The 34 identified molecules were subjected to reaction condition screening using a 24-well setup (Figure ) resulting in the formation of the desired product with ≥ 5% for all 34 selected substrate combinations. Based on these results, the most high-yielding examples were prioritized and, consequently, scale-up experiments for 14 novel MAGL inhibitors were performed following the synthesis route outlined in Figure (The detailed syn-thesis information can be found in SI5). By employing our late-stage Minisci-type building block alkylation procedure, we reduced the number of synthesis steps of the alkylated intermediate from the proposed seven steps required by a classical synthesis approach (orange) to just three steps (blue). This shortened route improved synthesis efficiency, leading to reduced cycle times. Subsequently, the left-hand side building blocks were coupled with various right-hand side residues, yielding 14 new compounds in a streamlined manner . The inhibitory potency of all newly synthesized compounds was evaluated in a biochemical assay against the human, mouse, rat and cynomolgus macaque MAGL enzyme. Compared to the human IC 50 value of starting point, hit compound 17 (IC 50 = 445 nM), all newly synthesized compounds exhibited enhanced potency (Table ). Six compounds To confirm the ligand binding mode, the crystal structures of four inhibitor-MAGL complexes (17, 23, 27, and 29) were determined (Figure ). These co-crystal structures reveal that the addition of cyclopentyl and cyclopropyl-cyclobutyl groups at the para position of the pyridine induces a flip of the pyridine, enabling a For the three compounds (23, 27, and 29), additional physicochemical and ADMET properties were obtained (SI2.2). High selectivity for MAGL over common safety-relevant off-targets and closely related brain hydrolases was confirmed through CEREP (SI2.3) and activity-based protein profiling (ABPP) (SI2.4), respectively.