Synthesis of Novel N4-Hydrocytidine Analogs as Potential Anti-SARS-CoV-2 Agents.

Coronavirus disease 2019 (COVID-19) is an emerging global pandemic with severe morbidity and mortality caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molnupiravir, an ester prodrug form of N4-hydroxycytidine (NHC), was recently emergency-use approved for the treatment of early SARS-CoV-2 infections. Herein, we report the synthesis and evaluation of a series of novel NHC analogs.

Diastereoselective Synthesis of 2'-Dihalopyrimidine Ribonucleoside Inhibitors of Hepatitis C Virus Replication.

We present a newly developed synthetic route to 2-bromo-2-fluoro ribolactone based on our published 2-chloro-2-fluoro ribolactone synthesis. Stereoselective fluorination is key to controlling the 2-diastereoselectivity. We also report a substantially improved glycosylation reaction with both the 2-bromo-2-fluoro and 2-chloro-2-fluoro sugars. These improvements allowed us to prepare 2'-dihalo nucleosides 13 and 14 in an overall 15-20% yield.

7-Deaza-7-fluoro-2'-C-methyladenosine inhibits Zika virus infection and viral-induced neuroinflammation.

Zika virus (ZIKV) has gained a lot of attention in the past few years due to its rapid spread worldwide and its close association to severe neurological outcomes, such as microcephaly and Guillain-Barre syndrome. In this study, the in vitro and in vivo anti-ZIKV activity of 7-deaza-7-fluoro-2'-C-methyl-adenosine (DFMA) was evaluated. In vitro, using primary mouse neuronal cells and human neural stem cells infected by ZIKV, treatment with DFMA resulted in impaired viral replication and protection against virus-induced cell death. In vivo, when administrated prior to infection, DFMA prevented lethality and markedly reduced viral loads and neuroinflammation, including microgliosis and overall brain damage. Additionally, as an early therapeutic treatment, DFMA increased survival rates in mice. Collectively, these findings demonstrate that the nucleoside analog DFMA inhibits ZIKV infection and viral-induced neuroinflammation in vitro and in vivo without apparent untoward effects, suggesting it may be useful in individuals infected with ZIKV.

Intracellular metabolism and potential cardiotoxicity of a ß-D-2'-C-methyl-2,6-diaminopurine ribonucleoside phosphoramidate that inhibits hepatitis C virus replication.

ß-D-2'-C-Methyl-2,6-diaminopurine ribonucleoside (2'-C-Me-DAPN) phosphoramidate prodrug (DAPN-PD) is a selective hepatitis C virus inhibitor that is metabolized intracellularly into two active metabolites: 2'-C-Methyl-DAPN triphosphate (2'-C-Me-DAPN-TP) and 2'-C-methyl-guanosine 5'-triphosphate (2'-C-Me-GTP). BMS-986094 and IDX-184 are also bioconverted to 2'-C-Me-GTP. A phase IIb clinical trial with BMS-986094 was abruptly halted due to adverse cardiac and renal effects. Herein, we developed an efficient large scale synthesis of DAPN-PD and determined intracellular pharmacology of DAPN-PD in comparison with BMS-986094 and IDX-184, versus Huh-7, HepG2 and interspecies primary hepatocytes and human cardiomyocytes. Imaging data of drug treated human cardiomyocytes was found to be most useful in determining toxicity potential as no obvious beating rate change was observed for IDX-184 up to 50 µM up at 48 h. However, with BMS-986094 and DAPN-PD at 10 µM changes to both beat rate and rhythm were noted.

Novel influenza polymerase PB2 inhibitors for the treatment of influenza A infection.

Exploration of the chemical space of known influenza polymerase PB2 inhibitor Pimodivir, was performed by our group. We synthesized and identified compounds 16a and 16b, two novel thienopyrimidine derivatives displaying anti-influenza A activity in the single digit nanomolar range in cell culture. Binding of these unique compounds in the influenza polymerase PB2 pocket was also determined using molecular modeling.

Nucleoside Analogs with Antiviral Activity against Yellow Fever Virus.

Yellow fever virus (YFV) is a human Flavivirus reemerging in parts of the world. While a vaccine is available, large outbreaks have recently occurred in Brazil and certain African countries. Development of an effective antiviral against YFV is crucial, as there is no available effective drug against YFV. We have identified several novel nucleoside analogs with potent antiviral activity against YFV with 50% effective concentration (EC50) values between 0.25 and 1 µM with selectivity indices over 100 in culture.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.

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.

2'-Chloro,2'-fluoro Ribonucleotide Prodrugs with Potent Pan-genotypic Activity against Hepatitis C Virus Replication in Culture.

Pan-genotypic nucleoside HCV inhibitors display a high genetic barrier to drug resistance and are the preferred direct-acting agents to achieve complete sustained virologic response in humans. Herein, we report, the discovery of a ß-d-2'-Cl,2'-F-uridine phosphoramidate nucleotide 16, as a nontoxic pan-genotypic anti-HCV agent. Phosphoramidate 16 in its 5'-triphosphate form specifically inhibited HCV NS5B polymerase with no marked inhibition of human polymerases and cellular mitochondrial RNA polymerase. Studies on the intracellular half-life of phosphoramidate 16-TP in live cells demonstrated favorable half-life of 11.6 h, suggesting once-a-day dosing. Stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes make phosphoramidate 16 a prospective candidate for further studies to establish its potential value as a new anti-HCV agent.

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.

Substrates and Inhibitors of SAMHD1.

SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'R)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'R)-2'-F and (2'R)-2'-OH nucleotides. However, arabinose ((2'S)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'S)-2' sugar moiety is increased to a (2'S)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1's dNTPase activity. Our computational modeling suggests that (2'S)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 in vivo.

Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094.

Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on ß-d-2'-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2'-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2'-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo Finally, we found that although both 2'-C-methyl-GTP and 2'-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2'-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2'-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.

Synthesis and Evaluation of 2,6-Modified Purine 2'-C-Methyl Ribonucleosides as Inhibitors of HCV Replication.

A variety of 2,6-modified purine 2'-C-methylribonucleosides and their phosphoramidate prodrugs were synthesized and evaluated for inhibition of HCV RNA replication in Huh-7 cells and for cytotoxicity in various cell lines. Cellular pharmacology and HCV polymerase incorporation studies on the most potent and selective compound are reported.

ß-D-2'-C-Methyl-2,6-diaminopurine Ribonucleoside Phosphoramidates are Potent and Selective Inhibitors of Hepatitis C Virus (HCV) and Are Bioconverted Intracellularly to Bioactive 2,6-Diaminopurine and Guanosine 5'-Triphosphate Forms.

The conversion of selected ß-D-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing for the generation of two bioactive nucleoside triphosphates (NTPs) in human hepatocytes. A variety of 2'-C-methyl DAPN-PDs were prepared and evaluated for inhibition of HCV viral replication in Huh-7 cells, cytotoxicity in various cell lines, and cellular pharmacology in both Huh-7 and primary human liver cells. The DAPN-PDs were pan-genotypic, effective against various HCV resistant mutants, and resistant variants could not be selected. 2'-C-Me-DAPN-TP and 2'-C-Me-GTP were chain terminators for genotype 1b HCV-pol, and single nucleotide incorporation assays revealed that 2'-C-Me-DAPN-TP was incorporated opposite U. No cytotoxicity was observed with our DAPN-PD when tested up to 50 µM. A novel, DAPN-PD, 15c, has been selected for further evaluation because of its good virologic and toxicologic profile and its ability to deliver two active metabolites, potentially simplifying HCV treatment.

Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition.

Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs' activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein-protein interactions at this membrane interface can explain potent inhibition of replication-complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.

Chutes and ladders in hepatitis C nucleoside drug development.

Chutes and Ladders is an exciting up-and-down-again game in which players race to be the first to the top of the board. Along the way, they will find ladders to help them advance, and chutes that will cause them to move backwards. The development of nucleoside analogs for clinical treatment of hepatitis C presents a similar scenario in which taking shortcuts may help quickly advance a program, but there is always a tremendous risk of being sent backwards as one competes for the finish line. In recent years the treatment options for chronic hepatitis C virus (HCV) infection have expand due to the development of a replicon based in vitro evaluation system, allowing for the identification of multiple drugable viral targets along with a concerted and substantial drug discovery effort. Three major drug targets have reached clinical study for chronic HCV infection: the NS3/4A serine protease, the large phosphoprotein NS5A, and the NS5B RNA-dependent RNA polymerase. Recently, two oral HCV protease inhibitors were approved by the FDA and were the first direct acting anti-HCV agents to result from the substantial research in this area. There are currently many new chemical entities from several different target classes that are being evaluated worldwide in clinical trials for their effectiveness at achieving a sustained virologic response (SVR) (Pham et al., 2004; Radkowski et al., 2005). Clearly the goal is to develop therapies leading to a cure that are safe, widely accessible and available, and effective against all HCV genotypes (GT), and all stages of the disease. Nucleoside analogs that target the HCV NS5B polymerase that have reached human clinical trials is the focus of this review as they have demonstrated significant advantages in the clinic with broader activity against the various HCV GT and a higher barrier to the development of resistant viruses when compared to all other classes of HCV inhibitors.

Adenosine Dioxolane Nucleoside Phosphoramidates as Antiviral Agents for Human Immunodeficiency and Hepatitis B Viruses.

There are currently six nucleoside reverse transcriptase inhibitors (NRTI) that are FDA approved for human clinical use and these remain the backbone of current HIV therapy. In order for these NRTIs to be effective they need to be phosphorylated consecutively by cellular kinases to their triphosphate forms. Herein, we report the synthesis of C-6 modified (-)-ß-D-(2R,4R)-1,3-dioxolane adenosine nucleosides and their nucleotides including our novel phosphoramidate prodrug technology. We have introduced a side chain moiety on the phenol portion of the phosphoramidate to reduce the toxicity potential. The synthesized phosphoramidates displayed up to a 3,600-fold greater potency versus HIV-1 when compared to their corresponding parent nucleoside and were up to 300-fold more potent versus HBV. No cytotoxicity was observed up to 100 µM in the various cell systems tested, except for compound 17 and 18 which displayed a CC50 of 7.3 and 12 µM respectively in Huh-7 cells. The improved and significant dual antiviral activity of these novel phosphoramidate nucleosides was partially explained by the increased intracellular formation of the adenosine dioxolane triphosphate.

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).

Scaleable processes for the synthesis of (-)-ß-D-2,6-diaminopurine dioxolane (Amdoxovir, DAPD) and (-)-ß-D-2-aminopurine dioxolane (APD).

An efficient and scalable synthesis of (-)-DAPD and (-)-APD has been developed. We discovered that t-butyl cyanoacetate can be used as a new additive for the sugar nucleoside base coupling step en route to DAPD with improved ß-selectivity and an isolated yield four fold greater than the original process scale method. Using this new process, (-)-DAPD has been prepared on greater than 20 g scale. In the synthesis of (-)-APD, a key enzyme-catalyzed hydrolysis reaction afforded the water-soluble deprotected α-anomer while leaving the ß-anomer completely untouched.