A series of azepino[3',4':4,5]pyrrolo[2,1-a]isoquinolin-12-ones (3a-f), that were conformationally restricted analogs of lead compound 2, were designed as potential cytotoxic compounds and synthesized using a radical oxidative aromatic substitution reaction as the key step. Compounds 3a-f were tested on five tumor cell lines to determine the conformational requirements for biological activity of compound 2. The results show that conformational restrictions on compound 2, generating the derivatives 3a-f, do not appreciably reduce the cytotoxic activity of 2, although compound 3d (R=Br) showed good activity against U-251 cells. Preliminary structure-activity relationship studies with these compounds revealed the importance of halogens bonded to the isoquinoline moiety. Additionally, derivatives 3f (R=NO(2)) and 3b (R=F) were cytotoxic to PC-3 and K-562 cells. However, none of the azepino[3',4':4,5]pyrrolo[2,1-a]isoquinolinones inhibited the enzymatic activity of CDK1/cyclin B, CDK5/p25, or GSK-3.