Evaluation of Larger Side‐Group Functionalities and the Side/End‐Group Interplay in Ritonavir‐Like Inhibitors of <scp>CYP3A4</scp>

A new series of 13 ritonavir-like inhibitors of human drug-metabolizing CYP3A4 was rationally designed to study the R₂ side-group and R₃ end-group interplay when the R₁ side-group is represented by phenyl. Spectral, functional, and structural characterization showed no improvement in the binding affinity and inhibitory potency of R₁/R₂-phenyl inhibitors upon elongation and/or fluorination of R₃-Boc (tert-butyloxycarbonyl) or its replacement with benzenesulfonyl. When R₃ is pyridine, the impact of R₂-phenyl-to-indole/naphthalene substitution was multidirectional and highly dependent on side-group stereo configuration. Overall, the R₂-naphthalene/R₃-pyridine containing 2f (R/S) was the series lead compound and one of the strongest binders/inhibitors designed thus far (K_s = 0.009 μM; IC₅₀ = 0.10 μM). Introduction of a larger biphenyl or fluorene as R₂ did not lead to any improvements. Contrarily, fluorene-containing 13 was the series weakest binder and inhibitor (K_s = 0.734 μM; IC₅₀ = 1.32 μM), implying that the fluorene moiety is too large to allow unrestricted access to the active site. The R₂-biphenyl, however, can switch positions with R₃-Boc to enable heme ligation. Thus, for small and chemically simple end-groups such as Boc and pyridine, the R₂/R₃ interplay could lead to conformational rearrangement that would be difficult to foresee without structural information.