Acyclic Nucleoside Research Articles

I CHEMIOTERAPICI ANTIVIRALI<sup>1</sup>

The concept of drug development with specific antiviral action is quite recent. The discovery, in the late ‘70s, that acyclic nucleoside analogs could inhibit the replication of the Herpes Simplex virus (HSV) DNA, was the fundamental step that opened the way to the research on drugs able to deal with diseases caused by viruses. In the ‘80s, the need to fight another disease related to a virus, AIDS caused by HIV, gave a further boost to the research in the field of viral chemotherapy. Currently, several classes of drugs are available for the treatment of viruses such as influenza virus, respiratory syncytial virus, herpes simplex virus, varicella-zoster virus, HBV and hepatitis viruses, cytomegalovirus and HIV. Nevertheless, we however need a continuous and progressive development of antiviral chemotherapy and of the research related to it that can cope with the problem of the effectiveness of antiviral vaccines, the emergence of new diseases related to immunosuppression. In this context, the research of professor Paolo Mantegazza has made a valuable contribution to the understanding of the transforming role of some viruses such as polyomaviruses, including the SV40 monkey vacuolar virus. Professor Mantegazza also made a significant contribution to the synthesis of compounds for transmissible spongiform encephalopathies caused by prions.
 1Trascrizione non corretta dall’Autore.

Intramolecular π-stacks in mixed-ligand copper(II) complexes formed by heteroaromatic amines and antivirally active acyclic nucleotide analogs carrying a hydroxy-2-(phosphonomethoxy)propyl residue‡

Acyclic nucleoside phosphonates (ANPs) are of medical relevance and deserve detailed chemical characterization. We focus here on 1-[2-(phosphonomethoxy)ethyl]cytosine (PMEC), (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC), 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison the nucleobase-free (phosphonomethoxy)ethane (PME) and (R)-hydroxy-2-(phosphonomethoxy)propane (HPMP). The acidity constants of H3(ANP)+ and H2(NP) (NP2– = nucleoside phosph(on)ate derivative) are needed to understand the properties of the ternary neutral Cu(Arm)(ANP/NP) [Arm = 2,2′-bipyridine (Bpy) or 1,10-phenanthroline (Phen)] and the monoprotonated Cu(Arm)(H;ANP)+ complexes. The Cu(Arm)(ANP) species are considerably more stable than the corresponding Cu(Arm)(R-PO3), where R- represents a phosph(on)ate ligand with a non-coordinating group R. The observed stability enhancements are due to intramolecular stack formation (st) between the aromatic rings of Arm and the nucleobase residues and also to the formation of five-membered chelates involving the ether oxygen of the –CH(R)–O–CH2– residue (cl/O) (R = H or CH2–OH). In intramolecular equilibria, three structurally different Cu(Arm)(ANP) isomers occur; for example, of Cu(Phen)(HPMPA) about 5% exist as an open (op) Cu(Phen)(HPMPA)op isomer, 17% as Cu(Phen)(HPMPA)cl/O, and 78% as Cu(Phen)(HPMPA)st. In Cu(Arm)(ANP) the stacking tendency decreases in the order PMEA2– > HPMPA2– > PMEC2– > HPMPC2–. In monoprotonated Cu(Arm)(H;ANP)+ both H+ and Cu(Arm)2+ are at undergoing similar intramolecular equilibria as indicated above.

Synthesis of Cyclic and Acyclic Pyrimidine Nucleosides Analogues with Anticipated Antiviral Activity.

A convenient method for preparation of cyclic and acyclic nucleosides was achieved by alkylation of 6-(2,4-dichlorophenoxymethyl) pyrimidine-2,4-dione (1) with a variety of acyclic and cyclic activated sugar analogues, namely (2-acetoxyethoxy)methyl acetate (3), 2-(acetoxymethoxy)propane-1,3-diyl dibenzoate (4), benzyloxymethyl acetate (5), 2-acetoxy-5-(benzoyloxymethyl)tetrahydrofuran-3,4-diyl dibenzoate (12), 5-chloro-2-((4-chlorobenzoyloxy)methyl) tetrahydrofuran-3-yl 4-chlorobenzoate (13) and 2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (14), respectively. Deprotection of the synthesized nucleosides was achieved by using methanolic ammonia. The structures of the newly synthesized nucleoside analogues were fully characterized by analytical methods (mass spectrometry, 1H NMR, 13C NMR, and elemental analysis).

Expedient synthesis and biological evaluation of alkenyl acyclic nucleoside phosphonate prodrugs

The importance of phosphonoamidate prodrugs (ProTides) of acyclic nucleoside phosphonate (ANPs) is highlighted by the approval of Tenofovir Alafenamide Fumarate for the treatment of HIV and HBV infections. In the present paper we are reporting an expedient, one-pot, two-steps synthesis of allyl phosphonoamidates and diamidates that offers a time saving strategy when compared to literature methods. The use of these substrates in the cross metathesis reactions with alkenyl functionalised thymine and uracil nucleobases is reported. ANPs prodrugs synthesized via this methodology were evaluated for their antiviral activities against DNA and RNA viruses. It is anticipated that the use of 5,6,7,8-tetrahydro-1-napthyl as aryloxy moiety is capable to confer antiviral activity among a series of otherwise inactive uracil ProTides.

Microwave in glycosylation reaction: design, and synthesis of highly substituted nicotinonitrile nucleosides

An efficient and rapid regiospecific approach for the synthesis of cyclic and acyclic nucleosides of 2-oxonicotinonitriles was performed. Whereas, glycosylation of 2-oxonicotinonitriles 1a, b with peracetylated sugars (namely, peracetylated glucose, galactose and ribose) under MWI tolerated exclusively the desired N-nucleosides 2a, b, 4a, b and 6a, b in significant yields (75–86%) and in short reaction time (5–7 min.). The same products were obtained under the conventional conditions, using halo-sugar with low yields in hard conditions. Similarly, the acyclic nucleosides 8a, b and 9a, b were obtained under MWI and conventional conditions via reaction of 1a, b with 4-bromo butyl acetate and 2-acetoxyethoxymethyl bromide. Finally, deprotection of the latter blocked N-nucleosides was performed via treatment with aqueous methanolic solution containing a catalytic amount of triethyl amine to give the desired free nucleosides 3a, b, 5a, b, 7a, b, 10a, b and 11a, b, respectively. The free nucleosides (3a, b, 5a, b, 7a, b and 11a, b) were evaluated against Gram (+ ve) bacteria, Gram (–ve) bacteria and one pathogenic Fungi namely, Aspergillus flavus. Good results were obtained for compounds 7a, b and 11a, b compared with the used standard drugs (Cefotaxime and Dermatin).

Insight into the recognition mechanism of DNA cytosine-5 methyltransferases (DNMTs) by incorporation of acyclic 5-fluorocytosine (FC) nucleosides into DNA

DNA cytosine-5 methyltransferase (DNMT) catalyzes methylation at the C5 position of cytosine in the CpG sequence in double stranded DNA to give 5-methylCpG (mCpG) in the epigenetic regulation step in human cells. The entire reaction mechanism of DNMT is divided into six steps, which are scanning, recognition, flipping, loop locking, methylation, and releasing. The methylation and releasing mechanism are well-investigated; however, few reports are known about other reaction steps. To obtain insight into the reaction mechanism, we planned the incorporation of acyclic nucleosides, which make it easy to flip out the target nucleobase, into oligodeoxynucleotides (ODNs) and investigated the interaction between the ODN and DNMT. Here, we describe the design and synthesis of ODNs containing new acyclic 5-fluorocytosine nucleosides and their physiological and biological properties, including their interactions with DNMT. We found that the ODNs containing the acyclic 5-fluorocytosine nucleoside showed higher flexibility than those that contain 5-fluoro-2′-deoxycytidine. The observed flexibility of ODNs is expected to influence the scanning and recognition steps due to the decrease in helicity of the B-form.

Molecular Mechanisms for Species Differences in Organic Anion Transporter 1, OAT1: Implications for Renal Drug Toxicity.

Species differences in renal drug transporters continue to plague drug development with animal models failing to adequately predict renal drug toxicity. For example, adefovir, a renally excreted antiviral drug, failed clinical studies for human immunodeficiency virus due to pronounced nephrotoxicity in humans. In this study, we demonstrated that there are large species differences in the kinetics of interactions of a key class of antiviral drugs, acyclic nucleoside phosphonates (ANPs), with organic anion transporter 1 [(OAT1) SLC22A6] and identified a key amino acid residue responsible for these differences. In OAT1 stably transfected human embryonic kidney 293 cells, the Km value of tenofovir for human OAT1 (hOAT1) was significantly lower than for OAT1 orthologs from common preclinical animals, including cynomolgus monkey, mouse, rat, and dog. Chimeric and site-directed mutagenesis studies along with comparative structure modeling identified serine at position 203 (S203) in hOAT1 as a determinant of its lower Km value. Furthermore, S203 is conserved in apes, and in contrast alanine at the equivalent position is conserved in preclinical animals and Old World monkeys, the most related primates to apes. Intriguingly, transport efficiencies are significantly higher for OAT1 orthologs from apes with high serum uric acid (SUA) levels than for the orthologs from species with low serum uric acid levels. In conclusion, our data provide a molecular mechanism underlying species differences in renal accumulation of nephrotoxic ANPs and a novel insight into OAT1 transport function in primate evolution.

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

Synthesis of Chiral Acyclic Pyrimidine Nucleosides

Key words pyrimidine nucleosides - quinine-derived thiourea catalyst - conjugate addition - protonation

Asymmetric Synthesis of Chiral Acyclic Purine Nucleosides Containing a Hemiaminal Ester Moiety via Three-Component Dynamic Kinetic Resolution.

An efficient route to construct chiral acyclic purine nucleosides containing a hemiaminal ester moiety is reported via three-component dynamic kinetic resolution of purines, aldehydes, and acid anhydrides. The procedure provides diverse chiral acyclic purine nucleoside analogues in a regioselective manner with good yields (up to 93% yield) and excellent enantioselectivities (up to 95% ee). Furthermore, the chiral (acyloxyalkyl)-5-fluorouracil could also be generated as a potential prodrug of 5-fluorouracil.

Synthesis of α-Branched Acyclic Nucleoside Phosphonates as Potential Inhibitors of Bacterial Adenylate Cyclases.

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC50 >10 μm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC50 values ranging from 55 to 362 nm.

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

ABSTRACTWe describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.

Organocatalytic enantioselective synthesis of acyclic pyrimidine nucleosides by aza-Michael reaction.

An efficient diarylprolinol triphenylsilyl ether-catalyzed enantioselective aza-Michael reaction of pyrimidines as N-centered nucleophiles to α,β-unsaturated aldehydes, followed by reduction, provided chiral acyclic pyrimidine nucleosides in good yields (51-78% yields for two steps) and excellent enantioselectivities (91-98% ee). In addition, the chiral acyclic pyrimidine nucleoside having the tert-butyldiphenylsilyl-protected hydroxyl substituent was successfully applied to the synthesis of the corresponding chiral cyclic pyrimidine nucleoside analogue bearing the tetrahydrofuranyl ring.

Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus.

While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC50 = 2.6 µM, TK+ Oka strain) and HCMV (EC50 = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC50 values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK– VZV 07–1 strain with EC50 = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.

Enantioselective and regiodivergent allylation of pyrimidines with terminal allenes: an approach to pyrimidine acyclic nucleosides

An atom-economic addition of pyrimidines to allenes has been developed for the diverse synthesis of branched or linearN-allylpyrimidine analogues.

Evaluation of ODE-Bn-PMEG, an acyclic nucleoside phosphonate prodrug, as an antiviral against productive HPV infection in 3D organotypic epithelial cultures

Evaluation of ODE-Bn-PMEG, an acyclic nucleoside phosphonate prodrug, as an antiviral against productive HPV infection in 3D organotypic epithelial cultures

The Anti-Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase.

Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy.

Synthesis of Chiral Acyclic Pyrimidine Nucleosides with a Sulfur‐Containing Side Chain via Enantioselective Tandem Conjugate Addition/Protonation

AbstractA direct route to chiral pyrimidine acyclic nucleoside analogues with a sulfur‐containing side chain is reported via enantioselective tandem conjugated addition‐protonation reactions of thioacetic acid to α‐pyrimidine substituted acrylates. In the presence of a quinine‐derived thiourea catalyst, diverse chiral sulfur‐containing pyrimidine acyclic nucleoside analogues could be obtained in excellent yields (95–99 %) and with moderate to excellent enantioselectivity values (up to 99 % ee).

The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin.

BackgroundFibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis.MethodsThioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5–10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5–10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA.ResultsTreatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1.ConclusionsThese studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.

Preparation of New Oxadiazole Acyclic Nucleoside and Thioglycoside Analogs Containing Chromene Moiety with Antimicrobial Evaluation

New sugar hydrazones linked to chroman ring system and their derived oxadiazole acyclic nucleoside analogs were synthesized from the substituted ethyl ester oxime derivative. The 2-sustituted 1,3,4-oxadiazole-5-thione prepared from acid hydrazide was glycosylated to afford the corresponding thioglycosides, not the N-linked glycosides. The novel compounds were evaluated for their antimicrobial activity and showed different degrees of activities.