Ribonucleotide reductase (RR), an established cancer target, is usually inhibited by antimetabolites, which display multiple cross-reactive effects. Recently, we discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH or E-3a) of human RR (hRR) binding at the catalytic site (C-site) and inhibiting hRR reversibly. We herein report the synthesis and biochemical characterization of 25 distinct analogs. We designed each analog through docking to the C-site of hRR based on our 2.7 Å X-ray crystal structure (PDB ID: 5TUS). Broad tolerance to minor structural variations preserving inhibitory potency is observed. E-3f (82% yield) displayed an in vitro IC50 of 5.3 ± 1.8 μM against hRR, making it the most potent in this series. Kinetic assays reveal that E-3a, E-3c, E-3t, and E-3w bind and inhibit hRR through a reversible and competitive mode. Target selectivity toward the R1 subunit of hRR is established, providing a novel way of inhibition of this crucial enzyme.