Design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides.

We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-Alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by (31)P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields.

Synthesis of phosphoramidate prodrugs: ProTide approach.

The ProTide (pronucleotide) approach is a prodrug strategy elaborated to deliver nucleoside monophosphate into the cell, circumventing the first and inefficient rate-limiting phosphorylation step of nucleosides and improving the cellular penetration of nucleotides. The ProTide of a nucleoside phosphate is a phosphoramidate prodrug consisting of an amino acid ester promoiety linked via P-N bond to a nucleoside aryl phosphate. Such prodrugs have increased lipophilicity and thus are capable of altering cell and tissue distribution. The ProTide technology was successfully and extensively applied to a wide variety of nucleoside phosphates, endowed with antiviral and anticancer activity. This unit describes two different synthetic strategies allowing the preparation of phosphoramidates of 6-O-methyl-2'-ß-C-methylguanosine as model compounds for nucleosides having only the 5'-OH as reactive hydroxyl group, and phosphoramidates of 2'-ß-D-arabinouridine (AraU) as model compounds for nucleosides containing two or more reactive hydroxyl groups.

Progress in the development of anti-hepatitis C virus nucleoside and nucleotide prodrugs.

The search for new anti-hepatitis C virus (HCV) therapeutics continues as the current treatment, consisting of PEGylated IFN-α and ribavirin, is of limited efficacy, nonspecific and can cause significant side effects. Modified nucleoside analogues with improved efficacy and selectivity, may become the backbone of the future standard of care for anti-HCV therapies. Several families of modified nucleoside are known to inhibit HCV RNA-dependent RNA polymerase, a vital enzyme for viral replication. Ongoing efforts are focused on improvement of potency, selectivity and delivery of antiviral nucleoside analogues, with several recent promising advances into clinical trials. This review summarizes the current progress in the development of new anti-HCV nucleoside and nucleotide prodrugs.

Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-HCV agents.

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 µM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus.

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.

INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties.

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus.

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2'-beta-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2'-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.

Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties.

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

The phosphoramidate ProTide approach greatly enhances the activity of beta-2'-C-methylguanosine against hepatitis C virus.

Beta-2'-C-methyl purines (1, 2) are known inhibitors of hepatitis C virus (HCV). We herein report the synthesis, biological and enzymatic evaluation of their 5'-phosphoramidate ProTides. Described herein are seven l-alanine phosphoramidate derivatives with variations to the amino acid ester. The 1-naphthyl phosphoramidate of beta-2'-methylguanosine containing the benzyl ester (20) was the most active at 0.12microM, an 84-fold of increase in activity compared to the parent nucleoside (2) with no increase of cytotoxicity. The carboxypeptidase mediated hydrolysis of several ProTides showed a predictive correlation with their activity versus HCV in replicon.