3.4. In Vitro Anticancer Activity

All the newly synthesized compounds were screened for their in vitro anticancer activity against six cancer cell lines: SK-MEL-2, MCF-7, IMR-32, MG-63, HT-29 and Hep-G2 by SRB assay, using adriamycin as a standard drug [57]. All the synthesized derivatives 4(a–n) were also tested for their cytotoxic effect on normal cell lines i.e., NIH/3T3 (murine embryonic fibroblast by the SRB assay method. The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM l-glutamine. For the present screening experiments, cells were inoculated into 96 well microtiter plates in 90 µL at 5000 cells per well. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO₂, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. Experimental drugs were solubilized in appropriate solvent to prepare stock of 10⁻² concentration. At the time of experiment four 10-fold serial dilutions were made using complete medium. Aliquots of 10 µL of these different drug dilutions were added to the appropriate micro-titer wells already containing 90 µL of medium, resulting in the required final drug concentrations. After compound addition, plates were incubated at standard conditions for 48 h. and assay was terminated by the addition of cold trichloroacetic acid (TCA). Cells were fixed in situ by the gentle addition of 50 µL of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant was discarded; the plates were washed five times with tap water and air dried.

Sulforhodamine B (SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 min at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on an ELISA plate reader at a wavelength of 540 nm with 690 nm reference wavelength. All the tests were repeated in at least three independent experiments at the concentrations of 10, 20, 40 and 80 µg/mL [57].