Correlation of indoleamine-2,3-dioxigenase 1 inhibitory activity of 4,6-disubstituted indazole derivatives and their heme binding affinity

[Display omitted] •4,6-Disubstituted indazoles we synthesized had stronger binding to ferrous heme than to ferric heme of IDO1.•We are the first to report a strong correlation between the heme-binding affinity and IDO1 inhibitory activity.•Good fit of the 6-substitutent of the indazole in Pocket-A of IDO1 is important for high inhibitory activity against IDO1. Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that acts on the first and rate-limiting step of the tryptophan/kynurenine pathway. Since the pathway is one of the means of cancer immune evasion, IDO1 inhibitors have drawn interest as potential therapeutics for cancers. We found a 4,6-disubstituted indazole 1 as a hit compound that showed both IDO1 inhibitory activity and binding affinity for IDO1 heme. Structural modification of 1 yielded compound 6, whose relatively large substituent at the 4-position and proper size substituent at the 6-position were found to be important for the enhancement of IDO1 inhibitory activity and heme affinity. A series of compounds synthesized in this work were evaluated by in silico docking simulations and by in vitro experiments using a C129Y mutant of the pocket-A of IDO1. Our results revealed that proper substituents at the 6- and 4-positions of the compounds interact with pockets A and B, respectively, and that, in particular, a good fit in pocket-A is important for the compounds’ biological activities. Absorption spectral analysis of these compounds showed that they strongly bound to the ferrous heme rather than its ferric heme. Furthermore, we observed that the heme affinities of these compounds strongly correlate with their IDO1 inhibitory activities.