When investigating the literature-reported HPK1 inhibitors (Fig. ), we found that there were usually large gaps between the enzymatic and cellular activities, which could result from the low apparent ATP Km of HPK1 (reported as 8.9 μM) . Thus, high potency might be of great importance for a HPK1 inhibitor to compete with high concentrations of cellular ATP so as to attain adequate target engagement. In light of this, we turned our attentions to a set of highly potent isoquinoline-3,8-diamine HPK1 inhibitors, which were previously disclosed in Genetech's patent and later reevaluated by Lacey et al . To better understand the binding mode of this chemotype, we selected compound 6 (Fig. ) as a representative and applied it to a molecular docking study. As Fig. depicts, the docking model suggested a type Ⅰ (DFG-in) binding mode, the isoquinoline-3,8-diamine hinge-binding motif could form classic donor-acceptor-donor hydrogen bonds with hinge residues Glu92 and Cys94, the nitrogen atom in 4-methylpyridine moiety could also interact with Lys46, and the cyclopropanecarbonyl group was positioned near the solvent-exposed region. Inspired by this amino-6,6-heterocyclic bicycle, we embedded a nitrogen atom in isoquinoline-3,8-diamine and generated the quinazoline-2,5-diamine skeleton (Fig. ). The subsequent SAR explorations would be performed at 2-amino group (substituent designated as R 1 ) and C7 position (substituent designated as R 2 ). Initially, to test the compatibility of carbonyl group with our quinazoline-2,5diamine core, we synthesized 7a-the quinazoline-2,5-diamine analogue of 6 (Table ). Disappointingly, while the IC50 value of compound 6 was measured as 3.1 nM in our internal ADP-Glo kinase assay, 7a exhibited an extremely low HPK1 inhibitory rate at 100 nM. To find out the intrinsic cause of such a great activity drop, we carried out a molecular modeling study using energy-dihedral angle scan, and the later result suggested a distinct conformational difference between 6 and 7a. While the lowest energy conformer of 6 harbored a trans-amide that identical to the predicted bioactive conformer (Fig. and Fig. ), 7a preferred to adopt a cis-conformation in its amide moiety (Fig. ). More importantly, conversion between the lowest energy conformation and bioactive conformation of 6 was free owing to the high similarity, however, the energy barrier was nearly 11.6 kcal/mol for 7a to accomplish such a conformational change, which means the energy penalty for the binding of 7a to HPK1 was much higher than that of 6 (when calculating, we assumed that the conformations of cyclopropanecarboxamide moiety in the bioactive conformer of 6 and 7a were identical; see Fig. for the contour plot of potential energy of 6 and 7a). We speculated that this conformational difference was attributed to repulsion of lone pair electrons between quinazoline N1 atom and amide O atom.