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1,2,3‐Triazole as a Peptide Surrogate in the Rapid Synthesis of HIV‐1 Protease Inhibitors

Given the ubiquitous nature of the peptide linkage in biological molecules, replacement of the amide bond with isosteres in potential drug candidates has been a continual goal of many laboratories. Successful replacements will provide improved stability, lipophilicity, and absorption. Many surrogates have been introduced already, yet the synthesis of many of these isosteres in a combinatorial way is difficult and requires several steps. Thus, the discovery of new peptide surrogates with easier syntheses is an important achievement that could open new opportunities for the study of amide-containing molecules and the development of inhibitors with novel physicochemical properties. We have used the copper(i)-catalyzed azide–alkyne [3+2] cycloaddition as a straightforward reaction for the preparation of inhibitor libraries. Over 100 compounds were synthesized in microtiter plates and screened in situ. Two of these compounds—AB2 (pdb-1zp8) and AB3 (pdb-1zpA)—showed the best activity against wild type and mutant HIV-1 proteases (Table 1). AB2 and AB3, were then computationally docked by using AutoDock3. The docking simulation produced two conformations of approximately equal energy. One conformation placed the triazole in the position normally adopted by the peptide unit—between P2’ and P1’—in peptidomimetic compounds. Furthermore, the central nitrogen of the triazole was perfectly positioned to form a hydrogen bond with the water molecule normally found under the protease flaps. This water molecule also formed a hydrogen bond with the sulfonamide as seen in the crystallographic structure of amprenavir when bound to HIV-1 protease. The other conformation positioned the compounds in a similar place, but with the triazole rotated by 180 8. This allowed for a slightly better fit of the triazole substituent but sacrificed the hydrogen bond with the water molecule. In this work we have solved the ambiguity in binding conformation by solving the crystal structure of two inhibitors derived from a library of triazole compounds with HIV-1 protease. Interestingly, the two structures show that the triazole ring is an effective amide surrogate that retains all hydrogen bonds in the active site (Figure 1). HIV-1 protease (3 mgmL 1 in 0.025m sodium acetate pH 5.4, 10 mm dithiothreitol, 1 mm EDTA) was combined with inhibitor (32 mm in 50% (v/v) dimethylsulfoxide and 2-methylpentane2,4-diol) at 4 8C to give a 2:1 molar ratio of inhibitor to protein, and the mixture was centrifuged to remove the precipitate. The complex was crystallized by the hanging-drop vapor-diffusion method by mixing 9.6 mL of protease solution with 4 mL of crystallization buffer (1.34m ammonium sulfate, 0.1m sodium acetate, pH 4.8–5.4). Plates were sealed at 20 8C for one to two weeks. Data were collected from frozen crystals at the Argonne National Laboratory SER-CAT beamline 22-ID and with a Rigaku Table 1. Binding constants of 1,2,3-triazole compounds to HIV-1 protease.