AZT 5'-Phosphonates: Achievements and Trends in the Treatment and Prevention of HIV Infection.

Despite the numerous drawbacks, 3'-azido-3'-deoxythymidine (AZT, Zidovudine, Retrovir) remains one of the key drugs used in the treatment and prevention of HIV infection in both monotherapy and HAART. A strategy in searching for new effective and safe AZT agents among latent (depot) forms of AZT has yielded its first positive results. In particular, the sodium salt of AZT 5'-H-phosphonate (Nikavir, phosphazide) has demonstrated clinical advantages over parent AZT: first and foremost, lower toxicity and better tolerability. It can be effectively used for the prevention of vertical transmission from mothers to babies and as an alternative drug for HIV-infected patients with low tolerance to Zidovudine. Preclinical studies of another phosphonate, AZT 5'-aminocarbonylphosphonate, have demonstrated that it releases AZT when taken orally. Pharmacokinetic studies have shown a prolonged action potential. Based on the analysis of both toxicological and pharmacological data, AZT 5'-aminocarbonylphosphonate has been recommended for clinical trials.

Aryl-containing esters of triphosphoric acid as substrates of terminal deoxynucleotidyl transferase.

A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.

[New derivatives of alkyl- and aminocarbonylphosphonic acids containing 3'-azido-3'-deoxythymidine].

New 5'-phosphonates of 3'-azido-3'-deoxythymidine were synthesized and shown to be low-toxic and markedly active in MT-4 cell cultures infected with HIV-1.

[The study of coupling agents for the phosphorylation of 3'-azido-3'-deoxythymidine with (P32)orthophosphoric acid].

The kinetics of 3'-azido-3'-deoxythymidine phosphorylation with [32P]orthophosphoric acid was studied in the presence of various coupling agents. The most effective method with the use of BrCN provided the isolation of the target 3'-azido-3'-deoxythymidine 5'-[32P]monophosphate in 46% yield and a high specific radioactivity (>100 Ci/mmol).

[New phosphonoformic acid derivatives of 3'-azido-3'-deoxythymidine]. / Novye proizvodnye 3'-azido-3'-dezoksitimidina i fosfonomurav'inoi kisloty.

New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of some of the compounds exceeded that of the parent AZT due to their higher antiviral activity. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Telomerase-dependent reactivation of DNA synthesis in macrophages implies alteration of telomeres.

In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2'-O-methyl-RNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors.

Modified dinucleoside tetraphosphonates, new potential inhibitors of HIV reverse transcriptase.

New gamma-substituted analogues of dNTP were synthesized and their enzymatic stability and antiviral properties were evaluated.

Anti-HIV activity of novel phosphonate derivatives of AZT, d4T, and ddA.

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.

Synthesis of [bis(inosine-5')]-tetraphosphate and [bis(inosine-5')]-pentaphosphate analogues bearing the residues of methylenediphosphonic acid.

Various methods of synthesis of metabolically stable phosphonate analogues of bisnucleoside oligophosphates containing two residues of methylenediphosphonic acid in the oligophosphate chain are studied. Phosphonate analogues of Ip4I and Ip5I are prepared.

P-(alkyl)-nucleoside 5'-hydrogenphosphonates as depot forms of antiviral nucleotide analogues.

P-(Alkyl)esters of AZT 5'-hydrogenphosphonate were synthesized and their stabilities in the phosphate buffer and human serum were evaluated. The esters bearing residues of primary and secondary alcohols were degraded to give AZT, whereas those containing tertiary alkyl groups yielded AZT 5'-hydrogenphosphonate. The corresponding derivatives of d2A and d4T showed the same properties.

Reasons and limits of substrate activity of modified L-dNTP in DNA biosynthesis.

Theoretical and experimental analysis of interaction of modified D- and L- dNTP as substrates for template-dependent and template-independent DNA polymerases was performed. It is shown that if the modified nucleoside 5'-triphosphates do not contain a substituent in position 3' DNA chains can be extended by both strereoisomeric series with the same kinetic parameters. But the presence of even a 3'-hydroxy group in L-dNTP prevents their incorporation into the DNA chain.

Effect of triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity towards various DNA polymerases.

Some natural and glycon-modified dNTPs with beta,gamma-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases alpha and beta, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of beta,gamma-methylenediphosphonate substituted dTTP and 3'-azido-3'-deoxy-dTTP decreased in the following order: CF2 = CHF > CBr2 > CFMe >> CH2. This order is individual for each DNA polymerase. It is interesting to mention that beta,gamma-CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase beta. This specificity distinguishes DNA polymerase beta from other DNA polymerases studied.

Modified substrates of DNA polymerases and design of antivirals.

The results obtained in our laboratory on investigating of substrate properties of a large number of compounds towards different DNA polymerases have been summarized. On the basis of systematic analysis a directed synthesis of nucleotides with antiviral properties was performed.

[Study of reverse transcriptase selectivity. Substrate properties of new 2',3'-unsaturated acyclic nucleotide analogs]. / Izuchenie selektivnosti obratnykh transkriptaz. Substratnye svoistva novykh 2',3'-nenasyshchennykh atsiklicheskikh nukleotidnykh analogov.

A new series of nucleotide analogs, (Z)-pyrophosphoryl (phosphonyloxymethyl) but-2-enyl derivatives of pyrimidines and purines, was synthesized. Their substrate and inhibitory properties toward some DNA polymerases and reverse transcriptases were evaluated. They were shown to be selective inhibitors of HIV reverse transcriptase. The structure-substrate properties relationships for nucleotide analogs were discussed.

Novel acyclic nucleotides and nucleoside 5'-triphosphates imitating 2',3'-dideoxy-2',3'-didehydronucleotides: synthesis and biological properties.

A series of pyrophosphoryl (Z)-(phosphonomethoxy)but-2-enyl derivatives of pyrimidines and purines 9a-d and the corresponding phosphonates 10a-d were synthesized. The prepared compounds contain the phosphonate group as an alpha-phosphate mimic as well as an acyclic residue emulating the sugar moiety in 2',3'-dideoxy-2',3'-didehydronucleoside 5'-triphosphates known as highly potent chain terminators of DNA polymerases. Phosphonates 10a-d were obtained by alternative alkylations of the nucleic bases followed by condensation with ethyl [[(p-tolylsulfonyl)oxy]methyl]phosphonate. Pyrophosphorylation of 10a-d afforded phosphonate diphosphates 9a-d. Their substrate properties were evaluated in cell-free systems containing various DNA polymerases including viral reverse transcriptases. Compounds 9a-d manifested good terminating substrate properties toward HIV-1 and AMV reverse transcriptases. They exhibited high selectivity and were not recognized by human DNA polymerases alpha and epsilon, DNA polymerase beta from rat liver, Escherichia coli DNA polymerase I, and HSV-1 and CMV DNA polymerases. Phosphonates 10b-d displayed no activity in HIV-1-infected MT-4 cells cultures; 10a was moderately effective (ED50 = 9 microM).