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A novel scaffold for HIV protease inhibitors based on a pyrrolidine core was developed using the co-crystal structure of a previously designed inhibitor in complex with the HIV-1 protease. The enantioselective preparation of 3,4-disubstituted pyrrolidines could be accomplished via a chiral-pool approach employing L-(+)- and D-(-)-tartaric acids as starting materials. On the basis of 3,4-pyrrolidine-diol as core structure, several diesters were prepared as a first series of inhibitors. The synthesis of 3S,4S-diamino-pyrrolidine via the corresponding diazide was optimized using a BOC-protection strategy. The facile and straightforward synthesis of this core structure gave way to the further decoration of the scaffold.
Utilization of arylsulphonamides as H-bond acceptor groups allowed the further introduction of substituents at the sulphonamid nitrogen. Hence, N-alkylation with reactive electrophiles like benzyl as well as allyl bromides followed by deprotection of the pyrrolidine nitrogen furnished compounds equipped with four substituents suitable to address the specificity pockets of the enzyme. The opportunity to introduce these moieties during the last steps of the synthesis enabled us to selectively vary the respective residues. Due to the enantioselective inhibitor synthesis employed, the unambiguous interpretation of the biological activity obtained for every tested inhibitor was possible. Usage of an optimized synthetic strategy as well as the obtained structure-activity relationship allowed the further optimization of the inhibitors.
The first series of inhibitors only showed affinity in the micromolar range against the HIV protease, but nevertheless yielded a first structure-activity relationship thus enabling us to postulate a putative binding mode. Although most inhibitors in this series only showed rather weak binding affinities, it is remarkable that one representative out of these inhibitors exhibited an affinity of 18 µM, which is striking for such a small molecule. Decoration of the pyrrolidine core with two benzyl moieties served as the starting point for the further development of putative inhibitors.
Starting from 3S,4S-diamino-pyrrolidine, condensation with benzene sulphonamide and subsequent alkylation with benzyl bromide yielded, after N-deprotection, the first inhibitor. The affinity of this compound was significantly better than the best representative of the first inhibitor series. The analysis of the obtained co-crystal structure in complex with the protease allowed the further structure-based optimization of this class of inhibitors.
Further substitution at the aromatic ring substituents in ortho or para position was chosen as optimization strategy. The resulting inhibitors of the 2nd generation all exhibited a considerably higher affinity compared to the previous lead compound. Analysis of several co-crystal structures and comparison with the initial crystal structure revealed a conserved binding mode and proved the higher affinity being a consequence of the predicted interactions.
Based on the crystal structures and the affinity data, the best combination of substituents was selected for the synthesis of the 3rd generation of inhibitors. The corresponding inhibitors exhibited affinities up to the two-digit nanomolar range. The determination of the crystal-structures in complex with the enzyme showed a binding mode comparable to those of similarly substituted representatives of the 2nd generation.
The inhibitors were also tested against mutant variants of the HIV protease and showed a intriguing activity-profile. The mutation of Isoleucin 84 to Valine, which is observed during antiviral chemotherapy with approved HIV protease inhibitors, dramatically reduces the affinity and clinical efficacy of all approved protease inhibitors. Remarkably, the pyrrolidine-based inhibitors even show an improved potency against this mutant in comparison to the wild-type. This phenomenon can easily be explained on a structural basis: The pyrrolidine-based inhibitors address the enzyme’s S1 and S1’ specificity pockets differently in comparison to peptidomimetic inhibitors, thus resulting in additional van der Waals contacts, additional polar interactions, and a better surface match.