In addition to indoles two

In addition to indoles –, two additional structural variants were synthesized to explore the synthetic flexibility and structure–activity relationships of this system. The first of these was the homologation of the acetic Sertraline HCl side chain to prepare compound (). The extension of the chain was expected to provide more flexibility in the binding of the two key functional groups to IN and to affect the hydrogen bonding of the compound. Compound was prepared through application of the chemistry of Larock. Upon coupling of alkyne to iodoaniline , the indole was cyclized and the phenyl group appended to the C3 position under microwave conditions in the presence of palladium. The formation of the -butyl ether and subsequent saponification were carried out in a manner analogous to the preparation of previous derivatives, generating a structural analogue of . The second structural variant took advantage of functional group reversal (), a technique previously seen in indole containing compounds like indomethacin/clometacin and serotonin. For the preparation of compound (), the anisole group was introduced directly onto 3-methylindole () via Ullmann coupling with iodoanisole (). At that stage, the introduction of the acetic acid side chain once again could be accomplished as shown for the transformation of – to provide compound , which possesses the same functional groups as analogue , in a total of five synthetic steps and with an overall yield of nearly 35% from inexpensive commercial reagents. The synthesized analogues were tested in a homogenous time-resolved fluorescence (HTRF)-based LEDGF/p75-dependent integration assay., The advantage of this assay is its ability to capture IN inhibitors with different modes of action. For example, compounds that impair IN catalytic activity, induce aberrant IN multimerization, or inhibit IN-LEDGF/p75 binding all can be identified using this approach., Comparative analysis of the IC values () for the indole-based compounds synthesized above have revealed (entry 3) as the most promising lead compound with an IC value of 4.5μM. Additional trends in this series of compounds, however, can also be observed to provide structure–activity relationships within this class. For example, entries 1–3 demonstrate the important role that substitution of the C3 aromatic ring plays on compound activity, with both the electron rich and slightly larger anisole and chromane rings resulting in increased activity over the unsubstituted phenyl ring. Surprisingly, the more flexible benzyl moiety was also reasonably well tolerated at the C3 position (, entry 4), giving similar results to the anisole in entry 2. Introduction of a benzyl group at the N1 position (, entry 5), however, was not well tolerated, resulting in a two-fold reduction in activity as compared to entry 2. The compounds containing the homologated acetic acid side chain (, entry 6) and the inversion of the indole ring system (, entry 7) failed to show any activity in the assay at concentrations up to 100μM. Although the relative lack of activity for compound is not clear, the extension of the acid chain in compound likely affects the ability of the molecule to achieve efficient hydrogen bonding with the protein through the carboxylic acid moiety while simultaneously maintaining the proper orientation of the aryl ring in the binding site, a potential consequence of the increased length and conformational flexibility in this linker. Based on the promising activity displayed by in the LEDGF/p75 dependent integration assay, however, further mechanism of action studies were carried out using this compound. This work included additional HTRF-based assays that allow monitoring of LEDGF/p75-independent IN 3′-processing activity, IN binding to LEDGF/p75, and aberrant IN multimerization. The results of these assays, summarized in , indicate that exhibits a multimodal mechanism of action. In particular, this compound not only effectively inhibited LEDGF/p75-dependent integration, but also LEDGF/p75-independent 3′-processing activity of IN. Furthermore, induced aberrant IN multimerization, which could explain its ability to inhibit LEDGF/p75-independent 3′-processing activity of IN. In addition, was able to inhibit IN-LEDGF/p75 binding, albeit at a somewhat higher concentration. As one may expect, this multimodal mechanism of action is reminiscent of the mechanism of action of quinoline-based ALLINI compounds such as BI-1001., Due to the similarity in activity to the quinoline-based ALLINIs, was also screened against the single amino-acid A128T substitution in HIV-1 IN that confers marked resistance to quinoline-based inhibitors, including BI-1001. In sharp contrast to BI-1001, promoted aberrant multimerization of both wild type and the A128T mutant INs with similar IC values (). Likewise, also showed similar inhibition of 3′-processing activity in both the wild type and A128T mutant with IC values of 3.2 and 0.9μM, respectively.