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 Potent DAG-Lactone Derivatives as HIV Latency Reversing Agents.

Toward human immunodeficiency virus type-1 (HIV-1) cure, cells latently infected with HIV-1 must be eliminated from people living with HIV-1. We previously developed a protein kinase C (PKC) activator, diacylglycerol (DAG)-lactone derivative 3, with high HIV-1 latency-reversing activity, based on YSE028 (2) as a lead compound and found that the activity was correlated with binding affinity for PKC and stability against esterase-mediated hydrolysis. Here, we synthesized new DAG-lactone derivatives not only containing a tertiary ester group or an isoxazole surrogate but also several symmetric alkylidene moieties to improve HIV-1 latency reversing activity. Compound 9a, with a dimethyl group at the α-position of the ester group, exerted twice higher HIV-1 latency reversing activity than compound 3, and compound 26, with the isoxazole moiety, was significantly active. In addition, DAG-lactone derivatives with moderate hydrophobicity and potent biostability showed high biological activity.

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.

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-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.

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.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Development of Novel Dihydrofuro[3,4-d]pyrimidine Derivatives as HIV-1 NNRTIs to Overcome the Highly Resistant Mutant Strains F227L/V106A and K103N/Y181C.

Here, we report the design, synthesis, structure-activity relationship studies, antiviral activity, enzyme inhibition, and druggability evaluation of dihydrofuro[3,4-d]pyrimidine derivatives as a potent class of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Compounds 14b (EC50 = 5.79-28.3 nM) and 16c (EC50 = 2.85-18.0 nM) exhibited superior potency against a panel of HIV-1-resistant strains. Especially, for the changeling mutations F227L/V106A and K103N/Y181C, both compounds exhibited remarkably improved activity compared to those of etravirine and rilpivirine. Moreover, 14b and 16c showed moderate RT enzyme inhibition (IC50 = 0.14-0.15 µM), which demonstrated that they acted as HIV-1 NNRTIs. Furthermore, 14b and 16c exhibited favorable pharmacokinetic and safety properties, making them excellent leads for further development.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 µM and 0.15-2 µM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Synthesis and anti-HIV activity of a new isoxazole containing disubstituted 1,2,4-oxadiazoles analogs.

Continuing on our antiviral drug discovery research, we intended to diversify our lead anti-HIV-1 inhibitor by non-classical isosteric replacement of amide to 1,2,4-oxadiazoles. The resulting molecules isoxazole-1,2,4-oxadiazole analogs were synthesized using mild bases in ethanol under microwave irradiation. The anti-HIV potential was checked in human CD4+ reporter cell lines, TZM-bl and CEM-GFP, at the highest non-cytotoxic concentration (HNC), demonstrating that 3-((3-(p-tolyl)isoxazol-5-yl)methyl)-1,2,4-oxadiazole and 3-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-1,2,4-oxadiazole inhibit HIV-1 replication significantly and could be considered as a new lead candidate against HIV-1.

Mononucleoside phosphorodithiolates as mononucleotide prodrugs.

The synthesis and in vitro anti-HIV activity of a novel series of pronucleotides are reported. These prodrugs were characterized by a phosphorodithiolate structure, incorporating two O-pivaloyl-2-oxyethyl substituents as biolabile phosphate protections. The compounds were obtained following an original one-pot three-step procedure, involving the formation of a phosphorodithioite intermediate which is in situ oxidized. In vitro, comparative anti-HIV evaluations demonstrate that such original prodrugs are able to allow the efficient intracellular release of the corresponding 5'-mononucleotide. The pronucleotide of 2',3'-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC50) values in nanomolar concentration range in various cell lines. In primary monocytes/macrophages, this derivative was 500 times more potent in inhibiting HIV replication (EC50 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.

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.

Expanding the known structure space for RNA binding: a test of 2,5-diketopiperazine.

As the importance of RNA as a therapeutic target has become increasingly recognized, the need for new chemotypes able to bind RNA has grown in significance. We hypothesized that diketopiperazines (DKPs), common substructures in natural products and protein-targeting therapeutic agents, could serve as effective scaffolds for targeting RNA. To confirm this hypothesis, we designed and synthesized two analogs, one incorporating a DKP and one not, of compounds previously demonstrated to bind an RNA critical to the life cycle of HIV-1 with high affinity and specificity. Prior to compound synthesis, calculations employing density functional methods and molecular mechanics conformational searches were used to confirm that the DKP could present functionality in a similar (albeit not identical) orientation to the non DKP-containing compound. We found that both the DKP-containing and parent compound had similar affinities to the target RNA as measured by surface plasmon resonance (SPR). Both compounds were found to have modest but equal anti-HIV activity. These results establish the feasibility of using DKPs to target RNA.

Development of Triazoles and Triazolium Salts Based on AZT and Their Anti-Viral Activity against HIV-1.

We report herein a set of 3'-azido-3'-deoxythymidine (AZT) derivatives based on triazoles and triazolium salts for HIV-1 infection. The compounds were synthesized via click chemistry with Cu(I) and Ru(II) catalysts. Triazolium salts were synthesized by reaction with methyl iodide or methyl triflate in good yields. The antiviral activity of the compounds was tested using two methodologies: In method one the activity was measured on infected cells; in method two a pre-exposure prophylaxis experimental model was employed. For method one the activity of the compounds was moderate, and in general the triazolium salts showed a decreased activity in relation to their triazole precursors. With method two the antiviral activity was higher. All compounds were able to decrease the infection, with two compounds able to clear almost all the infection, while a lower antiviral activity was noted for the triazolium salts. These results suggest that these drugs could play an important role in the development of pre-exposure prophylaxis therapies.

Design, Synthesis, and Antiviral activity of 1,2,3,4-Tetrahydropyrimidine derivatives acting as novel entry inhibitors to target at "Phe43 cavity" of HIV-1 gp120.

The HIV-1 invasion is initiated with the interaction of viral glycoprotein gp120 and cellular receptor CD4. The binding mechanism reveals two major hotspots involved in gp120-CD4 interaction. The first one is a hydrophobic cavity (Phe43 cavity) on gp120 capped with phenyl ring of phe43CD4 and the second is the electrostatic interaction between positive charge of Arg59CD4 and negative charge of Asp368gp120. Targeting these hotspots, small molecules for entry inhibition and HIV-1 neutralization were designed and tested. In the process, pyrimidine derivatives were identified as potent molecules to intercept gp120-CD4 binding by targeting both the hotspots. Herein, the synthesis, characterization of 1,2,3,4-Tetrahydropyrimidine derivatives, and biological evaluation on 93IN101, a clade C virus are presented. The paper presents a novel set of entry inhibitors to target dual hotspots on gp120 to inhibit protein-protein interactions.

Synthesis and Anti-HIV Activity of a Novel Series of Isoquinoline-Based CXCR4 Antagonists.

An expansion of the structure-activity relationship study of CXCR4 antagonists led to the synthesis of a series of isoquinolines, bearing a tetrahydroquinoline or a 3-methylpyridinyl moiety as head group. All compounds were investigated for CXCR4 affinity and antagonism in competition binding and calcium mobilization assays, respectively. In addition, the anti-HIV activity of all analogues was determined. All compounds showed excellent activity, with compound 24c being the most promising one, since it displayed consistently low nanomolar activity in the various assays.

Systematic Evaluation of Fluorination as Modification for Peptide-Based Fusion Inhibitors against HIV-1 Infection.

With the emergence of novel viruses, the development of new antivirals is more urgent than ever. A key step in human immunodeficiency virus type 1 (HIV-1) infection is six-helix bundle formation within the envelope protein subunit gp41. Selective disruption of bundle formation by peptides has been shown to be effective; however, these drugs, exemplified by T20, are prone to rapid clearance from the patient. The incorporation of non-natural amino acids is known to improve these pharmacokinetic properties. Here, we evaluate a peptide inhibitor in which a critical Ile residue is replaced by fluorinated analogues. We characterized the influence of the fluorinated analogues on the biophysical properties of the peptide. Furthermore, we show that the fluorinated peptides can block HIV-1 infection of target cells at nanomolar levels. These findings demonstrate that fluorinated amino acids are appropriate tools for the development of novel peptide therapeutics.

Design of the naphthyl-diarylpyrimidines as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) via structure-based extension into the entrance channel.

A series of novel naphthyl-diarylpyrimidine (DAPY) derivatives were designed and synthesized to explore the entrance channel of the non-nucleoside reverse transcriptase inhibitors binding pocket (NNIBP) by incorporating different flexible side chains at the C-6 position. The biological evaluation results showed that all analogues possessed promising HIV-1 inhibitory activity at the nanomolar concentration range. Three compounds (7, 9 and 39) displayed excellent potency against WT HIV-1 strain with EC50 values ranging from 5 to 10 nM and high selectivity indexes (SI = 3504, 30488 and 22846, respectively), which were higher than for nevirapine and comparable to the values for etravirine. The RT inhibition activity, preliminary structure-activity relationship and molecular docking study showed that the side chain at the C-6 position of the DAPYs occupied the entrance channel and significantly influenced anti-HIV activity and selectivity. Additionally, the physicochemical properties were investigated to evaluate the drug-like features, which indicated that introducing various substituents on the pyrimidine ring can improve solubility.

Design, synthesis, and mechanism study of dimerized phenylalanine derivatives as novel HIV-1 capsid inhibitors.

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 EC50 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.