Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals (original) (raw)
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Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues
Angewandte Chemie (International ed. in English), 2016
The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.
Febs Letters, 1993
Nucleoside analogues previously found to be inactive against the human immunodeficiency virus (HIV) may be activated by simple chemical derivatisation. As part of our effort to deliver masked phosphates inside living cells we have discovered that certain phosphate triester derivatives of inactive nucleoside analogues become inhibitors of HIV replication. This discovery underlies the importance of the masked phosphate approach, and has significant implications for the future design of chemotherapeutic nucleoside analogues. If highly modified nucleoside analogues may be active without the intervention of nucleoside kinase enzymes, major advantage may accrue in terms of low toxicity and enhanced selectivity. Moreover, the increased structural freedom may have implications for dealing with the emergence of resistance. The concept herein described as 'kinase bypass' may thus stimulate the discovery of a new generation of antiviral agents.
Tetrahedron Letters, 2010
Bis(dichlorophosphino)methane was converted to a β,γ-methylenetriphosphitylating reagent. The reagent was immobilized on aminomethyl polystyrene resin-bound linker of 4-acetoxy-3phenylbenzyl alcohol to afford a polymer-bound β,γ-methylenetriphosphitylating reagent, which was reacted with unprotected nucleosides followed by oxidation with tert-butyl hydroperoxide, deprotection of cyanoethoxy groups with DBU, and acidic cleavage, to produce 5′-O-β,γ-methylene triphosphate nucleosides in 53-82% overall yields. Among all the compounds, cytidine 5′-O-β,γmethylenetriphosphate inhibited completely RNase H activity of HIV-1 reverse transcriptase at 700 μM. Phosphate transfer is involved in several enzymatic catalyzed reactions 1-3 and therefore is a subject of considerable interest in biological systems. Triphosphate mimics, such as methylenetriphosphates, halogenated methylenetriphosphates, and imidotriphosphates, have been used to probe the mechanism of phosphoryl transfer in enzyme-catalyzed processes 2-4 and to target specific receptors or enzymes that bind or hydrolyze triphosphates. 5-8 Replacement of labile P-O-P bond in nucleoside triphosphates with a stable isosteric P-CH 2-P bond in nucleotide analogs results in enhanced metabolic stability. Synthesis of nonhydrolyzable triphosphate analogs of nucleosides is considered a challenge. A number of solution phase strategies have been previously reported for the synthesis of nucleoside 5′-O-β,γ-methylenetriphosphates including some of the compounds described here by the coupling reactions of nucleoside 5′-monophosphate salt forms or activated nucleoside monophosphates with diphosphonates (methylene diphosphonic acids). Some examples Correspondence to: Keykavous Parang. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary data: Supplementary data including experimental procedures and characterization of resins with IR and final compounds with NMR, high-resolution mass spectrometry, and quantitative phosphorus analysis, enzyme assay procedures can be found in the online version of this article.
Organic & Biomolecular Chemistry, 2010
A polymer-bound α, β-methylene-β-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5′-O-α, β-methylene-β-triphosphates. Among all the compounds, cytidine 5′-O-α, β-methylene-β-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a K i value of 225 μM. Modified nucleoside triphosphates have received much attention as mimics of naturally occurring deoxyribo-and ribonucleoside triphosphates, as probes in several biochemical pathways involving DNA and RNA synthesis, and as potential diagnostic and therapeutic agents. 1,2 The structural similarity of modified nucleotides to natural nucleoside triphosphates make them useful reagents as substrates or inhibitors for DNA or RNA polymerases. 3,4 Although most natural polymerase enzymes incorporate natural nucleoside triphosphates into nucleic acids, there are certain polymerases that are capable of incorporating unnatural nucleoside triphosphates into nucleic acids. 5-7 A number of approaches have been focused on modifications and/or substitution on the base, 8-9 carbohydrate, 10-13 and linear triphosphate moieties 14-17 to design modified nucleotides for diverse applications in nucleic acid and antiviral research. Early in the life cycle of human immunodeficiency virus type 1 (HIV-1), viral RNA is reverse transcribed into double stranded DNA for integration into the genome of the infected cell. 18 This process is catalyzed by reverse transcriptase (RT), a virus encoded heterodimeric enzyme composed of 66 and 51 kD subunits (p66 and p51), possessing DNA polymerase and ribonuclease H (RNase H) activities. 19 DNA polymerase activity is required for the synthesis of RNA: DNA heteroduplex from the single stranded viral RNA. Whereas, RNase H activity † Electronic supplementary information (ESI) available: Experimental procedures, characterization of resins with IR and final compounds with NMR, high-resolution mass spectrometry, and quantitative phosphorus analysis, DNA polymerase assay results.
European journal of medicinal chemistry, 2013
We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogues with antiviral or anticancer activity. Twentyfive symmetrical phosphorodiamidates were synthesised, 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 diasteroisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogues. 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.
Antimicrobial Agents and Chemotherapy, 2011
To investigate the mechanism of the antiviral activity, the active metabolites of HPMPC and HPMPA were studied for their effects on reactions catalyzed by HIV-1 RT. Incorporation of HPMPC and HPMPA into a DNA primer strand resulted in multiple inhibitory effects exerted on the enzyme and showed that neither compound acts as an absolute chain terminator. Further, inhibition of HIV-1 RT also occurred when these drugs were located in the template strand. These results indicate that HPMPC and HPMPA inhibit HIV-1 by a complex mechanism and suggest that this class of drugs has a broader spectrum of activity than previously shown.
Bis(benzoyloxybenzyl)-DiPPro Nucleoside Diphosphates of Anti-HIV Active Nucleoside Analogues
ChemMedChem, 2015
Nucleoside analogues are extensively used as antiviral and anticancer agents. Their efficiency is dependent on their metabolism into the ultimately active nucleoside triphosphates. Often one step or even more in the metabolism of the nucleoside to the triphosphate is inefficient. To overcome this hurdle, prodrugs of the nucleotides are needed. Bis(acyloxybenzyl)nucleoside diphosphates have been reported by us as a first example of an efficient nucleoside diphosphate prodrug (DiPPro nucleotides). Here, the synthesis and the properties of bis(benzoyloxybenzyl)nucleoside diphosphates of the nucleoside analogues d4T and AZT are disclosed. The synthesis was achieved by using a phosphoramidite/oxidation route. In chemical hydrolysis studies, most of the compounds formed a nucleoside diphosphate. This was confirmed in CEM cell extracts, although the prodrug stability in extracts was lower than in phosphate buffer. Furthermore, the stability and the amount of nucleoside diphosphate formed w...