Several chemical strategies have been developed to affect redox homeostasis by HIFs. However, broad-spectrum activities and off-target effects of e.g. proteasome inhibitors or iron chelators provided the impetus for an alternative approach, which is based on interfering with the binding of HIF-1α to VHL. This strategy relies on the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes and upstream of proteasomal degradation. Small-molecule VHL binders can act as competitors to the native substrate HIF-1α and stabilize HIF-1α levels, upregulating genes involved in the hypoxic response, consequently affecting hypoxia signaling. The successful development of VHL inhibitors has emphasized the importance of VHL as a therapeutic target for the treatment of conditions that occur in anemia, ischemic, inflammatory or mitochondrial diseases. The rational design of breakthrough VHL inhibitors exploited the structure of the native substrate, i.e. hydroxylated HIF-1α, and its molecular recognition by VHL as a starting point. The first co-crystal structure (PDB 1LM8) of a 20-residue HIF-1α peptide bound to the VHL-EloC-EloB complex (VCB) showed HIF-1α in an extended β strand-like conformation. Hydroxyproline Hyp564, which originated from post-translational hydroxylation, was inserted into a groove in a hydrophobic core formed by buried, mostly aromatic residues. 17 Hyp564 comprised the essential element for the recognition of HIF-1α derivatives by VHL and served as a central motif for the design of new VHL ligands. The molecular scaffold was extended to both sides of Hyp564 by appending right-hand side (RHS) fragments at the carbonyl group, and left-hand side (LHS) fragments at the nitrogen of Hyp564. Representative compounds that bind to and inhibit VHL are exemplified in Figure . Ligands I and II already contained an RHS benzylamine moiety equipped with a five-membered heteroaromatic ring, a feature that was maintained in the course of further structural optimizations. In ligand II, an anilinic LHS fragment was introduced, as well as methylthiazole, a characteristic RHS moiety found in several potent VHL inhibitors. Further structure-activity relationship (SAR) studies identified a tert-butyl residue to be advantageous as part of the LHS fragment. Acetylated amino acids other than tert-leucine in VH032 caused a reduced binding affinity to VHL. A constrained cyclopropyl ring with a cyano group or a fluorine substituent was employed, leading to the VHL inhibitors VH298 and VH101, respectively, with VH298 being a widely used benchmark compound, while application of VH101 is limited due to its cytotoxicity. Bioisosteric O-to-S replacements have been performed, e.g. to achieve the thioamide derivative III, which showed reduced affinity to VHL in comparison to its counterpart VH032. By introducing fluorohydroxyprolines, other derivatives of VH032 were generated, and all four 3-fluoro-4-hydroxyproline stereoisomers, e.g. the (3R,4S)-configured derivative IV, were investigated to study the influence of hydroxyproline fluorination on VHL binding. Trifluoromethyl groups were attached at different positions of the inhibitor scaffold, for example in reporters V and VI, to be used as F NMR spy molecules for the hydroxyproline binding site of VHL. An additional beneficial contribution arose from the stereoselective methylation at the benzylic position within the RHS fragment, and VII exhibited an improved IC50 value in comparison to the parent VH032. These examples of VHL inhibitors highlight a route of continuous improvement through structure-and SAR-based optimization. The developed compounds have been demonstrated to be applicable chemical probes and have been successfully employed to assemble proteolysistargeting chimeras (PROTACs), heterobifunctional molecules capable of exploiting the UPS machinery for targeted protein degradation. However, there are still further opportunities to improve the binding affinity of VHL ligands. In particular, the SAR of the RHS phenylene core has remained largely unexplored so far. In this study, we aimed to investigate a combinatorially generated library of VHL ligands with high structural diversity at the phenylene core. We conceived a structure-guided and bioactivity-driven design and devised a systematic survey to analyze the chemical space of VHL ligands.