Synthesis and antiviral evaluation of 4′-(1,2,3-triazol-1-yl) thymidines

Article abstract of DOI:10.1039/c4md00039k

Non-obligate chain terminating nucleosides with a linear substituent (azido or ethynyl group) at the 4′ position represent an important class of compounds in antiviral discovery, particularly against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). We have previously shown that 3′-azidothymidine (AZT)-derived 1,2,3-triazoles can be potent inhibitors of HIV-1. To gauge the medicinal chemistry impact of functionalizing the 4′-linear substituent and possibly generate novel antiviral nucleoside scaffolds, we have explored azide-alkyne cycloaddition reactions with 4′-azidothymidine (ADRT). The Ru-mediated reaction failed and the Cu-catalyzed variant generated 1,2,3-triazoles (9a-y) with only modest yields and efficiencies, indicating a substantial steric barrier around the 4′-azido group. Antiviral screening identified a few triazole analogues moderately active against HIV-1 (18-62% inhibition at 10 μM) and/or influenza A virus (15-50% inhibition at 10 μM), and none active against West Nile virus (WNV) or HCV. These results suggest that the linear 4′ azido group of ADRT may be essential for target binding and that its chemical manipulation could largely compromise antiviral potency. This journal is the Partner Organisations 2014.

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Synthesis and antiviral evaluation of
40-(1,2,3-triazol-1-yl)thymidines†
Cite this: DOI: 10.1039/c4md00039k
Sanjeev Kumar V. Vernekar, Li Qiu, Jeana Zacharias, Robert J. Geraghty
*
and Zhengqiang Wang
Non-obligate chain terminating nucleosides with a linear substituent (azido or ethynyl group) at the 40
position represent an important class of compounds in antiviral discovery, particularly against hepatitis C
virus (HCV) and human immunodeficiency virus (HIV). We have previously shown that 30-azidothymidine
(AZT)-derived 1,2,3-triazoles can be potent inhibitors of HIV-1. To gauge the medicinal chemistry impact
of functionalizing the 40-linear substituent and possibly generate novel antiviral nucleoside scaffolds, we
have explored azide–alkyne cycloaddition reactions with 40-azidothymidine (ADRT). The Ru-mediated
reaction failed and the Cu-catalyzed variant generated 1,2,3-triazoles (9a–y) with only modest yields and
efficiencies, indicating a substantial steric barrier around the 40-azido group. Antiviral screening identified
a few triazole analogues moderately active against HIV-1 (18–62% inhibition at 10 mM) and/or influenza A
virus (15–50% inhibition at 10 mM), and none active against West Nile virus (WNV) or HCV. These results
suggest that the linear 40 azido group of ADRT may be essential for target binding and that its chemical
manipulation could largely compromise antiviral potency.
Received 29th January 2014
Accepted 17th February 2014
DOI: 10.1039/c4md00039k
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antivirals, particularly against HCV, as major nucleoside scaf-
folds of HCV NS5B polymerase inhibitors, such as the Idenix
NM107 (1),5 the Pharmasset PSI-6130 (2)6,7 and the Roche R1479
Introduction
Synthetic nucleoside analogues provide an important platform
for drug discovery, especially as anticancer and antiviral agents.1
By mimicking endogenous nucleosides, these analogues exploit
cellular kinases for phosphorylation and compete against
nucleoside triphosphates (NTPs) or deoxynucleoside triphos-
phates (dNTPs) for active site binding and incorporation during
RNA or DNA polymerization. Mechanistically, nucleoside
analogues could act as obligate chain terminators when lacking
the 30-OH group,2 as exemplied by numerous nucleoside reverse
transcriptase inhibitors (NRTIs) for the treatment of HIV infec-
tion.3 The absence of the 30-OH, however, could reduce affinities
for cellular kinases and viral polymerases, and ultimately lead to
suboptimal antiviral efficacies.4 Alternatively, incorporated
analogues bearing the 30-OH group could also terminate the
growing RNA or DNA chain when chemical modications at the
20 or 40 position sterically hinder the proper alignment of the 30-
OH with the incoming NTP or dNTP.2 By virtue of the 30-OH
group, these non-obligate chain terminators presumably benet
binding to both kinases and polymerases. This critical advantage
has been successfully explored in the discovery of novel
(3),8,9 all have the 30-OH group (Fig. 1).10
HIV NRTIs bearing the 3-OH group are rare, with the notable
exception of two 40 modied deoxynucleosides: 40-ethynyl-2-
uoro-20-deoxyadenosine (EFdA, 4), an exceptionally potent
clinical candidate with an EC50 of 0.05 nM against HIV-1 in cell
culture;11 and ADRT (5)12,13 with an antiviral potency comparable
to that of AZT (6, Fig. 2). Mechanistically EFdA terminates the
chain via stabilizing RT at the N site to cause translocation
deciency14 and ADRT retains its activity against AZT-resistant
HIV mutants,13 suggesting a mechanism of RT inhibition
distinctive of typical NRTIs. Interestingly, both the non-obligate
chain terminating NRTIs 4–5 feature a linear 40 modier
(ethynyl and azido) which can be important for their unique
mechanism of inhibition. Chemical manipulation of these
linear functional groups into new chemical moieties of
completely different shapes and volumes might lead to novel
nucleoside scaffolds with distinct binding proles to both
kinases and polymerases. Recently, we have demonstrated for
the rst time that AZT can be clicked into 1,2,3-triazoles with
potent anti-HIV activities and interesting resistance proles
(Fig. 2a).15 Given the importance of the 40-modier in the
discovery of non-obligate chain terminating nucleoside antivi-
rals, we were prompted to extend our clicking efforts to ADRT
with the hope to generate 40-triazole antiviral scaffolds (Fig. 2b).
To the best of our knowledge synthetic or biological studies of
such 40-triazole nucleosides are currently unknown. Unlike the
Center for Drug Design, Academic Health Center, University of Minnesota,
Minneapolis, MN 55455, USA. E-mail: wangx472@umn.edu; Fax: +1 612 625-8154;
Tel: +1 612 626-7025
† Electronic supplementary information (ESI) available: Chemistry, experimental
details, and full characterization data (1H NMR, 13C NMR and HRMS) for newly
synthesized compounds; protocols for assays against HIV, inuenza virus, WNV
and HCV. See DOI: 10.1039/c4md00039k
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Fig. 1 Examples of non-obligate chain terminating antivirals. NM107 (1) and PSI-6130 (2) achieve chain termination through 20 modification while
R1479 (3), EFdA (4) and ADRT (5) act through 40 substitution. 1–3 are HCV NS5B polymerase inhibitors and 4–5 are HIV NRTIs.
Results and discussion
Chemistry
ADRT (5) was synthesized from thymidine (1) following the liter-
ature method16 as outlined in Scheme 1. The synthesis featured
an exocyclic alkene intermediate 13 which was prepared through
an E2 reaction of an iodide precursor 12 according to the proce-
dure reported by Verheyden and Moffatt.16 The iodide precursor
12 was readily prepared from thymidine with I2 and PPh3 in good
yields. The key step for the synthesis was the preparation of azide
14 in a stereoselective fashion. This was achieved in moderate
yield by treating 13 with benzyltriethylammonium chloride,
sodium azide and iodine to afford a diastereomeric mixture of 14
(de ¼ 90%), which can be separated by careful chromatography.
The desired isomer was then benzoylated at the 30-hydroxy group,
followed by an oxidative displacement of the 50-iodo group with
Fig. 2 Click azido nucleoside analogues into novel nucleoside antiviral
m-CPBA and m-chlorobenzoic acid to afford a dibenzoylated
scaffolds. (a) Clicking obligate chain terminator AZT generates novel
intermediate 16. Finally, removal of the benzoyl protecting groups
1,2,3-triazole nucleoside analogues active against HIV. (b) Can non-
with ammonia in MeOH delivered 5 in good yield. The NMR data
obligate chain terminator 5 be clicked into novel antiviral scaffolds?
of 5 were fully consistent with the literature.13
The aliphatic and aromatic alkynes (23) were either commer-
click chemistries with AZT where both 1,4 (7) and 1,5 (8)
disubstituted triazole nucleoside scaffolds can be easily
accessed,15 clicking of ADRT proved viable only via the 1,4 path
to yield scaffold 9. We report herein the synthesis and antiviral
evaluation of disubstituted 40-(1,2,3-triazol-1-yl)thymidines (9).
cially available or prepared from aldehydes via the Seyferth–
Gilbert homologation with the Bestmann reagent17 21 in good
yields (Scheme 2). The propargyl aryl ethers or thiol ethers (26)
were readily prepared via alkylation of phenols or thiophenols
with propargyl bromide and K2CO3 in good yields (Scheme 2).18
Scheme 1 Synthesis of ADRT 5. Reagents and conditions: (a) I2, PPh3, imidazole, THF, 15–25 ꢀC, 18 h, 77%; (b) NaOMe, MeOH, 60 ꢀC, 15 h, 81%;
(c) [Bn(Et)3N]Cl, NaN3, NMO, I2, THF, 0–5 ꢀC, 2 h, 52%; (d) BzCl, DMAP, pyridine, rt, 3 h, 81%; (e) (Bu4N)HSO4, K2HPO4, m-chlorobenzoic acid,
m-CPBA, DCM–H2O (4 : 1), rt, 15 h, 84%; and (f) 7 N NH3–MeOH, rt, 12 h, 79%.
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Scheme 2 Synthesis of alkynes 23 and 26. Reagents and conditions: (a) KI, P(OMe)3, acetone–CH3CN, rt–50 ꢀC, 12 h, 72%; (b) NaN3, acetone–
H2O, 0 ꢀC–rt, 14 h, 94%; (c) NaH, 20, THF–benzene, 0 ꢀC–rt, 12 h, 65%; (d) K2CO3, MeOH, rt, 12 h, 45–90%; and (e) propargyl bromide, K2CO3,
DMF, rt, 12–15 h, 60–76%.
The preparation of alkynes 23 and 26 allowed us to func- steric nature of the azide partner. The low efficiency of cyclo-
tionalize the azido group of ADRT (5) through click chemistry. It additions with tertiary azides is known.21
is well established that the Huisgen cycloaddition between an
It must be pointed out that incomplete consumption of 5 will
azide and an alkyne can be effected regioselectively through two render chromatographic separation extremely tedious as tri-
distinct catalytic pathways: the copper(I)-catalyzed azide–alkyne azole products tend to have very similar Rf to ADRT. Caution
cycloaddition (CuAAC)19 generates exclusively 1,4-disubstituted has to be taken to avoid any ADRT contamination which will
1,2,3-triazoles, while the ruthenium(II)-catalyzed variant likely cause false positive in the biological assays. Purity of the
(RuAAC)20 produces 1,5-disubstituted 1,2,3-triazoles. The nal 40-(1,2,3-triazol-1-yl)thymidines (9a–y) was conrmed by
combination of CuAAC and RuAAC enables the preparation of NMR and through HPLC analysis.
triazoles with diverse residues and substitution patterns (1,4 vs.
1,5). In our case, the CuAAC reaction of ADRT (5) with alkynes
(23a,b or 26a–d) produced 40-(1,2,3-triazol-1-yl)thymidines (9a–
y) in moderate yields (Scheme 3), whereas the RuAAC variant
failed and the expected 1,5-disubstituted triazoles 10 were not
observed (Scheme 3). Further investigation of reaction condi-
tions, including solvent effect, catalyst, co-catalyst, tempera-
ture, method of heating (microwave vs. conventional), yielded
no signicant improvement. These unsuccessful attempts
strongly suggest that nucleoside tertiary azides are essentially
non-clickable via the RuAAC pathway, presumably due to steric
hindrance around the azido group. The steric barrier for click-
ing tertiary azide 5 is further manifested through the Cu-
mediated click reaction, which proceeded with a substantially
lower efficiency than the same reaction with secondary azide
AZT (6). With ADRT (5) the completion of the CuAAC reaction
required prolonged time (typically 5–7 days), elevated temper-
ature (60 ꢀC) and intermittent addition of a fresh catalyst (every
24 h). Not surprisingly, the size of alkyne substrates also
appeared to impact the reaction as aromatic alkynes required
elongated time when compared to alkynyl aryl ethers, though
the overall success of the CuAAC reaction depends more on the
Antiviral testing
We rst screened all nal compounds against HIV-1 with a
cytoprotection assay based on viral cytopathic effects (CPE).22
The synthesized 40-(1,2,3-triazol-1-yl)thymidines (9a–y) were
evaluated against an IIIB strain of HIV-1 and the results are
summarized in Table 1. While the screening identied two
compounds 9d (62%) and 9h (60%) with signicant antiviral
activities and four others (9e, 9m, 9p and 9v) with modest (18–
31%) cell protection, the observation that most analogues in
this series were either marginally active or completely inactive
indicates a general trend of low potency and dampens our
enthusiasm on further testing the two active analogues. In the
meantime, parallel cell control with mock infection revealed
that these 40-triazole analogues generally do not exhibit cyto-
toxicity, which prompted us to conduct additional screenings
against three RNA viruses: inuenza A, WNV and HCV. As
aforementioned, 20-deoxy non-obligate chain terminators can
be potent inhibitors of RNA viruses, e.g. compound 2 (ref. 6 and
7) against HCV (Fig. 1). The inuenza A virus antiviral assay is a
cytoprotection assay while the WNV and HCV assays use repli-
con-containing cells to evaluate potential antivirals. Interest-
ingly, our initial inuenza A screening assay using A549 cells
identied seven compounds (9h–k, 9n, 9r, and 9y) with
apparent antiviral activities ranging from 15% to 50% at 10 mM.
However, these activities were not conrmed through further
testing in MDCK cells as all seven analogues were found inac-
tive. Finally, the screening of all 40-(1,2,3-triazol-1-yl)thymidine
analogues (9a–y) in replicon assays yielded no compound with
appreciable inhibitory activity against WNV or HCV. Altogether,
these screening results suggest that, with the exception of the
moderate inhibitory activities observed with a few analogues
Scheme 3 Clicking ADRT 5. Reagents and conditions: (a) 23 or 26,
sodium ascorbate, CuSO4$5H2O, THF–H2O (3 : 1), 60 ꢀC, 5–7 days,
33–72% and (b) 23 or 26, Cp*RuCl(PPh3)2, THF, 60 ꢀC.
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Table 1 Antiviral screening of 40-(1,2,3-triazol-1-yl)thymidine analogues (9a–y) against HIV, and influenza
HIV
Inuenza (inhibition% @ 10 mM)
Compd
9a
R
Inhibition% @ 10 mM
Viability% @ 10 mM
A549 cells
MDCK cells
0
5
99
0
NT
NT
9b
100
3.1
9c
0
96
94
2.2
0
NT
NT
9d
62
9e
19
100
0
NT
9f
0
0
99
95
0
0
NT
NT
4.0
3.0
1.0
1.0
9g
9h
9i
60
0
100
98
18
54
21
19
9j
12
0
100
84
9k
9l
0
100
5.0
NT
9m
9n
31
1
80
95
1.7
48
NT
1.0
9o
9p
0
95
95
5.3
0
NT
NT
18
9q
0
86
0
NT
9r
9s
0
2
95
15
0
0
100
NT
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Table 1 (Contd.)
HIV
Inuenza (inhibition% @ 10 mM)
Compd
R
Inhibition% @ 10 mM
Viability% @ 10 mM
A549 cells
MDCK cells
9t
8
0
100
97
0
NT
NT
9u
10
9v
20
0
99
97
3.3
0
NT
NT
9w
9x
9y
1
0
99
93
3.7
16
NT
1.0
against HIV-1 and/or inuenza A virus, ADRT-derived triazoles
generally lack the potency to be considered viable antiviral
scaffolds, a stark contrast to AZT-derived triazoles which
showed potent inhibitory activities against HIV-1.
ADRT
WNV
NTP
dNTP
RT
40-Azidothymidine
West Nile virus
Nucleoside triphosphate
Deoxynucleoside triphosphate
Reverse transcriptase
NRTI
EFdA
Nucleoside reverse transcriptase inhibitor
40-Ethynyl-2-uoro-20-deoxyadenosine
Conclusion
CuAAC Copper(I)-catalyzed azide–alkyne cycloaddition
RuAAC Ruthenium(II)-catalyzed azide–alkyne cycloaddition
In an attempt to study the medicinal chemistry impact of func-
tionalizing the linear 40 substituent of non-obligate chain termi-
nating nucleosides and possibly generate novel antiviral
nucleoside scaffolds, we have conducted both CuAAC and RuAAC
reactions with 40-azido nucleoside ADRT. The observed low effi-
ciency of these reactions reects severe steric hindrance around the
40-azido modier. Antiviral screening identied a few analogues
moderately active against HIV-1 (18–62% inhibition at 10 mM) and/
or inuenza A virus (15–50% inhibition at 10 mM), and none active
against WNV or HCV. These results suggest that chemically
modifying the linear 40 modier of non-obligate chain terminating
nucleosides may not be tolerated in antiviral drug discovery.
CPE
Cytopathic effect
Acknowledgements
This research was supported by the Research Development and
Seed Grant Program of the Center for Drug Design, University of
Minnesota. We thank Roger Ptak at Southern Research Institute
for the cytoprotection antiviral assay.
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