Inspired by our previous efforts to improve the drug-resistance profiles of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), a novel series of “dual-site” binding diarylpyrimidine (DAPY) derivatives targeting both the NNRTI adjacent site and NNRTIs binding pocket (NNIBP) were designed, synthesized, and evaluated for their anti-HIV potency in TZM-bl and MT-4 cells. Eight compounds exhibited moderate to excellent potencies in inhibiting wild-type (WT) HIV-1 replication with EC values ranging from 2.45 nM to 5.36 nM, and 14c (EC = 2.45 nM) proved to be the most promising inhibitor. Of note, 14c exhibited potent activity against the single mutant strain E138K (EC = 10.6 nM), being comparable with ETR (EC = 9.80 nM) and 3.5-fold more potent than that of compound 7 (EC = 37.3 nM). Moreover, 14c acted as a classical NNRTI with high affinity for WT HIV-1 RT (IC = 0.0589 μM). The detailed structure-activity relationships (SARs) of the representative compounds were also determined, and further supported by molecular dynamics simulation. Overall, we envision that the “dual-site”-binding NNRTIs have significant prospects and pave the way for the next round of rational design of potent anti-HIV-1 agents.Copyright © 2020 Elsevier Masson SAS. All rights reserved.