PMPA and PMEA prodrugs for the treatment of HIV infections and human papillomavirus (HPV) associated neoplasia and cancer.

The synthesis and in vitro biological evaluation of novel phosphonamidate and phosphonodiamidate prodrugs of adefovir and tenofovir are reported. The selected synthetic approach from free phosphonic acid via bis-trimethylsilyl ester intermediates affords (L)-alanine ester derivatives in 10-70% yields. When assessed for their anti-HIV activity, all the prodrugs showed submicromolar activity. Noteworthy, the most potent derivative in the adefovir series contained a 5,6,7,8-tetrahydronaphtyl group, herein reported for the first time as an aryl moiety in a ProTide. A pronounced cytostatic activity of the above prodrugs is also reported. Selected compounds were tested for their antiproliferative activity against HPV-transformed cells and they were found significantly more active in comparison to their parent compounds. In this study a slightly improved activity of the adefovir derivatives over those of tenofovir was also noticed. However, no specificity for naturally HPV-transformed cell lines was observed.

Synthesis and biological evaluation of purine 2'-fluoro-2'-deoxyriboside ProTides as anti-influenza virus agents.

2'-Fluoro-2'-deoxyguanosine has been reported to have potent anti-influenza virus activity in vitro and in vivo. Herein we describe the synthesis and biological evaluation of 6-modified 2'-fluoro-2'-deoxyguanosine analogues and their corresponding phosphoramidate ProTides as potential anti-influenza virus agents. Whereas the parent nucleosides were devoid of antiviral activity in two different cellular assays, the 5'-O-naphthyl(methoxy-L-alaninyl) ProTide derivatives of 6-O-methyl-2'-fluoro-2'-deoxyguanosine, 6-O-ethyl-2'-fluoro-2'-deoxyguanosine, and 2'-deoxy-2'-fluoro-6-chloroguanosine, and the 5'-O-naphthyl(ethoxy-L-alaninyl) ProTide of 6-O-ethyl-2'-fluoro-2'-deoxyguanosine displayed antiviral EC(99) values of ~12 µM. The antiviral results are supported by metabolism studies. Rapid conversion into the L-alaninyl metabolite and then 6-modified 2'-fluoro-2'-deoxyguanosine 5'-monophosphate was observed in enzymatic assays with yeast carboxypeptidase Y or crude cell lysate. Evidence for efficient removal of the 6-substituent on the guanine part was provided by enzymatic studies with adenosine deaminase, and by molecular modeling of the nucleoside 5'-monophosphates in the catalytic site of a model of ADAL1, thus indicating the utility of the double prodrug concept.

Novel phosphoramidate prodrugs of N-acetyl-(D)-glucosamine with antidegenerative activity on bovine and human cartilage explants.

(D)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis (OA), a chronic and degenerative articular joint disease. In our preceding paper, a series of novel O-6 phosphate N-acetyl (d)-glucosamine prodrugs aimed at improving the oral bioavailability of N-acetyl-(d)-glucosamine as its putative bioactive phosphate form were shown to have greater chondroprotective activity in vitro when compared to the parent agent. In order to extend the SAR studies, this work focuses on the O-3 and O-4 phosphate prodrugs of N-acetyl-(d)-glucosamine bearing a 4-methoxy phenyl group and different amino acid esters on the phosphate moiety. Among the compounds, the (l)-proline amino acid-containing prodrugs proved to be the most active of the series, more effective than the prior O-6 compounds, and well processed in chondrocytes in vitro. Data on human cartilage support the notion that these novel O-3 and O-4 regioisomers may represent novel promising leads for drug discovery for osteoarthritis.

Synthesis and biological evaluation of pyrimidine nucleoside monophosphate prodrugs targeted against influenza virus.

Uridine-based nucleoside analogues have often been found to have relatively poor antiviral activity. Enzymatic assays, evaluating inhibition of influenza virus RNA polymerase, revealed that some uridine triphosphate derivatives displayed inhibitory activity on UTP incorporation into viral RNA. Here we report the synthesis, antiviral activity and enzymatic evaluation of novel ProTides designed to deliver the activated (monophosphorylated) uridine analogues inside the influenza virus-infected cells. After evaluation of the activation profile we identified two ProTides with moderate antiviral activity in MDCK cells (23a, EC(99)=49 ± 38 µM and 23b, EC(99)≥81 µM) while the corresponding nucleoside analogue (2'-fluoro-2'-deoxyuridine) was inactive. Thus, at least in these cases the poor antiviral activity of the uridine analogues may be ascribed to poor phosphorylation.

Medicinal chemistry of nucleoside phosphonate prodrugs for antiviral therapy.

Considerable attention has been focused on the development of phosphonate-containing drugs for application in many therapeutic areas. However, phosphonate diacids are deprotonated at physiological pH and thus phosphonate-containing drugs are not ideal for oral administration, an extremely desirable requisite for the treatment of chronic diseases. To overcome this limitation several prodrug structures of biologically active phosphonate analogues have been developed. The rationale behind the design of such agents is to achieve temporary blockade of the free phosphonic functional group until their systemic absorption and delivery, allowing the release of the active drug only once at the target. In this paper, an overview of acyclic and cyclic nucleoside phosphonate prodrugs, designed as antiviral agents, is presented.

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.

Phenylboronic-acid-based carbohydrate binders as antiviral therapeutics: monophenylboronic acids.

BACKGROUND: The development of carbohydrate-binding agents as novel therapeutics for the inhibition of highly glycosylated enveloped viruses has generated much attention in recent literature. Possessing a potential dual mode of action by inhibiting virus entry and exposing the virion to neutralization by the host immune system upon the deletion of envelope glycans under drug pressure, these substances might provide a new direction in antiviral treatment. Phenylboronic acids are widely known to bind the cis-diol functionality of carbohydrate structures, thereby identifying themselves as potential lead structures. To date, few details have been disclosed of the structure-activity relationship of these substances in correlation to their antiviral activity. METHODS: In this study, a compound library of a diverse range of ortho-, meta- and para- ring-substituted monophenylboronic acids and glutamine phenylboronic acid analogues was prepared, characterized and evaluated to probe antiviral activity versus a broad range of (enveloped) viruses. RESULTS: The compounds described herein lack antiviral activity. They also did not show measurable binding to HIV type-1 (HIV-1) gp120, using surface plasmon resonance technology. However, of note is the general lack of toxicity, which suggests that further investigation of the compounds as potential therapeutics is needed. CONCLUSIONS: The monophenylboronic acids tested exhibited no antiviral activity as potential carbohydrate binders versus a broad range of enveloped and non-enveloped viruses. The compounds tested did not bind HIV-1 gp120, possibly because of their small size and lack of multivalency.

The application of phosphoramidate ProTide technology to the potent anti-HCV compound 4'-azidocytidine (R1479).

We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the potent anti-HCV compound 4'-azidocytidine (R1479), with variation on the ester, amino acid and aryl moiety of the ProTide. Sub-muM inhibitors of HCV emerge. The compounds are all non-cytotoxic in the replicon assay. We herein report detailed SARs for each of the regions of the ProTide.

Preferential pi-pi complexation between tamoxifen and borage oil/gamma linolenic acid: transcutaneous delivery and NMR spectral modulation.

The effect of different proportions of borage oil on the in vitro transcutaneous delivery of tamoxifen were studied, with the aim of developing a gel capable of the simultaneous delivery of tamoxifen and gamma linolenic acid across (breast) skin. Supplementary work probed 1H NMR spectral data for tamoxifen in the presence of different proportions of polyunsaturated or unsaturated fatty acids. Typical, non-aqueous gels were modified to contain 1% tamoxifen and three levels of borage oil ( approximately 25% gamma linolenic acid) and the transcutaneous delivery of both tamoxifen and GLA across full thickness skin determined in vitro. Both tamoxifen and gamma linolenic acid permeated the skin with the ratio of moles being consistent at approximately 4:1. This was irrespective of time, amount of borage oil contained in the formulation (above a minimum) and the presence of other (unsaturated) excipients: mineral oil, Miglyiol 810N, white soft paraffin, PEG400 and Cabosil M5. Dose-dependent downfield shifts of tamoxifen aromatic protons were observed in the presence of borage oil and linolenic acid (gamma and alpha), but not saturated triacyl glycerol. The permeation data suggested vehicular complexation between tamoxifen and polyunsaturated constituents of borage oil and that such complexes permeated the skin intact. The 1H NMR data supported the hypothesis that such complexation was a consequence of preferential pi-pi orbital interactions between the phenyl groups of tamoxifen and the multiple double bonds of GLA. The mechanism for the permeation of intact complexes across skin remains to be elucidated.

Phosphoramidate protides of carbocyclic 2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine with potent activity against HIV and HBV.

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.