Small-molecule PROTAC mediates targeted protein degradation to treat STAT3-dependent epithelial cancer

The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology–based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule–based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC — especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.


Synthesis of TSMs.
Scheme II: Synthetic route of preparing TSM-2.
3-(benzyloxy)-3-oxypropionic acid (233 mg, 1.2 mmol) was dissolved in anhydraous DCM (5 mL). Oxaloyl chloride (0.12 mL, 1.4 mmol) and the catalytic amount of anhydrous DMF were added into the reaction solution at 0 o C. The mixture was then stirred at room temperature until the reaction of raw materials was complete. When the solvent was evaporated, the crude was dissolved in 2 mL anhydrous DCM.
A mixture of TSN (574 mg, 1 mmol) and triethylamine (0.27 mL, 2 mmol) were dissolved in anhydrous DCM (5 mL), which was allowed to room temperature and stirred for 12 h. The reaction mixture was diluted with DCM and washed with water for three times. The organic layer was washed with saturated NaCl, dried over Na2SO4, concentrated and purified by column chromatography (1:2 PE/EA) to yield white solid product S5 (390 mg, 52%). ESI-MS: m/z 751.8 [M+H] + .
The above S5 was dissolved in methanol (10 mL) and 10% palladium carbon (100 mg). After being replaced with hydrogen for three times, the mixture was stirred at room temperature until the reaction was complete. The crude product S6 (310 mg, 90%) was obtained by adding diatomite for extraction and filtration, followed by washing with methanol for three times. ESI-MS: m/z 660.7 [M+H] + .

Scheme III: Synthetic route of preparing TSM-3.
The mixture of S3 (328 mg, 1 mmol) and 3-formylazacyclobutane-1carboxylate tert-butyl ester (370 mg, 2 mmol) were dissolved in 10 mL methanol. Then the sodium cyanoborohydride (315 mg, 5 mmol) and 0.1 mL acetic acid were added into the reaction solution and stirred at room temperature until the reaction was complete, followed by evaporating the solvent and being redissolved in ethyl acetate. The organic layer was washed with H2O and dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (20:1 DCM/MeOH) to yield white solid product S7 (350 mg, 70%), ESI-MS: m/z 498.6 [M+H] + .
S7 was dissolved in 10 mL DCM and dropped into 1 mL TFA, then the mixture was stirred at room temperature until the reaction was complete. When the solvent was evaporated, the crude product was re-dissolved in water and dropped into saturated NaHCO3 solution. The products were precipitated at 5 o C and filtered to obtain white solid S8 (266 mg, 95%), ESI-MS: m/z 398.5 [M+H] + .
TFA (1 mL) was added dropwise into a solution of S9 or S10 (347 mg) in DCM (10 mL) at 0 o C. The mixture was then allowed to room temperature and stirred for 1 h, followed by removing the solvent under vacuo. The residue was suspended in H2O and the saturated NaHCO3 was added with efficient stirring at 5 o C, the resultant precipitate was filtered off and the filtrate (260 mg, 98%) was concentrated to dryness as white solid powder S11 (277 mg, 97%) or S12(279 mg, 98%). ESI-MS: m/z 343.4 [M+H] + (S11), and m/z 357.4 [M+H] + (S12).
The TSN was dissolved in anhydrous DCM (10 mL), then the succinic anhydride (500 mg, 5 mmol) and 4-dimethylaminopyridine (244 mg, 2 mmol) were added. The mixture was then allowed to room temperature and stirred for 12 h. Then the solution was concentrated in vacuo and the residue was added EA (100 mL). The organic layer was washed with 1 N HCl (100 mL) and H2O, dried over anhydrous Na2SO4, and concentrated to yield white solid S4 (640 mg, 95%), which was used without further purification. ESI-MS: m/z 674 [M+H] + .

Scheme V: Synthetic route of preparing TSM-6
The S1 (260 mg, 1 mmol) was dissolved in HCl/H2O (5 mL/5 mL) and the solution was cooled to -5 o C, then the aqueous solution of sodium nitrite (138 mg, 2 mmol in 1 mL H2O) was added. The mixture was stirred at same temperature for 1 h and then the potassium iodide in aqueous solution (332 mg, 2 mmol in 1 mL H2O) was added into the reaction solution and stirred for 12 h at room temperature, the concentrated to yield brown soild S13 (250 mg, 67%).