Synthesis of novel methyl 7-[(hetero)arylamino]thieno[2,3-b] pyrazine-6-carboxylates and antitumor activity evaluation: effects in human tumor cells growth, cell cycle analysis, apoptosis and toxicity in non-tumor cells

Several novel methyl 7-[(hetero)arylamino]thieno[2,3- b ]pyrazine-6-carboxylates were synthesized by Pd-catalyzed C–N Buchwald–Hartwig cross-coupling of either methyl 7-aminothieno[3,2- b ]pyrazine-6-carboxylate with (hetero)arylhalides or 7-bromothieno[2,3- b ]pyrazine-6-carboxylate with (hetero)arylamines in good-to-excellent yields (50% quantitative yield), using different reaction conditions, namely ligands and solvents, due to the different electronic character of the substrates. The antitumoral potential of these compounds was evaluated in four human tumor cell lines: gastric adenocarcinoma (AGS), colorectal adenocarcinoma (CaCo-2), breast carcinoma (MCF7), and non-small-cell lung carcinoma (NCI-H460) using the SRB assay, and it was possible to establish some structure–activity relationships. Furthermore, they did not show relevant toxicity against a non-tumor cell line culture from the African green monkey kidney (Vero). The most promising compounds (GI 50 ≤ 11 µM), showed some selectivity either against AGS or CaCo-2 cell lines without toxicity at their GI 50 values. The effects of the methoxylated compounds 2b (2-OMeC 6 H 4 ), 2f and 2g (3,4- or 3,5-diOMeC 6 H 3 , respectively) on the cell cycle profile and induction of apoptosis were further studied in the AGS cell line. Nevertheless, even for the most active (GI 50 = 7.8 µM) and selective compound ( 2g ) against this cell line, it was observed that a huge number of dead cells gave rise to an atypical distribution on the cell cycle profile and that these cells were not apoptotic, which points to a different mechanism of action for the AGS cell growth inhibition. This research was funded by Fundação para a Ciência e Tecnologia (FCT)—Portugal, which financially supports CQUM (UID/QUI/686/2019), and also financed by the European Regional Develop- ment Fund (ERDF), COMPETE2020 and Portugal2020, and the PTNMR network also supported by Portugal2020. J.M.R. PhD grant (SFRH/BD/115844/2016) was financed by FCT, ESF (European Social Fund—North Portugal Regional Operational Program) and HCOP (Human Capital Operational Program). The authors are grateful to FCT, Portugal, for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). L.B. and R.C.C. thank the national funding by FCT, Portugal, through the institutional scientific employment program-contract for their contracts.