Benzouracil-coumarin-arene conjugates as inhibiting agents for chikungunya virus

Article abstract of DOI:10.1016/j.antiviral.2015.03.013

Chikungunya virus (CHIKV) is an arbovirus that was first recognized in an epidemic form in East Africa in 1952-1953. The virus is primarily transmitted through mosquitoes and the resulting disease, chikungunya fever, is found in nearly 40 countries. Neith

Full text of DOI:10.1016/j.antiviral.2015.03.013

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Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
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Benzouracil–coumarin–arene conjugates as inhibiting agents
for chikungunya virus
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a
b,
a
a
Jih Ru Hwu a,b, , Mohit Kapoor , Shwu-Chen Tsay , Chun-Cheng Lin , Kuo Chu Hwang ,
⇑
⇑
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Jia-Cherng Horng a, I-Chia Chen a, Fa-Kuen Shieh b, Pieter Leyssen c, Johan Neyts c,
⇑
a Department of Chemistry & Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan
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b Department of Chemistry, National Central University, Jhongli City 32001, Taiwan
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Chikungunya virus (CHIKV) is an arbovirus that was first recognized in an epidemic form in East Africa in
1952–1953. The virus is primarily transmitted through mosquitoes and the resulting disease, chikun-
gunya fever, is found in nearly 40 countries. Neither an effective vaccine nor a specific antiviral drug
exists for treatments of chikungunya fever. Thus 22 new conjugated compounds of uracil–coumarin–
arene were designed and synthesized as potential inhibiting agents. Their chemical structures were
determined unambiguously by spectroscopic methods, including single-crystal X-ray diffraction crystal-
lography. The three units in these conjugates were connected by specially designed –SCH2– and –OSO2–
joints. Five of these new conjugates were found to inhibit CHIKV in Vero cells with significant potency
Received 18 January 2015
Revised 24 March 2015
Accepted 25 March 2015
Available online xxxx
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Keywords:
Chikungunya virus
Benzouracil
Coumarin
Conjugate
(EC50 = 10.2–19.1 lM) and showed low toxicity (CC50 = 75.2–178 lM). The selective index values were
8.8–11.5 for three conjugates. By analysis of the data from the anti-viral assays, the structure–activity
relationship is derived on the basis of the nature of the uracil, the functional groups attached to the arene,
and the joints between the ring units.
Thiomethylene
Sulfonate ester
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Ó 2015 Published by Elsevier B.V.
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1. Introduction
drugs with activity against CHIKV is becoming a global concern
of the utmost importance.
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Chikungunya virus (CHIKV) causes severe persistent arthralgia
that has affected at least 41 countries with about one million sus-
pected and 22,500 confirmed cases (Scholte et al., 2015). These
cases are due to a recent massive outbreak of the Caribbean island
in October 2013. Over the past half century, a number of CHIKV
cases have emerged mainly in Africa and Asia (Gerardin et al.,
2008) since it was first recognized in East Africa in 1952–1953
(Schwartz et al., 2012). The imported transmission happened to
Italy in 2007 and to France in 2010 because of infected travelers
(Albulescu et al., 2014). As the competent vectors exist in many
regions, the CHIKV might continue to threat the Americans, includ-
ing parts of the United States (Scholte et al., 2015). Presently, there
is neither an effective vaccine nor a specific antiviral drug available
for this disease (Cavrini et al., 2009). Hence the development of
CHIKV belongs to the genus Alphavirus and is an enveloped
virus with positive sense, single stranded RNA genome
a
(Bassetto et al., 2013). With the growing available structural infor-
mation about the CHIKV genome, the viral nonstructural proteins
(particularly nsP2 acting as protease as well as helicase) and the
viral envelope proteins are identified as the most promising targets
for drug design through inhibitory binding of the targeted proteins
(Rashad et al., 2014). Several chemical compounds have been
reported to exhibit weak anti-CHIKV activity. They include
flavones and their analogs (e.g., apigenin, chrysin, (5,7-dihydroxy)
flavones, and silibinin), nucleoside derivatives (e.g., 6-azauridine
and ribavirin), heterocyclic compounds (e.g., chloroquine, myco-
phenolic acid, and prothipendyl), and interferon etc. (Briolant
et al., 2004; Ozden et al., 2008; Pohjala et al., 2011; Khan et al.,
2011; Kaur et al., 2013). In 2011, arbidol and its two derived
metabolites were tested in vitro on the CHIKV by use of two cell
lines in pre- and post-infection conditions (Delogu et al., 2011).
⇑
Corresponding authors at: Department of Chemistry & Frontier Research Center
on Fundamental and Applied Sciences of Matters, National Tsing Hua University,
Hsinchu 30013, Taiwan (J.R. Hwu).
Arbidol is found to have an IC50 of 12.2
reported that two purine-b-lactam hybrids possess anti-CHIKV
activity with EC50 of 17.11 and 13.01 M, respectively, and selec-
tivity indices (SIs) of >5.75 and >4, respectively (D’hooghe et al.,
lM. D’Hooghe et al.
l
E-mail addresses: jrhwu@mx.nthu.edu.tw (J.R. Hwu), tsay.susan@gmail.com
(S.-C. Tsay), johan.neyts@rega.kuleuven.be (J. Neyts).
http://dx.doi.org/10.1016/j.antiviral.2015.03.013
0166-3542/Ó 2015 Published by Elsevier B.V.
Please cite this article in press as: Hwu, J.R., et al. Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus. Antiviral Res.
(2015), http://dx.doi.org/10.1016/j.antiviral.2015.03.013

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2012). In 2013, Brancale et al. reported that benzylidene(cyclo-
propane)carbohydrazide inhibits the virus-induced cytopathic
effect with an EC50 of 5.0 lM and SI of 14 (Bassetto et al., 2013).
Results from a recent review indicate the broad pharmacological
activity of natural and synthetic coumarins (Riveiro et al., 2010). It
has drawn much attention to drug discovery. The substituent pat-
tern and structural feature within the coumarin family are vital to
the improved activity. Kostova and co-workers found that coumar-
ins and their derivatives have prominent inhibitory effect against
chymotrypsin-like proteases of HIV (Kostova et al., 2005). Based
on the structure–activity relationships of several 3-substituted-4-
hydroxycoumarins, the substituents at positions 5 and 7 of the cou-
marin ring play an important role on the inhibitory potency of the
HIV-1 protease (Kirkiacharian et al., 2002). Recently, coumarins
conjugated with various benzimidazoles (Hwu et al., 2008; Tsay
et al., 2013), benzothiazoles, or benzoxazoles (Neyts et al., 2009)
have been synthesized by our research group. These conjugates
exhibit appealing anti-HCV activity and become promising leads
for further development. Replacement of the benzimidazole unit
with nucleobases results in the related conjugates with enhanced
anti-HCV activity (Hwu et al., 2011a,b). The data indicate the
importance of the thiomethylene (–SCH2–joint used for the connec-
tion of coumarin moieties with various heterocycles on the inhibi-
tory potency of HCV (Hwu et al., 2011a,b).
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In this study, we designed a new molecular structure 1 primar-
ily composed of a ‘‘triply’’ conjugated skeleton of uracil–coumarin–
arene for the development of new anti-CHIKV agents. The C-40 and
C-70 positions of coumarins were deliberately linked to the uracil
and arene units with a thiomethylene (SCH2) and a sulfonate ester
(OSO2) (or replaced by an oxymethylene (OCH2)) joints to form tri-
ply conjugated compounds.
The uracil, thymine (i.e., 5-methyluracil), and benzouracil (i.e.,
quinazolinone) moieties exist in many biologically active com-
pounds (Lagoja, 2005). Recently, Wang et al. (2012) revealed that
the substituents on the C-5 and C-6 positions (i.e., R1 and R2,
respectively, in 1) of the uracil unit are important to anti-viral
activity. Abdel-Aal (2010) synthesized compounds with two
hydrophobic residues at the C-5 and C-6 positions. These com-
pounds exhibited potent activity against hepatitis B virus (HBV).
Ordonez et al. (2012) found that the introduction of a substituent
at the C-6 position of uracil resulted in elevated anti-HIV-1 activity.
Benzouracil and its derivatives have been extensively studied
because of their wide range of pharmacological activities.
Alogliptin containing a benzouracil moiety is a potent selective
inhibitor of serine protease (Feng et al., 2007). Moreover, the
benzouracil unit is widely present in biologically active natural
products (Horton et al., 2003).
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Compounds containing a sulfo (R–OSO2–R0) joint have been
identified as potent protease inhibitors (Shenai et al., 2003). The
reaction of sulfonate compounds with the hydroxyl site of an
active protease residue can form a stable enzyme derivative
(Powers et al., 2002). Nitrophenyl esters of benzenesulfonic acid
Scheme 1. PDF Syntheses of coumarin derivatives with benzenesulfonyl or benzyl fragments.
Please cite this article in press as: Hwu, J.R., et al. Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus. Antiviral Res.
(2015), http://dx.doi.org/10.1016/j.antiviral.2015.03.013

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Scheme 2. PDF synthesis of the benzouracil–, thymine–, and uracil–coumarin–arene conjugates bearing the –SCH2– and –OSO2– joints.
Scheme 3. PDF Synthesis of uracil–coumarin–arene conjugates bearing the –SCH2– and –OCH2– joints.
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and phenylmethanesulfonic acid bearing various positively
charged groups on the benzene ring act as inactivators of tryp-
sin-like proteases (Powers et al., 2002). For example, p-nitrophenyl
p-(amidinothiomethyl)benzenesulfonate inactivates thrombin.
Recently, Udommaneethanakit et al. (2014) reported the inhibition
Herein we report the scope of uracil–coumarin–arene conju-
gates as an unprecedented class of anti-CHIKV agents. In total, 22
compounds with the common skeleton 1 were synthesized, five
of which exhibited significant inhibitory effect against CHIKV.
Through analysis of their anti-CHIKV activities influenced by the
types of joints and substituents on the uracil and the arene rings,
their SAR guidelines were deduced accordingly.
of avian influenza
A virus with sulfonate drugs, such as
Oseltamivir, Peramivir–sulfonate, and Zanamivir.
Please cite this article in press as: Hwu, J.R., et al. Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus. Antiviral Res.
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generation of a nucleophile from 2-thiobenzouracil 9 resulted in
problems with either ring opening of the coumarin moiety or sub-
stitution at the allylic chloride or both. Workup conditions with
appropriate pH values between 10.0 and 11.5 were found to be
critical for sustaining the S–CH2 single bond in 10. Accordingly,
the desired triply conjugated compounds 10a–e were synthesized
through alkylation at the C-2 thione center of 9, which resulted
from the presence of a preferred negative charge on the exocyclic
sulfur atom instead of the exocyclic oxygen atom (Somers et al.,
2001). As a result, the triply conjugated compounds 10a–e,
12a–e, and 14a–e, which contained units of benzouracil,
methyluracil (i.e., thymine), and uracil, were obtained in 81–94%
yields (see Scheme 2). Potential by-products caused by the
additions of uracil to the lactone carbonyl group of coumarins 6
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2. Materials and methods
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A detailed description of the materials and methods is available
in the Supporting information file.
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3. Results and discussion
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For the synthesis of the triply conjugated compounds with scaf-
folds 1 (i.e., uracil–coumarin–arenes), three challenges had to be
overcome. First, the labile sulfo group connecting the coumarin
and arene moieties had to remain intact throughout the synthetic
steps. Second, the conditions to form the S–CH2 single bond had to
be sufficiently mild to maintain the bond intact. Third, the reagents
could not induce an undesired Michael addition to the a,b-unsatu-
or to the
a,b-unsaturated carbon to carbon double bond were
rated ester group or ring-opening of the d-lactone moiety in 1.
Given these three concerns, the sequential synthetic routes shown
in Schemes 1–4 were designed and achieved with success.
prohibited.
Under similar reaction conditions, the triply conjugated com-
pounds 15a–d, 16a–d, and 17a–d with –SCH2– and –OCH2– joints
were also obtained as shown in Scheme 3. The corresponding
doubly conjugated compounds 18–20 without the arene moiety
were produced (see Scheme 4) as the reference compounds for
deduction of their structure–activity relationship.
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3.1. Syntheses of triply conjugated uracil–coumarin–arene targets and
their analogs (Schemes 1–3)
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For the construction of the triply conjugated compounds 1, the
intermediates (arenesulfonyl)coumarins 6 and benzylcoumarins 8
were first produced by sulfonylation or benzylation of 7-hydroxy-
coumarin 4, prepared from resorcinol (2) and ethyl 4-chloroace-
toacetate (3) as the starting materials (Opanasenko et al., 2013),
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3.2. Structure identification of new conjugated compounds
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The structures of all new compounds were determined accord-
ing to their spectroscopic characteristics. Moreover, molecular
framework of the uracil–coumarin–arene conjugate 14a was deter-
mined by single X-ray diffraction analysis to confirm the connec-
tion of the uracil unit to the coumarin through the C@O rather
than the C@S site (Fig. 1). Its monoclinic crystals possessed the
space group P1 21/c 1 with a = 21.413(4) Å, b = 6.2583(12) Å,
with benzenesulfonyl chlorides
5
or benzyl bromides 7,
respectively (Scheme 1). Accordingly, two series of doubly conju-
gated coumarins 6 and 8 were obtained, in which the substituents
R included Me, F, Cl, Br, Me, and NO2 in the ortho or para position.
The coupling of 2-thiobenzouracil
9
with various
(4-chloromethyl)coumarins 6 to generate uracil–coumarin–arene
conjugates 10a–e were performed under different alkaline
conditions. At elevated temperatures (>65 °C), the sulfo group
(i.e., –OSO2–) in 10 was destroyed. Use of bases, such as (N,N-diiso-
propyl)ethylamine and (4-dimethylamino)pyridine for the
c = 14.392(3) Å,
a = 90°, b = 99°, and c = 90°. These results indicate
that the alkylation took place unequivocally at the C-2 position
of the uracil and thus confirm the formation of an NCS–CH2 single
bond.
Scheme 4. PDF syntheses of doubly conjugated compounds containing benzouracil–, thymine–, and uracil–coumarin units.
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Fig. 1. PDF molecular frameworks: ORTEP diagram of 14a obtained by X-ray diffraction analysis.
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window of the compound in the assay system. The antiviral effect of
compounds that adversely affected the host cell metabolism was
likely as a result of a pleiotropic or non-specific effect on the host
cell.
Among these 22 new conjugated compounds, 10a, 10b, 10e,
12e, and 14e exhibited appealing potency in CHIKV Vero cells with
EC50 values ranging from 10.2 to 19.1 lM (Table 1). They displayed
a significant window of selectivity with SI values between 5.6 and
11.5.
Molecular lipophilicity, quantified as logP, of chemical entities
often plays an important role on the development of drug leads
(Hann and Keserü, 2012). For druglike molecules, a good range
for their logP values is between ꢀ0.4 and 5.6 (Ghose et al.,
1999). Nevertheless, some hydrophilic drugs have the value less
than ꢀ5.0. Poor absorption or permeation is more likely to occur
when the calculated logP is greater than 5 (Leeson, 2012).
Chloroquine is a currently possible treatment for chronic chikun-
Table 1
Inhibitory effects of conjugated compounds on CHIKV (899 strain) in Vero cells
subtype A and their lipophilicity.
SIc
logP
a
b
Conjugates
CC50
(
lM)
EC50 (lM)
10a
10b
10c
10d
10e
12a
12b
12c
12d
12e
14a
14b
14c
14d
14e
15a
15b
16a
16b
16c
17a
17d
18
178
117
30
117
144
126
114
86.4
–
107
–
60.2
111
–
75.2
–
102
–
–
104
–
19.1
10.2
18.4
54.5
17.2
58
9.3
11.5
1.6
2.2
8.8
2.2
4.3
–
–
5.6
–
2.6
–
–
5.8
–
–
–
–
2.3
–
3.0
>1.5
–
1.91
2.13
–
–
ꢀ1.07
–
26.4
116
0.611
–
–
>199
19.0
57.4
23.1
128
>205
13.0
>45.2
>219
>246
>48
ꢀ2.60
–
0.467
–
–
ꢀ2.74
3.35
3.57
–
gunya arthritis with EC50 of 717 lM (Kaur et al., 2013), but this
compound has a logP value of 5.3. Consequently, the ‘‘shake-flask
method’’ (Kraszni et al., 2003) was applied to obtain the logP
values of the triply conjugated compounds 10a, b, e, 12b, e, 14b,
and 15a, b as well as the doubly conjugated compound 18 for
comparison (Table 1). The data showed the molecular lipophilicity
of compounds 10–18 fell into the range of ꢀ2.74 to 3.57.
–
–
–
–
1.32
–
–
45.1
>255
4.6
13.8
>284
192
19
20
–
–
>316
>331
–
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3.4. Structure activity relationship: essential moieties and functional
groups
a
The concentration of a compound with an adverse effect of 50% was observed
on the host cell metabolism, as determined by the MTS method.
b
The concentration of a compound at which virus replication was inhibited by
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To explore the SAR of conjugates in this new compound library,
two families of triply conjugated compounds and one family of
doubly conjugated compounds were synthesized. These included
pyrimidine–SCH2–coumarin–OSO2–arene (i.e., compounds 10, 12,
and 14), pyrimidine–SCH2–coumarin–OCH2–arene (i.e., compounds
15–17), and pyrimidine–SCH2–coumarin–OH (i.e., compounds
18–20). Different pyrimidine or its analogs (including uracil, thy-
mine, and benzouracil) and arene with different substituents
(including Me, F, Cl, Br, Me, and NO2) were used to investigate the
influence on their bioactivities.
50% was observed, as determined by real-time quantitative RT-PCR.
c
Selectivity index.
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3.3. Evaluation of the anti-CHIKV activity and measurement of
lipophilicity
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The biological activity of the conjugated compounds against the
CHIKV (899 strain) in Vero cells subtype A was evaluated according
to the established methods (Bassetto et al., 2013). From the
obtained dose–response curves, the concentration of a compound
that inhibited virus replication by 50% (EC50) and the concentration
of a compound that reduced host cell metabolism by 50% (CC50
were calculated. The results in Table 1 were used to determine
the selectivity index (SI = CC50/EC50), a measure for the therapeutic
According to their EC50, CC50, SI, and logP values (Table 1),
benzouracil–SCH2–coumarin–OSO2–arene triply conjugated com-
pounds 10 displayed the most desirable anti-CHIKV activity among
all new compounds. Successful examples include 10a, 10b, and 10e
)
with EC50 = 10.2–19.1 lM and SI = 8.8–11.5. The data showed the
Please cite this article in press as: Hwu, J.R., et al. Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus. Antiviral Res.
(2015), http://dx.doi.org/10.1016/j.antiviral.2015.03.013

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triply conjugated compounds with the –OSO2– joint between cou-
marin–arene moieties had greater anti-CHIKV activity than those
with the –OCH2– joint (cf. 10a versus 15a, 10b versus 15b, 12a ver-
sus 16a, 12b versus 16b, and 14a versus 17a). Additionally, the dou-
bly conjugated compounds 18–20 (i.e., pyrimidine–SCH2–
coumarin–OH) without a benzenesulfonyl moiety did not exhibit
significant activity.
For pyrimidine–SCH2–coumarin–OSO2–arene triply conjugated
compounds, attachment of an Me group to the benzenesulfonyl
moiety often increased the potency and SI value by a factor of
1.2–2.3 (cf. 10b, 12b, and 14b versus 10a, 12a, and 14a, respec-
tively). Compounds with an NO2 group at the ortho position
showed greater activity (>3.16–15.8-fold) than those with the
same group at the para position (cf. 10e > 10d, 12e > 12d, and
14e > 14d).
Furthermore, the conjugated compounds with the benzouracil
moiety showed a higher selectivity (i.e., the SI value) than those
with thymine and uracil moieties (cf. 10a–e > 12a–e > 14a–e). As
the size of this moiety increases (i.e., benzouracil > thymine >
uracil), the inhibition of CHIKV generally increases. This relation-
ship also exists in the conjugates with lipophilicity as exhibited
by the logP values shown in Table 1 (cf. 10b > 12b > 14b and
10e > 12e > 14e).
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References
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409
Abdel-Aal, M.T., 2010. Synthesis and anti-hepatitis B activity of new substituted
uracil and thiouracil glycosides. Arch. Pharm. Res. 33, 797–805.
Albulescu, I.C. et al., 2014. An in vitro assay to study chikungunya virus RNA
synthesis and the mode of action of inhibitors. J. Gen. Virol. 95, 2683–2692.
Bassetto, M. et al., 2013. Computer-aided identification, design and synthesis of a
novel series of compounds with selective antiviral activity against chikungunya
virus. Antiviral Res. 98, 12–18.
Briolant, S. et al., 2004. In vitro inhibition of chikungunya and semliki forest viruses
replication by antiviral compounds: synergistic effect of interferon-
ribavirin combination. Antiviral Res. 61, 111–117.
Cavrini, F. et al., 2009. Chikungunya: an emerging and spreading arthropod-borne
viral disease. J. Infect. Dev. Ctries. 3, 744–752.
a and
Delogu, I. et al., 2011. In vitro antiviral activity of arbidol against chikungunya virus
and characteristics of a selected resistant mutant. Antiviral Res. 90, 99–107.
D’hooghe, M. et al., 2012. Design, synthesis, and antiviral evaluation of purine-b-
lactam and purine-aminopropanol hybrids. J. Med. Chem. 55, 5637–5641.
Feng, J. et al., 2007. Discovery of alogliptin: a potent, selective, bioavailable, and
efficacious inhibitor of dipeptidyl peptidase IV. J. Med. Chem. 50, 2297–2300.
Gerardin, P. et al., 2008. Multidisciplinary prospective study of mother-to-child
chikungunya virus infections on the island of la réunion. PLOS Med. 5, 413–423.
Ghose, A.K. et al., 1999. A knowledge-based approach in designing combinatorial or
to be medicinal chemistry libraries for drug discovery. 1. A qualitative and
quantitative characterization of known drug databases. J. Comb. Chem. 1, 55–68.
Hann, M.M., Keserü, G.M., 2012. Finding the sweet spot: the role of nature and
nurture in medicinal chemistry. Nat. Rev. 11, 355–365.
Horton, D.A. et al., 2003. The combinatorial synthesis of bicyclic privileged
structures or privileged substructures. Chem. Rev. 103, 893–930.
Hwu, J.R. et al., 2008. Synthesis of new benzimidazole–coumarin conjugates as anti-
hepatitis C virus agents. Antiviral Res. 77, 157–162.
Hwu, J.R. et al., 2011a. Coumarin–purine ribofuranoside conjugates as new agents
against hepatitis C virus. J. Med. Chem. 54, 2114–2126.
Hwu, J.R. et al., 2011b. Development of new sulfur-containing conjugated
compounds as anti-HCV agents. Phosphorus Sulfur Silicon Relat. Elem. 186,
1144–1152.
Kaur, P. et al., 2013. Chikungunya virus: an update on antiviral development and
challenges. Drug Discovery Today 18, 969–983.
Khan, M. et al., 2011. Cellular IMPDH enzyme activity is a potential target for the
inhibition of chikungunya virus replication and virus induced apoptosis in
cultured mammalian cells. Antiviral Res. 89, 1–8.
Kirkiacharian, S. et al., 2002. Structure–activity relationships of some 3-substituted-
4-hydroxycoumarins as HIV-1 protease inhibitors. IL Farmaco 57, 703–708.
Kostova, I. et al., 2005. Structure–activity relationships of synthetic coumarins as
HIV-1 inhibitors. Bioinorg. Chem. Appl. 2006 (Article ID 68274).
Kraszni, M. et al., 2003. Determination of conformer-specific partition coefficients in
octanol/water systems. J. Med. Chem. 46, 2241–2245.
Lagoja, I.M., 2005. Pyrimidine as constituent of natural biologically active
compounds. Chem. Biodivers. 2, 1–50.
Leeson, P., 2012. Chemical beauty contest. Nature 481, 455–456.
Neyts, J. et al., 2009. Structure–activity relationship of new anti-hepatitis C virus
agents: heterobicycle–coumarin conjugates. J. Med. Chem. 52, 1486–1490.
Opanasenko, M. et al., 2013. Solid acid catalysts for coumarin synthesis by the
pechmann reaction: MOFs versus zeolites. ChemCatChem 5, 1024–1031.
Ordonez, P. et al., 2012. Anti-human immunodeficiency virus type 1 activity of
290
4. Conclusions
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294
295
296
297
298
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300
301
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304
305
306
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For the development of new compounds with anti-CHIKV activ-
ity, 22 triply conjugated uracil–coumarin–arenes were designed
and synthesized. Five of them (i.e., 10a, 10b, 10e, 12e, and 14e)
were found to impede CHIKV replication at EC50 values of 19.1,
10.2, 17.2, 19.0, and 13.0 lM, respectively.
The coumarin moiety in the conjugated compounds was essen-
tial to their antiviral activity. This central moiety was attached to a
pyrimidine unit at the C-40 position through a SCH2 joint on one
side and an arene group at the C-70 position through a –SO2– joint
on the other side. The extension of the doubly conjugated uracil–
coumarins to triply conjugated uracil–coumarin–arenes by use of
the –SO2– (not OCH2) joint was vital to their anti-CHIKV activity.
When coumarin–arenes conjugated with bezouracil (i.e., 10), they
exhibited better selectivity indexes than their kin with uracil or
5-methyluracil.
These guidelines will be useful for the future design and synthe-
sis of new anti-virals. The studies of mode of action on inhibitors of
CHIKV replication, i.e., in vitro RNA synthesis assay (Albulescu
et al., 2014), is currently under investigation. Moreover, we will
evaluate these new conjugated compounds against additional
viruses, such as dengue virus, West Nile virus, yellow fever virus,
etc. Results will be reported in due course.
novel
6-substituted
1-benzyl-3-(3,5-dimethylbenzyl)uracil
derivatives.
Antimicrob. Agents Chemother. 56, 2581–2589.
Ozden, S. et al., 2008. Inhibition of chikungunya virus infection in cultured human
muscle cells by furin inhibitors. J. Biol. Chem. 283, 21899–21908.
Pohjala, L. et al., 2011. Inhibitors of alphavirus entry and replication identified with
a stable chikungunya replicon cell line and virus-based assay. PLoS One 6,
e28923.
Powers, J.C. et al., 2002. Irreversible inhibitors of serine, cysteine, and threonine
proteases. Chem. Rev. 102, 4639–4750.
Rashad, A.A. et al., 2014. Chikungunya virus: emerging targets and new
opportunities for medicinal chemistry. J. Med. Chem. 57, 1147–1166.
Riveiro, M.E. et al., 2010. Coumarins: old compounds with novel promising
therapeutic perspectives. Curr. Med. Chem. 17, 1325–1338.
Scholte, F.E.M. et al., 2015. Stress granule components G3BP1 and G3BP2 play a
proviral role early in chikungunya virus replication. J. Virol. 89, 4457–4469.
Schwartz, O. et al., 2012. Biology and pathogenesis of chikungunya virus. Nat. Rev.
8, 491–500.
Shenai, B.R. et al., 2003. Structure–activity relationships for inhibition of cysteine
protease activity and development of plasmodium falciparum by peptidyl vinyl
sulfones. Antimicrob. Agents Chemother. 47, 154–160.
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Acknowledgments
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For financial support, we thank the Ministry of Science and
Technology (Grant Nos. NSC 103-2923-I-008-001 and MOST
103-2113-M-007-018-MY3), Ministry of Education of R.O.C.
(Grant Nos. 104N2011E1 and 104N2016E1), and National Central
University (Grant No. 103G603-14). The work in Leuven is
supported by the European Commission SILVER project within
the 7th Framework Programme as Cooperation Project Grant
Agreement (No. 260644).
Somers, F. et al., 2001. Original 2-alkylamino-6-halogenoquinazolin-4(3H)-ones
and KATP channel activity. J. Med. Chem. 44, 2575–2585.
Tsay, S.C. et al., 2013. Coumarins hinged directly on benzimidazoles and their
ribofuranosides to inhibit hepatitis C virus. Eur. J. Med. Chem. 63, 290–298.
Udommaneethanakit, T. et al., 2014. Drugs against avian influenza A virus: design of
novel sulfonate inhibitors of neuraminidase N1. Curr. Pharm. Des. 20, 3478–3487.
Wang, X. et al., 2012. Design, synthesis, and biological evaluation of
1-[(2-benzyloxyl/alkoxyl) methyl]-5-halo-6-aryluracils as potent HIV-1 non-
nucleoside reverse transcriptase inhibitors with an improved drug resistance
profile. J. Med. Chem. 55, 2242–2250.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.antiviral.2015.03.
013.
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Please cite this article in press as: Hwu, J.R., et al. Benzouracil–coumarin–arene conjugates as inhibiting agents for chikungunya virus. Antiviral Res.
(2015), http://dx.doi.org/10.1016/j.antiviral.2015.03.013