Design, Synthesis, and Mechanism Study of Benzenesulfonamide-Containing Phenylalanine Derivatives as Novel HIV-1 Capsid Inhibitors with Improved Antiviral Activities

The HIV-1 CA protein has gained remarkable attention as a promising therapeutic target for the development of new antivirals, due to its pivotal roles in HIV-1 replication (structural and regulatory). Herein, we report the design and synthesis of three series of benzenesulfonamide-containing phenylalanine derivatives obtained by further structural modifications of <b>PF-74</b> to aid in the discovery of more potent and drug-like HIV-1 CA inhibitors. Structure-activity relationship studies of these compounds led to the identification of new phenylalanine derivatives with a piperazinone moiety, represented by compound <b>11l</b>, which exhibited anti-HIV-1_NL4-3 activity 5.78-fold better than <b>PF-74</b>. Interestingly, <b>11l</b> also showed anti-HIV-2_ROD activity (EC₅₀ = 31 nM), with almost 120 times increased potency over <b>PF-74</b>. However, due to the higher significance of HIV-1 as compared to HIV-2 for the human population, this manuscript focuses on the mechanism of action of our compounds in the context of HIV-1. SPR studies on representative compounds confirmed CA as the binding target. The action stage determination assay demonstrated that these inhibitors exhibited antiviral activities with a dual-stage inhibition profile. The early-stage inhibitory activity of compound <b>11l</b> was 6.25 times more potent as compared to <b>PF-74</b> but appeared to work via the accelerating capsid core assembly rather than stabilization. However, the mechanism by which they exert their antiviral activity in the late stage appears to be the same as <b>PF-74</b> with less infectious HIV-1 virions produced in their presence, as judged p24 content studies. MD simulations provided the key rationale for the promising antiviral potency of <b>11l</b>. Additionally, <b>11l</b> exhibited a modest increase in HLM and human plasma metabolic stabilities as compared to <b>PF-74</b>, as well as a moderately improved pharmacokinetic profile, favorable oral bioavailability, and no acute toxicity. These studies provide insights and serve as a starting point for subsequent medicinal chemistry efforts in optimizing these promising HIV inhibitors.