Discovery,Design And Synthesis Of Novel STAT3 Dual Phosphorylation Inhibitors And Evaluation For Their Anti-Gastric Cancer Activity In Vitro And In Vivo

Gastric cancer（GC）remains the most common global malignant tumor with the fifth incidence and the third mortality.Currently,the first-and second-line treatment drugs for advanced gastric cancer mainly include the combination of various chemotherapy drugs,the targeted drugs Trastuzumab and Ramucirumab and the immunotherapy drug Pembrolizumab.No targeted small-molecule drugs have been approved for gastric cancer.Notably,the effectiveness of these approved drugs is undesirable,as most patients with advanced gastric cancer treated with these drugs have only a median overall survival of around 1 year.Hence,developing more effective treatment strategies for gastric cancer is urgently acquired.Signal transducer and activator of transcription 3（STAT3）,as a vital transcription factor,is often found to be aberrated activation and promote the progression of various cancers.The activation of STAT3 mainly involves the phosphorylation of two amino acid residues,Tyr⁷⁰⁵ and Ser⁷²⁷,which play different biological functions in cancer cells.Phosphorylation of Tyr⁷⁰⁵mainly mediates the nuclear transcriptional function of STAT3,while Ser⁷²⁷ phosphorylation mainly mediates the mitochondrial oxidative phosphorylation function of STAT3.In gastric cancer cells,STAT3 phosphorylation at Tyr⁷⁰⁵ and Ser⁷²⁷ is often found to be abnormal over-expression and continuously activated,which promotes the progression of gastric cancer and is closely associated with the poor prognosis of gastric cancer patients.To date,although few STAT3inhibitors have entered clinical trials for the treatment of cancers,these inhibitors still have issues such as insufficient anti-tumor efficacy in vivo,poor drugability,and safety concern.More importantly,most STAT3 inhibitors reported can only inhibit STAT3phosphorylation at the Tyr⁷⁰⁵,but not the phosphorylation of Ser⁷²⁷.Given the critical role of STAT3 dual sites phosphorylation in gastric cancer progression,we propose that developing novel and potent dual phosphorylation STAT3 inhibitors would be a potentially effective therapeutic strategy for gastric cancer.In this paper,we constructed function-based screening assays based on the distinct biological functions mediated by the STAT3 phosphorylation at Tyr⁷⁰⁵ and Ser⁷²⁷,which resulted in the discovery of the triaromatic heterocyclic hit compound 1a with low micromolar inhibitory activity.In order to further improve the potency of 1a,we performed rational designs,optimizations and SAR studies using the drug design strategy of CADD;the optimized directions consist of the methyl substituent,the 2 or3 positions on the piperazine ring,and the p-trifluoromethyl benzene ring,1,2,4-oxadiazole ring,and pyridine ring of the triaromatic heterocycle.On the basis of the great optimization efforts,a candidate compound 5j with the optimal activity among these compounds was determined.Compound 5j was selectively bound to the SH2domain of STAT3 with high affinity（K_D=111.4 n M）,resulting in the significant inhibition effect on the phosphorylation of Tyr⁷⁰⁵ and Ser⁷²⁷and the nuclear transcriptional and mitochondrial oxidative phosphorylation functions mediated by Tyr⁷⁰⁵ and Ser⁷²⁷phosphorylation,respectively.In vitro,compound 5j selectively inhibited gastric cancer cells proliferation and colony formation and promoted gastric cancer cell apoptosis at low nanomolar levels;notably,the compound 5j did not inhibit the proliferation of gastric cancer cells with STAT3 knockdown and normal cells with low expression of STAT3 phosphorylation,indicating that STAT3 was the main target of 5j for its antitumor activity.In vivo,5j had excellent oral bioavailability（F=54.4%）and metabolism stability(t_1/2,po=16.0 h),which exhibited more potent antitumor potency in the MKN45 xenogeneic model than positive compound BBI608 by inhibiting the dual phosphorylation of STAT3,without showing noticeable side effect.In summary,on the basis of the structural design,synthesis and structure-activity relationship studies for the hit compound 1a,a novel and highly potent STAT3 inhibitor5j with dual phosphorylation inhibitory activity was discovered.With excellent pharmacokinetic properties and fewer side effects,the orally available compound 5j significantly inhibited the proliferation of gastric cancer cells in vitro and in vivo,and its activities are significantly better than that of the positive compound BBI608.Thus,the dual phosphorylation STAT3 inhibitor 5j could be a potential therapeutic drug for gastric cancer to develop further.