Ficant drop in activity versus the Y188L mutant (13 ?0.5 nM). The
Ficant drop in activity versus the Y188L mutant (13 ?0.5 nM). The `flipped’ analogue (Figure 2, analogue 13) maintained potency similar to 1 for the WT and each of mutants with the exception of E138K (4.2 ?0.3 nM). Retaining the p-methoxybenzyl protecting group at the 9-position of the purine reduced the potency of both analogues (Figures 2 and 3, compare 10 to 11 and 12 to 13). Based upon these data, we used 13 as a scaffold and examined the effects of different substituents in place of the 4-benzonitrile moiety.The results obtained with these analogues (Figure 2, analogues 14?5) show that many of the compounds that are highly active against WT RT show considerable differences in their ability to inhibit the various mutants. Attempts to replace the nitrile functionality of the benzonitrile with other moieties resulted in compounds that were active against the WT and most mutants. However, these analogues (14?9) showed a significant loss of activity versus the Y188L mutant (Figure 3). Replacing the benzonitrile with alternate ring systems was generally successful against WT, but Y188L again conferred resistance (analogues 20?5). The benzo[d]thiazole analogue (21) possessed particularly good activity PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26740125 versus WT and the mutants, including an EC50 of 4.4 nM against Y188L. The derivatives incorporating 2,9-purine ring systems (analogues 26 and 27) were found to possess very different activities. Placement of the benzonitrile moiety on the 9-position resulted in an analogue (26) which showed a significant decrease in activity versus WT and all of the mutants. However, the incorporation of the (E)-3-(3,5-dimethylphenyl)acrylonitrile moiety atJohnson et al. Retrovirology 2012, 9:99 http://www.retrovirology.com/content/9/1/Page 5 ofthe 9-position resulted in a highly active analogue (27) with potencies akin to rilpivirine for WT and all of the mutants except Y188L, which caused a modest drop in activity (9.6 ?2.2 nM).Selected analogues assayed against additional mutantsBased on the results obtained with the small panel of mutants, eleven compounds were chosen for testing against a broader panel which included all the mutants listed above (K103N, E138K, Y181C and Y188L), and included rilpivirine (1) as a reference compound. The analogues 6?, 11, 13, 20, 21, 26 and 27 were selected based on their broad potency against the smaller panel of mutants and/or to determine how their differences from 1 affected their activities. The K103N/Y181C double mutant caused a significant loss of potency for 6, 8, 9, 20, 21, 26 and 27 (Figure 4 and Additional file 1: PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 Table S1), while the analogues 7, 11 and 13 retained good potency against this mutant. Of particular interest, the related analogues 7 and 9 differed dramatically in their ability to inhibit K103N/Y181C RT (EC50 values of 2.2 nM and 47 nM, respectively). These compounds both possess a central 5-aminopyrimidine moiety that is `flipped’ between the two analogues. Keeping the pyrimidine ring in the same conformation as in 1 allows 7 to retain activity while 9 suffers 67-fold loss of potency against the double mutant. A structural explanation for this difference will be discussed in a later section.Cytotoxicitylower CC50 than 1 (2.0 M for 20 versus 10 M for 1). CC50 values for analogues 6, 9, 13 and 26 were 1- to 3-fold greater than that of 1. The remaining compounds (7, 8, 11, 21 and 27) had CC50 values greater than 38 M (up to 115 M for 11). The resulting HMPL-013MedChemExpress Fruquintinib therapeutic indices (T.I., ratio of CC50 to EC50.