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.     

The kinetics of the alkaline hydrolysis of phosphotriesters in DNA.

The degradation in alkali of normal DNA and DNA alkylated with dimethyl sulphate (DMS), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) has been investigated using analytical ultracentrifugation techniques. For control T7-DNA (w.st. denatured form 12.5 - 10(6) daltons) the rate of degradation at 37 degrees varies from 0.14 breaks/molecule/h in 0.1 M NaOH to 1.2 breaks/molecule/h in 0.4 M NaOH. When DNA is alkylated with reagents known to produce phosphotriesters addition of alkali leads to an initial rapid degradation not observed with control DNA. Ethyl phosphotriesters are hydrolysed at about half the rate of methyl phosphotriesters. Approximately one third of the methyl or ethyl phosphotriesters present hydrolyse to give breaks in the DNA chain.     

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

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.     

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

Neighbouring group participation in the unblocking of phosphotriesters of nucleic acids.

Two examples of neighbouring group participation during the removal of protecting groups from phosphotriesters of partially or fully protected intermediates of nucleic acids are presented. The first example shows that ammonolysis of aryl groups from phosphotriesters of partially protected - 5'- hydroxy free - nucleic acids (e.g., 4b approximately to; Ar=2C1C 6H4) gives rise to the formation of unnatural nucleic acids (e.g., 7 approximately to and 8 approximately to). The second one illustrates that fluoride ion promoted hydrolysis of 2,2,2-trichloroethyl groups from phosphotriesters of fully protected nucleic acids (e.g., 18a approximately to), having t-butyldimethylsilyl groups at the 2'-positions, leads to the formation of a considerable amount of side-products (e.g., 20 approximately to and 21 approximately to).     

SATE (aryl) phosphotriester series. I. Synthesis and biological evaluation

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.     

New Series of Mixed Pronucleotides. Synthesis and Anti-HIV Activities of Mononucleoside Phenyl SATE Phosphotriesters

Abstract

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.     

Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli.

The E. coli ada+ gene product that controls the adaptive response to alkylating agents has been purified to apparent homogeneity using an overproducing expression vector system. This 39 kDa protein repairs 0(6)-methylguanine and 0(4)-methylthymine residues in alkylated DNA by transfer of the methyl group from the base to a cysteine residue in the protein itself. The Ada protein also corrects one of the stereoisomers of methyl phosphotriesters in DNA by the same mechanism, while the other isomer is left unrepaired. Different cysteine residues in the Ada protein are used as acceptors in the repair of methyl groups derived from phosphotriesters and base residues.     

Phosphotriesters in rat liver deoxyribonucleic acid after the administration of the carcinogen NN-dimethylnitrosamine in vivo.

After treatment with NN-di[14C]methylnitrosamine, samples of DNA were isolated from rat livers by a conventional phenol procedure and examined for the presence of phosphotriesters. A method of capable of detecting relatively small amounts of 14C-labelled phosphotriesters was developed and used to establish that these products account for 10-12% of the total methylation pattern found after treatment with this agent in vitro. The significance of the presence of phosphotriesters in DNA is discussed.     

Synthesis of the tBuSATE pronucleotide of AZT by two different synthetic approaches.

A large scale synthesis of the tBuSATE pronucleotide of AZT was required for in vivo studies. A comparative synthesis of this derivative by phosphoramidite and monophosphate approaches is reported.     

Prooligonucleotides exhibit less serum-protein binding than phosphodiester and phosphorothioate oligonucleotides

The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.     

Studies on the stability of trialkyl phosphates and di-(2'deoxythymidine) phosphotriesters in alkaline and neutral solution. A model study for hydrolysis of phosphotriesters in DNA and on the influence of a beta hydroxyethyl ester group

Various trialkyl phosphates were investigated as model compounds for DNA-phosphotriesters for their stability in neutral or alkaline conditions. The results show that phosphotriesters were highly stable even at strongly alkaline pH, with the exception of diethyl 2-hydroxyethyl phosphate (DHP). The extreme instability of the latter was found to be due to the 2-hydroxy function. In accordance with earlier interpretations the 2-hydroxyethyl group is proposed to participate in the formation of a highly reactive dioxaphospholane ring intermediate which decays rapidly by hydrolysis. Alkylation of 3'- and 5'-deoxythymidine monophosphates with methyl- or hydroxyethylnitrosourea (MNU, HENU) results in practically exclusive phosphate alkylation. In analogy with the model phosphotriesters, di(2'-deoxythymidine) phosphotriesters generated after reaction with MNU or HENU showed extreme dependence of their stabilities on the nature of the alkyl group transferred to phosphate. Whereas the methyl phosphotriester was highly stable, the corresponding hydroxyethyl analogue showed half lives of decay of less than 1 min (pH 12.5), 27 min (pH 9.1) and 60 min (pH 7). Thus the introduction of a 2-hydroxyethyl function into phosphate strongly decreases the stability of the phosphate link of DNA, resulting in DNA single strand breaks, in analogy to RNA phosphotriesters which have been found earlier to be highly unstable because of the presence of the ribose 2'-OH-group.     

Phosphotriester formation by the haloethylnitrosoureas and repair of these lesions by E. coli BS21 extracts.

The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers. Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation. A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA. An extract of E. coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.     

Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5′-monophosphates, and their efficiency correlated well with the pK_a values of the aryloxy groups present.     

The stability of methyl and ethyl phosphotriesters in DNA in vivo

C57BL male mice were injected with N-methyl-N-nitrosourea (MNUA) or N-ethyl-N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5-20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t1/2 = 2-3 days. During the multiple injections the observed t1/2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t1/2 = 10-15 weeks. Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.     

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.     

Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract.

The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract.     

Synthesis and anti-HIV activity of some S-acyl-2-thioethyl (SATE) phosphoramidate derivatives of 3'-azido-2',3'-dideoxythymidine.

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.     

DNA-synthesis with methylated poly(dA-dT) templates: possible role of O4-methylthymine as a pro-mutagenic base.

The alternating copolymer poly(dA-dT) has been methylated with either dimethyl sulphate (DMS) or N-methyl-N-nitrosourea (MNU) and the levels of the various methylation products determined. In addition to the methylated adenines formed by both methylating agents, MNU resulted also in the formation of 3-methylthymine, O4-methylthymine and phosphotriesters. The methylated polymers have been ution of complementary and non-complementary nucleotides determined. With the DMS methylated template no wrong nucleotide incorporation was detectable, but with the MNU methylated polymer the incorporation of dGMP was observed. The amount of dGMP incorporated correlated with the level of O4-methylthymine in the template over the range of methylation studied. The results indicate that O4-methylthymine is capable of miscoding on a one-to-one basis while the products of DMS methylation (1-, 3- and 7-methyladenines), and also possibly the phosphotriesters, do not lead to any misincorporation.     

Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate

Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents. To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters. A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue. The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD). Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules. This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA. Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer. The interactions of two enzymes with the alkylated oligonucleotides were examined. First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated. Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters. Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness. The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.