Design, synthesis, and biological evaluation of new 2′-deoxy- 2′-fluoro-4′-triazole cytidine nucleosides as potent antiviral agents

Article abstract of DOI:10.1016/j.ejmech.2013.02.042

A series of 4′-[1,2,3]triazole-2′-deoxy-2′-fluoro-β- d-arabinofuranosylcytosines (9-17) were prepared by Cu(I)-mediated [3 + 2] cycloaddition reactions (CuAAC) of 1-(4′-azido-2′-deoxy-2′- fluoro-β-d-arabinofuranosyl)cytosine (1) with appropriate alkynes in good yields. Their structures were fully established by ¹H NMR, ¹³C NMR, HRMS, and elemental analysis. Most of these nucleoside analogs exhibited potent anti-HIV-1 activity with no cytotoxicity observed at the highest tested concentration up to 25 μM. Among them, compounds 9, 10 and 13 exhibited extremely potent antiviral activity, thus had a great potential for further development as novel nucleoside reverse transcriptase inhibitors (NRTIs) for the treatment of HIV-1 infection. Besides, the anti-HBV activity of compounds 10, 11 and 17 had been investigated.

Full text of DOI:10.1016/j.ejmech.2013.02.042

Accepted Manuscript
Design, synthesis, and biological evaluation of new 2'-deoxy-2'-fluoro-4'-triazole
cytidine nucleosides as potent antiviral agents
Jie Wu, Wenquan Yu, Leixia Fu, Wu He, Yao Wang, Baoshan Chai, Chuanjun Song,
Junbiao Chang
PII:
S0223-5234(13)00189-X
10.1016/j.ejmech.2013.02.042
EJMECH 6067
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 January 2013
Revised Date: 26 February 2013
Accepted Date: 28 February 2013
Please cite this article as: J. Wu, W. Yu, L. Fu, W. He, Y. Wang, B. Chai, C. Song, J. Chang, Design,
synthesis, and biological evaluation of new 2'-deoxy-2'-fluoro-4'-triazole cytidine nucleosides as potent
antiviral agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.02.042.
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ACCEPTED MANUSCRIPT
Graphic abstract: pictogram

ACCEPTED MANUSCRIPT
•A series of 2'-deoxy-2'-fluoro-4'-triazole cytidine nucleosides were prepared.
•Compounds 9-17 were obtained via Cu(I)-mediated [3 + 2] cycloaddition reactions.
•Compounds 9, 10 and 13 exhibited extremely potent antiviral activity.
•No cytotoxicity observed at the highest tested concentration up to 25 µM.
•A preliminary structure–activity relationship was obtained.

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Design,
synthesis,
and
biological
evaluation
of
new
2'-deoxy-2'-fluoro-4'-triazole cytidine nucleosides as potent antiviral
agents
Jie Wua, Wenquan Yua, Leixia Fua, Wu Hea, Yao Wanga, Baoshan Chaia, Chuanjun Songa and
Junbiao Changa
a
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001,
PR China
Abstract
A series of 4'-[1,2,3]triazole-2'-deoxy-2'-fluoro-β-D-arabinofuranosylcytosines (9–17) were
prepared by Cu(I)-mediated [3
+
2] cycloaddition reactions (CuAAC) of
1-(4'-azido-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)cytosine (1) with appropriate alkynes in
1
good yields. Their structures were fully established by H NMR, 13C NMR, HRMS, and
elemental analysis. Most of these nucleoside analogs exhibited potent anti-HIV-1 activity with no
cytotoxicity observed at the highest tested concentration up to 25 ꢀM. Among them, compounds
9, 10 and 13 exhibited extremely potent antiviral activity, thus had a great potential for further
development as novel nucleoside reverse transcriptase inhibitors (NRTIs) for the treatment of
HIV-1 infection. Besides, the anti-HBV activity of compounds 10, 11 and 17 had been
investigated.
Keywords: 2'-deoxy-2'-fluoro-4'-triazole nucleosides䟜Anti-HIV activity䟜Nucleoside reverse
transcriptase inhibitor (NRTI)䟜Anti-HBV activity
1. Introduction
Nucleoside analogs have become essential agents for the treatment of various infectious
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; AIDS, acquired
immunodeficiency syndrome; FDA, US Food and Drug Administration; dN, 2'-deoxynucleoside; ddN, 2',3'-dideoxynucleoside;
4'sdN, 4'-C-substituted-2'-deoxynucleoside; CuAAC, Copper(I) catalyzed azide-alkyne [3 + 2] cycloaddition; NMR, nuclear
magnetic resonance; HRMS, high resolution mass spectrometry; NRTI, nucleoside reverse transcriptase inhibitor; AZT,
zidovudine; HMDS, hexamethyldisilazane; tBuOH, tert-butanol; VSVG, vesicular stomatitis virus glycoprotein.
Corresponding author. Tel.: +86 371 67781788; e-mail: chjsong@zzu.edu.cn (C. Song).
Corresponding author. Tel.: +86 371 67783017; fax: +86 371 67783017; e-mail: changjunbiao@zzu.edu.cn (J. Chang).

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diseases such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C
virus (HCV) over the past forty years. Their mechanism of action generally involves the direct or
delayed chain termination of viral DNA or RNA elongation by incorporation of nucleoside
triphosphate analogues. However, the use of nucleoside drugs has been relatively limited by their
toxicity, drug resistance development, and more worryingly, the fact that some newly
HIV-infected patients carry viruses that are already resistant to the currently approved acquired
immunodeficiency syndrome (AIDS) treatment [1]. One strategy for salvage therapy is to
develop nucleoside analogs with better therapeutic indices, new mechanisms of action, and
activity against HIV strains resistant to currently available drugs.
In the effort to discover effective antiviral agents against AIDS and viral hepatitis, a variety of
nucleoside analogs have now been discovered which expand the antiviral spectrum and/or modify
the pharmacological and/or pharmacokinetic properties of the parent compounds [2]. Although
structure–activity relationship studies have not led to a pharmacophore model for the antiviral
activities of nucleosides, some structural features have been particularly successful. For example,
all seven of the nucleoside reverse transcriptase inhibitors (NRTIs) approved by the US Food and
Drug Administration (FDA) for the treatment of AIDS can be considered as
2',3'-dideoxynucleoside (ddN) [3]. The useful modifications on nucleosides include acyclic
nucleosides [4], cyclonucleosides [5], introduction of electron-withdrawing groups such as fluoro
[6] and azido [7] substitution into the molecule, exchange of the ribose ring for a heterocyclic
ring containing sulfur or nitrogen [8], or exchange of the glycoside C–N bond for a C–C bond
(C-nucleosides) [9], and so on. Among them, our group has reported that the
4'-C-substituted-2'-deoxy-nucleosides (4'sdNs) 1-8 could prevent the emergence of drug-resistant
HIV variants [10]. The 4'sdNs have all of the functional groups of 2'-deoxynucleoside (dN),
making it difficult for HIV to discriminate between 4'sdNs and dN, therefore, the compounds
were active against multidrug-resistant strains of HIV [10b]. Besides, the 3'-OH of 4'sdNs would
not be useful for elongation of proviral DNA biosynthesis. Thus, 4'sdNs could be the chain
terminator of proviral DNA biosynthesis and active against HIV as well as HIV strains resistant
to the current ddN drugs. Furthermore, the modifications of ribofuranosyl moiety of nucleosides
with functional groups, such as azido, cyano, and ethynyl at the 4'-position can affect the

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nucleoside’s electronic properties and conformational shape, leading to dramatically improved
activity [11].
As a continuous research program of our laboratory developing 4'sdNs as antiviral agents, we
further explored their structure at 4'-position by incorporation of a substituted 1,2,3-triazole group
(9-17, Fig. 1). Although 1,2,3-triazoles do not occur naturally, compounds containing a
1,2,3-triazole moiety show various biological activities such as anti-HIV, anti-microbial,
anti-allergic, selective β3 adrenergic receptor agonist, and their potency as pharmacophores has
been reviewed [12]. Copper(I) catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC) has
emerged as the method of choice for generating a 1,4-disubstituted 1,2,3-triazolyl linkage that
functions as a chemical linker for conjugating two desirable chemical or biological entities [13].
Furthermore, the reaction has also proven useful in the nucleoside/nucleotide field for the
construction of different bioconjugates [14], modified bases [15], modified sugar residues at the
3'- or 5'-position [16], and altered phosphodiester backbones [17]. However, to the best of our
knowledge, introduction of a 1,2,3-triazoles moiety to the 4'-position of nucleosides has not been
reported till now.
Herein, we report the synthesis and evaluation of novel 4'sdNs containing a 1,2,3-triazoles
moiety in the 4'-position (4'-[1,2,3]triazole-2'-deoxy-2'-fluoro-β-D-arabinofuranosylcytosine) as
novel potent NRTIs.
2. Chemistry
1-(4'-Azido-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)cytosine (1), a potential antiviral agent
previously developed by our group based on the favorable antiviral profiles of 4'-substituted
nucleoside, is currently being developed as an excellent anti-AIDS clinical trial candidate [10a-b].
1 was converted into the 1,4-disubstituted 1,2,3-triazoles 9-17 through a CuAAC reaction with
the appropriate alkynes in a water/tBuOH/acetonitrile (1:1:1) solvent mixture. The desired
compounds 9-17 were obtained in a fully regioselective (1, 4) manner with a complete conversion
of the starting materials, without contamination by the 1,5-regioisomer, which is in agreement
with the proposed reaction mechanism that the copper(I) acetylide intermediate formed may

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undergo stepwise addition with the azide, resulting in the regioselective product (Table 1) [18].
All compounds were isolated in good yields, ranging from 66% to 96%, regardless of whether the
substituents on the alkynes were aliphatic (13-17) or aromatic (10-12). Although alkyne
homocoupling might be a possible side reaction of the copper(I)-catalyzed Huisgen reaction
[16b], no trace of this kind of product was isolated or identified in the present study.
3. Result and discussion
In 293T cell line, the inhibitory activity (EC50) and the cytotoxcity (CC50) were measured by an
anti-HIV (wild-type) replication assay [19] and MTT assay [20], respectively. The results were
summarized in Table 1. At the highest concentrations tested (25 ꢀM), no cytotoxicity was
observed for any of these compounds. The 4,5-unsubstituted triazolo-nucleoside analog (9)
showed a nanomolar level anti-HIV-1 activity (EC50 = 0.09 ꢀM), which is equally potent as the
control compound AZT did (EC50 = 0.084 ꢀM). Incorporation of a phenyl group (10) to the
4-position of triazole ring could maintain the potent antiviral activity (EC50 = 0.083 ꢀM).
However, further substitution of this aromatic ring (11 and 12) decreased compounds' potency
dramatically. Analog 13 with the bulky tert-butyl group exhibited good antiviral activity (EC50 =
0.08 ꢀM) as well, while replacement of this tert-butyl group with n-butyl led to the less potent
analog 14. Generally, replacement of aliphatic groups (13 and 14) with cycloalkyl substituents
(15–17) resulted in reduced antiviral activity. Summarily, this evaluation provided three novel
triazolo-nucleoside analogs 9, 10 and 13 with potent anti-HIV-1 activity (selective index, SI >
278) for further development as potential new anti-HIV drugs.
In order to explore the utility of these triazolo-nucleosides as potential antiviral agents, we
picked three analogs (10, 11 and 17) and further examined their antiviral activity against HBV
infection in HepG2.2.15 cell line. The inhibitory activity of tested compounds against the
production of HBsAg and HBeAg were examined by ELISA assay; and their cytotoxicty was
measured by MTT assay (Table 2). Consistent with the result in 293T cell line, all the three
compounds showed low toxicity in HepG2.2.15 cell line. All these compounds exhibited
nanomolar inhibitory activity against HBsAg production, with 11 being the best (EC50 = 0.01 µM,

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SI = 49800). Among them, analog 10 possesses the most potent activity in inhibition of HBeAg
production (EC50 = 0.25 µM, SI = 2264).
4. Conclusions
In summary, we report herein the first highly efficient synthesis of novel 4'sdN analogs 9-17
with 1,2,3-triazole moiety at the 4'-position through a CuAAC reaction and their anti-HIV-1
activity was evaluated in vitro. Initial biological evaluation indicated that most of these
compounds exhibited potent anti-HIV-1 activity without significant cytotoxicity at the highest
tested concentration up to 25 ꢀM. In particular, compounds 9, 10 and 13 were extremely potent
against HIV-1 wide-type strain without obvious cytotoxicity, and merits further development as
an anti-AIDS clinical trial candidate.
5. Experiment
5.1. Chemistry
All chemical reagents were commercially available and column chromatography was
performed on silica gel 200–300 mesh (Yantai Silica Gel Co. LTD). Analytical TLC was
performed on silica gel GF254. Melting points were recorded with an XT-4 melting point
1
apparatus and are uncorrected. IR spectra were obtained on a NICOLET 6700 spectrometer. H
and 13C NMR spectra were obtained with Bruker Avance-300 and -400 spectrometers. Chemical
shifts are reported as δ (ppm) downfield with respect to an internal standard of tetramethylsilane
(TMS). High-resolution mass spectra (HRMS-ESI) were obtained on a MicroTM Q-TOF Mass
Spectrometer. LC-MS spectra were measured on an Agilent MSD-1100 ESI-MS/MS system.
Elemental analyses were carried out on a Flash EA1112 element analyzer.
5.1.1 Procedures for the synthesis of triazoles
To a solution of acetylene (0.24 mmol) in 2 mL of tBuOH/H2O 1:1 (v:v) was added DIPEA (50

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ꢀL, 0.3 mmol), and FNC (56 mg, 0.2 mmol). The mixture was stirred for 15 min. A solution of
CuI (4 mg, 0.02 mmol) in CH3CN (1 mL) was added, and the resulting mixture was stirred at r.t.
(for 9-16) or at 45°C (for 17) until complete conversion of FNC. The solvent was removed under
reduced pressure and the crude residue was purified by flash chromatography on silica gel (1-5%
methanol in dichloromethane).
5.1.2 1-[4'-(1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine (9)
The reaction was complete within 48 h. White solid (47 mg, 75%): mp = 155-156 °C; 1H NMR
(400 MHz, CD3OD) δ: 8.16 (1H, s, H-5''), 7.94 (1H, d, J = 7.6 Hz, H-6), 7.76 (1H, s, H-4''), 6.84
(1H, dd, J = 11.2, 5.2 Hz, H-1'), 6.00 (1H, d, J = 6.8 Hz, H-5), 5.36 (1H, dt, J = 54.0, 4.8 Hz,
H-2'), 4.32 (1H, d, J = 13.7 Hz, H-5'a) and 4.29 (1H, d, J = 13.7 Hz, H-5'b); the H-3' signal was
obstacled by water peak; 13C NMR (100 MHz, CD3OD) δ: 166.3, 156.5, 141.9, 132.3, 124.1, 97.2
(J = 7.9 Hz), 94.8, 94.6 (J = 192.3 Hz), 84.8 (J = 16.4 Hz), 74.8 (J = 25.5 Hz) and 61.8;
ESI-TOF/MS for C11H13FN6O4: calcd: 335.0880 [M + Na]+; found 335.0794 [M + Na]+; Anal.
Calcd. For C11H13FN6O4: C, 42.31; H, 4.17; N, 26.92. Found: C, 42.53; H, 4.02; N, 26.74.
5.1.3
1-[4'-(4-Phenyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine
(10)
The reaction was stirred at r.t. for 16h. Yellow solid (74 mg, 96%): mp = 163-164 °C; 1H NMR
(300 MHz, CD3OD) δ: 8.51 (1H, s, H-5''), 7.97 (1H, d, J = 7.6 Hz, H-6), 7.85 (2H, d, J = 7.2 Hz,
HAr), 7.44-7.30 (3H, m, HAr), 6.91 (1H, dd, J = 11.9, 4.8 Hz, H-1'), 6.00 (1H, br, H-5), 5.43 (1H,
dt, J = 53.8, 4.5 Hz, H-2'), 4.41 (1H, d, J = 12.2 Hz, H-5'a) and 4.34 (1H, d, J = 12.2 Hz, H-5'b);
the H-3' signal was obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 167.8, 158.0, 148.2,
143.4, 131.6, 130.0, 126.8, 122.0, 99.0 (d, J = 7.1 Hz), 96.3, 96.1 (d, J = 193.2 Hz), 86.5 (d, J =
16.4 Hz), 76.4 (d, J = 25.5 Hz) and 63.1; ESI-TOF/MS for C17H17FN6O4: calcd: 411.1295 [M +
Na]+; found 411.1163 [M + Na]+; Anal. Calcd. For C17H17FN6O4: C, 52.58; H, 4.38; N, 21.65.
Found: C, 52.44; H, 4.17; N, 21.44.
5.1.4
1-[4'-(4-methylphenyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine
(11)
1
The reaction was complete within 16 h. Yellow solid (74 mg, 92%): mp = 171-172 °C; H

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NMR (300 MHz, CD3OD) δ: 8.46 (1H, s, H-5''), 7.98 (1H, d, J = 7.5 Hz, H-6), 7.73 (2H, d, J =
8.1 Hz, HAr), 7.23 (2H, d, J = 8.1 Hz, HAr), 6.91 (1H, dd, J = 11.3, 5.0 Hz, H-1'), 6.02 (1H, br,
H-5), 5.43 (1H, dt, J = 54.1, 4.7 Hz, H-2'), 4.37 (2H, m, H-5') and 2.34 (1H, s, CH3); the H-3'
signal was obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 167.8, 158.0, 148.3, 143.3,
139.5, 130.6, 128.7, 126.8, 121.6, 98.8 (d, J = 7.7 Hz), 96.1 (d, J = 193.4 Hz), 86.3, 76.4 (d, J =
25.6 Hz), 63.3 and 21.4; ESI-TOF/MS for C18H19FN6O4: calcd: 425.1452 [M + Na]+; found
425.1317 [M + Na]+; Anal. Calcd. For C18H19FN6O4: C, 53.73; H, 4.73; N, 20.90. Found: C,
54.02; H, 4.57; N, 21.02.
5.1.5
1-[4'-(4-ethylphenyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine
(12)
1
The reaction was complete within 16 h. Yellow solid (78 mg, 94%): mp = 153-154 °C; H
NMR (300 MHz, CD3OD) δ: 8.45(1H, s, H-5''), 7.98 (1H, d, J = 7.4 Hz, H-6), 7.76 (2H, d, J =
8.1 Hz, HAr), 7.26 (2H, d, J = 8.1 Hz, HAr), 6.90 (1H, dd, J = 11.4, 4.2 Hz, H-1'), 5.99 (1H, br,
H-5), 5.42 (1H, dd, J = 54.0, 4.2 Hz, H-2'), 4.40 (1H, d, J = 12.4 Hz, H-5'a), 4.33 (1H, d, J = 12.4
Hz, H-5'b), 2.66 (2H, q, J = 7.6 Hz, CH2) and 1.24 (3H, t, J = 7.6 Hz, CH3); the H-3' signal was
obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 167.8, 158.1, 148.4, 143.4, 129.5, 129.1,
126.9, 121.6, 99.0 (J = 7.3 Hz), 96.3, 96.1 (J = 193.5 Hz), 86.5 (J = 16.4 Hz), 76.4 (J = 25.5 Hz),
63.4, 29.7 and 16.1; ESI-TOF/MS for C19H21FN6O4: calcd: 439.1608 [M + Na]+; found 436.1496
[M + Na]+; Anal. Calcd. For C19H21FN6O4: C, 54.81; H, 5.05; N, 20.19. Found: C, 54.55; H, 4.87;
N, 20.46.
5.1.6 1-[4'-(4-tert-butyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine
(13)
The reaction was complete within 48 h. White solid (49 mg, 66%): mp = 159-161 °C; 1H NMR
(300 MHz, CD3OD) δ: 7.95 (1H, d, J = 7.5 Hz, H-6), 7.87 (1H, s, H-5''), 6.82 (1H, dd, J = 11.3,
5.1 Hz, H-1'), 5.99 (1H, br, H-5), 5.37 (1H, dt, J = 54.3, 4.9 Hz, H-2'), 4.31(1H, d, J = 12.2 Hz,
t
H-5'a), 4.26 (1H, d, J = 12.2 Hz, H-5'b) and 1.35 (9H, s, Bu); the H-3' signal was obstacled by
13
water peak; C NMR (75 MHz, CD3OD) δ: 167.9, 157.8, 143.4, 120.7, 98.4 (J = 7.8 Hz), 96.3,
96.2 (J = 193.4 Hz), 86.3 (J = 16.7 Hz), 76.4 (J = 25.5 Hz), 63.2, 31.8 and 30.8; ESI-TOF/MS for

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C15H21FN6O4: calcd: 391.1608 [M + Na]+; found 391.1472 [M + Na]+; Anal. Calcd. For
C15H21FN6O4: C, 48.91; H, 5.71; N, 22.83. Found: C, 48.75; H, 5.58; N, 23.05.
5.1.7 1-[4'-(4-butyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosine (14)
The reaction was complete within 48 h. White solid (51 mg, 70%): mp = 133-135 °C; 1H NMR
(300 MHz, CD3OD) δ: 7.95 (1H, d, J = 7.6 Hz, H-6), 7.90 (1H, s, H-5''), 6.83 (1H, dd, J = 11.4,
5.1 Hz, H-1'), 5.98 (1H, d, J = 7.6 Hz, H-5), 5.38 (1H, dt, J = 54.1, 4.8 Hz, H-2'), 4.32(1H, d, J =
13.0 Hz, H-5'a), 4.27 (1H, d, J = 13.0 Hz, H-5'b), 2.71 (2H, t, J = 7.5 Hz, CH2), 1.71-1.61 (2H, m,
CH2), 1.44-1.28 (2H, m, CH2) and 0.94 (3H, t, J = 7.3 Hz, CH3); the H-3' signal was obstacled by
water peak; 13C NMR (75 MHz, CD3OD) δ: 167.5, 157.6, 148.6, 143.5, 122.8, 98.6 (J = 7.7 Hz),
96.2, 96.1 (J = 193.4 Hz), 86.3 (J = 16.9 Hz), 76.3 (J = 25.7 Hz), 63.2, 32.8, 26.0, 23.3 and 14.2;
ESI-MS: m/z 391 [M + 23]+; Anal. Calcd. For C15H21FN6O4: C, 48.91; H, 5.71; N, 22.83. Found:
C, 48.80; H, 5.63; N, 22.64.
5.1.8
1-[4'-(4-cyclohexylmethyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytosi
ne (15)
The reaction was complete within 24 h. White solid (65 mg, 80%): mp = 148-150 °C; 1H NMR
(300 MHz, CD3OD) δ: 7.94 (1H, d, J = 7.5 Hz, H-6), 7.88 (1H, s, H-5''), 6.82 (1H, dd, J = 11.5,
4.6 Hz, H-1'), 5.98 (1H, br, H-5), 5.37 (1H, d, J = 54 Hz, H-2'), 4.32 (1H, d, J = 12.4 Hz, H-5'a),
4.26 (1H, d, J = 12.4 Hz, H-5'b), 2.59 (2H, d, J = 6.8 Hz, CH2) and 1.73-1.15 (11H, m,
H
cyclohexane); the H-3' signal was obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 167.9,
158.0, 147.1, 143.4, 123.4, 98.6 (d, J = 7.5 Hz), 96.3, 96.1 (d, J = 193.3 Hz), 86.4 (d, J = 16.7
Hz), 76.4 (d, J = 25.5 Hz), 63.3, 39.5, 34.1, 27.6 and 27.3; ESI-TOF/MS for C16H25FN6O4: calcd:
431.1921 [M + Na]+; found 431.1779 [M + Na]+; Calcd. For C16H25FN6O4: C, 47.06; H, 6.13; N,
20.59. Found: C, 46.89; H, 5.97; N, 20.66.
5.1.9
1-[4'-(4-hydroxycyclopentyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cyt
osine (16)
The reaction was complete within 48 h. White solid (68 mg, 86%): mp = 165-167 °C; 1H NMR
(300 MHz, CD3OD) δ: 7.98 (1H, s, H-5''), 7.93 (1H, d, J = 7.5 Hz, H-6), 6.81 (1H, dd, J = 12.0,

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5.1 Hz, H-1'), 5.97 (1H, d, J = 7.5 Hz, H-5), 5.38 (1H, dt, J = 54.3, 4.8 Hz, H-2'), 4.32(1H, d, J =
12.6 Hz, H-5'a), 4.26 (1H, d, J = 12.6 Hz, H-5'b) and 2.15-1.81 (8H, m, CH2); the H-3' signal was
obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 165.8, 155.9, 153.1, 141.5, 120.1, 96.7
(J = 7.6 Hz), 94.2 (J = 193.4 Hz), 84.4 (J = 16.6 Hz), 77.7, 74.4 (J = 25.6 Hz), 61.4, 39.9 (J = 4.1
Hz) and 22.6; ESI-MS: m/z 419 [M + 23]+; Calcd. For C16H21FN6O5: C, 48.48; H, 5.30; N, 21.21.
Found: C, 48.21; H, 5.16; N, 21.42.
5.1.10 1-[4'-(cyclopropyl-1,2,3-triazol-1-yl)-2'-deoxy-2'-fluoro-β-D-arabinofuranosyl]cytidine
(17)
The reaction was complete within 28 h. White solid (58 mg, 85%): mp = 156-158 °C; 1H NMR
(300 MHz, CD3OD) δ: 7.96 (1H, d, J = 7.5 Hz, H-6), 7.86 (1H, s, H-5''), 6.81 (1H, dd, J = 11.1,
5.0 Hz, H-1'), 5.99 (1H, d, J = 7.5 Hz, H-5), 5.37 (1H, dt, J = 54.0, 4.8 Hz, H-2'), 4.29 (1H, d, J =
12.5 Hz, H-5'a), 4.24 (1H, d, J = 12.5 Hz, H-5'b), 1.98 (1H, m) and 1.00-0.78 (5H, m); the H-3'
signal was obstacled by water peak; 13C NMR (75 MHz, CD3OD) δ: 167.5, 157.6, 150.8, 143.5,
121.7, 98.5 (d, J = 7.8 Hz), 96.2, 96.1 (d, J = 193.2 Hz), 86.2 (d, J = 16.5 Hz), 76.2 (d, J = 25.5
Hz), 63.1, 8.2 and 7.3; ESI-TOF/MS for C14H17FN6O4: calcd: 375.1295 [M + Na]+; found
375.1174 [M + Na]+; Calcd. For C14H17FN6O5: C, 47.73; H, 4.83; N, 23.86. Found: C, 47.54; H,
5.05; N, 23.97.
5.2. Biology
5.2.1 Anti-HIV (pseudotype virus) activity assay
Vesicular stomatitis virus glycoprotein (VSVG) plasmid was co-transfected with env-deficient
HIV vector, pNL4-3.luc.R-E- [21], into 293T cells by using modified Ca3(PO4)2 method [19].
Briefly, 293T cells (100 mm plate) were transfected with 8 ꢀg HIV vector alone or with 3 ꢀg
VSVG DNA. After 16 h, plates were washed with phosphate-buffered saline, and fresh medium
was added into the plates. After post-infection for 32 h, the supernatant was harvested and filtered
through a 0.45 ꢀm filter. The supernatant contains VSVG/HIV pseudotyped virions and can be
used directly or stored at -80 °C.

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One hour prior to infection, 293T cells were plated on 24-well plates at the density of 6 × 104
cells per well. Compound was incubated with target cells for 15 min prior to adding
VSVG/HIV-1 (0.5 mL/well) for infection. The same amount of solvent alone was used as control.
After post-infection for 48 h, cells were spin down and supernatant was discarded. Cells were
lysed in 50 ꢀL Cell Lysis Reagent (Promega). Luciferase activity of the cell lysate was measured
by an FB15 luminometer (Berthold Detection System according to the manufacturer’s
instructions.
5.2.2 Cell Viability Assay
Compounds 9-17 were evaluated for cell viability by MTT assay using MT-2 cell line [20].
Compounds 9-17 had no significant effect on cell growth or any cytotoxic effect on MT-2 cells at
the highest tested concentration up to 25 ꢀM.
5.2.3 Cell Culture and Treatment
HepG2.2.15 (clonal cells derived from human hepatoma cell line G2) cells were provided by
Wuhan Institute of Virology. The HepG2.2.15 cell line was maintained in our laboratory in RPMI
1640 medium supplemented with 10% FBS, 200ꢀg/mL G418 at 37°C in a humidified atmosphere
containing 5% CO2.
5.2.4 Cytotoxicity Assay䟉MTT assay䟊
MTT assays were used to detect the survival rates of HepG2.2.15 cells [22]. Cells were seeded
in 96-well culture plates at a density of 1×105 cells per well and cultured at 37°C for 24h. Then
the culture medium was removed and replaced with fresh medium supplemented with various
concentrations (cells were incubated in culture medium supplemented with 75, 150, 300, 600
ꢀg/mL in 96-well plates for 3 days) of compounds 10, 11 and 17 (5 wells/concentration). Cells
incubated without drug were used as negative control. After 3 days of culture, the cytotoxic effect
of compounds 10, 11 and 17 was evaluated by MTT assay. By the end of the incubation, 20 ꢀL
MTT (5mg/mL) was added to each well and incubation was allowed to continue for another 4 h
at 37°C. Finally, 200 ꢀL DMSO was added to each well to dissolve the formazan. The absorbance
(A) at 490 nm was measured by an automatic plate reader (BIORAD, 168-1000XC, USA). The
survival ratio of 2.2.15 cells (%) = (A490 of experimental group/A490 of negative control)×100%.

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Then, the 50% toxicity dose (CC50) was calculated according to the Reed-Muench method [23].
5.2.5 Detection of HBsAg and HBeAg
The HepG2.2.15 cells were plated at a density of 2×104 cells per well on 24-well cell culture
plates and routinely cultured. Different concentrations (0.012, 0.06, 0.3 ꢀg/mL) of compounds 10,
11 and 17 were supplemented to the medium in triplicate 24h after cells were plated. After
incubation with compounds 10, 11 and 17 for 9 days, the supernatants and cells were collected.
The supernatants were used for HBsAg or HBeAg assays immediately. The concentrations of
HBsAg and HBeAg were quantified by commercial ELISA kit (Kehua Bio-engineering
Corporation, China) according to the protocol of the manufacturer, the optical density (OD) value
at 490/630 nm was measured by using an automatic plate reader. Data were calculated as
percentage of the control by the formula [21,24]: Suppression rate (%) = [OD value (control) –
OD value (tested compound)]/[OD value (control)]×100%.
Acknowledgments
This project is supported by the National Science Foundation of China (#21172202,
#21201152), China Postdoctoral Science Foundation (#20090450095, #201003401) and the
Student Innovative Funded Projects of Zhengzhou University (#1210459088).
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Captions
Table 1. Anti-HIV-1 activity and cellular toxicity of compounds 9-17 in 293T cell line.
Table 2. Anti-HBV activity and cytotoxicity of compounds 10, 11 and 17 on HepG 2.2.15 cell
line.
Fig. 1 Structures of 4'-C-substituted-2'-deoxy-nucleosides (4'sdNs) mentioned in the text.

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Table 1. Anti-HIV-1 activity and cellular toxicity of compounds 9-17 in 293T cell line.
b
CC50
Compounds
R =
EC50a (ꢀM)
(ꢀM)
1
9
-
0.0003
0.09
> 10
H
≥ 25
10
11
12
0.083
3.7
≥ 25
≥ 25
≥ 25
0.6
13
0.08
≥ 25
14
15
0.3
4.8
≥ 25
≥ 25
16
1.3
≥ 25
17
0.6
≥ 25
≥ 25
AZT
–
0.084
a EC50: 50% effective concentration, measured by a modified Ca3(PO4)2 method.
b CC50: 50% cytotoxic concentration, measured by MTT assay; both CC50 and EC50 are the
average of at least two independent determinations.

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Table 2. Anti-HBV activity and cytotoxicity of compounds 10, 11 and 17 on HepG 2.2.15 cell
line.
EC50a (µM)
(HBsAg)
EC50a (µM)
(HBeAg)
CC50b (µM)
(HepG2.2.15)
Compounds
R =
10
11
17
0.1
0.25
> 1.0
1.0
566
498
834
0.01
0.12
a EC50: 50% effective concentration, measured by ELISA assay.
b CC50: 50% cytotoxic concentration, measured by MTT assay; both CC50 and EC50 are the average of
at least two independent determinations.

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Fig. 1 Structures of 4'-C-substituted-2'-deoxy-nucleosides (4'sdNs) mentioned in the text.

ACCEPTED MANUSCRIPT
A series of 4'-[1,2,3]triazole-2'-deoxy-2'-fluoro-β-D-arabinofuranosylcytosines
were prepared, most of which exhibited potent anti-HIV-1 activity with no
cytotoxicity observed at the highest tested concentration up to 25 µM.