Background: Hybrid molecules combining uracil skeleton with methylidene exo-cyclic group were
designed in the search for novel anticancer drug candidates.
Objective: Two series of racemic 5-methylidenedihydrouracils, either 1,3-disubstituted or 1,3,6-trisubstituted
were synthesized and tested for their possible cytotoxic activity against two cancer cell lines (HL-60 and MCF-7)
and two healthy cell lines (HUVEC and MCF-10A). The most cytotoxic analogs were re-synthesized as pure
enantiomers. The analog designated as U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil],
which had a very low IC50 value in HL-60 cell line (0.77μM) and was the most selective towards cancer cells
was chosen for further experiments on HL-60 cell line, in order to determine the possible mechanism involved
in its antineoplastic action.
Methods: Cytotoxic activities of compound was assessed by the MTT assay. In order to explore the mechanism
of U-332 activity, we performed quantitative real-time PCR analysis of p53 and p21 genes. Apoptosis, cell
proliferation and DNA damage in HL-60 cells were determined using the flow cytometry. The ability of U-332
to determine GADD45ɑ protein level in HL-60 cells incubated with U-332 was analyzed by ELISA test.
Results: U-332 was shown to generate excessive DNA damage (70% of the cell population), leading to p53
activation, resulting in p21 down-regulation and a significant increase of GADD45α protein, responsible for the
cell cycle arrest in G2/M phase.
Conclusion: U-332 can be used as a potential lead compound in the further development of novel uracil analogs
as anticancer agents.