In search of flavivirus inhibitors: Evaluation of different tritylated nucleoside analogues

Article abstract of DOI:10.1016/j.ejmech.2013.04.034

Following up on a hit that was identified in a large scale cell-based antiviral screening effort, a series of triphenylmethyl alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against the dengue virus (DENV) and the yellow fever virus (YFV). Hereto, trityl moieties were attached at various positions of the sugar ring combined with subtle variations of the heterocyclic base. Several triphenylmethyl modified nucleosides were uncovered being endowed with submicromolar in vitro antiviral activity against the YFV. The most selective inhibitor in this series was 3′,5′-bis-O-tritylated-5-chlorouridine (1b) affording a selectivity index of over 90, whereas the 3′,5′-bis-O-tritylated inosine congener (5b) displayed the highest activity, but proved more toxic. The finding of these lipophilic structures being endowed with high antiviral activity for flaviviruses, should stimulate the interest for further structureeactivity research.

Full text of DOI:10.1016/j.ejmech.2013.04.034

Accepted Manuscript
In search of Flavivirus inhibitors: evaluation of different tritylated nucleoside
analogues
Gré gory Chatelain, Yannick Debing, Tine De Burghgraeve, Joanna Zmurko, Milind
Saudi, Jef Rozenski, Johan Neyts, Arthur Van Aerschot
PII:
S0223-5234(13)00266-3
10.1016/j.ejmech.2013.04.034
EJMECH 6144
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 February 2013
Revised Date: 16 April 2013
Accepted Date: 17 April 2013
Please cite this article as: G. Chatelain, Y. Debing, T. De Burghgraeve, J. Zmurko, M. Saudi,
J. Rozenski, J. Neyts, A. Van Aerschot, In search of Flavivirus inhibitors: evaluation of different
tritylated nucleoside analogues, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.04.034.
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ACCEPTED MANUSCRIPT

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In search of Flavivirus inhibitors: evaluation of different
tritylated nucleoside analogues
Grégory Chatelaina, Yannick Debinga, Tine De Burghgraeveb, Joanna Zmurkob, Milind
Saudia, Jef Rozenskia, Johan Neytsb, Arthur Van Aerschota*
aMedicinal Chemistry and bVirology and Chemotherapy, Rega Institute for Medical Research,
KU Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium.
* Corresponding author: Dr. Arthur Van Aerschot, Rega Institute for Medical Research, KU
Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium
E-mail address: arthur.vanaerschot@rega.kuleuven.be
Tel.: +32 16 33 73 88
Fax: +32 16 33 73 40
Keywords: Flavivirus inhibitors, Dengue virus, yellow fever virus, bis-O-tritylated
nucleosides, 3’,5’-di-O-trityl-uridine

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ABSTRACT
Following up on a hit that was identified in a large scale cell-based antiviral screening effort,
a series of triphenylmethyl alkylated nucleoside analogues were synthesized and evaluated for
their in vitro antiviral activities against the dengue virus (DENV) and the yellow fever virus
(YFV). Hereto, trityl moieties were attached at various positions of the sugar ring combined
with subtle variations of the heterocyclic base. Several triphenylmethyl modified nucleosides
were uncovered being endowed with submicromolar in vitro antiviral activity against the
YFV. The most selective inhibitor in this series was 3’,5’-bis-O-tritylated-5-chlorouridine
(1b) affording a selectivity index of over 90, whereas the 3’,5’-bis-O-tritylated inosine
congener (5b) displayed the highest activity, but proved more toxic . The finding of these
lipophilic structures being endowed with high antiviral activity for flaviviruses, should
stimulate the interest for further structure-activity research.

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1. Introduction
Several flaviviruses cause severe disease in man. Important members of this genus
(family Flaviviridae) include the tick-borne encephalitis virus, the West Nile virus, the yellow
fever virus (YFV) and the dengue virus (DENV) [1]. There is today no therapy available for
the treatment of these potentially life-threatening viral infections [2,3]. We here report on a
series of tritylated nucleoside analogues of which some inhibit in vitro DENV and YFV
replication. Chemical synthesis of these congeners and antiviral evaluation herewith is
reported.
DENV is a flavivirus transmitted by mosquitoes (Aedes aegypti). Today the virus is
endemic in virtually all tropical and subtropical regions of the world [4]. Currently about 2.5
billion people are at risk for developing dengue fever and it is estimated that 50 to 100 million
infections occur every year. Half a million people develop dengue haemorrhagic fever
annually, of which ~ 25,000 of these cases are fatal [5]. Dengue haemorrhagic fever and
dengue shock syndrome are leading causes of hospitalization and death in children in several
South-East Asian countries [6]. Currently, there is neither vaccine nor an antiviral drug
available, even though dengue is the fastest progressing vector-borne disease worldwide. An
antiviral agent could be of good use in the prevention of epidemics and to avert worsening of
disease severity [4, 5, 7].
YFV is another mosquito-borne flavivirus, endemic in tropical Africa and South
America. It is estimated that 200,000 infections occur annually, leading to 30,000 deaths each
year (http://www.who.int/mediacentre/factsheets/fs100/en/ (November 2011)). A case fatality
rate of 20 to 50% has been observed in the severely infected. Although a highly effective and
safe vaccine (17D) is available [1], an antiviral drug would prove useful for treating the
severe infections that may occur in the non-vaccinated, especially during epidemics [8, 9].

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A regularly used strategy for development of antiviral drugs is the screening of large
compound libraries (in silico and/or in vitro) to identify hit molecules [10] followed by a hit-
optimization process. Efforts in our group to discover inhibitors of flavivirus replication
resulted in the identification of 2’,5’-bis-O-tritylated uridine as a hit. This discovery was
followed by synthesis of a series of close analogues in which different regio-isomers of trityl
containing nucleosides were synthesized as a first attempt for setting up a structure-activity
relationship (SAR) as reported here. In another paper [11], the in vitro anti-flavivirus activity
of the most selective compound identified in this series, i.e. 3’,5’-bis-O-tritylated uridine, is
described in more detail and the mechanism of action, i.e. inhibition of the viral RNA
polymerase, is reported.

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2. Results and Discussion
2.1. Synthesis and analysis of bis-O-tritylated congeners
Synthesis of 5-chlorouridine (9) was initially attempted on unprotected uridine (6) using
ceric(IV)ammonium nitrate (CAN) and concentrated HCl in MeOH at 70°C, according to the
procedure described by Asakura and Robins, Figure 1 [12]. Although 9 was obtained in 79%
yield, neutralization of the reaction mixture with NaOH proved necessary, resulting in
significant amounts of inorganic salts with concomitant solubility problems in several organic
solvents, yielding problems on subsequent tritylation attempts. Hence, synthesis of 9 was
reinitiated according to another protocol by Asakura using acetylated uridine (7) [12]. The
supplementary protection simplified workup and product isolation and avoided the use of
HCl. Treatment of 7 with ceric ammonium nitrate (CAN) and LiCl in anhydrous MeCN at
80°C gave 89% of 8, which was deprotected to afford 9 (85%).
Tritylation of unprotected ribonucleosides (uridine, chlorouridine, inosine, Fig. 1 and 2,
respectively) is straightforward [13, 14] and was done at 80°C overnight using 2.5 to 3 eq. of
triphenylmethyl chloride, affording the 5’-monotritylated (1a, 4a, 5a) and the 2’,5’- and 3’,5’-
bis-O-tritylated analogues (1b-c, 4b-c, 5b-c). The latter regioisomers were separated tediously
by column chromatography, and their structure was proven using 2D-NMR techniques
(COSY, HSQC, HMBC), with the 2’,5’-analogues proving the slightly more lipophilic ones.
Assignment for all compounds starts with unambiguous assignment of the 1H signals, using
COSY NMR (H-H correlation spectroscopy) starting from the easily identifiable H1’. As an
example, for 1b (3’,5’-bis-O-trityl uridine derivative) the H1’ signal is coupled to the H2’
signal at 3.67 ppm. Fortunately, also the OH signal at 2.90 ppm is coupled to the signal at
3.67 ppm, indicating already C2’-OH to be unalkylated (Fig. 4A). HSQC further allows
assignment of the carbon signals (Fig. 4B). Finally, HMBC analysis shows a correlation
between the quaternary carbons of the trityl moieties around 88 ppm with the proton signals

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for respectively H3’ and H5’, and absence of such correlation for H2’. Hence, the structure
for 1b can be unambiguously identified (Fig. 4C). The same strategy was adapted for 1c (Fig.
4D) and for all other tritylated species (additional spectra can be found in the suplementary
file).
In analogy, concomitant mono- and bis-O-tritylation of respectively thymidine and 2’-
deoxyuridine proved straightforward using 1.8 eq. of trityl chloride yielding compounds 2a-b,
3a-b. To obtain the ribonucleoside congeners monotritylated at a secondary alcohol, the more
labile monomethoxytrityl (MTr) moiety was used to temporarily protect the 5’-position,
followed by tritylation of one of the remaining alcohol moieties. Finally, acidic treatment with
TFA in DCM preferentially removed the more labile MTr moiety to afford 1d-e and 4d.
During the tritylation reaction however, the in situ formed pyridinium hydrochloride
combined with the increased temperature removed part of the MTr protecting group and
allowed substitution for a trityl moiety, resulting in low yields of the desired compounds.
Hence, selective 3’-tritylation of thymidine and 2’-deoxyuridine therefore was accomplished
following monobenzoylation of the primary alcohol (Figure 3). Hereto, the 5’-benzoylated
analogues were heated overnight with 1.6 eq. of trityl chloride at 85°C, and deprotected with
LiOH in aqueous methanol to afford the analogues 2c and 3c.
2.2. Assessment of the antiviral activity
The lipophilic uridine analogue 1c was found in an antiviral screen of an in house
available library of nucleosidic compounds to exert selective inhibition of YFV replication
[11] and was used as a lead compound for exploration of its structure activity relationship
properties. The 2’,5’-bis-O-trityl uridine (1c) therefore was resynthesized to confirm its
activity and at the same time the regioisomeric structures with lipophilic trityl
(triphenylmethyl, Tr) moieties attached at different positions on the sugar ring were

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synthesized and evaluated. The hit compound 1c inhibited YFV replication with an EC50 of
1.2 µM, and did not affect normal cell morphology at concentrations up to 100 µM, resulting
in a calculated selectivity index (SI) of around 80. A 25-fold weaker but still selective activity
(SI of ~3) was noticed as well for DENV. Hence, a series of nucleoside derivatives was
synthesized with different heterocyclic bases and with one or two lipophilic triphenylmethyl
moieties attached at different positions of the sugar ring. An overview of all new compounds
tested is given in Fig. 5, and the results are gathered in Table 1.
Whereas the 5’ monotritylated analogue 1a proved 5-fold more potent against DENV,
cytotoxicity increased drastically. Both 1d and 1e, having a single trityl group attached at
either the 2’ or 3’ hydroxyl position, likewise proved to be relatively toxic molecules.
Remarkably, the inhibitory activity of the 3’,5’-bis-O-tritylated regioisomer 1b for DENV
increased more than 15-fold in comparison with the lead compound 1c (EC50 of 1.75 and 30
µM, respectively), but cytotoxicity likewise increased. In contrast, both 1b and 1c exhibited
substantial selectivity for inhibition of YFV replication with a SI of >80.
Removal of the remaining 2’ hydroxyl moiety in 1b resulting in the bis-O-tritylated 2’-
deoxyuridine 2a endowed the compound with analogous inhibitory properties, resulting in a
SI above 17 for inhibition of DENV (EC50 3.7 µM) and above 70 for YFV (EC50 0.9 µM).
The monotrityl analogues 2b and 2c on the other hand resulted in more adverse effects on the
host cell and had lost activity against both the DENV and YFV. Further modification of the
base moiety with a 5-methyl group resulted in the thymidine series 3a-c which proved
considerably less active. Only the bis-O-tritylated derivative 3a resulted in some selectivity
for YFV with an EC50 of 10 µM (a 10-fold reduction in activity in comparison with 2a) and a
CC50 of 70 µM.
Chlorination of the uracil moiety as in the series 4a-d resulted in rather unexpected results.
While none or very weak inhibitory properties were recorded in the DENV screen, all three

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analogues, the mono- and bis-O-tritylated nucleosides 4a-c, were endowed with substantial
antiviral effect against YFV. The 2’,5’-bis-O-tritylated analogue 4c proved most selective
with an EC50 of 0.75 µM and a SI above 90; herein 4c Is markedly less toxic than its
congeners 4a and 4b and thus results in a higher SI. .
Next, the effect of expanding the base moiety to a two-ring heterocycle was evaluated by
analysing tritylated inosine analogues. Herewith, the O6 position of the hypoxanthine base
could be imagined as a possible mimic of the O4 position on the uracil moiety as in 1a-c. All
inosine analogues 5a-c proved relatively toxic. Only the 3’,5’-bis-O-tritylated inosine 5b
resulted in a pronounced antiviral effect (EC50 of <0.4 µM against YFV). In analogy with the
uridine series, again the 3’,5’-modified nucleoside resulted in the best inhibitory properties
but was more cytotoxic than the uridine congener 1b. Finally, a series of cytidine derivatives
either unprotected at the base moiety or N4-benzoylated, were prepared. None of these
molecules inhibited viral replication (data not shown).
To conclude, a general comment to the structure of these compounds is warranted.
Obviously the molecular weight is very high (>700) and the log P of some of the presented
compounds is very high, which are not the optimal properties for drug-like compounds one
might expect. As shown however, a nice inhibitory effect is noticed in a cellular assay and as
discussed by De Burghgraeve et al. [11], the polymerase can be clearly pinpointed as the real
target. Apparently uptake is thus not an issue in this cellular assay. In addition, while the trityl
group is known as a sugar protecting group, its acid stability is relatively high and hence
should not pose too many degradation problems. Nevertheless, we are currently exploring
strategies to substitute for one or both of the trityl moieties.

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3. Conclusion
In summary, several triphenylmethyl modified nucleosides were discovered that were
endowed with relatively pronounced in vitro anti-YFV activity. While the lead compound 1c
displayed an EC50 for YFV of 1.2 µM, the congener 1b inhibited YFV already at slightly
lower concentration and the inhibitory properties against DENV were even more in favor of
1b. Likewise, 4b, 4c and 5b proved submicromolar inhibitors for YFV proliferation. Antiviral
activity against DENV mostly proved less strong. These lipophilic compounds are exerting
their effect through inhibition of the RNA polymerase of the Flaviviridae. Overall, in view of
these simple lipophilic structures being endowed with strong YFV inhibiting properties,
further structure-activity research is warranted.

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4. Experimental protocols
4.1. General synthetic procedures
Starting nucleosides were obtained from ACROS. All other chemicals were provided by
Aldrich or ACROS and were of the highest quality. 1H and 13C NMR spectra were determined
with a 300 MHz Varian Gemini apparatus with tetramethylsilane as internal standard for the
1H NMR spectra (s = singlet, d = doublet, dd = double doublet, t = triplet, br. s = broad signal,
br. d = broad doublet, m = multiplet) and the solvent signal – DMSO-d6 (δ =39.6 ppm) or
CDCl3 (δ =76.9 ppm) – for the 13C NMR spectra. Exact mass measurements were performed
with a quadrupole/orthogonal acceleration time-of-flight tandem mass spectrometer (qTOF2,
Micromass, Manchester, UK) fitted with a standard electrospray ionization (ESI) interface.
All solvents were carefully dried or bought as such. Detailed procedures can be found in the
supplementary file.
4.2. Tritylated uridine analogues
Uridine (6) (1.1 g, 4.4 mmol) was coevaporated twice with dry pyridine and dissolved in dry
pyridine (4.5 mL/mmol). Triphenylmethyl chloride (2.8 eq) was added and the solution was
heated to 80°C overnight under argon. The reaction was quenched by adding methanol (3 mL)
at room temperature for 30 min. The solution was then concentrated and diluted in
dichloromethane. The organic solution was washed three times with a solution of saturated
NaHCO3 and the combined aqueous layers were extracted with dichloromethane. Combined
organic layers were dried over Na2SO4, filtered and concentrated under vacuum. The residue
was purified by column chromatography on silica (going from 20 to 0% hexane in
dichloromethane containing 0.5% triethylamine, followed by 0 to 2.5% MeOH/ DCM and
0.5% triethylamine) and afforded the tritylated compounds 1a (1184 mg, 2.43 mmol, 56%

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yield), 1b (262 mg, 0.36 mmol, 8% yield) and 1c (517 mg, 0.71 mmol, 16% yield) as white
foams.
1
5’-O-trityluridine (1a): H NMR (DMSO-d6, 300 MHz): δ 3.20 (m, 1H, 5a’-H); 3.26 (m,
1H, 5b’-H); 3.96 (m, 1H, 4’-H); 4.09 (m, 2H, 2’-H and 3’-H); 4.16 (d, 1H, J= 5.1 Hz, OH);
5.32 (d, 1H, J=5.7 Hz, 5-H); 5.50 (d, 1H, J= 4.4 Hz, OH); 5.75 (d, 1H, J= 4.0 Hz, 1’-H); 7.28-
7.39 (m, 15H, h,i,j,k,l-H); 7.71 (d, 1H, J=8.1 Hz, 6-H); 11.35 (br s, 1H, 3-NH). 13C (DMSO-
d6, 75 MHz): 63.23 (C5’); 59.51 (C3’); 73.33 (C2’); 82.30 (C4’); 88.91 (Ph3-C and C1’);
101.43 (C5); 127.15, 127.97, 128.27, 143.40 (arom-C); 140.58 (C6); 150.46(C2); 162.98(C4).
HRMS (thgly) calcd. for C28H26N2O6Na+ (M+Na+) 509.1683; found 509.1708.
3’-5’-bis-O-trityluridine (1b): 1H (CDCl3, 500Mhz): δ = 2.90 (d, J = 6.9Hz, 1H, 2’-OH),
3.04 (dd, J = 2.6 and 10.9Hz, 1H, 5’-H), 3.36 (dd, J = 2.1 and 10.9Hz, 1H, 5’-H), 3.65-3.69
(m, 1H, 2’-H), 3.75-3.78 (m, 1H, 4’-H), 4.32 (dd, J = 4.0 and 5.0Hz, 1H, 3’-H), 5.25 (dd, J =
8.2 and 2.2Hz, 1H, 5-H), 5.99 (d, J = 5.1Hz, 1H, 1’-H), 7.18-7.44 (m, 30H, Tr), 7.59 (d, J =
8.2Hz, 1H, 6-H), 8.54 (s, 1H, 3-NH) ppm. 13C (CDCl3, 500 MHz): δ = 63.08 (C5’), 73.38
(C3’), 74.80 (C2’), 82.82 (C4’), 87.77 (4°C of Tr), 88.19 (4°C of Tr), 89.69 (C1’), 102.21
(C5), 127.40, 127.73, 127.94, 128.19, 128.72, 143.11, 143.38 (Tr), 140.35 (C6), 150.38 (C2),
162.78 (C4) ppm. HRMS (thgly) calcd. for C47H40N2O6Na+ (M+Na+): 751.2779; found
751.2776.
1
2’,5’-bis-O-trityluridine (1c): H (CDCl3, 500Mhz): δ = 2.79 (d, J = 4.5Hz, 1H, 3’-H), 3.11
(d, J = 1.5Hz, 2H, 5’-H), 3.98 (s, 1H, 4’-H), 4.51 (dd, J = 4.5 and 7.5Hz, 1H, 2’-H), 5.11 (dd,
J = 2.1 and 8.1Hz, 1H, 5-H), 6.57 (d, J = 7.5Hz, 1H, 1’-H), 7.05-7.31 (m, 30H, Tr), 7.70 (d, J
= 8.1 Hz, 1H, 6-H), 8.85 (s, 1H, 3-NH) ppm. 13C (CDCl3, 500 MHz): δ = 64.37 (C5’), 70.87
(C3’), 77.77 (C2’), 84.32 (C4’), 86.22 (C1’), 87.84 (4°C of Tr), 87.98 (4°C of Tr), 102.68
(C5), 127.50, 127.81, 127.98, 128.28, 128.50, 128.55, 142.84, 143.14 (Tr), 140.98 (C6),

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150.72 (C2), 163.00 (C4) ppm. HRMS (thgly) calcd. for C47H40N2O6Na+ (M+Na+):
751.2779, found 751.2772.
4.4. 5-Chlorouridine (9)
Uridine (6) (3.0 g, 12.3 mmol) was coevaporated with dry pyridine (2x10 mL) and
dissolved in dry pyridine (20 mL). Acetic anhydride (4.1 mL, 43.5mmol) was added and the
mixture was stirred at room temperature overnight under argon. Work-up afforded the
acetylated uridine intermediate 7 (3.81 g, 10.3 mmol, 84% yield) as a white foam [ESI for
C15H18N2O9 (M+): 258.8 (2,3,5-tri-O-acetylribose+H+, 37.5%), 370.7 (M+H+, 13%), 392.9
(M+Na+, 100%)]. The obtained compound 7 (3.81 g, 10.3 mmol) was coevaporated with dry
acetonitrile (3x20 mL), followed by addition of acetonitrile (165 mL), LiCl (515 mg, 12.2
mmol) and dried ceric ammonium nitrate (CAN, 11.3 g, 20.6 mmol). The mixture was stirred
at 80°C for 44h, after which it was concentrated and partitioned between ethyl acetate (400
mL) and brine (200 mL). Further work-up of the organic layer yielded the peracetylated-5-
chlorouridine 8 as a white powder in 89% yield [ESI for C15H17ClN2O9 (M+): 258.8 (2,3,5-tri-
O-acetylribose+H+, 36%), 404.6 (M+H+, 9%), 426.9 (M+Na+, 100%)]. This latter compound
was dissolved in MeOH (25 mL) and ammonia (25% in H2O, 25 mL). The mixture was
stirred at room temperature for 90 min before an additional aliquot of ammonia (10 mL) was
added. The solution was finally stirred overnight, concentrated and coevaporated with toluene
(3x15 mL). The desired product 9 was obtained as a white foam (2.7g, 9.7 mmol, 94%) and
used for tritylation without any further purification. NMR and MS data were in agreement
with published results [12, 15]: ESI for C9H11ClN2O6 [(M-H)-]: 277.0 (100%).

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4.5. General tritylation procedures
The obtained chlorouridine 9 (298 mg, 1.07 mmol) was coevaporated twice with dry
pyridine and dissolved in dry pyridine (4.5 mL/mmol). Trityl chloride (2.8 eq; TrCl) was
added and the solution was heated to 80°C overnight under argon. The reaction was quenched
by adding methanol (3 mL) and work-up including column chromatography on silica afforded
the tritylated compounds 4a (243 mg, 0.48 mmol, 45% yield), 4b (90 mg, 0.12 mmol, 11%
yield) and 4c (174 mg, 0.23 mmol, 21% yield) as white foams.
An analogous procedure on inosine using a mixture of anhydrous pyridine and DMF for
dissolution, afforded after three days of heating the tritylated derivatives 5a (345 mg, 0.68
mmol, 34% yield), 5b (55 mg, 0.07 mmol, 4% yield) and 5c (240 mg, 0.32 mmol, 16% yield)
as white foams. Synthesis of the tritylated uridine derivatives 1a-c has been reported briefly in
the accompanying paper. Analogous procedures yielded the tritylated 2’-deoxynucleoside
derivatives. Selective tritylation at a secondary alcohol on the other hand was accomplished
following protection of the primary 5’-hydroxyl moiety with either a monomethoxytrityl or a
benzoyl moiety and followed by deprotection of the 5’-position.
Only for the tritylated uridine analogues complete NMR assignment has been provided as
an example. For all other compounds only HRMS data are given, and all 1H and 13C NMR
data can be found in the supplementary files, including figures highlighting complete
assignment for the bis-O-tritylated uridine analogues as well as their HPLC purity evaluation.
2’-O-Trityluridine (1d) [16]: HRMS (thgly) calcd. for C28H26N2O6Na [(M+Na)+]: 509.1683;
found 509.1671.
3’-O-Trityluridine (1e) [16]: HRMS (thgly) calcd. for C28H26N2O6Na [(M+Na)+]: 509.1683;
found 509.1689.
5’,3’-di-O-Trityl-2’-deoxyuridine (2a) [17]: HRMS (thgly) calcd. for C47H39N2O5 [(M-H)-]:
711.2864; found 711.2880.

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5’- O-Trityl-2’-deoxyuridine (2b) [18]: HRMS (thgly) calcd. for C28H25N2O5 [(M-H)-]:
469.1769; found 469.1772.
3’-O-Trityl-2’-deoxyuridine (2c): [17]: HRMS (thgly) calcd. for C28H26N2O5Na
[(M+Na)+]: 493.1734, found 493.1745.
5’,3’-di-O-Tritylthymidine (3a) [17, 19]: HRMS calcd. for C48H42N2O5Na [(M+Na)+]:
749.2986; found 749.2993.
5’- O-Tritylthymidine (3b) [20]: HRMS calcd. for C29H28N2O5Na [(M+Na)+]: 507.1890;
found 507.1907.
3’-O-Tritylthymidine (3c): [17, 19]: HRMS (thgly) calcd. for C29H28N2O5Na [(M+Na)+]:
507.1890; found 507.1884.
5’-O-Trityl-5-chlorouridine (4a): HRMS (thgly) calcd. for C28H24ClN2O6 [(M-H)-]:
519.1328; found 519.1339.
3’,5’-di-O-Trityl-5-chlorouridine (4b) [21]: HRMS (thgly) calcd. for C47H38ClN2O6 [(M-
H)-]: 761.2424; found 761.2437.
2’,5’-di-O-Trityl-5-chlorouridine (4c) [21]: HRMS (thgly) calcd. for C47H38ClN2O6 [(M-
H)-]: 761.2424; found 761.2426.
2’-O-Trityl-5-chlorouridine (4d): HRMS (thgly) calcd. for C28H25ClN2O6Na [(M+Na)+]:
543.1293; found 543.1271.
5’-O-Tritylinosine (5a) [22, 23]: HRMS (thgly) calcd. for C29H27N4O5 [(M+H)+]: 511.1976;
found 511.1997.
3’,5’-di-O-Tritylinosine (5b) [24, 25]: HRMS (thgly) calcd. for C48H41N4O5 [(M+H)+]:
753.3071; found 753.3074.
2’,5’-di-O-Tritylinosine (5c) [24, 25]: HRMS (thgly) calcd. for C48H41N4O5 [(M+H)+]:
753.3071; found 753.3089.

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4.6. Antiviral assays and activity determination
The YFV-17D vaccine strain (Stamaril) (Aventis Pasteur MSD, Brussels, Belgium) was
passaged once in Vero-B cells to prepare a working virus stock and stored at −80°C until
further use. DENV serotype 2 New Guinea C (NGC) strain (kindly provided by V. Deubel
[formerly at Institut Pasteur, Lyon, France]) was cultivated in C6/36 mosquito cells (ATCC:
CRL-1660). Green monkey kidney cells [Vero-B cells (ECACC for DENV assays and ATCC
CCL-81 for YFV assays)] were grown in minimum essential medium MEM Rega-3 (Gibco,
Merelbeke, Belgium) supplemented with 10% foetal calf serum (FCS), 1% L-glutamine and
1% sodium bicarbonate. Antiviral assays were performed in medium with 2% FCS. Vero-B
cells were seeded at a density of 7 x 10³ (DENV assay) or 2 x 104 (YFV assay) cells/well in
100 µl assay medium and allowed to adhere overnight. To each well of a 96-well plate, 100 ꢀl
of culture medium containing 100 50% cell culture infectious doses (i.e., CCID50) of virus
was added to each well, after which serial dilutions of the compounds were added. Following
a one-week incubation, culture medium was discarded and cultures were fixed with ethanol
and stained with 1% methylene blue. In parallel, cell cultures were incubated in the presence
of compound and absence of virus. The 50% effective concentration (EC50), which is defined
as the compound concentration that is required to inhibit the virus-induced CPE by 50%, and
50% cytotoxic concentration (CC50), which is defined as the compound concentration that is
required to inhibit the cell growth by 50%, was visually determined. Ribavirin was included
as a reference compound. Two independent experiments were carried out.

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Acknowledgments
Milind Saudi holds a scholarship of the Erasmus Mundus Cooperation Window. Mass
spectrometry was made possible by the support of the Hercules Foundation of the Flemish
Government (grant 20100225–7). The antiviral work was supported by EU FP7 project
SILVER (contract no HEALTH-F3-2010-260644) and the chemistry part was supported by
the Rega Foundation.
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559.

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Figure captions
Figure 1. Synthesis of the tritylated uridine and 5-chlorouridine analogues. Reaction
conditions: i) Acetic anhydride, pyridine, RT; ii) CAN, LiCl, CH3CN, 80°C; iii) aq. Ammonia
– methanol, RT; iv) 2.5 eq. TrCl, pyridine, 80°C overnight; v) 2.5 eq. TrCl, pyridine, 80°C,
48h; 3% TFA in DCM, 2h at °C. Ac: acetyl; Tr: trityl or triphenylmethyl; other abbreviations
see text.
Figure 2. Synthesis of the tritylated inosine congeners. Reaction conditions: 2.5 eq. TrCl,
pyridine, 80°C 48h.
Figure 3. Synthesis of the tritylated deoxynucleosides. Reaction conditions: i) 1.8 eq. TrCl,
pyridine, 80°C overnight; ii) for 2c and 3c: Benzoyl chloride, pyridine, 0°C; TrCl, pyridine,
18h 85°C; LiOH 1M in MeOH-water, 5h at RT. Trityl (Tr) structure is depicted at right.
Figure 4. NMR analysis is shown, corroborating the structure for 1b and 1c. Panel A shows
the COSY spectrum for 1b with assignment of the sugar proton signals. The HSQC spectrum
in Panel B allows assignment of the sugar carbon signals, with in Panels C and D the HMBC
correlation between the quaternary trityl signals and the corresponding sugar proton for
respectively compound 1b and 1c.
Figure 5. Structural overview of all evaluated tritylated nucleoside analogues.

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In search of Flavivirus inhibitors: evaluation of different tritylated
nucleoside analogues
Grégory Chatelaina, Yannick Debinga, Tine De Burghgraeveb, Joanna Zmurkob, Milind Saudia,
Jef Rozenskia, Johan Neytsb, Arthur Van Aerschota*
Highlights
At present there is no therapy for the treatment of Flavivirus infections
A series of tritylated nucleoside analogues was evaluated for antiviral activity
Several bis-O-tritylated nucleosides showed submicromolar YFV inhibiting properties
The most potent and selective inhibitor proved to be the 3’,5’-bis-O-tritylated uridine
The compound afforded a selectivity index of over 400 for inhibition of YFV.

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compound
DENV
YFV
EC50 CC50 SI
#
R1
Tr
Tr
Tr
H
R2
H
R3
H
H
Tr
Tr
H
-
Base EC50 CC50 SI
U
U
U
U
U
U
U
U
T
6
10
1.67
>5
4
18
4.5
1a
1b
1c
1d
1e
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
5a
5b
5c
Tr
H
1.75
30
>10
1
>85
>85
>100 > 3
1.2
25
>100 >80
H
ND
ND
3.7
ND
ND
ND
ND
ND
ND
ND
17
40
18
>65
8
-
35
18
>65
5
1.4
1.8
>70
1.7
2.7
7
H
Tr
Tr
H
-
10
Tr
Tr
H
>17
0.9
3
-
-
Tr
Tr
H
-
5
-
1.5
10
4
Tr
Tr
H
-
35
4
-
70
8
-
T
-
ND
10
-
Tr
H
-
T
10
5
-
18
5
1.8
2.5
2.9
>90
2
Tr
Tr
Tr
H
H
H
Tr
Tr
H
H
Tr
ClU
ClU
ClU
ClU
I
-
2
Tr
H
1
-
0.7
0.75
15
2
30
30
30
10
6
1.75
>70
30
30
10
10
H
ND
ND
1.5
ND
-
Tr
Tr
Tr
H
-
ND
<0,4
ND
-
Tr
H
I
6.6
-
>40
-
I
Table 1. Antiviral activity assesment of the tritylated nucleoside analogues versus Dengue virus and
yellow fever virus, determined as described in the Materials and Methods section.

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O
N
O
O
N
l
C
l
C
NH
NH
NH
r
T
O
R
O
R
O
N
O
O
O
O
O
O
v
i
ii
R
O
R
O
R
O
R
O
R
O
R
O
2
1
a
=
=
=
=
=
=
c
4
6 R
7 R
H
8 R
R1 R2 H
A
i
iii
c
A
4b R1
=
r
T
=
R
2
9
R
H
H
;
c
4
=
= r
R1 H R T
2
;
v
i
O
O
O
X
X
NH
NH
NH
r
T
r
MT
O
H
O
O
N
N
N
O
O
O
O
O
O
v
R
O
R
O
H
O
H
O
R
O
R
O
2
1
2
1
a
=
=
=
=
=
=
=
r
1
R1 R2
H
1
1d X R1 H R
T
0 X
H
Cl
;
2
=
r
T
=
=
e
1
=
=
=
r
T
1b R1
R
H
11
X
R2 H R
X
;
;
2
1
c
1
=
=
r
T
=
=
r
T
R1 H R
4d X
R
H R
;
2
Cl
;
;
2
1

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O
O
NH
NH
N
N
N
N
H
O
R
1O
N
N
O
O
r
T
Cl
80°C
H
O
H
O
R
O
R
O
2
3
a
= r
=
=
=
=
14
5
R1
T
R
R3
H
H
H
;
2
=
= r
T
;
5b R1 R2
R
3
c
5
=
= r
R1 R3 T
;
R
2

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O
N
O
X
X
NH
NH
H
O
R
1O
N
O
O
O
O
or
i
ii
r t
r
T
T
i l
,
y
H
O
R
O
2
a
a
R1
=
=
=
r
;
=
=
=
=
=
r
=
=
H X
C
12 X
13 X
H
2
R1
T
R
=
X
X
X
H
H
H
3
T
R
=
H
;
;
2
2
3
=
r
T
=
r
T
;
=
X H
C
H
C
2b R1 R2
3b R1 R2
;
3
3
c
2
=
r
=
c
3
=
=
r
T
;
=
X
R2
T
R
R1 H R
H
C
;
;
1
2
3

ACCEPTED MANUSCRIPT

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O
O
N
O
N
H
3C
NH
NH
NH
-
-
R1
O
R1
O
-
R1
O
N
O
O
O
O
O
O
-
-
-
R
O
2
R
R
O
O
-
R
O
2
3
2
a-e
1
a-c
2
a-c
3
O
O
N
l
C
N
N
NH
NH
-
R1
O
-
R1
O
N
O
O
O
-
-
R
O
3
R
O
-
-
R
O
2
R
O
2
3
a-c
5
a-
4 d

ACCEPTED MANUSCRIPT
In search of Flavivirus inhibitors: evaluation of different
tritylated nucleoside analogues
Grégory Chatelaina, Yannick Debinga, Tine De Burghgraeveb, Joanna Zmurkob, Milind
Saudia, Jef Rozenskia, Johan Neytsb, Arthur Van Aerschota*
aMedicinal Chemistry and bVirology and Chemotherapy, Rega Institute for Medical Research,
KU Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium.
Appendix A. Supplementary data
Supplementary data are given here regarding chemical synthesis of the different analogues
presented in the paper. The complete synthetic procedure and all analytical details are
provided.
1. Experimental protocols
1.1. General procedures
Uridine, 2’-deoxyuridine and inosine were obtained from ACROS. All other chemicals
were provided by Aldrich or ACROS and were of the highest quality. 1H and 13C NMR
spectra were determined with a 300 MHz Varian Gemini apparatus with tetramethylsilane as
internal standard for the 1H NMR spectra (s = singlet, d = doublet, dd = double doublet, t =
triplet, br. s = broad signal, br. d = broad doublet, m = multiplet) and the solvent signal –
DMSO-d6 (δ =39.6 ppm) or CDCl3 (δ =76.9 ppm) – for the 13C NMR spectra. For some
products a Varian Unity-500 spectrometer (500 MHz for 1H NMR) was used. Coupling
constant values were derived by first-order spectral analysis. Exact mass measurements were
performed with a quadrupole/orthogonal acceleration time-of-flight tandem mass
spectrometer (qTOF2, Micromass, Manchester, UK) fitted with a standard electrospray

ACCEPTED MANUSCRIPT
ionization (ESI) interface. Precoated Machery–Nagel Alugram SILG/ UV254 plates were
used for TLC, and the spots were examined under UV light and by use of sulfuric
acid/anisaldehyde spray. Column chromatography was performed on ACROS silica gel
(0.060–0.200 mm or 0.035–0.060 mm). Anhydrous solvents were obtained as follows:
dichloromethane was stored over calcium hydride, heated at reflux and distilled. Pyridine was
heated at reflux in the presence of potassium hydroxide pellets and distilled.
Dimethylformamide (DMF) was dried with activated molecular sieves (4 Å).
1.2. 5-Chlorouridine (9)
Uridine (6) (3.0 g, 12.3 mmol) was coevaporated with dry pyridine (2x10 mL) and dissolved
in dry pyridine (20 mL). Acetic anhydride (4.1 mL, 43.5mmol) was added and the mixture
was stirred at room temperature overnight under argon. The reaction mixture was cooled to
0°C, quenched with MeOH (10 mL) and stirred for 2h. The mixture was concentrated and
dissolved in ethyl acetate (100 mL), followed by washing with a saturated NaHCO3 solution
(50mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered and
concentrated. The residue was purified by column chromatography on silica (0-3% MeOH/
dichloromethane) yielding the acetylated uridine intermediate 7 (3.81 g, 10.3 mmol, 84%
yield) as a white foam [MS: ESI + for C15H18N2O9 (M): 258.8 (2,3,5-tri-O-acetylribose+H+,
37.5%), 370.7 (M+H+, 13%), 392.9 (M+Na+, 100%)].
The obtained compound 7 (3.81 g, 10.3 mmol) was coevaporated with dry acetonitrile (3x20
mL), followed by addition of acetonitrile (165 mL), LiCl (515 mg, 12.2 mmol) and dried ceric
ammonium nitrate (CAN, 11.3 g, 20.6 mmol). The mixture was stirred at 80°C for 44h, after
which it was concentrated and partitioned between ethyl acetate (400 mL) and brine (200
mL). The aqueous layer was extracted with ethyl acetate (2x200 mL) and the organic layers
were washed with water (300 mL). The organic layer was dried over Na2SO4, filtered and

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concentrated. The residue was purified by column chromatography on silica (0-10% acetone/
dichloromethane) yielding the peracetylated-5-chlorouridine 8 as a white powder in 89%
1
yield [NMR: H (CDCl3, 300MHz): δ = 2.12 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 4.33-4.38 (m, 2H, 5’-H), 4.38-4.42 (m, 1H, 3’-H), 5.32 (m, 1H, 4’-H), 5.33 (m, 1H, 2’-
H), 6.08-6.09 (d, J = 4.7Hz, 1H, 1’-H), 7.74 (s, 1H, 6-H), 8.25 (s, 1H, 3-NH); MS: ESI + for
C15H17ClN2O9 (M): 258.8 (2,3,5-tri-O-acetylribose+H+, 36%), 404.6 (M+H+, 9%), 426.9
(M+Na+, 100%)].
This latter compound was dissolved in MeOH (25 mL) and ammonia (25% in H2O, 25 mL).
The mixture was stirred at room temperature for 90 min before an additional aliquot of
ammonia (10 mL) was added. The solution was finally stirred overnight, concentrated and
coevaporated with toluene (3x15 mL). The desired product 9 was obtained as a white foam
(2.7g, 9.7 mmol, 94%) and used for tritylation without any further purification
1H NMR (DMSO-d6, 300MHz): δ = 3.58-3.71 (m, 2H, 5’-H), 3.87 (m, 1H, 4’-H), 3.98-4.02
(m, 1H, 3’-H), 4.07-4.12 (m, 1H, 2’-H), 5.06-5.08 (d, J = 5.0Hz, 1H, 3’-OH), 5.28-5.29 (t, J =
5.0Hz, 1H, 5’-OH), 5.41-5.43 (m, J = 5.4Hz, 1H, 2’-OH), 5.73-5.74 (d, J = 4.5Hz, 1H, 1’-H),
8.40 (s, 1H, 6-H), 11.86 (s, 1H, 3-NH); MS: ESI- for C9H11ClN2O6 (M): 277.0 (M-H-, 100%)
(CAS number 2880-89-9; Prystas and Sorm, 1964; Asakura and Robins, 1990).
1.3. General tritylation procedure for ribonucleosides
The obtained chlorouridine 9 (298 mg, 1.07 mmol) was coevaporated twice with dry
pyridine and dissolved in dry pyridine (4.5 mL/mmol). Trityl chloride (2.8 eq; TrCl) was
added and the solution was heated to 80°C overnight under argon. The reaction was quenched
by adding methanol (3 mL) at room temperature for 30 min. The solution was then
concentrated and diluted in dichloromethane. The organic solution was washed with a
solution of saturated NaHCO3 (three times) and combined aqueous layers were extracted with