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This work is a continuation of our initial discovery of a potent monocyclic carbamoyl pyridone human immunodeficiency virus type-1 (HIV-1) integrase inhibitor that displayed favorable antiviral and pharmacokinetic properties. We report herein a series of bicyclic carbamoyl pyridone analogues to address conformational issues from our initial SAR studies. This modification of the core unit succeeded to deliver low nanomolar potency in standard antiviral assays. An additional hydroxyl substituent on the bicyclic scaffold provides remarkable improvement of antiviral efficacies against clinically relevant resistant viruses. These findings led to additional cyclic tethering of the naked hydroxyl group resulting in tricyclic carbamoyl pyridone inhibitors to address remaining issues and deliver potential clinical candidates. The tricyclic carbamoyl pyridone derivatives described herein served as the immediate leads in molecules to the next generation integrase inhibitor dolutegravir which is currently in late stage clinical evaluation.

HIV-1 integrase (IN) is one of three enzymes encoded by the HIV genome and is essential for viral replication, and HIV-1 IN inhibitors have emerged as a new promising class of therapeutics. Recently, we reported the synthesis of orally bioavailable azaindole hydroxamic acids that were potent inhibitors of the HIV-1 IN enzyme. Here we disclose the design and synthesis of novel tricyclic N-hydroxy-dihydronaphthyridinones as potent, orally bioavailable HIV-1 integrase inhibitors displaying excellent ligand and lipophilic efficiencies.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

HIV-1 integrase (IN) catalyzes the integration of viral DNA into the host genome, involving several interactions with host proteins. We have previously identified peptide IN inhibitors derived from the alpha-helical regions along the dimeric interface of HIV-1 IN. Herein, we show that appropriate hydrocarbon-stapling of these peptides to stabilize their helical structure remarkably improves the cell permeability, thus allowing inhibition of the HIV-1 replication in cell culture. Furthermore, the stabilized peptides inhibit the interaction of IN with the cellular cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed in four different cell lines using a fluorescein-labeled analogue. Given their enhanced potency and cell permeability, these stapled peptides can serve as not only lead compounds of novel HIV-1 IN inhibitors but also prototypical biochemical probes or ‘nanoneedles’ for the elucidation of HIV-1 IN dimerization and host co-factor interactions within their native cellular environment.

We extended the previously described dynamic pharmacophore model studies of HIV-1 integrase (IN) by considering more key residues in the active site, including Mg2+. First, we applied a Monte Carlo sampling method to map the complementary features of the IN binding surface. Two types of dynamic pharmacophore models were generated. One considers Mg2+ as part of the IN and therefore as an excluded volume, and the other treats Mg2+ as a positively charged feature, representing a new type of pharmacophore model aimed to identify compounds potentially preventing Mg2+ binding. Second, we validated the models with 385 known active (IC5020 microM) and 235 (IC50100 microM) inactive IN inhibitors. Third, we used the derived models to screen our small molecule database. Twenty-two structurally novel compounds were tested in an in vitro assay specific for IN, and two of them showed IC50or = 10 microM for strand transfer reaction.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.