Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold

Article abstract of DOI:10.1016/j.bmc.2012.04.055

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K_i) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.

Full text of DOI:10.1016/j.bmc.2012.04.055

Bioorganic & Medicinal Chemistry 20 (2012) 4140–4148
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitors of Dengue virus and West Nile virus proteases based
on the aminobenzamide scaffold
Sridhar Aravapalli a, Huiguo Lai b, Tadahisa Teramoto b, Kevin R. Alliston a, Gerald H. Lushington c,
Eron L. Ferguson a, R. Padmanabhan b, William C. Groutas a,
⇑
a Department of Chemistry, Wichita State University, Wichita, KS 67260, USA
b Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
c Molecular Graphics and Modeling Laboratory, The University of Kansas, Lawrence, KS 66045, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant
morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this
study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and sub-
sequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds
were identified which showed comparable activity toward the two proteases and shared in common a
meta or para(phenoxy)phenyl group. The inhibition constants (Ki) for the most potent compound 7n
Received 23 February 2012
Revised 19 April 2012
Accepted 27 April 2012
Available online 10 May 2012
Keywords:
Protease
Dengue virus
West Nile virus
Aminobenzamide
against Dengue and West Nile virus proteases were 8.77 and 5.55 lM, respectively. The kinetics data sup-
port a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further sup-
ported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further
optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative
medicinal chemistry/structure–activity relationship studies and in vitro screening.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
RNA helicase, and a 50-RNA triphosphatase within the C-terminal
(161–618 amino acid residues) region of NS3.4–6 Activation of the
The Dengue virus (DENV) and West Nile Virus (WNV) are mem-
bers of the family Flaviviridae which comprise several human and
animal pathogens, including Yellow fever virus (YFV), Hepatitis C
virus (HCV), and Japanese encephalitis virus (JEV).1 Infection by
DENV1–4 serotypes results in Dengue fever which can progress
to life-threatening Dengue hemorrhagic fever (DHF) and Dengue
shock syndrome (DSS).2 Dengue infection is a global health threat
for which there are currently no small molecule antiviral therapeu-
tics or effective vaccines. Inhibition of DENV replication constitutes
a promising avenue of investigation for the development of thera-
peutics against DENV.3–5
Both Dengue and West Nile viruses are small, enveloped viruses
containing a positive-stranded 11-kb RNA genome which encodes
a polyprotein precursor which is co- and post-translationally pro-
cessed by host cell proteases and the viral encoded trypsin-like
protease, NS2B/NS3, into three structural proteins (C, prM, E) and
seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A,
NS4B, and NS5).6,7 NS3 is a multi-functional protein containing a
serine protease with a prototypical catalytic triad (H51, D75,
S135) within the N-terminal 185 amino acid residues, an NTPase/
serine protease domain of NS3 requires NS2B cofactor, a ꢀ130 ami-
no acid hydrophobic integral membrane protein in the endoplas-
mic reticulum (ER). A ꢀ45 amino acid conserved hydrophilic
segment from the NS2B sequence is sufficient for interaction with
and activation of the NS3 protease domain for protease activity
(hereafter referred to as ‘NS2B/NS3pro’) in vitro.5,7,8 Processing of
the polyprotein by NS2B/NS3pro is essential for viral replication,
consequently NS2B/NS3pro has emerged as an attractive target
for the discovery and development of small molecule therapeutics
for DENV infection.3–6
DENV NS2B/NS3pro has a substrate specificity for an –X–K–R–R–
G/S– sequence corresponding to the subsites –S4–S3–S2–S1–S01–.9
Cleavage is at the P1–P0 (R–G/S) scissile bond. The hydrophilic
1
b-hairpin segment of the NS2B protein cofactor wraps around the
NS3 protease core and is required for optimal catalytic effi-
ciency.10–12 Crystallographic12,13 and high-field NMR studies14 have
illuminated greatly our understanding of the role played by the
NS2B cofactor and the nature of the interaction of NS2B/NS3pro
with small-molecule inhibitors.
DENV NS2B/NS3pro plays a vital role in virus replication, conse-
quently, orally-bioavailable drug-like agents that inhibit NS2B/
NS3pro are of value as potential therapeutics for DENV infection.
Inhibitors of NS2B/NS3pro containing a highly charged peptidyl
recognition element with a dibasic motif at P1–P2 coupled to an
⇑
Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.055

S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
4141
array of electrophilic warheads (aldehydes, trifluoromethyl ke-
tones, and boronic acids),15–18 non-peptidyl -ketoamides,19
platform capable of orienting appended non-peptidyl recognition
elements in a precisely-defined vector relationship would lead to
agents capable of interacting with multiple active site residues.
Thus, the aminobenzamide platform was chosen for generating
the desired compounds and for conducting exploratory studies.
An added advantage is the flexibility afforded by the two points
of diversity in the chosen scaffold, augmenting synthetic tractabil-
ity; (c) a urea functionality was initially employed to lessen the
conformational flexibility of the appended recognition elements.
The latter included an electron-rich heterocycle having multiple
hydrogen bond acceptor sites linked to an aryl–alkyl group and,
(d) it was furthermore envisaged that the initial attachment of
an array of structurally-diverse aliphatic and aromatic amines to
the carboxyl group of the aminobenzamide scaffold would provide
additional binding sites.
The desired compounds were readily obtained as shown in
Scheme 1. These were subsequently screened against DENV
NS2B/NS3pro and WNV NS2B/NS3pro. Four of the compounds
based on (I) were found to exhibit activity against both proteases
and the results are summarized in Table 3 and Fig 2. It can be gen-
erally inferred from the SAR studies that the nature of both R1 and
R2 influence activity. Furthermore, activity is manifested when R1
is a meta or p-(phenoxy)phenyl group. All other amides were inac-
tive, suggesting that the meta or p-(phenoxy)phenyl group is
accommodated in a hydrophobic cleft. It should be noted that
some of the compounds were inactive despite the presence of a
meta or p-(phenoxy)phenyl group (compounds 7a and 8f).
The percent inhibition shown by compound 7n against both
DENV2 and WNV was found to be higher than any of the other se-
lected compounds (Fig 2). The apparent IC50 values of compound
a
phthalazine-based derivatives,20 arylcyanoacrylamides,21 retro
peptide-hybrids,22 benz[d]isothiazol-3(2H)-one derivatives,23 and
others24 have been reported. We describe herein the results of syn-
thetic and biochemical studies related to the inhibition of DENV
and WNV NS2B/NS3pro by functionalized para- and meta-substi-
tuted aminobenzamide derivatives represented by structure (I)
(Fig 1).
2. Chemistry
Compounds 7a–t (Table 1) and 8a–f (Table 2) were synthesized
starting with methyl p-aminobenzoate or methyl m-aminobenzo-
ate, respectively, as illustrated in Scheme 1. Thus, treatment with
trichloromethyl chloroformate yielded the corresponding isocya-
nate which was reacted with propargylamine to form urea deriva-
tives 2a–b. Click chemistry25 with an array of structurally diverse
azides yielded the corresponding functionalized urea derivatives
which were further elaborated to form the final compounds.
3. Biochemistry
The expression and purification of DENV NS2B/NS3pro and
WNV NS2B/NS3pro have been previously described.26–28 Enzyme
assays and inhibition studies were carried out as previously de-
scribed29–31 and the results are summarized in Fig. 2–4 and Tables
3 and 4.
4. Results and discussion
7n (Fig 3) were determined to be 6.82 0.09 and 5.51 0.08 lM
against DENV2 and WNV protease, respectively. We also per-
formed kinetics analyses to determine the Km, kcat and Vmax values
in the presence and absence of compound 7n at four different con-
centrations (Fig 4 and Table 4). The apparent Michaelis–Menten
constants (Km,app) increased and kcat/Km decreased proportionally
with increasing concentration of compound 7n. The results sup-
port a competitive mode of inhibition. Next, we employed molec-
ular modeling to identify a plausible binding mode for 7n. Based
on the molecular docking simulations, a high affinity bound con-
former that is similar in both the DENV and WNV proteases was
identified. Docking poses analysis yielded only one high potency
conformer (i.e., one of the top five scoring poses) that conserved
most of the key interactions in both DENV and WNV (Fig. 5A and
5B, respectively). Key features of this pose that are conserved
The world-wide health problem stemming from infection by
Dengue virus and related flaviviruses, as well as the paucity of
small-molecule therapeutics for combating flavivirus infection,
have provided the impetus for the research described herein. The
aminobenzamide scaffold was utilized in the synthesis of a series
of structurally-diverse meta and para-substituted derivatives, rep-
resented by general structure (I), (Fig 1). The design of (I) rested on
the following considerations: (a) the shallow active site of DENV
NS2B/NS3pro presents a formidable challenge in terms of the de-
sign of potent inhibitors of the enzyme, necessitating the use of a
suitably-embellished multifunctional molecule capable of engag-
ing in multiple favorable binding interactions with the enzyme in
order to attain high potency, without compromising oral bioavail-
ability and PK characteristics.32–39 The problem is further com-
pounded by the stringent substrate specificity requirements of
the protease for positively charged substrates/inhibitors; (b) based
on insights gained from examining the X-ray crystal structures of
DENV2 NS2B/NS3pro with bound ligands12,13 and molecular mod-
eling studies, it was hypothesized that the utilization of a planar
across the two receptors include
p–p stacking interactions be-
tween the ligand fluorobenzyl group and the side chain of
Tyr161, and hydrophobic interactions between the ligand phen-
oxyphenyl group and Val72. Visual inspection suggests that these
interactions are not only conserved but are also likely to comprise
most of the favorable binding features in both cases. Furthermore
confidence in this proposed pharmacophore is attained through
comparison with predicted binding modes for inactive compounds
7a and 8f. In the case of DENV NS2B/NS3pro in Fig 5A, neither of
the inactive compounds is predicted to exploit either of the two
lipophiles listed above with the same effectiveness as 7n: com-
pound 8f has poor overlap with both lipophiles, and while com-
O
NHR2
HN
HN
O
pound 7a is predicted to achieve some
its furan group is not situated anywhere near any lipophiles. In
the case of WNV NS2B/NS3pro (Fig 5B), reasonable stacking
p–p stacking with Tyr161,
N
p–p
N
is achieved with Tyr161 in all cases, but the furan group of 7a is
again poorly situated, while the phenoxyphenyl group of 8f is
not predicted to derive nearly as much a hydrophobic interaction
as is achieved by the corresponding phenoxyphenyl group of 7n,
whose terminal phenyl group is predicted to wedge favorably be-
tween Val72 and the lipophilic portion of neighboring Lys73.
N
R1
(I)
Figure 1. General structure of aminobenzamide derivatives (I).

4142
S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
Table 1
p-Aminobenzamide compounds
O
NH
NHR2
NH
O
R1
N
N
7a-t
N
Compound
R1
R2
Compound
R1
R2
7a
7b
7c
7d
7e
7f
7g
7h
7i
Phenylthiomethyl
Phenylthiomethyl
Phenylthiomethyl
Phenylthiomethyl
Phenylthiomethyl
Phenylthiomethyl
Benzyl
Benzyl
Benzyl
Benzyl
4-Phenoxyphenyl
Benzyl
Phenylethyl
Phenyl
2-Furanylmethyl
2-Morpholinoethyl
4-Phenoxyphenyl
Benzyl
Phenylethyl
Phenyl
7k
7l
Benzyl
Benzyl
Benzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
4-Fluorobenzyl
2-Furanylmethyl
2-Morpholinoethyl
3-Phenoxyphenyl
4-Phenoxyphenyl
Benzyl
Phenylethyl
Phenyl
2-Furanylmethyl
2-Morpholinomethyl
3-Phenoxyphenyl
7m
7n
7o
7p
7q
7r
7s
7t
7j
Table 2
m-Aminobenzamide compounds
5.2. Representative syntheses
5.2.1. Compound 1a
O
To methyl 4-aminobenzoate (30.23 g; 200 mmol) in anhydrous
1,4-dioxane (500 mL) was added trichloromethyl chloroformate
(59.35 g; 300 mmol) dropwise and the reaction mixture was re-
fluxed for 8 h. The solvent was removed on the rotary evaporator
to yield 1a as a yellow solid, which was purified by vacuum distil-
lation to give a yellow solid (34 g, 96% yield). IR (neat) mNCO
NHR2
NH
O
NH
N
R1
2268 cmꢁ1 1H NMR (CDCl3): d 3.88 (s, 3H), 7.14–7.18 (m, 2H),
.
8a-f
N
N
7.96–8.02 (m, 2H).
Compound
R1
R2
5.2.2. Compound 1b
8a
8b
8c
8d
8e
8f
Phenylthiomethyl
Phenylthiomethyl
Phenylthiomethyl
Benzyl
Benzyl
Benzyl
Benzyl
Phenylethyl
3-Phenoxyphenyl
Benzyl
Phenylethyl
Brown solid (94% yield). IR (neat) mNCO 2257 cmꢁ1 1H NMR
.
(CDCl3): d 3.93 (s, 3H), 7.28–7.29 (m, 1H), 7.37–7.44 (m, 1H),
7.76–7.78 (1H), 7.85–7.89 (m, 1H).
3-Phenoxyphenyl
5.2.3. Compound 2a
To a solution of 1a (15.06 g; 85 mmol) in anhydrous THF
(150 mL) was added propargylamine (4.68 g; 85 mmol). The reac-
tion mixture was stirred at room temperature overnight. The pre-
cipitate formed was collected by suction filtration to give 2a as a
white solid (18.5 g; 94% yield), mp 175–177 °C. 1H NMR (DMSO-
d6): d 3.13 (t, J = 2.45 Hz, 1H), 3.80 (s, 3H), 3.88 (dd, J = 5.70,
2.48 Hz, 2H), 6.71 (t, J = 5.69 Hz, 1H), 7.46–7.59 (m, 2H), 7.78–
7.91 (m, 2H), 9.11 (s, 1H).
In summary, the studies described herein have demonstrated
that the aminobenzamide scaffold can be employed in the synthe-
sis of inhibitors of DENV and WNV NS2B/NS3pro.
5. Experimental section
5.1. General
5.2.4. Compound 2b
The 1H spectra were recorded on a Varian XL-300 or XL-400
NMR spectrometer. Melting points were determined on a Mel-
Temp apparatus and are uncorrected. High resolution mass spectra
(HRMS) were performed at the University of Kansas Mass Spec-
trometry Lab. Reagents and solvents were purchased from various
chemical suppliers (Aldrich, Acros Organics, TCI America, and Ba-
chem). Silica gel (230–450 mesh) used for flash chromatography
was purchased from Sorbent Technologies (Atlanta, GA). Thin layer
chromatography was performed using Analtech silica gel plates.
The TLC plates for the final compounds were eluted using two dif-
ferent solvent systems and were visualized using iodine and/or UV
light. Each individual compound was identified as a single spot on
TLC plate (purity was greater than 95% by 1H NMR). DENV2 NS2B/
NS3 pro (or WNV NS2B/NS3 pro) substrate Bz-Nle-Lys-Arg-Arg-
AMC was purchased from Bachem, Torrance, CA or custom synthe-
sized by NeoBioScience, Cambridge, MA.
White solid (90% yield), mp 158–160 °C. 1H NMR (DMSO-d6): d
3.11 (t, J = 2.46 Hz, 1H), 3.84 (s, 3H), 3.88 (dd, J = 5.72, 2.46 Hz, 2H),
6.61 (t, J = 5.73 Hz, 1H), 7.37 (t, J = 7.87 Hz, 1H), 7.49–7.52 (m, 1H),
7.58–7.62 (m, 1H), 8.12 (t, J = 1.80 Hz, 1H), 8.94 (s, 1H).
5.2.5. Compound 3a
Compound 2a (11.61 g; 50 mmol) and phenylthiomethyl azide
(8.26 g; 50 mmol) were suspended in a 1:1 mixture of t-butanol
and water (100 mL). Sodium ascorbate (1.00 g; 5 mmol) and
CuSO4ꢂ5H2O (0.12 g; 0.5 mmol) were then added and the
heterogeneous mixture was stirred vigorously at room tempera-
ture for 2 days. The progress of the reaction was monitored by
TLC. The reaction mixture was diluted with water (200 mL) and
cooled in an ice bath. The precipitate formed was collected by suc-
tion filtration to give 3a as an off-white powder (19.47 g, 98%
yield), mp 155–157 °C. 1H NMR (CDCl3): d 3.88 (s, 3H), 4.48 (d,

S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
4143
COOCH3
COOCH3
COOCH3
O
COOCH3
i
ii
iii
HN
HN
HN
HN
NH2
O
NCO
1a, 1b
N
para isomers : 1a, 2a
meta isomers : 1b, 2b
2a, 2b
N
N
R1
O
3a-c, 4a-b
NHR2
COOH
O
iv
v
HN
HN
O
HN
HN
N
N
N
N
N
N
R1
R1
7a-t, 8a-f
5a-c, 6a-b
para isomers
R1 = phenylthiomethyl
benzyl
meta isomers
1 = phenylthiomethyl
benzyl
3a, 5a
3b, 5b
3c, 5c
4a, 6a
4b, 6b
R
4-fluorobenzyl
R2 = benzyl
phenylethyl
3-phenoxyphenyl
R2 = 4-phenoxyphenyl
benzyl
phenylethyl
phenyl
2-furanylmethyl
2-morpholinoethyl
3-phenoxyphenyl
Scheme 1. Reagents and conditions: (i) Trichloromethyl chloroformate/THF,
D
; (ii) propargl amine/THF; (iii) arklalkyl azide t-BuOH and H2O sodium ascorbate; (iv) aqLiOH/
1,4-dioxane; (V) CD/THF, followed by amine.
120
J = 5.84 Hz, 2H), 5.58 (s, 2H), 6.86 (t, J = 4.92 Hz, 1H), 7.21–7.34 (m,
5H), 7.39–7.42 (m, 2H), 7.66 (s, 1H), 7.91 (d, J = 8.73 Hz, 3H).
WNV protease
100
DENV2 protease
5.2.6. Compound 3b
Light green solid (93% yield), mp 175–177 °C. 1H NMR (DMSO-
d6): d 3.79 (s, 3H), 4.34 (d, J = 5.51 Hz, 2H), 5.57 (s, 2H), 6.79 (br
s, 1H), 7.28–7.40 (m, 5H), 7.50–7.53 (m, 2H), 7.82–7.84 (m, 2H),
8.02 (s, 1H), 9.03 (s, 1H).
80
60
40
20
0
5.2.7. Compound 3c
Light green solid (100% yield), mp 194–195 °C. 1H NMR (DMSO-
d6): d 3.79 (s, 3H), 4.33 (d, J = 5.55 Hz, 2H), 5.56 (s, 2H), 6.78 (t,
J = 5.61, Hz, 1H), 7.17–7.23 (m, 2H), 7.36–7.41 (m, 2H), 7.50–7.54
(m, 2H), 7.82–7.84 (m, 2H), 8.03 (s, 1H), 9.03 (s, 1H).
7g
7n
7t
8c
Aprotinin
5.2.8. Compound 4a
White solid (80% yield). 1H NMR (DMSO-d6): d 3.83 (s, 3H), 4.29
(d, J = 5.67 Hz, 2H), 5.92 (s, 2H), 6.69 (t, J = 5.67 Hz, 1H), 7.25–7.42
Compound
Figure 2. Inhibition of DENV2 and WNV NS2B/NS3pro by selected compounds at
25 M. The concentrations of WNV and DENV2 NS2B/NS3pro protease were 28 and
(m, 6H), 7.48–7.51 (m, 1H), 7.56–7.59 (m,1H), 7.91 (s, 1H), 8.13 (t,
J = 1.92 Hz, 1H), 8.89 (s, 1H).
l
25 nM, respectively. The buffer used contained 200 mM Tris–HCl, 6.0 mM NaCl, 30%
glycerol, and 0.1% CHAPS, pH 9.5. The percent values were calculated from the
relative fluorescence units obtained in the presence and absence of tested
compound. BPTI (Aprotinin) and DMSO were used as a positive and negative
control, respectively. All assays were performed in triplicate and the average values
are shown.
5.2.9. Compound 4b
White solid (66% yield), mp 158–160 °C. 1H NMR (DMSO-d6): d
3.83 (s, 3H), 4.33 (d, J = 5.47 Hz, 2H), 5.57 (s, 2H), 6.66 (t,

4144
S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
0.08
100
80
60
40
20
0.06
0.04
0.02
0.00
40
38
36
34
32
30
28
26
24
22
0
1
2
3
4
5
[7n] (μM)
-2
-1
0
1
0
10
20
30
40
50
Log[7n] (μΜ)
[Substrate] (μM)
Figure 3. Determination of IC50 value of the inhibitor 7n against DENV-2 and WNV
protease. The inhibitor was incubated with DENV2 NS2B/NS3pro (25 nM) or WNV
NS2B/NS3pro (28 nM) in buffer (200 mM Tris–HCl, 6 mM NaCl and 30% glycerol, pH
Figure 4. Inhibition of WNV NS2B/NS3pro protease activity by compound 7n.
Initial reaction rates of the substrate (Bz-Nle-Lys-Arg-Arg-AMC) cleavage catalyzed
by WNV NS2B/NS3pro protease (28 nM) in 200 mM Tris–HCl (pH 9.5), 6.0 mM NaCl,
30% glycerol and 0.1% CHAPS at 37 °C were determined by varying the substrate
9.5) for 15 min. Bz-Nle-Lys-Arg-Arg-AMC (5.0
lM) was added to the mixture in a
final volume of 100 L. The fluorescence intensity was measured at 460 nm with
l
concentrations in the range of 0, 1, 2, 4, 6, 8, 10, 15, 20, 30, 40 and 50
concentration of inhibitor fixed at 0 (solid circle), 1.0 M (open circle), 3.0
triangle) and 5.0 M (open triangle). The reactions were initiated by the addition of
lM at each
excitation at 380 nm and converted to the percentage of protease activity in the
absence and presence of inhibitors. The solid line is the theoretical fitting curve
based on the Sigmoidal equation.40 The apparent IC50 values for compound 7n were
l
l
M (solid
l
WNV NS2B/NS3pro protease and the fluorescence intensity at 460 nm was
monitored with an excitation at 380 nm. Reactions were less than 5% completion
in all cases to maintain valid steady-state measurements. The solid lines are fitted
lines using the Michaelis–Menten equation. Inset: Secondary plot of Km,app against
the concentration of compound 7n. Kinetics studies were carried out as described
6.82 0.09 and 5.51 0.08
respectively.
lM against DENV-2 (solid circle) and WNV (open circle),
utilizing substrate concentrations of 0–50 lM Bz-Nle-Lys-Arg-Arg-AMC. Each
J = 5.37 Hz, 1H), 7.29–7.40 (m, 6H), 7.48–7.50 (m, 1H), 7.56–7.59
(m, 1H), 8.02 (s, 1H), 8.13 (t, J = 1.76 Hz, 1H), 8.84 (s, 1H)
experiment was performed in duplicate and repeated three times. Data were
analyzed using SigmaPlot 2001 v7.0 software40 to determine values for apparent Km
and kcat
.
5.2.10. Compound 5a
To a solution of ester 3a (17 g; 42.5 mmol) in 1,4-dioxane
(500 mL) was added a solution of LiOH (12.21 g; 510 mmol) in
water (150 mL) and the reaction mixture was stirred at room tem-
perature overnight. The reaction mixture was cooled in an ice bath
and a 5% HCl solution was added dropwise until the pH was ꢀ3.
The precipitate formed was collected by suction filtration to give
acid 5a as a white solid (18.64 g, 97% yield), mp >230 °C. 1H NMR
(DMSO-d6): d 4.30 (d, J = 5.61 Hz, 2H), 5.93 (s, 2H), 6.77 (t,
J = 5.67 Hz, 1H), 7.23–7.42 (m, 5H), 7.48–7.51 (m, 2H), 7.80–7.83
(m, 2H), 7.92 (s, 1H), 9.01 (s, 1H), 12.49–12.72 (br s, 1H).
5.2.15. Compound 7a
To a solution of acid 5a (0.5 g; 1.30 mmol) in anhydrous THF
(20 mL) was added CDI (0.21 g; 1.304 mmol) and the mixture
was refluxed for 20 min. 4-Phenoxyaniline (0.24 g; 1.30 mmol)
was added and the mixture was stirred at room temperature for
2 h. The precipitate formed was collected by suction filtration to
give 7a as an off-white solid (0.23 g; 33% yield). 1H NMR (DMSO-
d6): d 4.32 (d, J = 5.60 Hz, 2H), 5.93 (s, 2H), 6.81 (t, J = 5.67 Hz,
1H), 6.96–7.14 (m, 5H), 7.23–7.43 (m, 8H), 7.50–7.57 (m, 2H),
7.74–7.81 (m, 2H), 7.93 (s, 2H), 9.01 (s, 1H), 10.09 (s, 1H). HRMS
(ESI): Calcd for C30H26N6O3SNa (M+Na) 573.1685; Found 573.1689.
5.2.11. Compound 5b
White solid (87% yield), mp >230 °C. 1H NMR (DMSO-d6): d 4.33
(s, 2H), 5.57 (s, 2H), 6.76–6.90 (br s, 1H), 7.28–7.40 (m, 5H), 7.47–
7.51 (m, 2H), 7.78–7.82 (m, 2H), 8.02 (s, 1H), 9.10 (s, 1H).
5.2.16. Compound 7b
White solid (89% yield), mp 182–183 °C. 1H NMR (DMSO-d6): d
4.30 (d, J = 5.63 Hz, 2H), 4.45 (d, J = 5.99 Hz, 2H), 5.93 (s, 2H), 6.90
(t, J = 5.40 Hz 1H), 7.20–7.49 (m,12H), 7.79 (d, J = 8.83 Hz, 2H), 7.92
(s, 1H), 8.86 (d, J = 5.98 Hz, 1H), 9.04 (s, 1H). HRMS (ESI): Calcd for
5.2.12. Compound 5c
White solid (99% yield), mp >230 °C. 1H NMR (DMSO-d6): d 4.33
(d, J = 5.61 Hz, 2H), 5.56 (s, 2H), 6.80 (t, J = 5.65 Hz, 1H), 7.16–7.24
(m, 2H), 7.35–7.42 (m, 2H), 7.46–7.50 (m, 2H), 7.78–7.82 (m, 2H),
8.03 (s, 1H), 9.03 (s, 1H), 12.25–12.84 (br s, 1H).
C25H24N6O2SNa (M+Na) 495.1579; Found 495.1596.
5.2.17. Compound 7c
White solid (68% yield), mp 201–202 °C. 1H NMR (DMSO-d6): d
2.82 (t, J = 7.45 Hz, 2H), 3.41–3.49 (m, 2H), 4.30 (d, J = 5.59 Hz, 2H),
5.92 (s, 2H), 6.83 (t, 5.64 Hz, 1H), 7.19–7.35 (m, 8H), 7.38–7.46 (m,
4H), 7.70–7.73 (m, 2H), 7.91 (s, 1H), 8.36 (t, J = 5.64 Hz, 1H), 8.96 (s,
1H). HRMS (ESI): Calcd for C26H26N6O2SNa (M+Na) 509.1736;
Found 509.1730.
5.2.13. Compound 6a
Off white solid (82% yield), mp 185–189 °C. 1H NMR (DMSO-d6):
d 4.29 (d, J = 5.56 Hz, 2H), 5.92 (s, 2H), 6.74 (t, J = 5.72 Hz, 1H),
7.24–7.49 (m, 7H), 7.57–7.61 (m,1H), 7.91 (s, 1H), 8.06 (t,
J = 1.78 Hz, 1H), 8.96 (s, 1H), 12.78–13.15 (br s, 1H).
5.2.14. Compound 6b
5.2.18. Compound 7d
Light green powder (88% yield), mp 192–194 °C. 1H NMR
(DMSO-d6): d 4.33 (d, J = 5.60 Hz, 2H), 5.57 (s, 2H), 6.65 (t,
J = 5.63 Hz, 1H), 7.25–7.39 (m, 6H), 7.45–7.49 (m, 1H), 7.55–7.60
(m, 1H), 8.02 (s, 1H), 8.04–8.06 (m,1H), 8.80 (s, 1H), 12.64–13.06
(br s, 1H).
Off white solid (88% yield), mp 190–191 °C. 1H NMR (DMSO-d6):
d 4.29–4.35 (m, 2H), 5.94 (s, 2H), 6.76 (t, J = 5.71 Hz, 1H), 7.04–7.10
(m, 2H), 7.24–7.36 (m, 5H), 7.39–7.43 (m, 2H), 7.48–7.55 (m, 2H),
7.74–7.93 (m, 4H), 8.98 (s, 1H), 10.05 (s, 1H). HRMS (ESI): Calcd for
C24H22N6O2SNa (M+Na) 481.1423; Found 481.1439.

S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
4145
Table 3
Inhibition of DENV2 and WNV NS2B/NS3pro protease by compounds 7g, 7n, 7t and 8c
Inhibitors
DENV2
WNV
% Inhibition at 10
lM
% Inhibition at 25
lM
% Inhibition at 10
lM
% Inhibition at 25 lM
7g
7n
7t
39.41 2.69
55.31 1.39
41.06 1.83
40.27 1.11
52.54 3.21
76.01 0.96
56.98 1.81
58.21 4.31
46.51 1.75
58.78 1.62
44.45 2.08
49.28 1.87
57.95 0.71
82.26 1.04
60.85 2.46
60.27 2.36
8c
WNV protease = 28 nM; DENV2 protease = 25 nM.
Table 4
Kinetics parameters for the tetra-peptide substrate and compound 7n against WNV
NS2B/NS3pro at 37 °C
7n (lM)
Km
(
lM)
kcat (sꢁ1
)
kcat/Km (Mꢁ1 sꢁ1
)
0
1
3
5
24.01 0.62
26.04 0.75
34.16 1.88
36.90 2.36
0.060 0.0013
0.059 0.0014
0.062 0.0018
0.055 0.0020
2499 99
2274 100
1811 163
1503 162
5.2.19. Compound 7e
Light brown solid (90% yield), mp 146–148 °C. 1H NMR (DMSO-
d6): d 4.31 (d, J = 5.55 Hz, 2H), 4.44 (d, J = 5.62 Hz, 2H), 5.93 (s, 1H),
6.86 (t, J = 5.50 Hz, 1H), 7.04 (br s, 2H), 7.22–7.92 (m, 11H), 8.79 (t,
J = 5.71 Hz, 1H), 9.02 (s, 1H). HRMS (ESI): Calcd for C23H22N6O3SNa
(M+Na) 485.1372; Found 485.1373.
5.2.20. Compound 7f
Off white solid (46% yield), mp 184–185 °C. 1H NMR (DMSO-d6):
d 2.40–2.45 (m, 8H), 3.56 (t, J = 4.45 Hz, 4H), 4.30 (d, J = 5.57 Hz,
2H), 5.93 (s, 2H), 6.89 (t, J = 5.18 Hz, 1H), 7.24–7.47 (m, 7H),
7.71–7.73 (m, 2H), 7.92 (s, 1H), 8.21 (t, J = 5.80 Hz, 1H), 9.02 (s,
1H). HRMS (ESI): Calcd for C24H30N3O7S (M+H) 496.2131; Found
496.2114.
Figure 5A. Computationally predicted conformation of compounds 7a, 7n and 8f
bound to the catalytic site of DENV2 NS2B/NS3 protease. Ligands are rendered as
CPK-colored sticks with the exception of carbon atoms (7a: orange, 7n: white, 8f:
black), while the receptor surface is colored as follows: yellow = hydrophobic,
white = polarized alkyl or aryl groups, cyan = polar hydrogens, blue = polar nitro-
gens and red = polar oxygens.
5.2.21. Compound 7g
Light pink solid (47% yield), mp >230 °C. 1H NMR (DMSO-d6): d
4.35 (d, J = 5.56 Hz, 2H), 5.58 (s, 2H), 6.77 (t, J = 5.67 Hz, 1H), 6.96–
7.05 (m, 4H), 7.08–7.14 (m, 1H), 7.31–7.41 (m, 7H), 7.53 (d,
J = 8.78 Hz, 2H), 7.75–7.80 (m, 2H), 7.87 (d, J = 8.77 Hz, 2H),
8.03(s, 1H), 8.95 (s, 1H), 10.08 (s, 1H). 13C NMR (DMSO-d6): d
164.71, 157.27, 154.63, 151.71, 145.55, 143.43, 136.04, 135.17,
129.85, 128.62, 128.00, 127.85, 126.88, 122.84, 122.70, 122.57,
121.88, 119.18, 117.75, 116.53, 52.61, 34.74. HRMS (ESI): Calcd
for C30H26N6O3SNa (M+Na) 541.1964; Found 541.1956.
5.2.22. Compound 7h
White solid (54% yield), mp 213–214 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.56 Hz, 2H), 4.45 (d, J = 5.96 Hz, 2H), 5.57 (s, 2H), 6.74
(t, J = 5.53 Hz, 1H), 7.30–7.40 (m, 10H), 7.44–7.48 (m, 2H), 7.76–
7.82 (m, 2H), 8.02 (s, 1H), 8.78–8.94 (m, 2H). HRMS (ESI): Calcd
for C25H24N6O2SNa (M+Na) 463.1858; Found 463.1844.
5.2.23. Compound 7i
White solid (62% yield), mp 230 °C. 1H NMR (DMSO-d6): d 2.82
(t, J = 7.0 Hz, 2H), 3.41–3.48 (m, 2H), 4.33 (d, J = 5.57 Hz, 2H), 5.57
(s, 2H), 6.74 (t, J = 5.59 Hz, 1H), 7.16–7.40 (m, 10H), 7.42–7.47 (m,
2H), 7.70–7.73 (m, 2H), 8.02 (s, 1H), 8.38 (t, J = 5.65 Hz,1H), 8.87 (s,
1H). HRMS (ESI): Calculated for C26H26N6O2SNa (M+Na) 477.2015;
found 477.2036.
Figure 5B. Computationally predicted conformation of compound 7n bound to the
catalytic site of WNV NS2B/NS3 protease. Ligands are rendered as CPK-colored
sticks with the exception of carbon atoms (7a: orange, 7n: white, 8f: black), while
the receptor surface is colored as follows: yellow = hydrophobic, white = polarized
alkyl or aryl groups, cyan = polar hydrogens, blue = polar nitrogens and red = polar
oxygens.
5.2.24. Compound 7j
White solid (35% yield), mp >230 °C. 1H NMR (DMSO-d6): d 4.35
(d, J = 5.56 Hz, 2H), 5.58 (s, 2H), 6.75 (t, J = 5.56 Hz, 1H), 7.07 (t,

4146
S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
J = 7.37 Hz, 1H), 7.29–7.40 (m, 7H), 7.50–7.54 (m, 2H), 7.74–7.78
(m, 2H), 7.85–7.90 (m, 2H), 8.03 (s, 1H), 8.94 (s, 1H), 10.04 (s,
5.2.32. Compound 7r
White solid (99% yield), mp 189–191 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.60 Hz, 2H), 4.43 (d, J = 5.65 Hz, 2H), 5.56 (s, 2H), 6.24–
6.25 (m, 1H), 6.37–6.39 (m, 1H), 6.79–6.86 (m, 1H), 7.02 (s, 1H),
7.16–7.24 (m, 2H), 7.34–7.50 (m, 4H), 7.72–7.83 (m, 2H), 8.03 (s,
1H), 8.76 (t, J = 5.70 Hz, 1H), 9.00 (s, 1H). HRMS (ESI): Calcd for
1H). HRMS (ESI): Calculated for
449.1702; found 449.1722.
C24H22N6O2SNa (M+Na)
5.2.25. Compound 7k
White solid (35% yield), mp 207–208 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.56 Hz, 2H), 4.43 (d, J = 5.66 Hz, 2H), 5.57 (s, 2H),
6.24–6.25 (m, 1H), 6.38–6.39 (m, 1H), 6.76 (t, J = 5.59 Hz, 1H),
7.28–7.40 (m, 5H), 7.42–7.46 (m, 2H), 7.56–7.57 (m, 1H), 7.74–
7.78 (m, 2H), 8.02 (s, 1H), 8.76(t, J = 5.80 Hz, 1H), 8.89 (s, 1H).
HRMS (ESI): Calcd for C23H22N6O3SNa (M+Na) 453.1651; Found
453.1651.
C23H21FN6O3Na (M+Na) 471.1557; Found 471.1555.
5.2.33. Compound 7s
(90% Yield), mp 163–166 °C. 1H NMR (DMSO-d6) d 2.35–2.47 (m,
8H), 3.51- 3.63 (m, 4H), 4.32 (d, J = 5.56 Hz, 2H), 5.56 (s, 2H), 6.83
(t, J = 5.62 Hz, 1H), 7.01 (s, 1H), 7.16–7.24 (m, 2H), 7.36–7.47 (m,
4H), 7.69–7.73 (m, 2H), 8.02 (s, 1H), 8.21 (t, J = 5.64 Hz, 1H), 9.01
(s, 1H). HRMS (ESI): Calcd for C24H29FN7O3 (M+Na) 482.2316;
Found 482.2297.
5.2.26. Compound 7l
White solid (45% yield), mp 204–206 °C. 1H NMR (DMSO-d6): d
2.36–2.45 (m, 6H), 3.33–3.37 (m, 2H), 3.56 (t, J = 4.59 Hz, 4H), 4.33
(d, J = 5.57 Hz, 2H), 5.57 (s, 2H), 6.77 (t, J = 5.63 Hz, 1H), 7.29–7.39
(m, 5H), 7.42–7.45 (m, 2 H), 7.70–7.73 (m, 2H), 8.02 (s, 1H), 8.21 (t,
J = 5.67 Hz, 1H), 8.91(s, 1H). HRMS (ESI): Calcd for C24H30N7O3
(M+H) 464.2410; Found 464.2422.
5.2.34. Compound 7t
Off white solid (16% yield), mp 211–212 °C. 1H NMR (DMSO-d6):
d 4.34 (d, J = 5.50 Hz, 2H), 5.56 (s, 2H), 6.69–6.80 (m, 2H), 7.00–7.09
(m, 2H), 7.10–7.27 (m, 3H), 7.29–7.46 (m, 5H), 7.47–7.62 (m, 4H),
7.84 (d, J = 8.78 Hz, 2H), 8.03 (s, 1H), 8.95 (s, 1H), 10.10 (s, 1H). 13
NMR (DMSO-d6): d 164.96, 162.97, 160.54, 156.75, 156.43, 154.61,
145.61, 143.56, 140.89, 132.34, 130.24, 129.95, 128.66, 126.71,
123.39, 122.63, 118.68, 116.46, 115.58, 115.37, 114.89, 109.95,
51.79, 34.74. HRMS (ESI): Calcd for
559.1870; Found 559.1862.
C
5.2.27. Compound 7m
Light pink solid (25% yield), mp 199–200 °C. 1H NMR (DMSO-
d6): d 4.34 (d, J = 5.55 Hz, 2H), 5.57 (s, 2H), 6.72–6.77 (m, 2H),
7.02–7.07 (m, 2H), 7.12–7.18 (m, 1H), 7.30–7.44 (m, 8H), 7.49–
7.58 (m, 4H), 7.82–7.85 (m, 2H), 8.02 (s, 1H), 8.94 (s, 1H), 10.10
(s, 1H). HRMS (ESI): Calcd for C30H26N6O3Na (M+Na) 541.1964;
Found 541.1932.
C30H25FN6O3Na (M+Na)
5.2.35. Compound 8a
White solid (21% yield), mp 156–157 °C. 1H NMR (DMSO-d6): d
4.30 (d, J = 5.64 Hz, 2H), 4.45 (d, J = 5.99 Hz, 2H), 5.93 (s, 2H), 6.65
(t, J = 5.67 Hz, 1H), 7.22–7.44 (m, 12H), 7.56–7.61(m, 1H), 7.85 (t, J
=1.79 Hz, 1H), 7.91(s, 1H), 8.75(s, 1H), 8.97(t, J = 6.07 Hz, 1H). HRMS
(ESI): Calcd for C25H24N6O2SNa (M+Na) 495.1579; Found 495.1567.
5.2.28. Compound 7n
White solid (49% yield), mp >230 °C. 1H NMR (DMSO-d6): d
4.32 (d, J = 5.59 Hz, 2H), 5.55 (s, 2H), 6.71 (t, J = 5.62 Hz, 1H),
6.93–7.04 (m, 4H), 7.05–7.12 (m, 1H), 7.14–7.24 (m, 2H), 7.32–
7.42 (m, 4H), 7.47–7.52 (m, 2H), 7.72–7.80 (m, 2H), 7.82–7.90
(m, 2H), 8.02 (s, 1H), 8.91 (s, 1H), 10.06 (s, 1H). 13C NMR
(DMSO-d6): d 164.72, 162.97, 160.54, 157.28, 154.64, 151.73,
145.62, 143.44, 135.18, 132.34, 130.24, 129.87, 128.54, 126.90,
122.86, 122.59, 121.85, 119.17, 117.77, 116.51, 115.58, 115.37,
5.2.36. Compound 8b
White solid (57% yield), mp 155–156 °C. 1H NMR (DMSO-d6): d
2.83 (t, J = 7.46 Hz, 2H), 3.45 (q, J = 6.30 Hz, 2H), 4.30 (s, 2H), 5.93
(s, 2H), 6.72 (br s, 1H), 7.19–7.42 (m, 12H), 7.55–7.59 (m, 1H),
7.79 (t, J = 1.80 Hz, 1H), 7.91 (s, 1H), 8.50 (t, J = 5.55 Hz, 1H), 8.83
(s, 1H). HRMS (ESI): Calcd for C26H26N6O2SNa (M+Na) 509.1736;
Found 509.1757.
51.80, 34.74. HRMS (ESI): Calcd for
559.1870; Found 559.1859.
C30H25FN6O3Na (M+Na)
5.2.37. Compound 8c
White solid (21% yield), mp 179–181 °C. 1H NMR (DMSO-d6): d
4.30 (d, J = 5.65 Hz, 2H), 5.93 (s, 2H), 6.72–6.82 (m, 2H), 7.02–7.08
(m, 2H), 7.12–7.20 (m, 1H), 7.22–7.47 (m, 10H), 7.50–7.65 (m, 3H),
7.87 (t, J = 1.76 Hz, 1H), 7.91 (s, 1H), 8.90 (s, 1H), 10.28 (s, 1H). 13C
NMR (DMSO-d6): d 165.79, 156.79, 156.37, 154.89, 145.89, 140.70,
140.42, 135.42, 132.50, 130.25, 129.95, 129.84, 129.14, 128.54,
127.47, 123.43, 122.37, 120.63, 120.09, 118.71, 116.98, 114.87,
113.52, 109.93, 51.36, 34.69. HRMS (ESI): Calcd for C30H26N6O3SNa
(M+Na) 573.1685; Found 573.1693.
5.2.29. Compound 7o
White solid (91% yield), mp 208–209 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.56 Hz, 2H), 4.45 (d, J = 5.94 Hz, 2H), 5.56 (s, 2H), 6.98
(t, J = 5.58 Hz, 1H), 7.15–7.26 (m, 3H), 7.28–7.33 (m, 4H), 7.34–7.43
(m, 2H), 7.46–7.49 (m, 2H), 7.77–7.80 (m, 2H), 8.03 (s, 1H), 8.85 (t,
J = 5.95 Hz, 1H), 9.10 (s, 1H). HRMS (ESI): Calcd for C25H23FN6O2Na
(M+Na) 481.1761; Found 481.1754.
5.2.30. Compound 7p
White solid (90% yield), mp 207–209 °C. 1H NMR (DMSO-d6): d
2.82 (t, J = 7.46 Hz, 2H), 3.43 (m, 2H), 4.33 (d, J = 5.55 Hz, 2H), 5.56
(s, 2H), 7.05 (t, J = 5.56 Hz, 1H), 7.16–7.41 (m, 9H), 7.46 (d,
J = 8.79 Hz, 2H), 7.71 (d, J = 8.75 Hz, 2H), 8.03 (s, 1H), 8.38 (t,
J = 5.55 Hz, 1H), 9.17 (s, 1H). HRMS (ESI): Calcd for C26H25FN6O2Na
(M+Na) 495.1921; Found 495.1921.
5.2.38. Compound 8d
White solid (80% yield), mp 162–164 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.58 Hz, 2H), 4.45 (d, J = 5.97 Hz, 2H), 5.57 (s, 2H), 6.67
(t, J = 5.61 Hz, 1H), 7.2–7.42 (m, 12H), 7.57–7.60 (m, 1H), 7.84 (t,
J = 1.64 Hz, 1H), 8.01 (s, 1H), 8.75 (s, 1H), 8.96 (t, J = 5.96 Hz, 1H).
HRMS (ESI): Calcd for C25H24N6O2Na (M+Na) 509.1736; Found
509.1757.
5.2.31. Compound 7q
White solid (46% yield), mp >230 °C. 1H NMR (DMSO-d6): d 4.35
(d, J = 5.55 Hz, 2H), 5.57 (s, 2H), 6.86 (t, J = 5.62 Hz, 1H), 7.04–7.09
(m, 1H), 7.17–7.43 (m, 7H), 7.53 (d, J = 8.78 Hz, 1H), 7.75–7.79 (m,
2H), 7.88 (d, J = 8.75 Hz, 2H), 8.04 (s, 1H), 9.12 (s, 1H), s (10.06, 1H).
HRMS (ESI): Calcd for C24H21FN6O2Na (M+Na) 467.1608; Found
467.1594.
5.2.39. Compound 8e
White solid (83% yield), mp 197–198 °C. 1H NMR (DMSO-d6): d
2.83 (t, J = 7.45 Hz, 2H), 3.41–3.49 (m, 2H), 4.33 (d, J = 5.56 Hz, 2H),
5.57 (s, 2H), 6.68 (t, J = 5.65 Hz, 1H), 7.17–7.40 (m, 12H), 7.55–7.58
(m, 1H), 7.78 (t, J = 1.65 Hz, 1H), 8.01 (s, 1H), 8.48 (t, J = 5.60 Hz,

S. Aravapalli et al. / Bioorg. Med. Chem. 20 (2012) 4140–4148
4147
1H), 8.74 (s, 1H). HRMS (ESI): Calcd for C26H26N6O2Na (M+Na)
477.2015; Found 477.2032.
active form crystal structure of Noble et al. (PDB ID: 3U1I)12 by
extracting all ligands and waters, and protonating the receptor
according to assumption of anionic aspartate and glutamate
groups, and cationic arginines and lysines). The WNV NS2B/NS3pro
receptor model was prepared in an analogous manner using the
crystal structure of Erbel et al. (PDB ID 2FP7).13 In both cases, the
co-crystallized ligand was used to define the Surflex protomol
structure (which guides the original ligand binding site prediction),
and five distinct randomized starting conformations and 100 final
conformations were specified for the ligand. Ligands were con-
structed and refined according to default molecular mechanics
constraints, force fields and optimization settings via the SYBYL
8.1 program (Tripos Associates, St. Louis, MO, 2009).
5.2.40. Compound 8f
White solid (64% yield), mp 201–202 °C. 1H NMR (DMSO-d6): d
4.33 (d, J = 5.53 Hz, 2H), 5.57 (s, 2H), 6.69–6.79 (m, 2H),7.02–7.08
(m, 2H), 7.13–7.19 (m, 1H), 7.29–7.46 (m, 10H), 7.51–7.64 (m,
3H), 7.86 (s, 1H), 8.01 (s, 1H), 8.83 (s, 1H), 10.28 (s, 1H). HRMS
(ESI): Calcd for C30H26N6O3Na (M+Na) 541.1945; Found 541.1945.
6. Biochemistry
6.1. DENV2 NS2B/NS3pro expression and purification
Acknowledgments
The construction of the pQE30-NS2BH(QR)NS3pro expression
plasmid has been described previously.26 The protease was ex-
pressed from Escherichia coli strain Top 10 F’ (Invitrogen) trans-
formed by pQE30-NS2BH(QR)–NS3pro plasmid and purified as
described.28 DENV2 NS2B/NS3pro contains the hydrophilic NS2B
cofactor peptide (NS2BH) linked to the N-terminal NS3 protease
domain via Q-R (P2 and P1 residues at the NS2B–NS3 junction site).
The generous financial support of this work by the National
Institutes of Health (AI082068) is gratefully acknowledged.
References and notes
1. Lindenbach, B. D.; Thiel, H.-J.; Rice, C. M., 5th ed. In Fields Virology; Knipe, D. M.,
Howley, P. M., Eds.; Lippincott, Williams and Wilkins: Philadelphia, 2007; Vol.
1, pp 1101–1152.
2. Stevens, A. J.; Gahan, M. E.; Mahalingam, S.; Keller, P. A. J. Med. Chem. 2009, 52,
7911.
3. Cregar-Hernandez, L.; Jiao, G.-S.; Johnson, A. T.; Lehrer, A. T.; Wong, T. A. S.;
Margosiak, S. A. Antiviral Chem. Chemother. 2011, 21, 209.
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6.2. WNV NS2B/NS3pro expression and purification
The expression and purification of the WNV NS2BH–NS3pro
containing the five amino acid spacer between the NS2B and
NS3pro domains was previously described.28
5. Sampath, A.; Padmanabhan, R. Antiviral Res. 2009, 81, 6.
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6.3. In vitro DENV2 and WNV NS2B/NS3pro assays and inhibition
studies
The compounds were dissolved in dimethyl sulfoxide (DMSO)
to make 50 mM stock solutions. The compounds were screened
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says were performed in triplicates in Greiner Black 96 well plates.
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lM in 1% v/v DMSO in the final reaction mixture. Protease as-
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l
200 mM Tris–HCl buffer, pH 9.5, 30% glycerol, 25 nM DENV2
NS2B–NS3 pro (or 28 nM WNV NS2B/NS3pro) and the compound.
The enzyme and the compound were pre-incubated at room tem-
perature for 15 min prior to addition of the substrate (5 lM), Bz-
Nle-Lys-Arg-Arg-AMC. The time course of the reaction at 37 °C
was followed at every 90 s intervals for up to 30 min in a monoch-
rometer-based spectrofluorometer (Molecular Devices, Sunnyvale,
CA) at excitation and emission wavelengths of 380 and 460 nm,
respectively. The percent inhibition for each compound at 25 lM
was first determined. For determining IC50 values, twelve data
points obtained from the range of 10, 50 nM, 0.1, 0.5, 1, 2, 4, 6, 8,
10, 20, and 25 lM inhibitor concentrations of selected compounds
were used. IC50 values were calculated using the SigmaPlot 2001
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v7.0 software.40
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7. Kinetics analysis
To determine the Km and Vmax values of compound 7n, four dif-
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l
M) were as-
sayed at varying concentrations (0–50
lM) of substrate. The
kinetics analysis methods have been previously described.41 Ki val-
ues were calculated from these data using a secondary plot of K
against the concentrations of selected compound 7n using
m,app
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