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Previously published S1P receptor modulator benzyl ether derivatives have shown potential as being viable therapeutics for the treatment of neurodegenerative diseases, however, two of the most S1P 1 -selective compounds are reported as being poorly phosphorylated by kinases in vivo. Phosphoramidates of BED compounds (2a, 2b) were synthesised with the aim of producing kinase-independent S1P receptor modulators. Carboxypeptidase, human serum and cell lysate processing experiments were conducted. ProTide BED analogues were found to have an acceptable level of stability in acidic and basic conditions and in vitro metabolic processing experiments showed that they are processed to the desired pharmacologically active monophosphate. The research describes the development of an entirely novel family of therapeutic agents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD 4 + T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The first copper-catalysed diastereoselective synthesis of P-chiral phosphoramidate prodrugs (ProTides) is reported. This procedure allows the synthesis of diastereomeric-enriched mixtures of ProTides. Application of this methodology to the asymmetric phosphorylation of purine and pyrimidine nucleoside analogues is presented.

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

7-Deazapurines are known to possess broad antiviral activity, however the 2'-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2'-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2'-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2'-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2'-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

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.

Background: Recently, the synthesis and antiviral activity of a series of 2'-fluoro derivatives of the most potent anti-varicella zoster virus (VZV) agent reported to date, the bicyclic nucleoside analogue Cf1743, have been reported., Methods: Here, we present molecular modelling studies for the interaction of these compounds with VZV-encoded thymidine kinase (TK) and we report the synthesis of a series of phosphoramidate ProTides of these compounds designed to bypass the nucleoside kinase dependence of the parent nucleoside analogues., Results: The phosphoramidate prodrugs were equally effective as their parent compounds against VZV in cell culture, but lost antiviral potency against TK-deficient VZV strains., Conclusions: ProTide-based kinase bypass is not successful in this case.

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.

Prodrug technologies aimed at delivering nucleoside monophosphates into cells (protides) have proved to be effective in improving the therapeutic potential of antiviral and anticancer nucleosides. In these cases, the nucleoside monophosphates are delivered into the cell, where they may then be further converted (phosphorylated) to their active species. Herein, we describe one of these technologies developed in our laboratories, known as the phosphoramidate protide method. In this approach, the charges of the phosphate group are fully masked to provide efficient passive cell-membrane penetration. Upon entering the cell, the masking groups are enzymatically cleaved to release the phosphorylated biomolecule. The application of this technology to various therapeutic nucleosides has resulted in improved antiviral and anticancer activities, and in some cases it has transformed inactive nucleosides to active ones. Additionally, the phosphoramidate technology has also been applied to numerous antiviral nucleoside phosphonates, and has resulted in at least three phosphoramidate-based nucleotides progressing to clinical investigations. Furthermore, the phosphoramidate technology has been recently applied to sugars (mainly glucosamine) in order to improve their therapeutic potential. The development of the phosphoramidate technology, mechanism of action and the application of the technology to various monophosphorylated nucleosides and sugars will be reviewed.

Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the L-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl variation was tolerated. ACV ProTides with other amino acids, other than L-phenylalanine, showed no detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure-activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV agents and vice versa, so the assay became predictive.

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.

As part of our studies on the anti-HIV activities of 2',3'-didehydro-2',3'-dideoxyuridine (d4U), 2',3'-dideoxyuridine (ddU) and their 'ProTides', we have prepared and evaluated the anti-HIV activity of a range of d4U and ddU aryl triester phosphoramidates. Besides elucidating SAR characteristics, we performed molecular modelling studies on both d4U and ddU in order to probe the first phosphorylation step required for the activation of these two nucleoside analogues. Overall, the application of the phosphoramidate approach turned the inactive ddU to a moderately active anti-HIV agent, while this was not the case with d4U. Enzymatic assays investigating the metabolism of d4U phosphoramidates revealed an efficient cleavage of the phosphoramidate motif to release the d4U monophosphate. Thus, a poor second and/or third phosphorylation step may be the most likely reason for the virtual lack of anti-HIV activity in this case.

Novel phosphoramidate ProTides derived from 4'-azidoinosine have been prepared and evaluated in the replicon assay against hepatitis C Virus (HCV). The parent nucleoside analogue is inactive in this assay, while the ProTides are active at low microM levels in some cases. This is a rare example of an inosine nucleoside analogue with potent antiviral activity and further supports the notion of ProTides as a drug discovery motif.

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.

Novel phosphoramidates of acyclovir have been prepared and evaluated in vitro against acyclovir-sensitive and -resistant herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV). Unlike the parent nucleoside these novel phosphate prodrugs retain antiviral potency versus the ACV-resistant virus strain, suggesting an efficient bypass of the viral thymidine kinase.

We report the synthesis of 2',3'-didehydro-2',3'-dideoxyuridine (d4U) and 2',3'-dideoxyuridine (ddU) phosphoramidate 'ProTide' derivatives and their evaluation against HIV-1 and HIV-2. In addition, we conducted molecular modeling studies on both d4U and ddU monophosphates to investigate their second phosphorylation process. The findings from the modeling studies provide compelling evidence for the lack of anti-HIV activity of d4U phosphoramidates, in contrast with the corresponding ddU phosphoramidates.

The aim of the present work is to investigate through molecular modelling the possible role of the human enzyme Hint1 in the final P-N bond cleavage of phosphoramidate ProTides, which would lead to the intracellular delivery of unmasked nucleoside analogue monophosphates. Herein, we report our preliminary analysis based on docking studies of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) related aminoacyl phosphates with Hint1 and the effect of the amino acid moiety on the enzyme-substrate binding affinity.

Based on our wide ranging knowledge of phosphoramidate ProTides as anti-viral agents we have tuned the lead anti-cancer agent thymectacin in the ester and amino acid regions and revealed a substantial enhancement in in vitro potency versus colon and prostate cancer cell lines. Twelve analogues have been reported, with yields of 29-78%. The compounds are fully characterised and data clearly reveal the presence of two phosphate diastereoisomers, as expected, in roughly equi-molar proportions. The compounds were evaluated in tissue culture versus three different tumour cell lines, using thymectacin as the control. It is notable that minor structural modification of the parent phenyl methoxyalaninyl structure of thymectacin leads to significant enhancements in potency. In particular, replacement of the methyl ester moiety in the lead by a benzyl ester gave a 175-fold boost in potency versus colon cancer HT115. This derivative emerges as a low micromolar inhibitor of HT115 cells and a new lead for further optimisation.

P-site inhibitors of adenyl cyclase, such as the dideoxynucleosides 2',3'-ddA and 2',5-ddA, have been shown to attenuate EDHF phenomenon in rabbit arteries and veins. In order to present the dideoxynucleosides as pre-activated nucleotides and bypass the kinase, as well as to prevent their metabolism to dideoxyinosine by adenosine deaminase, the aryloxyphosphoramidate approach has been successfully applied, initially on the 2',3'-ddA. In the present work a new series of 2',5'-ddA phosphoramidates has been synthesized, representing the first example of phosphoramidate protide not at the 5'-position.

The phosphoramidate technology we have developed has been recently applied to BVdU, leading to NB1011 (NewBiotics Inc., California), a novel potential anticancer compound recently entered into phase 2 of the clinical trials for colon cancer. We report in this work a new series of derivatives containing naphthol as aryl masking group on the phosphate moiety, which has shown a significant increase in anticancer activity in preliminary biological evaluations.

We herein describe the development of aryloxy phosphoramidate triesters as an effective pro-tide motif for the intracellular delivery of charged bio-active antiviral nucleoside monophosphates. The review covers the discovery of such aryl phosphoramidates, their mechanism of action and structure-activity relationships. The application of this strategy to a range of antiviral nucleosides is highlighted.

The phosphoramidate triester prodrug approach is widely used to deliver nucleotide forms of nucleoside analogues into target cells. We investigated the stereoselective stability of a series of prodrugs of the anti-HIV agent 2',3'-didehydro-2',3'-dideoxythymidine (d4T). Chemical stability was evaluated in phosphate buffer at pH values of biological relevance (i.e. pH 2.0, 4.6, 7.4). Enzymatic stability was tested in human plasma, in Caco-2 cell homogenates and monolayers and in rat liver. The compounds were relatively stable to chemical hydrolysis. Between 50 and 70% of unchanged prodrug was recovered after 16h incubation in human plasma, with no stereoselective preference for phosphate diastereoisomers. The p-OMe phenyl derivative, however, was an exception and only 5% of one diastereoisomer was recovered. In Caco-2 cells the stability and stereoselectivity largely depended on the experimental conditions: high enzymatic activity and stereoselectivity was observed in cell homogenates, but not in monolayers. In rat liver S9 fractions the stability profile was similar to that in Caco-2 cells and carboxyl ester cleavage appeared to be the sole mechanism of degradation in both media. The large and unpredictable differences in stereoselective metabolic rate of the pronucleotide series here presented suggest that in vivo circulating levels of intact prodrug could exert profoundly different activity or toxicity due to preferential body distribution of one diastereoisomeric form.

In rabbit arteries endogenous production of cAMP facilitates electrotonic signalling via gap junctions, thus explaining the ability of P-site inhibitors of adenylyl cyclase to attenuate EDHF-type responses. In the present study, we show that a lipophilic phosphoramidate pronucleotide derivative of dideoxyadenosine, 2',3'-ddA-PMAPh, exhibits enhanced activity as an inhibitor of EDHF-type smooth muscle hyperpolarizations induced by acetylcholine (ACh) compared to the parent nucleoside 2',3'-ddA, and that the effects of both compounds can be reversed by the cAMP phosphodiesterase inhibitor IBMX. Neither 2',3'-ddA nor 2',3'-ddA-PMAPh depress ACh-evoked endothelial hyperpolarization directly. Modifications in the lipophilicity of dideoxyadenosine and its direct intracellular delivery as a mononucleotide may thus enhance the ability to inhibit adenylyl cyclase and depress electrotonic signalling via myoendothelial gap junctions.

Synthesis of phosphoramidate protides of carbocyclic D- and L-2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleosides against both HIV and HBV.

Syntheses of phosphoramidate protides of several 2',3'-dideoxy-3'-fluoroadenosine derivatives by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleoside against HIV and HBV. Especially marked was the improvement in potency of phosphoramidate protides of 2',3'-dideoxy-3'-fluoroadenosine against both HIV and HBV.

We report the design, synthesis and antiviral evaluation of a number of lipophilic, masked phosphoramidate derivatives of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), designed to act as membrane soluble prodrugs of the free nucleotide. The phosphoramidate derivatives of BVDU that contain L-alanine exhibited potent anti herpes simplex virus type 1 and varicella-zoster virus activity but lost marked activity against thymidine kinase-deficient virus strains. The phosphoramidate derivative bearing the amino acid alpha,alpha-dimethylglycine showed poor activity in all cell lines tested. It appears that successful kinase bypass by phosphoramidates is highly dependent on the nucleoside analogue, amino acid and ester structure, as well as the cell line to which the drugs are exposed.

Some novel amidate prodrugs of PMEA and PMPA have been synthesised and tested in vitro for their biological activity. Compound 5 in particular showed greatly enhanced antiviral potency compared with the parent nucleotide analogue. In vitro enzymatic studies and structure-activity relationships indicate that the degradation mechanism of such prodrugs may be the same as that described for the phosphoramidate triesters of nucleotide analogues.

Some novel phosphoramidate derivatives of the nucleoside analogue stavudine have been prepared as membrane-soluble prodrugs of the bioactive free phosphate forms. Phenyl phosphates linked via nitrogen to methyl esterified amino acid analogues were studied, where the amino acid was an unnatural alpha-alkyl (or aryl) glycine or an alpha,alpha-dialkyl glycine. All compounds were characterized by a range of spectroscopic, spectrometric and analytical methods and were subjected to in vitro evaluation of their anti-human immunodeficiency virus efficacy. It is notable that certain unnatural amino acid derivatives could substitute for alanine with only a relatively small loss of activity and, moreover, that this activity did not fall-off with increasing alkyl chain length for the C2-C4 mono-alkyl series. These data are further probed by the application of our recently reported 31P-NMR-based carboxyl esterase assay, with informative results.

A range of polyether para-substituted phosphoramidates were synthesised and found to have substantially elevated aqueous solubilities compared to the underivatised parent prodrug. A 30-fold increase in aqueous solubility could be achieved without a substantial decrease of in vitro activity against HIV-1. Replacement of the aryl (i.e. phenolic) moiety by tyrosine led to a substantial enhancement in aqueous solubility but also to a decrease in antiviral potency. A previously unobserved trend was identified, relating increased aryl substituent steric bulk to decreased antiviral activity.

A series of new substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble nucleotide prodrugs, to extend and quantify the SAR observed for an earlier series of related derivatives. All of the compounds were found to be significantly more potent against HIV in cell culture than the nucleoside analogue d4T, and most were also found to be significantly more potent than the parent phosphoramidate. A Hansch type QSAR analysis was applied to the combined series of 21 compounds. The results of this analysis revealed anti-HIV activity to be principally dependent on lipophilicity in a quadratic manner, with terms representing substituent steric bulk and electronic effects having a minimal significance.