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Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'- dideoxythymidine) are reported herein. These mononucleotide prodrugs (pronucleotides) are characterized by the presence of polar (amino or hydroxyl) functions on the SATE biolabile phosphate protections. Whereas pronucleotides incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moiety confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, derivative 2, was able to cross a Caco-2 cell monolayer mainly in intact form, probing that its further development is warranted as a possible HIV-pronucleotide candidate.

The synthesis, anti-HIV activity and stability studies of a H-phosphonamidate derivative of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a N,N-diisopropylamino residue as first model of alkylamino group are reported. The results demonstrate that such phosphorylated structure exerts its biological effects via chemical hydrolysis into the corresponding H-phosphonate, precursor of the parent nucleoside.

The synthesis and the study of two phosphorothiolate derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and glucosyl residues associated to the phosphorus atom by a 2-oxyethyl link, are reported. These derivatives could be considered as prototypes of a new series of nucleotide prodrugs (pronucleotides).

Synthesis and biological activities of several phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and aryl residues derived from L-tyrosine are reported. All compounds showed marked anti-HIV activity in thymidine kinase-deficient CEM cells demonstrating their ability to deliver intracellularly the parent 5'-mononucleotide.

The synthesis, in vitro anti-HIV activity and stability studies of the 5'-fluorophosphate derivative of 3'-azido-3'-deoxythymidine (AZT) are reported. The results support the hypothesis that this phosphorylated entity exerts its biological effect via the delivery of the corresponding 5'-mononucleotide through an enzymatic process. However, the antiviral evaluation in thymidine kinase-deficient CEM cells as well as the stability studies in culture medium and cell extract showed that this bioconversion is not specific to the intracellular medium. Attempts to improve the biological activity of mononucleoside 5'-fluorophosphates by the use of the S-pivaloyl-2-thioethyl (tBuSATE) group as biolabile phosphate protection are reported., (Copyright 2001 Elsevier Science (USA).)

We report the synthesis, in vitro anti-HIV-1 activity and stability study of a mononucleoside phosphotriester derivative of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a new biolabile phosphate-protection, namely S-pivaloyl-2-thioisopropyl (tBuSATP). This transient protection was characterized by the presence of a methyl substituent at the Calpha-position of the previously described S-pivaloyl-2-thioethyl (tBuSATE) group. Results demonstrated that the new phosphotriester entity was able to deliver selectively the corresponding 5'-mononucleotide within the infected cells. The introduction of a methyl group at the Calpha-position of the tBuSATE chain decreased the rate of this delivery.

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.

The in vitro anti-HIV activity, stability, and potential for oral absorption of a phosphotriester derivative of AZT (zidovudine; 3'-azido-2',3'-deoxythymidine) bearing a new esterase-labile S-acyl-2-thioethyl (SATE) group as transient phosphate protection are reported. The biolabile protection is characterized by the presence of a hydroxyl function in the acyl chain. In accordance with previously reported data in the bis(SATE) prodrug series, the present results demonstrate that the studied bis(hydroxytBuSATE)phosphotriester exerts its biological effects via intracellular delivery of the 5'-monophosphate of AZT. The hydroxyl function confers a high resistance against esterase hydrolysis, and the studied prodrug is able to cross the Caco-2 cell monolayers in intact form, suggesting that its further development as a possible anti-HIV pronucleotide candidate is warranted., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:448-463, 2001)

The synthesis and biological activities of phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a phenyl group or L-tyrosinyl residues are reported. The target compounds were obtained via either P(V) or P(III) chemistry from the appropriate aryl precursors. All the derivatives were evaluated for their in vitro anti-HIV activity, and they appeared to be potent inhibitors of HIV-1 replication in various cell culture experiments, with EC(50) values between the micro- and nanomolar range. Furthermore, compounds incorporating an amino- and/or acid-substituted tyrosinyl residue demonstrated significant anti-HIV effects in thymidine kinase-deficient (TK(-)) cells showing their ability to act as mononucleotide prodrugs. The proposed decomposition process of these mixed mononucleoside aryl phosphotriesters may involve esterase activation followed by phosphodiesterase hydrolysis.

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

A simple and efficient protocol for the preparation of various symmetrical dinucleoside phosphoramidates derived from AZT, is presented. It consists of the phosphonylation of AZT with phosphonic acid in the presence of DCC to produce the symmetrical H-phosphonate diester, followed by its oxidative conversion to various phosphoramidate analogues. The synthesized compounds were evaluated for their anti-HIV activity in different cell cultures.

Phosphate and H-phosphonate derivatives of anti-HIV nucleoside analogues (AZT and d4T) were prepared as potential prodrugs of the bio-active free nucleotide and they were evaluated for their inhibitory effects on the replication of HIV-1 in several cell culture systems. One compound exhibited an important anti-HIV-1 activity and proved to be significantly more efficient than the parent nucleoside.

The synthesis and in vitro anti-HIV activity of tBuSATE phosphoramidate derivatives of AZT incorporating several methyl-esterified alpha-amino acids are reported. The biological evaluation strongly supports the hypothesis that such compounds exert their anti-HIV effects via intracellular delivery of the corresponding 5'-mononucleotide.

The synthesis, in vitro anti-HIV activity, and stability studies of AZT 5'-fluorophosphate (F-AZTMP) are reported. The present results demonstrate that such compound is a bioprecursor of its parent 5'-mononucleotide (AZTMP) but its biotransformation does not allow its selective intracellular delivery. Moreover, several attempts were carried out in order to improve the biological activity of this compound by the use of a SATE prodrug strategy.

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.

The synthesis and anti-HIV activities of phenyl S-pivaloyl-2-thioethyl (tBuSATE) phosphotriesters of AZT and d4T are reported. These compounds show similar activity compared to bis(tBuSATE) phosphotriesters and appear to be able to deliver the corresponding 5'-mononucleotides inside the cells.

MonoSATE aryl phosphotriesters of AZT are able to deliver intracellularly the corresponding 5'-mononucleotide. This process requires activation by an esterase followed by a phosphodiesterase. This finding opens the way to the design of new pronucleotide series.

The purpose of the present study was to compare the decomposition pathways in CEM cell extracts of various phenyl phosphoramidate derivatives of AZT. In addition, the structures of their metabolites were identified. Correlations with their anti-HIV activities in a thymidine kinase deficient (TK-) CEM cell line have been established with a rationale of designing phosphoramidate pronucleotides capable of delivering intracellularly their respective 5'-nucleoside monophosphate derivatives.

The synthesis of a range of di- and triester derivatives of phosphonoformate (PFA; foscarnet) as potential lipophilic, membrane-soluble prodrugs is described. In addition to normal alkyl esters in the carboxylate and phosphonate residues of PFA, the bioreversible S-(pivaloyl)thioethyl (t-butyl-SATE) group was introduced in an attempt to deliver PFA after bioactivation inside the cells. Furthermore, PFA-AZT conjugates were prepared in order to develop combinational drugs. The key synthetic step was in all cases the formation of the P-C bond to build up the different PFA esters. In contrast to the diester derivatives, the triesters of PFA showed high hydrolytic instability during chromatographic purification. The compounds were evaluated in vitro for their ability to inhibit viruses in several tissue culture systems. All PFA alkyl di- and triesters proved poorly active or inactive against human immunodeficiency virus (HIV) and inactive against hepatitis B virus. In contrast, the PFA-AZT conjugates exhibited significant anti-HIV activity. However, this activity was nearly completely lost in thymidine kinase-deficient cells, suggesting a fast unselective chemical hydrolysis of the conjugates to yield the nucleoside analogue AZT in the cell culture medium. Furthermore, no synergistic effect of PFA and AZT was observed.

The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.

It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway.

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.

The monomeric and symmetrical dimeric 5'-hydrogenphosphonate derivatives of AZT were prepared and evaluated for their inhibitory properties against HIV-1 in several cell lines. The synthesis of the compounds was achieved by reaction of AZT with in situ prepared phosphorus tris(imidazolide) or with phosphonic acid in the presence of pivaloyl chloride. The two title compounds showed in vitro anti-HIV activity similar to (but not better than) that of AZT in three cell lines which were not deficient in thymidine kinase. On the other hand they were inactive in CEM-TK- cells. Pharmacokinetic studies in several media corroborate the assumption that these compounds must not be considered as 'true antiviral agents', but that they act by releasing their nucleoside entity.