Synthesis and in vitro evaluation of west nile virus protease inhibitors based on the 1,3,4,5-tetrasubstituted 1H-Pyrrol-2(5H)-one scaffold

Article abstract of DOI:10.1002/cmdc.201300244

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a large number of human infections each year. There are currently no vaccines or antiviral therapies available for human use against WNV. Therefore, efforts to develop new chemotherapeutics against this virus are highly desired. In this study, a WNV NS2B-NS3 protease inhibitor with a 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold was identified by screening a small library of nonpeptidic compounds. Optimization of this initial hit by the synthesis and screening of a focused library of compounds with this scaffold led to the identification of a novel uncompetitive inhibitor ((-)-1a16, IC₅₀=2.2±0.7μM) of the WNV NS2B-NS3 protease. Molecular docking of the chiral compound onto the WNV protease indicates that the Renantiomer of 1a16 interferes with the productive interactions between the NS2B cofactor and the NS3 protease domain and is thus the preferred isomer for inhibition of the WNV NS2B-NS3 protease.

Full text of DOI:10.1002/cmdc.201300244

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DOI: 10.1002/cmdc.201300244
Synthesis and in vitro Evaluation of West Nile Virus
Protease Inhibitors Based on the 1,3,4,5-Tetrasubstituted
1H-Pyrrol-2(5H)-one Scaffold
Yaojun Gao,[a] Sanjay Samanta,[a] Taian Cui,[b] and Yulin Lam*[a]
West Nile virus (WNV), a member of the Flaviviridae family, is
a mosquito-borne pathogen that causes a large number of
human infections each year. There are currently no vaccines or
antiviral therapies available for human use against WNV. There-
fore, efforts to develop new chemotherapeutics against this
virus are highly desired. In this study, a WNV NS2B–NS3 pro-
tease inhibitor with a 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-
one scaffold was identified by screening a small library of non-
peptidic compounds. Optimization of this initial hit by the syn-
thesis and screening of a focused library of compounds with
this scaffold led to the identification of a novel uncompetitive
inhibitor ((ꢁ)-1a16, IC50 =2.2ꢀ0.7 mm) of the WNV NS2B–NS3
protease. Molecular docking of the chiral compound onto the
WNV protease indicates that the R enantiomer of 1a16 inter-
feres with the productive interactions between the NS2B cofac-
tor and the NS3 protease domain and is thus the preferred
isomer for inhibition of the WNV NS2B–NS3 protease.
Introduction
The West Nile virus (WNV), a neurotropic flavivirus, is a mosqui-
to-borne, reemerging pathogen causing outbreaks on multiple
continents.[1] While a larger proportion of infected people
suffer from milder symptoms, such as West Nile fever, there
has been an observed increase in the cases of severe neuro-in-
vasive diseases over the past decade, such as meningitis, flac-
cid paralysis, and encephalitis.[2] Close follow-up of WNV-infect-
ed humans have shown that patients who manifest sympto-
matic infections often suffer lifetime consequences, such as
long-term cognitive and neurological impairment.[3] Despite
the growing public health concern associated with WNV infec-
tion, to date there are no approved vaccines or antiviral thera-
peutics available for human use against WNV.
tial for the NS3 protease to exhibit its catalytic activity. The
WNV NS2B–NS3 protease cleaves peptide substrates at the C-
terminus, and substrate recognition is made possible by a pair
of basic amino acids (Arg and Lys) at the P1 and P2 positions.[6]
Site-directed mutagenesis studies have shown that mutation
of essential catalytic residues in the NS3 protease cleavage
sites leads to a halt in viral infectivity, indicating the impor-
tance of these sites in the WNV life cycle. In addition, produc-
tive interaction of the NS2B binding cavity with the NS3 pro-
tease domain is essential to increase NS3 protease catalytic ac-
tivity.[6a,7] Therefore, inhibitor interference with the productive
conformation of the NS2B cofactor is an important area for
drug discovery.[5] However, the design of WNV NS2B–NS3 pro-
tease inhibitors has met with only modest success thus far. Ear-
lier studies have identified various peptidic compounds con-
taining highly charged moieties which demonstrate good in-
hibitory activity against the WNV NS2B–NS3 protease in vitro.[8]
However, their short physiological half-lives and high clearance
rates in vivo curtail their potential for drug development. Thus,
there is a need to explore nonpeptidic compounds for their
WNV NS2B–NS3 protease inhibitory activities. The most potent
compounds known to date are several sulfonyl pyrazolyl deriv-
atives, the best of which is an uncompetitive inhibitor with an
IC50 value of 0.105 mm).[9] However, these small organic mole-
cules were unstable under physiological conditions and de-
graded within an hour in aqueous buffer. Attempts to improve
their stability without compromising biological activity proved
to be unsatisfactory.[10] Other nonpeptidic inhibitors with mod-
erate to good activities are compounds with isothiourea (IC50 =
184 mm, competitive inhibitor)[11] or guanidino (Ki =13ꢀ
1 mm)[12] moieties, as well as 8-hydroxyquinoline (Ki =3.2ꢀ
0.3 mm, competitive inhibitor),[13] isoquinoline (IC50 =30 mm),[14]
9,10-dihydro-3H,4aH-1,3,9,10a-tetraaza-phenanthren-4-one (Ki =
WNV is a small enveloped virus with a single-stranded posi-
tive-sense RNA genome that is translated to a polyprotein pre-
cursor which produces ten mature proteins by co- and post-
translation.[4] The RNA genome encodes three structural (C,
capsid; M, membrane; E, envelope) and seven nonstructural
(NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins that are
important for viral replication in the infected cell.[5] The full-
length NS3 protein represents a multifunctional protein in
which the N-terminus encodes a serine protease and the C-ter-
minus codes for a RNA helicase. Interaction between the NS2B
cofactor and the NS3 protease domain was found to be essen-
[a] Y. Gao, S. Samanta, Prof. Y. Lam
Department of Chemistry, National University of Singapore
3 Science Drive 3, Singapore 117543
E-mail: chmlamyl@nus.edu.sg
[b] Dr. T. Cui
School of Applied Science, Tampines Polytechnic
21 Tampines Avenue 1, Singapore 529757
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300244.
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4.47ꢀ0.37 mm, uncompetitive inhibitor),[15a] and 2-{6-[2-(5-
phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)acetylamino]benzothiazol-
2-ylsulfanyl}acetamide (Ki =2.8ꢀ0.1 mm, uncompetitive inhibi-
tor)[15b] derivatives. To develop more potent WNV inhibitors,
there is a need to examine other compounds for their activities
and suitability. We herein describe the identification of a new
scaffold with promising inhibitory activity against WNV NS2B–
NS3 protease, the library synthesis and biological evaluation of
39 analogues of the lead compound, and the structure–activity
relationships (SAR) and cytotoxicity properties of the most
potent inhibitors.
gave three fragments: 2,4-diketo ethyl ester 2, an aldehyde,
and 2-amino-5-sulfanyl-1,3,4-thiadiazole 3.
Compound 2 was prepared by reacting the respective
methyl aryl ketone with diethyl oxalate and sodium ethoxide
at room temperature for 14 h (Scheme 1). Compound 3 was
synthesized from 5-sulfanyl-1,3,4-thiadiazol-2-ylamine 4 using
Results and Discussion
Hit identification and analogue synthesis
Initially, a collection of 110 compounds from our in-house com-
pound library which have a history of antimicrobial, antiviral,
and anticancer activities[16] were screened by high-throughput
screening (HTS) at 100 mm for WNV NS2B–NS3 protease inhibi-
tion. The best eight compounds with regard to their inhibitory
activities toward WNV NS2B–NS3 protease, were further refined
by cell cytotoxicity assays. This resulted in the identification of
compound 1a1 (Figure 1) as a lead compound. To optimize the
potency of this compound, a library of structurally diverse ana-
logues of compound 1a1 was synthesized for biological evalu-
ation of their inhibitory activities.
Scheme 1. Synthesis of compounds 1a and 5a. Reagents and conditions:
a) (COOEt)2, NaOEt, EtOH, RT, 14 h, 70–85%; b) R3Br, KOH, EtOH, RT, 2 h, 69–
95%; c) R2CHO, 1,4-dioxane, 708C, 18 h, 35–64%; d) R4NH2, 1,4-dioxane,
reflux, 36–40%.
a reported procedure[17] in which compound 4 was suspended
in an EtOH solution with KOH and treated with different alkyl
and benzyl bromides at room temperature for 2 h. Compound
1a was then constructed via a three-component reaction in-
volving intermediates 2 and 3 and the corresponding aldehyde
in 1,4-dioxane at 708C.[18] To increase the diversity of com-
pound 1a, we made modifications by 1) replacing the enolic
hydroxy group with an amino group, which was achieved by
reacting compound 1a with the respective amine in anhydrous
1,4-dioxane at reflux to yield compound 5a; 2) oxidizing the
sulfide moiety to a sulfone (Scheme 2A); or 3) converting the
enolic hydroxy group to a methoxy moiety (Scheme 2B). To in-
vestigate the importance of the thiadiazole ring in compound
1a on the inhibitory activity, structural modifications that re-
placed the thiadiazole ring with different substituted amino
groups were achieved using a similar three-component reac-
tion as was used for the preparation of compound 1a
(Scheme 2C). This yielded compound 6a, which was subse-
quently treated with various amines to afford compound 7a.
Figure 1. Structure and IC50 value of lead compound against WNV NS2B–NS3
protease.
To the best of our knowledge, the synthesis of compound
1a has not been previously reported. To develop a synthetic
strategy to prepare this compound expediently, we carried out
a retrosynthetic analysis of the compound (Figure 2). Discon-
nection of compound 1a at the amide moiety and CꢁN bonds
SAR studies
A representative set of 41 analogues of compound 1a1 was
synthesized and tested in a WNV assay at 200 mm concentra-
tion to identify potential inhibitors. Compounds exhibiting
greater than 50% inhibition were further evaluated for their ef-
fects at different concentrations, and the IC50 values for each
compound were calculated using the GraphPad Prism software
(Table 1). A fluorogenic peptide (Pyr-RTKR-AMC) was used as
a substrate for the WNV NS2B–NS3 protease, and the KM value
was determined to be 3.5ꢀ0.4 mm using the protocol from the
SensoLyte 440 West Nile virus protease assay kit.
Figure 2. Retrosynthesis of compound 1a.
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replaced by an alkyl or benzyl group were synthe-
sized and tested for their inhibitory activities. Un-
fortunately, these compounds also showed no inhibi-
tory activity against WNV NS2B–NS3 protease.
Next, we proceeded to establish the stabilities of
compounds 1a16 and 1a17, the two strongest candi-
dates identified from our SAR studies. This was ach-
ieved using LCMS–IT-TOF detection to analyze the
amount of compound remaining over time in the
assay buffer (pH 8). Quantification of the compounds
was determined by Shimadzu’s LC solution software.
Compounds 1a16 and 1a17 showed excellent stabili-
ty in pH 8 buffer at 378C.
Considering that the compounds tested thus far
were always racemic mixtures, we proceeded to in-
vestigate the influence of stereochemistry on the bio-
logical activities of the compounds. To investigate
the inhibitory activity of each enantiomer, the (+)-
and (ꢁ)-enantiomers of compounds 1a16 and 1a17
were obtained via chiral HPLC separation of the re-
spective racemic mixture. Screening results of these
Scheme 2. Modification of compound 1a. Reagents and conditions: a) m-CPBA, CH2Cl2, 0–
58C, 2.5 h, 76%; b) MeOH, PPh3, DIAD, 08C!RT, 15 h, 83%; c) R2CHO, R3NH2, EtOH, RT,
15 h, 45–60%; d) R4NH2, 1,4-dioxane, reflux, 70–90%.
In our preliminary SAR study, R1, R2, and R3 in compound
1a1 were replaced with phenyl groups containing electron-do-
nating or electron-withdrawing groups (Table 1, compounds
1a2–1a13). Although these changes did not provide any signif-
icant improvement in inhibitory activity, the presence of a p-
chlorophenyl group at R1 and R2 and a benzyl group at R3 re-
sulted in improved activity (Table 1, compound 1a13) relative
to other changes. Hence, we carried out further SAR studies by
varying the R3 position whilst retaining p-chlorophenyl sub-
stituent at R1 and R2. Analogues of 1a13 were synthesized in
which R3 was an alkyl group of various sizes or chain lengths.
Screening results (Table 1, compounds 1a14–1a23) showed
that the highest inhibition was observed for compound 1a16,
which contains an n-propyl group at the R3 position. Further
tuning of compound 1a16 by varying the halide moiety or the
position of the halide on the phenyl ring in R1 or R2 (Table 1,
compounds 1a24–1a29) did not result in further improve-
ments in inhibitory activity. In addition, evaluation of ana-
logues of 1a16 with an electron-donating or electron-with-
drawing group in the phenyl ring at the R1 or R2 position did
not result in any improvement in biological activity (Table 1,
compounds 1a29–1a32). This indicates the importance of the
p-chlorophenyl moiety for the inhibitory activity of the com-
pound.
enantiomers showed that the (ꢁ)-enantiomer demonstrated
much better inhibitory potency than the corresponding
(+)-enantiomer (Table 2). Furthermore, amongst the four enan-
tiomers evaluated, (ꢁ)-1a16 exhibited the best inhibitory activ-
ity, with an IC50 value of 2.2ꢀ0.7 mm.
Next, we evaluated the cytotoxicity of compounds (ꢁ)-1a16
and (ꢁ)-1a17 against baby hamster kidney fibroblast (BHK21)
cells at three different incubation times. Dimethyl sulfoxide
(DMSO) was also included to serve as a vehicular control, as
the test compounds were dissolved in DMSO. The amount of
DMSO used in the assay was controlled at 0.1% of the total
volume. This was to ensure that DMSO did not lead to cytotox-
icity in the cells. From the results (Figure 3), compounds (ꢁ)-
1a16 and (ꢁ)-1a17 were shown to be non-cytotoxic to BHK21
cells.
To investigate the effects of the sulfide moiety and the
enolic hydroxy group on the inhibitory activity of compound
1a16, we oxidized the sulfide to the corresponding sulfone
and replaced the enolic hydroxy group with a methoxy group.
However, compounds 1a34 and 1a35 did not exhibit any in-
hibitory activities. Similarly, replacement of the enolic hydroxy
group with a 2-hydroxyethylamino or NH2 group (Table 1, com-
pounds 5a1–5a2) also resulted in a complete loss of the inhibi-
tory potency against WNV NS2B–NS3 protease. Finally we in-
vestigated the importance of the thiadiazole ring for inhibiting
the protease activity. A series of compounds (Table 1, com-
pounds 6a1–6a2, 7a1–7a3) in which the thiadiazole ring was
Figure 3. MTS assay results obtained at incubation times of 24, 48, and 72 h
with the BHK21 cell line. Compounds were added at a final concentration of
25 mm.
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tive inhibition mode, with a Ki value of 1.8ꢀ0.6 mm
(where Ki =IC50/(1+KM/[S])[18] for uncompetitive inhibi-
tion, Table 2).
Table 1. IC50 values of compounds 1a and 5a–7a.
Compd
R1
R2
R3
R4
IC50 [mm][a]
WNV NS2B–NS3 protease and the dengue virus
DENV2 NS2B–NS3 protease are different serocomplex
subgroups with ~70% amino acid sequence identity.
To determine the selectivity of the inhibitory activity,
compound (ꢁ)-1a16 was analyzed in vitro using the
recombinant DENV2 NS2B–NS3 protease. Compound
(ꢁ)-1a16 did not show any inhibition of DENV2 pro-
tease activity (IC50 >200 mm).
1a2
1a3
1a4
1a5
1a6
1a7
1a8
1a9
4-ClC6H4
C6H5
C6H5
C6H5
3-MeOC6H4
C6H5
3,4-Cl2C6H3
C6H5
C6H5
4-MeSC6H4
C6H5
4-FC6H4
4-FC6H4
4-MeSC6H4
C6H5
4-FC6H4CH2
C6H5CH2
4-FC6H4CH2
4-FC6H4CH2
4-FC6H4CH2
C6H5CH2
4-MeOC6H4CH2
4-MeOC6H4CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
CH3
CH(CH3)2
(CH2)2CH3
(CH2)3CH3
CH(CH2)4
(CH2)4CH3
CH2CH3
CH2CH2OH
CH2CO2Et
CH2CH2CN
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
SO2(CH2)2CH3
(CH2)2CH3
4-FC6H4CH2
(CH2)2CH3
CH3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
118ꢀ8.4
151ꢀ4.6
44.8ꢀ5.7
26.8ꢀ4.5
79.6ꢀ6.2
24.5ꢀ4.0
41.3ꢀ3.7
31.1ꢀ2.5
35.5ꢀ4.1
38.3ꢀ5.1
34.4ꢀ3.4
21.4ꢀ3.1
124.0ꢀ5.1
42.5ꢀ5.5
6.1ꢀ1.9
11.4ꢀ2.6
11.9ꢀ1.5
70.0ꢀ7.1
94.7ꢀ6.2
>200
C6H5
C6H5
1a10
1a11
1a12
1a13
1a14
1a15
1a16
1a17
1a18[b]
1a19
1a20
1a21
1a22
1a23
1a24
1a25
1a26
1a27
1a28
1a29
1a30
1a31
1a32
1a33
1a34
1a35[d]
5a1[e]
5a2[e]
6a1
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-FC6H4
4-BrC6H4
4-IC6H4
4-NO2C6H4
4-NH2C6H4
4-ClC6H4
2-benzofuryl
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
CH3
2-furyl
4-MeOC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-FC6H4
4-BrC6H4
3-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-NO2C6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-FC6H4
4-ClC6H4
Ph
Docking analysis
A docking study was performed to rationalize the in-
hibitory activity and kinetics results, as well as to
identify the possible binding sites of compound (ꢁ)-
1a16, on the WNV NW2B–NS3 protease. The crystal
structure of WNV NS2B–NS3 protease in complex
with peptide Bz-Nle-Lys-Arg-Arg-H (PDB: 2FP7) has
been reported previously.[19] To identify the potential
binding site of compound (ꢁ)-1a16 on the NS2B–
NS3 protease, in silico docking studies were per-
formed with Molecular Operating Environment (MOE)
using the reported crystal structure coordinates. As
compound (ꢁ)-1a16 is an uncompetitive inhibitor of
WNV NSB–NS3 protease, we focused our analysis on
regions beyond the substrate binding sites. Earlier
studies showed that the integration of residues 78–
87 of NS2B into the protease–cofactor complex af-
fected the formation of the catalytic active
sites.[6,19–20] Deletion or mutagenesis of these residues
produced an inert enzyme. Therefore, the region on
the NS3 protease where key interactions with the
NS2B cofactor (residues 78–87) occur is important for
NS2B–NS3 protease activity, and particular attention
was paid to this region during our docking studies.
As compound 1a16 contains a chiral center, the R-
and S-enantiomers were analyzed separately for their
interactions with WNV NS2B–NS3 protease. Our dock-
ing analysis suggested that the guanidine group in
Arg76 of the NS3 protease is bound to the thiadia-
zole group of both enantiomers via a p–cation type
interaction. The R-enantiomer could also potentially form hy-
drogen bonds with residues Glu169 and Glu120, as well as p–
cation type interactions with Lys88, on the NS3 protease (Fig-
ure 5a). A p–cation type of interaction was also observed be-
tween the acetophenyl group of the S-enantiomer and the
protonated amino group from Lys73 on the NS3 protease (Fig-
ure 5b). Both enantiomers closely interact with amino acid resi-
due Leu79 of NS2B, which may interfere with the productive
associations of the NS2B and the NS3 protease. Docking analy-
sis predicted that the binding energy of the R-enantiomer
(28.56 kcalmolꢁ1) is higher than that of the S-enantiomer
(24.04 kcalmolꢁ1). This is in agreement with experimental re-
sults, which showed that one enantiomer is a stronger inhibi-
tor than the other.
>200
>200
24.5ꢀ3.4
30.6ꢀ0.8
51.5ꢀ2.3
32.0ꢀ2.1
58.6ꢀ7.3
NA
32.6ꢀ1.1
38.8ꢀ7.5
19.7ꢀ5.3
21.3ꢀ0.6
NA[c]
NA
NA
NA
NA
NA
NA
NA
NA
–
–
(CH2)2OH
H
–
–
H
CH3
(CH2)2OH
6a2
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
CH2C6H4
CH2C6H4
CH2C6H4
CH2C6H4
7a1[e]
7a2[e]
7a3[e]
[a] Data are concentrations required to inhibit WNV NS2B–NS3 protease activity by
50% and are the mean ꢀSE of triplicate experiments; compound purities ꢂ95%.
[b] R3 =cyclopentyl. [c] NA: no activity. [d] Enolic OH group replaced by OMe.
[e] Enolic OH group replaced by NH2.
Table 2. Inhibitory activities of enantiomers of compounds 1a16 and
1a17.
Compd
IC50 [mm]
Ki [mm]
(ꢁ)-1a16
(+)-1a16
(ꢁ)-1a17
(+)-1a17
2.2ꢀ0.7
90.4ꢀ8.3
7.2ꢀ0.3
1.8ꢀ0.6
5.9ꢀ0.3
22.3ꢀ0.5
To determine the mode of inhibition of WNV NS2B–NS3 pro-
tease activity by compound (ꢁ)-1a16, we performed a kinetics
of inhibition study using Pyr-RTKR-AMC as a substrate. The in-
hibition mode of compound (ꢁ)-1a16 was determined from
a Lineweaver–Burk plot (Figure 4) and revealed an uncompeti-
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Conclusions
In this study, primary screening of 110 compounds led to the
discovery of an initial hit compound, 1a1, with moderate inhib-
itory activity against the WNV NS2B–NS3 protease. Refinement
of this hit by synthesizing a focused library of 1a1 derivatives
for SAR studies provided compound (ꢁ)-1a16, which inhibited
the interaction of the NS2B cofactor and the NS3 protease
with a Ki value of 1.8ꢀ0.6 mm in an uncompetitive manner.
Compound (ꢁ)-1a16 is stable under physiological conditions
and is not cytotoxic to baby hamster kidney fibroblast cells.
Compared with the other physiologically stable, nonpeptidic
inhibitors which were reported earlier, compound (ꢁ)-1a16 is
also a stronger inhibitor of WNV NS2B–NS3 protease.
Experimental Section
Figure 4. Uncompetitive inhibition of (ꢁ)-1a16 with WNV NS2B–NS3 pro-
tease. WNV protease assays were performed at various concentrations of
(ꢁ)-1a16 (0, 2.5, 5.0, 10, and 25 mm) and substrate (3.3, 8.3, 16.7, 33.3, and
83.3 mm). Dixon plot of 1/v versus [I] for (ꢁ)-1a16 at each substrate concen-
tration were plotted using OriginPro 8.
Chemical synthesis: All chemical reagents were obtained from
Sigma–Aldrich, Merck, Lancaster, or Fluka and were used without
further purification. Analytical TLC was carried out on pre-coated
plates (Merck silica gel 60, F254) and visualized with UV light or
stained with ninhydrin. Column chromatography was performed
with silica (Merck, 70–230 mesh). Final compounds for biological
assays were purified on a Gilson preparative HPLC system with
a Waters C18 column (250ꢁ10.0 mm, 5 mm). Detection was con-
ducted at 254 nm. The mobile phase was a gradient, with solvent B
increasing from 5% to 100% over 40 min and a flow rate of
5.0 mLminꢁ1. Solvent A was 0.1% trifluoroacetic acid (TFA) in H2O,
and solvent B was 0.1% TFA in CH3CN. Purities of the compounds
were determined via analytical HPLC using a Shimadzu LCMS–IT-
TOF system with a Phenomenex Luma C18 column (50ꢁ3.0 mm,
5 mm). Detection was conducted at 254 nm, and integration was
obtained using Shimadzu LCMS solution software. The mobile
phase was a gradient, with solvent B increasing from 0% to 100%
over 7 min and with a flow rate of 0.8 mLminꢁ1. Compounds used
in biological assays had purities of ꢂ95%. Racemic compound sep-
aration was performed on a Shimadzu HPLC system with a Chiral-
pak AD-H column (250ꢁ4.6 mm, 5 mm), eluting with hexanes/
iPrOH (80:20), or a Chiralpak IC column (250ꢁ4.6 mm, 5 mm), elut-
ing with 100% CHCl3 and 0.1% TFA at 1.0 mLminꢁ1, g=254 nm. H
1
and 13C NMR spectra were measured at 298 K on a Bruker ACF 300
or AMX 500 Fourier transform spectrometer. Chemical shifts are re-
ported in d (ppm) relative to the residual undeuterated solvent
that was used as internal reference. The signals observed are de-
scribed as: s (singlet), d (doublet), t (triplet), m (multiplet). The
number of protons (n) for a given resonance are indicated as nH.
Mass spectra were performed on a Finnigan MAT 95/XL-T spec-
trometer under electron impact (EI) and electrospray ionization
(ESI) techniques. Optical rotations were determined with a JASCO
DCP-1000 digital polarimeter and were the average of at least 10
measurements with an RSD value of less than ꢁ100%.
General procedure for the synthesis of 2: A 21% sodium ethox-
ide solution in EtOH (11.33 mL, 30 mmol) was added to a mixture
of the corresponding acetophenone (10 mmol) and diethyl oxalate
(2.03 mL, 15 mmol) in anhydrous EtOH (40 mL). The resulting mix-
ture was stirred at room temperature for 12 h, after which the reac-
tion mixture was neutralized with 4 n HCl, and the solvent was re-
moved under rotary evaporation. The resulting brown solid residue
was extracted with EtOAc/hexane, and the combined organic
layers were dried over Na2SO4, concentrated, purified by column
chromatography using EtOAc/hexane (starting from 1:20 to 1:9) as
Figure 5. Molecular docking of compounds a) (R)-1a16 and b) (S)-1a16 onto
WNV NS2B–NS3 protease. The WNV protease crystal structure coordinates
(PDB: 2FP7) were used for docking. The protease Ca backbone is shown as
a grey ribbon with key residues highlighted; the enantiomers of compound
1a16 are shown as stick models. Images were generated with the Discovery
Studio 3.1 client.
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ChemMedChem 2013, 8, 1554 – 1560 1558

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the eluent system, and dried under vacuum at 508C to yield com-
pound 2 in 70–85% yield.
were dried over Na2SO4, concentrated, purified by column chroma-
tography using EtOAc/hexane (from 1:1 to 3:1) as the eluent, and
dried under vacuum at 508C to yield compound 1a34 in 83%
yield.
General procedure for the synthesis of 3: 5-amino-1,3,4-thiadia-
zole-2-thiol 4 (2.664 g, 20 mmol) was dissolved in a mixture of KOH
(2.64 g, 40 mmol) in absolute EtOH (20 mL). After 10 min, the corre-
sponding alkyl bromide (20 mmol) was added dropwise with vigo-
rous stirring. The reaction progress was monitored by TLC and was
completed within 2 h. The reaction mixture was concentrated and
then extracted with EtOAc/H2O. The combined organic layers were
dried over anhydrous Na2SO4, concentrated, purified by column
chromatography using EtOAc/Hexane as an eluent, and dried in
a vacuum oven (at 508C) to afford solid 3 in 69–95% yield.
General procedure for the synthesis of 5a and 7a: The corre-
sponding amine (0.2 mmol) was added to a solution of compound
1a or 6a (1 mmol) in anhydrous 1,4-dioxane (10 mL). The mixture
was stirred at reflux, and the reaction was monitored by TLC for re-
action completion. When the reaction was completed, the mixture
was concentrated and extracted with an EtOAc/H2O mixture. The
combined organic layers were concentrated and purified by
column chromatography using EtOAc/hexane (3:1) as the eluent to
afford products 5a (36–40%) and 7a (70–90%). For the synthesis of
compound 7a3, AcOH was used as the reaction solvent.
General procedure for the synthesis of 1a: A suspension of 2,4-
diketo ethyl ester 2 (1.0 mmol), the corresponding amine 3
(1 mmol), and the aldehyde (1.0 mmol) in 1,4-dioxane (8 mL) was
stirred at 708C for 18 h. The reaction mixture was then concentrat-
ed and washed with H2O and cold Et2O. The residual crude product
was purified either by column chromatography (using EtOAc/
CH2Cl2 1:1 as the eluent) or by reverse phase preparative HPLC
using 0.1% TFA in H2O (solvent A) and 0.1% TFA in CH3CN (sol-
vent B) as the mobile phase. The mobile phase was a gradient,
with solvent B increasing from 5% to 95% over 30 min and at
a flow rate of 8.0 mLminꢁ1. Detection was conducted at 254 nm.
Fractions containing the pure product were combined, concentrat-
ed, and dried overnight under vacuum at 508C to give compound
1a in 35–64% yield.
Enzyme inhibition tests: All compounds used in biological testing
were at least 95% pure. Compounds were tested against the WNV
NS2B–NS3 protease using the SensoLite 440 WNV protease assay
kit (Cat. #72079) and the active recombinant WNV protease (Cat.
#72081), which were purchased from Anaspec (Fremont, CA, USA).
This protease assay kit uses a fluorogenic peptide Pyr-RTKR-AMC as
the substrate. According to the kit protocol, the KM value of pro-
tease on the substrate was determined to be 3.45ꢀ0.41 mm.
Therefore, substrate (eight substrate concentrations [S] ranging
from 1 mm to 80 mm) were incubated in 96-well plates, with
0.3 mgmLꢁ1 recombinant WNV protease at 378C in the buffer pro-
vided. The increase in fluorescence intensity was monitored contin-
uously using an Infinite F200 microplate reader (Tecan, Switzerland)
at an excitation wavelength of 354 nm (ꢀ10 nm) and emission
wavelength of 442 nm (ꢀ15 nm). The initial velocity was deter-
mined from the linear region of the progress curve, and KM was
calculated from the Michaelis–Menten equation, for which v=Vmax
[S]/([S]+KM). Triplicate measurements were taken at each data
point, and the data were reported as mean ꢀSE. In the preliminary
WNV NS2B–NS3 protease inhibition assay, analogues of 1a were
screened at a fixed concentration of 50 mm to filter out potential
inhibitors. Compounds which showed greater than 50% inhibitory
activity were further investigated for their IC50 determination.
4-(4-Aminobenzoyl)-5-(4-chlorophenyl)-3-hydroxy-1-(5-(propylth-
io)-1,3,4-thiadiazol-2-yl)-1H-pyrrol-2(5H)-one (1a30): SnCl2·H2O
(57 mg, 0.25 mmol) was added to a solution of compound 1a29
(26 mg, 0.05 mmol) in absolute EtOH (8 mL). The mixture was
stirred at reflux, and the reaction was monitored by TLC for reac-
tion completion. When the reaction was completed, the mixture
was concentrated and extracted with an EtOAc/H2O mixture. The
combined organic layers were concentrated, purified by column
chromatography using EtOAc/hexane (3:1), and dried under
vacuum at 508C to yield compound 1a30 in 87% yield.
4-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-3-hydroxy-1-(5-(propyl-
sulfonyl)-1,3,4-thiadiazol-2-yl)-1H-pyrrol-2(5H)-one (1a33):: A so-
lution of compound 1a16 (150 mg, 0.29 mmol) in CH2Cl2 (10 mL) at
08C was treated with m-CPBA (110 mg, 0.64 mmol), and the mix-
ture was stirred at 0–58C for 2.5 h. The reaction mixture was dilut-
ed with another 10 mL of CH2Cl2 and then washed three times
with saturated Na2CO3 solution. The organic layer was dried over
Na2SO4, concentrated and purified by reverse phase preparative
HPLC using 0.1% TFA in H2O (solvent A) and 0.1% TFA in CH3CN
(solvent B) as the mobile phase. The mobile phase was a gradient
with solvent B increasing from 5% to 95% over 30 min and at
a flow rate of 8.0 mLminꢁ1. Detection was conducted at 254 nm.
Fractions containing the pure product were combined, concentrat-
ed, and dried overnight under vacuum at 508C to give compound
1a33 in 76% yield.
Determination of IC50 values: For the IC50 calculations, recombinant
protease concentration of 0.3 mgmLꢁ1 and seven different concen-
trations of the inhibitor, ranging from 10 nm to 200 mm, were used.
For each experiment, the protease was pre-incubated with the in-
hibitor at 378C for 15 min in separate wells, and the enzyme kinet-
ics were initiated by adding substrate Pyr-RTKR-AMC to a final con-
centration 16.7 mm. The increase in fluorescence intensity was
monitored continuously using an Infinite F200 microplate reader at
an excitation wavelength of 354 nm and emission wavelength at
442 nm. Fluorescence values obtained from the positive control
(no inhibitor) were considered as 100% complex formation, and
those values obtained in the presence of inhibitors were calculated
as a percentage of inhibition of the control. Triplicate measure-
ments were obtained for each data point. IC50 values were calculat-
ed using a sigmoidal dose–response curve using the GraphPad
Prism 3.0 software (San Diego, USA). Triplicate measurements were
recorded as the mean ꢀSE.
4-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-3-methoxy-1-(5-(pro-
pylthio)-1,3,4-thiadiazol-2-yl)-1H-pyrrol-2(5H)-one (1a34): To a so-
lution of compound 1a16 (100 mg, 0.2 mmol) in THF (5 mL) at 08C
was added triphenylphosphine (78.5 mg, 0.3 mmol) and DIAD
(59 mL, 0.3 mmol). The mixture was stirred for 15 min at 08C, then
MeOH (12 mL, 0.3 mmol)) was added. The resulting reaction mix-
ture was stirred at room temperature for 15 h, then the solvent
was removed via rotary evaporation. The crude mixture was dis-
solved in EtOAc (30 mL) and washed with 1 m NaOH followed by
saturated sodium chloride solution. The resultant organic layers
Determination of inhibition mechanism: Three different inhibitor
concentrations (0 to 25 mm) and a no-inhibitor control were each
assayed at five substrate concentrations ranging from 3.3 mm to
83.3 mm. In each assay, the enzyme and inhibitor were incubated at
378C for 15 min, followed by the addition of the substrate to start
the kinetic measurement. The rate of substrate cleavage (v) was
monitored using the F200 microplate reader. To illustrate the inhib-
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FULL PAPERS
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Docking: All docking studies were performed using the Molecular
Operating Environment (MOE 2009.10) software system designed
by the Chemical Computing Group. The program was run using
the Windows Vista operating system installed on an Intel Core2
Duo PC with a 2.4 MHz processor and 4.0 GB RAM. The docked
compound was built using the MOE builder, and the energy was
minimized using molecular mechanics (MM) with the MMFF94x
force field. The active site of the WNV protease was prepared
using the crystal structure of WNV NS2B–NS3 protease in complex
with peptide Bz-Nle-Lys-Arg-Arg-H (PDB: 2FP7). All water molecules
and the bound ligand were removed from the structure, followed
by the addition of valance hydrogen atoms to the protein. By
keeping in mind the amino acid residues in NS2B which are re-
sponsible for the activation of the catalytic triad, the active site in
NS2B–NS3 was identified using the Alpha site finder tool. The trian-
gle matcher placement method was used to generate docking
poses, and each conformation was scored based on the London
DG scoring function, which estimates the free energy of binding
(kcalmolꢁ1). Refinement and geometry of the resulting complex
was studied using the Discovery Studio 3.1 client.
Acknowledgements
We thank the National University of Singapore for financial sup-
port of this work (ARF: R-143-000-399-112). We also thank the
Novartis Institute for Tropical Diseases for the DENV2 NS2B–NS3
protease.
Keywords: antiviral agents · bioorganic chemistry · docking
studies · inhibitors · NS2B–NS3 protease
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Received: May 30, 2013
Published online on July 18, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 1554 – 1560 1560