Thymidylate synthase targeting PROTAC molecule and application thereof

Abstract

The invention provides preparation and application of a PROTAC molecule for targeted degradation of thymidylate synthetase, and relates to the field of medicine. The invention relates to a novel folic acid PROTAC molecule which is prepared based on a pyrrolo [2, 3-d] pyrimidine multi-target folic acid antagonist by utilizing connection chains with different lengths and types to be connected with an E3 ligase Cerebron (CRBN) ligand, and an application of the novel folic acid PROTAC molecule in folic acid related diseases such as cancers. The compound has a structure as shown in a general formula I,

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the preparation and application of a PROTAC molecule that targets thymidine synthase. Background Technology

[0002] Cancer remains a stubborn disease that seriously threatens human health. Tumor cells have the characteristic of unlimited proliferation, a process that requires large amounts of DNA as raw materials for synthesis. Therefore, inhibiting key enzymes in the DNA synthesis pathway can effectively block the growth process of tumor cells. Folic acid antagonists are an important class of drugs in clinical cancer treatment. Their core function is to specifically inhibit the enzymes required for DNA synthesis. For example, methotrexate (MTX) targets and inhibits dihydrofolate reductase (DHFR), while pemetrexed (PMX) specifically inhibits thymidylate synthase (TS). However, these drugs generally face the challenge of drug resistance in clinical application, a problem that significantly limits their therapeutic efficacy and clinical application.

[0003] Proteolysis-targeting chimeras (PROTACs), with their unique mechanism of action, have become a research hotspot and important technological direction in the field of cancer treatment, providing a new path to overcome the limitations of traditional drugs. The concept was first proposed in 2001 by Professor Crews and his team at Yale University. Its core principle is to leverage the body's inherent ubiquitin-proteasome degradation pathway to exert therapeutic effects by downregulating target protein levels. This differs from the traditional drug mechanism of merely inhibiting protein function, giving it an irreplaceable advantage. The PROTAC molecule consists of three core domains: a "warhead" structure that specifically binds to the target protein, a ligand that binds to the E3 ubiquitin ligase, and a linker connecting the two domains. Its specific mechanism of action is as follows: one end of the PROTAC molecule binds to the E3 ubiquitin ligase, and the other end binds to the target protein. The ligase shortens the spatial distance between the two, thereby inducing the E3 ubiquitin ligase to modify the ubiquitination tag for the target protein. The ubiquitinated target protein is recognized and degraded by the proteasome, while the PROTAC molecule itself can be recycled and participate in the next round of ubiquitination and target protein degradation.

[0004] Given these advantages, PROTAC technology has demonstrated enormous application potential in overcoming tumor drug resistance and improving the specificity of targeted therapy. It has attracted major pharmaceutical companies worldwide to actively invest in related research, and has driven multiple PROTAC molecules into clinical trials. According to the PROTAC online database (PROTAC DB), 6111 PROTAC molecules have been developed, covering 442 targets, including androgen receptor (AR), estrogen receptor (ER), Bruton's tyrosine kinase (BTK), anaplastic lymphoma kinase (ALK), fusion protein BCR-ABL, bromide domain protein 4 (BRD4), mitogen-activated protein kinase 1 (MEK1), cyclin-dependent kinase 4 (CDK4), and epidermal growth factor receptor (EGFR), among others. However, it is worth noting that PROTAC molecules related to the targets of folic acid antagonists are extremely scarce, with only one related report to date—a study published by Rai Ganesha et al. in 2024, in which a PROTAC molecule based on methotrexate was constructed and used as a DHFR-specific chemical probe; while there are currently no reports on PROTAC molecules targeting TS, indicating a significant research gap in this field.

[0005] Pyrrolo[2,3-d]pyrimidine multi-target folate antagonists, with their unique structural advantages, possess excellent tumor targeting properties, making them an ideal basis for constructing TS-targeting PROTAC molecules. These compounds are specifically transported into tumor cells via the highly expressed folate receptor (FR) and proton-coupled folate transporters (PCFTs), with minimal entry into normal cells, thus significantly reducing toxicity to normal tissues. Furthermore, after entering tumor cells, these compounds simultaneously inhibit the activities of TS, glycine ribonucleotide formoyltransferase (GARFTase), and 5-aminoimidazole-4-carboxamide ribonucleotide formoyltransferase (AICARFTase), blocking the synthesis of thymine nucleotides and purine nucleotides required for DNA synthesis through multiple pathways, thereby leading to impaired DNA synthesis in tumor cells and ultimately inducing tumor cell apoptosis. In addition, these compounds have shown significant cell proliferation inhibitory activity in various tumor cell lines, including KB, MCF7, and SW620.

[0006] Based on the aforementioned research foundation and clinical needs, this invention utilizes a pyrrolo[2,3-d]pyrimidine multi-target folic acid antagonist as its core to construct a PROTAC molecule targeting TS, providing a novel mechanism of action for the treatment of TS-mediated cancers. This PROTAC molecule exerts its therapeutic effect by targeting and degrading TS target proteins, rather than simply inhibiting their enzymatic activity. It is expected to overcome the drug resistance problem of traditional folic acid antagonists at its root, providing new strategies and technological directions for tumor treatment. Summary of the Invention

[0007] This invention relates to a novel folic acid-based PROTAC molecule prepared by linking a pyrrolo[2,3-d]pyrimidine multi-target folic acid antagonist with an E3 ligase Cereblon (CRBN) ligand using linker chains of different lengths and types, and its application in folic acid-related diseases such as cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a PROTAC degrading agent targeting TS, having the structure shown in general formula I below:

[0010]

[0011] In Formula I, E3 ligand is a protein ligand in the ubiquitin ligase complex, specifically a Cereblon (CRBN) ligand.

[0012] Preferably, the CRBN ligand has the following structure:

[0013]

[0014] In Formula I, Linker is the linking group between the target protein ligand and the E3 ligase ligand, representing -alkylene, -alkoxy, -piperazinyl, -piperidinyl, or -1,2,3-triazolyl, wherein -alkylene, -alkoxy, -piperazinyl, -piperidinyl, or -1,2,3-triazolyl is selected from any one or more combinations of the following groups, where n represents a natural number from 1 to 15.

[0015]

[0016] In Formula I, X is one of -CONH- or -CH2SCH2-, and R is one of H, methyl, ethyl, isopropyl, or tert-butyl.

[0017] Preferably, the compound is a compound as shown below or a stereoisomer, geometric isomer, tautomer, hydrate, pharmaceutically or physiologically acceptable salt, or prodrug:

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] The pharmaceutically or physiologically acceptable salts referred to in this invention are salts formed by the PROTAC chimera referred to in this invention and a pharmaceutically or physiologically acceptable acid or base.

[0027] The present invention also proposes a pharmaceutical composition comprising a PROTAC compound targeting TS or a stereoisomer, geometric isomer, tautomer, hydrate, pharmaceutically or physiologically acceptable salt or prodrug thereof.

[0028] The pharmaceutical composition further includes pharmaceutically acceptable carriers, excipients, diluents, excipients, mediators, or combinations thereof.

[0029] The pharmaceutical composition is an injection or an oral preparation.

[0030] The pharmaceutical composition further includes other drugs that have therapeutic or preventative effects on tumors.

[0031] The present invention also provides the use of a PROTAC chimera targeting TS or a pharmaceutical composition comprising the chimera.

[0032] The aforementioned PROTAC chimera targeting TS or a pharmaceutical composition containing the chimera is used in the treatment of folic acid-related diseases.

[0033] Preferably, the use of the PROTAC chimera targeting TS or a pharmaceutical composition containing the chimera in antitumor activity.

[0034] Preferably, the tumor is gastric cancer, breast cancer, lung cancer, gastric cancer, colonic adenocarcinoma, pancreatic cancer, bladder cancer, hepatocellular carcinoma, or cervical cancer. Further, the tumor is a tumor with high FR expression.

[0035] This invention also proposes a synthetic route for the PROTAC chimera targeting TS, as shown in Formula I, specifically including the following steps:

[0036] The compounds represented by general formula I are synthesized by linking thalidomide or pomalidomide with a pyrrolo[2,3-d]pyrimidine multi-target folic acid antagonist via a Click reaction, amide condensation, or hydrolysis reaction. The pyrrolo[2,3-d]pyrimidine multi-target folic acid antagonist is constructed from protected glutamate with either pyrrolo[2,3-d]pyrimidine acetylglycine or pyrrolo[2,3-d]pyrimidine ethioalanine. The amide condensation reaction uses the condensing agents HATU and TCFH.

[0037] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0038] This invention presents a novel and diverse chimeric structure for targeting and degrading TS proteins, based on a pyrrolo[2,3-d]pyrimidine multi-target folic acid antagonist and the E3 ubiquitin ligand CRBN. Western blot experiments confirmed that this protein-targeting degradation chimeric structure can bind to TS proteins and induce effective degradation, thereby triggering apoptosis. Attached Figure Description

[0039] Figure 1 Synthetic route for Gly-C1~C14 chimeras with TS degradation activity:

[0040] Figure 2 Synthetic route for Gly-C15~C20 chimera with TS degradation activity:

[0041] Figure 3 Synthetic route for Gly-C21~C24 chimeras with TS degradation activity:

[0042] Figure 4 Synthetic route for Gly-C25~C34 chimeric compound with TS degradation activity:

[0043] Figure 5 Synthetic route for Gly-C35~C38 chimeras with TS degradation activity:

[0044] Figure 6 Synthetic route for Gly-C39~C46, a chimeric compound with TS degradation activity. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments, but these embodiments do not limit the scope of the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0046] Example 1: The synthetic routes for Gly-C1 and Gly-C2 are shown below. Figure 1 As shown.

[0047] Dissolve S0 (1.5 g, 5.4 mmol), tert-butyl (4-aminobutyl)carbamate (1.65 g, 8.2 mmol), and DIPEA (1.9 ml, 1.09 mmol) in DMF (20 mL). Under N2 protection, heat to 90 °C and stir until the reactants have reacted completely. Add water (60 mL) and ethyl acetate (20 mL), separate the organic layer, extract the aqueous phase with ethyl acetate, combine the organic phases, wash with saturated sodium chloride aqueous solution, dry to anhydrous sodium sulfate, filter, and concentrate. Purify the crude product by column chromatography to give a green oily liquid S1800 mg, with a yield of 67.8%.

[0048] S1 (800 mg, 1.71 mmol), TFA (4.00 mL, 52 mmol), and DCM (10 mL) were added sequentially to a 50 mL round-bottom flask. The mixture was reacted at room temperature for 2 h. The solvent was removed by vacuum evaporation to obtain a pale green oily substance S2 (580 mg), with a yield of 95.2%. This substance was used directly in the next reaction without purification.

[0049] Fluorenylmethoxycarbonyl-L-glutamic acid-1-tert-butyl ester (610 mg, 1.43 mmol), HATU (540 mg, 1.43 mmol), DIPEA (560 mg, 5.20 mmol), and DMF (6.00 mL) were added to a 50 mL reaction flask. The mixture was reacted at 0 °C for 30 min, then S2 (550 mg) was added, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction mixture was concentrated, and water (20 mL) and ethyl acetate (7 mL) were added. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give 889 mg of green oily liquid S3, with a yield of 73.3%.

[0050] S3 (889 mg, 1.18 mmol), DEA (3.61 mL, 34.8 mmol), and DCM (10.0 mL) were added to a 50 mL flask and reacted at room temperature for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography to give 529 mg of white solid S4, with a yield of 84.5%.

[0051] S12 (44 mg, 0.19 mmol), HATU (57 mg, 0.17 mmol), DIPEA (39 mg, 0.34 mmol), and DMF (3 mL) were added to a 10 mL reaction flask and reacted at 0 °C for 30 min. S4 (80 mg, 0.17 mmol) was then added, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography to obtain a yellow solid Gly-C1 weighing 38 mg, with a yield of 32.5%.

[0052] NMR data for compound Gly-C1:

[0053] 1 H NMR (600 MHz, DMSO-d6): δ 11.09 (s, 1H), 10.79 (s, 1H), 10.17 (s,1H), 8.21 (d, J = 7.5 Hz, 1H), 8.04 (s, 1H), 7.82 (s, 1H), 7.56 (t, J = 8.0Hz, 1H), 7.05 (dd, J = 44.5, 7.9 Hz, 2H), 6.54 (s, 1H), 6.01 (d, J = 14.1 Hz, 3H), 5.10-4.97 (m, 1H), 4.17-4.03 (m, 1H), 3.87-3.63 (m, 3H), 3.42 (s, 2H),3.06 (s, 2H), 2.88 (t, J = 15.7 Hz, 1H), 2.58 (d, J = 18.7 Hz, 1H), 2.12 (t,J = 8.0 Hz, 2H), 2.07-1.98 (m, 1H), 1.96-1.87 (m, 1H), 1.82-1.70 (m, 1H), 1.54 (s, 2H), 1.45 (s, 3H), 1.38 (s, 9H).

[0054] S5 (38 mg, 0.049 mmol), TFA (0.44 mL, 5.88 mmol), and DCM (3 mL) were added to a 10 mL round-bottom flask and reacted at room temperature for 12 h. The mixture was then concentrated. After preparative chromatographic purification, a pale yellow solid, Gly-C6, weighing 10 mg, was obtained, with a yield of 30%.

[0055] NMR data for compound Gly-C2:

[0056] 1H NMR (600 MHz, DMSO-d6): δ 8.32-8.26 (m, 1H), 7.90-7.84 (m, 1H), 7.62-7.54 (m, 2H), 7.08 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 6.54(t, J = 6.1 Hz, 1H), 6.37-6.28 (m, 2H), 6.01 (s, 1H), 5.04 (dd, J = 12.8, 5.5Hz, 1H), 3.85 (q, J = 6.9 Hz, 1H), 3.71-3.65 (m, 2H), 3.45 (s, 2H), 3.28 (q,J = 6.8 Hz, 2H), 3.04 (q, J = 6.6 Hz, 2H), 2.60-2.93 (m, 1H), 2.63-2.51 (m,2H), 2.01 -2.25(m, 3H), 1.93-1.87 (m, 1H), 1.74-1.69 (m, 1H), 1.54 (p, J =7.2 Hz, 2H), 1.44 (p, J = 7.1 Hz, 2H).

[0057] Example 2: Synthetic routes of Gly-C15 and Gly-C16:

[0058] In a 100 mL round-bottom flask, S5 (2.00 g, 9.56 mmol), 2-(2-chloroethoxy)ethanol (2.38 g, 19.12 mmol), potassium carbonate (3.97 g, 28.68 mmol), potassium iodide (0.32 g, 1.91 mmol), and DMF (20 mL) were added sequentially, and the mixture was reacted at 90 °C for 12 h. After cooling, the solvent was removed by vacuum distillation, and ethyl acetate (10 mL) and water (10 mL) were added. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic layers were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give S6, a white solid weighing 2.28 g, in 80.5% yield.

[0059] Compound S6 (2.27 g, 7.63 mmol), DMAP (0.186 g, 1.526 mmol), TEA (1.54 g, 15.26 mmol), and DCM (20 mL) were added sequentially to a 100 mL p-bell-shaped flask. The mixture was stirred at 0 °C for 30 min, and then p-toluenesulfonyl chloride (1.16 g, 11.45 mmol) was added. The mixture was stirred at room temperature for 12 h. The reaction solution was diluted with 60 mL of water, the organic layer was separated, extracted with DCM, and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S7, a colorless oily liquid, 2.62 g, in a yield of 76.5%.

[0060] S7 (2.62 g, 5.80 mmol), K2CO3 (1.20 g, 8.70 mmol), 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (3.18 g, 11.6 mmol), and DMF (26 mL) were added sequentially to a 100 mL round-bottom flask. The mixture was heated to 40 °C and stirred for 12 h. After cooling, the solvent was removed by vacuum distillation. Ethyl acetate (15 mL) and water (15 mL) were added, and the organic layer was separated. The aqueous phase was extracted with ethyl acetate, and the organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give S8, a white solid of 1.44 g, in a yield of 45.8%.

[0061] S8 (1.44 g, 2.61 mmol), TFA (6.00 mL, 78.30 mmol), and DCM (15 mL) were added to a 50 mL round-bottom flask and reacted at room temperature for 2 h. The mixture was then concentrated. Water (30 mL) and DCM (10 mL) were added, and the pH was adjusted to 7-8 by adding saturated NaHCO3 aqueous solution. After adjustment, the organic layer was separated, and the aqueous phase was extracted with DCM. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. S9 was obtained as a white solid, 1.09 g, with a yield of 93.2%. This was used directly in the next reaction.

[0062] Fluorenylmethoxycarbonyl-L-glutamic acid-1-tert-butyl ester (0.74 g, 1.73 mmol), HATU (0.66 g, 1.73 mmol), DIPEA (0.67 g, 5.19 mmol), and DMF (8 mL) were added to a 50 mL reaction flask. The mixture was reacted at 0 °C for 30 min. S9 (1.18 g, 2.60 mmol) was added, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction mixture was concentrated, and water (30 mL) and ethyl acetate (10 mL) were added. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give S10, a white solid, 1.00 g, with a yield of 67.8%.

[0063] Compound S10 (1.00 g, 1.16 mmol), DEA (3.61 mL, 34.8 mmol), and DMF (10.0 mL) were added to a 50 mL round-bottom flask. The mixture was reacted at room temperature for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography. S11 was obtained as a white solid with a yield of 70.5%.

[0064] S12 (0.22 g, 0.81 mmol), HATU (0.31 g, 0.81 mmol), DIPEA (0.21 g, 1.62 mmol), and DMF (3 mL) were added to a 50 mL reaction flask and reacted at 0 °C for 30 min. S11 (0.78 g, 1.22 mmol) was then added and stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography to obtain Gly-C15, a white solid, in a yield of 0.34 g (32.5%).

[0065] NMR data for Gly-C15:

[0066] 1H NMR (600 MHz, DMSO-d6): δ 11.10 (s, 1H), 10.78 (d, J = 2.3 Hz, 1H), 10.16 (s, 1H), 9.73 (s, 1H), 8.24 (d, J = 7.7 Hz, 1H), 8.04 (t, J = 5.7 Hz,1H), 7.83-7.77 (m, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.45 (dd, J = 8.1, 5.7 Hz,3H), 6.88-6.82 (m, 2H), 6.07-5.97 (m, 3H), 5.08 (dd, J = 12.9, 5.5 Hz, 1H), 4.37 (t, J = 4.5 Hz, 2H), 4.16 (td, J = 8.5, 5.5 Hz, 1H), 4.05 (t, J = 4.6Hz, 2H), 3.86 (dt, J = 11.3, 4.5 Hz, 4H), 3.83-3.78 (m, 1H), 3.72 (dd, J =16.7, 5.6 Hz, 1H), 3.42 (s, 2H), 2.84-2.92 (m, 1H), 2.63-2.54 (m, 2H), 2.34(t, J = 7.7 Hz, 2H), 2.07-1.98 (m, 2H), 1.88-1.81 (m, 1H), 1.40 (s, 9H).

[0067] Gly-C15 (0.03 g, 0.03 mmol), TFA (0.08 mL, 4.07 mmol), and DCM (2 mL) were added to a 10 mL round-bottom flask and reacted at room temperature for 12 h. The mixture was then concentrated. After preparative chromatographic purification, 20 mg of Gly-C16, a white solid, was obtained, with a yield of 30%.

[0068] NMR data for Gly-C16:

[0069] 1H NMR (600 MHz, DMSO-d6): δ 10.88 (s, 1H), 10.01 (d, J = 14.7 Hz,1H), 8.25 (t, J = 5.6 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.66 (d, J = 6.9 Hz,1H), 7.53 (d, J = 8.5 Hz, 1H), 7.45 (dd, J = 8.1, 3.7 Hz, 3H), 6.83 (d, J =8.7 Hz, 2H), 6.19 (s, 2H), 6.02 (s, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H),4.37 (t, J = 4.6 Hz, 2H), 4.05 (t, J = 4.7 Hz, 2H), 3.90 (t, J = 6.4 Hz, 1H), 3.87-3.82 (m, 4H), 3.70 (d, J = 5.7 Hz, 2H), 3.45 (s, 2H), 2.92-2.77 (m, 1H), 2.25 (q, J = 6.7 Hz, 3H), 2.05-2.00 (m, 1H), 1.96 (d, J = 13.8 Hz, 1H), 1.81 (d, J = 13.6 Hz, 2H).

[0070] Example 3: Synthetic routes of Gly-C21 and Gly-C23:

[0071] In a 100 mL round-bottom flask, S13 (1.00 g, 4.36 mmol), DMAP (106 mg, 0.87 mmol), TEA (0.88 g, 8.72 mmol), and DCM (10 mL) were added sequentially. The mixture was stirred at 0 °C for 30 min, and then p-toluenesulfonyl chloride (1.24 g, 6.54 mmol) was added. The mixture was stirred at room temperature for 12 h. The reaction solution was diluted with 30 mL of water, the organic layer was separated, extracted with DCM, and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain S14, a colorless oily liquid weighing 1.90 g, with a yield of 116.8%. The product was used directly in the next reaction without purification.

[0072] In a 100 mL round-bottom flask, 2-(2,6-dioxadiazine-3-yl)-4-hydroxyisoindoline-1,3-dione (1.60 g, 5.84 mmol), K₂CO₃ (0.8 g, 5.8 mmol), S₁₄ (1.9 g, 5.0 mmol), and DMF (20 mL) were added sequentially. The mixture was heated to 40 °C and stirred for 12 h. After cooling, the solvent was removed by vacuum distillation. Ethyl acetate (20 mL) and water (60 mL) were added, and the organic layer was separated. The aqueous phase was extracted with ethyl acetate, and the organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give S₁₈, a white solid, 2.27 g, with a yield of 94.6%.

[0073] S18 (2.27 g, 4.68 mmol), TFA (10 mL, 140 mmol), and DCM (20 mL) were added to a 50 mL round-bottom flask and reacted at room temperature for 2 h. The solvent was removed by vacuum evaporation to obtain S16, a colorless oily liquid weighing 1.62 g, with a yield of 87.6%. The product was used directly in the next step without purification.

[0074] In a 50 mL reaction flask, fluorenemethyloxycarbonyl-L-glutamic acid-1-tert-butyl ester (1.98 g, 4.66 mmol), HATU (183 g, 4.81 mmol), DIPEA (1.63 g, 12.62 mmol), and DMF (16 mL) were added. The reaction was carried out at 0 °C for 30 min. S16 (1.62 g, 4.20 mmol) was added, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction solution was concentrated, and water (60 mL) and ethyl acetate (20 mL) were added. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to give S17, a white solid weighing 1.50 g, with a yield of 45.5%.

[0075] S17 (1.50 g, 1.89 mmol), DEA (5.80 mL, 56.8 mmol), and DCM (15 mL) were added to a 50 mL round-bottom flask and reacted at room temperature for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography. S18 was obtained as a white solid, weighing 700 mg, with a yield of 65.2%.

[0076] S12 (51 mg, 0.19 mmol), HATU (66 mg, 0.18 mmol), DIPEA (45 mg, 0.35 mmol), and DMF (3 mL) were added to a 50 mL reaction flask and reacted at 0 °C for 30 min. S36 (100 mg, 0.18 mmol) was then added, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and reacted for 2 h. The reaction solution was concentrated, and the crude product was purified by column chromatography to obtain Gly-C21, a pale yellow solid weighing 66 mg, with a yield of 46.1%.

[0077] NMR data for Gly-C21:

[0078] 1 H NMR (600 MHz, DMSO-d6): δ 11.10 (s, 1H), 10.77 (s, 1H), 10.16 (s,1H), 8.16 (d, J = 7.6 Hz, 1H), 8.06 (t, J = 5.8 Hz, 1H), 7.81 (t, J = 7.9 Hz,1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 6.00 (d, J = 6.2 Hz,3H), 5.08 (dd, J = 12.9, 5.5 Hz, 1H), 4.34 (d, J = 13.0 Hz, 1H), 4.24 (s,2H), 4.15 (p, J = 7.0 Hz, 1H), 3.82-3.65 (m, 3H), 3.42 (s, 2H), 2.96-2.83 (m,2H), 2.63-2.56 (m, 1H), 2.53 (s, 1H), 2.37-2.26 (m, 2H), 2.06-1.99 (m, 1H), 1.98-1.90 (m, 1H), 1.81-1.66 (m, 6H), 1.48 (d, J = 20.9 Hz, 1H), 1.39 (s,9H), 1.10-0.99 (m, 2H).

[0079] Gly-C21 (46 mg, 0.056 mmol), TFA (0.50 mL, 6.75 mmol), and DCM (5 mL) were added to a 10 mL flask and reacted at room temperature for 12 h. The mixture was then concentrated and purified by preparative chromatography to obtain Gly-C23, a pale yellow solid weighing 30 mg, with a yield of 70%.

[0080] Gly-C23 NMR data:

[0081] 1 H NMR (600 MHz, DMSO-d6): δ 8.40 (dt, J = 21.5, 5.9 Hz, 1H), 7.85-7.77 (m, 1H), 7.60 (t, J = 6.6 Hz, 1H), 7.54 (dd, J = 8.6, 6.1 Hz, 1H), 7.44(d, J = 7.2 Hz, 1H), 6.41 (d, J = 8.2 Hz, 2H), 6.02 (s, 1H), 5.08 (dd, J =12.9, 5.5 Hz, 1H), 4.32 (d, J = 12.9 Hz, 1H), 4.24 (d, J = 7.4 Hz, 2H), 3.85(p, J = 5.6 Hz, 1H), 3.66 (q, J = 6.4, 4.5 Hz, 3H), 2.96-2.80 (m, 2H), 2.64-2.53 (m, 2H), 2.48-2.42 (m, 1H), 2.30-2.18(m, 1H), 2.07 (s, 1H), 2.05-1.99(m, 1H), 1.93 (dt, J = 18.0, 5.2 Hz, 1H), 1.76 (s, 1H), 1.74-1.62 (m, 5H), 1.11-0.94 (m, 2H).

[0082] Example 4: Synthetic routes of Gly-C25 and Gly-C26:

[0083] 2,2'-Oxadiethylamine (205 mg, 1.97 mmol) and methanol (5 mL) were placed in a reaction flask, and a methanol (5 mL) solution of di-tert-butyl dicarbonate (180 μL, 0.78 mmol) was slowly added dropwise at 0 °C. The reaction was carried out at room temperature for 6 h. The solvent was removed by vacuum distillation, and saturated sodium bicarbonate solution (5 mL) was added. The mixture was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed by vacuum distillation, and the solution was purified by column chromatography to give S19, a pale yellow oily liquid, 0.11 g, with a yield of 71.6%.

[0084] 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (470 mg, 1.70 mmol) and DMF (2 mL) were placed in a reaction flask and heated to 90 °C. A solution of compound S19 (521 mg, 2.55 mmol) in DMF (3 mL) and DIPEA (0.89 mL, 5.1 mmol) were added, and the reaction was carried out overnight at 90 °C. After cooling to room temperature, saturated brine (20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to give S20, a yellow viscous substance, 0.31 g, with a yield of 40.3%.

[0085] Compound S20 (170 mg, 0.37 mmol) was dissolved in dichloromethane (5 mL). After the reaction solution was cooled to 0 °C, trifluoroacetic acid (0.825 mL, 11.1 mmol) was added dropwise. The mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the solution was purified by column chromatography to obtain S21, a brownish-yellow viscous liquid, in a yield of 0.04 g, with a yield of 60.4%.

[0086] Fluorenylmethoxycarbonyl-L-glutamic acid-1-tert-butyl ester S21 (97.47 mg, 0.229 mmol) was dissolved in DMF (2 mL), followed by the addition of HATU (113.2 mg, 0.298 mmol) and DIPEA (0.16 mL, 0.916 mmol). The mixture was stirred at room temperature for 2 h. Then, S21 (99 mg, 0.275 mmol) was dissolved in DMF (8 mL) and slowly added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. Saturated brine (10 mL) was added, and the mixture was extracted with ethyl acetate and washed with saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to obtain S22, a yellow oily liquid, 0.076 g, with a yield of 43.2%.

[0087] Compound S22 (146.7 mg, 0.191 mmol) was dissolved in DMF (3 mL), and DEA (0.6 mL, 5.74 mmol) was added. The mixture was stirred at room temperature for 2 h. The solvent DMF was evaporated, and saturated brine (5 mL) was added. The mixture was extracted with ethyl acetate and washed with saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was purified by column chromatography to give S23, a yellow solid of 45.9 mg, with a yield of 48.7%.

[0088] Compound S23 (0.53 g, 0.972 mmol) was dissolved in DMF (2 mL). Compounds S12 (0.26 g, 0.972 mmol), HATU (0.74 g, 1.94 mmol), and DIPEA (0.85 mL, 4.86 mmol) dissolved in DMF (10 mL) were added sequentially. The mixture was stirred at room temperature for 2 h. The solvent was removed by vacuum evaporation, and the solution was purified by column chromatography to obtain Gly-C25, a yellow solid of 218 mg, with a yield of 28.3%.

[0089] NMR data for Gly-C25:

[0090] 1 H NMR (600 MHz, Methanol-d4): δ 7.53-7.56 (m, 1H), 7.05-7.07 (m, 2H), 6.28 (s, 1H), 5.09-5.06 (dd, J = 12.8, 5.5 Hz, 1H), 4.30 (s, 1H), 3.85-3.92(m, 2H), 3.69 (m, 2H), 3.62 (s, 2H), 3.56 (m, 2H), 3.48 (m, 2H), 3.39 (m,2H), 2.88 (d, J = 18.6 Hz, 1H), 2.72-2.78 (m, 2H), 2.29 (d, J = 7.0 Hz, 2H), 2.14 (s, 2H), 1.90 (s, 1H), 1.45 (s, 9H).

[0091] Compound Gly-C25 (44.5 mg, 0.056 mmol) was placed in a reaction flask, and trifluoroacetic acid (0.42 mL, 0.56 mmol) and dichloromethane (2 mL) were added simultaneously. The mixture was stirred at room temperature, and after 2 h of reaction, the solvent was removed by vacuum distillation. The mixture was purified by column chromatography to obtain Gly-C26, a yellow solid of 23.9 mg, with a yield of 13.7%.

[0092] NMR data for Gly-C26:

[0093] 1H NMR (600 MHz, DMSO- d6): δ 10.83 (s, 1H), 8.16 (d, J = 6.1 Hz, 1H), 7.75-7.90 (m, 2H), 7.56 (q, J = 5.9, 3.8 Hz, 1H), 7.12-7.14 (dd, J = 8.7, 4.4Hz, 1H), 7.02 (d, J = 3.7 Hz, 1H), 6.60 (d, J = 5.8 Hz, 1H), 6.14 (d, J = 7.0Hz, 1H), 6.01 (s, 1H), 5.05-5.08 (dd, J = 12.8, 5.5 Hz, 1H), 3.97 (s, 1H),3.82 (s, 1H), 3.70 (t, J = 5.5 Hz, 2H), 3.18 (s, 3H), 2.87 (s, 2H), 2.50-2.60(m, 2H), 2.01-2.11 (m, 4H), 1.92 (d, J = 14.7 Hz, 2H), 1.73-1.77 (m, 2H), 1.23 (d, J = 14.4 Hz, 1H).

[0094] Example 5: Synthetic routes of Gly-C35 and Gly-C36:

[0095] 1.47 g (5.4 mmol) of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione was dissolved in 15 mL of anhydrous THF and placed in a reaction flask. The mixture was heated to reflux, and 3.00 g (16.2 mmol) of 4-bromobutyryl chloride was added. The mixture was refluxed for 4 h. After the reaction was completed, the mixture was cooled to room temperature, the solvent was removed under reduced pressure, 20 mL of saturated brine was added, and the mixture was extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give S24, a white solid, 2.07 g, in a yield of 90.8%.

[0096] Compound S24 (2.07 g, 4.92 mmol) was dissolved in THF (20 mL) in a reaction flask. TMSN3 (0.85 g, 7.37 mmol) was added to the flask, and the mixture was placed under nitrogen protection. TBAF (1.93 g, 7.37 mmol) was added, and the mixture was stirred overnight at room temperature. The solvent was removed by vacuum distillation, and the mixture was washed with 40 mL of dichloromethane and dried over anhydrous sodium sulfate. After filtration and vacuum distillation, the solvent was removed again, and the mixture was purified by column chromatography to give S25 as 1.20 g of a white solid, in a yield of 63.5%.

[0097] (S)-4-((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-tert-butoxy-5-oxovaleric acid (2.00 g, 4.70 mmol) was dissolved in DMF (10 mL) and placed in a reaction flask. Propryl-2-yn-1-amine (388 mg, 7.05 mmol), HATU (3.57 g, 9.40 mmol), and DIPEA (2.5 mL, 14.10 mol) were added sequentially, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the solvent was removed under reduced pressure, and saturated brine (10 mL) was added. The mixture was extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give S26, a pale yellow solid, 1.81 g, in a yield of 83.4%.

[0098] Compounds S26 (1.37 g, 2.96 mmol) and S25 (1.14 g, 2.96 mmol) were dissolved in THF (30 mL) and placed in a reaction flask. CuSO4•5H2O (0.89 g, 3.55 mmol) was added to the flask, and the mixture was kept under nitrogen protection. Sodium ascorbate dissolved in H2O (8 mL) (1.76 g, 8.88 mmol) was then injected, and the mixture was stirred overnight at room temperature. After the reaction was complete, saturated brine (20 mL) and dichloromethane were added simultaneously for extraction. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed under reduced pressure, and the solution was purified by column chromatography to give S27, a pale red solid, 1.03 g, in a yield of 40.9%.

[0099] Compound S27 (204.8 mg, 0.242 mmol) was dissolved in DCM (2.5 mL), and DEA (2.5 mL, 24.20 mmol) was added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was evaporated and purified directly by column chromatography to obtain S28, a pale yellow solid of 90 mg, with a yield of 59.6%.

[0100] Compound S28 (31.2 mg, 0.0450 mmol) was dissolved in DMF (2 mL). Compounds S12 (13.25 mg, 0.0450 mmol), HATU (38 mg, 0.099 mmol), and DIPEA (0.026 mL, 0.149 mmol) dissolved in DMF (3 mL) were added sequentially. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed by vacuum distillation. The crude product was purified by preparative liquid chromatography under medium pressure to obtain 11 mg of Gly-C35, a gray powder solid, with a yield of 22.9%.

[0101] NMR data for Gly-C35:

[0102] 1 H NMR (600 MHz, Methanol- d4): δ 10.78 (s, 1H), 9.78 (s, 1H), 8.39 (d,J = 8.4 Hz, 1H), 8.32 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 8.06 (s, 1H), 7.94(s, 1H), 7.83 (s, 1H), 7.62 (d, J = 7.3 Hz, 1H), 6.06 (s, 2H), 5.99 (s, 1H),5.12-5.15 (dd, J = 12.9, 5.5 Hz, 1H), 4.40 (s, 2H), 4.28 (d, J = 5.6 Hz, 2H),4.10-4.11 (m, 1H), 3.77-3.81 (dd, J = 16.8, 6.0 Hz, 1H), 3.69-3.72 (m, 2H),2.86-2.90 (m, 1H), 2.59-2.62 (m, 1H), 2.54 (d, J = 4.1 Hz, 1H), 2.14-2.17 (m,5H), 2.07 (s, 2H), 1.94 (d, J = 7.9 Hz, 2H), 1.75 (s, 1H), 1.38 (s, 9H).

[0103] Compound Gly-C35 (93.4 mg, 0.107 mmol) was placed in a reaction flask, and trifluoroacetic acid (0.8 mL, 1.07 mmol) and dichloromethane (3.2 mL) were added simultaneously. The mixture was stirred at room temperature and reacted for 2 h. The solvent was removed by vacuum distillation, and the product was purified by column chromatography to obtain Gly-C36, 50 mg of gray solid, with a yield of 11.2%.

[0104] NMR data for Gly-C36:

[0105] 1H NMR (600 MHz, DMSO-d6): δ 10.86 (s, 1H), 9.87 (s, 1H), 8.37 (d, J =8.6 Hz, 2H), 8.21 (s, 1H), 7.94 (s, 1H), 7.81-7.83 (dd, J = 8.4, 7.3 Hz, 1H),7.74 (s, 1H), 7.61-7.65 (m, 2H), 7.20 (s, 1H), 6.15 (s, 2H), 6.00 (s, 1H),5.12-5.15 (m, 1H), 4.40 (t, J = 7.0 Hz, 2H), 4.26 (d, J = 5.6 Hz, 2H), 3.86(d, J = 6.3 Hz, 1H), 3.68 (d, J = 5.9 Hz, 2H), 2.86-2.92 (m, 2H), 2.58-2.62(m, 1H), 2.53-2.54 (m, 1H), 2.12-2.14 (m, 3H), 2.07 (d, J = 2.8 Hz, 2H), 1.98-2.00 (m, 2H), 1.75 (s, 2H), 1.45 (s, 1H).

[0106] Example 6: Synthetic routes of Gly-C39 and Gly-C40:

[0107] Compound 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)isoindoline-1,3-dione (403 mg, 0.95 mmol) was dissolved in DMF (5 mL), cooled to 0 °C, and HATU (469 mg, 1.23 mmol) and DIPEA (0.66 mL, 3.80 mmol) were added sequentially. The mixture was stirred for 1 h. Then, (S)-4-((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-tert-butoxy-5-oxovaleric acid (500 mg, 1.14 mmol) was dissolved in DMF (8 mL) and slowly added dropwise to the reaction mixture. The mixture was stirred overnight at room temperature. Add saturated brine (40 mL), extract with ethyl acetate, wash with saturated brine (40 mL), dry with anhydrous sodium sulfate, filter, remove solvent under reduced pressure, and purify by column chromatography to obtain 998 mg of yellow solid (94%). Dissolve the solid in dichloromethane (15 mL), add DEA (3.6 mL, 35.37 mmol), stir at room temperature for 2 h, remove solvent after reaction, and purify directly by column chromatography to obtain S29, a pale yellow solid of 430 mg, with a yield of 58.0%.

[0108] Compound S12 (20 mg, 0.06 mmol) was dissolved in DMF (1 mL), cooled to 0 °C, and HATU (24 mg, 0.06 mmol) and DIPEA (0.02 mL, 0.13 mmol) were added sequentially. The mixture was stirred for 1 h, and then compound S29 (40 mg, 0.06 mmol) dissolved in DMF (2 mL) was added. The mixture was transferred to room temperature and reacted for 4 h. The solvent was removed by vacuum evaporation, and the mixture was purified by preparative chromatography to obtain Gly-C39, a yellow solid of 11 mg, with a yield of 20%.

[0109] NMR data for Gly-C39:

[0110] 1H NMR (600 MHz, DMSO-d6): δ 11.08 (s, 1H), 10.77 (s, 1H), 10.17 (s,1H), 8.16 (t, J = 7.3 Hz, 1H), 8.07 (t, J = 5.8 Hz, 1H), 7.70 (t, J = 7.8 Hz,1H), 7.34 (dd, J = 16.5, 7.8 Hz, 2H), 6.01 (d, J = 8.0 Hz, 3H), 5.09 (dd, J =12.8, 5.5 Hz, 1H), 4.35 (d, J = 12.7 Hz, 1H), 4.11–4.19 (m, 1H), 3.65–3.84(m, 3H), 3.42 (s, 2H), 3.29 (s, 4H), 2.83–2.96 (m, 2H), 2.56 (dd, J = 34.2,13.4 Hz, 7H), 2.32 (t, J = 7.5 Hz, 2H), 2.17 (d, J = 7.2 Hz, 2H), 1.88–2.06(m, 4H), 1.65–1.82 (m, 4H), 1.40 (s, 9H).

[0111] Compound Gly-C39 (30 mg, 0.03 mmol) and dichloromethane (5 mL) were placed in a reaction flask, and TFA (0.255 mL, 3.44 mmol) was added. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the solvent was removed by vacuum distillation, and the product was purified by preparative chromatography to obtain Gly-C40, a yellow viscous substance of 7 mg, with a yield of 25%.

[0112] NMR data for Gly-C40:

[0113] 1H NMR (600 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.29 (s, 1H), 7.93 (d, J =7.7 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.34 (dd, J = 12.7, 7.8 Hz, 2H), 3.39 (q, J = 15.2 Hz, 3H), 3.29(s, 5H), 2.82–2.95 (m, 2H), 2.59 (d, J = 17.7 Hz, 6H), 2.30 (q, J = 10.2, 6.4Hz, 2H), 2.1–2.21 (m, 3H), 2.00–2.08 (m, 1H), 1.95 (d, J = 4.9 Hz, 3H), 1.56–1.85 (m, 5H).

[0114] Example 7: Degradation efficiency of the compound on TS cells

[0115] KB cells were seeded into 6-well plates at a density of 300,000 cells / well. They were incubated at 37 °C in a 5% CO2 incubator for 24 h. Then, 10 μM of the target compound (stored in DMSO at -20 °C) was added to each well, and the plates were incubated at 37 °C in 5% CO2 for 72 h. Cells were collected, centrifuged, and RIPA lysis buffer was added. The lysed cell proteins were centrifuged again, and the supernatant was collected to obtain a protein solution. The protein concentration in the protein solution was determined using a BCA protein quantification kit. The loading volume was calculated based on the protein concentration, and the cells were separated by SDS-PAGE with sodium dodecyl sulfate gel. The resulting membrane was then transferred to a membrane, incubated overnight at 4 °C with the antibody, and then incubated for two hours with the corresponding secondary antibody. The membrane was then visualized using Odyssey imaging.

[0116] The degradation effects of the compounds of this invention on TS protein in KB cells are shown in the table below:

[0117]

[0118] Western blot (WB) results showed that the target compounds had a certain degradation effect on TS, with compounds Gly-C25, Gly-C27 and Gly-C41 significantly degrading TS protein at 10 μM.

Claims

1. A PROTAC chimera targeting TS or a pharmaceutically or physiologically acceptable salt thereof, having the structure shown in general formula I: ； E3 ligand is the protein ligand in the ubiquitin ligase complex, which is a Cereblon (CRBN) ligand; Linker is the linking group between the target protein ligand and the E3 ligase ligand; X is one of -CONH- or -CH2SCH2-; and R is one of H, methyl, ethyl, isopropyl, or tert-butyl.

2. The chimera according to claim 1, characterized in that... The CRBN ligand is selected from thalidomide or its derivatives.

3. The chimera according to claim 1, characterized in that, The preferred structure of the CRBN ligand is shown below: 。 4. The chimera according to claim 1, characterized in that, Linker is -alkylene or -alkoxy or -piperazinyl or -piperidinyl or -1,2,3-triazolyl, wherein the -alkylene or -alkoxy or -piperazinyl or -piperidinyl or -1,2,3-triazolyl is selected from any one or more combinations of the following groups, where n represents a natural number from 1 to 15; 。 5. The PROTAC chimera for targeted TS degradation according to claim 1, or a pharmaceutically or physiologically acceptable salt thereof, characterized in that, The PROTAC chimera for targeted TS degradation is preferably any one of the following compounds: Gly-C1 to Gly-C46. ； ； ； ； ； ； ； 。 6. The method for preparing the PROTAC chimera for targeted TS degradation or a pharmaceutically or physiologically acceptable salt thereof according to claim 5, characterized in that, The preparation methods employ different synthetic routes, the specific synthetic routes are as follows: Synthesis Route 1: ； Synthesis Route 2: ； Synthesis Route 3: ； Synthesis Route 4: ； Synthesis Route 5: ； Synthesis Route 6: 。 7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the PROTAC chimera targeting TS as described in any one of claims 1-5, or a pharmaceutically or physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium, or combination thereof.

8. The use of a PROTAC chimera targeting TS as described in any one of claims 1-5, or a pharmaceutically or physiologically acceptable salt thereof, or the pharmaceutical composition of claim 7, in the preparation of a TS-degrading drug.

9. The use of a PROTAC chimera targeting TS as described in any one of claims 1-5, or a pharmaceutically or physiologically acceptable salt thereof, or the pharmaceutical composition of claim 7, in the treatment of folic acid-related diseases.

10. The application according to claim 9, characterized in that... Folic acid-related diseases include cancer.

11. The use of a PROTAC chimera targeting TS as described in any one of claims 1-5, or a pharmaceutically or physiologically acceptable salt thereof, or the pharmaceutical composition of claim 7, in the treatment of tumors, said tumors being gastric cancer, breast cancer, lung cancer, gastric cancer, colon adenocarcinoma, pancreatic cancer, bladder cancer, hepatocellular carcinoma, and cervical cancer.

12. The application according to claim 11, characterized in that... Tumors with high FR expression.

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