Rapid diversity-oriented synthesis in microtiter plates for in situ screening of HIV protease inhibitors.

Since the early days of the discovery of HIV-1 protease (HIV-1 PR), this enzyme has been selected as an important target for the inhibition of viral replication. The enormous effort over the past two decades to develop effective molecules that inhibit the HIV1 PR has resulted in the discovery of drugs that have dramatically improved the quality of life and survival of the patients infected with HIV-1. To date there are six different HIV-1 PR inhibitors (PI) that are commercially available. These drugs are administered in combination with the reverse transcriptase inhibitors in what is called TMhighly active anti-retroviral therapy (HAART)∫. Unfortunately, many drug-resistant and cross-resistant mutant HIV-1 PRs have been identified, thus hampering long term suppression of the virus and resulting in return of AIDS symptoms. Therefore, the development of new protease inhibitors, which are efficacious against both the wild type and drug resistant HIV-1 PR and less prone to development of resistance, is urgently needed. During the last decade, the number and throughput of biological assays of protease activity has notably increased. However, the high rates of HIV-1 PR mutation still outpace conventional drug discovery efforts, mostly because of limitations associated with identification of the lead structures and, to a greater extent, slow structure ± activity profiling. While the former can be improved by rational design and computational studies, rapid synthesis of diverse analogues and their optimization still remains a challenge. We have recently developed a new strategy to facilitate the drug discovery process: diversityoriented organic synthesis in microtiter plates followed by in situ screening without product isolation and protecting group manipulation. This strategy was demonstrated with the use of amide-forming reaction in a rapid identification of new potent HIV protease inhibitors. Click chemistry has emerged as a strategy for the rapid and efficient assembly of molecules with diverse functionality on both laboratory and production scales. Enabled by a few nearly perfect reactions, it guarantees reliable synthesis of the desired products in high yield and purity. Modularity, selectivity, and wide scope make click chemistry ideal for achieving diversity in just a few steps and with no need for further purification. Advantages of click chemistry in biological studies have recently been demonstrated in several applications: construction of fluorescent oligonucleotides for DNA sequencing, in situ assembly of acetylcholinesterase inhibitors, chemically orthogonal high fidelity bioconjugation, and activity-based protein profiling in whole proteomes. In principle, this type of chemistry is well suited for microscale synthesis and for biological screening in situ. To demonstrate its feasibility we have used the copper(I)-catalysed triazole formation for the synthesis of sugar arrays in the above mentioned microtiter plate format, followed by in situ screening of glycosyltransferase inhibitors and enzyme glycosylation. Herein, we report an expedient approach to the discovery of novel HIV-1 PR inhibitors based on the latest advance in the copper(I)-catalyzed 1,2,3-triazole synthesis. 11] This highly reliable process, which proceeds well in aqueous solvents and tolerates virtually all functional groups without the need for protection, made it possible to quickly generate the desired libraries of potential inhibitors and to screen them directly in microtiter plates, without any purification, against HIV-1 PR and its mutants. The efficacy of hydroxyethylamine isosteres as transition-state mimics and as backbone replacements of amide bonds in the P1/P1 position of aspartyl protease inhibitors has been well documented, most notably in incorporation in the structures of three commercially available drugs, amprenavir, nelfinavir, and saquinavir. We, therefore, envisioned a library of compounds which retained this core, while diversifying the P2/P2 residues to generate new inhibitors. Starting from the optically active epoxy amine 1, two different azide cores were prepared as summarized in Scheme 1. Epoxy amine 1 in H2O/EtOH was