Antiviral Acyclic Nucleoside Phosphonates Research Articles

Lyotropic liquid crystal emulsions of LAVR-289: Influence of internal mesophase structure on cytotoxicity and in-vitro antiviral activity

Emerging and reemerging viruses pose significant public health threats, underscoring the urgent need for new antiviral drugs. Recently, a novel family of antiviral acyclic nucleoside phosphonates (ANP) composed of a 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl phosphonic acid skeleton (O-DAPy nucleobase) has shown promise. Among these, LAVR-289 stands out for its potent inhibitory effects against various DNA viruses. Despite its efficacy, LAVR-289s poor water solubility hampers effective drug delivery. To address this, innovative delivery systems utilizing lipidic derivatives have been explored for various administration routes. Submicron lyotropic liquid crystals (LLCs) are particularly promising drug carriers for the encapsulation, protection, and delivery of lipophilic drugs like LAVR-289.This study focuses on developing submicron-sized lipid mesophase dispersions, including emulsified L2 phase, cubosomes, and hexosomes, by adjusting lipidic compounds such as Dimodan® U/J, Lecithins E80, and Miglyol® 812 N. These formulations aim to enhance the solubility and bioavailability of LAVR-289. In vitro evaluations demonstrated that LAVR-289-loaded LLCs at a concentration of 1 µM efficiently inhibited vaccinia virus in infected human cells, with no observed cytotoxicity. Notably, hexosomes exhibited the most favorable antiviral outcomes, suggesting that the internal mesophase structure plays a critical role in optimizing the therapeutic efficacy of this drug class.

1,2,4-Thiadiazole acyclic nucleoside phosphonates as inhibitors of cysteine dependent enzymes cathepsin K and GSK-3β

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N5-protected phosphonic acids. The subsequent attempts to remove the protecting group from N5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes – human cathepsin K (CatK) and glycogen synthase kinase 3β (GSK-3β). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3β was similar to that of the thiadiazole GSK-3β inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity.

Enhanced Topical and Transdermal Delivery of Antineoplastic and Antiviral Acyclic Nucleoside Phosphonate cPr-PMEDAP

Acyclic nucleoside phosphonates possess unique antiviral and antineoplastic activities; however, their polar phosphonate moiety is associated with low ability to cross biological membranes. We explored the potential of transdermal and topical delivery of 2,6-diaminopurine derivative cPr-PMEDAP. In vitro diffusion of cPr-PMEDAP was investigated using formulations at different pH and concentration and with permeation enhancer through porcine and human skin. Ability of 0.1-5% cPr-PMEDAP to cross human skin barrier was very low with flux values ~40 ng/cm(2)/h, the majority of compound found in the stratum corneum. The highest permeation rates were found at pH 6; increased donor concentration had no influence. The permeation enhancer dodecyl 6-dimethylaminohexanoate (DDAK, 1%) increased flux of cPr-PMEDAP (up to 61 times) and its concentration in nucleated epidermis (up to ~0.5 mg of cPr-PMEDAP/g of the tissue). No deamination of cPr-PMEDAP into PMEG occurred during permeation studies, but N-dealkylation into PMEDAP mediated by skin microflora was observed. Transdermal or topical application of cPr-PMEDAP enabled by the permeation enhancer DDAK may provide an attractive alternative route of administration of this potent antitumor and antiviral compound.

Synthesis of Peptidomimetic Conjugates of Cyclic Nucleoside Phosphonates

Cyclic nucleoside phosphonates connected through a P-O-C linkage to a promoiety represent a class of prodrugs designed to overcome the low oral bioavailability of parent antiviral acyclic nucleoside phosphonates. In our prodrug approach, a nontoxic promoiety, such as an amino acid or dipeptide, is conjugated to the cyclic form of the parent drug by esterification of the phosphonic acid moiety with an alcoholic amino acid side chain (Ser, Tyr, and Thr) or a glycol linker. For the biological evaluation and investigation of the pharmacokinetic profiles of these modified nucleoside phosphonates, a reliable synthetic procedure that allows preparation of sufficient amount of potential prodrugs is needed. This unit provides a procedure for synthesizing peptidomimetic conjugates of two broad-spectrum antiviral acyclic nucleoside phosphonates: (S)-HPMPC and (S)-HPMPA. Two alternate strategies allowing synthesizing selected amino acid, dipeptide, or ethylene glycol-linked amino acid prodrugs of (S)-HPMPC and (S)-HPMPA in solution and using a solid-phase approach are presented.

Chiral analysis of anti‐acquired immunodeficiency syndrome drug, 9‐(R)‐[2‐(phosphonomethoxy)propyl]adenine (tenofovir), and related antiviral acyclic nucleoside phosphonates by CE using β‐CD as chiral selector

A new CZE method has been developed for chiral analysis of an important anti-acquired immunodeficiency syndrome drug, 9-(R)-[2-(phosphonomethoxy)propyl]adenine ((R)-PMPA, tenofovir), and six related antiviral acyclic nucleoside phosphonates using beta-CD as a chiral selector. The influence of the composition, concentration and pH of the BGE and the type and concentration of chiral selector on enantiomer resolution was investigated. Complete separations of (R,S)-enantiomers of PMPA with very good resolution (R(s)=1.50-3.64) were achieved within a short time (4-15 min) in 20-50 mM sodium borate or sodium tetraborate BGEs, pH 10.0, at 20 mg/mL concentration of beta-CD. (R,S)-enantiomers of five similar PMPA analogs containing purine bases (adenine, diaminopurine or guanine) and hydroxyl or fluor substituents at C3 carbon atom of propyl chain were baseline separated within 10-17 min in 35 mM sodium tetraborate BGE, pH 10.0, at 20 mg/mL beta-CD concentration. Another important antiviral used by acquired immunodeficiency syndrome patients, derived from pyrimidine base cytosine, 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (cidofovir), and the (R)-enantiomer of this drug were successfully separated in 50 mM sodium tetraborate BGE, pH 10.5, at 20 mg/mL beta-CD concentration within 45 min. Using the UV-absorption detection at 206 nm, the concentration detection limits of the analyzed acyclic nucleoside phosphonates were determined in the submicromolar to micromolar range (0.15-2.51 microg/mL level).

Synthesis of the 5-phosphono-pent-2-en-1-yl nucleosides: A new class of antiviral acyclic nucleoside phosphonates

A new class of acyclic nucleoside phosphonates, the 5-phosphono-pent-2-en-1-yl nucleosides and their hexadecyloxypropyl esters, were synthesized from butyn-1-ol. Only the hexadecyloxypropyl esters showed antiviral activity against herpes simplex virus type 1, in vitro. Hexadecyloxypropyl 1-(5-phosphono-pent-2-en-1-yl)-thymine was the most active and selective compound among the synthesized nucleotides with an EC 50 value of 0.90 μM.

Antiviral acyclic nucleoside phosphonates structure activity studies

This review concerns acyclic nucleoside phosphonates (ANP) and describes the concept of the design of isopolar and isosteric nucleotide analogues resistant towards degradation by enzymes in vivo. It describes the development of research which led to the discovery of several structurally related potent antivirals and ultimately resulted in the development of drugs directed against HIV, HBV and DNA-virus infections in general, namely adefovir, cidofovir and tenofovir. In addition to these “classical compounds” the review describes the present development in the field of ANP, the “open-ring ANP” and discusses the present achievements, concept of prodrug design and application.

In vivo effects of antiviral acyclic nucleoside phosphonate 9-[2-(phosphonomethoxy)ethyl]adenine (adefovir) on cytochrome P450 system of rat liver microsomes

Interference of antiviral agent adefovir, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) with microsomal drug metabolizing system was investigated in rats. The content of total liver cytochrome P450 (CYP) was lowered while that of its denaturated form, P420, was elevated in animals intraperitoneally treated with PMEA (25 mg/kg). Similar effect was observed after treatment with a prodrug of adevofir, adefovir dipivoxil (bisPOM-PMEA). The CYP2E1-dependent formation of 4-nitrocatechol from p-nitrophenol was diminished, though the specific activity of p-nitrophenol hydroxylase remained unchanged. PMEA had no influence on expression of CYP2E1 protein and mRNA and mRNAs of other P450 isoenzymes (1A1, 1A2, 2C11, 3A1, 3A2, and 4A1). It may be concluded that repeated systemic administration of higher doses of PMEA results in a partial degradation of rat CYP protein to inactive P420.

Inhibition of human telomerase by diphosphates of acyclic nucleoside phosphonates

Diphosphates of the antiviral acyclic nucleoside phosphonates (ANPs) were evaluated in telomeric repeat amplification protocol (TRAP) for their ability to inhibit the extension of telomeres by human telomerase. Extracts from human leukaemia HL-60 cells were used as a source of the enzyme. Data show that the most effective compound studied was the guanine derivative PMEGpp (IC 50 12.7 ± 0.5 μmol L −1 at 125 μmol L −1 deoxynucleoside triphosphates (dNTPs)). The inhibitory effects of other PME, PMP and HPMP diphosphates on telomerase reverse transcriptase decreased in the order: ( R)-PMPGpp > ( R)-HPMPGpp > PMEDAPpp > ( S)-PMPGpp > ( S)-HPMPApp > PMEO-DAPypp > ( R)-6-cyprPMPDAPpp > ( R)-PMPApp > ( R)-PMPDAPpp ≥ PMEApp ≥ PMECpp > PMETpp > ( S)-PMPApp ∼ 6-Me 2PMEDAPpp. These results are consistent with the observed antineoplastic activities of the parental guanine (PMEG) and 2,6-diaminopurine (PMEDAP) PME-derivatives. Moreover, structure–activity relationship indicates enantioselectivity of some of these human telomerase inhibitors: ( R)-isomers of the PMP-derivatives possess stronger inhibitory potency towards the enzyme than ( S)-isomers. The data may contribute to the rational design of telomerase inhibitors based on the structure of acyclic nucleotide analogues.

Cytostatic activity of antiviral acyclic nucleoside phosphonates in rodent lymphocytes

Acyclic nucleoside phosphonates (ANPs) inhibit replication of both DNA viruses and retroviruses, including HIV. The major mechanism of their antiviral action is inhibition of virus-induced DNA polymerases and/or of reverse transcriptases. We investigated the effects of ANPs on proliferation of mitogen-stimulated mouse and rat splenocytes. Included in the study were compounds differing at the heterocyclic base, i.e., adenine (A) and 2,6-diaminopurine (DAP), and at the N 9-side chain, i.e., 9-[2-(phosphonomethoxy)ethyl] (PME) and ( R)- or ( S)-enantiomers of 9-[2-(phosphonomethoxy)propyl] (PMP) moieties, and their numerous N 6-substituted derivatives. The medial inhibitory concentrations (IC50) of N 6-nonsubstituted compounds range from 0.13 (PMEDAP) to 354 μM (( R)-PMPA). Antiproliferative effects are more pronounced in PME than in PMP series, and they are more prominent in DAP compared to A analogs. The ( S)-enantiomers of PMP series are more effective than corresponding ( R)-congers. The highest cytostatic potential is exhibited by N 6-allyl-PMEDAP (IC50 = 0.017 μM) and N 6-cyclopropyl-PMEDAP (IC50 = 0.036 μM). The N 6-substituted derivatives of ( S)-PMPA are virtually devoid of cytostatic activity. No tight correlation between the cytostatic and reported antiviral effects could be detected.

Macrophage activation by antiviral acyclic nucleoside phosphonates in dependence on priming immune stimuli

Acyclic nucleoside phosphonates (ANPs) are potent broad-spectrum antivirals, also effective against immunodeficiency viruses and hepatitis viruses. Effects of several ANPs on in vitro cytokine gene expression and nitric oxide (NO) production by murine peritoneal macrophages were investigated. Included in the study were 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; Adefovir), 9-( R)-[2-(phosphonomethoxy)propyl]adenine [( R)-PMPA; Tenofovir], 9-( S)-[2-(phosphonomethoxy)propyl]adenine; ( S)-PMPA), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), 9-( R)-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP), and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG). Some of them, i.e. ( R)-PMPA, ( S)-PMPA, and PMEG, stimulate secretion of TNF-α and IL-10 in a concentration-dependent manner, and enhance the IFN-γ-induced secretion of TNF-α. Although unable to activate production of nitric oxide (NO) on their own, these compounds substantially augment NO formation induced by IFN-γ. Analysis of the expression of inducible NO synthase mRNA indicates that the NO-enhancing effect of ANPs is mediated posttranscriptionally. In contrast to IFN-γ, production of NO triggered by lipopolysaccharide (LPS) alone, or synergistically by LPS+IFN-γ, remains unaltered by ANPs. The immunomodulatory effects have been differentially expressed in distinct genotypes of inbred strains of mice, including the low NO-responders Balb/c and high NO-responders C3H/HeN. Although less effectively, PMEG and ( R)-PMPA also increase production of TNF-α and NO by the IFN-γ- but not LPS-co-stimulated macrophages from C3H/HeJ mice, which are otherwise hypo-responsive to major immune stimuli provided by IFN-γ and LPS. It can be concluded that the expression of immunomodulatory properties of ANPs depends on the immune state of cells and its activation by distinct priming signals.

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Antiviral Acyclic Nucleoside Phosphonate Inhibitors: Kinetics and Emission Studies

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

Antiviral acyclic nucleoside phosphonate analogues as inhibitors of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP, E.C.2.4.2.1) catalyses reversible phosphoro-lysis of purine ribo- and deoxyribonucleosides. Mammalian phosphorylases are specific for 6-ketopurines while some bacterial PNPs (e.g. from E. coli and S. typhimurium) have very broad specificty.l–4 Genetic deficiency of PNP leads to a loss of cellular immunity.5 The potential clinical applications of PNP inhibitors include treatment of T-cell leukemias, suppression of the host-vs-graft response in organ transplantation and potentation of the chemotherapeutic action of PNP-cleavable nucleosides.6