Structure-based discovery of novel piperidine-biphenyl-DAPY derivatives as non-nucleoside reverse transcriptase inhibitors featuring improved potency, safety, and selectivity: From piperazine-biphenyl-DAPYs to piperidine-biphenyl-DAPYs.

Starting from our previously reported nonnucleoside reverse transcriptase inhibitor (NNRTI, 3), continuous efforts were made to enhance its potency and safety through a structure-based drug design strategy. This led to the discovery of a series of novel piperidine-biphenyl-diarylpyrimidines (DAPYs). Compound 10p, the most active compound in this series, exhibited an EC50 value of 6 nM against wide-type HIV-1 strain, which was approximately 560-fold more potent than the initial compound 3 (EC50 = 3.36 µM). Furthermore, significant improvements were observed in cytotoxicity and selectivity (CC50 > 202.17 µM, SI > 33144) compared to compound 3 (CC50 = 14.84 µM, SI = 4). Additionally, compound 10p demonstrated increased inhibitory activity against clinically mutant virus strains (EC50 = 7-63 nM). Further toxicity evaluation revealed that compound 10p exhibited minimal CYP enzyme and hERG inhibition. Importantly, single-dose acute toxicity testing did not result in any fatalities or noticeable pathological damage in mice. Therefore, compound 10p can be regarded as a lead candidate for guiding further development of biphenyl-diarylpyrimidine NNRTIs with favorable druggability for HIV therapy.

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.

Picomolar inhibitor of reverse transcriptase featuring significantly improved metabolic stability.

Considering the undesirable metabolic stability of our recently identified NNRTI 5 (t1/2 = 96 min) in human liver microsomes, we directed our efforts to improve its metabolic stability by introducing a new favorable hydroxymethyl side chain to the C-5 position of pyrimidine. This strategy provided a series of novel methylol-biphenyl-diarylpyrimidines with excellent anti-HIV-1 activity. The best compound 9g was endowed with remarkably improved metabolic stability in human liver microsomes (t1/2 = 2754 min), which was about 29-fold longer than that of 5 (t1/2 = 96 min). This compound conferred picomolar inhibition of WT HIV-1 (EC50 = 0.9 nmol/L) and low nanomolar activity against five clinically drug-resistant mutant strains. It maintained particularly low cytotoxicity (CC50 = 264 µmol/L) and good selectivity (SI = 256,438). Molecular docking studies revealed that compound 9g exhibited a more stable conformation than 5 due to the newly constructed hydrogen bond of the hydroxymethyl group with E138. Also, compound 9g was characterized by good safety profiles. It displayed no apparent inhibition of CYP enzymes and hERG. The acute toxicity assay did not cause death and pathological damage in mice at a single dose of 2 g/kg. These findings paved the way for the discovery and development of new-generation anti-HIV-1 drugs.

Development of fluorine-substituted NH2-biphenyl-diarylpyrimidines as highly potent non-nucleoside reverse transcriptase inhibitors: Boosting the safety and metabolic stability.

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC50 = 1.0 nmol/L), but the poor metabolic stability in human liver microsomes (t 1/2 = 14.6 min) and insufficient selectivity (SI = 2059) with high cytotoxicity (CC50 = 2.08 µmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH2-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC50 = 1.8-349 nmol/L). The best compound 5t in this collection (EC50 = 1.8 nmol/L, CC50 = 117 µmol/L) was 32-fold in selectivity (SI = 66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t 1/2 = 74.52 min), approximately 5-fold higher than JK-4b in human liver microsomes (t 1/2 = 14.6 min). Also, 5t possessed good stability in both human and monkey plasma. No significant in vitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

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.

Development of fluorine-substituted NH2-biphenyl-diarylpyrimidines as highly potent non-nucleoside reverse transcriptase inhibitors: Boosting the safety and metabolic stability

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC50 = 1.0 nmol/L), but the poor metabolic stability in human liver microsomes (t 1/2 = 14.6 min) and insufficient selectivity (SI = 2059) with high cytotoxicity (CC50 = 2.08 μmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH2-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC50 = 1.8-349 nmol/L). The best compound 5t in this collection (EC50 = 1.8 nmol/L, CC50 = 117 μmol/L) was 32-fold in selectivity (SI = 66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t 1/2 = 74.52 min), approximately 5-fold higher than JK-4b in human liver microsomes (t 1/2 = 14.6 min). Also, 5t possessed good stability in both human and monkey plasma. No significant in vitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

Picomolar inhibitor of reverse transcriptase featuring significantly improved metabolic stability

Considering the undesirable metabolic stability of our recently identified NNRTI 5 (t1/2 = 96 min) in human liver microsomes, we directed our efforts to improve its metabolic stability by introducing a new favorable hydroxymethyl side chain to the C-5 position of pyrimidine. This strategy provided a series of novel methylol-biphenyl-diarylpyrimidines with excellent anti-HIV-1 activity. The best compound 9g was endowed with remarkably improved metabolic stability in human liver microsomes (t1/2 = 2754 min), which was about 29-fold longer than that of 5 (t1/2 = 96 min). This compound conferred picomolar inhibition of WT HIV-1 (EC50 = 0.9 nmol/L) and low nanomolar activity against five clinically drug-resistant mutant strains. It maintained particularly low cytotoxicity (CC50 = 264 μmol/L) and good selectivity (SI = 256,438). Molecular docking studies revealed that compound 9g exhibited a more stable conformation than 5 due to the newly constructed hydrogen bond of the hydroxymethyl group with E138. Also, compound 9g was characterized by good safety profiles. It displayed no apparent inhibition of CYP enzymes and hERG. The acute toxicity assay did not cause death and pathological damage in mice at a single dose of 2 g/kg. These findings paved the way for the discovery and development of new-generation anti-HIV-1 drugs.

Design, synthesis, and antiviral evaluation of novel piperidine-substituted arylpyrimidines as HIV-1 NNRTIs by exploring the hydrophobic channel of NNIBP.

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 (EC50 = 19 nM) was found to be the most active molecule, which is better than NVP (EC50 = 0.10 µM). In addition, most of the compounds displayed micromolar activity against K103N and E138K mutant strains, while FT1 (EC50(K103N) = 50 nM, EC50(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).

Design, synthesis and anti-HIV evaluation of novel 5-substituted diarylpyrimidine derivatives as potent HIV-1 NNRTIs.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely used in combination therapies against HIV-1. As a continuation of our efforts to discover and develop "me-better" drugs of DAPYs, novel diarylpyrimidine derivatives were designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells. All the compounds demonstrated strong inhibition activity against wide-type HIV-1 strain (IIIB) with EC50 values in the range of 2.5 nM ~ 0.93 µM. Among them, compounds IVB-5-4 and IVB-5-8 were the most potent ones which showed anti-HIV-1IIIB activity much superior than that of Nevirapine, comparable to Efavirenz and Etravirine. What's more, some compounds also showed low nanomole activity against some mutant strains such as K103N and E138K. The selected compound IVB-5-4 was also evaluated for the activity against reverse transcriptase (RT), and exhibited submicromolar IC50 values indicating that this series compounds are specific RT inhibitors. Preliminary structure-activity relationships and modeling studies of these new analogues provide valuable avenues for future molecular optimization.

Chemical space exploration of novel naphthyl-carboxamide-diarylpyrimidine derivatives with potent anti-HIV-1 activity.

Fifteen naphthyl-carboxamide-DAPYs were generated to explore chemical space in reverse transcriptase (RT) binding site via lead optimization strategy. They displayed up to single-digit nanomolar activity against wild-type (WT) and rilpivirine-associated resistant mutant E138K viruses, as well as potent inhibitory ability toward the RT enzyme. Compound a1 showed exceptionally inhibitory effects with an EC50 value of 3.7 nM against HIV-1 wt strain, and an EC50 of 11 nM targeting mutant E138K. The structure-activity relationships (SARs) of the newly obtained DAPYs were also investigated. Molecular docking analysis elucidated the biological activity and offered a structural insight for follow-up research.

Development of non-nucleoside reverse transcriptase inhibitors (NNRTIs): our past twenty years.

Human immunodeficiency virus (HIV) is the primary infectious agent of acquired immunodeficiency syndrome (AIDS), and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are the cornerstone of HIV treatment. In the last 20 years, our medicinal chemistry group has made great strides in developing several distinct novel NNRTIs, including 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), thio-dihydro-alkoxy-benzyl-oxopyrimidine (S-DABO), diaryltriazine (DATA), diarylpyrimidine (DAPY) analogues, and their hybrid derivatives. Application of integrated modern medicinal strategies, including structure-based drug design, fragment-based optimization, scaffold/fragment hopping, molecular/fragment hybridization, and bioisosterism, led to the development of several highly potent analogues for further evaluations. In this paper, we review the development of NNRTIs in the last two decades using the above optimization strategies, including their structure-activity relationships, molecular modeling, and their binding modes with HIV-1 reverse transcriptase (RT). Future directions and perspectives on the design and associated challenges are also discussed.

Scaffold Hopping in Discovery of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors: From CH(CN)-DABOs to CH(CN)-DAPYs.

Scaffold hopping is a frequently-used strategy in the development of non-nucleoside reverse transcriptase inhibitors. Herein, CH(CN)-DAPYs were designed by hopping the cyano-methylene linker of our previous published CH(CN)-DABOs onto the etravirine (ETR). Eighteen CH(CN)-DAPYs were synthesized and evaluated for their anti-HIV activity. Most compounds exhibited promising activity against wild-type (WT) HIV-1. Compounds B4 (EC50 = 6 nM) and B6 (EC50 = 8 nM) showed single-digit nanomolar potency against WT HIV-1. Moreover, these two compounds had EC50 values of 0.06 and 0.08 µM toward the K103N mutant, respectively, which were comparable to the reference efavirenz (EFV) (EC50 = 0.08 µM). The preliminary structure-activity relationship (SAR) indicated that introducing substitutions on C2 of the 4-cyanophenyl group could improve antiviral activity. Molecular docking predicted that the cyano-methylene linker was positioned into the hydrophobic cavity formed by Y181/Y188 and V179 residues.

Pharmacophore-fusing design of pyrimidine sulfonylacetanilides as potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Twenty-seven derivatives (40-66) were generated by pharmacophore fusing of sulfonylacetanilide-diarylpyrimidine (1) with rilpivirine or biphenyl-diarylpyrimidines. They displayed up to single-digit nanomolar activity against wild-type (WT) virus and various drug-resistant mutant strains in HIV-1-infected MT-4 cells, thereby targeting the reverse transcriptase (RT) enzyme. Compound 51 displayed exceptionally potent activity against WT virus (EC50 = 6 nM) and several mutant strains (L100I, EC50 = 8 nM, K103N, EC50 = 6 nM, Y181C, EC50 = 26 nM, Y188L, EC50 = 122 nM, E138K, EC50 = 26 nM). The structure-activity relationships of the newly obtained pyrimidine sulfonylacetanilides were also elucidated. Molecular docking analysis explained the activity and provided a structural insight for follow-up research.

Fragment hopping-based discovery of novel sulfinylacetamide-diarylpyrimidines (DAPYs) as HIV-1 nonnucleoside reverse transcriptase inhibitors.

The fragment hopping approach is widely applied in drug development. A series of diarylpyrimidines (DAPYs) were obtained by hopping the thioacetamide scaffold to novel human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitors (NNRTIs) to address the cytotoxicity issue of Etravirine and Rilpivirine. Although the new compounds (11a-l) in the first-round optimization possessed less potent anti-viral activity, they showed much lower cytotoxicity. Further optimization on the sulfur led to the sulfinylacetamide-DAPYs exhibiting improved anti-viral activity and a higher selectivity index especially toward the K103N mutant strain. The most potent compound 12a displayed EC50 values of 0.0249 µM against WT and 0.0104 µM against the K103N mutant strain, low cytotoxicity (CC50 > 221 µM) and a high selectivity index (SI WT > 8873, SI K103N > 21186). In addition, this compound showed a favorable in vitro microsomal stability across species. Computational study predicted the binding models of these potent compounds with HIV-1 reverse transcriptase thus providing further insights for new developments.

Development of non-nucleoside reverse transcriptase inhibitors (NNRTIs): our past twenty years

Human immunodeficiency virus (HIV) is the primary infectious agent of acquired immunodeficiency syndrome (AIDS), and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are the cornerstone of HIV treatment. In the last 20 years, our medicinal chemistry group has made great strides in developing several distinct novel NNRTIs, including 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), thio-dihydro-alkoxy-benzyl-oxopyrimidine (-DABO), diaryltriazine (DATA), diarylpyrimidine (DAPY) analogues, and their hybrid derivatives. Application of integrated modern medicinal strategies, including structure-based drug design, fragment-based optimization, scaffold/fragment hopping, molecular/fragment hybridization, and bioisosterism, led to the development of several highly potent analogues for further evaluations. In this paper, we review the development of NNRTIs in the last two decades using the above optimization strategies, including their structure-activity relationships, molecular modeling, and their binding modes with HIV-1 reverse transcriptase (RT). Future directions and perspectives on the design and associated challenges are also discussed.

Follow on-based optimization of the biphenyl-DAPYs as HIV-1 nonnucleoside reverse transcriptase inhibitors against the wild-type and mutant strains.

The present work follows our preliminary discovery of biphenyl diarylpyrimidines (DAPYs) as HIV-1 nonnucleoside reverse transcriptase inhibitors. Further structural optimization of biphenyl-DAPYs led to the identification of a new series of biphenyl-substituted thiophene[3,2-d]pyrimidine analogues by a scaffold-hopping strategy. Biological evaluation of this series showed that these compounds possessed up to single-digit nanomolar potency (EC50 = 7.8-526.2 nM) and prominently low toxicity (CC50 = 18.5-280.8 µM) against wild-type (WT) HIV-1-infected cells. Furthermore, the results also demonstrated that compounds 29-32 exhibited high, broad-spectrum antiviral effects against clinically observed HIV-1 mutants. Specifically, compound 30, which had the highest selectivity index (SI = 16094) and the best anti-reverse transcriptase ability (IC50 = 39 nM), displayed marked inhibitory activity (EC50 = 13.5, 9.4, 17.0, 52.0, and 58.2 nM) against WT, K103N, E138K, L100I, Y181C mutants and moderate activity against double mutants. This study provides important avenues for the further design of HIV-1 inhibitors.

Synthesis and biological evaluation of dihydroquinazoline-2-amines as potent non-nucleoside reverse transcriptase inhibitors of wild-type and mutant HIV-1 strains.

A novel series of dihydroquinazolin-2-amine derivatives were synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell cultures. All of the molecules were active against wild-type HIV-1 with EC50 values ranging from 0.61 µM to 0.84 nM. The most potent inhibitor, compound 4b, had an EC50 value of 0.84 nM against HIV-1 strain IIIB, and thus was more active than the reference drugs efavirenz and etravirine. Moreover, most of the compounds maintained high activity (low-micromolar EC50 values) against strains bearing the reverse transcriptase (RT) E138K mutation. Compound 4b had EC50 values of 3.5 nM and 66 nM against non-nucleoside reverse transcriptase inhibitor-resistant strains bearing the RT E138K and RES056 mutations. In enzyme activity assays, compound 4b exhibited an IC50 value of 10 nM against HIV-1 RT. Preliminary SARs and molecular docking studies provide valuable insights for further optimization.

Docking-based 3D-QSAR and pharmacophore studies on diarylpyrimidines as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

Diarylpyrimidines (DAPYs), a type of effective HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs), have been considered as one of the most successful agents for treating AIDS. A number of structurally diverse DAPYs have been designed and synthesized in the past decade, and most of them exhibited potent anti-HIV-1 activities; however, the structure-activity relationships of recently reported DAPYs and their pharmacophore features that interacted with HIV-1 reverse transcriptase (RT) remain to be studied. In the present study, molecular docking studies were first performed on three novel classes of DAPYs to study their binding pattern in the HIV-1 RT. Based on the docking conformations of these DAPYs, 3D-QSAR models were constructed using CoMSIA and Topomer CoMFA methods, and pharmacophore models were also built using distance comparison technique. All selected DAPYs presented preferred U- or L-shaped conformations while being docked into the non-nucleoside inhibitor-binding pocket of the HIV-1 RT. The best CoMSIA model exhibited powerful predictivity, with satisfactory statistical parameters such as a q2 of 0.572, an r2 of 0.952, and an [Formula: see text] of 0.728. Contour maps of the best CoMSIA model were in accordance with those of the Topomer CoMFA model, giving the insight into the feature requirements of DAPYs for the anti-HIV-1 activity. Three potential pharmacophore models were constructed, and each of them was consisted of five hypothesis features. All results suggested that the aromatic ring on the left wing of DAPYs and the central pyrimidine ring contained key pharmacophore features for the anti-HIV-1 activity, and also indicated that the right wing of DAPYs had potential for further structural modification to improve activity. Eight novel DAPY molecules with potential anti-HIV-1 activities were designed on the basis of the obtained results. The findings in this study might provide important information for further design and development of novel HIV-1 NNRTIs.

Discovery of uracil-bearing DAPYs derivatives as novel HIV-1 NNRTIs via crystallographic overlay-based molecular hybridization.

A novel series of uracil-bearing DAPYs derivatives were designed and synthesized via structure-based molecular hybridization to discover compounds with improved anti-resistance profiles. Anti-HIV activity of the designed compounds was tested in MT-4 cell cultures. The most promising compound 16d showed excellent activity with EC50 value of 5.6 nM against wide-type HIV-1 and low cytotoxicity (SI > 50000). Activity against the clinic prevalent mutant strains was also tested, suggesting that 16d was sensitive to E138K (EC50 = 34.2 nM). Primary drug-like properties, such as water solubility and logP, were evaluated by experiment or calculation, which indicated that introducing an uracil can improve solubility. The molecular modeling accompanied with the preliminary SAR correlations paved the way for the next round of rational design of potent anti-HIV agents.

Synthesis and biological evaluation of DAPY-DPEs hybrids as non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A series of new DAPY-DPEs hybrids, combined the important pharmacophores of DAPYs and DPEs, has been synthesized and biologically evaluated for their anti-HIV activities against wild-type HIV-1 strain IIIB, double RT mutant (K103N+Y181C) strain RES056 and HIV-2 strain ROD in MT-4 cell cultures. Many promising candidates with potent inhibitory activity (wild-type) within the EC50 range from 0.16 to 0.013 µM were obtained. In particular, 3c, 3p, 3r and 3s displayed low nM level EC50 values (35, 13, 50 and 17 nM, respectively) and high selectivity (9342, 25131, 2890 and 11338, respectively), which were much more potent than NVP (EC50=0.31 µM, SI=48), 3TC (EC50=2.24 µM, SI>39), DDI (EC50=23.20 µM, SI>9) and DLV (EC50=0.65 µM, SI>67), and comparable to AZT (EC50=0.0071 µM, SI>13144) and EFV (EC50=0.0062 µM, SI>1014). The HIV-1 reverse transcriptase inhibitory assay confirmed that these DAPY-DPEs hybrids targeted HIV-1 RT. Molecular simulation was performed to investigate the potential binding mode of the newly synthesized compounds. And reasonable explanation for the activity results was discussed with docking method.