During the integration step, HIV-1 integrase (IN) interacts with viral DNA and the cellular cofactor LEDGF/p75 to effectively integrate the reverse transcript into the host chromatin. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a new class of antiviral agents that bind at the dimer interface of the IN catalytic core domain and occupy the binding site of LEDGF/p75. While originally designed to block IN-LEDGF/p75 interactions during viral integration, several of these compounds have been shown to also severely impact viral maturation through an IN multimerization mechanism. In this study, we tested the hypothesis that these dual properties of ALLINIs could be decoupled toward late stage viral replication effects by generating additional contact points between the bound ALLINI and a third subunit of IN. By sequential derivatization at position 7 of a quinoline-based ALLINI scaffold, we show that IN multimerization properties are enhanced by optimizing hydrophobic interactions between the compound and the C-terminal domain of the third IN subunit. These features not only improve the overall anti-viral potencies of these compounds but also significantly shift the ALLINIs selectivity toward the viral maturation stage. Thus, we demonstrate that in order to fully maximize the potency of ALLINIs, the interactions between the inhibitor and all three IN subunits need to be simultaneously optimized.

Allosteric HIV-1 integrase (IN) inhibitors, or ALLINIs, are a new class of antiviral agents that bind at the dimer interface of the IN, away from the enzymatic catalytic site and block viral replication by triggering an aberrant multimerization of the viral enzyme. To further our understanding of the important binding features of multi-substituted quinoline-based ALLINIs, we have examined the IN multimerization and antiviral properties of substitution patterns at the 6 or 8 position. We found that the binding properties of these ALLINIs are negatively impacted by the presence of bulky substitutions at these positions. In addition, we have observed that the addition of bromine at either the 6 (6-bromo) or 8 (8-bromo) position conferred better antiviral properties. Finally, we found a significant loss of potency with the 6-bromo when tested with the ALLINI-resistant IN A128T mutant virus, while the 8-bromo analog retained full effectiveness.

This paper describes the three-step synthesis of TBS-MAC, a masked acyl cyanide (MAC) and a versatile one-carbon oxidation state three synthon. We have developed a scalable and detailed synthesis that involves: (1) acetylation of malononitrile to form the sodium enolate, (2) protonation of the enolate to form acetylmalononitrile, and (3) epoxidation of the enol, rearrangement to an unstable alcohol, and TBS-protection to form the title compound. Both the sodium enolate and acetylmalononitrile are bench-stable precursors to the intermediate hydroxymalononitrile, which can be converted to other MAC reagents beyond TBS by varying the protecting group (Ac, MOM, EE, etc.).