Oral USC-093, a novel homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087 has decreased nephrotoxicity while maintaining antiviral efficacy against human adenovirus infection of Syrian hamsters.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.

Discovery of a potent and selective human AC2 inhibitor based on 7-deazapurine analogues of adefovir.

Adefovir based acyclic nucleoside phosphonates were previously shown to modulate bacterial and, to a certain extent, human adenylate cyclases (mACs). In this work, a series of 24 novel 7-substituted 7-deazaadefovir analogues were synthesized in the form of prodrugs. Twelve analogues were single-digit micromolar inhibitors of Bordetella pertussis adenylate cyclase toxin with no cytotoxicity to J774A.1 macrophages. In HEK293 cell-based assays, compound 14 was identified as a potent (IC50 = 4.45 µM), non-toxic, and selective mAC2 inhibitor (vs. mAC1 and mAC5). Such a compound represents a valuable addition to a limited number of small-molecule probes to study the biological functions of individual endogenous mAC isoforms.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization.

Activation of Vγ9Vδ2 T cells with butyrophilin 3A1 (BTN3A1) agonists such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) has the potential to boost the immune response. Because HMBPP is highly charged and metabolically unstable, prodrugs may be needed to overcome these liabilities, but the prodrugs themselves may be limited by slow payload release or low plasma stability. To identify effective prodrug forms of a phosphonate agonist of BTN3A1, we have prepared a set of diesters bearing one aryl and one acyloxymethyl group. The compounds were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase production of interferon γ, resist plasma metabolism, and internalize into leukemia cells. These bioassays have revealed that varied aryl and acyloxymethyl groups can decouple plasma and cellular metabolism and have a significant impact on bioactivity (>200-fold range) and stability (>10 fold range), including some with subnanomolar potency. Our findings increase the understanding of the structure-activity relationships of mixed aryl/acyloxymethyl phosphonate prodrugs.

Phosphonate and Thiasugar Analogues of Glucosamine-6-phosphate: Activation of the glmS Riboswitch and Antibiotic Activity.

The glmS riboswitch is a motif found in 5'-untranslated regions of bacterial mRNA that controls the synthesis of glucosamine-6-phosphate (GlcN6P), an essential building block for the bacterial cell wall, by a feedback mechanism. Activation of the glmS riboswitch by GlcN6P mimics interferes with the ability of bacteria to synthesize its cell wall. Accordingly, GlcN6P mimics acting as glmS activators are promising candidates for future antibiotic drugs that may overcome emerging bacterial resistance against established antibiotics. We describe the synthesis of a series of phosphonate mimics of GlcN6P as well as the thiasugar analogue of GlcN6P. The phosphonate mimics differ in their pKa value to answer the question of whether derivatives with a pKa matching that of GlcN6P would be efficient glmS activators. We found that all derivatives activate the riboswitch, however, less efficiently than GlcN6P. This observation can be explained by the missing hydrogen bonds in the case of phosphonates and is valuable information for the design of future GlcN6P mimics. The thiasugar analogue of GlcN6P on the other hand turned out to be a glmS riboswitch activator with the same activity as the natural metabolite GlcN6P. The nonphosphorylated thiasugar displayed antimicrobial activity against certain bacilli. Therefore, the compound is a promising lead structure for the development of future antibiotics with a potentially novel mode of action.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Cyclopentenylcytosine (CPE-C): In Vitro and In Vivo Evaluation as an Antiviral against Adenoviral Ocular Infections.

Adenoviruses are the major cause of ocular viral infections worldwide. Currently, there is no approved antiviral treatment for these eye infections. Cyclopentenylcytosine (CPE-C) is an antiviral that has demonstrated activity against more than 20 viruses. The goals of the current study were to determine the in vitro and in vivo antiviral activity of CPE-C as well as its ocular toxicity. Antiviral activity was evaluated in vitro using standard plaque reduction assays to determine the 50% effective concentrations (EC50s) and in vivo in the Ad5/NZW rabbit ocular replication model. Ocular toxicity was determined in uninfected rabbit eyes following topical ocular application. The in vitro EC50s for CPE-C ranged from 0.03 to 0.059 µg/mL for nine adenovirus types that commonly infect the eye. Ocular toxicity testing determined CPE-C to be non-irritating or practically non-irritating by Draize scoring. In vivo, 3% CPE-C topically administered 4X or 2X daily for 7 days to adenovirus-infected eyes demonstrated effective antiviral activity compared with the negative control and comparable antiviral activity to the positive control, 0.5% cidofovir, topically administered twice daily for 7 days. We conclude CPE-C was relatively non-toxic to rabbit eyes and demonstrated potent anti-adenoviral activity in vitro and in vivo.

N2'-Branched Acyclic Nucleoside Phosphonates Containing 9-Deazahypoxanthine as Inhibitors of Plasmodium falciparum 6-Oxopurine Phosphoribosyltransferase.

Twelve N2'-branched acyclic nucleoside phosphonates and bisphosphonates were synthesized as potential inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), the key enzyme in the purine salvage pathway for production of purine nucleotides. The chemical structures were designed with the aim to study selectivity of the inhibitors for PfHGXPRT over human HGPRT. The newly prepared compounds contain 9-deazahypoxanthine connected to a phosphonate group via a five-atom-linker bearing a nitrogen atom (N2') as a branching point. All compounds, with the additional phosphonate group(s) in the second aliphatic linker attached to N2' atom, were low micromolar inhibitors of PfHGXPRT with low to modest selectivity for the parasite enzyme over human HGPRT. The effect of the addition of different chemical groups/linkers to N2' atom on the inhibition constants and selectivity is discussed.

Synthesis, Bioactivity and Molecular Docking of Nereistoxin Derivatives Containing Phosphonate.

Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via 31P, 1H and 13C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) against Mythimna separata Walker, Myzus persicae Sulzer and Rhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound 7f displayed good activity against all three insect species, showing LC50 values of 136.86 µg/mL for M. separata, 138.37 µg/mL for M. persicae and 131.64 µg/mL for R. padi. Compound 7b had the highest activity against M. persicae and R. padi, with LC50 values of 42.93 µg/mL and 58.19 µg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches.

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24-40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26-28, 30, 33-36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

Enzymatic Synthesis of a Novel Coumarin Aminophosphonates: Antibacterial Effects and Oxidative Stress Modulation on Selected E. coli Strains.

The objective of the present study was to evaluate the synergistic effect of two important pharmacophores, coumarin and α-amino dimethyl phosphonate moieties, on antimicrobial activity toward selected LPS-varied E. coli strains. Studied antimicrobial agents were prepared via a Kabachnik-Fields reaction promoted by lipases. The products were provided with an excellent yield (up to 92%) under mild, solvent- and metal-free conditions. A preliminary exploration of coumarin α-amino dimethyl phosphonate analogs as novel antimicrobial agents was carried out to determine the basic features of the structure responsible for the observed biological activity. The structure-activity relationship revealed that an inhibitory activity of the synthesized compounds is strongly related to the type of the substituents located in the phenyl ring. The collected data demonstrated that coumarin-based α-aminophosphonates can be potential antimicrobial drug candidates, which is particularly crucial due to the constantly increasing resistance of bacteria to commonly used antibiotics.

Possible Interaction between ZnS Nanoparticles and Phosphonates on Mediterranean Clams Ruditapes decussatus.

This study aims to evaluate the toxicity of ZnS nanoparticles (ZnS NP50 = 50 µg/L and ZnS NP100 = 100 µg/L) and diethyl (3-cyano-1-hydroxy-2-methyl-1-phenylpropyl)phosphonate or P (P50 = 50 µg/L and P100 = 100 µg/L) in the clams Ruditapes decussatus using chemical and biochemical approaches. The results demonstrated that clams accumulate ZnS NPs and other metallic elements following exposure. Moreover, ZnS NPs and P separately lead to ROS overproduction, while a mixture of both contaminants has no effect. In addition, data showed that exposure to P100 resulted in increased levels of oxidative stress enzyme activities catalase (CAT) in the gills and digestive glands. A similar trend was also observed in the digestive glands of clams treated with ZnS100. In contrast, CAT activity was decreased in the gills at the same concentration. Exposure to ZnS100 and P100 separately leads to a decrease in acetylcholinesterase (AChE) levels in both gills and digestive glands. Thus, AChE and CAT after co-exposure to an environmental mixture of nanoparticles (ZnS100) and phosphonate (P100) did not show any differences between treated and non-treated clams. The outcome of this work certifies the use of biomarkers and chemical assay when estimating the effects of phosphonate and nanoparticles as part of an ecotoxicological assessment program. An exceptional focus was given to the interaction between ZnS NPs and P. The antioxidant activity of P has been demonstrated to have an additive effect on metal accumulation and antagonistic agents against oxidative stress in clams treated with ZnS NPs.

Antivirals With Activity Against Mpox: A Clinically Oriented Review.

Mpox virus is an emergent human pathogen. While it is less lethal than smallpox, it can still cause significant morbidity and mortality. In this review, we explore 3 antiviral agents with activity against mpox and other orthopoxviruses: cidofovir, brincidofovir, and tecovirimat. Cidofovir, and its prodrug brincidofovir, are inhibitors of DNA replication with a broad spectrum of activity against multiple families of double-stranded DNA viruses. Tecovirimat has more specific activity against orthopoxviruses and inhibits the formation of the extracellular enveloped virus necessary for cell-to-cell transmission. For each agent, we review basic pharmacology, data from animal models, and reported experience in human patients.

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.

Phosphonate Derivatives of Paracetamol and Valproic Acid.

Paracetamol and valproic acid are standalone drugs with leading position in the world drug market. The phosphonate analogues of these drugs were synthesized and were tested in vivo. N-(4-hydroxyphenylcarbamoyl)phosphonic acid was four times more potent than paracetamol in preventing acetic acid-induced writhing. Phosphonate derivative of valproic acid, (2-propylpentanoyl)phosphonic acid, had similar in vivo activity to valproic acid in the pentylenetetrazole-induced kindling mouse model.

Novel coumarin aminophosphonates as potential multitargeting antibacterial agents against Staphylococcus aureus.

Unique coumarin aminophosphonates as new antibacterial agents were designed and synthesized to combat severely bacterial resistance. Bioactivity assessment identified that 3-hydroxylphenyl aminophosphonate 6f with low hemolytic activity not only exhibited excellent inhibition potency against Staphylococcus aureus at low concentration (0.5 µg/mL) in vitro but also showed considerable antibacterial potency in vivo. Meanwhile, the active compound 6f was capable of eradicating the S. aureus biofilm, thus alleviating the development of S. aureus resistance. Furthermore, the drug combination of compound 6f with norfloxacin could enhance the antibacterial efficacy. Mechanistic explorations manifested that molecule 6f was able to destroy the integrity of cell membrane, which resulted in the leakage of protein and metabolism inhibition. The cellular redox homeostasis was interfered through inducing the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to the reduction of glutathione (GSH) activity and lipid peroxidation. Furthermore, compound 6f could intercalate into DNA base pair to hinder normal biological function. The above results provided powerful information for the further development of coumarin aminophosphonates as antibacterial agents.

Silver(I) Complexes Based on Oxadiazole-Functionalized α-Aminophosphonate: Synthesis, Structural Study, and Biological Activities.

Two silver(I) complexes, bis{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN3:κN4-amino) (4-trifluoromethylphenyl)methyl]phosphonate-(tetrafluoroborato-κF)}-di-silver(I) and tetrakis-{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN3-amino)(4-trifluoromethylphenyl)methyl]phosphonate} silver(I) tetrafluoroborate, were prepared starting from the diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-amino)(4-trifluoromethylphenyl)methyl]phosphonate (1) ligand and AgBF4 salt in Ag/ligand ratios of 1/1 and 1/4, respectively. The structure, stoichiometry, and geometry of the silver complexes were fully characterized by elemental analyses, infrared, single-crystal X-ray diffraction studies, multinuclear NMR, and mass spectroscopies. The binuclear complex ([Ag2(1)2(BF4)2]; 2) crystallizes in the monoclinic asymmetric space group P21/c and contains two silver atoms adopting a {AgN2F} planar trigonal geometry, which are simultaneously bridged by two oxadiazole rings of two ligands, while the mononuclear complex ([Ag(1)4]BF4; 3) crystallizes in the non-usual cubic space group Fd-3c in which the silver atom binds to four distinct electronically enriched nitrogen atoms of the oxadiazole ring, in a slightly distorted {AgN4} tetrahedral geometry. The α-aminophosphonate and the monomeric silver complex were evaluated in vitro against MCF-7 and PANC-1 cell lines. The silver complex is promising as a drug candidate for breast cancer and the pancreatic duct with half-maximal inhibitory concentration (IC50) values of 8.3 ± 1.0 and 14.4 ± 0.6 µM, respectively. Additionally, the interactions of the ligand and the mononuclear complex with Vascular Endothelial Growth Factor Receptor-2 and DNA were evaluated by molecular docking methods.

Oroxylin-A and its phosphonate derivative potentiate eNOS/NO-mediated relaxation and attenuate vasoconstrictor-induced contraction in the mouse aorta.

Oroxylin-A (OroA), a flavonoid isolated from Scutellariae baicalensis, alleviates cardiovascular dysfunction. Several procedures for synthesizing OroA have been developed but show low production yield and regioselectivity. We synthesized OroA from baicalin using a one-pot reaction to increase its overall yield. We also determined the chemical properties and mechanism of action of the synthesized OroA and OroA phosphate diethyl ester (OroA-OET) in vascular function. The induction of vascular reactivity by OroA and OroA-OET was evaluated using blood vessel myography and biochemical analysis to assess nitric oxide synthase-mediated nitric oxide production in mouse aortic arteries. OroA and OroA-OET (0.1-30 µM) induced sustained vasorelaxation, which was partly mediated by the endothelium in isolated normal arteries pre-contracted with phenylephrine. OroA and OroA-OET significantly attenuated vasoconstrictors-induced contractile responses. Dilation effects were blocked by the non-selective nitric oxide synthase inhibitor N (omega)-nitro-l-arginine methyl ester but not by tetraethylammonium or 1H-(1,2,4)oxadiazolo [4,3-a]quinoxalin-1-one. Notably, preincubation with OroA and OroA-OET potentiated acetylcholine-induced relaxation and endothelial nitric oxide production in the arteries with the endothelium. OroA and OroA-OET protected against cardiovascular dysfunction. The synthesis and lead compounds used not only improved the yield of OroA from natural sources but also potentially regulated vascular tone.

The Synthesis and Evaluation of Diethyl Benzylphosphonates as Potential Antimicrobial Agents.

The impact of substituent at phenyl ring of diethyl benzylphosphonate derivatives on cytotoxic activity was studied. The organophosphonates were obtained based on developed palladium-catalyzed α, ß-homodiarylation of vinyl esters protocol. The new synthetic pathway toward 1,2-bis(4-((diethoxyphosphoryl)methyl)phenyl)ethyl acetate was proposed which significantly improves the overall yield of the final product (from 1% to 38%). Several newly synthesized organophosphonates were tested as new potential antimicrobial drugs on model Escherichia coli bacterial strains (K12 and R2-R3). All tested compounds show the highest selectivity and activity against K12 and R2 strains. Preliminary cellular studies using MIC and MBC tests and digestion of Fpg after modification of bacterial DNA suggest that selected benzylphosphonate derivatives may have greater potential as antibacterial agents than typically used antibiotics such as ciprofloxacin, bleomycin and cloxacillin. These compounds are highly specific for pathogenic E. coli strains based on the model strains used and may be engaged in the future as new substitutes for commonly used antibiotics, which is especially important due to the increasing resistance of bacteria to various drugs and antibiotics.

Synthesis and Anti-influenza Activity of Vinylphosphonic Acid (Co)polymers.

New copolymers of vinylphosphonic acid (VPA) with 2-deoxy-2-methacrylamido-D-glucose, 4-acryloylmorpholine (4-AM), and acrylamide have been synthesized, and their antiviral activity against influenza virus has been studied in in vitro and in vivo experiments. Optimal antiviral characteristics and low cytotoxicity were exhibited by the copolymer of VPA with 4-AM, composition 56 : 44 mol %, molecular weight 33 000. The polymer exhibited virus-inhibiting properties with an IC50 = 1 µg/mL and a selectivity index of 302. Prophylactic intranasal administration of the polymer in a murine model of influenza pneumonia completely prevented the virus-induced death of animals, whereas the level of mortality in the placebo group was 90%. The results of this study indicate a high antiviral potential of polymeric compounds based on VPA.

Synthesis and Evaluation of Prodrugs of α-Carboxy Nucleoside Phosphonates.

A range of lipophilic prodrugs of α-carboxy nucleoside phosphonates, potent inhibitors of HIV-1 reverse transcriptase without requiring prior phosphorylation, were synthesized to evaluate their in vivo potency against HIV in cell culture. A series of prodrug derivatives bearing a free carboxylic acid where the phosphonate was masked with bispivaloyloxymethyl, diisopropyloxycarbonyloxymethyl, bisamidate, aryloxyphosphoramidate, hexadecyloxypropyl, CycloSal, and acycloxybenzyl moieties were synthesized, adapting existing methodologies for phosphonate protection to accommodate the adjacent carboxylic acid moiety. The prodrugs were assayed for anti-HIV activity in CEM cell cultures─the bispivaloyloxymethyl free acid monophosphonate prodrug exhibited some activity (inhibitory concentration-50 (IC50) 59 ± 17 µM), while the other prodrugs were inactive at 100 µM. A racemic bispivaloyloxymethyl methyl ester monophosphonate prodrug was also prepared to assess the suitability of the methyl ester as a carboxylic acid prodrug. This compound exhibited no activity against HIV in cellular assays.