The tankyrases are poly(ADP-ribose)polymerases that are overexpressed in many cancers. They operate through three distinct mechanisms; they regulate the elong¬ation of telomeres, are a feature of Wnt-signalling (aberrant in many cancers) and are vital for the correct functioning of the mitotic spindle during mitosis. Thus the tank¬yrases are an intriguing target for a new therapy. A library of iso¬quin¬olin-1-ones with a variety of 3-substituents was synthesised using Pd-catalysed couplings, to probe the nico¬tin¬amide-binding site and adjacent hydro¬phobic pocket. Positions-4 and -5 were also accessed using carbanion chemistry. Preliminary ranging studies were conducted against tankyrase-2. Key Structure-Activity Relationship (SAR) features for potent inhibition included a para-substituted 3-aryl group and a small hydrophobic group in position-5. New inhibitors were des¬ig¬ned with extensions and steric bulk in the 3-aryl region, designed to reach further into and test the capacity of the hydrophobic pocket. Removal of the 5-amine improved potency but worsened water-solubility. This was re-introduced through a basic tertiary aliphatic amine tethered to the 3-phenyl group.Molecular modelling and small-molecule crystal structures facilitated the under¬standing of the SAR. Steric clashes were apparent for inhibitors with bulky ortho-substituted 3-aryl groups, whereas para-substituted 3-aryl groups fitted well into the hydrophobic pocket. Although ortho-substituents on the 3-aryl group confer a dihedral twist between the isoquinolin-1-one and 3-aryl group, this is not responsible for the loss of activity. Fifteen isoquinolin-1-ones were selected for fuller biological evaluation, based on potent activity, SAR features and superior pharmaceutical properties. IC50 values were measured against tankyrases-1 and -2 and a counter-screen conducted against PARP-1. A number of analogues were highly anti-proliferative in the human colon carcinoma HT29 cells and human FEK4 fibroblasts; however, inhibition of tankyrases did not always correlate with this activity. A subset of potent tankyrase-2 inhibitors demonstrated good selectivity for HT29 over FEK4 cells; these also showed good inhibition of PARP-1.