Leukemia is a malignant disease that originates in the bone marrow, local of blood cells production. Chemotherapy treatment is one of the alternatives to improve the survival of patients. In this context, the search for new compounds with potential cytotoxic action is justified. The present work evaluated the cytotoxic activity of 16 new synthetic analogs of 3-alkylpyridine alkaloids. The cytotoxic profiles were determined by MTT in vitro assay against THP-1 (acute monocytic leukemia), K562 (chronic myeloid leukemia) and PBMC (peripheral blood mononuclear) human cells. To investigate the mechanism of action, cell cycle analysis and alterations of TP53, p21, Bax, Bcl2, NOX-1, NOX-2, NOX-4 and p47-phox gene expressions were performed by qPCR, along with the measurement of reactive oxygen species (2’,7’-dichlorodihydrofluorescein diacetate and dihydroethidium). Compounds 4c, 5b, 5c and 6d were the most active and selective for THP-1 and compounds 7c and 11 were found to be more active for K562. All of this induced apoptosis in the tested strains. Concerning the investigation of the mechanism of action, it was observed that the pathway being activated is a p53-independent pathway. The data presented in this work indicate that 3-alkylpyridine alkaloid analogues are a potential class of compounds with cytotoxic action. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.