The correlation of binding affinity and cellular potency of selected compounds is shown in Figure . The monofluoro substitution pattern of 8 was maintained en route to 25 and 27, including either the benzylic (S)-methylation or cyano-to-fluoro replacement. These modifications were accompanied by ~2-fold lower binding affinity, yet consistently higher capability for HIF-1α stabilization (Figure ), probably arguing for a better cell permeability in cases of 25 and 27 compared to 8. The readjustment of the second point of diversity resulted in 31 with improved properties. The second design pathway (Figure ) provided an even clearer picture. Both structural modifications were additive and advantageous for VHL affinity as well as cellular HIF-1α stabilization. In compound 30, both features were realized to generate an exceptional VHL inhibitor capable of most potently increasing the intracellular level of HIF-1α. Moreover, we characterized 30 as an efficacious, small molecule inhibitor of VHL inducing the HIF transcriptional activation pathway and demonstrated the superiority of 30 above and beyond both previously reported VHL ligands and all other new ones reported herein. Inhibitor 30 is expected to serve as a valuable tool compound to study the hypoxia signaling pathway, which is predominantly governed by HIF-1α regulation. Inhibitor 30 may also act as a lead compound for developing drugs to be applied under conditions where the adaptive cell response against hypoxic conditions would be beneficial. Our work aimed to employ an extended series of compounds to scan the ligand binding site of VHL. An additional essential facet of this study arose from the opportunity to utilize the structural variability of ligands for expanding the repertoire of VHL-recruiting PROTACs. We explored SARs of ligands whose LHS-terminal amino moiety is blocked by the well-described fluoro-or cyanocyclopropyl capping group. However, there are further exit vectors on VHL ligands accessible for linker attachment which can be employed for PROTAC design. Such approaches, beyond the linkage at the LHS amino moiety or at a phenolic group of the RHS phenylene core, have recently emerged as attractive strategies to efficiently hijack VHL. These alternative exit vectors include a thioether connection at the side chain of tert-leucine or the appendage of the linker via an acetamide portion at the benzylic position. For future PROTAC design, such linker attachments might be pursued by exploiting the structure of the optimized VHL ligand 30. The assembly of so-designed compounds is currently under investigation in our laboratories.