Bisnaphthalimides represent a promising group of
DNA-targeted
anticancer agents. In this series, the lead compounds
elinafide and
bisnafide have reached clinical trials, and the search for more potent analogues remains a priority. In the course of a medicinal chemistry program aimed at discovering novel
antitumor drugs based on the
naphthalimide skeleton, different dimeric molecules containing two tetracyclic neutral
DNA intercalating chromophores were synthesized. The
naphthalimide unit has been fused to a
benzene ring (
azonafide derivatives), an
imidazole, a
pyrazine, or, as reported here, a
furan ring which increases the planar surface of the chromophore and enhances its stacking properties. We report a detailed investigation of the
DNA binding capacity of the dimeric molecule MCI3335 composed of two furonaphthalimide units connected by a 12 A long amino alkyl linker [(CH(2))(2)-NH-(CH(2))(3)-NH-(CH(2))(2)] identical to that of
elinafide. Qualitative and quantitative binding studies, in particular using surface plasmon resonance, establish that the dimer binds considerably more tightly to
DNA (up to 1000 times) than the corresponding monomer and exhibits a higher sequence selectivity for GC-rich sequences.
DNase I footprinting experiments attest that the dimer, and to a lesser extent the monomer, preferentially intercalate at GC sites. The strong binding interaction between the drugs and
DNA perturbs the relaxation of
supercoiled DNA by topoisomerases, but the test compounds do not promote DNA cleavage by
topoisomerase I or II. Despite the lack of
poisoning effect toward
topoisomerase II, MCI3335 displays a very high cytotoxicity toward CEM human
leukemia cells, with an IC(50) in the low nanomolar range, approximately 4 times inferior to that of the reference drug
elinafide. Confocal microscopy observations indicate that the monomer shows a stronger tendency to accumulate in the cell nuclei than the dimer. The extremely high cytotoxic potential of MCI3335 is attributed to its enhanced capacity to bind to
DNA and to inhibit
DNA synthesis, as evidenced by flow cytometry experiments using the
BrdU assay. The results provide novel mechanistic information that furthers the understanding of the structure-activity relationships in the bisnaphthalimide series and identify MCI3335 as a novel lead compound for further preclinical investigations.