In this study, the pyrrolo[2,1- a ]isoquinolines 4a – n were synthesized in good yields in a three steps synthesis from the corresponding α,β-unsaturated esters starting materials. These compounds were tested on six human cancer cells lines to measure the cytotoxic activity as a function of the electronic properties and aromaticity of the substituent at the C-2 position of the pyrroloisoquinoline. Our results reveal that the cytotoxic activity could be explained in terms of the distribution of electronic density across the ring joined to C-2. Also, this study identified 3-hydroxy ( 4d ) and 3-chloro ( 4j ) derivatives with powerful cytotoxic activities. The IC₅₀ values of these compounds were found to be comparable to those of the commercially available Topotecan, Irinotecan, Etoposide, Tamoxifen, and Cisplatin.