Alexander A. Krayevsky

Design of anti-HIV compounds: from nucleoside to nucleoside 5'-triphosphate analogs. Problems and perspectives.

To date, human immunodeficiency virus infection remains incurable although a variety of antiviral agents have been identified and characterized. Even though nucleoside analogs have been the most successful prodrugs, there remains the need to develop new compounds that exhibit a more favorable toxicity profile, less susceptible to cross-resistance, and greater efficacy. As prodrugs, the nucleoside analogs should be sequentially phosphorylated by cellular kinases to yield triphosphate form before they can inhibit HIV replication at the reverse transcriptase level. The efficiency of phosphorylation of nucleoside analogs is a key factor in their antiviral activity and strongly depends on nucleoside structure and cell type. In recent years, several attempts have been made to improve therapeutic potential of nucleoside analogs by the use of nucleotide prodrugs (pronucleotides), that can avoid the first step of phosphorylation. This review focuses on problems of intracellular phosphorylation of nucleoside analogs and perspectives of developing of a new class of nucleotide analogs modified at phosphate group as a form for the delivery of nucleotide analogs into the cell.

Properties of 2′,3′-dideoxy-2′,3′-dehydrothymidine 5′-triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases

2′‐3′‐Dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase β, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3′‐termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase α. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.

Synthesis and antiviral activity of some fluorinated nucleotide derivatives

Abstract A number of 3′-fluoro-3′-deoxythymidine 5′-phosphonates and nucleoside 5′-phosphorofluoridates were prepared to study their ability to inhibit replication of HIV-1. Compounds, the 5′-phosphorofluoridates of 3′-azido-3′-deoxythymidine (VIIIc), 3′-fluoro-3′-deoxythymidine (VIIId) and 3′-deoxy-2′,3′-didehydrothymidine (VIIIe), exhibit potent anti-HIV-1 activities.

Modified triphosphates of carbocyclic nucleoside analogues: Synthesis, stability towards alkaline phosphatase and substrate properties for some DNA polymerases

Abstract New triphosphate derivatives of carbocyclic nucleoside analogues have been synthesized and shown to be potent substrates for terminal deoxynucleotidyltransferase and/or HIV reverse trascriptase; the compounds are stable to dephosphorylation with human placental alkaline phosphatase.

dNTP modified at triphosphate residues: substrate properties towards DNA polymerases and stability in human serum.

Substrate and terminating substrate properties of dNTP with phosphate groups replaced by phosphonates at alpha-, gamma-, beta, gamma-, and alpha, beta, gamma-positions towards different human DNA polymerases and retroviral reverse transcriptases are reviewed. Substitution of the phosphate group by the phosphonate at any of the three phosphate positions of dNTP increased their stability towards dephosphorylating enzymes of human blood. In some cases hydrophobicity of these compounds was markedly enhanced.

New modified nucleoside 5′-triphosphates: synthesis, properties towards DNA polymerases, stability in blood serum and antiviral activity

A series of new nucleoside 5′-triphosphate mimetics, 2, 3, 5, 6, 8–10, modified at the glycone and all three phosphate residues, have been synthesised and studied. These compounds only bear the enzymatically labile anhydride bond between the α and β phosphorus atoms. The preparative chemistry involved the preparation of phosphonic salts 30, 31 and 32 and coupling of these species to the morpholidate 33. The mechanism of formation of some of the intermediates ‘en route’ to 27 and 28 is discussed. All of the target compounds demonstrated high stability in human blood serum with half lives towards hydrolysis of up to 4.5 days. Some of these nucleoside triphosphonates have been shown to be selective inhibitors of DNA synthesis catalysed by retroviral reverse transcriptases and terminal deoxynucleotidyl transferases. They inhibited replication of the artificial virus containing Moloney murine leukemia virus reverse transcriptase in infected cell culture, probably due to the inhibition of a reverse transcription step of a genomic RNA. Compared to the triphosphonates, the corresponding monophosphonates demonstrated decreased antiviral activity by 1–2 orders of magnitude. This implies that the triphosphonates inhibit virus replication directly, rather than by a two-step mechanism based on their hydrolysis to the monophosphonates and subsequent intracellular diphosphorylation. Being totally independent of the enzymatic phosphorylation pathways of the host cell, the compounds under study may also be able to inhibit retrovirus reproduction both in kinase deficient cell lines and in the intercellular blood media.

Inhibition of reverse transcription in rat liver intracisternal A-particles by thymidine derivatives.

The thymidine derivatives araAzT, dTTP(3′N3), TTP(3′NH2), and araTTP(3′N3), were studied as inhibitors of the reverse transcription taking place within endogenous retroviral A‐type particles, where retroviral RNAs served as templates and primers. dTTP(3′N3) was shown to be the most efficient inhibitor of retroviral particle reverse transcription. Termination of DNA chain elongation is the basic mechanism of the inhibitory action of dTTP(3′N3). The compound has a very low inhibitory effect on mammalian DNA‐dependent DNA polymerases α, β and γ.