Synthesis of 21,31,51-trideoxyuridine-51-methylphosphonic acid and its diphosphate derivative. Study of the interaction with HIV-1reverse transcriptase

2′,3′,5′-Trideoxyuridine-5′-methylphosphonate, [8], was synthesized from ddU. The 5′,6′ carbon-carbon bond was formed by condensing the 5′-aldehyde of ddU and a Wittig reagent. The binding interaction of the diphosphate derivative [10] of the phosphonate [8] with HIV-1 reverse transcriptase (RT) was studied using methods based primarily on fluorescence spectroscopy. From the quenching of intrinsic tryptophan fluorescence of RT during its titration against [10], a dissociation constant of 24 μm was calculated at 25°C. In the presence of a DNA/DNA template/primer of defined sequence and RT, [10] and a fluorescent derivative of ddTTP competed for binding to RT without incorporation of ddU-5′-methylphosphonate. In the presence of a 0.5 mm concentration of [10], the RT activity measured with Poly(rA)/(dT)15 and [3H] dTTP was lowered to 65%. All our observations are consistent with suppression of the catalysis of bond formation between the OH at the primer 3′-end and α-P of [10] after formation of the complex between RT, template/primer and [10]

Synthesis of 2′,3′,5′-trideoxyuridine-5′-methylphosphonic Acid and its Diphosphate Derivative. Study of the Interaction with HIV-1 reverse Transcriptase

2′,3′,5′-Trideoxyuridine-5′-methylphosphonate, [8], was synthesized from ddU. The 5′,6′ carbon-carbon bond was formed by condensing the 5′-aldehyde of ddU and a Wittig reagent. The binding interaction of the diphosphate derivative [10] of the phosphonate [8] with HIV-1 reverse transcriptase (RT) was studied using methods based primarily on fluorescence spectroscopy. From the quenching of intrinsic tryptophan fluorescence of RT during its titration against [10], a dissociation constant of 24 μm was calculated at 25°C. In the presence of a DNA/DNA template/primer of defined sequence and RT, [10] and a fluorescent derivative of ddTTP competed for binding to RT without incorporation of ddU-5′-methylphosphonate. In the presence of a 0.5 mm concentration of [10], the RT activity measured with Poly(rA)/(dT)15 and [3H] dTTP was lowered to 65%. All our observations are consistent with suppression of the catalysis of bond formation between the OH at the primer 3′-end and α-P of [10] after formation of the complex between RT, template/primer and [10]

Comparison of the disposition of ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] in Caco-2 monolayers

PURPOSE: To evaluate the potential of several bis-ester prodrugs of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) to enhance the oral absorption of PMEA. METHODS: Caco-2 monolayers were used to estimate intestinal transport and metabolism of the bis(pivaloyloxymethyl)-ester [bis(POM)-] and a series of bis(S-acyl-2-thioethyl)-esters [bis(SATE)-] of PMEA. An LC-MS method was used for the identification of unknown metabolites which were formed from the SATE-esters. RESULTS: During transport across Caco-2 monolayers, all esters were extensively degraded as could be concluded from the appearance of the mono-ester and free PMEA in apical as well as basolateral compartments. Incubation of SATE-esters with the monolayers resulted in the formation of two additional metabolites, which were identified as 2-thioethyl-PMEA and its dimerisation product. All ester prodrugs resulted in enhanced transepithelial transport of total PMEA (i.e. the bis-esters and their corresponding metabolites, including PMEA), but significant differences could be observed between the various esters. Transport of total PMEA ranged from 0.4 +/- 0.1% for the bis[S(methyl) ATE]-ester to 15.3 +/- 0.9% for the more lipophilic bis[S(phenyl)ATE]-PMEA. A relationship between total transport of the esters and their lipophilicity (as estimated by their octanol/water partition coefficient) was established (r2 = 0.87). Incubation of prodrug esters with homogenates from Caco-2 cells showed large differences in susceptibility of the compounds to esterases, the half-lives of the bis-esters varying from 4.3 +/- 0.3 min for the bis[S(phenyl)ATE]-PMEA to 41.5 +/- 0.8 min for its methyl analogue. In addition, intracellularly formed PMEA was observed to be further converted by the cells to the diphosphorylated PMEA (PMEApp). CONCLUSIONS: Several SATE-esters of PMEA can be considered as potential alternatives to bis(POM)-PMEA, due to enhanced epithelial transport, sufficient chemical and enzymatic stability and adequate release of PMEA. Toxicological studies as well as in vivo experiments are required in order to further explore the potential of those SATE-esters as prodrugs for oral delivery of PMEA.status: publishe

2’-C-methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of RNA virus replication

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain 'natural' pyrimidine bases, but possess a beta-methyl substituent at the 2'-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA+), single-stranded negative (ssRNA-) or double-stranded (dsRNA), revealed potent activities for D-2'-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5'-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2'-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development