5′-silylated 3′-1,2,3-triazolyl thymidine analogues as inhibitors of West Nile Virus and Dengue virus

Article abstract of DOI:10.1021/acs.jmedchem.5b00327

West Nile virus (WNV) and Dengue virus (DENV) are important human pathogens for which there are presently no vaccine or specific antivirals. We report herein a 5′-silylated nucleoside scaffold derived from 3′-azidothymidine (AZT) consistently and selectively inhibiting WNV and DENV at low micromolar concentrations. Further synthesis of various triazole bioisosteres demonstrated clear structure-activity relationships (SARs) in which the antiviral activity against WNV and DENV hinges largely on both the 5′-silyl group and the substituent of 3′-triazole or its bioisosteres. Particularly interesting is the 5′ silyl group which turns on the antiviral activity against WNV and DENV while abrogating the previously reported antiviral potency against human immunodeficiency virus (HIV-1). The antiviral activity was confirmed through a plaque assay where viral titer reduction was observed in the presence of selected compounds. Molecular modeling and competitive S-adenosyl-l-methionine (SAM) binding assay suggest that these compounds likely confer antiviral activity via binding to methyltransferase (MTase).

Full text of DOI:10.1021/acs.jmedchem.5b00327

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Article
5'-Silylated 3'-1,2,3-triazolyl thymidine analogues
as inhibitors of West Nile virus and Dengue virus
Sanjeev Kumar V Vernekar, Li Qiu, Jing Zhang, jayakanth
kankanala, Hongmin Li, Robert J. Geraghty, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00327 • Publication Date (Web): 24 Apr 2015
Downloaded from http://pubs.acs.org on April 26, 2015
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'-Silylated 3'-1,2,3-triazolyl thymidine analogues as inhibitors of
West Nile virus and Dengue virus
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a§
a§
b
a
b,c
Sanjeev Kumar V. Vernekar Li Qiu , Jing Zhang , Jayakanth Kankanala , Hongmin Li ,
,
a*
a
Robert J. Geraghty and Zhengqiang Wang *
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455, USA
b
Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, New York 12208, USA
c
Department of Biomedical Sciences, School of Public Health, University at Albany, Albany NY 12201, USA
Key Words
West Nile virus, Dengue virus, inhibitor, 5'-silyl- 3'-1,2,3-triazolyl thymidine, MTase
Abstract
West Nile virus (WNV) and Dengue virus (DENV) are important human pathogens for which
there are presently no vaccine or specific antivirals. We report herein a 5'-silylated nucleoside
scaffold derived from 3’-azidothymidine (AZT) consistently and selectively inhibiting WNV and
DENV at low micromolar concentrations. Further synthesis of various triazole bioisosteres
demonstrated clear structure-activity-relationships (SARs) in which the antiviral activity against
WNV and DENV hinges largely on both the 5'-silyl group and the substituent of 3'-triazole or its
bioisosteres. Particularly interesting is the 5' silyl group which turns on the antiviral activity
against WNV and DENV while abrogating the previously reported antiviral potency against
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human immunodeficiency virus (HIV-1). The antiviral activity was confirmed through a plaque
assay where viral titer reduction was observed in the presence of selected compounds. Molecular
modeling and competitive S-adenosyl-L-methionine (SAM) binding assay suggest that these
compounds likely confer antiviral activity via binding to methyltransferase (MTase).
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Introduction
WNV and DENV are two important members of the genus Flavivirus in the family Flaviviridae.
Endemic in many tropical and sub-tropical regions of the world and transmitted by infected
mosquitos, these viruses infect a large human population and cause significant human morbidity
and mortality. WNV is a neurotropic virus with outbreaks on multiple continents. Particularly the
epidemics of 1999 and 2012 in USA have resulted in thousands of reported human cases, with
clinical manifestations ranging from asymptomatic to severe neuroinvasive diseases such as
meningitis, flaccid paralysis and encephalitis. On the other hand, DENV endangers 2.5 billion
people worldwide with 50 to 100 million annual infections and can cause dengue fever, dengue
hemorrhagic fever and dengue shock syndrome. Despite these grave public health threats,
currently there are no effective antiviral therapies against either virus. Developing antivirals for
the treatment of WNV and DENV infections addresses a critical medical need. Current efforts
towards this end target either the nucleoside triphosphate biosynthesis as exemplified by
1
,2
1,3
4
mycophenolic acid (MPA), ribavirin, and 6-azauridine; or viral proteins including both the
5,6
7-15
16-19
helicase and the protease
activities of NS3; the RNA dependent RNA polymerase
and
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the MTase
functions of NS5. We have previously reported the first AZT-derived 1,2,3-
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triazole scaffold (1, Figure 1) potently inhibiting HIV-1. Key to the unprecedented antiviral
activity with these 1,2,3-triazoles is the incorporation of a bulky group at the C5 position of the
triazole ring. A key SAR trend was that the C5 bulk substituent conferred significantly better
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antiviral potency than the C4 one, reflecting a critical requirement of bulkiness in the region
between 3' and 5' positions (highlighted). Interestingly, the bulkiness in scaffold 1 is highly
reminiscent of the unique TSAO-T chemotype (2, Figure 1), a well-known HIV non-nucleoside
reverse transcriptase inhibitor (NNRTI). This observation led us to explore the impact of the 5’
silyl protecting group on the antiviral activity of our scaffold 1. With this aim we synthesized a
new series of 5' silyl protected AZT 3'-1,2,3-triazoles (scaffold 3). Unfortunately, none of the
silylated analogues inhibited HIV-1. However, when the antiviral evaluation was extended to a
panel of other viruses, these silylated analogues were found to selectively inhibit WNV and
DENV without inhibiting influenza virus, human cytomegalovirus, or hepatitis C virus. Such a
flavivirus-specific inhibition profile prompted us to expand the SAR by synthesizing a few types
of triazole bioisosteres. We report herein the synthesis, antiviral and biochemical studies of these
new scaffolds as inhibitors of WNV and DENV.
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Figure 1. Generation of scaffold 3: AZT-derived 1,2,3-triazole 1 showed potent antiviral activity
against HIV-1. Structural comparison between 1 and NNRTI TSAO-T (2) led to the introduction
of a 5' silyl group to generate scaffold 3 which was identified to selectively inhibit WNV and
DENV.
Results and Discussion
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Chemistry. The 3' azido group of AZT (4) provides an excellent synthetic handle for chemical
modifications. All inhibitor scaffolds studied herein were synthetically accessed from AZT as
outlined in scheme 1. The first set of inhibitors were generated via click chemistry between the
azido functional group of AZT 4 and alkynes to yield 1,4 (8) and 1,5 (10) disubstituted triazoles
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according to our reported procedure. As previously noted, the copper(I)-catalyzed azide–alkyne
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cycloaddition (CuAAC) worked with substantially higher efficiency than the ruthenium(II)-
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catalyzed variant (RuAAC). All triazole analogues (8a–e, 10a–i) were further derivatized by
protecting the 5'-OH group with tert-butyldimethylsilyl chloride (TBSCl) in the presence of
imidazole to produce the corresponding 5′-silylated 1,4 and 1,5 disubstituted 1,2,3-triazoles (9a–
b, 11a–i) in good yields. Additional sets of inhibitors all feature a bioisostere of the 3' triazole
functionalities. The common 3'-amino intermediate 6 required for the synthesis of these
bioisoteres was easily prepared by 5' TBS protection of AZT followed by reducing the 3' azido
group via catalytic hydrogenation. The thiazolidinone derivatives 13 were synthesized via a one-
pot two-step reaction sequence with an aromatic aldehyde and 2-mercaptoacetic acid following a
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reported procedure. The amide analogues 15 were prepared through coupling with a carboxylic
acid
mediated
by
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI)
and
hydroxybenzotriazole (HOBt). All other scaffolds were readily accessed by reacting the amino
intermediate with various electrophiles, including sulfonyl chloride for sulfonamides 17,
isocyanate / isothiocyanate for ureas or thioureas 19, and sulfonyl isocyanate for sulfonylureas
2
1. Finally, deprotection of the 5' TBS group using TBAF efficiently generated corresponding 5'
OH analogues (12a–c), (14a–i), (16a–c), (18a–d) and (20a).
To explore the effect of 5' OH protecting group, eight different analogues of triazole 10a were
prepared as depicted in Scheme 2. The protecting group featured in these analogues ranges from
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small methyl (25) and acyl (24) groups to medium sized acetal functionalities (26 and 27) to
bulky dimethoxytrityl (28) and silyl groups (11a, 22 and 23). Synthetically, silylation of the 5'-
OH was effected by treating compound 10a with silylating agents TBSCl, TIPSCl and TBDPSCl
to yield corresponding 5'-O-tertbutyldimethylsilyl ether (TBS) 11a, 5'-O-triisopropylsilyl ether
(TIPS) 22 and 5'-O-tertbutyldiphenylsilyl ether (TBDPS) 23 respectively in good yields.
However, 5'-O-trimethylsilyl ether (TMS) and 5'-O-triethylsilyl ether (TES) were found to be
unstable and could not be isolated in the pure form. The protection of the 5'-OH with various
alkyl ethers was achieved with ethoxymethyl chloride in the presence of DIPEA, 3,4-
dihydropyran in the presence of catalytic amount of pTSA and 4,4’-dimethoxytrityl chloride in
pyridine solvent, resulting in corresponding 5'-O-ethoxymethyl ether 26, 5'-O-tetrahydropyranyl
ether (THP) 11a and 5'-O-dimethoxytrityl ether (DMT) 28 respectively in moderate yields. It
must be pointed out that reacting 6 with methyl iodide in the presence of sodium hydride resulted
in the 5'-O and N-3 bismethylated analogue 25. Lastly, 5'-OH of 10a was easily acylated using
acetic anhydride in pyridine to furnish 5'-O-acyl compound 24 in 87% yield.
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a,b
Scheme 1. Synthesis of AZT-derived scaffolds 8–21
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Reagents and conditions: a) R' substituted alkyne, sodium ascorbate, CuSO ·5H O, THF/H O
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(3:1), rt, 12 h, 54–81%; b) R' substituted alkyne, Cp*RuCl(PPh ) , THF, 60 ºC, 1–2 d, 30–52%;
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c) TBSCl, imidazole, THF, rt, 12 h, 91–76%; d) Pd/C, H , MeOH, rt, overnight, 78%; e) 1)
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R'CHO, MeOH, 50 °C, 2–3 h 2) SHCH COOH, tolune, reflux, 12 h, 51–65%; f) R'COOH,
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HOBt, EDCI, DMF, rt, 6–10 h, 70–80%; g) R'SO Cl, Et N, CH Cl , 0 °C–rt, 81–86%; h)
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R'NCO/ R'NCS, CH Cl , 0 °C–rt, overnight, 63–72%; i) R'SO NCO, CH Cl , 40 °C, 1 h, then rt,
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0 h, 60–70%; j) 1N TBAF, THF, rt, 5–10 h, 80–92%.
R' for all analogues is defined in Tables 2 and 3.
b
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a
Scheme 2. Preparation of 5' OH protected analogues of 10a
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a
Reagents and conditions: a) TIPSCl/ TBSCl/ TBDPSCl, imidazole, THF, rt, 12 h, 76–81%; b)
CH I, NaH, CH Cl , 0 °C–rt, 6 h, 78%; c) C H OCH Cl, DIPEA, CH Cl , rt, 2 h, 66%; d) 3,4-
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dihydropyran, pTSA, CH Cl , 8 h, 72%; e) DMTrCl, pyridine, 60 °C, 6 h, 78%; f) Ac O,
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pyridine, rt, 5 h, 87%.
Antiviral Screening. Since the introduction of a 5' silyl protecting group was to mimic the
bulkiness of NNRTI TSAO-T (2, Figure 1), all analogues of scaffold 3 were first screened in an
HIV-1 antiviral assay as well as a biochemical assay against HIV RT. Unfortunately, none of
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these compounds showed any appreciable activity in either assay (data not shown). Additional
antiviral testing against influenza virus, hCMV and HCV did not yield any hit either (data not
shown); however, when the antiviral screening was expanded to WNV and DENV, the 5'-silyl-
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'-1,2,3-triazole series of scaffold 3 demonstrated consistent inhibitory activities. This
observation led to further efforts on antiviral SAR against WNV and DENV in which
compounds were evaluated for antiviral properties using a viral subgenomic replicon-containing
baby hamster kidney (BHK) replicon cell line. In these assays the level of replicon RNA
produced by the respective viral proteins was monitored by measuring the activity of the renilla
luciferase that is embedded and expressed within each of the WNV and DENV subgenomic
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replicons. Lycorine, a natural product and a reported WNV inhibitor was used at 1 M for
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experiments with the WNV replicon-containing cells. MPA, a published DENV inhibitor, was
used at 1 M as a control inhibitory compound for experiments with the DENV replicon-
containing cells. Initial screening was done at a single concentration (10 M) and an inhibition%
was calculated after three days. In parallel, the cell viability under the same concentration was
determined.
It was observed early on during this study that protecting the 5' OH of the 1, 2, 3-triazole
scaffold 1 with TBS completely flipped its antiviral profile. For example compound 10a
demonstrated exceptionally potent antiviral activity against HIV-1 (EC = 67 nM) as reported
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previously, whereas no appreciable anti-HIV-1 activity was observed with its 5' TBS analogue
11a; in contrast, 10a did not show any inhibition against WNV or DENV while 11a inhibited
both viruses almost completely at 10 M (Table 1). To confirm this SAR trend and further
establish the effect of the 5' protecting group on antiviral potency against WNV and DENV, we
synthesized a panel of seven additional analogues with different 5' OH protecting groups and
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tested them against WNV and DENV at 10 M. The results are summarized in Table 1. The
most striking SAR from this series is that analogues with a bulky silyl protecting group including
TBS (11a), triisopropylsilyl (TIPS, 22), tert-butyldiphenylsilyl (TBDPS, 23) all potently
inhibited both WNV and DENV with 85–100% inhibition at 10 M, whereas small non-silyl
protecting groups such as acetyl (24), methyl (25), ethoxymethyl (26) and tetrahydropyran (THP,
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7) did not yield appreciable antiviral activity. Clearly, a certain level of bulkiness is required at
the 5' position to achieve antiviral activity. In addition, the nature of the protecting group also
appears to substantially impact antiviral potency as a particularly bulky non-silyl dimethoxytrityl
(DMTr, 28) conferred only modest antiviral potencies (23% against WNV and 46% against
DENV). The exact reason why Si-based protecting groups offer drastically better antiviral
activities than C-based ones is presently unclear. Finally, both TIPS (22) and TBDPS (23)
analogues are associated with significant cytotoxicity, rendering TBS as the choice of 5’
protecting group for optimal antiviral activities.
Table 1. Effect of the 5' protecting group (R) on antiviral activities against WNV and DENV.
WNV
DENV
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R
a
a
a
a
Inhibition %
Viability %
Inhibition %
Viability %
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The SAR around the substituent on the 3' triazole ring (R’ group) was explored by testing
another series of synthetic triazole compounds (Table 2). The 5' unprotected analogue (R= H) of
each compound was also included in the assay for comparison purpose. Significantly these 5'-
TBS triazole analogues typically do not show cytotoxicity at 10 M (Table 2) except for 11c and
11e, further substantiating TBS as the optimal 5' protecting group. As for antiviral activity, again
none of the 5' unprotected compounds showed any activity against WNV or DENV while large
inhibition was observed with all 5' protected analogues, with the lone exception of 11h which
has a small cyclopropyl substituent on the triazole ring. Another interesting observation was that
when the bulky group is connected to the triazole ring through a linker, the resulting compound
(e.g. 11g) showed considerably lower potency against both WNV and DENV (11g vs 11b).
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Furthermore, while the substituent is an aromatic ring in most cases, a bulky alkyl group appears
to also confer antiviral activity effectively (compound 11i). Finally, both C5 and C4 substituents
seem to confer nearly equal antiviral activities (9a vs 11a and 9b vs 11d), an SAR trend in stark
contrast with the previously observed anti-HIV SAR where the antiviral potency was
significantly reduced when the bulky substituent is relocated from C5 to C4 triazoles. The
overall SAR from this series appears to indicate that the substitution on the 3' triazole ring may
require a bulkiness threshold, and that once the bulkiness is satisfied, antiviral activity may not
be sensitive to size changes.
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Effect of the bulky substituent (R' group) on antiviral activities against WNV and
DENV.
Substitution
WNV
DENV
Compound
R
R'
Position
(C4 / C5)
Inhibition Viability Inhibition Viability
a
a
a
a
%
%
%
%
1
1a
TBS
H
5
5
97
100
100
85
0
86
10a
11b
7.4
100
TBS
H
5
5
93
99
99
51
0
93
1
0b
1c
4.0
100
1
TBS
5
100
8.0
99
60
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0c
H
5
5
3.3
99
100
69
0
90
11d
TBS
21
100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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5
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1
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8
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1
2
3
4
5
6
7
8
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0
1
0d
H
5
0
100
0
95
1
1
1e
0e
TBS
H
5
5
100
23
10
99
47
92
100
2.3
1
1
1f
0f
TBS
H
5
5
5
5
95
9.0
50
00
79
99
95
0
73
100
84
1
1g
TBS
H
100
96
28
0
10g
1h
10h
100
1
TBS
H
5
5
21
00
100
97
6.4
4.7
93
100
1
1
1i
0i
TBS
H
5
5
85
00
100
100
75
10
93
100
9
a
TBS
TBS
4
4
92
97
100
100
57
77
96
81
9
b
8c
H
4
0
100
8.2
100
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d
H
H
4
4
14
0
100
100
10
0
100
88
8
e
a
10
11
12
13
14
15
16
17
18
19
20
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41
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43
44
45
46
47
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49
50
51
52
53
54
55
56
57
58
59
60
Single concentration assay at 10 M.
Another important aspect of the current SAR concerns the bioisosterism of the 3' 1,2,3-triazole
ring. Replacing a functional group with its bioisosteres for improved target binding is a common
medicinal chemistry practice. Towards this end we synthesized and tested a large number of
compounds with 3' linkers of a few distinct functionalities generally considered as 1,2,3-triazole
bioisosteres, including thiazolidinone, amide, sulfonamide, urea / thiourea and sulfonyl urea.
Again the 5' unprotected analogue for each compound in this series was included in antiviral
assays. These efforts resulted in a few notable observations. First, bioisosteric replacement by
these functionalities did not appear to negatively impact the cytotoxicity profile as the vast
majority of compounds in this series remain non-cytotoxic. Second, just like the 3'-1,2,3-triazole
series, the 5' TBS protecting group is required for antiviral activity as none of the unprotected
analogues were active. Third, many of the TBS protected analogues demonstrated strong
inhibition at 10 M, though some appeared to have noticeably different antiviral potencies
between the WNV and DENV assays. For example, thiazolidinones 13b, 13c, and urea 19c
showed substantially higher inhibition against WNV than DENV, whereas amides 15f, 15h, urea
19a, 19b, and sulfonylurea 21a were considerably more active against DENV than WNV. This
differs from the 3'-triazole series where compounds showed largely similar potencies against
WNV and DENV.
Table 3. Effect of triazole bioisosteres on antiviral activities against WNV and DENV.
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2
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2
2
2
2
3
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3
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3
4
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5
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5
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5
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0
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0
1
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0
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0
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0
1
2
3
4
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8
9
0
WNV
DENV
Compound
R
R'
Inhibition
Viability
Inhibition
Viability
a
a
a
a
%
%
%
%
6
TBS
TBS
NH
2
0
100
0
100
1
1
3a
2a
26
94
22
0
98
H
TBS
H
0
99
99
100
99
1
3b
92
9.7
49
13
12b
100
95
1
1
3c
2c
TBS
H
85
12
90
46
0
97
96
100
1
1
5a
4a
TBS
95
0
50
92
0
8.0
H
100
100
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15b
14b
TBS
H
0
0
100
7.6
0
98
95
10
11
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13
14
15
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19
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21
22
23
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46
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49
50
51
52
53
54
55
56
57
58
59
60
92
1
1
5c
4c
TBS
H
54
00
100
95
28
16
100
94
1
1
5d
4d
TBS
H
37
00
94
87
11
15
99
98
1
1
5e
4e
TBS
H
99
00
51
92
98
45
10
97
1
5f
TBS
19
93
90
27
14f
H
0
97
89
4.3
51
96
98
15g
TBS
24
1
4g
H
0
91
95
23
87
97
26
1
5h
TBS
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
4h
H
0
85
3.3
56
95
1
5i
TBS
44
98
97
100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14i
H
0
6.3
95
1
7a
TBS
H
94
0
84
91
98
0
83
99
16a
1
1
7b
6b
TBS
H
94
0
92
88
73
10
99
100
1
1
7c
6c
TBS
H
33
0
94
99
42
22
100
98
1
1
9a
8a
TBS
H
7.0
0
88
96
37
0
96
98
1
1
9b
8b
TBS
H
5.0
0
94
91
69
21
97
73
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1
1
9c
8c
TBS
H
65
0
92
9.0
8.3
98
99
96
75
10
11
12
13
14
15
16
17
18
19
20
21
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49
50
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52
53
54
55
56
57
58
59
60
1
1
9d
8d
TBS
H
99
0
90
56
92
83
1.3
2
1a
TBS
H
0
0
0
96
84
95
65
3.0
14
71
96
89
20a
2
1b
TBS
a
Single concentration assay at 10 M.
Dose-Response Antiviral Potency. To confirm the observed antiviral potency and gauge activity
profile with these scaffolds, we further tested 12 compounds in dose-response fashion. Amongst
selected compounds, 11 were tested against WNV and 7 against DENV using the same assays
and control compounds as for single dose testing. The selection of compounds was based on their
activity and cell viability against each corresponding replicon cell line at 10 M. As summarized
in Table 4, all compounds tested inhibited WNV and / or DENV in low micromolar range with
the majority showing an EC of single-digit M. In addition, all compounds but one (19b) tested
50
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
against DENV also showed similar level of antiviral activity against WNV, suggesting that our
compounds tend to inhibit both flaviviruses. The dose-response inhibition of our compounds
against WNV and DENV is further manifested in curves depicted in Figure 2. The challenge
with these scaffolds is that, although no or marginal toxicity was observed at 10 M, most
compounds showed only a modest 1–5 fold of selectivity in dose-response testing. The lack of
antiviral selectivity represents a common issue in flavivirus antiviral discovery. Nevertheless,
our dose-response testing did identify two analogues compound (9a and 15d) that did not exhibit
any cytotoxicity at the highest tested concentration (200 M), suggesting that it is possible to
address the toxicity concern of our scaffolds through chemical modifications.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Dose-response testing of selected compounds against WNV and DENV
WNV
DENV
Compound
a
b
a
b
EC50 (M)
CC50 (M)
EC50 (M)
CC50 (M)
c
9a
7.4±1.3
2.9±2.1
8.4±3.7
7.1±0.4
3.4±0.7
9.0±0.5
9.9±0.1
33±2.8
15±2.1
8.4±1.6
--
>200
8.4±0.8
7.3±1.0
14±2.8
7.5±0.7
--
21±7.8
7.9±0.7
31±1.4
22±7.0
--
11a
13±1.4
32±11
24±3.5
15±3.5
27±9.2
15±7.1
1
1
1b
1
1i
3b
1
1
3c
5c
--
--
--
--
c
1
5d
>200
--
--
15i
23±2.1
12±2.1
--
9.6±0.6
7.4±1.4
11±0
--
22±4.9
14±4.2
23±4.9
--
17b
1
9b
9c
1
10±0
16±1.4
a
Concentration inhibiting virus replication by 50%; mean value ± standard deviation from two
b
separate experiments. Concentration resulting in 50% cell death; mean value ± standard
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c
deviation from two separate experiments. No cytotoxicity observed at the highest dose tested
(200 M).
(a)
10
11
12
13
14
15
16
17
18
19
20
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59
60
(b)
Figure 2. Dose response curves of selected compounds in antiviral assays: (a) WNV assay; (b)
DENV assay.
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6
DENV yield reduction assay. To verify the observed dose-response antiviral potency, we also
tested two selected compounds (9a and 15i) in a DENV yield reduction assay. This assay directly
measures the ability of a compound to inhibit viral production. The assay was done by
inoculating Vero cells with DENV and adding compound. The titer of the virus produced was
determined by plating serial dilutions on fresh Vero cells and counting the corresponding number
of plaques. As shown in Figure 3, both compounds tested significantly reduced the titer of
DENV at 10 M, with compound 9a almost completely suppressing viral production (98%
0
1
2
3
4
5
6
7
8
9
0
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0
1
2
3
4
5
6
7
8
9
0
inhibition) and 15i inhibiting DENV (67%) as effectively as the lower dosed MPA (1.0 M).
These results strongly indicate that our compounds indeed directly impact viral replication.
Figure 3. Impact of selected compounds on DENV viral production. Percent reduction in viral
titer at 10 M (average of two separate experiments): 98% for compound 9a and 67% for 15i.
Percent reduction at 1.0 M (one experiment): 68% for MPA.
Antiviral mechanism of action. Since the 5' OH is protected by a silyl group, it is unlikely that
these nucleoside derivatives could get phosphorylated in cells and act as chain terminators.
While the exact antiviral mechanism of action for our compounds remains unclear, we noticed
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that another 5' silylated thymidine-based scaffold as represented by GRL-002 was recently
2
0
reported as an inhibitor type of WNV MTase. Specifically, these analogues were found to
mimic the methyl donor SAM and competitively bind to the SAM binding site of the MTase.
Since the northern part of our compounds is similar to that of the reported chemotype (Figure 4,
a, highlighted), we were prompted to look at the ability of our compounds to bind to MTase.
Toward this end, compound 9a was docked into WNV MTase using the reported crystal
10
11
12
13
14
15
16
17
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2
8
structure of WNV MTase co-crystallized with Sinefungin (SIN) (PDB code: 3LKZ). In our
docking, the predicted binding mode of GRL-002 was found to be identical as reported by
20
Hongmin Li et al. The predicted binding mode of compound 9a and its overlay with GRL-002
is shown in Figure 4b. The thymine and sugar moiety in compound 9a were found to be bound
identical to that of GRL-002 and SIN. The dimethyl-t-butylsilyl core is predicted to bind in the
hydrophobic pocket in which methionine group of SIN occupies which is predicted to confer
selective inhibition of viral MTase over human MTase. The 3’ triazole core of compound 9a is
found to occupy an additional pocket adjacent to the sugar bound region which is not utilized by
earlier reported ligands (GRL-002 and SIN). These additional interactions obtained through the
novel designs of the current analogues in the current study would result in the design of potent
flavivirus inhibitors. Furthermore, the southern part of 9a, the 3’ triazole substituent, appears to
occupy a lot of empty space in the binding groove, which might provide highly beneficial
binding interactions absent from GRL-002. Collectively, molecular modeling suggests that it is
likely that our novel antiviral compounds target the SAM binding site of the MTase.
(a)
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(
b)
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Docking of compound 9a into the crystal structure of WNV MTase (PDB code: 3LKZ
2
8
). (a) Structure of 9a and GRL-002; (b) overlay with GRL-002 (Yellow) and the predicted
binding mode of compound 9a (Magenta) within the MTase SAM-binding pocket. Residues
lining the pocket are highlighted in green sticks.
To confirm the predicted binding mode of our compounds, we tested 12 selected compounds in a
2
9
previously reported SAM competition assay. This assay measures the ability of the compounds
3
to compete against H-labeled SAM–MTase complex formation (Fig. 5). SIN, a close analogue
of SAM and a reported potent inhibitor of SAM binding, was used as a positive control.
3
Remarkably, at 20 M all tested compounds reduced the formation of the H-labeled SAM–
MTase complex to the same level as without MTase, suggesting a complete inhibition of SAM
binding. Interestingly, the competition of cold SAM or SIN at 20 M also led to the same level
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of reduction on the formation of H-labeled SAM–MTase complex, implying that our
compounds could be competitive inhibitors of SAM binding and that the observed potency
against WNV and DENV could contribute to validating viral MTase as a unique antiviral target.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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50
51
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53
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55
56
57
58
59
60
2
1
0000
5000
SAM binding inhibition
10000
5
000
0
Figure 5. Inhibition of the [3H]-SAM-MTase complex formation by SAM, SIN and selected
compounds at 20 µM concentration. The biotinylated DNV3 MTase and 3H-labeled SAM were
incubated with or without compounds AdoMet, SIN, and each compound. The reaction mixtures
29
were mixed with the streptavidin-coated SPA beads and quantified using a Microbeta
scintillation counter.
Conclusions
5'-Silylated AZT-derived 3'-1,2,3-triazole nucleoside bioisosteric scaffolds were found to
consistently inhibit WNV and DENV at low micromolar concentrations without inhibiting HIV
or any other viruses tested. SAR showed that both the 5' silyl protecting group and the 3' bulky
substituent are essential for antiviral activity against WNV and DENV. That none of the 5'-
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6
desilylated, potently HIV-inhibiting analogues showed any activity against WNV or DENV
indicates that a simple silylation-desilylation process can serve as a switch beween inhibiting
WNV / DENV and HIV-1. The antiviral activity in the primary replicon assays was confirmed
through a plaque assay where viral titer reduction was observed. Molecular modeling and SAM-
binding assay indicate that the observed antiviral activity is likely due to binding to flavivirus
MTase.
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Experimental
Chemistry
General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents were either purchased (toluene and MeOH) or dispensed under argon
from an anhydrous solvent system with two packed columns of neutral alumina or molecular
sieves. Flash chromatography was performed on a Teledyne Combiflash RF-200 with RediSep
columns (silica) and indicated mobile phase. All moisture sensitive reactions were performed
1
13
under an inert atmosphere of ultra-pure argon with oven-dried glassware. H and C NMR
spectra were recorded on a Varian 600 MHz spectrometer. Mass data were acquired on an
Agilent TOF II TOS/MS spectrometer capable of ESI and APCI ion sources. Analysis of sample
purity was performed on a Varian Prepstar SD-1 HPLC system with a Phenomenex Gemini, 5
micron C18 column (250mm x 4.6 mm). HPLC conditions: solvent A = H O, solvent B =
2
MeCN; flow rate = 1.0 mL/min; compounds were eluted with a gradient of 20% MeCN/H O for
2
5 min then to 100% MeCN for 40 min. Purity was determined by total absorbance at 254 nm. All
tested compounds have a purity ≥ 96.
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General procedure 1 for silylation. To a solution of 5’-hydroxy nucleoside (1.12 mmol, 1.0
equiv.) and imidazole (2.24 mmol, 2.0 equiv.) in DMF (10 mL) was added appropriate silyl
chloride (1.34 mmol, 1.2 equiv.) and the mixture was stirred at room temperature for 10-12 h.
The reaction progress was monitored by TLC. The solvent was removed in vacuo, diluted with
water and extracted with EtOAc (3 x 20 mL). The organic phase was dried over Na SO , filtered
10
11
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60
2
4
and concentrated. The crude product was purified by column chromatography, eluted with 2-
0% MeOH in CH Cl , yielded the desired compound.
1
2
2
1-((2R,4S,5S)-4-Azido-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-
1
methylpyrimidine-2,4(1H,3H)-dione (5). Yield 91%. H NMR (600 MHz, CD OD) δ 7.55 (s,
3
1
H), 6.15 (t, J = 6.6 Hz, 1H), 4.32-4.31 (m, 1H), 3.95-3.86 (m, 3H), 2.38-2.35 (m, 2H), 1.87 (s,
1
3
3H), 0.94 (s, 9H), -0.13 (s, 6H); C NMR (150 MHz, CD OD) δ 164.8, 150.7, 135.9, 110.1,
3
8
3
4.6, 84.5, 62.7, 60.7, 36.8, 25.0, 17.8, 11.2, -6.6; HRMS-ESI(-) m/z calcd for C H N O Si
16 26 5 4
-
80.1754 [M-H] , found 380.1768.
General procedure 2 for the synthesis of 1,4-triazoles derivatives via CuACC (8a-e). To the
mixture of AZT (0.375 mmol, 1.0 equiv.) and alkyne (0.375 mmol, 1.0 equiv.) in 4.0 mL of
THF/H O (3:1) was added freshly prepared 1 M solution of sodium ascorbate (0.1 equiv.) in
2
water, followed by the addition of freshly prepared 1 M solution of CuSO • 5H O (0.06 equiv.)
4
2
in water. The heterogeneous reaction mixture was stirred at room temperature for 12 h and
monitored by TLC and MS. After the completion, the reaction was evaporated to dryness. The
crude product was purified by column chromatography, eluted with 2-10% MeOH in CH Cl ,
2
2
yielded desired 1,4-triazole.
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(6-methoxynaphthalen-2-yl)-1H-1,2,3-triazol-1-
1
yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8a). Yield 91%. H NMR
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6
(
600 MHz, DMSO-d ) δ 11.35 (s, 1H, 3-NH), 8.83 (s, 1H), 8.31 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H),
6
7
.88 (t, J = 7.2 Hz, 2H), 7.83 (s, 1H), 7.33 (s, 1H), 7.18 (dd, J = 2.6 Hz, J = 9.0 Hz, 1H), 6.45 (t,
J = 6.6 Hz, 1H), 5.40-5.42 (m, 1H), 5.30 (t, J = 5.0 Hz, 1H, 5’-OH), 4.28-4.29 (m, 1H), 3.87 (s,
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13
3
H, OMe), 3.66-3.75 (m, 2H), 2.69-2.83 (m, 2H), 1.81 (s, 3H, CH ); C NMR (150 MHz,
3
DMSO-d ) δ 164.6, 157.9, 150.9, 147.2, 136.8, 134.4, 130.0, 128.9, 127.9, 125.9, 124.0, 121.3,
6
1
19.6, 110.3, 106.4, 84.8, 84.5, 61.4, 61.1, 55.6, 37.5, 12.5; HRMS-ESI(+) m/z calcd for
+
C H N O 450.1777 [M+H] , found 450.1775.
23
24
5
5
General procedure 3 for the synthesis of 1,5-triazoles derivatives via RuACC (10a–i). To the
mixture of AZT (0.5 mmol, 1.0 equiv.) and alkyne (0.75 mmol, 1.5 equiv.) in dry THF (5.0 mL)
was added catalytic amount of Cp*RuCl(PPh ) (0.05 equiv.) and stirred at 60 ºC for 1-2 days.
3 2
The reaction was monitored by TLC and MS. The reaction mixture was evaporated to dryness
and the crude product was purified by column chromatography, eluted with 2-10% MeOH in
CH Cl , yielded desired 1,5-triazole.
2
2
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-(6-methoxynaphthalen-2-yl)-1H-1,2,3-triazol-1-yl)
tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (10a). Yield 39%. The reaction
of AZT (150 mg, 0.56 mmol) with alkyne (143 mg, 0.83 mmol) yielded compound 18e (98 mg,
1
3
8
9%) as a yellow solid. mp 134–136 ºC; H NMR (600 MHz, DMSO-d ) δ 11.36 (s, 1H, 3-NH),
6
.03 (s, 1H), 7.95-7.96 (m, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.56 (d, J = 8.4 Hz, 1H),
7.41 (s, 1H), 7.25 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 6.57 (t, J = 6.8 Hz, 1H), 5.21-5.23 (m, 2H),
.38-4.39 (m, 1H), 3.88 (s, 3H, OMe), 3.56 (dd, J = 1.8 Hz, J = 12.0 Hz, 1H), 3.46 (dd, J = 2.4
4
1
3
Hz, J = 12.0 Hz, 1H), 2.58-2.64 (m, 2H), 1.73 (s, 3H, CH ); C NMR (150 MHz, DMSO-d ) δ
3
6
1
64.1, 158.7, 150.9, 138.6, 136.5, 134.8, 133.4, 130.3, 129.1, 128.5, 128.0, 127.2, 121.6, 120.1,
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10.1, 106.3, 85.4, 85.0, 61.8, 58.7, 55.8, 38.2, 12.7; HRMS-ESI(+) m/z calcd for C H N O
5
2
3
24
5
+
50.1777 [M+H] , found 450.1811.
General procedure 4 for the synthesis of 4-thiazolidinone derivatives (13a–c). The mixture
amine 6 (0.14 mmol, 1.0 equiv.) and appropriate aldehyde (0.28 mmol, 2.0 equiv.) in MeOH (10
ml) was heated at 50 ºC for 2-3 h and the solvent was evaporated under reduced pressure. The
solid obtained was dissolved in toluene (10 mL) and dropwise thioglycolic acid (0.72 mmol, 5.0
equiv.) was added and the reaction was carried out at reflux temperature for 12 h. The reaction
progress was monitored by TLC. The solvent was removed in vacuo, diluted with water and
10
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extracted with EtOAc (3 x 15 mL), washed with an aqueous solution of NaHCO . The combined
3
organic phase was dried over Na SO , filtered and concentrated. The crude product was purified
2
4
by column chromatography, eluted with CH Cl and 4-10% MeOH in CH Cl , yielded the
2
2
2
2
desired 4-thiazolidinone.
-((2R,4S,5S)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-4-(4-oxo-2-phenylthiazolidin-3-
1
yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione
(13a).
Yield
65%.
1
Diastereomeric mixture (1:1.5), H NMR (600 MHz, CD OD) δ 7.65-7.48 (m, 6H), 6.52 (t, J =
3
6.6 Hz, 1H), 5.97-5.95 (m, 1H), 4.46-4.44 (m, 1H), 4.11-4.05 (m, 1H), 3.94-3.84 (m, 2H), 3.71
(dd, J = 1.8 Hz, J = 11.6 Hz, 1H), 3.16 (dd, J = 2.4 Hz, J = 11.6 Hz, 1H), 2.22-2.20 (m, 2H), 1.92
1
3
(s, 3H), 0.94 (s, 9H), 0.06 (s, 3H), -0.00 (s, 3H); C NMR (150 MHz, CD OD) δ 172.6, 164.8,
3
150.8, 139.9, 136.0, 129.2, 127.3, 127.1, 110.0, 86.3, 81.0, 65.0, 63.0, 55.4, 37.5, 32.2, 25.0,
-
1
5
7.7, 11.2, -6.5, -6.6; HRMS-ESI(-) m/z calcd for C H N O SSi 516.1988 [M-H] , found
2
5
34
3
5
16.1975.
General procedure 5 for amide coupling (15a–i). To the mixture of acid (0.21 mmol, 1.0
equiv.), EDCI (0.23 mmol, 1.1 equiv.) and HOBt (0.23 mmol, 1.1 equiv.) in CH Cl : DMF (4:1,
2
2
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6
1
0 mL) was added amine 6 (0.21 mmol, 1.0 equiv.) and stirred for 6-10 h at room temperature
under nitrogen atmosphere. The reaction progress was monitored by TLC. The solvent was
removed in vacuo and extracted with EtOAc (3 x 20 mL). The organic phase was dried over
Na SO , filtered and concentrated. The crude product was purified by column chromatography,
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eluted with CH Cl and 2-10% MeOH in CH Cl , yielded the desired compound.
2
2
2
2
N-((2S,3S,5R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-6-methoxy-2-naphthamide (15a). Yield
1
72%. mp 125–129 ºC; H NMR (600 MHz, CD OD) δ 8.31 (s, 1H), 7.86-7.84 (m, 3H), 7.70 (s,
3
1
1
H), 7.29 (s, 1H), 7.21-7.19 (m, 1H), 6.33 (t, J = 6.6 Hz, 1H), 4.78-4.76 (m, 1H), 4.14-4.13 (m,
H), 4.03-4.01 (m, 1H), 3.96 (dd, J = 1.2 Hz, J = 11.4 Hz, 1H), 3.93 (s, 3H), 2.47-2.41 (m, 2H),
1
3
1.91 (s, 3H), 0.95 (s, 9H), 0.14 (s, 3H), 0.14 (s, 3H); C NMR (150 MHz, CD OD) δ 169.0,
3
1
6
59.4, 150.8, 136.6, 136.0, 130.0, 128.7, 127.3, 126.7, 124.0, 119.3, 110.0, 105.2, 85.0, 84.6,
3.0, 54.4, 49.9, 37.6, 33.3, 25.0, 17.9, 11.2, -6.5. -6.6; HRMS-ESI(-) m/z calcd for
-
C H N O Si 538.2373 [M-H] , found 538.2358.
28
36
3
6
General procedure 6 for synthesis of sulfonamide (17a–c). To a solution of amine 6 (0.17
mmol, 1.0 equiv.) and triethyl amine (0.34 mmol, 2.0 equiv.) in CH Cl (10 mL) was added
2
2
sulfonyl chloride (0.21 mmol, 1.2 equiv.) at 0 °C and slowly warmed to room temperature and
stirred under a nitrogen atmosphere for 12 h. The reaction progress was monitored by TLC. The
reaction was stopped by adding water and extracted with CH Cl (3 x 20 mL). The organic layer
2
2
was dried over Na SO , filtered and concentrated. The crude product was purified by column
2
4
chromatography, eluted with 1-10% MeOH in CH Cl , yielded the desired sulfonamide.
2
2
N-((2S,3S,5R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-4-chlorobenzenesulfonamide (17a). Yield
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8
4%. mp 179–180 ºC; H NMR (600 MHz, CD OD) δ 7.85 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 9.0
3
Hz, 2H), 7.46 (s, 1H), 6.11 (t, J = 6.6 Hz, 1H), 3.92-3.89 (m, 1H), 3.88-3.86 (m, 1H), 3.83 (d, J =
1
2.0 Hz, 1H), 3.65 (dd, J = 3.0 Hz, J = 12.0 Hz, 1H), 2.15-2.13 (m, 1H), 2.10-2.07 (m, 1H), 1.84
10
11
12
13
14
15
16
17
18
19
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46
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50
51
52
53
54
55
56
57
58
59
60
13
(
s, 3H), 0.88 (s, 9H), 0.06 (s, 6H); C NMR (150 MHz, CD OD) δ 162.4, 150.7, 139.6, 138.7,
3
135.8, 129.2, 128.3, 110.1, 84.9, 84.4, 62.1, 52.3, 37.8, 25.0, 17.8, 11.2, -6.6; HRMS-ESI(-) m/z
-
calcd for C H N O SSiCl 528.1391 [M-H] , found 528.1398.
2
2
31
3
6
General procedure 7 for synthesis of urea and thiourea (19a–d). To a solution of amine 6
(0.17 mmol, 1.0 equiv.) in CH Cl (10 mL) was added isocyanate/ thioisocyanate (0.25 mmol,
2
2
1
.5 equiv.) at 0 °C and stirred for 1 h and then slowly warmed to room temperature and stirred
under a nitrogen atmosphere for overnight. The reaction progress was monitored by TLC and
MS. The reaction was stopped by adding water and extracted with CH Cl (3 x 20 mL). The
2
2
organic layer was dried over Na SO , filtered and concentrated. The crude product was purified
2
4
by column chromatography, eluted with 1-10% MeOH in CH Cl , yielded the desired urea or
2
2
thiourea.
N-((2S,3S,5R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
1-((2S,3S,5R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-3-phenylurea (19a). Yield 66%. mp 112–
1
1
15 ºC; H NMR (600 MHz, CD OD) δ 7.65 (s, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.24 (t, J = 7.8
3
Hz, 2H), 6.97 (t, J = 7.8 Hz, 1H), 6.23 (t, J = 6.6 Hz, 1H), 4.42-4.40 (m, 1H), 3.98-3.95 (m, 2H),
13
3
.90-3.88 (m, 1H), 2.34-2.29 (m, 2H), 1.89 (s, 3H), 0.93 (s, 9H), -0.14 (s, 3H), -0.13 (s, 3H); C
NMR (150 MHz, CD OD) δ 164.9, 156.2, 150.8, 139.1, 136.0, 128.4, 122.2, 118.9, 110.0, 85.5,
3
8
4
4.5, 63.0, 50.0, 38.2, 25.0, 17.9, 11.3, -6.5, -6.6; HRMS-ESI(-) m/z calcd for C H N O Si
23 33 4 5
-
73.2220 [M-H] , found 473.2236.
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