Porphyrins with combinations of 4-carboxyphenyl and 4-hydroxyphenyl substituents in meso-positions as anti-HIV-1 agents.

A series of 4-carboxyphenyl/4-hydroxyphenyl meso-substituted porphyrins were synthesized, purified, and characterized. The compounds exhibited anti-HIV-1 activities, in vitro, under both non-photodynamic (non-PDT) and photodynamic (PDT) conditions. Specifically, the porphyrins inhibited HIV-1 virus entry, with c-PB2(OH)2 and PB(OH)3 showing significant anti-HIV-1 activity. All of the porphyrins inhibited HIV-1 subtype B and C virus entry under PDT conditions. Our study demonstrated that the compounds bearing combinations of 4-carboxyphenyl/4-hydroxyphenyl moieties were not toxic even at higher concentrations, as compared to the reference porphyrins 5,10,15,20-tetra-(4-carboxyphenyl)porphyrin (TCPP) and 5,10,15,20-tetra-(4-hydroxyphenyl)porphyrin (THPP), under PDT conditions. This study underscores the promising potential of these compounds as HIV entry inhibitors in both non-PDT and PDT scenarios.

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.

Discovery of Novel Aryl Triazolone Dihydropyridines (ATDPs) Targeting Highly Conserved Residue W229 as Promising HIV-1 NNRTIs.

NNRTI is an important component of the highly active antiretroviral therapy (HAART), but the rapid emergence of drug resistance and poor pharmacokinetics limited their clinical application. Herein, a series of novel aryl triazolone dihydropyridines (ATDPs) were designed by structure-guided design with the aim of improving drug resistance profiles and pharmacokinetic profiles. Compound 10n (EC50 = 0.009-17.7 µM) exhibited the most active potency, being superior to or comparable to that of doravirine (DOR) against the whole tested viral panel. Molecular docking was performed to clarify the reason for its higher resistance profiles. Moreover, 10n demonstrated excellent pharmacokinetic profile (T1/2 = 5.09 h, F = 108.96%) compared that of DOR (T1/2 = 4.4 h, F = 57%). Additionally, 10n was also verified to have no in vivo acute or subacute toxicity (LD50 > 2000 mg/kg), suggesting that 10n is worth further investigation as a novel oral NNRTIs for HIV-1 therapy.

Seco-cyclic phorbol derivatives and their anti-HIV-1 activities.

Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (Kd) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 µmol·L-1 and a CC50 of 153.92 µmol·L-1, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.

Design and synthesis of Fsp3-enriched spirocyclic-substituted diarylpyrimidine derivatives as novel HIV-1 NNRTIs.

In this study, a novel series of diarylpyrimidine derivatives with Fsp3-enriched spirocycles were designed and synthesized to further explore the chemical space of the hydrophobic channel of the NNRTI-binding pocket. The biological evaluation results showed that most of the compounds displayed effective inhibitory potency against the HIV-1 wild-type strain, with EC50 values ranging from micromolar to submicromolar levels. Among them, TT6 turned out to be the most effective inhibitor with an EC50 value of 0.17 µM, demonstrating up to 47 times more active than that of reference drug 3TC (EC50 = 8.01 µM). More encouragingly, TT6 was found to potently inhibit the HIV-1 mutant strain K103N with an EC50 value of 0.69 µM, being about 6-fold more potent than 3TC (EC50 = 3.68 µM) and NVP (EC50 = 4.62 µM). Furthermore, TT6 exhibited the most potent inhibitory activity toward HIV-1 reverse transcriptase with an IC50 value of 0.33 µM. Additionally, molecular simulation studies were conducted to investigate the binding modes between TT6 and NNRTI-binding pocket, which may provide valuable clues for the follow-up structural optimizations.

Antiviral Activity of Lipophilic Nucleoside Tetraphosphate Compounds.

We report on the synthesis and characterization of three types of nucleoside tetraphosphate derivatives 4-9 acting as potential prodrugs of d4T nucleotides: (i) the δ-phosph(on)ate is modified by two hydrolytically stable alkyl residues 4 and 5; (ii) the δ-phosph(on)ate is esterified covalently by one biodegradable acyloxybenzyl moiety and a nonbioreversible moiety 6 and 7; or (iii) the δ-phosphate of nucleoside tetraphosphate is masked by two biodegradable prodrug groups 8 and 9. We were able to prove the efficient release of d4T triphosphate (d4TTP, (i)), δ-monoalkylated d4T tetraphosphates (20 and 24, (ii)), and d4T tetraphosphate (d4T4P, (iii)), respectively, by chemical or enzymatic processes. Surprisingly, δ-dialkylated d4T tetraphosphates, δ-monoalkylated d4T tetraphosphates, and d4T4P were substrates for HIV-RT. Remarkably, the antiviral activity of TetraPPPPro-prodrug 7 was improved by 7700-fold (SI 5700) as compared to the parent d4T in CEM/TK- cells, denoting a successful cell membrane passage of these lipophilic prodrugs and an intracellular delivery of the nucleotide metabolites.

Synthesis and anti-HIV activities of phorbol derivatives.

In this study, 37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated, building upon our previous synthesis of 51 phorbol derivatives. 12-Para-electron-acceptor-trans-cinnamoyl-13-decanoyl phorbol derivatives stood out, demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation. These derivatives exhibited a higher safety index compared with the positive control drug. Among them, 12-(trans-4-fluorocinnamoyl)-13-decanoyl phorbol, designated as compound 3c, exhibited the most potent anti-HIV-1 activity (EC50 2.9 nmol·L-1, CC50/EC50 11 117.24) and significantly inhibited the formation of syncytium (EC50 7.0 nmol·L-1, CC50/EC50 4891.43). Moreover, compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor. Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C (PKC). Therefore, compound 3c emerges as a potential candidate for developing new anti-HIV drugs.

Development of Small-Molecule Anti-HIV-1 Agents Targeting HIV-1 Capsid Proteins.

The capsid of human immunodeficiency virus type 1 (HIV-1) forms a conical structure by assembling oligomers of capsid (CA) proteins and is a virion shell that encapsulates viral RNA. The inhibition of the CA function could be an appropriate target for suppression of HIV-1 replication because the CA proteins are highly conserved among many strains of HIV-1, and the drug targeting CA, lenacapavir, has been clinically developed by Gilead Sciences, Inc. Interface hydrophobic interactions between two CA molecules via the Trp184 and Met185 residues in the CA sequence are indispensable for conformational stabilization of the CA multimer. Our continuous studies found two types of small molecules with different scaffolds, MKN-1 and MKN-3, designed by in silico screening as a dipeptide mimic of Trp184 and Met185 have significant anti-HIV-1 activity. In the present study, MKN-1 derivatives have been designed and synthesized. Their structure-activity relationship studies found some compounds having potent anti-HIV activity. The present results should be useful in the design of novel CA-targeting molecules with anti-HIV activity.

Medicinal chemistry perspectives on the development of piperazine-containing HIV-1 inhibitors.

The Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS), one of the most perilous diseases known to humankind. A 2023 estimate put the number of people living with HIV around 40 million worldwide, with the majority benefiting from various antiretroviral therapies. Consequently, the urgent need for the development of effective drugs to combat this virus cannot be overstated. In the realm of medicinal and organic chemistry, the synthesis and identification of novel compounds capable of inhibiting HIV enzymes at different stages of their life cycle are of paramount importance. Notably, the spotlight is on the progress made in enhancing the potency of HIV inhibitors through the use of piperazine-based compounds. Multiple studies have revealed that the incorporation of a piperazine moiety results in a noteworthy enhancement of anti-HIV activity. The piperazine ring assumes a pivotal role in shaping the pharmacophore responsible for inhibiting HIV-1 at critical stage, including attachment, reverse transcription, integration, and protease activity. This review also sheds light on the various opportunities that can be exploited to develop effective antiretroviral targets and eliminate latent HIV reservoirs. The advancement of highly potent analogues in HIV inhibitor research has been greatly facilitated by contemporary medicinal strategies, including molecular/fragment hybridization, structure-based drug design, and bioisosterism. These techniques have opened up new avenues for the development of compounds with enhanced efficacy in combating the virus.

Membrane-permeable tenofovir-di- and monophosphate analogues.

The development of new antiviral agents such as nucleoside analogues or acyclic nucleotide analogues (ANPs) and prodrugs thereof is an ongoing task. We report on the synthesis of three types of lipophilic triphosphate analogues of (R)-PMPA and dialkylated diphosphate analogues of (R)-PMPA. A highly selective release of the different nucleotide analogues ((R)-PMPA-DP, (R)-PMPA-MP, and (R)-PMPA) from these compounds was achieved. All dialkylated (R)-PMPA-prodrugs proved to be very stable in PBS as well as in CEM/0 cell extracts and human plasma. In primer extension assays, both the monoalkylated and the dialkylated (R)-PMPA-DP derivatives acted as (R)-PMPA-DP as a substrate for HIV-RT. In contrast, no incorporation events were observed using human polymerase γ. The dialkylated (R)-PMPA-compounds exhibited significant anti-HIV efficacy in HIV-1/2 infected cells (CEM/0 and CEM/TK-). Remarkably, the dialkylated (R)-PMPA-MP derivative 9a showed a 326-fold improved activity as compared to (R)-PMPA in HIV-2 infected CEM/TK- cells as well as a very high SI of 14,000. We are convinced that this study may significantly contribute to advancing antiviral agents developed based on nucleotide analogues in the future.

Discovery, Crystallographic Studies, and Mechanistic Investigations of Novel Phenylalanine Derivatives Bearing a Quinazolin-4-one Scaffold as Potent HIV Capsid Modulators.

Optimization of compound 11L led to the identification of novel HIV capsid modulators, quinazolin-4-one-bearing phenylalanine derivatives, displaying potent antiviral activities against both HIV-1 and HIV-2. Notably, derivatives 12a2 and 21a2 showed significant improvements, with 2.5-fold over 11L and 7.3-fold over PF74 for HIV-1, and approximately 40-fold over PF74 for HIV-2. The X-ray co-crystal structures confirmed the multiple pocket occupation of 12a2 and 21a2 in the binding site. Mechanistic studies revealed a dual-stage inhibition profile, where the compounds disrupted capsid-host factor interactions at the early stage and promoted capsid misassembly at the late stage. Remarkably, 12a2 and 21a2 significantly promoted capsid misassembly, outperforming 11L, PF74, and LEN. The substitution of easily metabolized amide bond with quinolin-4-one marginally enhanced the stability of 12a2 in human liver microsomes compared to controls. Overall, 12a2 and 21a2 highlight their potential as potent HIV capsid modulators, paving the way for future advancements in anti-HIV drug design.

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.

Design, Synthesis, Docking Studies, and Biological Evaluation of Novel 2-Hydroxyacetophenone Derivatives as Anti-HIV-1 Agents.

BACKGROUND: The persistence of HIV mutations and the existence of multidrug resistance have produced an opportunity for an array of innovative anti-HIV medicines with a variety of structures that target HIV key enzymes. OBJECTIVE: The goal of this work was to find a new class of anti-HIV drugs founded on HIV integrase inhibitor pharmacophores. METHODS: A novel class of 2-hydroxy acetophenone analogs featuring substituted benzamide or N-phenylthiourea groups was designed and synthesized based on the general pharmacophore of HIV-1 integrase inhibitors (INs). RESULTS: Most of the synthesized analogs were found to be moderately active against the virus, with EC50 values ranging from 40 to 140 µM. Additionally, it was found that most of the compounds presented no considerable cytotoxicity (CC50 > 500 µΜ). The most potent compounds substituting with 4-fluorobenzamide (compound 7) and 4-methylbenzamide (compound 9) rings inhibited the HIV-1 replication by EC50 values of 40 and 45 µΜ, respectively. Docking studies using the crystallographic data available for PFV IN indicated that the Mg2+ coordination might be the possible mechanism of the anti-viral activity. CONCLUSION: Our findings proved that the synthesized analogs may suggest a very good basis for the development of new anti-HIV-1 agents.

Covalent and noncovalent strategies for targeting Lys102 in HIV-1 reverse transcriptase.

Reverse transcriptase (RT) is one of three key proteins responsible for the replication cycle of HIV-1 in the host. Several classes of inhibitors have been developed to target the enzyme, with non-nucleoside reverse transcriptase inhibitors forming first-line treatment. Previously, covalent RT inhibitors have been identified and found to bind irreversibly to commonly mutated residues such as Y181C. In this work we aim to circumvent the issue of NNRTI resistance through targeting K102, which has not yet been identified to confer drug resistance. As reported here, 34 compounds were synthesized and characterized biochemically and structurally with wild-type (WT) HIV-1 RT. Two of these inhibitors demonstrate covalent inhibition as evidenced by protein crystallography, enzyme kinetics, mass spectrometry, and antiviral potency in HIV-1 infected human T-cell assays.

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.

Exploring novel HIV-1 reverse transcriptase inhibitors with drug-resistant mutants: A double mutant surprise.

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.

Lipophilic Nucleoside Triphosphate Prodrugs of Anti-HIV Active Nucleoside Analogs as Potential Antiviral Compounds.

Nucleoside analogs require three phosphorylation steps catalyzed by cellular kinases to give their triphosphorylated metabolites. Herein, the synthesis of two types of triphosphate prodrugs of different nucleoside analogs is disclosed. Triphosphates comprising: i) a γ-phosphate or γ-phosphonate bearing a bioreversible acyloxybenzyl group and a long alkyl group and ii) γ-dialkyl phosphate/phosphonate modified nucleoside triphosphate analogs. Almost selective conversion of the former TriPPPro-compounds into the corresponding γ-alkylated nucleoside triphosphate derivatives is demonstrated in CEM/0 cell extracts that proved to be stable toward further hydrolysis. The latter γ-dialkylated triphosphate derivatives lead to the slow formation of the corresponding NDPs. Both types of TriPPPro-compounds are highly potent in wild-type CEM/0 cells and more importantly, they exhibit even better activities against HIV-2 replication in CEM/TK- cell cultures. A finding of major importance is that, in primer extension assays, γ-phosphate-modified-NTPs, γ-mono-alkylated-triphosphates, and NDPs prove to be substrates for HIV-RT but not for cellular DNA-polymerases α,γ.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

5-Cyano substituted diarylpyridines as potent HIV-1 NNRTIs: Rational design, synthesis, and activity evaluation.

To develop more potent HIV-1 inhibitors against a variety of NNRTIs-resistant strains, a series of 5-cyano substituted diarylpyridines was designed based on the cocrystal structural analysis. Among them, I-5b showed the greatest potency (EC50 = 5.62-171 nM) against the wild-type (WT) and mutant HIV-1 strains. Especially for K103 N, I-5b exhibited outstanding activity with EC50 values of 9.37 nM, being much superior to that of NVP (EC50 = 5128 nM) and EFV (EC50 = 114 nM) and comparable to that of ETR (EC50 = 3.45 nM). In addition, the target of all compounds was turned out to be HIV-1 RT with moderate RT enzyme inhibitory activity (IC50 = 0.094-12.0 µM). Moreover, the binding mode of representative compounds with RT was elaborated via molecular docking.

Discovery of novel diarypyrimidine derivatives bearing six-membered non-aromatic heterocycles as potent HIV-1 NNRTIs with improved anti-resistance and drug-like profiles.

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 EC50 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.