The azanaphthoquinone annelated pyrrole derivatives were synthesized and characterized with an effort to develop novel DNA intercalating agents as anticancer compounds. To model the binding of these compounds in the intercalation site of the DNA duplex, molecular dynamics (MD) simulations using AMBER program were performed. Moreover, molecular mechanics/Poisson–Boltzmann surface area method was used to calculate the binding free energy of azanaphthoquinone annelated pyrrole derivatives in the intercalation site of the DNA duplex. On the basis of MD simulations, the π–π stacking interaction of azanaphthoquinone annelated pyrrole derivatives with base pairs of C and G is the crucial interaction for binding of these DNA intercalating agents. Moreover, the hydrogen bond interaction of side chain could enhance the binding affinity of azanaphthoquinone annelated pyrrole derivatives in the intercalation site. Therefore, the obtained results should be helpful for better understanding the binding of azanaphthoquinone annelated pyrrole derivatives in the intercalation site. © 2012 Wiley Periodicals, Inc.