Synthesis and anti-HIV evaluation of 3′-triazolo nucleosides

Article abstract of DOI:10.1080/15257770.2011.580291

A series of hitherto unknown 3′-α-[1,2,3]-substituted triazolo-2′,3′-dideoxypyrimidine nucleoside analogues of the anti-HIV 3′-azido-3′-deoxythymidine (AZT) were synthesized through catalyzed alkyne-azide 1,3-dipolar cycloaddition (Huisgen reaction). Those 3′-[1,2,3]-triazolo analogues bearing an azido alkyl chain were evaluated for their anti-HIV activity against HIV-1 in primary human lymphocytes as well as for their cytotoxicity in different cells. None of them inhibit HIV replication (EC50 > 20 μM); two of them were converted to their triphosphate form to evaluate their HIV-RT inhibition. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.580291

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Synthesis and Anti-HIV Evaluation of 3′-
Triazolo Nucleosides
Vincent Roy a b , Aleksandr Obikhod b , Hong-Wang Zhang b , Steven
J. Coats c , Brian D. Herman d , Nicolas Sluis-Cremer d , Luigi A.
Agrofoglio a & Raymond F. Schinazi b
a Institut de Chimie Organique et Analytique, UMR 6005, Université
d′Orléans, Orléans, France
b Laboratory of Biochemical Pharmacology, Department of
Pediatrics, Emory University School of Medecine and Veterans Affairs
Medical Center, Atlanta, Georgia, USA
c RFS Pharma LLC, Tucker, Georgia, USA
d University of Pittsburgh School of Medicine, Department of
Medicine, Division of Infectious Diseases, Pittsburgh, Pennsylvania,
USA
Version of record first published: 27 May 2011.
To cite this article: Vincent Roy , Aleksandr Obikhod , Hong-Wang Zhang , Steven J. Coats , Brian D.
Herman , Nicolas Sluis-Cremer , Luigi A. Agrofoglio & Raymond F. Schinazi (2011): Synthesis and Anti-
HIV Evaluation of 3′-Triazolo Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 30:4, 264-270
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Nucleosides, Nucleotides and Nucleic Acids, 30:264–270, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.580291
SYNTHESIS AND ANTI-HIV EVALUATION OF 3ꢀ-TRIAZOLO
NUCLEOSIDES
Vincent Roy,1,2 Aleksandr Obikhod,2 Hong-Wang Zhang,2 Steven J. Coats,3
Brian D. Herman,4 Nicolas Sluis-Cremer,4 Luigi A. Agrofoglio,1
and Raymond F. Schinazi2
1Institut de Chimie Organique et Analytique, UMR 6005, Universite´ dꢁOrle´ans, Orle´ans,
France
2Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University
School of Medecine and Veterans Affairs Medical Center, Atlanta, Georgia, USA
3RFS Pharma LLC, Tucker, Georgia, USA
4University of Pittsburgh School of Medicine, Department of Medicine, Division of Infectious
Diseases, Pittsburgh, Pennsylvania, USA
A series of hitherto unknown 3ꢁ-α-[1,2,3]-substituted triazolo-2ꢁ,3ꢁ-dideoxypyrimidine nucle-
2
oside analogues of the anti-HIV 3ꢁ-azido-3ꢁ-deoxythymidine (AZT) were synthesized through cat-
alyzed alkyne-azide 1,3-dipolar cycloaddition (Huisgen reaction). Those 3ꢁ-[1,2,3]-triazolo ana-
logues bearing an azido alkyl chain were evaluated for their anti-HIV activity against HIV-1 in
primary human lymphocytes as well as for their cytotoxicity in different cells. None of them inhibit
HIV replication (EC50 > 20 µM); two of them were converted to their triphosphate form to evaluate
their HIV-RT inhibition.
Keywords 3ꢁ-triazolo nucleosides; CuAAC reaction; “click” chemistry; antiviral activity
Pharmacomodulation has become central to drug discovery and has played
a major role in the research of new treatments for viral infectious diseases.
However, the discovery and process optimization of potential agents is often
slow, expensive and often involves complex synthesis. The “click chemistry”
proposed by Sharpless et al.[1] has emerged as a fast and efficient approach
to simplify compound synthesis. The Huisgen 1,3-dipolar cycloaddition of
azides and terminal alkynes is one of the best known and powerful click re-
actions.[2] This highly specific, irreversible, and chemo-selective reaction is
also termed the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC),
Received 25 February 2011; accepted 7 April 2011.
This work was supported in part by NIH grants 5R37-AI-041980, 4R37-AI-025899, 5P30-AI-50409
(CFAR), and the Department of Veterans Affairs (to R. F. S.).
Address correspondence to Luigi A. Agrofoglio, Institut de Chimie Organique et Analytique, UMR
6005, Universite´ dꢁOrle´ans, 45067 Orle´ans, France. E-mail: luigi.agrofoglio@univ-orleans.fr
264

Synthesis and Anti-HIV Evaluation of 3ꢁ-Triazolo Nucleosides
265
FIGURE 1 Some 3ꢁ-triazolo- or 3ꢁ-azidomethyl nucleosides and target compounds.
which offers exclusively the 1,4-isomer if catalyzed by copper (I).[3] Re-
cently, several research teams have also explored some experiments that
afford the selective 1,5-regioisomer 1,2,3-triazol via the use of azides and
1-bromomagnesium acetylenes[4] or 1-trimethylsilyl acetylenes[5] and palla-
dium[6] or ruthenium[7] as catalyst. The use of 1,3-dipolar cycloaddition has
been recently reviewed by Amblard et al.[8] to enhance the discovery of new
nucleoside analogues. Several teams have reported the synthesis and HIV
evaluation of 3ꢁ-heterocyclic[9] 9 and 3ꢁ-C-branched-azido-chain[10] 10 substi-
tuted 3ꢁ-deoxythimidines, with no antiviral activity. Thus, as part of our drug
discovery program on nucleosides containing a 1,2,3-triazolo moiety,[11]
we report herein a full account of a new generation of 3ꢁ-substituted-2ꢁ,3ꢁ-
dideoxy-nucleoside analogues bearing an azido-alkyl-chain functionalized-
[1,2,3]-triazolo moiety at the 3ꢁ-position (Figure 1) through a CuAAC or
trimethylsilyl-directed cyclo-addition reactions.
In this work, we tried to study the influence of the length of the azido-
alkyl-chain functionalized heterocycle ring and its effect on the regioselec-
tivity (1,4 and 1,5). All synthesized compounds were evaluated for their
anti-HIV activity and their cytotoxicities.
The synthesis of 3ꢁ-substituted deoxynucleosides substituted at the 3ꢁ-
position with an functionalized 1,2,3-triazol, is summarized in Scheme 1.
Starting from the known 5ꢁ-benzoyl-AZT[12] or 5ꢁ-silylated-3ꢁ-azido-2ꢁ,3ꢁ-
dideoxyuridine[13] and commercial alkynes, the 1,3-dipolar cyclo-addition
was catalyzed by sodium ascorbate/CuSO4[14] in a 1/1 mixture water/tBuOH
at room temperature, to afford the regioselectivity (1,4) 2 and 2ꢁa,b,c
with no contamination by the 1,5-regioisomer. Reaction of 1ꢁ with N,O-
bis(trimethylsilyl) acetamide in toluene was stirred at room temperature,
then addition of 3-(trimethylsilyl)propargyl alcohol (110◦C, 16 hours)
gave the 1,4,5-trisubstituted-[1,2,3]-triazole. Cleavage of trimethylsilyl (TMS)
group via with aqueous HF (48%) for 3 hours, provide the 1,5-substituted
triazole 7ꢁa in 26% yield. The regioselectivity of the ligation leading to 1,5
1
or 1,4-disubstitued-[1,2,3]-triazole moiety was confirmed by H, 13C NMR

266
V. Roy et al.
SCHEME 1 Reagents and conditions: (i) sodium ascorbate, CuSO4, alkyne, tert-Butanol/H2O; (ii) a) MsCl,
Et3N, CH2Cl2, 0◦C to room temperature, b) NaN3, DMF; (iii) a) 2,4,6-Triisopropylbenzenesulfonyl
chloride, DMAP, Et3N, CH2Cl2, b) NH4OH; (iv) NH3/MeOH, 3◦C for benzoyl group; TBAF/THF for
silyl group; (v) a) BSA, 3-(trimethylsilyl)propargyl alcohol, toluene, 110◦C, b) 48% HF(aq), 26% for two
steps; (vi) a) MsCl, pyridine, room temperature, 2 hours, b) NaN3, DMF, 85◦C, 3 hours, c) NH3/CH3OH,
49%.
long range correlation spectra (HMBC). Mesylation of free hydroxyl group
followed by treatment with NaN3 afforded azide derivatives 3, 3ꢁa,b,c and 7ꢁa,
respectively.
Several methods have been reported for the conversion of uracil to
cytosine analogues[15] and are based on (1) a first activation of the 4-
carbonyl moiety either (mainly by heating with P2S5,[15a] by tris-(1,2,4-
triazolyl)phosphate,[15b] as o-nitrophenol derivative,[15c] with SOCl2,[15d] or
2,4,6-triisopropylbenzensulfonic chloride[15e]) then (2) a subsequent treat-
ment with ammonia, primary or secondary amine to give the desired N4-
modified cytosine analogues. Thus, uridines 4c and 4ꢁa-c, were reacted with
2,4,6-triisopropylbenzenesulfonyl chloride, Et3N, and DMAP in CH2Cl2,
then treated by a solution of NH4OH. The desired cytidines 5c and 5-
methylcytidines 5ꢁa-c were isolated in good yields.

Synthesis and Anti-HIV Evaluation of 3ꢀ-Triazolo Nucleosides
267
The standard deprotection of benzoyl group with NH3/MeOH and silyl
group with a solution of TBAF/THF, led to the free 3ꢁ-substituted-2ꢁ,3ꢁ-
dideoxynucleoside analogues[16–20] 4a, 4ꢁa, 6c, 6ꢁc, and 8ꢁa.
All final compounds were screened against HIV-1 for their biological
activity. The antiviral[21] assay was performed in peripheral blood mono-
nuclear (PBM) cells and compared to AZT activity. None of the synthesized
compounds showed significant activities and displayed toxic effects on un-
infected PHA-stimulated primary human peripheral blood mononuclear
(PBM), CEM (T-lymphoblastoid cell line), or Vero (African green monkey
kidney) cells.[22]
In order to measure the inhibitory activity of those compounds on HIV-
1 RT DNA polymerase, compounds 4ꢁa and 8ꢁa were converted to their
corresponding triphosphates.[23] The HIV-1 RT inhibition mediated DNA
polymerization on a DNA/DNA T/P is reported in Figure 2. In this exper-
iment, 3ꢁ-azido-3ꢁ-deoxy-thymidine triphosphate (AZT-TP) was included as
a control.[24,25] Unlike AZT-TP, no 3ꢁ-triazole-thymidine triphosphate ana-
logues were incorporated into the nascent DNA chain by wildtype (WT)
HIV-1 RT up to 50 µM. Accordingly, chain-termination and inhibition of
HIV-1 RT were not observed. Those analogues do not inhibit the HIV-1 RT.
The large 3ꢁ-triazole group may prevent proper positioning in the HIV-1 RT
nucleotide binding site, thus preventing efficient incorporation.
In conclusion, we have synthesized several hitherto unknown 3ꢁ-[1,2,3]-
triazolo substituted-2ꢁ,3ꢁ-dideoxy-nucleosides via catalyzed regioselective
azide-alkyne 1,3-dipolar Huisgenꢁs cycloaddition. Two compounds were con-
verted to their triphosphate analogues and the HIV-1 RT inhibition mediated
DNA polymerization on a DNA/DNA T/P realized. The synthesized com-
pounds were evaluated in human PBM cells infected by HIV-1 and none of
them showed significant activities.
FIGURE 2 HIV-1 RT inhibition mediated DNA polymerization on a DNA/DNA T/P.

268
V. Roy et al.
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16. Selected data for compound 4a: 1H NMR (400 MHz, CD3OD): 8.17 (s, 1H), 8.07 (d, J = 8.1 Hz, 1H),
6.47 (t, J = 6.4 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.43 (dt, J = 5.6 and 8.5 Hz, 1H), 4.49 (s, 2H),
4.39 (dt, J = 3.2 and 5.6 Hz, 1H), 3.89 (dd, J = 3.0 and 12.2 Hz, 1H), 3.77 (dd, J = 3.2 and 12.2 Hz,
1H), 2.95 (m, 1H), 2.76 (ddd, J = 6.0, 8.4 and 14.4 Hz, 1H). 13C NMR (400 MHz, CD3OD): 166.24,
152.18, 144.15, 142.65, 124.72, 102.82, 87.19, 86.62, 62.19, 61.26, 46.09, 39.26.
17. Selected data: compound 4ꢁa: 1H NMR (400 MHz, CD3OD): 8.15 (s, 1H), 7.89 (d, J = 1.2 Hz, 1H),
6.45 (t, J = 6.4 Hz, 1H), 5.40 (m, 1H), 4.47 (s, 2H), 4.33 (m, 1H), 3.86 (dd, J = 3.2 and 12.4 Hz,
1H), 3.73 (dd, J = 3.6 and 12.4 Hz, 1H), 2.86 (m, 1H), 2.69 (m, 1H), 1.88 (d, J = 1.2 Hz, 3H). 13
C
NMR (400 MHz, CD3OD): 165.21, 151.10, 142.87, 137.06, 123.49, 110.48, 85.48, 85.16, 60.91, 59.95,
44.82, 37.82, 11.29.
18. Selected data for compound 6c: 1H NMR (400 MHz, CD3OD): 8.09 (d, J = 7.2 Hz, 1H), 7.93 (s, 1H),
6.41 (t, J = 6.4 Hz, 1H), 5.93 (d, J = 7.2 Hz, 1H), 5.35 (dt, J = 5.6 and 8.4 Hz, 1H), 4.39 (dt, J =
3.2 and 6.0 Hz, 1H), 3.89 (dd, J = 3.2 and 12.4 Hz, 1H), 3.76 (dd, J = 3.2 and 12.4 Hz, 1H), 3.37
(t, J = 6.8 Hz, 2H), 2.97 (m, 1H), 2.80 (t, J = 7.6 Hz, 2H), 2.65 (ddd, J = 5.6, 8.4 and 14.0 Hz, 1H),
1.95 (quin, J = 6.8 Hz, 2H). 13C NMR (400 MHz, CD3OD): 167.78, 158.20, 148.34, 142.82, 123.07,
96.12, 87.97, 86.55, 62.08, 60.78, 51.73, 39.90, 29.67, 23.48.
1
19. Selected data for compound 6ꢁc: H NMR (400 MHz, CD3OD): 7.95 (d, J = 0.8 Hz, 1H), 7.93 (s, 1H),
6.43 (t, J = 6.0 Hz, 1H), 5.36 (dt, J = 6.0 and 8.8 Hz, 1H), 4.38 (dt, J = 3.2 and 6.0 Hz, 1H), 3.92
(dd, J = 2.8 and 12.4 Hz, 1H), 3.76 (dd, J = 3.2 and 12.4 Hz, 1H), 3.36 (t, J = 6.8 Hz, 2H), 2.95 (m,
1H), 2.80 (t, J = 7.2 Hz, 2H), 2.65 (ddd, J = 6.0, 8.8 and 14.0 Hz, 1H), 1.99 (d, J = 0.8 Hz, 3H),
1.94 (quin, J = 7.2 Hz, 2H). 13C NMR (400 MHz, CD3OD): 167.43, 158.26, 148.32, 140.21, 123.07,
104.46, 87.67, 86.46, 62.46, 60.69, 51.73, 39.82, 29.68, 23.49, 13.40.
1
20. Selected data for compound 8ꢁa: H NMR (400 MHz, CD3OD): 7.90 (d, J = 1.2 Hz, 1H), 7.78 (s, 1H),
6.58 (t, J = 6.8 Hz, 1H), 5.30 (m, 1H), 4.66 (d, J = 3.6 Hz, 2H), 4.36 (q, J = 2.8 Hz, 1H), 3.85
(dd, J = 3.2 and 11.8 Hz, 1H), 3.74 (dd, J = 3.2 and 12.2 Hz, 1H), 2.79 (m, 1H), 2.66 (m, 1H), 1.90
(d, J = 1.2 Hz, 3H). 13C NMR (400 MHz, CD3OD): 184.63, 151.18, 137.05, 133.40, 133.10, 110.54,
85.97, 85.47, 61.32, 58.38, 41.81, 38.11, 11.29.
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2ꢁ,3ꢁ-dideoxy-2ꢁ,3ꢁ-didehydrocytidine analogues. Antimicrob. Agents Chemother. 2002, 46, 3854–3860.
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25. Wildtype (WT) HIV-1 (LAI) reverse transcriptase (RT) was purified as described previously. The
protein concentration of the purified enzyme was determined spectrophotometrically at 280 nm
using an extinction coefficient (ꢀ280) of 260450 M−1 cm−1, and by Bradford protein assays (Sigma-
Aldrich, St. Louis, MO, USA). AZT-TP was purchased from TriLink Biotechnologies, Inc (San Diego,
CA, USA), dNTPs were acquired from GE Healthcare (Piscataway, NJ, USA), and [γ -32P] ATP was
obtained from PerkinElmer Life Sciences (Boston, MA, USA). DNA oligonucleotides were synthe-
sized by IDT (Coralville, IA, USA). The ability of 3ꢁ-triazole thymidine analogues to inhibit HIV-1
RT DNA synthesis was evaluated using a DNA/DNA template/primer (T/Ps). The sequences of the
T/P substrate are provided in Figure 2, and the DNA primers were 5ꢁ-radiolabeled with [γ -32P]ATP
as described previously.[3] Briefly, reactions were carried out in 50 mM Tris (pH 7.5), 50 mM KCl,
10 mM MgCl2 containing 20 nM T/P, 0.5 µM each dNTP and various concentrations of AZT-TP,

270
V. Roy et al.
RS-467a-TP, or RS-689-TP (0–50 µM). Reactions were initiated by the addition of 200 nM WT RT, in-
cubated for 10 minutes at 37◦C and then quenched using 20 µL of gel loading buffer (98% deionized
formamide containing 1 mg/mL each of bromophenol blue and xylene cyanol). Samples were then
denatured at 95◦C for 10 minutes and polymerization products were separated from substrates by
denaturing gel electrophoresis using 14% acrylamide containing 7 M urea. Gels were analyzed using
phosphorimaging with a GS525 Molecular Imager and Quantity One Software (Biorad Laboratories,
Inc., Hercules, CA, USA).