Tankyrase 1 and tankyrase 2 are members of PARP family. They can use NAD+ as substrates to transfer ADP-ribose polymers onto target proteins. The tankyrases bind and PARSylate axin proteins which are the negative regulator of Wnt pathway. Thus tankyrasesa re promising targets for treatment for adenomatous polyposis coli. Tankyrase inhibitor 49 is an optimization of the previous hit compound inhibitor 8 with improved potency and selectivity. It has excellent effects in both tankyrase assay and cellular assay (total β-catenin degradation assay in SW480 cells) with IC50 values of 0.1 nM and 1.9 nM, respectively. In addition, it is found to be a dual binder with both the nicotinamide pocket and the induced pocket of the enzymes. In the in vivo studies in rodents, tankyrase inhibitor 49 is found to potently inhibit TNKS2 autoparsylation with IC50 = 7.6 nM. It also causes stabilization and accumulation of axin protein in SW480 cells with EC50= 4 nM. In DLD-1 cells with truncated APC, the inhibitor inhibits the STF reporter transcription with IC50 = 0.3 nM, suggesting its downstream inhibitory activity on Wnt-associated transcription.