However, substituting the intracyclic carbon with a methyl group led to a decrease of the anti-hsTYR effect (IC50 = 8.2 µM for racemic 24), while introducing a second carbonyl group caused a complete loss of activity (IC50 >100 µM for 25). Nitrogen-and sulfur-based counterparts, namely hemiindigos (HI) and hemithioindigos (HTI), produced mixed results, but generally managed to retain the inhibition potency of the best aurones and indanones (IC50 = 2.3-5.9 µM for 27-29). Surprisingly, the 7-hydroxy HTI 30 did not share the greater potency of aurone and indanone counterparts (IC50 = 48 µM, vs. 1.57-1.6 µM for 10 and 23). Finally, several derivatives were synthesized in order to identify elements that are crucial for activity in the hemiindigoid scaffolds (Figure ). The evaluation of chalcone and flavone analogues of 2 confirmed the need to retain a five-membered C-ring, as both compounds were found less active against melanogenesis (IC50 >60 µM for 31 and 32, vs. 18 µM for 2). Similarly, the removal of the exocyclic double bond or the replacement of the ketone at position 3 afforded inactive products in all cases (IC50 >100 µM for 33-37), highlighting the impact of hemiindigoid scaffolds themselves beyond substitution patterns. The B-ring resorcinol moiety was also questioned, but phloroglucinol analogues such as 38 and 4'-hydroxy-2'-methoxy derivatives such as 39 always led to a significant activity decrease, compared to parent compound 5 (IC50 = 15 µM and >100 µM, vs. Inhibition of purified human tyrosinase and in silico modeling. The next step was to confirm the biomolecular target of resorcinol-based hemiindigoids. Thus, the best compounds identified in the human melanoma MNT-1 cell lysates assay were evaluated in an inhibition test involving purified hsTYR. The expression of a construct of hsTYR (intra-melanosomal domain, residues 1-456 with a C-terminal HisTag) was performed in human HEK293T cells and the enzyme was purified using a HisTrap TM column. The obtained molecular weight was 60-75 kDa, a range attesting multiple glycosylations, as the amino-acid sequence alone is ~53 kDa. In addition, the use of a human expression system ensured the production of proper mammalian N-glycans, while only paucimannose structures are generated by insect cells. The kinetic parameters measured for hsTYR-mediated oxidation of L-DOPA were found as relevant (Km = 0.32 ± 0.01 mM, Vmax = 5.35 ± 0.10 µmol.min -1 .mg -1 , kcat = 5.95 s -1 , Figure ), in regard to those reported previously. Then, kinetic studies were performed with the two best compounds identified during the previous screening assay, 10 and 23, using L-DOPA as the substrate and a readout at 475 nm. Both molecules displayed a very high level of anti-hsTYR activity, associated with inhibition constants in the submicromolar range (Ki = 0.55 µM and 0.25 µM, respectively, Table , Figure ). Thus, the hemiindigoid series showed a similar inhibition potency as the reference thiamidol (Ki = 0.25 µM). In addition, as expected the inhibition mechanisms of compounds 10 and 23 were identified as partially competitive, with alpha values of 3.0-3.5, consistent with a direct binding to the dicopper center. In comparison, kojic acid was found as a very weak inhibitor (Ki = 1.1 mM) with a mixed mechanism, as already revealed in different hsTYR constructs and expression systems. Overall, these results tend to confirm hsTYR as the main target of the hemiindigoid series in the MNT-1 lysates assay. Thus, we relied on a homology model of hsTYR, based on the crystal structure of hsTYRP1, for proposing a binding geometry and a rationalization of interactions at the active site through molecular docking. The use of LeDock was endorsed by a recent report that pointed out the superior accuracy obtained by this docking software for metalloproteins. The best pose identified for compound 23 is represented in Figure . As expected, the phenol group at position 4' is predicted to interact directly with copper atom A. In addition, several hydrogen bonds could occur, between the phenol group at position 2' and the backbone of Ser375, or between the 7-hydroxy substituent and the carboxylate of Glu203 (Figure ). These elements could explain the higher potency of resorcinol compounds, and of 7-substituted compounds. The A-ring of hemiindigoid scaffolds was found well-positioned for the building of an edge-to-face π-stacking interaction with the aromatic ring of Phe347. Finally, the B-ring could establish a face-to-face π-stacking with His367, reminiscently of the interaction observed between the aromatic ring of L-tyrosine and His208 of tyrosinase from Bacillus megaterium in a reported crystallographic study (Figure ). Indeed, the superimposition of 23 with L-tyrosine was almost perfect, with a similar interaction with the metallic center and an identical aromatic ring orientation (Figure ). In addition, the intracyclic ketone of compound 23 was found very close to the carboxylate group of L-tyrosine. Human melanoma whole cells assay and cytotoxicity. As hsTYR was confirmed as the biomolecular target of resorcinol hemiindigoids, we next evaluated the potential of the two best inhibitors of the series to operate in cell-based environments. Indeed, compound 1 revealed a very low activity against whole MNT-1 cells, despite its promising profile against the isolated enzyme. The cytotoxicity of compounds 10 and 23 on MNT-1 cells was explored first, and no significant effect was recorded until ~100 µM. The CC50 values obtained (108 µM for 10 and 91 µM for 23, Table ) were consistent with previously reported polyhydroxylated aurones and provided room for operating concentrations in the micromolar range. Thus, the melanin content was measured after a 96 h incubation of MNT-1 whole cells with either 10, 23 or kojic acid. The hemiindigoids induced a 50% inhibition of melanogenesis at 32 µM and 29 µM respectively (Table ), a range of concentration where both compounds were found completely non-cytotoxic (see Supporting Information). While these values are still higher than those obtained from the MNT-1 lysates assay, compounds 10 and 23 exhibited a ~3-fold improvement of cellular activity, compared to the parent compound 1. It is worth noting that kojic acid was found as a far weaker inhibitor in this assay, yielding an IC50 of 15 mM. Altogether, these results demonstrate a correlation between the values obtained from assays involving the purified hsTYR enzyme, MNT-1 cell lysates and MNT-1 whole cells, and endorse the development of more active hsTYR inhibitors as a way to find more efficient melanogenesis suppressors in cellbased environments.