Synthesis and anti-HCV activity of ß-d-2'-deoxy-2'-α-chloro-2'-ß-fluoro and ß-d-2'-deoxy-2'-α-bromo-2'-ß-fluoro nucleosides and their phosphoramidate prodrugs.

We report herein the synthesis and evaluation of a series of ß-d-2'-deoxy-2'-α-chloro-2'-ß-fluoro and ß-d-2'-deoxy-2'-α-bromo-2'-ß-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.

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 of (2S)-2-Chloro-2-fluororibolactone via Stereoselective Electrophilic Fluorination.

A novel and efficient route for the preparation of (2S)-2-chloro-2-fluorolactone 29 is described. This approach takes advantage of a highly efficient diastereoselective electrophilic fluorination reaction (94% yield; >50:1 dr).

Synthesis and antiviral evaluation of novel heteroarylpyrimidines analogs as HBV capsid effectors.

New modifications to the scaffold of previously reported HBV capsid assembly effectors such as BAY 41-4109, HAP-12 and GLS4 were explored. The anti-HBV activity in the HepAD38 system, and cytotoxicity profiles of each of the new compounds has been assessed. Among them, five new iodo- and bromo-heteroarylpyrimidines analogs displayed anti-HBV activity in the low micromolar range.

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.

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.

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.

Synthesis of carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases.

New carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases were synthesized and evaluated as potential anti-HIV and anti-HCV agents. Among the synthesized carbocyclic nucleoside analogs, the pyrazole amide 15f exhibited modest selective anti-HIV-1 activity (EC50 = 24 µM).

Synthesis and antiviral evaluation of 2',3'-dideoxy-2',3'-difluoro-D-arabinofuranosyl 2,6-disubstituted purine nucleosides.

The synthesis of new 2,6-disubstituted purine 2',3'-dideoxy-2',3'-difluoro-D-arabino nucleosides is reported. Their ability to block HIV and HCV replication along with their cytotoxicity toward Huh-7 cells, human lymphocyte, CEM and Vero cells was also assessed. Among them, ß-2,6-diaminopurine nucleoside 25 and guanosine derivative 27 demonstrate potent anti-HIV-1 activity (EC50 = 0.56 and 0.65 µM; EC90 = 4.2 and 3.1 µM) while displaying only moderate cytotoxicity in primary human lymphocytes.

Synthesis and antiviral evaluation of 2-amino-6-carbamoylpurine dioxolane nucleoside derivatives and their phosphoramidates prodrugs.

The synthesis of 9-(ß-d-1,3-dioxolan-4-yl)2,6-diaminopurine nucleoside phosphoramidate prodrugs as well as various 2-amino-6-carbamoylpurine dioxolane derivatives and their phosphoramidates prodrugs is reported. Their ability to block HIV and HBV replication along with their cytotoxicity toward HepG2, human lymphocyte, CEM and Vero cells was also assessed.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Synthesis of cyclopentanyl carbocyclic 5-fluorocytosine ((-)-5-fluorocarbodine) using a facially selective hydrogenation approach.

An efficient synthetic route to biologically relevant (-)-5-fluorocarbodine 6 was developed. Direct coupling of N(6)-protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring-closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4'-ß stereoisomer.

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.

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.

HIV nucleoside reverse transcriptase inhibitors.

More than 40 years into the pandemic, HIV remains a global burden and as of now, there is no cure in sight. Fortunately, highly active antiretroviral therapy (HAART) has been developed to manage and suppress HIV infection. Combinations of two to three drugs targeting key viral proteins, including compounds inhibiting HIV reverse transcriptase (RT), have become the cornerstone of HIV treatment. This review discusses nucleoside reverse transcriptase inhibitors (NRTIs), including chain terminators, delayed chain terminators, nucleoside reverse transcriptase translocation inhibitors (NRTTIs), and nucleotide competing RT inhibitors (NcRTIs); focusing on their history, mechanism of action, resistance, and current clinical application, including long-acting regimens.