Human melanoma cell lysates assay and structure-activity relationship. Successive generations of potential inhibitors were first screened against tyrosinase activity from human melanoma cell lysates. In a previous study, compound 1 was found only moderately active in this screening test (IC50 = 16.6 µM). A similar value was obtained from our slightly modified protocol (IC50 = 15 µM, Table ). The direct replacement of the HOPNO-embedded part of aurone 1 by a 2',4'-resorcinol-based B-ring was successful, as it led to a ~2.5-fold improved activity (IC50 = 6.4 µM for 5). As already seen with the previous HOPNO series, 4-hydroxy and 4,6-dihydroxy substitution patterns were slightly less effective (IC50 = 25 µM for 3, 7.7 µM for 12), as were several other alternative phenolic (IC50 = 19 µM for 4, 11.8 µM for 17) or naked (IC50 = 18 µM for 2) A-rings. If the methylation of the 6-hydroxy group maintained a similar potency (IC50 = 6.9 µM for 6), the introduction of a longer chain dramatically compromised the activity (IC50 >100 µM for O-butyl 7). Similarly, replacing the phenol group by a halogen or amino derivative at position 6 produced inactive analogues (IC50 >100 µM for 8-9), including 4,6-disubstituted compounds such as 13-15. Finally, the 7-hydroxy analogue 10 was found as the best inhibitor in the aurone series, with an IC50 value of 1.57 µM (a 10-fold gain of activity starting from compound 1). Modifying the nature of the substituent at position 7 and combining it with OH or OMe groups at position 6 afforded underwhelming results in all cases (IC50 >11.8 µM for 16-20). As expected, the well-known TYR inhibitor kojic acid was completely devoid of biological activity in the studied range of concentrations. Thus, given the superior potential of the 7-hydroxy substitution and, to a lesser extent, of the 6-hydroxy counterpart, we decided to explore the impact of C-ring modifications of 2, 5 and 10, first by replacing the intracyclic oxygen atom of the aurone skeleton with a carbon-, nitrogen or sulfur-based moiety. The methylene-based indanone series provided overall better results than the previous aurone series, and maintained the same order of inhibition potency (Table ): the 7-hydroxy analogue 23 (IC50 = 1.6 µM, vs. 1.57 µM for 10) still appeared as superior to the 6-hydroxy 22 (IC50 = 3.4 µM, vs.