Compounds synthesized were tested for their inhibitory activity against digestive vacuole plms II and IV, endoplasmic reticulum protease plm V, and selectivity cross-marker -human catD (see SI for experimental details). The plm II inhibition potency (IC50) of the compounds tested varied in a range from 3.5 µM down to 64 nM depending on the length of hydrophobic substituent and position of aromatic ring (see Figure and Table for full inhibition data). There were minor variations between plm II and plm IV inhibition potencies, whereas the potency against plm V and human catD was considerably lower. The catD inhibition potency followed plms II and IV inhibition pattern with 10 to 70 fold lower activities (55 to 2 µM), whereas potency against plm V varied in a narrow range (90 to 25 µM) and size of the flap pocket substituent seems to play no role in inhibitor activity against plm V. More detailed analysis of structure activity relationships (SAR) of the inhibitor series indicates that installation of the flap pocket substituent greatly increases the inhibitor potency (15-fold activity increase from compound 1 to 2). By increasing the distance between the ligand core (2-aminoquinazolin-4(3H)-one group) and the terminal aromatic ring, the inhibitory activity initially decreases, reaching lowest activity for inhibitors with methylene and ethylene linker (3, 4), whereas further increase of linker length led to increase in inhibition potency. The highest potency was achieved for the compound bearing phenylpentyl group (7). Linker length increase beyond pentylene group led to reduced activity (8, 9). The inhibition potency of the compound 10 suggests that presence of the terminal aromatic group in the flap pocket substituent is not necessary for high activity, as inhibition potency of both compounds (7 and 10) was comparable. A ligand subset (11-15) bearing the linkers with the same overall length as compound 7, but varied in positions of aromatic ring was tested in order to understand, whether the activity of the inhibitor is affected solely by the length of the hydrophobic flap pocket substituent or position of the aromatic ring. The general inhibitory activity trend was the same as in initial inhibitor set, however, these inhibitors were 5 to 20 times more potent. The last ligand subset had terminal benzene ring fixed at a specific distance, and an additional aromatic ring was installed in the flap pocket substituent to introduce stacking interactions with Trp41, Tyr77, Phe111, Tyr115 and/or Phe120. Inhibitors bearing two benzene rings in the flap pocket substituent showed low micromolar/nanomolar potency, however, no clear SAR was observed for these compounds.