showing slightly reduced potency with IC 50 values of 11.97 μM and 13.95 μM, respectively. When mono-halogen groups such as chlorine and fluorine were introduced (compound 8 and 9), they also slightly reduced potency if comparison with compound 5. However, single trifluoromethyl-substituent and methyl-substituent compounds (10 and 11) showed equal potency to compound 5. When another methyl group was introduced to yield compound 12, the potency was significantly improved to an IC 50 value of 0.57 μM, about 8-fold more potent than of compound 5. This encouraged us to further explore the effect of other parts of this chemotype inhibitors on NSD2-PWWP1 binding. To verify the design, we performed the crystallization experiment and solved the cocrystal structure of NSD2-PWWP1 domain with compound 5. As illustrated in Figure , compound 5 bound to the NSD2 in a mode very similar to BI-9321 bound to the NSD3: the N-methylimidazole situated into the aromatic cage and the benzylamine group interacted with the nearby Glu272; while the benzonitrile motif represents as an essential pharmacophore for PWWP1 of NSD2, which, as predicted, interacts with residues Asp269 and Ala270. Clearly, the nitrile group would be collided with the serine residue in NSD3.