Gid4-based protein degradation targeting chimera compound and application thereof

Abstract

The application discloses a kind of protein degradation targeting chimeric compounds based on GID4 or its pharmaceutically acceptable salt, structural general formula is selected from one of the following structures:;The protein degradation targeting chimeric compound based on GID4 prepared in the application adopts new E3 ligase ligand, can obtain high yield, high-purity protein degradation targeting chimeric compound, can effectively degrade various proteins in cell, expands the type of protein degradation targeting chimeric, has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a GID4-based protein degradation-targeting chimeric compound and its application. Background Technology

[0002] Targeted protein degradation (TPD) is an emerging therapeutic approach that has attracted significant attention due to its ability to modulate proteins that are difficult to target with traditional small molecules. Protein degradation-targeting chimeras (PROTACs) utilize the ubiquitin-proteasome system (UPS), a natural intracellular protein degradation system, to achieve targeted degradation of a point of interest (POI). PROTACs are heterobifunctional molecules composed of three parts: a target protein ligand, an E3 ligase ligand, and a linker connecting the two. Intracellularly, one end of the PROTAC molecule binds to the target protein, and the other end binds to the E3 ligase, forming a target protein-PROTACs-E3 ligase ternary complex. This complex brings the target protein and the E3 ligase closer, prompting the E3 ligase to catalyze the transfer of ubiquitin to the target protein. Subsequently, the ubiquitinated target protein is recognized and degraded by the proteasome, and the physiological function of the target protein disappears with its destruction. Due to its catalytic mechanism, PROTACs have shown significant therapeutic potential in areas such as cancer treatment and neurodegenerative diseases.

[0003] Although over 600 E3 ligases have been identified in the human genome, only a few have been successfully used in PROTAC development to date, primarily VHL, CRBN, MDM2, and IAPs. However, E3 ligases exhibit significant differences in tissue distribution, expression levels, and regulatory mechanisms. Furthermore, deletion, mutation, or functional inhibition of VHL or CRBN can lead to drug resistance and off-target toxicity. Therefore, there is an urgent need to expand the library of E3 ligases and their ligands available for PROTAC design. Summary of the Invention

[0004] The purpose of this invention is to provide a GID4-based protein degradation-targeting chimeric compound.

[0005] Another object of the present invention is to provide the application of the GID4-based protein degradation-targeting chimeric compound in the preparation of a medicament for treating tumors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a GID4-based protein degradation-targeting chimeric compound or a pharmaceutically acceptable salt thereof, wherein the general structural formula is selected from one of the following structures:

[0008] ;

[0009] L1 is selected from one of the following structures:

[0010] -NH(CH2)n1NH-, -NH(CH2CH2O)m(CH2)n2NH-, ;

[0011] n1 is selected from positive integers from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

[0012] m is selected from positive integers from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8);

[0013] n2 is selected from positive integers from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6).

[0014] L2 is selected from -(CH2)n3-;

[0015] n3 is selected from positive integers from 1 to 20 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14).

[0016] Most preferably, the structure of the GID4-based protein degradation-targeting chimeric compound is selected from one of the following structures:

[0017] ; ;

[0018] .

[0019] The pharmaceutical salt is an acid addition salt formed by the GID4-based protein degradation targeting chimeric compound and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid.

[0020] In a second aspect, the present invention provides the use of the GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt in the preparation of a medicament for treating tumors.

[0021] The tumor was selected from kidney cancer.

[0022] A third aspect of the present invention provides a pharmaceutical formulation made from the GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt and medically acceptable excipients.

[0023] The pharmaceutical preparation is selected from oral liquids, injections, and tablets.

[0024] The drug formulation can be administered orally or intravenously.

[0025] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0026] The protein degradation-targeting chimeric compounds prepared by this invention utilize a novel E3 ligase ligand, resulting in high-yield and high-purity protein degradation-targeting chimeric compounds that can effectively degrade various intracellular proteins, thus expanding the types of protein degradation-targeting chimeric compounds and showing broad application prospects.

[0027] This invention designed and synthesized 16 GID4-based BRD4 protacid degraders, all of which showed varying degrees of potential to degrade BRD4, a common cancer target. Among the synthesized compounds, compound M-11 can efficiently and selectively degrade BRD4 and DC... 50 =0.21±0.04 μM, Dmax=77±4%. Furthermore, compound M-11 exhibited superior in vitro antiproliferative effects (IC50) compared to JQ1 and GID4 ligands in VHL-native deficient 786-O renal cell carcinoma (RCC). 50 =5.32 μM), and exhibited superior in vitro anti-migration ability compared to the VHL-recruiting PROTAC—MZ1. Furthermore, it achieved significant tumor growth inhibition (TGI=67%) in the 786-O xenograft model, outperforming the corresponding CRBN and VHL-recruiting degraders (dBET1, MZ1), highlighting the broader applicability of the compounds of this invention in overcoming drug resistance caused by E3 ligase deficiency. Simultaneously, this invention designed and synthesized compound M-17, DC, by targeting another oncogenic target, VEGFR2, with GID4-based PROTACs. 50 =0.34±0.06 μM, Dmax=71±7%, IC50 in 786-O cells 50 =3.59 μM, further indicating that the compounds of the present invention are promising as an alternative therapeutic strategy for cancers with E3 ligase mutations or defects. Attached Figure Description

[0028] Figure 1 This is a schematic diagram showing the degradation results of compounds M-3 to M-11.

[0029] Figure 2 This is a schematic diagram showing the degradation results of compounds M-12 to M-16.

[0030] Figure 3 This is a schematic diagram showing the degradation results of compound M-17.

[0031] Figure 4This is a schematic diagram showing the in vitro cytotoxicity test results of compounds M-11 and M-17.

[0032] Figure 5 This is a schematic diagram illustrating the in vitro anti-tumor cell migration effect of compound M-11.

[0033] Figure 6 This is a schematic diagram illustrating the effect of compound M-11 in a mouse xenograft model of renal cell carcinoma.

[0034] Figure 7 This is a schematic diagram illustrating the effects of compound M-11 on major organs in a mouse xenograft model of renal cell carcinoma.

[0035] Figure 8 This is a schematic diagram of the immunohistochemical effects of compound M-11 on tumor tissue in a mouse xenograft model of renal cell carcinoma. Detailed Implementation

[0036] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038]

[0039] 1H-indole-2-carboxaldehyde (7.40 g, 50.98 mmol), glycine methyl ester hydrochloride (16.0 g, 127 mmol), and triethylamine (7.57 mL, 56.5 mmol) were dissolved in methanol (50 mL). Sodium borohydride (3.52 g, 56.1 mmol) was added at 0 °C. The resulting mixture was heated to room temperature and stirred for 12 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and subjected to column chromatography to give compound 1 (7.4 g, 67%).

[0040]

[0041] Compound 1 (3 g, 13.7 mmol) and di-tert-butyl dicarbonate (10.5 g, 48.1 mmol) were dissolved in DCM (60 mL), and DMAP (336 mg, 2.75 mmol) and triethylamine (4.17 g, 41.2 mmol) were added. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated ammonium chloride and saturated sodium chloride solutions, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound 2 (3 g, 52%).

[0042]

[0043] Compound 2 (3 g, 7.17 mmol) was dissolved in a mixture of methanol (30 mL), THF (30 mL), and water (10 mL) at 0 °C, and lithium hydroxide (602 mg, 14.34 mmol) was added. The mixture was stirred at room temperature for 3 hours, and the pH was adjusted to slightly acidic with 1 N HCl. The mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and directly dissolved in DMF (30 mL). Methyl (1s,4s)-4-aminocyclohexane-1-carboxylic acid (1.13 g, 7.17 mmol), HATU (4.09 g, 10.76 mmol), and DIPEA (1.85 g, 14.34 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and purified by column chromatography to give compound 3 (1.56 g, 40%).

[0044]

[0045] Compound 3 (1.56 g, 2.87 mmol) was dissolved in a mixture of methanol (10 mL), THF (10 mL), and water (3.3 mL) at 0 °C, and lithium hydroxide (138 mg, 5.74 mmol) was added. After stirring at room temperature for 3 hours, the pH was adjusted to slightly acidic with 1 N HCl. The mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound 4 (896 mg, 59%).

[0046]

[0047] Example 2

[0048]

[0049] JQ1-COOH (250 mg, 0.62 mmol), tert-butyl(2-aminoethyl)carbamate (99 mg, 0.62 mmol), HATU (354 mg, 0.93 mmol), and DIPEA (160 mg, 1.24 mmol) were dissolved in DMF (15 mL) and stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound 5 (218 mg, 65%).

[0050]

[0051] Example 3

[0052] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (3-aminopropyl) carbamate, yielding compound 6 (67% yield).

[0053]

[0054] Example 4

[0055] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (4-aminobutyl) carbamate, yielding compound 7 (71% yield).

[0056]

[0057] Example 5

[0058] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (5-aminopentyl) carbamate, yielding compound 8 (69% yield).

[0059]

[0060] Example 6

[0061] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (6-aminohexyl) carbamate, yielding compound 9 (60% yield).

[0062]

[0063] Example 7

[0064] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (7-aminoheptyl) carbamate, yielding compound 10 (61% yield).

[0065]

[0066] Example 8

[0067] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (8-aminooctyl) carbamate, yielding compound 11 (62% yield).

[0068]

[0069] Example 9

[0070] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (9-aminononyl) carbamate, yielding compound 12 (62% yield).

[0071]

[0072] Example 10

[0073] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (10-aminodecyl) carbamate to obtain compound 13 (60% yield).

[0074]

[0075] Example 11

[0076] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (11-aminoundecane) carbamate, yielding compound 14 (yield 59%).

[0077]

[0078] Example 12

[0079] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (12-aminododecyl) carbamate, yielding compound 15 (yield 59%).

[0080]

[0081] Example 13

[0082] The difference from Example 2 is that tert-butyl(2-aminoethyl)carbamate was replaced with tert-butyl(2-(2-aminoethoxy)ethyl)carbamate, yielding compound 16 (70% yield).

[0083]

[0084] Example 14

[0085] The difference from Example 2 is that tert-butyl(2-aminoethyl)carbamate was replaced with tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate, yielding compound 17 (65% yield).

[0086]

[0087] Example 15

[0088] The difference from Example 2 is that tert-butyl(2-aminoethyl)carbamate was replaced with tert-butyl(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate, yielding compound 18 (63% yield).

[0089]

[0090] Example 16

[0091] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl (14-amino-3,6,9,12-tetraoxotetradecyl) carbamate, to obtain compound 19 (60% yield).

[0092]

[0093] Example 17

[0094] The difference from Example 2 is that tert-butyl (2-aminoethyl) carbamate was replaced with tert-butyl 4-(piperidine-4-methylene)piperazine-1-carboxylate, yielding compound 20 (66% yield).

[0095]

[0096] Example 18

[0097]

[0098] Compound 5 (200 mg, 0.37 mmol) was dissolved in DCM (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. After reacting for 1 hour, the mixture was evaporated to dryness using a water pump, and the pH was adjusted to slightly alkaline with the addition of DIPEA. Subsequently, it was dissolved in DMF (10 mL), and compound 4 (196 mg, 0.37 mmol), HATU (209 mg, 0.55 mmol), and DIPEA (96 mg, 0.74 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and dissolved again in DCM (4 mL). Trifluoroacetic acid (2 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to dryness, extracted with ethyl acetate, washed with saturated sodium bicarbonate solution and sodium chloride solution, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound M-1 (67 mg, 25%).

[0099]

[0100] Example 19

[0101] The difference from Example 18 is that compound 5 was replaced with compound 6 to obtain compound M-2 (74 mg, yield 26%).

[0102]

[0103] Example 20

[0104] The difference from Example 18 is that compound 5 was replaced with compound 7 to obtain compound M-3 (64 mg, yield 22%).

[0105]

[0106] Example 21

[0107] The difference from Example 18 is that compound 5 was replaced with compound 8 to obtain compound M-4 (65 mg, yield 22%).

[0108]

[0109] Example 22

[0110] The difference from Example 18 is that compound 5 was replaced with compound 9 to obtain compound M-5 (57 mg, yield 19%).

[0111]

[0112] Example 23

[0113] The difference from Example 18 is that compound 5 was replaced with compound 10 to obtain compound M-6 (52 mg, yield 17%).

[0114]

[0115] Example 24

[0116] The difference from Example 18 is that compound 5 was replaced with compound 11 to obtain compound M-7 (46 mg, yield 15%).

[0117]

[0118] Example 25

[0119] The difference from Example 18 is that compound 5 was replaced with compound 12 to obtain compound M-8 (41 mg, yield 13%).

[0120]

[0121] Example 26

[0122] The difference from Example 18 is that compound 5 was replaced with compound 13 to obtain compound M-9 (32 mg, yield 10%).

[0123]

[0124] Example 27

[0125] The difference from Example 18 is that compound 5 was replaced with compound 14 to obtain compound M-10 (33 mg, yield 10%).

[0126]

[0127] Example 28

[0128] The difference from Example 18 is that compound 5 was replaced with compound 15 to obtain compound M-11 (33 mg, yield 10%).

[0129]

[0130] Example 29

[0131] The difference from Example 18 is that compound 5 was replaced with compound 16 to obtain compound M-12 (47 mg, yield 16%).

[0132]

[0133] Example 30

[0134] The difference from Example 18 is that compound 5 was replaced with compound 17 to obtain compound M-13 (50 mg, yield 16%).

[0135]

[0136] Example 31

[0137] The difference from Example 18 is that compound 5 was replaced with compound 18 to obtain compound M-14 (43 mg, yield 13%).

[0138]

[0139] Example 32

[0140] The difference from Example 18 is that compound 5 was replaced with compound 19 to obtain compound M-15 (34 mg, yield 10%).

[0141]

[0142] Example 33

[0143] The difference from Example 18 is that compound 5 was replaced with compound 20 to obtain compound M-16 (45 mg, yield 14%).

[0144]

[0145] Example 34

[0146]

[0147] Compound 21 (250 mg, 0.83 mmol), tert-butyl (12-aminododecyl)carbamate (250 mg, 0.83 mmol), HATU (3475 mg, 1.25 mmol), and DIPEA (163 mg, 1.25 mmol) were dissolved in DMF (15 mL) and stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound 22 (141 mg, 29%).

[0148]

[0149] Compound 22 (141 mg, 0.24 mmol) was dissolved in DCM (4 mL), and trifluoroacetic acid (2 mL) was added dropwise. After reacting for 1 hour, the solution was evaporated to dryness using a water pump, and the pH was adjusted to slightly alkaline with DIPEA. Subsequently, the solution was dissolved in DMF (10 mL), and compound 4 (127 mg, 0.24 mmol), HATU (137 mg, 0.36 mmol), and DIPEA (47 mg, 0.36 mmol) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over sodium sulfate, concentrated, and dissolved again in DCM (4 mL). Trifluoroacetic acid (2 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to dryness, extracted with ethyl acetate, washed with saturated sodium bicarbonate and sodium chloride solutions, dried over sodium sulfate, concentrated, and then subjected to column chromatography to give compound M-17 (29 mg, 15%).

[0150]

[0151] Application Example 1

[0152] The degradation effects of compounds M-3 to M-17 on target proteins were detected by immunoblotting.

[0153] The detection method is as follows: Renal cell carcinoma 786-O cells were treated with DMSO or different concentrations of compounds for 24 hours. BRD4 protein and internal control β-Tubulin were detected by Western blotting. The detection results for compounds M-3 to M-11 are shown below. Figure 1 As shown, Figure 1 This diagram illustrates the degradation results of compounds M-3 to M-11. As shown, compounds M-3 to M-11 can all degrade the oncogenic target BRD4 in renal cell carcinoma cells 786-O to varying degrees. Compound M-11 exhibits the best degradation activity, demonstrating highly efficient and selective degradation of BRD4 and DC. 50 =0.21±0.04 μM, Dmax=77±4%.

[0154] The detection results of compounds M-12 to M-16 are as follows: Figure 2 As shown, Figure 2 This is a schematic diagram showing the degradation results of compounds M-12 to M-16. As can be seen from the figure, compounds M-12 to M-16 can all degrade the oncogenic target BRD4 in renal cell carcinoma cells 786-O to varying degrees.

[0155] The detection results of compound M-17 are as follows Figure 3 As shown, Figure 3 This is a schematic diagram showing the degradation results of compound M-17. As can be seen from the figure, compound M-17 can efficiently and selectively degrade the oncogenic target VEGFR2 and DC in renal cell carcinoma cells 786-O. 50 =0.34±0.06 μM, Dmax=71±7%.

[0156] from Figures 1 to 3 The results show that the compounds prepared in this invention can effectively degrade BRD4 and VEGFR2 proteins. Further research can be conducted on the degradation of other proteins, including but not limited to EGFR, WDR5, and SMARCA2 proteins. The compounds prepared in this invention have broad application prospects in the field of protein degradation.

[0157] Application Example 2

[0158] The killing effects of compounds M-11 and M-17 on tumor cells were detected by in vitro cytotoxicity assays.

[0159] The detection method is as follows: Renal cell carcinoma 786-O cells (3000 cells / well) were seeded in 96-well plates. After culturing for 24 hours, the cells were treated with different concentrations of the compound for 3 days. Cell viability was detected using 10% CCK8 reagent. The results are shown below. Figure 4 As shown, Figure 4This is a schematic diagram showing the in vitro cytotoxicity assay results for compounds M-11 and M-17. As can be seen from the figure, in 786-O renal cell carcinoma (RCC), compared to JQ1 (IC... 50 =24.96 μM) and GID4 ligand (IC) 50 =99.39 μM), compounds M-11 and M-17 exhibited superior in vitro antitumor proliferation effects, IC50 = 99.39 μM. 50 The values ​​were 5.32 μM and 3.59 μM, respectively.

[0160] The structures of JQ1 and GID4 ligands are shown below:

[0161] .

[0162] Application Example 3

[0163] Compound M-11 was tested for its ability to inhibit tumor cell migration in vitro using scratch assays and transwell assays.

[0164] The detection method is as follows:

[0165] Will contain 5×10 6 Serum-free RPMI-1640 medium was added to six-well plates for 786-O cells. After the cell density reached over 90%, scratching was performed, and the cells were observed and photographed using a fluorescence microscope. After incubation with a suitable compound for 12 and 24 hours, the cells were observed and photographed again using a fluorescence microscope. The results are as follows: Figure 5 As shown, Figure 5 This diagram illustrates the in vitro anti-tumor cell migration effect of compound M-11. In the diagram, A represents the cell scratch assay.

[0166] 200 μL containing 1.2 × 10 5 Serum-free RPMI-1640 medium for 786-O cells was added to the upper chamber of a Transwell plate, and 600 μL of medium containing 20% ​​serum was added to the lower chamber. After incubation with a suitable compound for 24 hours, the medium was discarded, the cells were washed with PBS, and then fixed with 4% paraformaldehyde for 10 minutes. The paraformaldehyde was discarded, the cells were washed again with PBS, and stained with 0.1% crystal violet for 5 minutes. The cells were then washed three times with PBS, and the cells in the upper chamber were gently wiped away with a cotton swab. Finally, the cells were observed and photographed using a fluorescence microscope. The results are as follows: Figure 5 As shown in B, B is a schematic diagram of the cell transwell experiment. (From...) Figure 5It is known that in VHL-deficient 786-O renal cell carcinoma (RCC), compared with VHL recruiting PROTAC-MZ1, the compound M-11 of the present invention has a more significant ability to inhibit the lateral and longitudinal migration of renal cell carcinoma cells, indicating that the compound of the present invention has a wider applicability in overcoming drug resistance caused by E3 ligase deficiency.

[0167] Application Example 4

[0168] The in vivo antitumor activity of compound M-11 was detected in a 786-O renal cell carcinoma xenograft model constructed in female BALB / c nude mice.

[0169] The detection method is as follows: 1×10 7 786-O cells were subcutaneously injected into the axillary region of 5-week-old female BALB / c nude mice. When the tumor volume reached approximately 150 mm... 3 The compound was administered via intraperitoneal injection, once every other day, for 14 days (7 injections in total). Tumor growth was monitored by measuring tumor volume and body weight every other day, and the test results were as follows: Figure 6 As shown, Figure 6 This diagram illustrates the effect of compound M-11 in a mouse xenograft model of renal cell carcinoma. A shows the in vivo study design, B shows the endpoint anatomical tumor control, C shows the tumor growth curve and endpoint tumor weight, and D shows the weight change during treatment. As shown in the figure, compound M-11 exhibits superior tumor treatment efficacy compared to the corresponding CRBN and VHL recruiting degraders (dBET1, MZ1). It achieved tumor growth inhibition rates (TGI) of 54% and 67% at doses of 15 mg / kg and 30 mg / kg, respectively, significantly better than dBET1 (TGI=33%) and MZ1 (TGI=30%). Furthermore, compared to MZ1 and dBET1, compound M-11 had minimal impact on mouse weight throughout the experiment, indicating better safety profiles. In conclusion, compound M-11 of this invention can overcome the limitations of traditional PROTACs in the treatment of 786-O renal cell carcinoma (RCC).

[0170] The structures of dBET1 and MZ1 are shown below:

[0171] .

[0172] Histopathological analysis was performed on mice treated with compound M-11.

[0173] The detection methods are as follows: After the administration of the drug, major organs (heart, liver, spleen, lung, and kidney) were collected for histopathological analysis using hematoxylin-eosin (H&E) staining. The results are as follows. Figure 7 As shown, Figure 7 This diagram illustrates the effects of compound M-11 on major organs in a mouse model of renal cell carcinoma xenograft. As shown in the figure, all examined organs from mice treated with compound M-11 exhibited normal tissue structure and no obvious pathological changes, indicating that compound M-11 possesses good in vivo tolerability.

[0174] Histological and immunohistochemical analyses were performed on the tumor tissue, and the results were as follows: Figure 8 As shown. Figure 8 This diagram illustrates the immunohistochemical effects of compound M-11 on tumor tissue in a mouse xenograft model of renal cell carcinoma. Figure A shows H&E staining and Ki67 immunohistochemistry, while figure B shows BRD4 immunofluorescence staining in tumor tissue. As shown, the number of Ki67-positive proliferating cells in the tumors of mice treated with compound M-11 was significantly reduced, indicating that compound M-11 effectively inhibits tumor cell proliferation in vivo. Immunofluorescence staining further confirmed that compound M-11 significantly induced the degradation of the oncogenic target BRD4 protein in tumor tissue. In conclusion, compound M-11 of this invention exhibits good therapeutic effects on renal cell carcinoma.

[0175] The results of this invention demonstrate that the compounds prepared in this invention can effectively degrade BRD4 and VEGFR2 proteins. In 786-O renal cell carcinoma (RCC) with a natural VHL deficiency, the compounds exhibit superior in vitro antitumor activity compared to JQI and GID4 ligands, and superior in vitro anti-lateral and longitudinal migration ability of renal cell carcinoma cells compared to MZ1. Furthermore, in a 786-O renal cell carcinoma xenograft model constructed in female BALB / c nude mice, tumor growth inhibition superior to the corresponding CRBN and VHL recruiting degraders (dBET1, MZ1) was achieved, overcoming the limitations of traditional PROTACs in the treatment of 786-O renal cell carcinoma (RCC), demonstrating excellent in vivo antitumor efficacy and good in vivo tolerability. Further research can be conducted on the degradation of other proteins, including but not limited to EGFR, WDR5, and SMARCA2 proteins. In summary, the compounds of this invention have broader applicability in overcoming drug resistance caused by E3 ligase deficiency and are expected to become an alternative therapeutic strategy for cancers with E3 ligase mutations or defects.

[0176] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt, characterized in that, The general structural formula is selected from one of the following structures: ； L1 is selected from one of the following structures: -NH(CH2)n1NH-, -NH(CH2CH2O)m(CH2)n2NH-, ; n1 is selected from positive integers from 1 to 20; m is selected from positive integers from 1 to 8; n2 is selected from positive integers from 1 to 6; L2 is selected from -(CH2)n3-; n3 is selected from positive integers from 1 to 20.

2. The GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt according to claim 1, characterized in that, n1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

3. The GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt according to claim 1, characterized in that, m is selected from 1, 2, 3, 4, 5, 6, 7, 8.

4. The GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt according to claim 1, characterized in that, n2 is selected from 1, 2, 3, 4, 5, 6.

5. The GID4-based protein degradation-targeting chimeric compound or its pharmaceutical salt according to claim 1, characterized in that, The structure of the GID4-based protein degradation-targeting chimeric compound is selected from one of the following structures: ； ； 。 6. The use of a GID4-based protein degradation-targeting chimeric compound or a pharmaceutical salt thereof as described in any one of claims 1 to 5 in the preparation of a medicament for treating tumors.

7. The application according to claim 6, characterized in that, The tumor was selected from kidney cancer.

8. A pharmaceutical preparation, characterized in that, It is made from the GID4-based protein degradation targeting chimeric compound as described in any one of claims 1 to 5, or its pharmaceutical salt and medically acceptable excipients.

9. The pharmaceutical preparation according to claim 8, characterized in that, The pharmaceutical preparation is selected from oral liquids, injections, and tablets.

10. The pharmaceutical preparation according to claim 8, characterized in that, The drug formulation can be administered orally or intravenously.

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