Synthesis and Antiviral Evaluation of (1,4-Disubstituted-1,2,3-Triazol)-(E)-2-Methyl-but-2-Enyl Nucleoside Phosphonate Prodrugs.

A series of hitherto unknown (1,4-disubstituted-1,2,3-triazol)-(E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs bearing 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as the key synthetic step. All novel compounds were evaluated for their antiviral activities against HBV, HIV and SARS-CoV-2. Among these molecules, only compound 15j, a hexadecyloxypropyl (HDP)/(isopropyloxycarbonyl-oxymethyl)-ester (POC) prodrug, showed activity against HBV in Huh7 cell cultures with 62% inhibition at 10 µM, without significant cytotoxicity (IC50 = 66.4 µM in HepG2 cells, IC50 = 43.1 µM in HepG2 cells) at 10 µM.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase.

Nucleoside analog inhibitors (NAIs) are an important class of antiviral agents. Although highly effective, some NAIs with activity against hepatitis C virus (HCV) can cause toxicity, presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT was studied in order to explore structure-activity relationships that can facilitate the identification of nontoxic NAIs. These findings have important implications for the development of all anti-RNA virus NAIs.

Synthesis and anti-HCV activity of a series of ß-d-2'-deoxy-2'-dibromo nucleosides and their corresponding phosphoramidate prodrugs.

Several ß-d-2'-deoxy-2'-substituted nucleoside analogs have displayed potent and selective anti-HCV activities and some of them have reached human clinical trials. In that regard, we report herein the synthesis of a series of 2'-deoxy,2'-dibromo substituted U, C, G and A nucleosides 10a-d and their corresponding phosphoramidate prodrugs 13a-d. The synthesized nucleosides 10a-d and prodrugs 13a-d were evaluated for their inhibitory activity against HCV as well as cellular toxicity. The results showed that the most potent compound was prodrug 13a, which exhibited micromolar inhibitory activity (EC50 = 1.5 ±â€¯0.8 µM) with no observed toxicity. In addition, molecular modeling and free energy perturbation calculations for the 5'-triphosphate formed from 13a and related 2'-modified nucleotides are discussed.

Synthesis and antiviral evaluation of 2',2',3',3'-tetrafluoro nucleoside analogs.

Herein, we report the synthesis of novel 2',2',3',3'-tetrafluorinated nucleoside analogs along with their phosphoramidate prodrugs. A tetrafluoro ribose moiety was coupled with different Boc/benzoyl-protected nucleobases under Mitsunobu conditions. After deprotection, tetrafluorinated nucleosides 13b, 14b, 20b-22b were reacted with phenyl-(isopropoxy-L-alaninyl)-phosphorochloridate to afford corresponding monophosphate prodrugs 24b-28b. All synthesized compounds were evaluated against several DNA and RNA viruses including HIV, HBV, HCV, Ebola and Zika viruses.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Synthesis and evaluation of Janus type nucleosides as potential HCV NS5B polymerase inhibitors.

The synthesis of new ribo and 2'-ß-C-methyl ribo Janus type nucleosides J-AA, J-AG and J-AU is reported along with their ability to block HCV and HIV replication. Their toxicity was also assessed in Huh7, human lymphocytes, CEM and Vero cells.

Synthesis and evaluation of non-dimeric HCV NS5A inhibitors.

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI > 90,000).

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors.

Herein, we report the synthesis and structure-activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC50=0.8 µM).

Synthesis and evaluation of novel potent HCV NS5A inhibitors.

Judicious modifications to the structure of the previously reported HCV NS5A inhibitor 1, resulted in more potent anti-HCV compounds with similar and in some cases improved toxicity profiles. The synthesis of 19 new NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. For the most potent compounds chemical stability, stability in liver microsomes and inhibition of relevant CYP450 enzymes is also presented.

Synthesis and biological evaluation of new potent and selective HCV NS5A inhibitors.

NS5A inhibitors are a new class of direct-acting antiviral agents which display very potent anti-HCV activity in vitro and in humans. Rationally designed modifications to the central biphenyl linkage of a known NS5A series led to selection of several compounds that were synthesized and evaluated in a HCV genotype 1b replicon. The straight triphenyl linked compound 11a showed similar anti-HCV activity to the clinical compound BMS-790052 and a superior cytotoxicity profile in three different cell lines, with an EC(50) value of 26 pM and a therapeutic index of over four million in an HCV replicon assay. This triphenyl analog warrants further preclinical evaluation as an anti-HCV agent.

Synthesis, evaluation of anti-HIV-1 and anti-HCV activity of novel 2',3'-dideoxy-2',2'-difluoro-4'-azanucleosides.

A series of 2',3'-dideoxy-2',2'-difluoro-4'-azanucleosides of both pyrimidine and purine nucleobases were synthesized in an efficient manner starting from commercially available L-pyroglutamic acid via glycosylation of difluorinated pyrrolidine derivative 15. Several 4'-azanucleosides were prepared as a separable mixture of α- and ß-anomers. The 6-chloropurine analogue was obtained as a mixture of N(7) and N(9) regioisomers and their structures were identified based on NOESY and HMBC spectral data. Among the 4'-azanucleosides tested as HIV-1 inhibitors in primary human lymphocytes, four compounds showed modest activity and the 5-fluorouracil analogue (18d) was found to be the most active compound (EC(50)=36.9µM) in this series. None of the compounds synthesized in this study demonstrated anti-HCV activity.

Discovery of a 2'-Fluoro,2'-Bromouridine Phosphoramidate Prodrug Exhibiting Anti-Yellow Fever Virus Activity in Culture and in Mice.

Yellow fever virus (YFV) is a potentially lethal, zoonotic, blood-borne flavivirus transmitted to humans and non-human primates by mosquitoes. Owing to multiple deadly epidemics, the WHO classifies YFV as a "high impact, high threat disease" with resurgent epidemic potential. At present, there are no approved antiviral therapies to combat YFV infection. Herein we report on 2'-halogen-modified nucleoside analogs as potential anti-YFV agents. Of 11 compounds evaluated, three showed great promise with low toxicity, high intracellular metabolism into the active nucleoside triphosphate form, and sub-micromolar anti-YFV activity. Notably, we investigated a 2'-fluoro,2'-bromouridine phosphate prodrug (C9), a known anti-HCV agent with good stability in human blood and favorable metabolism. Predictive modeling revealed that C9 could readily bind the active site of the YFV RdRp, conferring its anti-YFV activity. C9 displayed potent anti-YFV activity in primary human macrophages, 3D hepatocyte spheroids, and in mice. In an A129 murine model, shortly after infection, C9 significantly reduced YFV replication and protected against YFV-induced liver inflammation and pathology with no adverse effects. Collectively, this work identifies a potent new anti-YFV agent with strong therapeutic promise.

Synthesis of purine modified 2'-C-methyl nucleosides as potential anti-HCV agents.

Based on the anti-hepatitis C activity of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine, a series of new modified purine 2'-C-methyl nucleosides was prepared as potential anti-hepatitis C virus agents. Herein, we report the synthesis of both 6-modified and 2-modified purine 2'-C-methyl-nucleosides along with their anti-HCV replication activity and cytotoxicity in different cells.

Synthesis and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleosides, their phosphoramidate prodrugs and 5'-triphosphates.

Thirty novel α- and ß-d-2'-deoxy-2'-fluoro-2'-C-methyl-7-deazapurine nucleoside analogs were synthesized and evaluated for in vitro antiviral activity. Several α- and ß-7-deazapurine nucleoside analogs exhibited modest anti-HCV activity and cytotoxicity. Four synthesized 7-deazapurine nucleoside phosphoramidate prodrugs (18-21) showed no anti-HCV activity, whereas the nucleoside triphosphates (22-24) demonstrated potent inhibitory effects against both wild-type and S282T mutant HCV polymerases. Cellular pharmacology studies in Huh-7 cells revealed that the 5'-triphosphates were not formed at significant levels from either the nucleoside or the phosphoramidate prodrugs, indicating that insufficient phosphorylation was responsible for the lack of anti-HCV activity. Evaluation of anti-HIV-1 activity revealed that an unusual α-form of 7-carbomethoxyvinyl substituted nucleoside (10) had good anti-HIV-1 activity (EC(50)=0.71±0.25 µM; EC(90)=9.5±3.3 µM) with no observed cytotoxicity up to 100 µM in four different cell lines.

Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside.

Remdesivir, a monophosphate prodrug of nucleoside analog GS-441524, is widely used for the treatment of moderate to severe COVID-19. It has been suggested to use GS-441524 instead of remdesivir in the clinic and in new inhalation formulations. Thus, we compared the anti-SARS-CoV-2 activity of remdesivir and GS-441524 in Vero E6, Vero CCL-81, Calu-3, Caco-2 â€‹cells, and anti-HCoV-OC43 activity in Huh-7 â€‹cells. We also compared the cellular pharmacology of these two compounds in Vero E6, Vero CCL-81, Calu-3, Caco-2, Huh-7, 293T, BHK-21, 3T3 and human airway epithelial (HAE) cells. Overall, remdesivir exhibited greater potency and superior intracellular metabolism than GS-441524 except in Vero E6 and Vero CCL-81 â€‹cells.

3'-(1,2,3-Triazol-1-yl)-3'-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure-activity investigations.

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3 -azido-3'-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3'-alpha-[1,2,3]triazol-3'-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3'-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K(m)/V(max) values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.

Discovery of a Series of 2'-α-Fluoro,2'-ß-bromo-ribonucleosides and Their Phosphoramidate Prodrugs as Potent Pan-Genotypic Inhibitors of Hepatitis C Virus.

Hepatitis C virus (HCV) nucleoside inhibitors display pan-genotypic activity, a high barrier to the selection of resistant virus, and are some of the most potent direct-acting agents with durable sustained virologic response in humans. Herein, we report, the discovery of ß-d-2'-Br,2'-F-uridine phosphoramidate diastereomers 27 and 28, as nontoxic pan-genotypic anti-HCV agents. Extensive profiling of these two phosphorous diastereomers was performed to select one for in-depth preclinical profiling. The 5'-triphosphate formed from these phosphoramidates selectively inhibited HCV NS5B polymerase with no inhibition of human polymerases and cellular mitochondrial RNA polymerase up to 100 µM. Both are nontoxic by a variety of measures and display good stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes. Ultimately, a preliminary oral pharmacokinetics study in male beagles showed that 28 is superior to 27 and is an attractive candidate for further studies to establish its potential value as a new clinical anti-HCV agent.