Alain Pompon

Intracellular delivery of nucleoside monophosphates through a reductase-mediated activation process

On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.

Decomposition Pathways of the Mono- and Bis(Pivaloyloxymethyl) Esters of Azidothymidine 5′-Monophosphate in Cell Extract and in Tissue Culture Medium: An Application of the ‘on-line ISRP-Cleaning’ HPLC Technique

Bis(pivaloyloxymethyl) azidothymidine 5′-monophosphate (piv2-AZTMP) was designed as a cell membrane-permeable precursor of AZTMP. We have reported previously that when incubated with CEM cells deficient in thymidine kinase, piv2-AZTMP gives rise to intracellular AZTMP and the corresponding diphosphate (AZTDP) and triphosphate (AZTTP). Under similar conditions, no intracellular nucleotides were formed with AZT. However, the mechanism by which piv2-AZTMP is converted to AZTMP has not been established. To address this question, we have used the recently developed ‘on-line ISRP-cleaning’ HPLC technique to investigate the stability and metabolic fate of piv2-AZTMP (1) in RPMI 1640 medium, (2) in RPMI containing 10% heat-inactivated fetal calf serum, and (3) in CEM cell extracts. Similar studies were conducted starting with mono(pivaloyloxymethyl) azidothymidine 5′-monophosphate (piv2 AZTMP). From the kinetics of these reactions, it appears that piv2-AZTMP is slowly hydrolyzed to piv1-AZTMP in RPMI and that the metabolic sequence in cell extract and in tissue culture medium is clearly: piv2-AZTMP→ piv1AZTMP→ AZTMP→ AZT. The rate constants are quite different in these three media. Although it is evident that the first step in the metabolism of piv2-AZTMP is catalysed by carboxylate esterase, the enzyme(s) responsible for the second step, piv1-AZTMP→ AZTMP, is less apparent, as carboxylate esterases and/or phosphodiesterases can be taken in account. However, analysis of the kinetic data strongly suggests that carboxylate esterase does not play a significant role and that this second step is mediated by phosphodiesterases. Collectively, these studies demonstrate that piv2-AZTMP is an effective prodrug of AZTMP. They also establish that prv1-AZTMP is an intermediate in this process, and define the sequence of the overall metabolic reaction. With this increased understanding of the metabolism of piv2-AZTMP, it should be possible rationally to design analogues with optimal structural and pharmacological properties for use in vivo.

Kinetics study of the biotransformation of an oligonucleotide prodrug in cells extract by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. The oligonucleotide prodrug was fully metabolized to yield the T12 phosphorothioate likely through a carboxyesterase mediated mechanism.

DESIGN OF NEW MONONUCLEOTIDE PRODRUGS: ARYL (SATE) PHOSPHOTRIESTER DERIVATIVES

Synthesis, biological activities and decomposition kinetics of novel phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-tButyl-2-thioethyl (tBuSATE) group and L-tyrosinyl residues are reported. All the derivatives appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments. The proposed decomposition process of these mixed phosphotriesters may involve successively an esterase and then a phosphodiesterase activation.

Anti-HIV Pronucleotides: SATE Versus Phenyl as a Protecting Group of AZT Phosphoramidate Derivatives

We comparatively studied the decomposition pathways in CEM cell extract of several PHENYL phosphoramidate diesters of AZT. A correlation between anti-HIV activities in TK- cell lines and pharmacokinetic data has been observed. This study would help to design corresponding SATE phosphoramidate diesters which revealed potent anti-HIV properties.

Chemical Synthesis of Fully and Partially Xyloadenosine-Substituted 2′, 5′-Oligoadenylates Designed as New Potential Antiviral and Antitumor Agents

Abstract Seven 2′, 5′-oligoadenylate analogues containing 9-β-D-xylofuranosyladenine (xyloaadenosine, XyloA) at the 2′-end or at other positions were synthesized by the phosphotriester method. These new analogues, from dimer to tetramers, exhibit antiproliferative activity which is probably due to their degradation to xyloadenosine units.

Synthesis, Decomposition Pathways and “In Vitro” Evaluation of Bioreversible Phosphotriesters of Azt

Abstract The synthesis, pharmacokinetic data and biological evaluation of a series of phosphotriesters containing S-acyl-2-thioethyl groups as enzyme-labile phosphate protecting groups and AZT as a model are described. A comparison of pharmacokinetic data and “in vitro” experiments show that such bioreversible phosphotriesters of AZT are able to cross cell membranes and deliver the corresponding nucleoside monophosphate inside the cell. Moreover, kinetic data show that modification of the protecting groups can allow to modulate both the extracellular stability of the parent compond and the delivery of nucleoside monophosphate inside the cell.

Study of the Enantioselectivity of Enzymes Involved in Nucleoside Analogue Metabolism: Deoxycytidine Kinase

Abstract The substrate properties of recombinant human deoxycytidine kinase (dCK) with regard to a series of D- or L-enantiomers of cytidine, 2′-deoxycytidine, and 2′,3′-dideoxycytidine analogues were studied using HPLC analysis. Our results suggest that dCK has a remarkably relaxed enantioselectivity with respect to a large number of cytidine derivatives in the β configuration.

The Pronucleotide Approach. III. Synthesis, Anti-HBV Activity and Stability Studies of the Bis(S-pivaloyl-2-thioethyl) Phosphotriester Derivative of Acyclovir

Abstract The nucleoside analog Acyclovir (ACV) is used in the treatment of herpes simplex (HSV) and varicella-zoster (VZV) diseases. The possibility to extend the application field of ACV by using the bis[SATE] pronucleotide approach in order to deliver ACVMP inside the cell was investigated. And actually, the title compound has potent anti-hepatitis B activity in cell culture experiments. Here, we also report its synthesis and stability in various media.

SATE (aryl) phosphotriester series. II. Stability studies and physicochemical parameters

Abstract The stability of phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.