A series of 58 3-arylisoquinoline antitumor agents were investigated for defining the pharmacophore model using comparative molecular field analysis (CoMFA) program. The studied compounds related to bioisostere of benzophenanthridine alkaloid were synthesized and evaluated for antitumor cytotoxicity against human lung tumor cell (A 549). In order to perform the systematic molecular modeling study of these compounds, the conformational search was carried out based on the single X-ray crystallographic structure of 7,8-dimethoxy-3-phenylisoquinolin-(2H)-one (2). Interestingly, two types of structures having different dihedral angles between the isoquinoline ring and 3-aryl ring were found in the crystals. Therefore, CoMFA was performed two different, overlapping ways. The alignments of the structures were based on the common isoquinoline ring and 3-aryl ring. The 3-D-quantitative structure-activity relationship study resulted in significant cross-validated, conventional r(2) values equal to 0.715 and 0.927, respectively.