In search of new inhibitors of HIV-1 replication: Synthesis and study of 1-(2′-deoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide as a selective viral mutagenic agent

Article abstract of DOI:10.1080/15257770701490902

With the emergence of HIV strains resistant or cross-resistant to nearly all antiretroviral regimen, novel therapy approaches have to be considered. As a part of our current work on viral mutagenic compounds, we prepared 1-(2′-deoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2′-deoxy-ribavirin) and its 5′-triphosphate derivative. The nucleoside mutagenic activity was evaluated on HIV-1 NL4-3 in CEMx174 cell culture. After 2.5 months, no reduction on HIV-1 viability was observed. On the other hand, in vitro experiments with purified HIV-1 RT demonstrated that the triphosphate analog can be incorporated opposite to several natural nucleosides. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701490902

Nucleosides, Nucleotides, and Nucleic Acids, 26:743–746, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490902
IN SEARCH OF NEW INHIBITORS OF HIV-1 REPLICATION:
SYNTHESIS AND STUDY OF 1-(2’-DEOXY-β-D-RIBOFURANOSYL)-
1,2,4-TRIAZOLE-3-CARBOXAMIDE AS A SELECTIVE VIRAL
MUTAGENIC AGENT
V. Vivet-Boudou and J.-C. Paillart
Architecture et Re´activite´ de l’ARN, Universite´
2
Louis Pasteur, CNRS, IBMC, Strasbourg, France
A. Burger Laboratoire de Chimie Bioorganique, Universite´ de Nice Sophia Antipolis,
2
Parc Valrose, Nice Cedex, France
R. Marquet Architecture et Re´activite´ de l’ARN, Universite´ Louis Pasteur, CNRS,
2
IBMC, Strasbourg, France
With the emergence of HIV strains resistant or cross-resistant to nearly all antiretroviral regi-
2
men, novel therapy approaches have to be considered. As a part of our current work on viral mu-
tagenic compounds, we prepared 1-(2ꢁ-deoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2ꢁ-
deoxy-ribavirin) and its 5ꢁ-triphosphate derivative. The nucleoside mutagenic activity was evaluated
on HIV-1 NL4-3 in CEMx174 cell culture. After 2.5 months, no reduction on HIV-1 viability was
observed. On the other hand, in vitro experiments with purified HIV-1 RT demonstrated that the
triphosphate analog can be incorporated opposite to several natural nucleosides.
Keywords HIV-1; nucleoside analogues; deoxy-ribavirin; triphosphate analogue
Significant progress has been accomplished in the discovery of new antiviral
agents and therapeutic approaches against human immunodeficiency virus
(HIV) infection. However, none of them is fully effective to eradicate the
virus, due in part to the high capacity of this virus to mutate. However, re-
cent literature data has shown that the high mutation rate observed with
RNA viruses and retroviruses can be transformed into an advantage for the
host (for a general review see.[1] These viruses replicate close to the thresh-
old of error catastrophe and a moderate increase in mutation rate during
proviral DNA synthesis dramatically reduces their infectivity and viability.
This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS), France, and
by Sidaction.
Address correspondence to V. Vivet-Boudou, Architecture et Re´activite´ de l’ARN, Universite´ Louis
Pasteur, CNRS, IBMC, 15 Rue Rene´ Descartes, Strasbourg 67084, France. E-mail: v.vivet@bmc.u-strasbg.fr
743

744
V. Vivet-Boudou et al.
This strategy has been validated against HIV-1 in 1999 with 5-OH-dC[2] and
a molecule (SN 1212) is under phase I clinical trials.[3] Within the frame-
work of our research program, we designed and synthesized several com-
pounds that are able to base pair with a maximum of natural nucleosides
in order to create hyper-variability in proviral DNA.[4] Based on the antivi-
ral activity reported for ribavirin[5] we decided to evaluate the mutagenic
potency of its 2ꢁ-deoxy-analog, 1-(2ꢁ-deoxy-β-D-ribofuranosyl)-1,2,4-triazole-
3-carboxamide 3. Here, we report the preparation of the nucleoside analog
3 and its 5ꢁ-triphosphate 4 as well as the preliminary results of their evalua-
tion as mutagenic compounds.
From a synthetic viewpoint, the 1-(2ꢁ-deoxy-β-D-ribofuranosyl)-1,2,4-
triazole-3-carboxamide 3 was prepared by the strategy described in L-
series.[6] However, reagents for the radical deoxygenation were slightly mod-
ified to improve the global yield from 10.5 to 50% (Scheme 1). In our
protocol, the 3ꢁ- and 5ꢁ-OH of commercially available ribavirin were selec-
tively protected by the bidentate reagent 1,3-dichloro-1,1,3,3-tetraisopropyl-
1,3-O-disiloxane (TIPSiCl2) to give compound 1 in 94% yield. Then, the
2ꢁ-hydroxyl group was treated with O-phenyl chlorothionoformate. The
crude material was deoxygenated with tris(trimethylsilyl)silane in the pres-
ence of AIBN in dioxane to give compound 2. A change of silane reagent
((Me3Si)3SiH was used instead of Ph2SiH2[6]) increased the yield from 31
to 68% for the two steps. Finally, pure compound 3 was obtained after
elimination of the protecting group with triethylamine trihydrofluoride in
dichloromethane.
SCHEME 1 Chemical synthesis of 2ꢁ-deoxyribavirin and its 5ꢁ-triphosphate derivative.

New Inhibitors of HIV-1 Replication
745
The triphosphate derivative (dRTP) 4 was prepared without further pro-
tection of the 3ꢁ-hydroxyl of compound 3 by the ”one pot” protocol initially
developed by Ludwig[7] and more recently described in an online free ac-
cess patent.[8] Nucleoside 3 was treated with phosphorous oxychloride in
triethyl phosphate for 1 h 30 at 0◦C then tributylamine and a 0.5M solution
of bis-tri-n-butylammonium pyrophosphate in dry DMF were added. After 1
min, the reaction was quenched with TEAB 1M and the mixture was stirred
at room temperature for 10 min. The crude triphosphate was easily purified
by HPLC chromatography on C18 Zorbax SB column with TEAB 0.5M as
eluant. The final compound was converted to its sodium salt by exchange
on Dowex 50WX2 resin.
The 2ꢁ-deoxy-ribavirin (dR) was tested for its potency to reduce HIV-1
replication. HIV-1 NL4-3 virions were cultured on CEM × 174 cell lines in
the presence or absence of dR. Replication (RT activity) has been moni-
tored every 4 days. Over a period of 2.5 months, we did not detect any dif-
ference of virus replication in the presence or absence of 1mM dR. The lack
of activity can possibly be attributed to a lack of nucleoside phosphorylation
by cellular kinases, or the triphosphate dRTP is not substrate of RT. Alter-
natively, once incorporated into nascent DNA, deoxyribavirin is not able
to induce mutations in viral genome or induces silent mutations. In order
to answer the last question, the sequence analysis of genes obtained at the
end of the ex vivo experiments is underway. We also used the nucleoside 5ꢁ-
triphosphate (dRTP) to check its incorporation into the DNA primer strand
by purified reverse transcriptase (RT). We found that dRTP is introduced by
RT in front of the 4 natural bases with an efficiency such as: dR :dU ꢂ
∼
dR :dG dR :dC > dR :dA. To complete this study, incorporation efficiency
=
of dRTP versus natural dNTP (Kpol/Kd) will be evaluated. We also plan to
synthesize oligonucleotides modified with dR in order to determine the in-
corporation of natural dNTP opposite dR when the modification is placed
in the template strand and to measure the melting temperature of duplexes
including a mismatch (dR:dU, dR:dC, dR:dG, or dR:dA).
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