EWS-FLI1 is an oncogenic fusion protein implicated in the development of Ewing's 
sarcoma family tumors (ESFT). Using our previously reported lead compound 2 
(YK-4-279), we designed and synthesized a focused library of analogues. The 
functional inhibition of the analogues was measured by an EWS-FLI1/NR0B1 
reporter luciferase assay and a paired cell screening approach measuring effects 
on growth inhibition for human cells containing EWS-FLI1 (TC32 and TC71) and 
control PANC1 cell lines devoid of the oncoprotein. Our data revealed that 
substitution of electron donating groups at the para-position on the phenyl ring 
was the most favorable for inhibition of EWS-FLI1 by analogs of 2. Compound 9u 
(with a dimethylamino substitution) was the most active inhibitor with GI50 = 
0.26 ± 0.1 μM. Further, a correlation of growth inhibition (EWS-FLI1 expressing 
TC32 cells) and the luciferase reporter activity was established (R(2) = 0.84). 
Finally, we designed and synthesized a biotinylated analogue and determined the 
binding affinity for recombinant EWS-FLI1 (Kd = 4.8 ± 2.6 μM).