REACTIVITY ON DNA

DNA is the main target for most of the anticancer agents. We did some preliminary
experiments to test the activity of compound 1 with the macromolecule

1.- Affinity for DNA:

The affinity constant was determined by fluorescence, using the ethidium bromide
displacement technique. Compound 1 binds to DNA with a 50,000 M-1 affinity constant.


2.- Covalent binding:

The reaction of compound 1 with simple nucleophiles (methanol, 2-mercaptoethanol)
clearly indicated that 1 may act as an electrophile and this result prompted us to check if it
was able to covalently bind to DNA.

We used the following technique: compound 1 was incubated in water with calf thymus
DNA in the dark at 37¡C. The ratio Drug/DNA base pairs was close to 2. At different time
intervals, aliquots were taken. To remove the drug that was not covalently bound to
DNA, the aliquots were extracted with phenol and chloroform and DNA was then
precipitated. After re-solubilisation of the DNA in water, the same treatment was repeated
3 times. The amount of drug bound to the macromolecule was calculated from the UV
absorbance at 480 nm (maximum absorption for 11-aminobenzo[b][1,7]phenanthroline).

We observed an increase of the absorption at 480 nm with time, the maximum bein g
reached after 1 day of reaction as shown in the following scheme:

       

This result is still preliminary and has to be confirmed. But it shows that compound 1
may act as a DNA intercalating-alkylating agent.