Structure-activity relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase

Article abstract of DOI:10.1016/j.bmcl.2018.04.069

To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA polymerase, a series of 2′- and/or 4′-ribose sugar modified uridine nucleoside phosphoramidate prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of 2′-OH with 2′-F led to be a poor substrate for both dengue virus and human mitochondrial RNA polymerases. Instead of 2′-fluorination, the introduction of fluorine at the ribose 4′-position was found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by mitochondrial RNA polymerase. 2′-C-ethynyl-4′-F-uridine phosphoramidate prodrug displayed potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2018.04.069

Accepted Manuscript
Structure-activity relationship of uridine-based nucleoside phosphoramidate
prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase
Gang Wang, Siew Pheng Lim, Yen-Liang Chen, Jürg Hunziker, Ranga Rao,
Feng Gu, Cheah Chen Seh, Nahdiyah Abdul Ghafar, Haoying Xu, Katherine
Chan, Xiaodong Lin, Oliver L. Saunders, Martijn Fenaux, Weidong Zhong, Pei-
Yong Shi, Fumiaki Yokokawa
PII:
S0960-894X(18)30387-1
https://doi.org/10.1016/j.bmcl.2018.04.069
BMCL 25818
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 April 2018
25 April 2018
29 April 2018
Please cite this article as: Wang, G., Lim, S.P., Chen, Y-L., Hunziker, J., Rao, R., Gu, F., Seh, C.C., Ghafar, N.A.,
Xu, H., Chan, K., Lin, X., Saunders, O.L., Fenaux, M., Zhong, W., Shi, P-Y., Yokokawa, F., Structure-activity
relationship of uridine-based nucleoside phosphoramidate prodrugs for inhibition of dengue virus RNA-dependent
RNA polymerase, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.04.069
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Structure-activity relationship of uridine-
based nucleoside phosphoramidate prodrugs
for inhibition of dengue virus RNA-
dependent RNA polymerase
Leave this area blank for abstract info.
Gang Wang, Siew Pheng Lim, Yen-Liang Chen, Jürg Hunziker, Ranga Rao, Feng Gu, Cheah Chen Seh, Nahdiyah Abdul
Ghafar, Haoying Xu, Katherine Chan, Xiaodong Lin, Oliver L. Saunders, Martijn Fenaux, Weidong Zhong, Pei-Yong Shi,
Fumiaki Yokokawa*
O
DENV RdRp IC50 0.65 M as triphosphate
O
P
mt-RNA POL substrate incorporation 3.0 %
DNA POL substrate incorporation 0 %
DENV EC50 0.57 M (PBMC)
NH
O
N
O
N
O
H
O
O
F
O
CC50 >50 M (HepG2, K562, MT-4)
HO
OH
CC50 >200 M (PC-3, mitochondrial protein synthesis)

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Structure-activity relationship of uridine-based nucleoside phosphoramidate
prodrugs for inhibition of dengue virus RNA-dependent RNA polymerase
Gang Wanga, Siew Pheng Lima, Yen-Liang Chena,c, Jürg Hunzikerb, Ranga Raoa, Feng Gua,c, Cheah Chen
Seha, Nahdiyah Abdul Ghafara, Haoying Xua, Katherine Chana,c, Xiaodong Linc, Oliver L. Saundersc,
Martijn Fenauxc, Weidong Zhongc, Pei-Yong Shia,d, Fumiaki Yokokawaa,c*
aNovartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670
bNovartis Institutes for BioMedical Research, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland
cNovartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, CA 94608, United States
dDepartment of Biochemistry & Molecular Biology, Department of Phamarcology & Toxicology, Sealy Center for Structural Biology & Molecular Biophysics,
University of Texas Medical Branch, Galveston, TX 77555, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
To identify a potent and selective nucleoside inhibitor of dengue virus RNA-dependent RNA
polymerase, a series of 2’- and/or 4’-ribose sugar modified uridine nucleoside phosphoramidate
prodrugs and their corresponding triphosphates were synthesized and evaluated. Replacement of
2’-OH with 2’-F led to be a poor substrate for both dengue virus and human mitochondrial RNA
polymerases. Instead of 2’-fluorination, the introduction of fluorine at the ribose 4’-position was
found not to affect the inhibition of the dengue virus polymerase with a reduction in uptake by
mitochondrial RNA polymerase. 2’-C-Ethynyl-4’-F-uridine phosphoramidate prodrug displayed
potent anti-dengue virus activity in the primary human peripheral blood mononuclear cell-based
assay with no significant cytotoxicity in human hepatocellular liver carcinoma cell lines and no
mitochondrial toxicity in the cell-based assay using human prostate cancer cell lines.
Revised
Accepted
Available online
Keywords:
Dengue virus
Nucleoside
RNA-dependent RNA polymerase
Phosphoramidate prodrug
Mitochondrial RNA polymerase
2009 Elsevier Ltd. All rights reserved
.
Dengue fever (DF) is an acute mosquito-borne viral disease
that causes flu-like symptoms, and occasionally develops
potential life-threatening dengue hemorrhagic fever (DFS) and
dengue shock syndrome (DSS).1 It is caused by the four
serotypes of dengue viruses (DENV-1 to -4) and is widely spread
throughout tropical and sub-tropical countries with an estimated
390 million people infected annually, including 20,000 deaths.2
Secondary infection by a different DENV serotype can increase
the risk of developing severe dengue diseases, therefore the ideal
treatment for DF requires pan-serotype activity.3 The first dengue
vaccine, Dengvaxia® by Sanofi, was first registered in Mexico in
2015 and has been approved in 19 countries for use in endemic
areas. However recent analysis found that Dengvaxia® could
cause more cases of severe disease for those who had never been
infected by dengue virus.5 On the other hand, no antivirals are
currently available for the treatment of DENV infection, despite
many medicinal chemistry efforts on drug discovery being
pursued.6
catalyzes replication of RNA from an RNA template. It is
essential for viral replication and is a proven antiviral target.7
Nucleoside analogs are a clinically proven chemical class of viral
polymerase inhibitors such as for HIV and HCV therapies.8 It is
anticipated that nucleoside DENV polymerase inhibitors can
provide an increased barrier to developing resistance and have
pan-serotype activity, given that they bind to the highly
conserved active site of the RdRp.9 As part of our research for
dengue drug discovery,10 we therefore investigated nucleoside
class DENV RdRp inhibitors. We previously reported the
adenosine-based nucleoside (NITD-008 (1), 7-deaza-2’-C-
ethynyl-adenosine) which potently inhibited DENV both in vitro
and in vivo. However, development of NITD-008 was terminated
due to its insufficient safety profile.11,12 4’-Azido-cytidine (R-
1479 (2)) and its ester prodrug, balapiravir (3), were originally
developed by Roche for the treatment of HCV, but their
development was terminated due to hematologic adverse events
such as lymphopenia.13 4’-Azido-cytidine (2) was found to be
potent against DENV in both cellular and polymerase enzymatic
assays.14 Balapiravir (3) was repurposed for a phase II clinical
trial for the treatment of dengue, but it failed to reduce viral load
in patients, though reasons remain to be clarified (Figure 1).15
DENV is a single stranded RNA virus of the flavivirus genus
in the family Flaviviridae such as West Nile virus, Yellow fever
virus, and Zika virus. The DENV genome encodes three
structural and seven nonstructural proteins, of which the NS5
possesses methyltransferase and RNA-dependent RNA
polymerase (RdRp) activities. The RdRp is a viral enzyme that

Figure 1. Structures and in vitro biological profile of NITD-008 (1), R-1479
(2), and balapiravir (3). POLRMT = mitochondrial DNA-dependent RNA
polymerase, SNIR = single nucleotide incorporation rate in comparison with
the natural NTP of the nucleoside inhibitor (e.g. NITD-008 triphosphate
versus ATP and R-1479 triphosphate versus CTP).
Scheme 1. Synthesis of 2’-C-substituted uridine analogs. Reactions
conditions: (a) Dess-Martin periodinane, CH2Cl2; (b) RMgBr, THF, -70 ~ -40
ºC; (c) BzCl, DMAP, CH2Cl2; (d) uracil, N,O-bis(trimethylsilyl)acetamide,
SnCl4, 60 ºC; (e) NH3, MeOH.
Synthesis of 2’-C-ethynyl/propynyl-2’-F-uridine analogs
began with uridine 11 by applying the methods to introduce 2’-F
reported in the literature.20 Selective protection of 3’,5’-hydroxy
of uridine 11 and oxidation of 2’-OH of 12 gave the 2’-keto
intermediate 13. This intermediate was reacted by the alkynyl
Grignard reagents to provide 2’-C-alkynylarabino derivative 14.
After change of the protective group, nucleophilic fluorination of
the C-2′ tertiary alcohol 16 with (diethylamino)sulfur trifluoride
(DAST) gave the desired 2’-F intermediate 17, which was treated
with ammonia to afford the 2’-C-ethynyl/propynyl-2’-F-uridine
analogs 18 (Scheme 2).
Nucleoside viral RdRp inhibitors, which are chemical analogs
of RNA ribonucleosides, require intracellular metabolism to their
respective nucleoside triphosphates by cellular kinases. The
triphosphate metabolites serve as competing substrates for a viral
polymerase and are subsequently incorporated into the growing
RNA chain, resulting in the termination of the RNA chain
elongation. However, they can be also incorporated by the host
polymerases, such as mitochondrial DNA-dependent RNA
polymerase (POLRMT), potentially leading to mitochondrial
dysfunction in vivo.16 For the discovery of a new class of
nucleoside DENV polymerase inhibitor, it is therefore critical to
evaluate not only the inhibition of viral RNA replication by
nucleotide triphosphate analogs but also the selectivity for their
incorporation by POLRMT. R-1479 triphosphate (4’-azido-CTP)
was reported to be a potent inhibitor for HCV and DENV
RdRp.14 However, 4’-Azido-CTP was incorporated by POLRMT
at a rate similar to its corresponding natural counterpart, CTP
(Figure 1).16 Likewise, NITD-008 triphosphate was observed to
be incorporated by POLRMT at 14.2 % of the single nucleotide
incorporation rate (SNIR) measured at 500 µM (Figure 1) and the
failure of NITD-008 in the pre-clinical safety studies was
suspected to be associated with mitochondrial toxicity based on
histopathological analysis of the tissues.12 In contrast, the
triphosphate metabolite 4 of sofosbuvir (2’-fluoro-2’-C-methyl
uridine phosphoramidate prodrug),17 which has gained approval
for use as a HCV NS5B inhibitor, was incorporated by POLRMT
at a rate of 1.8 %, equivalent to background signal, however it
was not efficacious against DENV RdRp (IC50 18 µM) (Table
1).18 In order to discover a potent and selective nucleoside DENV
polymerase inhibitor, we synthesized 2’- and/or 4’- ribose sugar
modified uridine-based nucleoside analogs and evaluated their
anti-DENV activity in primary human peripheral blood
mononuclear cells (PBMCs), which is one of the major target
cells for dengue viral replication. In addition, the corresponding
triphosphates were evaluated for their inhibition of RNA
replication by DENV polymerase and incorporation by
POLRMT. Herein we report the results of our studies on the
structure-activity relationship (SAR) of uridine-based nucleotide
analogs.
Scheme 2. Synthesis of 2’-C-ethynyl/propynyl-2’-F-uridine analogs.
Reagents and conditions: (a) 1,1,3,3-tetraisopropyl-1,3-dichloro-disiloxane,
pyridine; (b) Dess-Martin periodinane, CH2Cl2; (c) RMgBr, THF; (d)
3HF·Et3N, THF; (e) BzCl, pyridine; (f) DAST, CH2Cl2; (g) NH3, MeOH.
Synthesis of 2’-C-Substituted-4’-F-uridine analogs started
with the 2’-C-ethynyl/propynyl-uridines 10, which were
converted to the 4’,5’-olefins 19. Iodofluorination of 19 using N-
iodosuccinimide (NIS) and 3HF·Et3N yielded 4’-F-5’-iodo
intermediates 20. Benzoylation of 20 followed by oxidative
hydrolysis or displacement of the 5’-iodine gave 22, which was
finally treated with ammonia to afford the desired free 4’-F-
nucleosides 23 (Scheme 3).21,22
2’-C-Substituted uridine analogs were synthesized using a
route described in the literature.19 Dess-Martin oxidation of 5
gave 2-keto ribofuranose 6, which was treated with the
corresponding Grignard reagent to provide 7. After conversion of
the 2’-OH to its benzoate 8, the sugar was coupled with uracil
under Vorbrüggen glycosylation conditions, followed by
deprotection of the benzoate to afford the 2’-C-substituted
uridine analogs 10 (Scheme 1).
Scheme 3. Synthesis of 2’-C-ethynyl/propynyl-4’-F-uridine analogs.
Reactions conditions: (a) i) I2, imidazole, pyridine, THF, ii) DBU, THF, 60

ºC; (b) NIS, 3HF-Et3N, THF; (c) BzCl, DMAP, CH2Cl2; (d) m-CPBA, n-
Bu4NOH aq., TFA or BzONa, 15-crown-5, DMSO, 100 ºC; (e) NH3, MeOH.
(Method A). Alternatively, the free 4’-F-uridine nucleoside was
directly coupled with the more reactive phosphoryl chloride
agent in the presence of weak base, N-methylimidazole (NMI) to
provide the diastereomeric mixture of phosphoramidate (Method
B).
The free nucleosides were converted to their corresponding
triphosphates as shown in Scheme 4.
Scheme 4. Synthesis of triphosphates. Reactions conditions: (a) POCl3,
proton sponge® (1,8-Bis(dimethylamino)naphthalene), P(O)(OMe)3, then
P2O7H2·(n-Bu3NH)2, n-Bu3N, DMF.
As modified uridine analogs are often poor substrates for the
intracellular mono-phosphorylation, these newly synthesized free
uridine analogs were converted to their phosphoramidate
prodrugs to bypass the non-productive phosphorylation step and
subjected to test in the dengue cell-based assay. The free
nucleoside was reacted with the diastereomerically pure
pentafluorophenyl ester agent in the presence of tert-BuMgCl as
a base to afford the single diastereomer phosphoramidate product
(Scheme 5).24 As free 4’-F substituted nucleoside was
decomposed by treating with tert-BuMgCl, 2’,3’-O-
methoxyethylidene protection was required for the 5’-O-
phosphoramidation using the pentafluorophenyl ester agent
Scheme 5. Synthesis of phosphoramidate prodrugs. Reagents and conditions:
For Y=H; PFP agent, t-BuMgCl, THF. For Y= F, X=OH; Method A: i)
CH(OMe)3, p-TsOH, 1,4-dioxane, ii) PFP agent, t-BuMgCl, THF, iii) 80 %
aq. HCOOH. or Method B: phosphoryl chloride agent, NMI, THF.
Table 1.
Anti-dengue activity of the uridine-based nucleoside phosphoramidate prodrugs and inhibition of DENV RdRp and selectivity over POLRMT by their
triphosphates.
Phopsphor
amidate
h-PBMC DENV-
2 EC50 (µM)a
HepG2 CC50
(µM)b
RdRp IC50
(µM)
POLRMT
SNIR (%)d
Y
H
H
R
X
Triphosphate
24e
Me
OH
OH
0.19
0.45
>50
>50
26
28
5.0
1.6
29.4
16.8
27
29
H
H
H
H
F
OH
F
1.9
1.2
>50
>50
>50
>50
>50
>50
30
4
2.0
18
6.7
1.8
2.0
2.2
5.7
3.0
sofosbuvir
Me
31
33
F
>25
>100c
1.1
32
34
36
38
15.9
>20
6.6
F
35e
37
Me
OH
OH
F
0.57
0.65
39
F
OH
9.0
>50
40
2.8
1.7
b
aDENV-2 plaque assay in h-PBMC. 4 days cytotoxicity. cDENV-2 replicon assay in Huh-7. dPOLRMT SNIR was determined at 500 µM of the tested
triphosphate. eTested as phosphorous diastereomixture (Rp/Sp).
The results of the SAR study of uridine-based nucleos(t)ide
analogs and their triphosphates are summarized in Table 1. All
free uridine nucleosides were inactive in the DENV cell-based
assay (EC50 >25 µM). 2’-C-Methyl-uridine phosphoramidate 24
gained an improved cellular anti-DENV activity with an EC50 of
0.19 µM with no significant cytotoxicity in the human
hepatocellular liver carcinoma cell lines (HepG2, CC50 >50 µM),
however the corresponding triphosphate (2’-C-Me-UTP) was
found to be a substrate for incorporation by POLRMT with SNIR
value of 29.4 %. 2’-C-Ethynyl-uridine triphosphate (28) had an
improved potency for DENV RdRp (IC50 1.6 µM) and selectivity
over POLRMT (SNIR 16.8 %), and its phosphoramidate 27

8. Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Nat. Rev.
Drug Discovery 2013, 12, 447.
showed comparable cellular activity to 24 (EC50 0.45 µM).
However, compound 27 was reported to cause adverse effects
due to mitochondrial dysfunction in 8-days toxicity studies in
dogs at >150 mg/kg/day oral administration, suggesting that even
moderate SNIR level by POLRMT increases the risk of in vivo
mitochondrial toxicity.24 2’-C-Propynyl-UTP (30) showed further
reduced SNIR level (6.7 %) while maintaining inhibition of
DENV RdRp (IC50 2.0 µM). However, 2’-C-propynyl uridine
phosphoramidate 29 suffered from 10-fold loss in anti-DENV
cellular activity as compared to 24.
9. Chen, Y.-L.; Yokokawa, F.; Shi, P.-Y. Antivir. Res. 2015, 122, 12.
10. Lim, S. P.; Wang, Q.-Y.; Noble, C. G.; Chen, Y.-L.; Dong, H.;
Zou, B.; Yokokawa, F.; Nilar, S.; Smith, P.; Beer, D.; Lescar, J.;
Shi, P.-Y. Antivir. Res. 2013, 100, 500.
11. Yin, Z.; Chen, Y.-L.; Schul, W.; Wang, Q.-Y.; Gu, F.;
Duraiswamy, J.; Kondreddi, R. R.; Niyomrattanakit, P.;
Lakshminarayana, S. B.; Goh, A.; Xu, H. Y.; Liu, W.; Liu, B.;
Lim, J. Y. H.; Ng, C. Y.; Qing, M.; Lim, C. C.; Yip, A.; Wang, G.;
Chan, W. L.; Tan, H. P.; Lin, K.; Zhang, B.; Zou, G.; Bernard, K.
A.; Garrett, C.; Beltz, K.; Dong, M.; Weaver, M.; He, H.; Pichota,
A.; Dartois, V.; Keller, T. H.; Shi, P. Y. Proc. Natl. Acad. Sci. U.
S. A. 2009, 106, 20435.
2’-C-Ethynyl-2’-F-UTP (32) resulted in
a
significant
reduction in POLRMT SNIR like sofosbuvir trisphosphate 4, but
it lost DENV RdRp inhibitory activity (IC50 15.9 µM). Similarly,
2’-C-propynyl-2’-F-UTP (34) was found to be a poor substrate
for both POLRMT and DENV RdRp.
12. Chen, Y.-L.; Yin, Z.; Duraiswamy, J.; Schul, W.; Lim, C. C.; Liu,
B.; Xu, H. Y.; Qing, M.; Yip, A.; Wang, G.; Chan, W. L.; Tan, H.
P.; Lo, M.; Liung, S.; Kondreddi, R. R.; Rao, R.; Gu, H.; He, H.;
Keller, T. H.; Shi, P. Y. Antimicrob. Agents Chemother. 2010, 54,
2932.
13. Klumpp, K.; Smith, D. B. In Antiviral Drugs: From Basic
Discovery Through Clinical Trials; 1st edition, Kazmierski, W. M.,
Ed.; John Wiley & Sons: Hoboken, 2011; pp 287-304.
In contrast to 2’-F, 4’-F substitution was found to be less
susceptible to DENV RdRp IC50, while reducing POLRMT
SNIR. In particular, 2’-C-ethynyl-4’-F-uridine triphosphate (38)
reduced SNIR by POLRMT up to background level and
displayed 2-fold improvement in DENV RdRp inhibition as
compared to 2’-C-ethynyl UTP (28). In addition, 2’-C-ethynyl-
14. Chen, Y.-L.; Ghafar, N. A.; Karuna, R.; Fu, Y.; Lim, S. P.; Schul,
W.; Gu, F.; Herve, M.; Yokokawa, F.; Wang, G.; Cerny, D.; Fink,
K.; Blasco, F.; Shi, P.-Y. J. Virol. 2013, 88, 1740.
15. Nguyen, N. M.; Tran, C. N.; Phung, L. K.; Duong, K. T.; Huynh,
Hle. A.; Farrar, J.; Nguyen, Q. T.; Tran, H. T.; Nguyen, C. V.;
Merson, L.; Hoang, L. T.; Hibberd, M. L.; Aw, P. P.; Wilm, A.;
Nagarajan, N.; Nguyen, D. T.; Pham, M. P.; Nguyen, T. T.;
Javanbakht, H.; Klumpp, K.; Hammond, J.; Petric, R.; Wolbers,
M.; Nguyen, C. T.; Simmons, C. P. J. Infect. Dis. 2013, 207, 1442.
16. Arnold, J. J.; Sharma, S. D.; Feng, J. Y.; Ray, A. S.; Smidansky,
E. D.; Kireeva, M. L.; Cho, A.; Perry, J.; Vela, J. E.; Park, Y.; Xu,
Y.; Tian, Y.; Babusis, D.; Barauskus, O.; Peterson, B. R.; Gnatt,
A.; Kashlev, M.; Zhong, W.; Cameron, C. E. PLoS Pathog. 2012,
8, e1003030.
4’-F-UTP (38) was shown to be
a poor substrate for
mitochondrial DNA polymerase γ (0 % SNIR by DNA POL γ at
100 µM). The phosphoramidate prodrug 37 of 2’-C-ethynyl-4’-F-
uridine exhibited potent anti-DENV cellular activity with EC50 of
0.57 µM. Compound 37 did not show cytotoxicity in three
different cell lines tested (CC50 >50 µM in HepG2, K562, MT-4).
Moreover, 37 had no effect on mitochondrial protein synthesis in
the PC-3 (prostate metastatic carcinoma) cell-based
mitochondrial toxicity assay (CC50 >200 µM).25
17. Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.;
Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-
R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer, H.
M. M.; Niu, C.; Otto, M. J.; Furman, P. A. J. Med. Chem. 2010,
53, 7202.
In conclusion, a systematic investigation of the SAR at 2’-and
4’-substitution of uridine-based nucleosides led to the finding
that 4’-F modification of the 2’-C-substituted uridine resulted in
a significant reduction in SNIR by POLRMT without affecting
DENV RdRp inhibition. Based on the combination of high
DENV RdRp potency and selectivity over POLRMT and DNA
18. DENV EC50 and IC50 values of sofosbuvir (4) as described in
Table 1 were measured by us.
19. Harry-O’kuru, R. E.; Smith, J. M.; Wolfe, M. S. J. Org. Chem.
1997, 62, 1754.
20. Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish,
P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R. F.; Watanabe, K.
A.; Otto, M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.;
Pankiewicz, K. W. J. Med. Chem. 2005, 48, 5504.
21. Martínez-Montero, S.; Deleavey, G. F.; Kulkarni, A.; Martín-
Pintado, N.; Lindovska, P.; Thomson, M.; González, C.; Götte,
M.; Damha, M. J. J. Org. Chem. 2014, 79, 5627.
POL
γ with potent anti-DENV activity lacking cellular
cytotoxicity, 2’-C-ethynyl-4’-F-uridine phosphoramidate prodrug
was considered to be a promising lead for a nucleoside inhibitor
of DENV RdRp.26
Acknowledgments
22. Wang, G.; Wan, J.; Hu, Y.; Wu, X.; Prhavc, M.; Dyatkina, N.;
Rajwanshi, V. K.; Smith, D. B.; Jekle, A.; Kinkade, A.; Symons,
J. A.; Jin, Z.; Deval, J.; Zhang, Q.; Tam, Y.; Chanda, S.; Blatt, M.;
Beigelman, M. J. Med. Chem. 2016, 59, 4611.
We also thank Dr. Krishna Reddy Valluru and his colleagues
in GVK Biosciences for their technical assistance.
23. Ross, B. S.; Reddy, P. G.; Zhang, H.-R.; Rachakonda, S.; Sofia,
M. J. J. Org. Chem. 2011, 76, 8311.
References and notes
24. Fenaux, M.; Lin, X.; Yokokawa, F.; Sweeney, Z.; Saunders, O.;
Xie, L.; Lim, S. P.; Uteng, M.; Uehara, K.; Warne, R.; Gang, W.;
Jones, C.; Yendluri, S.; Gu, H.; Mansfield, K.; Boisclair, J.;
Heimbach, T.; Catoire, A.; Bracken, K.; Weaver, M.; Moser, H.;
Zhong, W. Antimicrob. Agents Chemother. 2016, 60, 7077.
25. Feng, J. Y.; Xu, Y.; Barauskas, O.; Perry, J. K.; Ahmadyar, S.;
Stepan, G.; Yu, H.; Babusis, D.; Park, Y.; McCutcheon, K.;
Perron, M.; Schultz, B. E.; Sakowicz, R.; Ray, A. S. Antimicrob.
Agents Chemother. 2016, 60, 806.
1. Whitehorn, J.; Simmons, C. P. Vaccine 2011, 29, 7211.
2. Bhatt, S.; Gething, P. W.; Brady, O. J.; Messina, J. P.; Farlow, A.
W.; Moyes, C. L.; Drake, J. M.; Brownstein, J. S.; Hoen, A. G.;
Sankoh, O.; Myers, M. F.; George, D. B.; Jaenisch, T.; Wint, G.
R. W.; Simmons, C. P.; Scott, T. W.; Farrar, J. J.; Hay, S. I.
Nature 2013, 496, 504.
3. Guzman, M. G.; Alvarez, M.; Halstead, S. B. Arch. Virol. 2013,
158, 1445.
26. During our research, it was reported in WO2014100505 by Alios
Biopharma that 2’-C-ethynyl-4’-F-UTP was not an efficient
substrate for incorporation by POLRMT. Inhibitory activity of
DENV RdRp by 2’-C-ethynyl-4’-F-UTP and cell-based anti-
dengue activity by 2’-C-ethynyl-4’-F-urdine mono-phosphate
prodrug were later reported in US2015015341 by also Alios
Biopharma.
4. Capeding, M. R.; Tran, N. H.; Hadinegoro, S. R.; Ismail, H. I.;
Chotpitayasunondh, T.; Chua, M. N.; Luong, C. Q.; Rusmil, K.;
Wirawan, D. N.; Nallusamy, R.; Pitisuttithum, P.; Thisyakorn, U.;
Yoon, I.-K.; van der Vliet, D.; Langevin, E.; Laot, T.; Hutagalung,
Y.; Frago, C.; Boaz, M.; Wartel, T. A.; Tornieporth, N. G.;
Saville, M.; Bouckenooghe, A. Lancet 2014, 384, 1358.
5. Normile, D. Science 2017, 358, 1514.
6. Behnam, M. A. M.; Nitsche, C.; Boldescu, V.; Klein, C. D. J.
Med. Chem. 2016, 59, 5622.
7. Lim, S. P.; Noble, C. G.; Shi, P. Y. Antivir. Res. 2015, 119, 57.

Supplementary Material
Supplementary data (synthetic procedures, spectral
characterization and the antiviral assays) associated with this
article can be found, in the online version, at http:// --
Click here to remove instruction text...

• Nucleoside triphosphate is a prerequisite
for polymerase-mediated pharmacology
• SAR of ribose sugar modified uridine
triphosphates for dengue RdRp polymerase
• 2’-or 4’-F-uridine triphosphates reduced in
uptake by mitochondrial RNA polymerase
• Monophosphate prodrug bypasses the
nonproductive intracellular 1st
phosphorylation
• 2’-C-Ethynyl-4’-F-uridine phosphoramidate
displayed potent anti-dengue activity