Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.

Structure-based design of diarylpyrimidines and triarylpyrimidines as potent HIV-1 NNRTIs with improved metabolic stability and drug resistance profiles.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important component of anti-acquired immunodeficiency syndrome treatment regimen. In the present work, with the previously reported compound K-16c as lead, a series of novel 2,4,5-trisubstituted pyrimidine derivatives were designed based on the cocrystal structure of K-16c/RT, with the aim to improve the anti-human immunodeficiency virus type-1 (HIV-1) activities and metabolic stability properties. Compound 11b1 exhibited the most potent antiviral activity against wild-type (WT) and a panel of single mutant HIV-1 strains (EC50 = 2.4-12.4 nM), being superior to or comparable to those of the approved drug etravirine. Meanwhile, 11b1 exhibited moderate cytotoxicity (CC50 = 4.96 µM) and high selectivity index (SI = 1189) toward HIV-1 WT strain. As for HIV-1 RT inhibition test, 11b1 possessed excellent inhibitory potency (IC50 = 0.04 µM) and confirmed its target was RT. Moreover, the molecular dynamics simulation was performed to elucidate the improved drug resistance profiles. Moreover, 11b1 was demonstrated with favorable safety profiles and pharmacokinetic properties in vivo, indicating that 11b1 is a potential anti-HIV-1 drug candidate worthy of further development.

Discovery of low-molecular-weight phenylalanine derivatives as novel HIV capsid modulators with improved antiretroviral activity and metabolic stability.

The HIV capsid (CA) protein is a promising target for anti-AIDS treatment due to its critical involvement in viral replication. Herein, we utilized the well-documented CA inhibitor PF74 as our lead compound and designed a series of low-molecular-weight phenylalanine derivatives. Among them, compound 7t exhibited remarkable antiviral activity with a high selection index (EC50 = 0.040 µM, SI = 2815), surpassing that of PF74 (EC50 = 0.50 µM, SI = 258). Furthermore, when evaluated against the HIV-2 strain, 7t (EC50 = 0.13 µM) demonstrated approximately 14-fold higher potency than that of PF74 (EC50 = 1.76 µM). Insights obtained from surface plasmon resonance (SPR) revealed that 7t exhibited stronger target affinity to the CA hexamer and monomer in comparison to PF74. The potential interactions between 7t and the HIV-1 CA were further elucidated using molecular docking and molecular dynamics simulations, providing a plausible explanation for the enhanced target affinity with 7t over PF74. Moreover, the metabolic stability assay demonstrated that 7t (T1/2 = 77.0 min) significantly outperforms PF74 (T1/2 = 0.7 min) in human liver microsome, exhibiting an improvement factor of 110-fold. In conclusion, 7t emerges as a promising drug candidate warranting further investigation.

Structure-guided design of novel biphenyl-quinazoline derivatives as potent non-nucleoside reverse transcriptase inhibitors featuring improved anti-resistance, selectivity, and solubility.

In pursuit of enhancing the anti-resistance efficacy and solubility of our previously identified NNRTI 1, a series of biphenyl-quinazoline derivatives were synthesized employing a structure-based drug design strategy. Noteworthy advancements in anti-resistance efficacy were discerned among some of these analogs, prominently exemplified by compound 7ag, which exhibited a remarkable 1.37 to 602.41-fold increase in potency against mutant strains (Y181C, L100I, Y188L, F227L + V106A, and K103N + Y181C) in comparison to compound 1. Compound 7ag also demonstrated comparable anti-HIV activity against both WT HIV and K103N, albeit with a marginal reduction in activity against E138K. Of significance, this analog showed augmented selectivity index (SI > 5368) relative to compound 1 (SI > 37764), Nevirapine (SI > 158), Efavirenz (SI > 269), and Etravirine (SI > 1519). Moreover, it displayed a significant enhancement in water solubility, surpassing that of compound 1, Etravirine, and Rilpivirine. To elucidate the underlying molecular mechanisms, molecular docking studies were undertaken to probe the critical interactions between 7ag and both WT and mutant strains of HIV-1 RT. These findings furnish invaluable insights driving further advancements in the development of DAPYs for HIV therapy.

Design, synthesis and biological evaluation of Thiazolo[3, 2-a]Pyrimidine derivatives as novel RNase H inhibitors.

Targeting Ribonuclease H (RNase H) has been considered a viable strategy for HIV therapy. In this study, a series of novel thiazolo[3, 2-a]pyrimidine derivatives were firstly designed and synthesized as potential inhibitors of HIV-1 RNase H. Among these compounds, A28 exhibited the most potent inhibition against HIV-1 RNase H with an IC50 value of 4.14 µM, which was about 5-fold increase in potency than the hit compound A1 (IC50 = 21.49 µM). To gain deeper insights into the structure-activity relationship (SAR), a CoMFA model was constructed to yield reasonable statistical results (q2 = 0.658 and R2 = 0.969). Results from magnesium ion chelation experiments and molecular docking studies revealed that these thiazolopyrimidine inhibitors may exert their inhibitory activity by binding to an allosteric site on RNase H at the interface between subunits p51 and p66. Furthermore, this analog demonstrated favorable physicochemical properties. Our findings provide valuable groundwork for further development of allosteric inhibitors targeting HIV-1 RNase H.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 µM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors.

In situ click chemistry-based discovery of 1,2,3-triazole-derived diarylpyrimidines as novel HIV-1 NNRTIs by exploiting the tolerant region I in binding pocket.

HIV-1 reverse transcriptase (RT) is considered as one of the most significant targets for the anti-HIV-1 drug design due to their determined mechanism and well-decoded crystal structure. As a part of our continuous efforts towards the development of potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) by exploiting the tolerant region I of NNRTIs binding pocket (NNIBP), the miniaturized parallel synthesis via CuAAC click chemistry reaction followed by in situ biological screening have been performed in this work. The in situ enzyme inhibition screening results showed that 14 compounds exhibited higher or equivalent inhibitory activity compared to the lead K-5a2 and ETR. Anti-HIV-1 activity results indicated that C1N51 displayed the most potent activity (EC50 = 0.01-0.26 µM) against wild-type and a panel of NNRTIs-resistant strains. Moreover, the molecular simulation demonstrated that the newly introduced triazole ring could develop new hydrogen bonds with Lys103 and Pro236, which explained the feasibility of introducing triazole in the tolerant region I of the RT binding pocket.

Development of novel HEPT analogs featuring significantly improved anti-resistance potency against HIV-1 through chemical space exploration of the tolerant region I.

Our recent great interest in developing 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs for HIV therapy identified a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) 3 (EC50 = 0.01681 µM), but its therapeutic efficacy was limited by its poor anti-resistance potency. This prompted us to search for potential HEPT analogs with broad-spectrum activities, leading to the generation of a series of novel HEPT analogs through exploring the chemical space of the solvent - protein interface. Encouraging improvements in anti-resistance efficacy were observed in some of these analogs, with the most promising compound 7 g being 3 to 26 - fold more potent than 3 against five mutant strains (E138K, Y181C, L100I, K103N, and Y188L). This analog surpassed the activity and selectivity of compound 3 by approximately 2-fold (EC50 = 0.007468 µM, SI = 4260). Furthermore, it was found to demonstrate feeble inhibition of CYP and hERG in vitro, and no in vivo acute toxicity. This study will further enrich the structure-activity relationships (SARs) of the HEPT scaffold, providing new guidance for the development of NNRTIs.

Structure-directed linker optimization of novel HEPTs as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPTs) have been previously described as an important class of HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs). In our continuously pursuing HEPT optimization efforts, a series of novel HEPTs, featuring -C(OH)CH2R, -CC, or -CHCH2R linker at the benzylic α-methylene unit, were developed as NNRTIs. Among these new HEPTs, the compound C20 with -CHCH3 group at the benzylic α-methylene unit conferred the highest potency toward WT HIV-1 and selectivity (EC50 = 0.23 µM, SI = 150.20), which was better than the lead compound HEPT (EC50 = 7 µM, SI = 106). Also, C20 was endowed with high efficacy against clinically relevant mutant strains (EC50(L100I) = 1.07 µM; EC50(K103N) = 4.33 µM; EC50(Y181C) = 5.57 µM; EC50(E138K) = 1.06 µM; EC50(F227L+V106A) = 5.45 µM) and wild-type HIV-1 reverse transcriptase (RT) with an IC50 value of 0.55 µM. Molecular docking and molecular dynamics simulations, as well as preliminary structure-activity relationship (SAR) analysis of these new compounds, provided a deeper insight into the key structural features of the interactions between HEPT analogs and HIV-1 RT and laid the foundation for further modification on HEPT scaffold.

Improving the anti-HIV-1 activity and solubility of poorly water-soluble DAPYs by heteroaromatic replacement strategy: From naphthalene-DAPYs to quinoline-DAPYs.

To enhance the anti-HIV-1 efficacy and solubility of our previously documented NNRTI 1, a collection of innovative quinoline-substituted DAPY derivatives were devised using heteroaromatic replacement strategy. The results of biological evaluation revealed that the representative compound 5h possessed the highest inhibitory activity against wild-type HIV-1 and selectivity index (EC50 = 0.0018 µM, SI > 166667), which were obviously better than that of 1 (EC50 = 0.00978 µM, SI > 37764), NVP (EC50 = 0.059 µM, SI > 158), EFV (EC50 = 0.028 µM, SI > 269), and ETR (EC50 = 0.0029 µM, SI > 1519). The water solubility of compound 5h was remarkably improved, surpassing that of 1, ETR and RPV. Additionally, this compound exerted significantly enhanced anti-resistance potency, compared to 1, and displayed comparable activity to ETR against WT RT of HIV-1 (IC50 = 0.011 µM). To elucidate the underlying molecular mechanisms, molecular docking studies were conducted to investigate the crucial interactions between 5h and WT/mutant strains of HIV-1. These findings provide valuable insights and drive further advancements in the development of DAPYs for HIV therapy.

4-Phenylcoumarin derivatives as new HIV-1 NNRTIs: Design, synthesis, biological activities, and computational studies.

A series of 4-phenylcoumarin derivatives was synthesized and evaluated for their cellular anti-HIV-1 and HIV-2 activities as well as their inhibitory effects against HIV-1 reverse transcriptase (RT). The hydrazone compound 8b and the ethylthiosemicarbazide derivative 4c showed the best inhibition activity against wild-type (WT) HIV-1. The promising compounds were further evaluated against HIV-1 RT and exhibited significant inhibitory activity with compound 8b showing comparable effect to the reference NNRTI Efavirenz (IC50 = 9.01 nM). Structure activity relationship study revealed the importance of 6-chloro and 4-phenyl substituents for optimum activity, as well as the 5-atoms linker (=N-NH-CO-CH2-O-) at position 7 of coumarin scaffold that can support the rotation and flexibility of compound 8b to fit well in the binding pocket. The molecular docking of compound 8b demonstrated a typical seahorse binding mode with better binding interactions that covered more residues when compared to Efavirenz.

Covalently Targeted Highly Conserved Tyr318 to Improve the Drug Resistance Profiles of HIV-1 NNRTIs: A Proof-of-Concept Study.

This study presents proof of concept for designing a novel HIV-1 covalent inhibitor targeting the highly conserved Tyr318 in the HIV-1 non-nucleoside reverse transcriptase inhibitors binding pocket to improve the drug resistance profiles. The target inhibitor ZA-2 with a fluorosulfate warhead in the structure was found to be a potent inhibitor (EC50 = 11-246 nM) against HIV-1 IIIB and a panel of NNRTIs-resistant strains, being far superior to those of NVP and EFV. Moreover, ZA-2 was demonstrated with lower cytotoxicity (CC50 = 125 µM). In the reverse transcriptase inhibitory assay, ZA-2 exhibited an IC50 value of 0.057 µM with the ELISA method, and the MALDI-TOF MS data demonstrated the covalent binding mode of ZA-2 with the enzyme. Additionally, the molecular simulations have also demonstrated that compounds can form covalent binding to the Tyr318.

Anti-SARS-CoV-2 Activity and Cytotoxicity of Amaryllidaceae Alkaloids from Hymenocallis littoralis.

The Amaryllidaceae species are well-known as a rich source of bioactive compounds in nature. Although Hymenocallis littoralis has been studied for decades, its polar components were rarely explored. The current phytochemical investigation of Amaryllidaceae alkaloids from H. littoralis led to the identification of three previously undescribed compounds: O-demethyl-norlycoramine (1), (-)-2-epi-pseudolycorine (2) and (+)-2-epi-pseudolycorine (3), together with eight known compounds: 6α-hydroxyhippeastidine (4), 6ß-hydroxyhippeastidine (5), lycorine (6), 2-epi-lycorine (7), zephyranthine (8), ungeremine (9), pancratistatin (10) and 9-O-demethyl-7-O-methyllycorenine (11). Among the eight previously reported compounds, five were isolated from H. littoralis for the first time (compounds 4, 5, 7, 8, and 9). Compounds 1, 4, 5, 7, 8, and 11 exhibited weak anti-SARS-CoV-2 activity (EC50 = 40-77 µM) at non-cytotoxic concentrations. Assessment of cytotoxicity on the Vero-E6 cell line revealed lycorine and pancratistatin as cytotoxic substances with CC50 values of 1.2 µM and 0.13 µM, respectively. The preliminary structure-activity relationship for the lycorine-type alkaloids in this study was further investigated, and as a result ring C appears to play a crucial role in their anti-SARS-CoV-2 activity.

Discovery of novel 1,2,4-triazole phenylalanine derivatives targeting an unexplored region within the interprotomer pocket of the HIV capsid protein.

Human immunodeficiency virus (HIV) capsid (CA) protein is a promising target for developing novel anti-HIV drugs. Starting from highly anticipated CA inhibitors PF-74, we used scaffold hopping strategy to design a series of novel 1,2,4-triazole phenylalanine derivatives by targeting an unexplored region composed of residues 106-109 in HIV-1 CA hexamer. Compound d19 displayed excellent antiretroviral potency against HIV-1 and HIV-2 strains with EC50 values of 0.59 and 2.69 µM, respectively. Additionally, we show via surface plasmon resonance (SPR) spectrometry that d19 preferentially interacts with the hexameric form of CA, with a significantly improved hexamer/monomer specificity ratio (ratio = 59) than PF-74 (ratio = 21). Moreover, we show via SPR that d19 competes with CPSF-6 for binding to CA hexamers with IC50 value of 33.4 nM. Like PF-74, d19 inhibits the replication of HIV-1 NL4.3 pseudo typed virus in both early and late stages. In addition, molecular docking and molecular dynamics simulations provide binding mode information of d19 to HIV-1 CA and rationale for improved affinity and potency over PF-74. Overall, the lead compound d19 displays a distinct chemotype form PF-74, improved CA affinity, and anti-HIV potency.

Conformationally Locked Carbocyclic Nucleosides Built on a 4'-Hydroxymethyl-3'-hydroxybicyclo[4.1.0]heptane Template. Stereoselective Synthesis and Antiviral Activity.

Two new families of enantiomerically pure carbocyclic nucleoside analogues based on a cyclohexane moiety with five chiral centers and a fused cyclopropyl ring have been synthesized. A highly regio- and stereoselective synthetic approach for the modular construction of the functionalized bicyclo[4.1.0]heptyl azide intermediate 6 has been established. Key steps to achieve this asymmetric synthesis involved highly diastereoselective allylic oxidation and hydroboration reactions. The first family of compounds, 1a,b and 2, presents different natural nucleobases, whereas the second one 3a-e bears functionalized 1,2,3-triazoles. These derivatives have been tested as antiviral agents, and compound 3d has shown to display moderate activity against coxsackie B4 virus.

Indolylarylsulfones bearing phenylboronic acid and phenylboronate ester functionalities as potent HIV­1 non-nucleoside reverse transcriptase inhibitors.

To explore the chemical space around the entrance channel of the HIV-1 reverse transcriptase (RT) binding pocket, we innovatively designed and synthesized a series of novel indolylarylsulfones (IASs) bearing phenylboronic acid and phenylboronate ester functionalities at the indole-2-carboxamide as new HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) through structure-based drug design. All the newly synthesized compounds exhibited excellent to moderate potency against wild-type (WT) HIV-1 with EC50 values ranging from 6.7 to 42.6 nM. Among all, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide and (4-ethylphenyl) boronate ester substituted indole-2-carboxamide were found to be the most potent inhibitors (EC50 = 8.5 nM, SI = 3310; EC50 = 6.7 nM, SI = 3549, respectively). Notably, (3-ethylphenyl)boronic acid substituted indole-2-carboxamide maintained excellent activities against the single HIV-1 mutants L100I (EC50 = 7.3 nM), K103N (EC50 = 9.2 nM), as well as the double mutant V106A/F227L (EC50 = 21.1 nM). Preliminary SARs and molecular modelling studies are also discussed in detail.

Structure-Based design of [(2-Hydroxyethoxy)methyl]-6-(phenylthio)-thymine derivatives as nonnucleoside HIV-1 reverse transcriptase Inhibitors: From HEPTs to Sulfinyl-substituted HEPTs.

The [(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs were reported to be a kind of promising lead compounds as nonnucleoside HIV-1 reverse transcriptase inhibitors. In this work, a series of novel sulfinyl-substituted analogs were designed by structure-based design strategy with the purpose of improving the activity of HEPT, followed by evaluating their anti-HIV-1 activity in MT-4 cells. Most of the final compounds had moderate to strong activity against wild-type HIV-1 strain (IIIB) with EC50 values in the range of 0.21-1.91 µM, which were around 4 âˆ¼ 32-fold better than the reference compound HEPT. Some of them showed higher sensitivity toward clinically relevant mutant L100I and E138K viruses than NVP. Selected compounds were further evaluated for their activity against wild-type reverse transcriptase (RT), and most of them exhibited nanomolar activity, suggesting a good correlation with the cell-based activity. The compounds 11h, 11l, and 11ab displayed the best anti-HIV-1 activity against wild-type HIV-1 strain (EC50 = 0.280, 0.209, and 0.290 µM) and nanomolar activity against mutant strains (L100I and E138K), superior to HEPT and NVP. Molecular modeling studies were also performed to elucidate the biological activity, providing a structural insight for follow-up research on HEPT optimization.

Medicinal chemistry strategies for discovering antivirals effective against drug-resistant viruses.

During the last forty years we have witnessed impressive advances in the field of antiviral drug discovery culminating with the introduction of therapies able to stop human immunodeficiency virus (HIV) replication, or cure hepatitis C virus infections in people suffering from liver disease. However, there are important viral diseases without effective treatments, and the emergence of drug resistance threatens the efficacy of successful therapies used today. In this review, we discuss strategies to discover antiviral compounds specifically designed to combat drug resistance. Currently, efforts in this field are focused on targeted proteins (e.g. multi-target drug design strategies), but also on drug conformation (either improving drug positioning in the binding pocket or introducing conformational constraints), in the introduction or exploitation of new binding sites, or in strengthening interaction forces through the introduction of multiple hydrogen bonds, covalent binding, halogen bonds, additional van der Waals forces or multivalent binding. Among the new developments, proteolysis targeting chimeras (PROTACs) have emerged as a valid approach taking advantage of intracellular mechanisms involving protein degradation by the ubiquitin-proteasome system. Finally, several molecules targeting host factors (e.g. human dihydroorotate dehydrogenase and DEAD-box polypeptide 3) have been identified as broad-spectrum antiviral compounds. Implementation of herein described medicinal chemistry strategies are expected to contribute to the discovery of new drugs effective against current and future threats due to emerging and re-emerging viral pandemics.

Identification of Boronate-Containing Diarylpyrimidine Derivatives as Novel HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors.

In this study, privileged boronic acid ester was introduced into the right wing of etravirine (ETR) to obtain a series of novel boronate-containing derivatives. These newly synthesized derivatives were evaluated for their anti-HIV potency in MT-4 cells using the MTT method, and their inhibitory activity to HIV-1 reverse transcriptase (RT) was assayed by the ELISA method. Most of the synthesized compounds displayed promising antiviral activity against the wild-type and a wide range of HIV-1 mutant strains. In particular, 4a exhibited the most potent activity against the wild-type and a panel of single mutations (L100I, K103N, Y181C, and E138K) with EC50 values ranging from 0.005 to 0.648 µM, which were much superior to those of nevirapine (EC50 = 0.151 µM). Moreover, 4b turned out to be an effective inhibitor against the double-mutant strains F227L + V106A and RES056 with EC50 values of 3.21 and 2.30 µM, respectively. RT inhibition activity and molecular docking were also investigated.

Discovery and Mechanistic Investigation of Piperazinone Phenylalanine Derivatives with Terminal Indole or Benzene Ring as Novel HIV-1 Capsid Modulators.

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, F2-7f exhibited moderate anti-HIV-1 activity with an EC50 value of 5.89 µM, which was slightly weaker than the lead compound PF74 (EC50 = 0.75 µM). Interestingly, several compounds showed a preference for HIV-2 inhibitory activity, represented by 7f with an HIV-2 EC50 value of 4.52 µM and nearly 5-fold increased potency over anti-HIV-1 (EC50 = 21.81 µM), equivalent to PF74 (EC50 = 4.16 µM). Furthermore, F2-7f 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 F2-7f and PF74 bound at the same site. Additionally, we computationally analyzed the ADMET properties for 7f and F2-7f. Based on this analysis, 7f and F2-7f 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 F2-7f. Finally, the experimental metabolic stability results of F2-7f in human liver microsomes and human plasma moderately correlated with our computational prediction. Our findings show that F2-7f is a promising small molecule targeting the HIV-1 CA protein with considerable development potential.