 g, 40.0 mmol, 2.0 eq) was added at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was diluted with 10 mL of water and acidified to pH 3 with 1 M hydrochloric acid (aqueous solution). The product was extracted with ethyl acetate, the organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 19a. Synthesis of intermediates 19b-d: Refer to the preparation of intermediate 19a for specific procedures and proportions.

[0080] Example 14 Synthesis of compound SK17-20:

[0081] Synthesis of compound SK17: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.7 Hz, 1H), 7.46 (s, 1H), 7.36 – 7.33 (m, 2H),7.26 (dd, J = 7.7, 1.6 Hz, 1H), 6.70 (d, J = 9.4 Hz, 2H), 5.57 (s, 2H), 4.73 (s, 2H), 4.40 (s, 2H), 3.81 (dt, J = 13.5, 4.4 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 3.40 (s, 4H), 3.16 – 3.12 (m, 2H), 2.63 (s, 2H), 2.33 (s, 2H), 2.25 (dd, J =16.4, 8.9 Hz, 4H), 2.14 – 2.07 (m, 4H), 1.83 (s, 2H), 1.62 (p, J = 7.4 Hz, 2H), 1.43 (ddd, J = 14.1, 10.5, 4.1 Hz, 2H), 1.27 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d 172.68, 166.22, 165.71, 165.21, 153.68, 151.17, 143.29, 139.92, 132.57, 132.06, 131.15, 130.24, 128.77, 125.48, 125.30, 119.94, 117.17, 114.74, 112.69, 66.59, 57.71, 55.42, 53.35, 52.93, 51.31, 49.14, 44.74, 41.80, 40.72, 35.29, 34.54, 26.43, 23.79, 23.52, 23.07. HRMS (ESI + ): m / z Calculated for C 37H 45 Cl3N8O4[M + H] + , 771.2708, found 771.2703.

[0082] Synthesis of compound SK18: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 8.03 (d, J = 8.4 Hz, 1H), 7.61 – 7.59 (m, 1H), 7.50 (s, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.39 – 7.36 (m, 2H), 7.30 (d, J = 7.5 Hz, 1H), 6.72 (d, J = 9.0Hz, 2H), 5.61 (s, 2H), 4.76 (d, J = 7.4 Hz, 2H), 3.86 (dt, J = 13.5, 4.3 Hz, 2H), 3.62 (t, J = 6.3 Hz, 2H), 3.42 (q, J = 5.6, 5.2 Hz, 6H), 3.19 – 3.13 (m, 2H), 2.66 (s, 1H), 2.35 (s, 2H), 2.28 (s, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.17 (d, J =14.0 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.87 (s, 1H), 1.76 (s, 1H), 1.52 (t, J =7.4 Hz, 2H), 1.47 – 1.39 (m, 4H), 1.30 (s, 3H), 1.26 (t, J = 7.3 Hz, 2H). 13 C NMR (150 MHz, DMSO-) d 6) d172.88, 166.18, 165.21, 153.67, 151.17, 143.29, 139.93, 132.58, 132.06, 131.14, 130.22, 128.77, 125.46, 125.31, 119.94, 117.16, 111.10, 66.59, 58.22, 55.43, 53.42, 52.93, 51.28, 44.76, 41.82, 40.70, 36.64, 35.30, 27.02, 26.51, 26.43, 25.98, 23.53. HRMS (ESI + ): m / z Calculated for C 39 H 49 Cl3N8O4[M + H] + , 799.3021, found 799.3016.

[0083] Synthesis of compound SK19: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 8.03 (d, J = 8.4 Hz, 1H), 7.61 (dd, J = 8.0, 1.8 Hz, 1H), 7.51 (s, 1H),7.48 – 7.43 (m, 1H), 7.39 (dd, J = 7.7, 1.4 Hz, 1H), 7.37 (s, 1H), 7.31 (dd, J =7.7, 1.6 Hz, 1H), 6.75 (d, J = 13.8 Hz, 2H), 5.61 (s, 2H), 4.77 (s, 2H), 3.87(dt, J = 13.6, 4.4 Hz, 2H), 3.67 (t, J = 6.4 Hz, 1H), 3.62 (t, J = 6.3 Hz, 1H), 3.43 (s, 5H), 3.16 (ddd, J = 13.6, 10.7, 3.0 Hz, 2H), 2.67 (s, 1H), 2.35 (q, J=4.5 Hz, 2H), 2.31 – 2.27 (m, 2H), 2.24 (dd, J = 9.7, 4.9 Hz, 2H), 2.17 (d, J =14.0 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.88 (s, 1H), 1.52 – 1.44 (m, 4H), 1.43– 1.37 (m, 2H), 1.31 (s, 3H), 1.29 – 1.21 (m, 8H). 13 C NMR (150 MHz, DMSO- d 6) d 172.94, 166.19, 165.20, 153.66, 151.17, 143.29, 139.93, 132.59, 132.07, 131.13, 130.22, 128.76, 125.45, 125.29, 119.93, 117.16, 112.68, 111.10, 66.60, 58.23, 55.41, 53.42, 52.94, 51.28, 44.76, 41.82, 40.70, 36.68, 35.30, 29.18, 29.12, 27.37, 26.62, 26.50, 26.00, 23.79, 23.53. HRMS (ESI + ): m / z Calculated for C 41 H 53 Cl3N8O4[M + H] + , 827.3334, found 827.3327.

[0084] Synthesis of compound SK20: For specific procedures and proportions, refer to the preparation of compound SK12. 1 H NMR (600 MHz, DMSO- d 6) d 7.62 (dd, J = 8.0, 1.3 Hz, 1H), 7.50 (s, 1H), 7.41 – 7.37 (m, 2H),7.31 (d, J = 7.4 Hz, 1H), 6.75 (d, J= 9.2 Hz, 2H), 5.60 (s, 2H), 4.77 (s, 2H), 4.45 (s, 2H), 3.86 (dt, J = 13.5, 4.4 Hz, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.15(ddd, J = 13.5, 10.5, 3.0 Hz, 2H), 2.67 (s, 2H), 2.37 (t, J = 5.0 Hz, 2H), 2.33 – 2.28 (m, 2H), 2.26 (t, J = 7.4 Hz, 2H), 2.17 (dt, J = 13.9, 3.9 Hz, 2H), 2.09 (t, J = 7.3 Hz, 2H), 1.88 (s, 2H), 1.52 – 1.37 (m, 5H), 1.34 (dt, J = 11.1, 5.9 Hz, 3H), 1.30 (s, 3H), 1.25 (p, J = 4.8 Hz, 12H). 13 C NMR (150 MHz, DMSO- d 6) d 173.03, 166.23, 165.75, 153.67, 151.15, 139.88, 132.58, 132.03, 131.12, 130.25, 128.77, 125.27, 117.15, 114.75, 112.68, 66.57, 58.24, 55.39, 53.38, 52.91, 51.28, 44.71, 41.78, 40.69, 36.68, 35.28, 29.47, 29.41, 29.22, 29.08, 27.38, 26.80, 26.60, 26.48, 26.02, 23.78, 22.61. HRMS (ESI + ): m / z calculated for C 43 H 57 Cl3N8O4[M + H] + , 855.3647, found 855.3640。

[0085] Example 15 Synthesis of intermediate 24:

[0086] KB02(8) (400 mg, 1.78 mmol, 1.0 eq) and potassium carbonate (736 mg, 5.32 mmol, 3.0 eq) were dissolved in acetonitrile, and bromopropyne (254 mg, 2.14 mmol, 1.2 eq) was added dropwise. The reaction mixture was stirred at 60 °C for 4 hours. The mixture was extracted with 20 mL of water and 20 mL of ethyl acetate. The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to give intermediate 24. Yield: 34%. 1 H NMR (600 MHz, Chloroform- d ) d 7.11 (d, J = 11.3 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 4.67 (s, 2H), 4.18 (s, 2H), 3.79 (t, J = 6.9 Hz, 2H), 2.68 (s, 2H), 2.53 (s, 1H), 1.96 (s, 2H).

[0087] Example 16 Synthesis of intermediate 26a-d:

[0088] Synthesis of intermediate 26a: Compound 25a, namely azidoacetic acid (35 mg, 0.34 mmol, 1.2 eq), was dissolved in dichloromethane. EDCI (107 mg, 0.56 mmol, 2.0 eq) and HOBt (79 mg, 0.56 mmol, 2.0 eq) were added sequentially, and the mixture was stirred at 0 °C for 15 min. Then, SHP099(6) (100 mg, 0.28 mmol, 1.0 eq) and DIPEA (146 μL, 0.84 mmol, 3.0 eq) were added to the above reaction mixture, and the mixture was stirred at room temperature for 8 h. The mixture was extracted with 20 mL of water and 20 mL of dichloromethane. The organic layers were combined, dried over sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane to methanol volume ratio 80:1) to obtain intermediate 26a. The yield was 76%. 1 H NMR (600 MHz, DMSO- d 6) d 7.67 (s, 1H), 7.57 (dd,J = 8.0, 1.6 Hz, 1H), 7.47 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 7.6, 1.6 Hz, 1H), 5.59 (s, 2H), 3.81 (dt, J = 13.6, 4.4 Hz, 2H), 3.76 (s,2H), 3.16 (ddd, J = 13.5, 10.4, 3.0 Hz, 2H), 2.09 (dt, J = 13.8, 3.9 Hz, 2H),1.48 (ddd, J = 14.1, 10.5, 4.1 Hz, 2H), 1.30 (s, 3H).

[0089] Synthesis of intermediate 26b: Refer to the preparation of intermediate 26a for specific operations and proportions. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 7.6, 1.6 Hz, 1H), 5.58 (s, 2H), 3.81 (dt, J = 13.5, 4.4 Hz, 2H), 3.28 (t, J = 6.8 Hz, 2H), 3.14 (ddd, J = 13.5, 10.5, 3.0 Hz, 2H),2.16 (t, J = 7.3 Hz, 2H), 2.11 (dt, J = 13.1, 4.3 Hz, 2H), 1.71 (p, J = 7.1 Hz, 2H), 1.43 (ddd, J = 14.1, 10.6, 4.1 Hz, 2H), 1.27 (s, 3H).

[0090] Synthesis of intermediate 26c: For specific procedures and proportions, refer to the preparation of intermediate 26a. 1H NMR (600 MHz, DMSO- d 6) d 7.60 (dd, J = 8.0, 1.6 Hz, 1H), 7.48 (s, 1H), 7.38 (s, 1H), 7.36 (d, J =7.8 Hz, 1H), 7.29 (dd, J = 7.6, 1.6 Hz, 1H), 5.60 (s, 2H), 3.84 (dt, J = 13.6, 4.3 Hz, 2H), 3.29 (t, J = 6.9 Hz, 2H), 3.15 (ddd, J = 13.6, 10.6, 3.0 Hz, 2H),2.17 – 2.12 (m, 2H), 2.09 (t, J = 7.3 Hz, 2H), 1.51 (qd, J = 7.6, 2.9 Hz, 4H), 1.44 (ddd, J = 14.2, 10.8, 4.1 Hz, 2H), 1.33 – 1.29 (m, 2H), 1.29 (s, 3H).

[0091] Synthesis of intermediate 26d: For specific procedures and proportions, refer to the preparation of intermediate 26a. 1 H NMR (600 MHz, DMSO- d 6) d 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.35 – 7.33 (m, 2H),7.26 (dd, J = 7.7, 1.6 Hz, 1H), 5.57 (s, 2H), 3.82 (dt, J = 13.5, 4.4 Hz, 2H), 3.24 (t, J = 6.9 Hz, 2H), 3.12 (ddd, J = 13.5, 10.6, 3.0 Hz, 2H), 2.13 (dt, J =14.0, 3.0 Hz, 2H), 2.05 (t, J= 7.3 Hz, 2H), 1.50 – 1.38 (m, 7H), 1.26 (s, 3H), 1.25 – 1.21 (m, 5H).

[0092] Example 17 Synthesis of compound SK21-24:

[0093] Synthesis of compound SK21: Intermediate 24 (73 mg, 0.28 mmol, 1.2 eq), intermediate 26a (100 mg, 0.23 mmol, 1.0 eq), anhydrous copper sulfate (40 mg, 0.26 mmol, 1.1 eq), and sodium ascorbate (137 mg, 0.69 mmol, 3.0 eq) were dissolved in tetrahydrofuran and water (V / V = 1:1) and stirred at room temperature until the starting material was completely consumed. The mixture was extracted with 20 mL of water and 20 mL of ethyl acetate, the organic layers were combined and dried over sodium sulfate, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to give compound SK21. The yield was 27%. 1 H NMR (600 MHz, DMSO- d 6) d 8.14 (s, 1H), 8.01 (s, 1H), 7.58 (dd, J = 8.0, 1.6 Hz, 1H), 7.49 (s, 1H), 7.35(t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 23.3 Hz, 2H), 5.61 (s,2H), 5.10 (d, J = 7.5 Hz, 4H), 4.41 (s, 2H), 3.84 (dt, J = 14.0, 4.3 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 3.20 (t, J = 11.3 Hz, 2H), 2.65 (s, 2H), 2.10 (d, J =13.4 Hz, 2H), 1.84 (s, 2H), 1.52 – 1.46 (m, 2H), 1.30 (s, 3H). 13 C NMR (150MHz, DMSO-d 6) d 165.52, 153.67, 151.22, 142.85, 139.88, 132.58, 132.08, 131.82,131.16, 130.26, 128.77, 126.71, 125.36, 117.14, 114.74, 112.84, 61.66, 55.44,52.53, 52.13, 40.63, 35.18, 26.86, 26.25, 23.80. HRMS (ESI + ): m / z calculated for C 32 H 34 Cl3N9O3[M + H] + , 698.1928, found 698.1927.

[0094] Synthesis of compound SK22: For specific procedures and proportions, refer to the preparation of compound SK21. 1 H NMR (600 MHz, DMSO- d 6) d 8.19 (s, 1H), 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.44 (s,1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.90 – 6.78 (m,2H), 5.58 (s, 2H), 5.07 (s, 2H), 4.34 (t, J = 7.0 Hz, 2H), 3.80 (dt, J = 13.6, 4.5 Hz, 2H), 3.63 (t, J = 6.3 Hz, 2H), 3.15 (ddd, J = 13.4, 10.5, 3.0 Hz, 2H),2.64 (s, 2H), 2.10 (t, J = 7.1 Hz, 4H), 2.02 (q, J = 7.1 Hz, 2H), 1.83 (s, 2H), 1.43 (ddd, J = 14.2, 10.6, 4.0 Hz, 2H), 1.27 (s, 3H).13 C NMR (150 MHz, DMSO- d 6) d 171.68, 165.73, 153.64, 151.18, 143.22, 139.90, 132.57, 132.07, 131.86, 131.16, 130.24, 128.76, 125.21, 124.94, 117.12, 114.77, 112.87, 61.84, 55.43, 51.46, 49.63, 40.68, 35.29, 33.26, 26.84, 26.55, 26.36, 23.79.

[0095] Synthesis of compound SK23: For specific procedures and proportions, refer to the preparation of compound SK21. 1 H NMR (600 MHz, DMSO- d 6) d 8.17 (s, 1H), 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.34 (t, J =7.9 Hz, 1H), 7.33 (s, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.87 (d, J = 11.1 Hz,1H), 6.82 (s, 1H), 5.57 (s, 2H), 5.06 (s, 2H), 4.32 (t, J = 7.0 Hz, 2H), 3.80(dt, J = 13.6, 4.4 Hz, 2H), 3.63 (t, J = 6.3 Hz, 2H), 3.12 (ddd, J = 13.5, 10.6,3.1 Hz, 2H), 2.64 (s, 2H), 2.12 – 2.08 (m, 2H), 2.05 (t, J = 7.3 Hz, 2H), 1.83(s, 2H), 1.79 (q, J = 7.4 Hz, 2H), 1.49 (p, J = 7.4 Hz, 2H), 1.41 (ddd, J= 13.9,10.6, 4.1 Hz, 2H), 1.25 (s, 3H), 1.23 – 1.16 (m, 4H). 13 C NMR (150 MHz, DMSO- d 6) d 172.67, 153.62, 151.19, 143.12, 139.89, 132.57, 132.07, 131.16, 130.23,128.75, 125.19, 124.86, 117.10, 114.78, 112.83, 61.84, 55.44, 51.32, 49.82,40.70, 36.44, 35.29, 30.37, 30.02, 26.84, 26.39, 26.01, 25.40, 23.79. + ): m / z Calculated for C 36 H 42 Cl3N9O3[M + H] + , 754.2554, found 754.2550.

[0096] Synthesis of compound SK24: For specific procedures and proportions, refer to the preparation of compound SK21. 1 H NMR (600 MHz, DMSO- d 6) d 8.16 (s, 1H), 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J =7.8 Hz, 1H), 7.33 (d, J = 1.7 Hz, 1H), 7.26 (dd, J = 7.6, 1.6 Hz, 1H), 6.82 (d, J =7.3 Hz, 2H), 5.58 (s, 2H), 5.07 (s, 2H), 4.30 (t, J = 7.1 Hz, 2H), 3.81 (dt, J =13.4, 4.4 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 3.12 (ddd, J= 13.7, 10.6, 3.1 Hz,2H), 2.64 (s, 2H), 2.12 (d, J = 14.2 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.83 (s,2H), 1.76 (p, J = 7.2 Hz, 3H), 1.44 (dt, J = 14.9, 7.9 Hz, 5H), 1.26 (s, 3H), 1.23 – 1.13 (m, 6H). 13 C NMR (150 MHz, DMSO- d 6) d 172.90, 165.70, 153.67,151.20, 143.13, 139.86, 132.59, 132.08, 131.14, 130.25, 128.75, 125.20,124.86, 117.09, 114.80, 112.88, 61.84, 55.42, 51.28, 49.88, 40.71, 36.65,35.29, 30.21, 28.97, 28.62, 26.85, 26.45, 26.28, 25.92, 23.80. HRMS (ESI + ): m / z Calculated for C 38 H 46 Cl3N9O3[M + H] + , 782.2867, found 782.2861.

[0097] Example 18 Synthesis of intermediate 27:

[0098] SHP099(6) (200 mg, 0.57 mmol, 1.0 eq) was dissolved in acetonitrile, and bromopropyne (58 μL, 0.68 mmol, 1.2 eq), potassium carbonate (236 mg, 1.71 mmol, 3.0 eq), and KI (46 mg, 0.28 mmol, 0.5 eq) were added sequentially. The mixture was stirred at 80 °C for 6 hours. The mixture was extracted with 20 mL of water and 20 mL of ethyl acetate. The organic phases were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 70:1) to give intermediate 27. The yield was 45%.1 HNMR (600 MHz, Chloroform- d ) d 7.59 (s, 1H), 7.47 (dd, J = 7.9, 1.7 Hz, 1H), 7.31(dd, J = 7.7, 1.7 Hz, 1H), 7.29 – 7.24 (m, 2H), 4.21 (s, 2H), 3.62 (dt, J = 10.4, 5.0 Hz, 4H), 3.41 (d, J = 2.5 Hz, 2H), 2.20 (t, J = 2.5 Hz, 1H), 1.68 – 1.58 (m, 4H), 1.19 (s, 3H).

[0099] Example 19 Synthesis of intermediates 29a-b:

[0100] Synthesis of intermediate 29a: Intermediate 11 (100 mg, 0.35 mmol, 1.0 eq) was dissolved in dichloromethane, and EDCI (101 mg, 0.53 mmol, 1.5 eq) and HOBt (57 mg, 0.42 mmol, 1.2 eq) were added sequentially. The mixture was stirred at 0 °C for 15 min. Compound 28a, namely 2-(2-azidoethoxy)ethylamine HCl; 2-(2-azidoethoxy)ethylamine hydrochloride (55 mg, 0.42 mmol, 1.2 eq) and DIPEA (184 μL, 1.06 mmol, 3.0 eq), was added to the above reaction mixture. The resulting reaction system was stirred at room temperature for 8 h. The mixture was extracted with 20 mL of water and 20 mL of dichloromethane. The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 80:1) to obtain intermediate 29a. Synthesis of intermediate 29b: Refer to the preparation of intermediate 29a for specific operations and proportions.

[0101] Example 20 Synthesis of compound SK25-26:

[0102] Synthesis of compound SK25: For specific procedures and proportions, refer to the preparation of compound SK21. 1 H NMR (600 MHz, DMSO- d 6) d8.03 (t, J = 5.8 Hz, 1H), 7.94 (s, 1H), 7.61 (dd, J = 8.0, 1.6 Hz, 1H),7.48 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.6, 1.6 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 5.58 (s, 2H), 4.48 (t, J = 5.3 Hz, 3H), 4.45 (s, 2H), 3.79 (t, J =5.3 Hz, 2H), 3.75 (s, 2H), 3.66 (t, J = 6.3 Hz, 3H), 3.56 (s, 4H), 3.46 (t, J =5.8 Hz, 2H), 3.28 (q, J = 5.8 Hz, 3H), 2.67 (s, 2H), 1.87 (s, 2H), 1.63 (d, J =12.4 Hz, 2H), 1.48 (s, 2H), 1.15 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d 168.25,165.72, 153.67, 151.17, 139.97, 132.57, 132.18, 132.07, 131.16, 130.21,128.75, 125.14, 117.16, 114.90, 112.86, 69.12, 69.04, 67.64, 55.44, 49.79,40.71, 38.57, 37.08, 30.37, 26.83, 23.77. HRMS (ESI + ): m / z calculated forC 36 H 43 Cl3N 10 O4[M + H] + , 785.2613, found 785.2608。

[0103] Synthesis of compound SK26: For specific procedures and proportions, refer to the preparation of compound SK21. 1 H NMR (600 MHz, DMSO- d 6) d 8.04 (t, J = 5.8 Hz, 1H), 7.94 (s, 1H), 7.61 (dd, J = 8.0, 1.6 Hz, 1H),7.48 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.30 (dd, J = 7.6, 1.6 Hz, 1H), 6.79 (d, J = 9.9 Hz, 2H), 5.59 (s, 2H), 4.48 (t, J = 5.3 Hz, 3H), 4.46 (s, 2H), 3.80 (t, J =5.3 Hz, 3H), 3.77 (s, 1H), 3.66 (t, J = 6.3 Hz, 2H), 3.58 (s, 3H), 3.52 (dd, J =6.2, 3.5 Hz, 3H), 3.48 (dd, J = 5.9, 3.5 Hz, 2H), 3.42 (t, J = 5.9 Hz, 2H), 3.28(q, J = 5.9 Hz, 3H), 3.17 (s, 1H), 2.67 (s, 2H), 1.87 (s, 2H), 1.65 (dt, J =10.9, 4.6 Hz, 2H), 1.50 (s, 2H), 1.17 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d168.22, 165.72, 153.69, 151.17, 139.98, 132.57, 132.07, 131.16, 130.20,128.74, 125.09, 123.58, 117.16, 114.89, 112.85, 70.05, 69.93, 69.35, 67.66,55.44, 49.80, 49.13, 40.72, 38.76, 37.17, 29.56, 26.83, 23.76. HRMS (ESI + ): m / z Calculated for C 38 H 47 Cl3N 10 O5[M + H] + , 829.2875, found 829.2868.

[0104] Example 21 CCK8 assay for cell viability: HeLa cells in logarithmic growth phase and in good condition were seeded in 96-well plates at a density of 5000 cells per well. After attachment, the cells were treated with different concentrations of SHP2 PROTACs, DCAF16 ligand KB02 (8), and SHP2 inhibitor SHP099 (6) for 24 h, and cell viability was then assessed using a CCK-8 assay kit. The results showed that most SHP2 PROTACs (except SK18, SK19, SK21, and SK24) exhibited significant cytotoxicity against HeLa cells. Figure 1 Of the compounds tested, SK8 exhibited the strongest antitumor activity in HeLa cells (IC50). 50 =2.317±0.089 μM), its potency is 10 times higher than that of SHP099(6). Subsequently, further research was conducted using SK8 as a representative compound.

[0105] Example 22 In HeLa cells, SK8 induces SHP2 degradation via the ubiquitin-proteasome system: 1. Western blot analysis: 1.1 After collecting the cells, add cell lysis buffer containing protease inhibitors and phosphatase inhibitors, and lyse on ice for 30 min. Centrifuge the lysate at 13,000 rpm for 15 min at 4°C and collect the supernatant containing total protein.

[0106] 1.2 Protein concentration was determined using the BCA protein quantification kit (Beyotime, P0011). The sample was mixed with 5× loading buffer and then denatured at 100°C for 5 min.

[0107] 1.3 Equal amounts of protein samples were separated by electrophoresis using 10% SDS-PAGE (Epizyme, PG112) according to their molecular weight.

[0108] 1.4 After electrophoresis, the proteins were transferred to a PVDF membrane (Merck Millipor, ISEQ00010) and blocked with 5% skim milk powder. The membrane was then incubated overnight at 4°C with primary antibodies (both purchased from ABclonal). The next day, the membrane was washed with TBST for 30 min and then incubated with secondary antibodies at room temperature for 2 h. After washing again, the membrane was placed in high-sensitivity ECL chemiluminescence solution (MeilunBio, MA0186), and protein bands were detected using a chemiluminescence imaging system. Relative quantification of their grayscale intensity was performed using ImageJ software.

[0109] 2.1 HeLa cells were treated with different concentration gradients of SHP2 proteins for 24 h, and the changes in SHP2 protein levels were analyzed by Western blot. The results showed that SK8 cells exhibited the strongest SHP2 degradation activity, capable of degrading SHP2 in a concentration-dependent manner. Figure 2 A, 2D). Meanwhile, the degradation activities of SHP099(6) and KB02(8) on SHP2 were evaluated using the same method. Neither SHP099(6) nor KB02(8) could induce the degradation of SHP2. Figure 2 B, 2C).

[0110] 2.2 HeLa cells were treated with 15 µM SK8, and samples were taken at 3, 6, 12, 24, and 36 h for Western blot analysis. The results showed that SK8-mediated SHP2 degradation gradually increased over time. Figure 2 E).

[0111] 2.3 Pretreatment of HeLa cells with MG132 for 4 h significantly reversed SK8-induced SHP2 degradation ( Figure 2 F). The results showed that SK8 induces SHP2 degradation through the ubiquitin-proteasome system.

[0112] 2.4 Ligand competition experiments were conducted using SHP2 ligand SHP099 (6) and DCAF16 ligand KB02 (8) to compete with SK8 for binding to SHP2 and DCAF16, respectively. The results showed that treatment of HeLa cells with SK8 alone significantly induced SHP2 degradation, but pretreatment with SHP099 (6) or KB02 (8) significantly inhibited the degradation of SHP2 by SK8. Figure 2 G). These findings confirm that SK8 can simultaneously recruit SHP2 and DCAF16, thereby inducing SHP2 to be ubiquitin-labeled and subsequently degraded by the proteasome, making it a true PROTAC degrader.

[0113] Example 23 SK8 inhibits the clonogenic formation of HeLa cells: 1.1 Cells in the logarithmic growth phase were seeded in 6-well plates at 1000 cells per well.

[0114] 1.2 After the cells adhered, 2 mL of complete drug-containing culture medium was added and the cells were cultured in an incubator. The culture medium was replaced with fresh drug-containing culture medium every 2 days.

[0115] 1.3 On day 10, discard the culture medium and rinse the cells with PBS.

[0116] 1.4 Fix cells with 4% paraformaldehyde for 15 min, rinse again with PBS, and stain with crystal violet (Beyotime, C0121) for 30 min.

[0117] 1.5 Wash away excess dye with PBS until the background is clear. After the culture plate dries, take a picture and use ImageJ software to calculate the number of cell clones.

[0118] The results showed that SK8 was more effective than SHP099 in inhibiting the clonal growth of HeLa cells. Figure 3 A).

[0119] Example 24 SK8 induces S-phase arrest in HeLa cells: 1.1 After SK8 treatment for 24 h, HeLa cells were digested with EDTA-free trypsin, centrifuged, and the supernatant was discarded.

[0120] 1.2 Resuspend the cell pellet in ice-cold PBS, centrifuge again to precipitate the cells, and discard the supernatant.

[0121] 1.3 Add 1 mL of PBS to fully resuspend the cells into single cells. Slowly add 3 mL of anhydrous ethanol to the cell suspension until the final concentration is 75%. Fix the cells overnight at 4°C.

[0122] 1.4 Centrifuge the fixed cell suspension, wash with PBS, add propidium iodide (PI) staining solution according to the instructions of the cell cycle detection kit (Meilun Biotechnology, MA0334), incubate for 30 min in the dark, and then analyze the cell cycle distribution by fluorescence detection using flow cytometry.

[0123] The results showed that both SHP099 and SK8 could arrest the cell cycle in the S phase (…). Figure 3 B), and the accumulation of S-phase cells caused by SK8 was more significant, suggesting that the cells may have suffered DNA damage.

[0124] Example 25 SK8 inhibits the migration and invasion of HeLa cells: 1. Scratch test: 1.1 Take out the six-well plate and draw five parallel lines on the bottom of the petri dish with a marker as reference marks for subsequent observation.

[0125] 1.2 Cells in the logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 5 The cells / well were seeded in a six-well plate at a density of cells / well.

[0126] 1.3 After the cells form a monolayer, use a 10 μL pipette tip to make a cut along the parallel line perpendicular to the direction.

[0127] 1.4 After washing away the scraped cells with PBS, serum-free culture medium containing the drug was added. The cells were observed and photographed under an electron microscope at 0, 12, and 24 h.

[0128] 2. Transwell migration and invasion experiments 2.1 Matrigel (Corning, 356234) was thawed overnight at 4°C. Matrigel was diluted with serum-free medium at a ratio of 1:8, and 50 μL was evenly distributed to the bottom of each well. The wells were then incubated for 2 h.

[0129] 2.2 For migration experiments, the Matrigel coating is omitted. Pretreated cells are resuspended in serum-free medium to prepare 2.5 × 10⁻⁶ cells. 5 Cells were seeded in a suspension of 2.5 × 10⁶ cells / mL into the upper chamber of a Transwell (Costar, 3422) at a migration assay density of 2.5 × 10⁶ cells / well. 4 5 × 10 cells per well for invasion assay. 4 Each cell.

[0130] 2.3 Add 600 μL of DMEM medium containing 20% ​​FBS to the lower chamber. Culture the cells for 24 h, remove the chamber, wipe the cells on the upper surface of the membrane with a moistened cotton swab, wash twice with PBS, and fix the cells on the lower surface of the membrane with 4% paraformaldehyde for 30 min.

[0131] 2.4 After staining the cells with crystal violet for 30 min, wash with PBS until the background is clean, and then take pictures under a microscope.

[0132] The results showed that, at the same concentration, SK8 had a stronger inhibitory effect on the migration and invasion of HeLa cells than SHP099(6). Figure 3 C, 3D).

[0133] Example 26 SK8 induces apoptosis in HeLa cells: 1.1 After SK8 treatment for 24 h, HeLa cells were digested with EDTA-free trypsin and centrifuged to collect the cell pellet.

[0134] 1.2 After washing once with PBS, stain the cells according to the manufacturer's instructions for the Annexin V-FITC apoptosis detection kit (Beyotime, C1062L) for 10-15 min.

[0135] 1.3 Flow cytometry was used to detect the cells, and the proportions of Annexin V-positive and PI-positive cells were quantified using FlowJo v10.8.1 software.

[0136] The results showed that SK8 induced HeLa cell apoptosis in a concentration-dependent manner, while SHP099(6) did not significantly increase apoptosis at the same concentration. Figure 3 E). Furthermore, Western blot analysis showed that SK8 treatment increased the protein levels of the apoptosis biomarkers Cleaved Caspase-3 and Cleaved PARP, while SHP099(6) treatment did not show this upregulation. Figure 3 F). The increase in these cleavage products suggests that SK8 triggers the mitochondrial-mediated endogenous apoptosis pathway in HeLa cells.

[0137] Example 27 Preliminary study on the anti-tumor mechanism of SK8: 1. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using the RNA Rapid Extraction Kit (SparkJade, AC0202-B) according to the manufacturer's instructions. cDNA was then synthesized using the cDNA Synthesis Kit (LABLEAD, F0202). ArtiCan was used for analysis. ATM SYBR qPCR Mix (Qingke Biotechnology, DLQ102) was used for real-time quantitative PCR (qRT-PCR). Two... -ΔΔCt The relative mRNA expression level was calculated using a method with GAPDH as an internal control.

[0138] 2. Co-cultivation system 2.1 Mouse spleens were gently ground and filtered through a 70 μm cell sieve. The resulting suspension was centrifuged at 500 g for 5 min, and the supernatant was discarded. Red blood cell lysis buffer (TBD, NH4CL2009) was added, five times the volume of the cell pellet, and incubated at room temperature for 2 min. After lysis, the sample was centrifuged at 500 g for 5 min and washed twice with PBS to obtain a single-cell suspension of mouse spleen.

[0139] 2.2 T cells in spleen cell suspension were analyzed using ActBeads TM Mouse CD3 / CD28 activating magnetic beads (EynosLifeScience, AM2001) were activated according to the manufacturer's instructions. Cells were cultured at 37°C and 5% CO2 for 72 hours before expansion. T cell culture medium formulation: RPMI-1640 medium containing 10% fetal bovine serum supplemented with 10 ng / mL IL-2 (Novoprotein, CK24), 1% penicillin-streptomycin, and 50 μM β-mercaptoethanol (Macklin, M6230).

[0140] 2.3 Harvest 4T1 cells, count them, and seed them into 24-well plates. After cell adhesion, cells were treated with 10 ng / mL IFN-γ (Bioswamp, RPH00476) alone, or with a combination of IFN-γ + SHP099(6) (15 μM) or IFN-γ + SK8 (15 μM). After 12 h of drug treatment, the drug-containing culture medium was discarded, and the cells were washed once with PBS. T cell suspension was then added for co-culture at a ratio of 10:1 (T cells:tumor cells). After 24 h of co-culture, cells from each well were collected, stained with flow cytometry antibodies, and analyzed by flow cytometry. CD8 was assessed using FITC-CD8a (Biolegend, 100705) and APC-CD69 (Biolegend, 104513). +The activation status of T cells. 7-AAD (Biolegend, 420403) was used to distinguish between live and dead cells. CD69 was detected using flow cytometry (Agilent Novocyte). + CD8 + The proportion of T cells was analyzed using Novo Express software.

[0141] 3.1 The effect of SK8-mediated SHP2 degradation on the RAS / MAPK pathway was investigated by analyzing the phosphorylation levels of MAPK pathway-related proteins JNK, p38, and ERK. The results showed that, compared with the vector control group, both SK8 and SHP099(6) treatments significantly downregulated the ratios of p-JNK / JNK, p-p38 / p38, and p-ERK / ERK. Figure 4 A). Furthermore, at the same concentration, SK8 showed better inhibitory effects than SHP099 (6).

[0142] 3.2 Investigating the effect of SHP2 degradation on the PI3K / AKT / mTOR pathway. HeLa cells were treated with different concentrations of SK8 or SHP099(6), and key signaling molecules were analyzed by Western blotting. The results showed that SK8 treatment significantly and in a concentration-dependent manner reduced the phosphorylation level of AKT (Figure 4B). At the same time, the phosphorylation of its downstream effector molecule mTOR also decreased accordingly (Figure 4B). In contrast, the allosteric inhibitor SHP099(6) was less effective than SK8 in inhibiting this pathway.

[0143] 3.3 Investigating the effect of SHP2 degradation on the JAK / STAT1 pathway. HeLa cells or 4T1 cells (SK8's antitumor and SHP2 degradation activity was previously observed in 4T1 cells) were pretreated with 15 μM SK8 or SHP099(6) for 12 h, followed by stimulation with 10 ng / mL IFN-γ for 10 min. STAT1 phosphorylation levels were then analyzed by Western blotting. Results showed that, compared with the vector control group, pretreatment with both SK8 and SHP099(6) significantly enhanced IFN-γ-induced STAT1 phosphorylation levels (…). Figure 4 C), among which SK8 was more effective than SHP099(6). Activation of the JAK / STAT1 pathway promotes the expression of interferon regulatory factor 1 (IRF1), thereby enhancing the transcription of major histocompatibility complex class I (MHC-I) components. qRT-PCR results showed that both SHP099(6) and SK8 treatments upregulated [the expression of IRF1]. IRF1 , HLA and B2M transcription level ( Figure 4 D).

[0144] 3.4 Study on the role of SHP2 inhibition in enhancing tumor immune killing. 4T1 cells were pretreated with IFN-γ alone or in combination with SHP099(6) or SK8 for 12 h, and then co-cultured with mouse spleen immune cells in vitro for 24 h. CD8 was identified by anti-CD69 staining. + T cell activation status. Flow cytometry results showed that, compared with IFN-γ alone, treatment with IFN-γ in combination with SHP099(6) or SK8 significantly increased CD69. + CD8 + The proportion of T cells ( Figure 4 E). It is worth noting that the SK8 combination induces CD8. + T cell activation was most intense. Overall, these data suggest that SK8-mediated SHP2 degradation more effectively enhances the IFN-γ / JAK / STAT1 pathway than SHP099(6) enzyme inhibition, thereby promoting superior CD8 activation. + T cell activation.

[0145] The above description of specific embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Claims

1. A bifunctional molecular compound of general formula (I) based on DCAF16 ligand-induced degradation of SHP2 protein, or a pharmaceutically acceptable salt thereof: in, L can be any of the following structures: Where n is selected from integers between 1 and 10; R is one or two of hydroxyl, cyano, nitro, amino, halogen, hydrogen atom, C1-C6 alkyl, halogenated C1-C6 alkyl, 5-6 aryl or heteroaryl, wherein the aryl or heteroaryl is substituted by one or more substituents, wherein the substituents are one or more of nitro, hydroxyl, halogen, and amino; wherein the heteroaryl contains 1-3 heteroatoms of O, N or S. X is one or two of halogen, hydroxyl, nitro, amino, and C1-C6 alkyl.

2. The bifunctional molecular compound based on DCAF16 ligand-induced SHP2 protein degradation according to claim 1, characterized in that, Choose from any of the following structural formulas: 。 3. A method for preparing the bifunctional molecular compound based on DCAF16 ligand-induced SHP2 protein degradation as described in claim 2, characterized in that, Includes the following steps: Step 1: Starting with 2,3-dichlorophenylboronic acid (1) and 2-amino-3-bromo-6-chloropyrazine (2), intermediate (3) was obtained by Suzuki-Miyaura coupling reaction. Then, intermediate (5) was obtained by nucleophilic substitution reaction with (4-methylpiperidin-4-yl)carbamate tert-butyl ester (4). Finally, the Boc group was removed to obtain SHP099 (6). Step 2: 1,2,3,4-tetrahydroquinoline-6-ol (7) was treated with chloroacetyl chloride to obtain KB02 (8). KB02 (8) was alkylated with tert-butyl 2-bromoacetate (9) to obtain intermediate (10), and then deprotected to obtain intermediate (11). SHP099 (6) was amidated with acids (12a-j, 14) of different chain lengths to obtain the corresponding intermediates (13a-j) and (15). Subsequently, it was deprotected by N- and condensed with intermediate (11) to obtain SK1-SK11. Step 3: Compound (22a-d) undergoes nucleophilic substitution of piperazine-1-carboxylic acid tert-butyl ester (21) to generate intermediate (23a-d), which is then hydrolyzed to obtain intermediate (19a-d); SHP099 (6) undergoes amidation reaction with acids of different chain lengths (16a-d, 19a-d) to obtain the corresponding intermediates (17a-d) and (20a-d); subsequently, after N-deprotection, it is condensed with intermediate (11) to obtain SK12, SK14-SK20; intermediate (17a) is N-deprotected and reacted with intermediate (12b) to produce intermediate (18), which is then N-deprotected and condensed with intermediate (11) to obtain SK13; Step 4: KB02 (8) was etherified with propyne bromide to obtain intermediate (24); SHP099 (6) was reacted with azide 25a-d to generate intermediate 26a-d, and then copper-catalyzed azide-alkyne cycloaddition reaction was carried out with intermediate (24) to obtain compound SK21-SK24; Compound (28a-d) was condensed with intermediate (11) to obtain intermediate (29a-b), and SHP099 (6) was etherified with propyne bromide to obtain intermediate (27), which was then subjected to a copper-catalyzed cycloaddition reaction with intermediate (29a-b) to generate compound SK25-SK26; 。 4. The bifunctional molecular compound based on DCAF16 ligand-induced SHP2 protein degradation according to any one of claims 1-2, characterized in that, The pharmaceutically acceptable salts include their inorganic acid salts, organic acid salts, inorganic base salts, and organic base salts.

5. A pharmaceutical composition, characterized in that, The active ingredient is a bifunctional molecular compound based on DCAF16 ligand-induced SHP2 protein degradation as described in any one of claims 1-2, or a pharmaceutically acceptable salt thereof, combined with one or more pharmaceutically acceptable diluents, excipients, or carriers.

6. The pharmaceutical composition according to claim 5, characterized in that, Dosage forms include oral tablets, injectable preparations, topical preparations, and respiratory preparations; oral preparations include tablets, capsules, granules, powders, oral solutions, suspensions, and pills; injectable preparations include sterile powders for injection, injection solutions, and concentrated solutions for injection; topical preparations include ointments, creams, gels, patches, lotions, and liniments; and respiratory preparations include aerosols, powder inhalers, and sprays.

7. The use of the bifunctional molecular compound based on DCAF16 ligand-induced SHP2 protein degradation as described in any one of claims 1-2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in any one of claims 5-6, in the preparation of a medicament for treating or preventing tumors.

8. The application according to claim 7, characterized in that, The tumors include cervical cancer, lung cancer, breast cancer, colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, leukemia, melanoma, and glioma.

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