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The 35th International Conference on Antiviral Research (ICAR), sponsored by the International Society for Antiviral Research (ISAR), was held in Seattle, Washington, USA, on March 21-25, 2022 and concurrently through an interactive remote meeting platform. This report gives an overview of the conference on behalf of the society. It provides a general review of the meeting and awardees, summarizing the presentations and their main conclusions from the perspective of researchers active in many different areas of antiviral research and development. Through ICAR, leaders in the field of antiviral research were able to showcase their efforts, as participants learned about key advances in the field. The impact of these efforts was exemplified by many presentations on SARS-CoV-2 demonstrating the remarkable response to the ongoing pandemic, as well as future pandemic preparedness, by members of the antiviral research community. As we address ongoing outbreaks and seek to mitigate those in the future, this meeting continues to support outstanding opportunities for the exchange of knowledge and expertise while fostering cross-disciplinary collaborations in therapeutic and vaccine development. The 36th ICAR will be held in Lyon, France, March 13-17, 2023.

As a result of the multiple gathering and travels restrictions during the SARS-CoV-2 pandemic, the annual meeting of the International Society for Antiviral Research (ISAR), the International Conference on Antiviral Research (ICAR), could not be held in person in 2021. Nonetheless, ISAR successfully organized a remote conference, retaining the most critical aspects of all ICARs, a collegiate gathering of researchers in academia, industry, government and non-governmental institutions working to develop, identify, and evaluate effective antiviral therapy for the benefit of all human beings. This article highlights the 2021 remote meeting, which presented the advances and objectives of antiviral and vaccine discovery, research, and development. The meeting resulted in a dynamic and effective exchange of ideas and information, positively impacting the prompt progress towards new and effective prophylaxis and therapeutics., The author(s) received no financial support for the research, authorship, and/or publication of this article

The current focus for many researchers has turned to the development of therapeutics that have the potential for serving as broad-spectrum inhibitors that can target numerous viruses, both within a particular family, as well as to span across multiple viral families. This will allow us to build an arsenal of therapeutics that could be used for the next outbreak. In that regard, nucleosides have served as the cornerstone for antiviral therapy for many decades. As detailed herein, many nucleosides have been shown to inhibit multiple viruses due to the conserved nature of many viral enzyme binding sites. Thus, it is somewhat surprising that up until very recently, many researchers focused more on "one bug one drug," rather than trying to target multiple viruses given those similarities. This attitude is now changing due to the realization that we need to be proactive rather than reactive when it comes to combating emerging and reemerging infectious diseases. A brief summary of prominent nucleoside analogues that previously exhibited broad-spectrum activity and are now under renewed interest, as well as new analogues, that are currently under investigation against SARS-CoV-2 and other viruses is discussed herein.

Nucleoside analogues have long served as key chemotherapeutic drugs for the treatment of viral infections and cancers. Problems associated with the development of drug resistance have led to a search for the design of nucleosides capable of bypassing point mutations in the target enzyme's binding site. As a possible answer to this, the Seley-Radtke group developed a flexible nucleoside scaffold (fleximers), where the heterocyclic purine base is split into its two components, i.e. pyrimidine and imidazole. Herein, we present a series of new pyrazole-containing flex-bases and the corresponding fleximer analogues of 8-aza-7-deaza nucleosides. Subsequent studies found that pyrazole-containing flex-bases are substrates of purine nucleoside phosphorylase (PNP). We have compared the chemical synthesis of fleximers and enzymatic approaches with both isolated enzymes and the use of E. coli cells overproducing PNP. The latter provided stereochemically pure pyrazole-containing β-D-ribo- and β-D-2′-deoxyribo-fleximers and are beneficial in terms of environmental issues, are more economical, and streamline the steps required from a chemical approach. The reaction is carried out in water, avoiding hazardous chemicals, and the products are isolated by ion-exchange chromatography using water/ethanol mixtures for elution. Moreover, the target nucleosides were obtained on a multi-milligram scale with >97–99% purity, and the reactions can be easily scaled up.

Graphical abstract, Anti-HIV-1 drug design has been notably challenging due to the virus’ ability to mutate and develop immunity against commercially available drugs. The aims of this project were to develop a series of fleximer base analogues that not only possess inherent flexibility that can remain active when faced with binding site mutations, but also target a non-canonical, highly conserved target: the nucleocapsid protein of HIV (NC). The compounds were predicted by computational studies not to function via zinc ejection, which would endow them with significant advantages over non-specific and thus toxic zinc-ejectors. The target fleximer bases were synthesized using palladium-catalyzed cross-coupling techniques and subsequently tested against NC and HIV-1. The results of those studies are described herein.

This is the second of two invited articles reviewing the development of nucleoside analogue antiviral drugs, written for a target audience of virologists and other non-chemists, as well as chemists who may not be familiar with the field. As with the first paper, rather than providing a chronological account, we have chosen to examine particular examples of structural modifications made to nucleoside analogues that have proven fruitful as various antiviral, anticancer, and other therapeutics. The first review covered the more common, and in most cases, single modifications to the sugar and base moieties of the nucleoside scaffold. This paper focuses on more recent developments, especially nucleoside analogues that contain more than one modification to the nucleoside scaffold. We hope that these two articles will provide an informative historical perspective of some of the successfully designed analogues, as well as many candidate compounds that encountered obstacles.

Over the past two and a half years the world has seen a desperate scramble to find a treatment for SARS-CoV-2 and COVID. In that regard, nucleosides have long served as the cornerstone to antiviral treatments due to their resemblance to the naturally occurring nucleosides that are involved in numerous biological processes. Unlike other viruses however, it was found early on during the search for drugs to treat SARS-1 and later MERS, that the coronaviruses possess a unique repair enzyme, an exonuclease (ExoN)[3] which rendered nucleoside analogues useless, thus negating their use.[4] During the current outbreak however, as both well-known and new nucleoside analogues were investigated or reinvestigated as a possible cure for SARS-CoV-2, several novel and/or lesser-known mechanisms of action were uncovered. This review briefly describes these mechanisms.

Emerging flaviviruses are causative agents of severe and life-threatening diseases, against which no approved therapies are available. Among the nucleoside analogues, which represent a promising group of potentially therapeutic compounds, fluorine-substituted nucleosides are characterized by unique structural and functional properties. Despite having first been synthesized almost 5 decades ago, they still offer new therapeutic opportunities as inhibitors of essential viral or cellular enzymes active in nucleic acid replication/transcription or nucleoside/nucleotide metabolism. Here, we report evaluation of the antiflaviviral activity of 28 nucleoside analogues, each modified with a fluoro substituent at different positions of the ribose ring and/or heterocyclic nucleobase. Our antiviral screening revealed that 3'deoxy-3'-fluoroadenosine exerted a low-micromolar antiviral effect against tick-borne encephalitis virus (TBEV), Zika virus, and West Nile virus (WNV) (EC50 values from 1.1 +/- 0.1 mu M to 4.7 +/- 1.5 mu M), which was manifested in host cell lines of neural and extraneural origin. The compound did not display any measurable cytotoxicity up to concentrations of 25 mu M but had an observable cytostatic effect, resulting in suppression of cell proliferation at concentrations of > 12.5 mu M. Novel approaches based on quantitative phase imaging using holographic microscopy were developed for advanced characterization of antiviral and cytotoxic profiles of 3'-deoxy-3'-fluoroadenosine in vitro. In addition to its antiviral activity in cell cultures, 3'-deoxy-3'-fluoroadenosine was active in vivo in mouse models of TBEV and WNV infection. Our results demonstrate that fluoro-modified nucleosides represent a group of bioactive molecules with excellent potential to serve as prospective broad-spectrum antivirals in antiviral research and drug development.

The current focus for many researchers has turned to the development of therapeutics that have the potential for serving as broad-spectrum inhibitors that can target numerous viruses, both within a particular family, as well as to span across multiple viral families. This will allow us to build an arsenal of therapeutics that could be used for the next outbreak. In that regard, nucleosides have served as the cornerstone for antiviral therapy for many decades. As detailed herein, many nucleosides have been shown to inhibit multiple viruses due to the conserved nature of many viral enzyme binding sites. Thus, it is somewhat surprising that up until very recently, many researchers focused more on "one bug one drug," rather than trying to target multiple viruses given those similarities. This attitude is now changing due to the realization that we need to be proactive rather than reactive when it comes to combating emerging and reemerging infectious diseases. A brief summary of prominent nucleoside analogues that previously exhibited broad-spectrum activity and are now under renewed interest, as well as new analogues, that are currently under investigation against SARS-CoV-2 and other viruses is discussed herein.

Graphical abstract, Flaviviruses, such as Dengue (DENV) and Zika (ZIKV) viruses, represent a severe health burden. There are currently no FDA-approved treatments, and vaccines against most flaviviruses are still lacking. We have developed several flexible analogues (“fleximers”) of the FDA-approved nucleoside Acyclovir that exhibit activity against various RNA viruses, demonstrating their broad-spectrum potential. The current study reports activity against DENV and YFV, particularly for compound 1. Studies to elucidate the mechanism of action suggest the flex-analogue triphosphates, especially 1-TP, inhibit DENV and ZIKV methyltransferases. The results of these studies are reported herein.

The structurally unique “fleximer” nucleosides were originally designed to investigate how flexibility in a nucleobase could potentially affect receptor–ligand recognition and function. Recently they have been shown to have low‐to‐sub‐micromolar levels of activity against a number of viruses, including coronaviruses, filoviruses, and flaviviruses. However, the synthesis of distal fleximers in particular has thus far been quite tedious and low yielding. As a potential solution to this issue, a series of proximal fleximer bases (flex‐bases) has been successfully coupled to both ribose and 2′‐deoxyribose sugars by using the N‐deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purine nucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on a thieno‐expanded tricyclic heterocyclic base, as well as several distal and proximal flex‐bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion, thus potentially significantly shortening the route to these biologically significant compounds. The results of those studies are reported herein., A thieno‐expanded tricyclic base and several distal and proximal flex‐bases have been converted into their corresponding fleximer nucleosides by enzymatic transglycosylation reactions catalyzed by N‐deoxyribosyltransferase from L. leichmannii (LlNDT) or E. coli purine nucleoside phosphorylase (PNP).

For decades, nucleosides and nucleotides have formed the cornerstone of antiviral, antiparasitic and anticancer therapeutics and have been used as tools in exploring nucleic acid structure and function [...]

This is the first of two invited articles reviewing the development of nucleoside-analogue antiviral drugs, written for a target audience of virologists and other non-chemists, as well as chemists who may not be familiar with the field. Rather than providing a simple chronological account, we have examined and attempted to explain the thought processes, advances in synthetic chemistry and lessons learned from antiviral testing that led to a few molecules being moved forward to eventual approval for human therapies, while others were discarded. The present paper focuses on early, relatively simplistic changes made to the nucleoside scaffold, beginning with modifications of the nucleoside sugars of Ara-C and other arabinose-derived nucleoside analogues in the 1960’s. A future paper will review more recent developments, focusing especially on more complex modifications, particularly those involving multiple changes to the nucleoside scaffold. We hope that these articles will help virologists and others outside the field of medicinal chemistry to understand why certain drugs were successfully developed, while the majority of candidate compounds encountered barriers due to low-yielding synthetic routes, toxicity or other problems that led to their abandonment.

C-nucleosides have intrigued biologists and medicinal chemists since their discovery in 1950's. In that regard, C-nucleosides and their synthetic analogues have resulted in promising leads in drug design. Concurrently, advances in chemical syntheses have contributed to structural diversity and drug discovery efforts. Convergent and modular approaches to synthesis have garnered much attention in this regard. Among them nucleophilic substitution at C1' has seen wide applications providing flexibility in synthesis, good yields, the ability to maneuver stereochemistry as well as to incorporate structural modifications. In this review, we describe recent reports on the modular synthesis of C-nucleosides with a focus on D-ribonolactone and sugar modifications that have resulted in potent lead molecules.

Graphical abstract, 1-[tert-Butyl(diphenyl)silyl]pyrrol-3-ylboronic acid was obtained from pyrrole in three steps. Its Suzuki–Miyaura cross-coupling with functionalized pyridinyl and pyrimidinyl bromides afforded new promising 3-hetaryl-1H-pyrroles.

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as &ldquo, fleximers&rdquo, have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds for the development of antiproliferative agents. Much of the focus has been on modifications to the pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast, this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or RNA alkylators. In addition, N5 substitution reduced the EC50 against CCRF-CEM leukemia cells by up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.

A series of 1,6-bis[(benzyloxy)methyl]uracil derivatives combining structural features of both diphenyl ether and pyridone types of NNRTIs were synthesized. Target compounds were found to inhibit HIV-1 reverse transcriptase at micro- and submicromolar levels of concentrations and exhibited anti-HIV-1 activity in MT-4 cell culture, demonstrating resistance profile similar to first generation NNRTIs. The synthesized compounds also showed profound activity against influenza virus (H1N1) in MDCK cell culture without detectable cytotoxicity. The lead compound of this assay appeared to exceed rimantadine, amantadine, ribavirin and oseltamivir carboxylate in activity. The mechanism of action of 1,6-bis[(benzyloxy)methyl]uracils against influenza virus is currently under investigation.

As far back as Melville Wolfrom's acyclic sugar synthesis in the 1960's, synthesis of flexible nucleoside analogues have been an area of interest. This concept, however, went against years of enzyme-substrate binding theory. Hence, acyclic methodology in antiviral drug design did not take off until the discovery and subsequent FDA approval of such analogues as Acyclovir and Tenofovir. More recently, the observation that flexible nucleosides could overcome drug resistance spawned a renewed interest in the field of nucleoside drug design. The next generation of flexible nucleosides shifted the focus from the sugar moiety to the nucleobase. With analogues such as Seley-Radtkequot;fleximersquot;, and Herdewijn's C5 substituted 2'-deoxyuridines, the area of base flexibility has seen great expansion. More recently, the marriage of these methodologies with acyclic sugars has resulted in a series of acyclic flex-base nucleosides with a wide range of antiviral properties, including some of the first to exhibit anti-coronavirus activity. Various flexible nucleosides and their corresponding nucleobases will be compared in this review.

Graphical abstract, The need for novel therapeutic options to fight herpesvirus infections still persists. Herein we report the design, synthesis and antiviral evaluation of a new family of non-nucleoside antivirals, derived from 1-[ω-(4-bromophenoxy)alkyl]uracil derivatives – previously reported inhibitors of human cytomegalovirus (HCMV). Introduction of the N-(4-phenoxyphenyl)acetamide side chain at N3 increased their potency and widened activity spectrum. The most active compounds in the series exhibit submicromolar activity against different viral strains of HCMV and varicella zoster virus (VZV) replication in HEL cell cultures. Inactivity against other DNA and RNA viruses, including herpes simplex virus 1/2, points to a novel mechanism of antiviral action.

Fleximers, a novel type of flexible nucleoside that have garnered attention due to their unprecedented activity against human coronaviruses, have now exhibited highly promising levels of activity against filoviruses. The Flex-nucleoside was the most potent inhibitor against recombinant Ebola virus in Huh7 cells with an EC50 = 2 μM, while the McGuigan prodrug was most active against Sudan virus-infected HeLa cells with an EC50 of 7 μM.

Graphical abstract, The title compounds were prepared from 1-[5-(4-bromophenoxy) pentyl]uracil by the introduction of N-arylacetamide moiety at the 3-position, the better approach involving the use of N-aryl-2-chloroacetamides as the reactants. Antiviral activity of the obtained compounds was estimated.

A series of novel 5&prime, norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 &mu, g/mL (mc2155) and a MIC99 of 6.7&ndash, 67 &mu, g/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28&ndash, 61 &mu, g/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50&ndash, 60 &mu, g/mL), as well as two virulent strains of M. tuberculosis, a laboratory strain H37Rv (MIC99 20&ndash, 50 &mu, g/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20&ndash, g/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.

Nucleoside analogs represent the largest class of small molecule-based antivirals, which currently form the backbone of chemotherapy of chronic infections caused by HIV, hepatitis B or C viruses, and herpes viruses. High antiviral potency and favorable pharmacokinetics parameters make some nucleoside analogs suitable also for the treatment of acute infections caused by other medically important RNA and DNA viruses. This review summarizes available information on antiviral research of nucleoside analogs against arthropod-borne members of the genus Flavivirus within the family Flaviviridae, being primarily focused on description of nucleoside inhibitors of flaviviral RNA-dependent RNA polymerase, methyltransferase, and helicase/NTPase. Inhibitors of intracellular nucleoside synthesis and newly discovered nucleoside derivatives with high antiflavivirus potency, whose modes of action are currently not completely understood, have drawn attention. Moreover, this review highlights important challenges and complications in nucleoside analog development and suggests possible strategies to overcome these limitations. ispartof: Antiviral Chemistry & Chemotherapy vol:26 pages:1-28 ispartof: location:England status: published

A series of doubly flexible nucleoside analogues were designed based on the acyclic sugar scaffold of acyclovir and the flex-base moiety found in the fleximers. The target compounds were evaluated for their antiviral potential and found to inhibit several coronaviruses. Significantly, compound 2 displayed selective antiviral activity (CC50 >3× EC50) towards human coronavirus (HCoV)-NL63 and Middle East respiratory syndrome-coronavirus, but not severe acute respiratory syndrome-coronavirus. In the case of HCoV-NL63 the activity was highly promising with an EC50 100μM. As such, these doubly flexible nucleoside analogues are viewed as a novel new class of drug candidates with potential for potent inhibition of coronaviruses. publisher: Elsevier articletitle: Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity journaltitle: Bioorganic & Medicinal Chemistry Letters articlelink: http://dx.doi.org/10.1016/j.bmcl.2015.05.039 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved. ispartof: Bioorganic & Medicinal Chemistry Letters vol:25 issue:15 pages:2923-2926 ispartof: location:England status: published

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors.

Given the impressive success of Gilead's Sofosbuvir, many laboratories, including ours, have explored the unique 2'-sugar modification (2'-Me, 2'-F) of nucleoside analogues in the hopes of exploiting the biological activity that this unique modification has imparted to the nucleoside scaffold. In that regard, we have combined our tricyclic "expanded" purine base motif with the 2'-Me, 2'-F sugar modification. Although the synthesis of these complex molecules proved to be nontrivial, with the best results coming from a linear approach, the overall strategy resulted in highly promising biological results for several of the target compounds, including their corresponding McGuigan ProTides. Modest activity against HCV was observed with inhibitory concentrations of as low as 20 μM.

In order to identify new antiretroviral agents, a series of novel uracil derivatives have been synthesized. Optimized conditions for coupling of Weinreb amides with aromatic Grignard reagents allow the convenient preparation of key benzophenone intermediates in high yields and purities. The use of a modified silyl Hilbert–Johnson reaction affords the target compounds under mild conditions.

In order to identify novel nonnucleoside inhibitors of HIV-1 reverse transcriptase two series of amide-containing uracil derivatives were designed as hybrids of two scaffolds of previously reported inhibitors. Subsequent biological evaluation confirmed acetamide uracil derivatives 15a–k as selective micromolar NNRTIs with a first generation-like resistance profile. Molecular modeling of the most active compounds 15c and 15i was employed to provide insight on their inhibitory properties and direct future design efforts.

Halogenated pyrrolo[3,2-d]pyrimidine analogues have shown antiproliferative activity in recent studies, with cell accumulation occurring in the G2/M stage without apoptosis. However, the mechanism of action and pharmacokinetic (PK) profile of these compounds has yet to be determined. In order to investigate the PK profile of these compounds, a series of halogenated pyrrolo[3,2-d]pyrimidine compounds was synthesized and first tested for activity in various cancer cell lines followed by a mouse model. EC50 values ranged from 0.014 – 14.5 μM, and maximum tolerated doses (MTD) in mice were between 5–10 mg/kg. This indicates a wide variance in activity and toxicity that necessitates further study. To decrease toxicity, a second series of compounds was synthesized with N5 alkyl substitutions in an effort to slow the rate of metabolism, which was thought to be leading to the toxicity. The N-substituted compounds demonstrated comparable cell line activity (EC50 values between 0.83 – 7.3 μM) with significantly decreased toxicity (MTD = 40 mg/kg). Finally, the PK profile of the active N5-substituted compound shows a plasma half-life of 32.7 minutes, and rapid conversion into the parent unsubstituted analogue. Together, these data indicate that halogenated pyrrolo[3,2-d]pyrimidines present a promising lead into potent antiproliferative agents with tunable activity and toxicity, and rapid metabolism.

Carbocyclic nucleoside analogues have a distinguished history as anti-infectious agents, including key antiviral agents. Toxicity was initially a concern but this was reduced by the introduction of 5′-nor variants. Here, we report the result of our preliminary screening of a series of 5′-norcarbocyclic uridine analogues against protozoan parasites, specifically the major pathogens Leishmania mexicana and Trypanosoma brucei. The series displayed antiparasite activity in the low to mid-micromolar range and establishes a preliminary structure-activity relationship, with the 4′,N3-di-(3,5-dimethylbenzoyl)-substituted analogues showing the most prominent activity. Utilizing an array of specially adapted cell lines, it was established that this series of analogues likely act through a common target. Moreover, the strong correlation between the trypanocidal and anti-leishmanial activities indicates that this mechanism is likely shared between the two species. EC50 values were unaffected by the disabling of pyrimidine biosynthesis in T. brucei, showing that these uridine analogues do not act directly on the enzymes of pyrimidine nucleotide metabolism. The lack of cross-resistance with 5-fluorouracil, also establishes that the carbocyclic analogues are not imported through the known uracil transporters, thus offering forth new insights for this class of nucleosides. The lack of cross-resistance with current trypanocides makes this compound class interesting for further exploration.

Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.

Graphical abstract, HCMV infection represents a life-threatening condition for immunocompromised patients and newborn infants and novel anti-HCMV agents are clearly needed. In this regard, a series of 1-[ω-(phenoxy)alkyl]uracil derivatives were synthesized and examined for antiviral properties. Compounds 17, 20, 24 and 28 were found to exhibit highly specific and promising inhibitory activity against HCMV replication in HEL cell cultures with EC50 values within 5.5–12 μM range. Further studies should be undertaken to elucidate the mechanism of action of these compounds and the structure–activity relationship for the linker region.

Graphical abstract, A series of phenyloxyethyl and cinnamyl derivatives of substituted uracils were synthesized and found to exhibit potent activity against HIV-RT and HIV replication in cell culture. In general, the cinnamyl derivatives proved superior to the phenyloxyethyl derivatives, however 1-[2-(4-methylphenoxy)ethyl]-3-(3,5-dimethylbenzyl)uracil (19) exhibited the highest activity (EC50 = 0.27 μM) thus confirming that the 3-benzyluracil fragment in the NNRTI structure can be regarded as a functional analogue of the benzophenone pharmacophore typically found in NNRTIs.

Graphical abstract, Non-nucleoside reverse transcriptase inhibitors (NNRTI) are key components in highly active antiretroviral therapy for treating HIV-1. Herein we present the synthesis for a series of N1-alkylated uracil derivatives bearing ω-(2-benzyl- and 2-benzoylphenoxy)alkyl substituents as novel NNRTIs. These compounds displayed anti-HIV activity similar to that of nevirapine and several of them exhibited activity against the K103N/Y181C RT mutant HIV-1 strain. Further evaluation revealed that the inhibitors were active against most nevirapine-resistant mono- and di-substituted RTs with the exception of the V106A RT. Thus, the candidate compounds can be regarded as potential lead compounds against the wild-type virus and drug-resistant forms.

Pyrimidine analogs have long found use over a broad chemotherapeutic spectrum. In an effort to further explore the antiviral potential of several uracil derivatives previously synthesized in our laboratories, a series of benzylated pyrimidines were designed and synthesized. Introduction of the benzyl residue onto the 5-phenylaminouracil scaffold was carried out using 2,4-bis(trimethylsilyloxy)pyrimidine with the corresponding benzyl bromides. Similarly, 1-benzyl-5-(benzylamino)- and 1-benzyl-5-(phenethylamino)uracils were obtained via amination of 1-benzyl-5-bromouracils with benzylamine or phenylethylamine. The results of the broad screen antiviral studies revealed that compounds 5 and 11 exhibit promising inhibitory activity against HIV-1 in CEM-SS culture. A 50% protective effect was observed at concentrations of 11.9 and 9.5 μМ, respectively. Moreover, compounds 8 and 3 exhibited good inhibitory effects against EBV in АKАТА cell culture with EC50 values of 2.3 and 12 μM, respectively. The synthesis and biological studies are detailed herein.

New N1-benzyl esters of N1-oxide analogues of 5′-noraristeromycin were synthesized and tested as potential inhibitors of S-adenosyl-L-homocysteine hydrolase in Vaccinia virus-infected cell systems.

Over the past few years, nucleosides have maintained a prominent role as one of the cornerstones of antiviral and anticancer therapeutics, and many approaches to nucleoside drug design have been pursued. One such approach involves flexibility in the sugar moiety of nucleosides, for example, in the highly successful anti-HIV and HBV drug tenofovir. In contrast, introduction of flexibility to the nucleobase scaffold has only more recently gained significance with the invention of our fleximers. The history, development, and some biological relevance for this innovative class of nucleosides are detailed herein.

The discovery of azidothymidine’s (AZT) activity against human immunodeficiency virus (HIV) provided strong rationale for the design of additional thymidine analogues. One drawback of many nucleoside analogues is the toxicity that often arises due to phosphorylation by cellular kinases. In order to overcome this problem, a number of truncated nucleosides lacking the 4 ′-hydroxymethyl group have been synthesized. In that regard, the synthesis and preliminary biological results for two truncated carbocyclic thymidine analogues are presented herein.

The chemotherapeutic drug Gemcitabine, 2′,2′-difluoro-2′-deoxycytidine, has long been the standard of care for a number of cancers. Gemcitabine’s chemotherapeutic properties stem from its 2′,2′-difluoro-2′-deoxyribose sugar, which mimics the natural nucleoside, but also disrupts nucleic acid synthesis, leading to cell death. As a result, numerous analogues have been prepared to further explore the biological implications for this structural modification. In that regard, a thieno-expanded guanosine analogue was of interest due to biological activity previously observed for the tricyclic heterobase scaffold. Several analogues were prepared, including the McGuigan ProTide, however the parent nucleoside exhibited the best chemotherapeutic activity, specifically against breast cancer cell lines (89.53% growth inhibition).

In vitro evaluation of the halogenated pyrrolo[3,2-d]pyrimidines identified antiproliferative activities in compounds 1 and 2 against four different cancer cell lines. Upon screening of a series of pyrrolo[3,2-d]pyrimidines, the 2,4-Cl compound 1 was found to exhibit antiproliferative activity at low micromolar concentrations. Introduction of iodine at C7 resulted in significant enhancement of potency by reducing the IC50 into sub-micromolar levels, thereby suggesting the importance of a halogen at C7. This finding was further supported by an increased antiproliferative effect for 4 as compared to 3. Cell-cycle and apoptosis studies conducted on the two potent compounds 1 and 2 showed differences in their cytotoxic mechanisms in triple negative breast cancer MDA-MB-231 cells, wherein compound 1 induced cells to accumulate at the G2/M stage with little evidence of apoptotic death. In contrast, compound 2 robustly induced apoptosis with concomitant G2/M cell cycle arrest in this cell model. ispartof: Bioorganic & Medicinal Chemistry vol:23 issue:15 pages:4354-4363 ispartof: location:England status: published

Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-5-(phenylamino)uracils, and 1,3-di-(4′-hydroxy-2′-cyclopenten-1′-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4′-hydroxy-2′-cyclopenten-1′-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

Flexible nucleosides such as Holý’s sugar-modified acyclics have seen great utility in antiviral therapeutics. Another area of modification to the nucleoside scaffold that has only recently garnered attention involves imparting flexibility to the nucleobase instead of the sugar moiety. This flexibility can be achieved by splitting the purine base into its respective imidazole and pyrimidine components or by increasing the number of aromatic rings. These modifications also allow installation of additional substituents to the nucleobase scaffold that can potentially enhance biological activity and enzyme recognition. Nucleobase flexibility can also provide other advantages such as enhancing fluorescence properties that can be utilized for bioprobes as well as to assist in exploring the effects of the nucleobase on RNA and DNA helix stability.

In an effort to study the effects of flexibility on enzyme recognition and activity, we have developed several different series of flexible nucleoside analogues in which the purine base is split into its respective imidazole and pyrimidine components. The focus of this particular study was to synthesize the truncated neplanocin A fleximers to investigate their potential anti-protozoan activities by inhibition of S-adenosylhomocysteine hydrolase (SAHase). The three fleximers tested displayed poor anti-trypanocidal activities, with EC50 values around 200 μM. Further studies of the corresponding ribose fleximers, most closely related to the natural nucleoside substrates, revealed low affinity for the known T. brucei nucleoside transporters P1 and P2, which may be the reason for the lack of trypanocidal activity observed.