Discovery, Synthesis, and Evaluation of Highly Selective Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) Inhibitor for the Potential Treatment of Metastatic Triple-Negative Breast Cancer

We herein report the identification, structural optimization, and structure-activity relationship of thieno[2,3-d]pyrimidine derivatives as a novel kind of selective vascular endothelial growth factor receptor 3 (VEGFR3/Flt-4) inhibitors. N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(6-(4-(4-methylpiperazin-1-yl)phenyl)thieno[2,3-d]pyrimidin-4-yl)piperazine-1-carboxamide (38k) was the most potent VEGFR3/Flt-4 inhibitor (IC₅₀ = 110.4 nM) among developed compounds. Compared with VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, VEGFR3/Flt-4 was approximately 100 times more selective. Here, compound 38k significantly inhibited proliferation and migration of VEGF-C-induced human dermal lymphatic endothelial cells (HDLEC), MDA-MB-231, and MDA-MB-436 cells by inactivating the VEGFR3/Flt-4 signaling pathway. Additionally, 38k induced cell Apoptosis and a prolonged G1/S-phase in MDA-MB-231 and MDA-MB-436 cells. It also presented acceptable pharmacokinetic characteristics in Sprague-Dawley (SD) rats with an oral bioavailability of 30.9%. In the xenograft model in vivo, 38k effectively inhibited breast Cancer growth by suppressing the VEGFR3/Flt-4 signaling pathway. 38k pronouncedly resisted the formation of pulmonary metastatic nodules in mice. Collectively, 38k may be a promising therapeutic agent of metastatic breast Cancer.