The two binding modes observed for 5 in VRK1 suggested that the 2-amino moiety has no binding preference for each of the hinge carbonyl groups it can interact with (Figures and). This led us to hypothesize that these two interactions were either equally productive or equally weak in the binding process. To address these hypotheses, we synthesized the following analogues: (i) 23, with two amino groups that could interact with both hinge carbonyl groups simultaneously; (ii) 24, with a 2-amino and a space-filling 6-methyl group; (iii) 25, with the 2amino group removed; and (iv) 26 with the 2-amino group substituted by a 2-methyl group (Table ). The last two compounds were synthesized to eliminate the desolvation penalty of the 2amino group.