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Taking our previously reported HIV-1 NNRTIs BH-11c and XJ-10c as lead compounds, series of novel diarypyrimidine derivatives bearing six-membered non-aromatic heterocycles were designed to improve anti-resistance and drug-like profiles. According to the three rounds of in vitro antiviral activity screening, compound 12g was the most active inhibitor against wild-type and five prevalent NNRTI-resistant HIV-1 strains with EC 50 values ranging from 0.024 to 0.0010 μM. This is obviously better than the lead compound BH-11c and the approved drug ETR. Detailed structure-activity relationship was investigated to provide valuable guidance for further optimization. The MD simulation study indicated that 12g could form additional interactions with residues around the binding site in HIV-1 RT, which provided reasonable explanations for its improved anti-resistance profile compared to ETR. Furthermore, 12g showed significant improvement in water solubility and other drug-like properties compared to ETR. The CYP enzymatic inhibitory assay indicated that 12g was unlikely to induce CYP-mediated drug-drug interactions. 12g pharmacokinetics parameters were investigated and it displayed a long half-life of 6.59 h in vivo. The properties of compound 12g make it a promising lead compound for the development of new generation of antiretroviral drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

HIV-1 capsid (CA) performs multiple roles in the viral life cycle and is a promising target for antiviral development. In this work, we describe the design, synthesis, assessment of antiviral activity, and mechanistic investigation of 20 piperazinone phenylalanine derivatives with a terminal indole or benzene ring. Among them, F 2 -7f exhibited moderate anti-HIV-1 activity with an EC 50 value of 5.89 μM, which was slightly weaker than the lead compound PF74 (EC 50 = 0.75 μM). Interestingly, several compounds showed a preference for HIV-2 inhibitory activity, represented by 7f with an HIV-2 EC 50 value of 4.52 μM and nearly 5-fold increased potency over anti-HIV-1 (EC 50 = 21.81 μM), equivalent to PF74 (EC 50 = 4.16 μM). Furthermore, F 2 -7f preferred to bind to the CA hexamer rather than to the monomer, similar to PF74 , according to surface plasmon resonance results. Molecular dynamics simulation indicated that F 2 -7f and PF74 bound at the same site. Additionally, we computationally analyzed the ADMET properties for 7f and F 2 -7f . Based on this analysis, 7f and F 2 -7f were predicted to have improved drug-like properties and metabolic stability over PF74 , and no toxicities were predicted based on the chemotype of 7f and F 2 -7f . Finally, the experimental metabolic stability results of F 2 -7f in human liver microsomes and human plasma moderately correlated with our computational prediction. Our findings show that F 2 -7f is a promising small molecule targeting the HIV-1 CA protein with considerable development potential.

Further clinical development of PF74, a lead compound targeting HIV-1 capsid, is impeded by low antiviral activity and inferior metabolic stability. By modifying the benzene (region I) and indole of PF74, we identified two potent compounds (7m and 7u) with significantly improved metabolic stability. Compared to PF74, 7u displayed greater metabolic stability in human liver microsomes (HLMs) with half-life (t 1/2 ) 109-fold that of PF74. Moreover, mechanism of action (MOA) studies demonstrated that 7m and 7u effectively mirrored the MOA of compounds that interact within the PF74 interprotomer pocket, showing direct and robust interactions with recombinant CA, and 7u displaying antiviral effects in both the early and late stages of HIV-1 replication. Furthermore, MD simulation corroborated that 7u was bound to the PF74 binding site, and the results of the online molinspiration software predicted that 7m and 7u had desirable physicochemical properties. Unexpectedly, this series of compounds exhibited better antiviral activity than PF74 against HIV-2, represented by compound 7m whose anti-HIV-2 activity was almost 5 times increased potency over PF74. Therefore, we have rationally redesigned the PF74 chemotype to inhibitors with novel structures and enhanced metabolic stability in this study. We hope that these new compounds can serve as a blueprint for developing a new generation of HIV treatment regimens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Masson SAS.)

HIV-1 capsid (CA) plays indispensable and multiple roles in the life cycle of HIV-1, become an attractive target in antiviral therapy. Herein, we report the design, synthesis, and mechanism study of a novel series of dimerized phenylalanine derivatives as HIV-1 capsid inhibitors using 2-piperazineone or 2,5-piperazinedione as a linker. The structure-activity relationship (SAR) indicated that dimerized phenylalanines were more potent than monomers of the same chemotype. Further, the inclusion of fluorine substituted phenylalanine and methoxyl substituted aniline was found to be beneficial for antiviral activity. From the synthesized series, Q-c4 was found to be the most potent compound with an EC 50 value of 0.57 μM, comparable to PF74. Interestingly, Q-c4 demonstrated a slightly higher affinity to the CA monomer than the CA hexamer, commensurate with its more significant effect in the late-stage of the HIV-1 lifecycle. Competitive SPR experiments with peptides from CPSF6 and NUP153 revealed that Q-c4 binds to the interprotomer pocket of hexameric CA as designed. Single-round infection assays showed that Q-c4 interferes with the HIV-1 life cycle in a dual-stage manner, affecting both pre-and post-integration. Stability assays in human plasma and human liver microsomes indicated that although Q-c4 has improved stability over PF74, this kind of inhibitor still requires further optimization. And the results of the online molinspiration software predicted that Q-c4 has desirable physicochemical properties but some properties still have some violation from the Lipinski rule of five. Overall, the dimerized phenylalanines are promising novel platforms for developing future HIV-1 CA inhibitors with considerable potential for optimization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Herein, alkenylpiperidine and alkynylpiperidine moieties were introduced into the left wing of DAPYs (diarylpyrimidines) to explore the new site of the NNIBP (non-nucleoside inhibitor binding pocket) protein-solvent interface region via the structure-based drug design strategy. All the synthesized compounds displayed nanomolar to submicromolar activity against WT (wild-type) HIV-1. Among all, compound FT1 (EC 50  = 19 nM) was found to be the most active molecule, which is better than NVP (EC 50  = 0.10 μM). In addition, most of the compounds displayed micromolar activity against K103N and E138K mutant strains, while FT1 (EC 50(K103N)  = 50 nM, EC 50(E138K)  = 0.19 µM) still has the most effective activity. The molecular dynamics simulation studies revealed that the presence of pyridine moiety of FT1 was essential and played a significant role in its binding with RT (reverse transcriptase)., (Copyright © 2021. Published by Elsevier Inc.)

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 PF-74 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 11l , which exhibited anti-HIV-1 NL4-3 activity 5.78-fold better than PF-74 . Interestingly, 11l also showed anti-HIV-2 ROD activity (EC 50 = 31 nM), with almost 120 times increased potency over PF-74 . 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 11l was 6.25 times more potent as compared to PF-74 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 PF-74 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 11l . Additionally, 11l exhibited a modest increase in HLM and human plasma metabolic stabilities as compared to PF-74 , 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.

HIV-1 CA is involved in different stages of the viral replication cycle, performing essential roles in both early (uncoating, reverse transcription, nuclear import, integration) and late events (assembly). Recent efforts have demonstrated HIV-1 CA protein as a prospective therapeutic target for the development of new antivirals. The most extensively studied CA inhibitor, PF-3450074 (PF-74, discovered by Pfizer), that targets an inter-protomer pocket within the CA hexamer. Herein we reported the design, synthesis, and biological evaluation of a series of 4-phenyl-1H-1,2,3-triazole phenylalanine derivatives as HIV-1 CA inhibitors based on PF-74 scaffold. Most of the analogues demonstrated potent antiviral activities, among them, the anti-HIV-1 activity of 6a-9 (EC 50  = 3.13 μM) is particularly prominent. The SPR binding assay of selected compounds (6a-9, 6a-10, 5b) suggested direct and effective interaction with recombinant CA proteins. The mechanism of action studies also demonstrated that 6a-9 displays the effects in both the early and late stages of HIV-1 replication. To explore the potential binding mode of the here presented analogues, 6a-9 was analyzed by MD simulation to predict its binding to the active site of HIV-1 CA monomer. In conclusion, this novel series of antivirals can serve as a starting point for the development of a new generation of HIV-1 treatment regimen and highlights the potentiality of CA as a therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of HIV-1 replication and is considered an important, clinically unexploited therapeutic target. As such, small drug-like molecules that inhibit this critical HIV-1 protein have become a priority for several groups. Therefore, in this study we explore small molecule targeting of the CA protein, and in particular a very attractive inter-protomer pocket. We report the design, parallel synthesis, and anti-HIV-1 activity evaluation of a series of novel phenylalanine derivatives as HIV-1 CA protein inhibitors synthesized via Cu(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. We demonstrate robust inhibitory activity over a range of potencies against the HIV-1 NL 4-3 reference strain. In particular, compound 13m exhibited the greatest potency and lowest toxicity within this new series with an EC 50 value of 4.33 μM and CC 50 value of >57.74 μM (SI > 13.33). These values are very similar to the lead compound PF-74 (EC 50  = 5.95 μM, CC 50  > 70.50 μM, SI > 11.85) in our assay, despite significant structural difference. Furthermore, we demonstrate via surface plasmon resonance (SPR) binding assays that 13m interacts robustly with recombinant HIV-1 CA and exhibits antiviral activity in both the early and late stages of HIV-1 replication. Overall, the novel parallel synthesis and structure-activity relationships (SARs) identified within this study set the foundation for further rational optimization and discovery of CA-targeting compounds with improved potency., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)