Targeting HER2/VEGFR2 dual tyrosine kinases with novel Lapatinib and Neratinib hybrid analogues lead to potential apoptotic induction in HER2 positive breast cancers: Design, synthesis, in-vitro, in-vivo and molecular docking studies.

Abstract A high percentage of women worldwide will develop breast cancer during their lifetime, and there will always be a need to look for novel breast cancer treatment possibilities. The co-expression of HER2 and VEGFR2 in some breast cancers has been associated with a more aggressive tumour phenotype and poorer prognosis. As part of continuing research focusing on the possibility of simultaneously targeting HER2 and VEGFR2, we describe the design and synthesis of new lapatinib and neratinib hybrid analogues and their in vitro and in vivo evaluation for anti-cancer activity. We used the drug extension strategy to tailor the designed compounds to fit the RTKs, such as EGFR VEGFR2 and HER2 hydrophobic subpocket and cleft regions. The designed lapatinib and neratinib derivatives were successfully synthesized using established synthetic procedures and characterized using 1H, 13C-NMR, HRMS, and elemental analysis. The synthesized compounds were initially tested for their RTK inhibition capabilities, and compounds 15i and 15g were found to possess potential HER2 and VEGFR2 kinase inhibition abilities in-vitro with an IC50 less than the standards lapatinib and sorafenib used. The anti-proliferative capability of all derivatives demonstrated that compounds 15i and 15g potentially suppressed the growth of HER2 positive T-47D and BT-474 cells having a differential expression of HER2 and VEGFR2 with superior activity than lapatinib and sorafenib. SAR revealed that the trifluoromethyl group on the pyridinyl moiety of the side chain at the fourth position of the scaffold made compound 15i the most promising candidate among the other candidates. Flowcytometric apoptotic evaluation of compound 15i demonstrated potential induction of apoptosis at its IC50 in both T-47D and BT-474 cells, which was proved by examining the caspases (Caspase-3, 8, and 9) and Cytochrome-c release. Western blot analysis further determined HER2, VEGFR2, and their downstream signalling partner’s inhibition by the treatment of 15i. Further in-vivo tumour growth reduction by 15i was assessed in the T-47D xenograft mice model stating its potential anti-tumour capability. Based on docking studies, compound 15i was confirmed as a new lead candidate for the dual inhibition of HER2 and VEGFR2.