Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)- one and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

Article abstract of DOI:10.1016/j.bmc.2012.10.058

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one - 1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC₅₀ values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure-activity relationship studies, in vitro screening and X-ray crystallography.

Full text of DOI:10.1016/j.bmc.2012.10.058

Bioorganic & Medicinal Chemistry 21 (2013) 102–113
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one
and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue
and West Nile virus NS2B/NS3 proteases
Huiguo Lai a, , Dengfeng Dou b, , Sridhar Aravapalli b, , Tadahisa Teramoto a, Gerald H. Lushington c,
Tom M. Mwania b, Kevin R. Alliston b, David M. Eichhorn b, Radhakrishnan Padmanabhan a,
William C. Groutas b,
⇑
a Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA
b Department of Chemistry, Wichita State University, Wichita, KS 67260, 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:
1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable
interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of func-
tionalized 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives were synthesized and sub-
sequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds
Received 29 August 2012
Revised 22 October 2012
Accepted 31 October 2012
Available online 15 November 2012
showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10
of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 0.06 and 4.22 0.07
l
M). The IC50 values
M,
l
Keywords:
1,2-Benzisothiazol-3(2H)-ones
1,3,4-Oxadiazoles
Dengue virus protease
West Nile virus protease
respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular
modeling studies were performed to delineate the putative binding mode of this series of compounds.
This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable
platform for conducting a hit-to-lead optimization campaign via iterative structure–activity relationship
studies, in vitro screening and X-ray crystallography.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Both Dengue and West Nile viruses contain a single-strand sense
RNA (ssRNA) genome with positive polarity, approximately 11 kb in
Dengue virus (DENV) and West Nile virus (WNV) are the most
prevalent mosquito-borne viruses affecting humans.1 DENV and
WNV belong to the family Flaviviridae (genus Flavivirus) which
comprises several human and animal pathogens. Other notewor-
thy members in this family include Yellow fever virus (YFV), Japa-
nese encephalitis virus (JEV) and Hepatitis C virus (HCV).2 Four
distinct Dengue serotypes exist based on their antigenic properties
and/or sequence similarities. Each serotype can cause Dengue dis-
ease. Only one serotype exists for WNV. Dengue fever is a relatively
mild illness with rash; however, infection by Dengue virus can
potentially progress to a more severe illness, called Dengue hemor-
rhagic fever. Approximately 2.5 billion people worldwide are at
risk for Dengue infection alone. Indeed, Dengue virus is estimated
to infect 50–100 million individuals every year and causes approx-
imately 1.5 million clinical cases of Dengue disease annually.3,4
Currently, there is no effective chemotherapeutic agent or vaccine
for the management and treatment of Dengue infection.5
length flanked by a 50 cap but no poly(A) tail at the 30 end non-trans-
lated regions. After endocytosis and release of the viral nucleocapsid
from the viral RNA, the single long open reading frame (ORF) of the
viral RNA encodes a polyprotein, which is processed by both a cellu-
lar signal peptidase and a viral serine protease at the surface of the
endoplasmic reticulum (ER), resulting in the formation of three
structural proteins, capsid (C), precursor membrane (prM) and
envelope (E), and seven non-structural (NS) proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B and NS5).6,7 NS3 is a multi-functional pro-
tein containing a serine protease with a prototypical catalytic triad
(H51, D75, S135) within the N-terminal 185 amino acid residues,
an NTPase/RNA helicase, and a 50-RNA triphosphatase within the
C-terminal (161–618 amino acid residues) region of NS3.6 However,
NS3 alone is not active. NS2B is an ER-resident integral membrane
protein consisting of three hydrophobic domains and a conserved
hydrophilic domain. The hydrophobic sequence of NS2B is essential
for co-translational insertion of the protease cofactor into ER
membranes for efficient cleavage of the NS2B/NS3 junction. The
hydrophilic region of 49–92 amino acids of NS2B serves as an activa-
tor and is sufficient for interaction with and activation of the NS3
protease domain (hereafter referred to as ‘NS2B/NS3pro’) in vitro.7,8
⇑
Corresponding author. Tel.: +1 (316) 978 7374; fax: +1 (316) 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
These authors contributed equally in this study.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.058

H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
103
O
has found use in a variety of applications,34 including as an essen-
tial component of the pharmacophore and as a bioisosteric replace-
ment for carbonyl-containing compounds, such as esters, amides
and others. Recently, sulfonamide-1,3,4-oxadiazole derivatives
have been shown to exhibit noteworthy antibacterial and anti-
fungal activity.35
R1
N
S
O
N
R2
N
(I)
In this study, we describe the design, synthesis, and structure-
activity relationship (SAR) studies of a series of novel 1,2-ben-
zisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives,
represented by general structure (I) (Fig. 1) as potential inhibitors
of DENV2 and WNV NS2B/NS3 proteases. Inhibition percentage
and steady state kinetics against DENV2 and WNV NS2B/NS3 pro-
teases were also carried out, leading to the identification of a low
micromolar inhibitor.
Figure 1. General structure of inhibitor (I).
There are many Dengue and West Nile viral proteins that have
been targeted for drug discovery, including helicase,9,10 methyl
transferase,11–13 serine protease14–18 and viral RNA.19,20 In general,
the blockage of NS2B/NS3pro activity provides an effective means
for designing DENV and WNV small-molecule therapeutics.21–23
Small-molecule inhibitors of DENV2 and WNV proteases are of
interest both as therapeutic agents and as chemical probes for
investigating biological functions related to virus replication and
reproduction.24,25 To date only a small number of non-peptidyl
inhibitor scaffolds for DENV and WNV proteases have been de-
scribed and the available structure-activity relationship data are
rather limited.5,23,25
1,2-Benzisothiazol-3(2H)-one derivatives play a significant role
in various pharmaceutical applications.26–28 As an important class
of heterocyclic compounds, 1,2-benzisothiazol-3(2H)-ones show
broad spectrum bioactivity.29–32 Among these, suitably-substituted
1,2-benzisothiazol-3(2H)-ones have been shown to exhibit potent
antifungal33 and antivirus activity.17 Compounds containing a
1,3,4-oxadiazole template have also received significant attention
in medicinal and pharmaceutical research as this structural motif
2. Results and discussion
2.1. Chemistry
In this study, 24 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiaz-
ole hybrid derivatives were synthesized. The synthetic routes
leading to compounds 7a–x (Table 1) are illustrated in the syn-
thetic Scheme 1. Compounds 7a–d were synthesized from com-
pound 2, which was in turn synthesized33 by the sequential
treatment of 1,2-benzisothiazolin-3(2H)-one with NaH and t-bu-
tyl bromoacetate in trifluoroethanol, followed by hydrolysis of
the resulting ester with trifluoroacetic acid (Scheme 1). Coupling
of 2 with a series of hydrazides36 followed by p-toluenesulfonyl
chloride-mediated cyclodehydration of the corresponding prod-
ucts, yielded the desired compounds. Compounds 7e–l and
Table 1
Inhibition of DENV2 and WNV NS2B/NS3pro by compounds 7a–7x at 10 and 25 lM
O
R1
*
N
S
O
N
R2
N
(I)
Inhibitor
R1
R2
DENV2
WNV
% Inhibition at 10
lM
% Inhibition at 25
lM
% Inhibition at 10
lM
% Inhibition at 25 lM
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
H
H
H
m-Fluorophenyl
m-Chlorophenyl
Benzyl
p-Methoxybenzyl
p-Methoxybenzyl
Benzyl
p-Methoxybenzyl
m-Fluorophenyl
m-Chlorophenyl
p-Fluorophenyl
p-Chlorophenyl
p-Cyanophenyl
Phenyl
p-Methoxyphenyl
p-Ethoxyphenyl
p-Pyridyl
m-Pyridyl
2-Furyl
2-Thiophenyl
Methyl
Ethyl
Propyl
m-Chlorophenyl
m-Chlorophenyl
3.24 0.93
5.21 2.17
3.56 1.09
5.27 1.46
8.08 1.56
22.38 0.87
28.49 1.82
39.03 1.06
34.27 2.07
60.34 1.12
53.25 1.64
63.86 1.71
56.73 0.95
65.49 1.18
40.65 1.79
62.43 1.82
58.06 1.55
52.28 1.74
52.34 2.23
19.98 1.45
22.54 1.55
25.58 1.07
15.97 1.72
26.06 1.15
9.11 1.76
16.74 1.97
16.43 1.84
7.90 1.94
7.68 0.98
6.15 1.11
8.27 1.37
5.96 1.07
4.17 0.63
20.54 0.91
24.23 0.61
30.36 0.84
29.01 0.75
51.12 1.47
53.46 1.27
63.57 1.59
51.71 1.16
68.58 1.38
56.09 1.18
63.13 1.49
55.83 1.32
44.32 1.53
43.68 1.36
22.32 0.82
25.07 1.75
34.94 1.83
27.44 0.59
23.86 0.84
10.07 0.87
9.15 0.79
14.73 1.04
8.68 0.95
Methyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
m-Fluorobenzyl
Phenethyl
17.82 1.99
31.23 1.23
38.64 1.69
50.87 1.55
42.12 1.89
75.74 1.95
70.52 2.37
72.94 1.24
69.24 1.42
80.01 1.73
61.27 1.71
70.87 1.27
69.97 1.02
64.74 1.51
69.81 1.64
31.59 1.37
34.22 2.43
36.68 1.91
31.61 2.06
35.01 2.21
9.79 1.02
30.12 1.02
34.86 0.45
38.91 0.61
43.11 0.71
59.09 1.35
63.24 1.24
70.87 1.32
64.09 0.91
73.42 1.22
71.84 2.67
72.68 0.88
62.52 0.98
57.67 1.02
59.66 1.46
40.39 2.19
44.26 1.69
46.19 2.25
35.65 0.22
33.85 0.54
7s
7t
7u
7v
7w
7x
[WNV protease] = 28 nM; [DENV2 protease] = 25 nM. ⁄(DL) methyl, (L) benzyl, (DL) m-fluorobenzyl, (DL) phenethyl.

104
H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
O
O
O
iii
i, ii
N
N
NH
S
OH
S
S
O
O
O
2
1
O
R1
O
O
O
R1
R1
COOCH3
vi
v
N
H
COOCH3
OH
S)2
iv
N
N
2
S
COOH
S
S)2
5a-d
3a-d
4a-d
R1 = (DL) methyl
(L) benzyl
3a
3b
3c
3d
(DL) m-fluorobenzyl
(DL) phenethyl
O
O
R1
R1
viii
vii
N
N
S
S
NH
HN
R2
O
O
O
N
N
R2
7a-l, 7w-x
6a-l, 6w-x
O
O
(L)
ix,x
(L)
N
N
O
S
S
COOH
N
R2
N
5b
7m-v
R1 = H, R2 = m-fluorophenyl
= m-chlorophenyl
= benzyl
R1 = (L) benzyl, R2 = phenyl
6a/7a
6m/7m
6b/7b
6c/7c
6d/7d
6e/7e
6f/7f
= p-methoxyphenyl
= p-ethoxyphenyl
= p-pyridyl
= m-pyridyl
6n/7n
6o/7o
6p/7p
6q/7q
6r/7r
6s/7s
6t/7t
6u/7u
6v/7v
6w/7w
6x/7x
= p-methoxybenzyl
R1 = (DL) methyl, R2 = p-methoxybenzyl
R1 = (L) benzyl, R2 = benzyl
= p-methoxybenzyl
= 2-furyl
= 2-thiophenyl
= methyl
= ethyl
= propyl
6g/7g
6h/7h
6i/7i
6j/7j
6k/7k
6l/7l
= m-fluorophenyl
= m-chlorophenyl
= p-fluorophenyl
= p-chlorophenyl
R1 = (DL) m-fluorobenzyl, R2 = m-chlorophenyl
R1 = (DL) phenethyl, R2 = m-chlorophenyl
= p-cyanophenyl
Reagents and conditions: i) NaH/CH3CN; ii) BrCH2COOC(CH3)3 /CF3CH2OH; iii) TFA; iv) CDI/THF, followed by amino
acid and TEA; v) Br2/CH2Cl2, TEA; vi) LiOH/H2O, THF; vii) CDI/THF, R2CONHNH2; viii) p-TsCl/CH2Cl2, TEA; ix)
R2CONHNH2, DIPEA, TBTU, dry CH3CN; x) DIPEA, p-TsCl
Scheme 1. Synthesis of compounds 3a–d, 4a–d, 5a–d, 6a–x and 7a–x.
7w–x were prepared using 2,20-dithiodibenzoic acid as the start-
ing material (Scheme 1) via treatment with 1,10-carbonyldiimid-
azole (CDI) and an appropriate amino acid methyl ester
hydrochloride to afford the corresponding ester 3. Intermediate
4 was obtained by reacting 3 with bromine, followed by the addi-
tion of triethylamine. The corresponding acids 5 were obtained
following hydrolysis with LiOH in aqueous THF. Substituted
hydrazide derivatives 6 were obtained by reacting 5 with 1,10-car-
bonyldiimidazole in THF and an array of substituted hydrazides.
Finally, treatment of hydrazides 6 with p-toluenesulfonyl chloride
in the presence of triethylamine in CH2Cl2 under ambient temper-
ature gave cyclodehydration products 7. Compounds 7m–v were
conveniently prepared using a one-pot synthesis, as described
in the literature37 (Scheme 1). The synthesized compounds are
listed in Table 1.
2.2. X-ray crystallography
The structure of a representative member of this series (com-
pound 7p) was determined (Fig. 2), and the crystal and structure
refinement data are listed in Table 2.
2.3. Biochemistry
The expression and purification of DENV2 NS2B/NS3pro and
WNV NS2B/NS3pro have been previously described.38 Enzyme as-
says and inhibition studies were carried out as previously de-
scribed14,39 and the results are summarized in Tables 1 and 3
and Figures 3–5.
The armamentarium of antiviral therapeutics for the treat-
ment of Dengue and West Nile virus infections is currently very

H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
105
100
DENV2 protease
WNV protease
80
60
40
20
0
Compound
Figure 3. Inhibition of DENV2 and WNV NS2B/NS3pro by selected compounds at
25 M. The concentrations of DENV2 and WNV proteases were 25 and 28 nM,
respectively. Substrate concentration was 5.0 M. The buffer used contained
Figure 2. ORTEP drawing of compound 7p showing the 30% thermal ellipsoids.
Hydrogen atoms have been omitted for clarity.
l
l
200 mM TrisꢀHCl, 6.0 mM NaCl, 30% glycerol, and 0.1% CHAPS, pH 9.5 and the
reactions were run at 37 °C. 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.
Table 2
X-ray data collection and processing parameters for compound 7p
Molecular formula
Formula weight
Diffractometer
Radiation/k (Å)
Temperature (K)
Color/habit
Crystal size (mm3)
Crystal system
Space group
a (Å)
C22H16N4O2S
400.45
Bruker Kappa Apex II
(Fig. 1) were synthesized and their activity against DENV2 and
WNV NS2B/NS3 proteases investigated. The design of this series
of inhibitors was based on the following considerations: (a) it
was envisaged that an entity comprised of two heterocyclic scaf-
folds would provide multiple loci for hydrogen bond donor and/
or acceptor interactions between the protease and inhibitors,
leading to enhanced pharmacological activity. This approach
was anticipated to be advantageous in the case of DENV2
NS2B/NS3pro because of its rather shallow active site; (b) molec-
ular modeling using the known X-ray crystal structures of DENV2
NS2B/NS3pro23,40,41 suggested that derivatives of (I) can be
accommodated at the protease active site and, (c) linking the
two heterocyclic scaffolds could potentially result in the creation
of a novel entity that displays superior drug-like characteristics.
Thus, the 1,2-benzisothiazolin-2(3H)-one-1,3,4-oxadiazole hybrid
platform was chosen for generating a focused library of com-
pounds and for conducting exploratory studies.
The desired compounds were readily obtained as shown in the
synthetic Scheme 1 and the structure and stereochemistry of a rep-
resentative member was established using X-ray crystallography
(Fig. 2). The compounds were subsequently screened against
DENV2 NS2B/NS3pro and WNV NS2B/NS3pro. It is evident from
Figure 3 and Table 1 that several derivatives of (I) are active against
both proteases, with nine out of twenty-four compounds exhibit-
Mo K
150
a/0.71073
Colorless/
0.288 ꢁ 0.302 ꢁ 0.318
Orthorhombic
P212121
10.397(3)
11.313(3)
16.606(5)
1953.1(10)
4
1.362
h, k, l
12, 13, 20
3.60–25.99
0.192
b (Å)
c (Å)
V (Å3)
Z-value
Dcalc (g cmꢂ3
)
Octants collected
Max. h, k, l
H
Range (°)
Absorption coefficient (mmꢂ1
)
F(000)
Reflections/unique (Rint
Observed (>2r)/parameters
Robs/Rall
832
)
35,207/3812 (0.0405)
3812/262
0.0266/0.0307
1.030
0.02(5)
0.159–0.178
Goodness-of-fit
Absolute structure parameter
Largest diff. peak and hole (e Åꢂ3
)
Table 3
Kinetic parameters for the tetra-peptide substrate and compound 7n against DENV2
NS2B/NS3pro at 37 °C
ing more than 50% inhibitory activity ([Inhibitor] = 10 lM). It can
be generally inferred from the SAR studies that the nature of both
R1 and R2 influences activity. Furthermore, R1 exerts a more dom-
inant effect on activity than R2. Variation of R1 while keeping R2
constant reveals that inhibitory activity follows the order for R1
benzyl > H, corresponding to 7i, and 7b, respectively. This hierar-
chy is further confirmed when R2 is substituted with a p-methoxy-
benzyl group: benzyl is better than H (7g > 7d). Similarly, when R2
is a benzyl group, compound 7f (R1 = benzyl) is a better inhibitor
than 7c (R1 = H). The inhibition studies indicate that highest inhib-
itory activity is manifested where R1 is a benzyl group, while low-
7n (
l
M)
Km,
(
l
M)
kcat (sꢂ1
)
kcat/Km (Mꢂ1 sꢂ1
)
app
0
1
3
5
11.69 0.41
17.59 0.54
20.98 1.05
31.47 3.25
0.064 0.0008
0.065 0.0009
0.0623 0.0014
0.062 0.0034
5751 275
3879 169
3176 225
2327 349
limited;
consequently,
small-molecule
anti-Dengue
and
anti-West Nile therapeutics are urgently needed. A series of
structurally-diverse 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiaz-
ole hybrid derivatives represented by general structure (I)
est inhibition is observed where R1 is hydrogen.
comparison between 7w and 7x versus 7i cannot be made because
A direct

106
H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
0.08
100
80
0.06
0.04
0.02
0.00
35
30
25
20
15
10
60
DENV2 protease
WNV protease
40
0
1
2
3
4
5
[7n] (μM)
20
-2
-1
0
1
0
10
20
30
40
50
Log [7n (μM)]
[Substrate] (μΜ)
Figure 4. Determination of IC50 values of inhibitor 7n against DENV2 and WNV
proteases. Inhibitor 7n was incubated with DENV2 NS2B/NS3pro (25 nM) or WNV
NS2B/NS3pro (28 nM) in buffer (200 mM TrisꢀHCl, 6 mM NaCl and 30% glycerol, pH
Figure 5. Inhibition of DENV2 NS2B/NS3 protease activity by compound 7n. Initial
reaction rates of the substrate (Bz-Nle-Lys-Arg-Arg-AMC) cleavage catalyzed by
DENV2 NS2B/NS3 protease (25 nM) in 200 mM TrisꢀHCl (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 at 37 °C. Bz-Nle-Lys-Arg-Arg-AMC (5.0
lM) was added to the
mixture in a final volume of 100
lL. The fluorescence intensity was measured at
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
460 nm with 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. The apparent IC50 values for
l
l
M (solid
l
DENV2 NS2B/NS3 protease and the fluorescence intensity at 460 nm was monitored
with an excitation at 380 nm. Reactions were less than 5% complete 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 concentra-
tion of selected compound 7n. Kinetics studies were carried out as described
compounds 7n were 3.75 0.06 and 4.22 0.07 lM against DENV2 (solid circle)
and WNV (open circle), respectively.
utilizing substrate concentrations of 0–50 lM Bz-Nle-Lys-Arg-Arg-AMC. Each
experiment was performed in duplicate and repeated three times. Data were
analyzed using SigmaPlot 2001 v7.0 software to determine values for apparent Km
the former compounds were screened as racemic mixtures while
the latter compound was screened as the pure (L) enantiomer.
One of the most potent compounds (compound 7n), was cho-
sen for further kinetics studies. The percent inhibition of com-
pound 7n (R1 = benzyl and R2 = p-methoxyphenyl) against both
DENV2 and WNV proteases is higher than the rest of the com-
pounds (Fig. 3). The apparent IC50 values of compound 7n
(Fig. 4) against DENV2 and WNV proteases were determined to
and kcat
.
resolved within the manifold of top five poses of these ligands
for the DENV2 NS2B/NS3pro model based on the 2FOM40 crystal
structure. For consistency of interpretation, we have thus chosen
to focus on the docking poses generated for the 3U1I (DENV),23
3E90 (WNV)42 and 2FP7 (WNV)41 structure models.
be 3.75 0.06 and 4.22 0.07 lM, respectively. We performed
kinetics analysis to determine the Km, kcat and Vmax values in
the presence and absence of compound 7n at four different con-
centrations (Fig. 5 and Table 3). The apparent Michaelis-Menten
constants (Km, app) increased and kcat/Km decreased proportion-
ally with increasing concentration of compound 7n. These results
are supportive of a competitive mode of inhibition. The inhibi-
tion constant (Ki) of 7n against DENV2 NS2B/NS3pro was deter-
In a preliminary sense, the docking results corroborate some
general activity trends observed in the tested compounds. Varia-
tion of R1 with constant R2 (Fig. 6A) suggests that the significant
penalty incurred by absence of a hydrophobic R1 group (com-
pound 7b) likely arises from lost hydrophobic interactions which
our model suggests as arising for DENV2 NS2B/NS3pro from con-
tact with His 51, resulting in a failure to dock to the receptor in
a pose analogous to those of other more potent inhibitors. A
benzyl R1 group as per compound 7i, appears to fit well in this
hydrophobic pocket, whereas the larger fluorobenzyl (7w) and
phenethyl (7x) groups, while not sterically forbidden, may incur
a marginal receptor desolvation penalty by protruding into a
more polar region of the cavity. Variation of R2 (Fig. 6B) suggests
that, relative to benzyl analogs, the substituted phenyl groups in
our library achieve a marginally better steric fit within the cavity
around Tyr 161 and Val/Ile 155 for DENV2/WNV, respectively,
which could explain a binding preference for the latter com-
pounds. The p-methoxy group located on the phenyl group of
7n seems to possess an excellent steric fit for this cavity, which
may help to explain the strong potency of this compound. Fur-
thermore, the 7n pose solved for the 3U1I23 crystal structure
(Fig. 6B) suggests the possibility that the methoxy group may
serve as a H-acceptor for the receptor hydroxy proton on Tyr
150, although this is not corroborated by the other crystal
structures.
mined to be 3.61 lM. Next, we employed molecular modeling to
identify a plausible binding mode for a series of compounds,
including 7n and analogs thereof, with the goal of identifying
the binding domains of 1,2-benzisothiazol-3(2H)-one and 1,3,4-
oxadiazole moieties as a basis for pharmacophore perception.
In the case of DENV2 NS2B/NS3pro, we compared the distribu-
tion of docked conformers for compounds 7b, 7i, 7g, 7n, 7w
and 7x within models produced from the 3U1I23 and 2FOM40
crystal structures to determine whether a consistent ligand bind-
ing mode was evident for either case. For the 3U1I receptor
model, all compounds except 7b exhibited at least one predicted
binding mode within the five top-scoring poses that satisfied
both of the following two pharmacophore features: H-bond
acceptance of the Gly 153 backbone amide proton by the 1,2-
benzisothiazol-3(2H)-one O, and H-bond acceptance of the Ser
135 side chain hydroxyl proton by one or two nitrogens on
the 1,3,4-oxadiazole ring. A similar binding mode was predicted
for these compounds in docking to WNV NS2B/NS3pro with
additional conservation of ligand hydrophobic interactions with
His 51 and Tyr 161, however, no consistent binding mode was

H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
107
Figure 6. Putative binding mode of selected compounds interacting with DENV2 (A and B) and WNV (C) proteases, as predicted by molecular modeling. All compounds have
CPK-colored heteroatoms, and are distinguished according to the color of their carbon atoms as follows: black = 7i, cyan = 7w, purple = 7x, magenta = 7g and orange = 7n. The
protease receptor surfaces are colored as follows: yellow = lipophilic, white = weakly polar, cyan = polar H, blue = polar N and red = polar O. Apparent H-bonding features of
the receptor are marked with ‘‘⁄’’ and hydrophobic interactions with ‘‘#’’.
Bachem). 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 differ-
ent solvent systems and were visualized using iodine and/or UV
light. Each individual compound was identified as a single spot on
TLC plate. DENV2 NS2B/NS3pro (or WNV NS2B/NS3pro) substrate
Bz-Nle-Lys-Arg-Arg-AMC was purchased from Bachem, Torrance,
CA or custom synthesized by NeoBioScience, Cambridge, MA.
3. Conclusion
In summary, we have synthesized a focused library of 1,2-ben-
zisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives and
have investigated their inhibitory activity against DENV2 NS2B/
NS3pro and WNV NS2B/NS3pro. Several members of this series
of compounds were found to inhibit the two enzymes, and a low
micromolar inhibitor capable of inhibiting both enzymes has been
identified. Importantly, biochemical studies indicate that the
compounds act as competitive inhibitors. Furthermore, the interac-
tion of the compounds with the two enzymes was probed using
molecular graphics and modeling and a mode of binding that is
congruent with the results of SAR studies is proposed. The findings
reported herein have laid the groundwork for conducting hit-to-
lead optimization studies43–45 encompassing iterative medicinal
chemistry, in vitro screening, and X-ray crystallography.
4.2. Representative syntheses
Compounds 1 and 2 were synthesized using a previously pub-
lished procedure.33
4.2.1. t-Butyl (3-oxo-1,2-benzisothiazol-3(2H)-2-yl)-acetate (1)
Yellowish oil (20.5 g; 77% yield) which was used in the next
step without further purification. 1H NMR (CDCl3): d 1.51 (s, 9H),
4.57 (s, 2H), 7.20–8.29 (m, 4H).
4. Experimental section
4.1. General
4.2.2. (3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-acetic acid (2)
White solid (11.2 g; 69% yield), mp 229–230 °C. 1H NMR
(CDCl3): d 4.57 (s, 2H), 7.40–8.12 (m, 4H).
The 1H NMR 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 spectrometry
(HRMS) was performed using an LCT Premier mass spectrometer
(Waters, Milford MA) equipped with a time of flight mass analyzer
and an electrospray ion source, at the University of Kansas Mass
Spectrometry Lab. Reagents and solvents were purchased from var-
ious chemical suppliers (Aldrich, Acros Organics, TCI America, and
4.2.3. 2,20-Dithiodibenzoyl bis-alanine methyl ester (3a)
Compound 3a was prepared as described previously.33 Briefly, a
solution of 2,20-dithiodibenzoic acid (1.53 g; 5 mmol) in 10 mL THF
and 6 mL DMF was added dropwise a suspension of 1,10-carbonyl
diimidazole (1.70 g; 10 mmol) in 10 mL THF. The reaction mixture

108
H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
was stirred at room temperature for 20 min and refluxed for
20 min. The reaction mixture was allowed to cool to room temper-
ature and (DL) alanine methyl ester hydrochloride (1.40 g;
10 mmol) and triethylamine (1.5 mL; 12 mmol) were added. The
reaction mixture was stirred at room temperature overnight and
concentrated. The residue was taken up in ethyl acetate (40 mL)
and water (30 mL). The organic layer was washed with 5% HCl
(3 ꢁ 30 mL), saturated NaHCO3 (3 ꢁ 30 mL) and brine (30 mL).
The organic layer was dried over anhydrous sodium sulfate, fil-
tered, and concentrated, leaving behind 3a as an yellow solid
(1.8 g; 76% yield), mp 139–141 °C. 1H NMR (DMSO-d6): d 1.44 (d,
J = 7.3 Hz, 6H), 3.68 (s, 6H), 4.46–4.59 (m, 2H), 7.25–7.33 (m, 2H),
7.43–7.53 (m, 2H), 7.65–7.79 (m, 4H), 8.98 (d, J = 6.3 Hz, 2H).
4.2.11. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-propanoic acid
(5a)
Compound 5a was prepared as described previously.33 Briefly, a
solution of compound 4a (2.37 g; 10 mmol) was dissolved in 15 mL
1,4-dioxane and treated with 20 mL 1 M LiOH at 0 °C for 1 h. The
solution was neutralized and the solvent was removed. The residue
was adjusted to pH 1, forming a precipitate which was collected
with vacuum filtration and washed with diethyl ether to give pure
compound 5a as a gray solid (1.73 g; 77% yield), mp 201–202 °C. 1H
NMR (DMSO-d6): d 1.57 (d, J = 6.0 Hz, 3H), 5.14 (q, J = 6.5 Hz, 1H),
7.45 (t, J = 6.2 Hz, 1H), 7.70 (t, J = 6.0 Hz, 1H), 7.88 (d, J = 6.5 Hz,
1H), 7.97 (d, J = 6.5 Hz, 1H).
4.2.12. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid (5b)
4.2.4. 2,20-Dithiodibenzoyl bis-phenylalanine methyl ester (3b)
White solid (68% yield), mp 165–167 °C. 1H NMR (CDCl3) d 3.24
(dd, J = 13.9, 5.3 Hz, 2H), 3.36 (dd, J = 13.7, 5.8 Hz, 2H), 3.79 (s, 6H),
5.11 (dd, J = 10, 5.8 Hz, 2H), 6.58 (d, J = 7.6 Hz, 2H), 7.13–7.22 (m,
8H), 7.25–7.43 (m, 8H), 7.73–7.78 (m, 2H).
White solid (2.43 g; 44% yield), mp 180–182 °C. 1H NMR
(DMSO-d6): d 3.23 (dd, J = 15.1, 11.0 Hz, 1H), 3.45 (dd, J = 15.1,
4.7 Hz, 1H), 5.42 (dd, J = 10.4, 4.7 Hz, 1H), 7.12–7.32 (m, 5H),
7.37–7.44 (m, 1H), 7.63–7.70 (m, 1H), 7.77–7.82 (m, 1H), 7.90–
7.95 (m, 1H), 13.40 (br s, 1H).
4.2.5. 2,20-Dithiodibenzoyl bis-(m-fluoro)phenylalanine methyl
ester (3c)
4.2.13. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-(3-
fluorophenyl)propanoic acid (5c)
Light brown solid (75% yield). mp 141–143 °C. 1H NMR (CDCl3):
d. 3.25 (dd, J = 14.1, 5.1 Hz, 2H), 3.38 (dd, J = 15.8, 5.9 Hz, 2H),
3.80 (s, 6H), 5.14 (q, J = 5.4 Hz, 2H), 6.61 (d, J = 7.6 Hz, 2H), 6.82–
7.00 (m, 6H), 7.17–7.28(m, 4H), 7.33–7.44 (m, 4H), 7.76 (d,
J = 8.1 Hz, 2H).
Yellow oil (1.87g; 98% yield). 1H NMR (CDCl3): d 3.29–3.39 (m,
1H), 3.52–3.60 (m, 1H), 5.52–5.58 (m, 1H), 6.80–7.72 (m, 7H),
7.98–8.03 (m, 1H)
4.2.14. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-4-
phenylbutanoic acid (5d)
4.2.6. 2,20-Dithiodibenzoyl bis-benzylalanine methyl ester (3d)
White solid (55% yield), mp 190–192 °C. 1H NMR (DMSO-d6): d
2.09 (q, J = 14.7 Hz, 4H), 2.62–2.82 (m, 4H), 3.65 (s, 6H), 4.38 (q,
J = 7.3 Hz, 2H), 7.14–7.37 (m, 12H), 7.43–7.51 (m, 2H), 7.65–7.74
(m, 4H), 9.07 (d, J = 7.4 Hz, 2H).
Yellow solid (1.36 g; 80% yield), mp 149–150 °C. 1H NMR
(DMSO-d6): d 2.20–2.48 (m, 2H), 2.61 (t, J = 6.7 Hz, 2H), 5.05–5.11
(m, 1H), 7.18–7.36 (m, 5H), 7.46 (t, J = 6.7 Hz, 1H), 7.71 (t,
J = 6.7 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H).
4.2.7. Methyl 2-(3-oxo-1,2-benzisothiazol-3(2H)-2-yl)-
propanoic acetate (4a)
4.2.15. (3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-acetic acid, 20-(3-
fluorobenzoyl)hydrazide (6a)
Compound 4a was prepared as described previously.33 Briefly, a
chilled solution of 3a (1.8 g; 3.78 mmol) in 25 mL CH2Cl2 was
added bromine (0.60 g; 3.78 mmol) and the reaction mixture was
stirred for 30 min. Triethylamine (0.77 g; 7.5 mmol) was then
added and the resulting mixture was stirred for 5 min. The reaction
mixture was washed with water (2 ꢁ 15 mL) and the organic layer
was dried over anhydrous sodium sulfate, dried and concentrated,
leaving a pure compound 4a as yellowish oil (1.8 g; 100% yield). 1H
NMR (CDCl3): d 1.70 (d, J = 7.8 Hz, 3H), 3.77 (s, 3H), 5.50 (q,
J = 8.2 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 7.56–7.66 (m, 2H), 8.09 (d,
J = 8.8 Hz, 1H).
To a stirred solution of compound 2 (0.58 g; 2 mmol) in dry
DMF (6 mL) was added EDCI (0.46 g; 2.4 mmol), followed by m-flu-
orobenzhydrazide (0.31 g; 2 mmol), and the reaction mixture was
stirred at room temperature overnight. The solvent was removed
and the residue was taken up in ethyl acetate (50 mL). The organic
layer was washed with brine (15 mL), dried over anhydrous so-
dium sulfate, filtered, and concentrated. The crude product was
purified using flash chromatography (silica gel/ethyl acetate/hex-
anes) to give pure compound 6a, as a white solid (0.27 g; 32%
yield), mp 207–209 °C. 1H NMR (DMSO-d6): d 4.63 (s, 2H), 7.40–
8.01 (m, 8H), 10.41 (s, 1H), 10.60 (s, 1H).
4.2.8. Methyl 2-(3-oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acetate (4b)
4.2.16. (3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-acetic acid, 20-(3-
chlorobenzoyl)hydrazide (6b)
Yellow oil (14.92 g; 70% yield). 1H NMR (CDCl3): d 3.24 (dd,
J = 13.69, 9.83 Hz, 1H), 3.51 (dd, J = 14.52, 5.79 Hz, 1H), 3.74 (s,
3H), 5.68 (dd, J = 10.09, 5.80 Hz, 1H), 7.17–7.25 (m, 5H), 7.33–
7.40 (m, 1H), 7.50–7.63 (2H), 7.95–7.99 (m, 1H).
Yellow solid (42% yield), mp 88–90 °C. 1H NMR (DMSO-d6): d
4.66 (s, 2H), 7.35–7.97 (m, 8H), 10.40 (s, 1H), 10.58 (s, 1H).
4.2.17. (3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-acetic acid, 2-(2-
phenylacetyl)hydrazide (6c)
4.2.9. Methyl 2-(3-oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-(3-
fluorophenyl)propanoic acetate (4c)
White solid (28% yield), mp 230–232 °C. 1H NMR (DMSO-d6): d
3.47 (s, 2H), 4.56 (s, 2H), 7.20–7.35 (m, 5H), 7.43 (t, J = 5.3 Hz, 1H),
7.69 (t, J = 5.3 Hz, 1H), 7.86 (d, J = 6.7 Hz, 1H), 7.98 (d, J = 6.7 Hz,
1H), 10.21 (s, 1H), 10.29 (s, 1H).
Reddish oil (2.3 g; 92% yield). 1H NMR (CDCl3): d 3.23 (dd,
J = 11.2, 8.9 Hz, 1H), 3.49 (dd, J = 11.2, 8.9 Hz, 1H), 3.76 (s, 3H),
5.62–5.69 (m, 1H), 6.82–7.00 (m, 3H), 7.10–8.00 (m, 5H).
4.2.18. (3-Oxo-1,2-benzisothiazol-(3(2H)-2-yl)-acetic acid, 2-[2-
(4-methoxyphenyl)acetyl]hydrazide (6d)
4.2.10. Methyl 2-(3-oxo-1,2-benzisothiazol-3(2H)-2-yl)-4-
phenylbutanoic acetate (4d)
Graysolid(47%yield), mp203–205 °C. 1HNMR(DMSO-d6):d 3.40
(s, 2H), 3.74 (s, 3H), 4.56 (s, 2H), 6.88 (d, J = 8.0 Hz, 2H), 7.20 (d,
J = 8.0 Hz, 2H), 7.45 (t, J = 7.3 Hz, 1H), 7.71 (t, J = 7.3 Hz, 1H), 7.89
(d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 10.18 (s, 1H), 10.28 (s, 1H).
Yellow oil (1.61 g; 90% yield). 1H NMR (CDCl3): d 2.23–2.35 (m,
1H), 2.43–2.55 (m, 1H), 2.64 (t, J = 8.3 Hz, 2H), 3.75 (s, 3H), 5.40–
5.46 (m, 1H), 7.16–7.68 (m, 5H), 8.03–8.08 (m, 1H).

H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
109
4.2.19. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-propanoic acid,
2-[2-(4-methoxyphenyl) acetyl]hydrazide (6e)
the resulting reaction mixture was stirred at room temperature
overnight. CH2Cl2 (30 mL) was added and the solution was washed
with water (30 mL), and brine (30 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated. The
crude product was purified by flash chromatography (silica gel/
ethyl acetate/hexanes) to give 7a as a white solid (0.1 g; 39% yield),
mp 149–151 °C. 1H NMR (CDCl3): d 5.40 (s, 2H), 7.20–7.82 (m, 7H),
8.09–8.12 (m, 1H). 13C NMR (CDCl3): d 165.4, 164.0, 161.9, 161.5,
140.5, 132.7, 131.0, 127.2, 126.0, 125.2, 123.2, 122.9, 120.6,
119.3, 119.1, 114.3, 37.7. HRMS (ESI): Calcd for C16H10FN3O2SNa,
m/z [M+Na]+: 350.0375. Found 350.0366.
White solid (21% yield), mp 135–137 °C. 1H NMR (DMSO-d6): d
1.46 (d, J = 4.7 Hz, 3H), 3.40 (s, 2H), 3.72 (s, 3H), 5.30–5.40 (m, 1H),
6.82 (d, J = 9.3 Hz, 2H), 7.19 (d, J = 9.3 Hz, 2H), 7.40 (t, J = 6.7 Hz,
1H), 7.65 (t, J = 6.7 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.95 (d,
J = 7.3 Hz, 1H), 10.11 (s, 1H), 10.30 (s, 1H).
4.2.20. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(2-phenylacetyl)hydrazide (6f)
Yellow oil (52% yield). 1H NMR (DMSO-d6): d 3.18–3.42 (m, 2H),
3.56 (s, 2H), 5.81–5.87 (m, 1H), 7.00–7.60 (m, 14H).
4.2.30. 2-[(2-m-Chlorophenyl-1,3,4-oxadiazol-5-yl)methyl]-1,2-
benzisothiazol-3(2H)-3-one (7b)
4.2.21. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-[2-(4-methoxyphenyl)acetyl]hydrazide
(6g)
White solid (57% yield), mp 136–137 °C. 1H NMR (CDCl3): d 5.40
(s, 2H), 7.40–7.68 (m, 5H), 7.90–8.12 (m, 3H). 13C NMR (CDCl3): d
165.4, 164.8, 161.9, 140.5, 135.3, 132.7, 132.2, 130.4, 129.1, 127.2,
127.2, 126.0, 125.2, 125.0, 123.3, 120.6, 37.7. HRMS (ESI): Calcd
for C16H10ClN3O2SNa, m/z [M+Na]+: 366.0080. Found 366.0068.
Yellow oil (53% yield). 1H NMR (DMSO-d6): d 3.16–3.45 (m, 2H),
3.51 (s, 2H), 3.75 (s, 3H), 5.76–5.81 (m, 1H), 6.78–7.82 (m, 13H).
4.2.22. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(3-fluorobenzoyl)hydrazide (6h)
Brown oil (53% yield). 1H NMR (DMSO-d6): d 3.10–3.50 (m, 2H),
5.82–5.91 (m, 1H), 6.97–7.65 (m, 13H).
4.2.31. 2-[(2-Benzyl-1,3,4-oxadiazol-5-yl)methyl]-1,2-
benzisothiazol-3(2H)-3-one (7c)
Yellow solid (0.10 g; 35% yield), mp 93–95 °C. 1H NMR (CDCl3):
d 4.16 (s, 2H), 5.24 (s, 2H), 7.23–7.35 (m, 5H), 7.43 (t, J = 5.1 Hz,
1H), 7.53 (d, J = 5.5 Hz, 1H), 7.64 (t, J = 5.1 Hz, 1H), 8.05 (d,
J = 5.5 Hz, 1H). 13C NMR (CDCl3): d 166.7, 165.4, 162.2, 140.5,
133.3, 132.6, 128.9, 128.8, 127.6, 127.1, 126.0, 123.3, 120.6, 37.7,
4.2.23. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(3-chlorobenzoyl)hydrazide (6i)
Brown solid (50% yield), mp 96–97 °C. 1H NMR (DMSO-d6): d
3.20–3.60 (m, 2H), 5.64–5.75 (m, 1H), 7.20–7.98 (m, 13H).
31.7. HRMS (ESI): Calcd for
346.0626. Found 346.0635.
C
17H13N3O2SNa, m/z [M+Na]+:
4.2.24. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(4-fluorobenzoyl)hydrazide (6j)
White solid (77% yield), mp 189–191 °C. 1H NMR (CDCl3): d 3.55
(dd, J = 41.8, 13.5 Hz, 2H), 5.85–5.90 (m, 1H), 7.00–7.47 (m, 10H),
7.71–7.83 (m, 2H), 8.05–8.13 (m, 1H), 9.08 (s, 1H), 9.50 (s, 1H).
4.2.32. 2-[(2-p-Methoxybenzyl-1,3,4-oxadiazol-5-yl)methyl]-
1,2-benzisothiazol-3(2H)-3-one (7d)
Yellow solid (52% yield), mp 92–93 °C. 1H NMR (CDCl3): d 3.79
(s, 3H), 4.11 (s, 2H), 5.26 (s, 2H), 6.83 (d, J = 8.8 Hz, 2H), 7.20 (d,
J = 8.8 Hz, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H),
7.65 (t, J = 7.7 Hz, 1H), 8.08 (d, J = 8.9 Hz, 1H). 13C NMR (CDCl3): d
167.1, 165.4, 162.1, 159.0, 140.5, 132.6, 129.9, 127.1, 125.9,
125.2, 123.2, 120.6, 114.3, 55.3, 37.7, 30.9. HRMS (ESI): Calcd for
4.2.25. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(4-chlorobenzoyl)hydrazide (6k)
Yellow-green solid (92% yield), mp 110–112 °C. 1H NMR
(CDCl3): d 3.29 (dd, J = 13.9, 9.1 Hz, 1H), 3.51 (dd, J = 14.3, 6.3 Hz,
1H), 5.82 (dd, J = 9.0, 6.6 Hz, 1H), 7.09–7.19 (m, 4H), 7.27–7.37
(m, 4H), 7.49–7.59 (m, 2H), 7.68–7.74 (m, 2H), 7.86–7.91 (m,
2H), 9.10 (s, 1H), 10.06 (s, 1H).
C
18H15N3O3SNa, m/z [M+Na]+: 376.0732. Found 376.0735.
4.2.33. 2-[(2-p-Methoxybenzyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-3-one (7e)
White solid (48% yield), mp 139–141 °C. 1H NMR (CDCl3): d 1.86
(d, J = 7.7 Hz, 3H), 3.76 (s, 3H), 4.09 (dd, J = 19.6, 13.4 Hz, 2H), 6.20
(q, J = 7.2 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H),
7.40 (t, J = 6.7 Hz, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.61 (t, J = 6.7 Hz,
1H), 8.03 (d, J = 8.3 Hz, 1H). 13C NMR (CDCl3): d 166.8, 165.4,
165.1, 158.9, 140.4, 132.4, 129.8, 127.0, 125.8, 125.3, 124.0,
120.6, 114.2, 55.2, 44.6, 30.8, 17.9. HRMS (ESI): Calcd for
4.2.26. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-
phenylpropanoic acid, 2-(4-cyanobenzoyl)hydrazide (6l)
White solid (42% yield), mp 168–170 °C. 1H NMR (DMSO-d6): d
3.55 (dd, J = 26.0, 14.3 Hz, 2H), 5.75 (dd, J = 9.6, 4.7 Hz, 1H), 7.20–
7.45 (m, 6H), 7.49–7.55 (m, 1H), 7.63–7.69 (m, 1H), 7.87–8.03
(m, 4H), 8.73 (br s, 1H), 10.51 (br s, 1H), 10.75–10.87 (m, 1H)
C
19H17N3O3SNa, m/z [M+Na]+: 390.0888. Found 390.0869.
4.2.27. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-3-(3-
fluorophenyl)propanoic acid, 2-(3-fluorobenzoyl)hydrazide
(6w)
4.2.34. 2-[2-Phenyl-1-(2-benzyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-3-one (7f)
White solid (63% yield), mp 93–95 °C. 1H NMR (CDCl3): d 3.15–
3.40 (m, 2H), 5.39 (t, J = 7.1 Hz, 1H), 6.78–7.99 (m, 12H).
Colorless oil (56% yield). 1H NMR (CDCl3): d 3.47 (dd, J = 13.9,
8.6 Hz, 1H), 3.67 (dd, J = 13.9, 8.6 Hz, 1H), 4.12 (d, J = 12.2 Hz,
2H), 6.38 (dd, J = 8.9, 6.9 Hz, 1H), 7.12–7.62 (m, 13H), 7.96 (d,
J = 9.1 Hz, 1H). 13C NMR (CDCl3): d 166.3, 165.3, 164.6, 140.4,
134.8, 133.3, 132.4, 129.1, 129.0, 128.8, 128.7, 127.5, 127.3,
127.0, 125.7, 123.6, 120.5, 49.8, 38.2, 31.7. HRMS (ESI): Calcd for
4.2.28. 2-(3-Oxo-1,2-benzisothiazol-3(2H)-2-yl)-4-
phenylbutanoic acid, 2-(3-fluorobenzoyl)hydrazide (6x)
White solid (47% yield), mp 105–106 °C. 1H NMR (CDCl3): d
2.45–2.72 (m, 4H), 5.11–5.17 (m, 1H), 7.00–8.02 (m, 13H).
C
24H19N3O2SNa, m/z [M+Na]+: 436.1096. Found 436.1077.
4.2.29. 2-[(2-m-Fluorophenyl-1,3,4-oxadiazol-5-yl)methyl]-1,2-
benzisothiazol-3(2H)-3-one (7a)
To a stirred solution of compound 6a (0.27 g; 0.63 mmol) in
CH2Cl2 (10 mL) was added p-toluenesulfonyl chloride (0.12 g;
0.63 mmol) followed by triethylamine (0.20 mL; 1.5 mmol) and
4.2.35. 2-[2-Phenyl-1-(2-p-mehoxybenzyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-3-one (7g)
Colorless oil (64% yield). 1H NMR (CDCl3): d 3.48 (dd, J = 13.6,
9.9 Hz, 1H), 3.68 (dd, J = 13.6, 9.9 Hz, 1H), 3.77 (s, 3H), 4.06 (d,
J = 3.1 Hz, 2H), 6.37 (dd, J = 9.1, 7.0 Hz, 1H), 6.77–7.62 (m, 12H),

110
H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
7.92–7.98 (m, 1H). 13C NMR (CDCl3): d 152.3, 150.8, 150.1, 126.0,
120.5, 117.9, 117.6, 115.4, 114.7, 114.2, 112.9, 112.6, 111.3,
110.9, 109.3, 106.1, 99.9, 40.9, 35.5, 23.8, 16.4. HRMS (ESI): Calcd
for C25H21N3O3SNa, m/z [M+Na]+: 466.1201. Found 466.1178.
added and the reaction mixture was stirred at room temperature
overnight. Removal of the solvent left a crude residue which was
adsorbed on silica gel and purified using flash chromatography (sil-
ica gel/ethyl acetate/hexanes) to give compound 7m as an yellow
oil (0.37 g; 23% yield). 1H NMR (CDCl3): d 3.55 (dd, J = 14.3,
8.7 Hz, 1H), 3.81 (dd, J = 14.8, 6.9 Hz, 1H), 6.53 (dd, J = 8.8, 6.9 Hz,
1H), 7.18–7.29 (m, 3H), 7.31–7.35 (m, 2H), 7.38–7.42 (m, 1H),
7.44–7.55 (m, 4H), 7.59–7.65 (m, 1H), 7.97–8.04 (m, 3H). 13C
NMR (CDCl3): d 165.6, 165.2, 163.9, 140.4, 135.0, 132.4, 132.0,
129.2, 129.0, 128.7, 127.4, 127.1, 127.0, 125.7, 123.7, 123.3,
120.6, 49.9, 38.2. HRMS (ESI): Calcd for C23H17N3O2SNa, m/z
[M+Na]+: 422.0939; found 422.0923.
4.2.36. 2-[2-Phenyl-1-(2-m-fluorophenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7h)
Colorless oil (33% yield). 1H NMR (CDCl3): d 3.54 (dd, J = 12.9,
9.2 Hz, 1H), 3.78 (dd, J = 12.9, 9.2 Hz, 1H), 6.52 (dd, J = 8.8, 7.0 Hz,
1H), 7.19–8.03 (m, 13H). 13C NMR (CDCl3): d 165.3, 164.4, 164.2,
161.1, 134.9, 132.5, 131.0, 129.2, 128.8, 127.4, 127.1, 125.8,
122.9, 120.6, 119.3, 119.0, 114.3, 114.0, 49.9, 38.2. HRMS (ESI):
Calcd for C23H17N3O2SF, m/z [M+Na]+: 440.0845. Found 440.0840.
4.2.42. 2-[2-Phenyl-1-(2-p-methoxyphenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7n)
4.2.37. 2-[2-Phenyl-1-(2-m-chlorophenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7i)
White solid (20% yield), mp 187–189 °C. 1H NMR (CDCl3): d 3.54
(dd, J = 13.3, 8.7 Hz, 1H), 3.79 (dd, J = 14.3, 7.0 Hz, 1H), 3.85 (s, 3H),
6.49 (dd, J = 8.9, 7.0 Hz, 1H), 6.93–6.98 (m, 2H), 7.17–7.35 (m, 5H),
7.37–7.43 (1H), 7.51–7.55 (m, 1H), 7.59–7.64 (m, 1H), 7.90–7.95
(m, 2H), 7.99–8.03 (m, 1H). 13C NMR (CDCl3): d 165.6, 165.2, 163.4,
162.5, 140.4, 135.1, 132.4, 129.2, 128.9, 128.7, 127.3, 127.0, 125.7,
123.7, 120.6, 115.7, 114.4, 55.5, 49.8, 38.2. HRMS (ESI): Calcd for
Colorless oil (26% yield). 1H NMR (CDCl3): d 3.55 (dd, J = 13.1,
8.2 Hz, 1H), 3.81 (dd, J = 13.1, 8.2 Hz, 1H), 6.53 (dd, J = 8.8, 7.0 Hz,
1H), 7.20–7.63 (m, 11H), 7.97–8.02 (m, 2H). 13C NMR (CDCl3): d
165.6, 165.4, 164.2, 164.0, 135.1, 132.4, 132.0, 129.4, 129.2,
129.0, 128.7, 127.4, 127.2, 125.8, 125.1, 123.8, 123.4, 120.6, 50.0,
38.3. HRMS (ESI): Calcd for
456.0550. Found 456.0559.
C
23H17N3O2SCl, m/z [M+Na]+:
C
24H19N3O3SNa, m/z [M+Na]+: 452.1045. Found 452.1042.
4.2.38. 2-[2-Phenyl-1-(2-p-fluorophenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7j)
4.2.43. 2-[2-Phenyl-1-(2-p-ethoxyphenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7o)
Light yellow solid (23% yield), mp 161–163 °C. 1H NMR (CDCl3):
d 3.56 (dd, J = 14.3, 8.9 Hz, 1H), 3.8 (dd, J = 14.2, 7.0 Hz, 1H), 6.51
(dd, J = 8.7, 7.0 Hz, 1H), 7.12–7.44 (m, 9H), 7.51–7.66 (m, 2H),
7.96–8.04 (m, 2H). 13C NMR (CDCl3): d 166.2, 165.2, 164.8, 140.4,
134.9, 132.4, 129.5, 129.2, 128.7, 127.4, 127.1, 125.8, 123.7,
120.6, 119.6, 116.5, 116.3, 49.8, 38.2. HRMS (ESI): Calcd for
White solid (28% yield), mp 98–100 °C. 1H NMR (CDCl3): d 1.43
(t, J = 7.0 Hz, 3H), 3.54 (dd, J = 14.0, 8.9 Hz, 1H), 3.79 (dd, J = 14.7,
6.9 Hz, 1H), 4.07 (q, J = 7.0, 2H), 6.49 (dd, J = 8.5, 6.9 Hz, 1H),
6.91–6.96 (m, 2H), 7.17–7.28 (m, 3H), 7.30–7.35 (m, 2H), 7.37–
7.42 (m, 1H), 7.51–7.55 (m, 1H), 7.59–7.64 (m, 1H), 7.89–7.93
(m, 2H), 7.99–8.03 (m, 1H). 13C NMR (CDCl3): d 165.6, 165.2,
163.3, 162.0, 140.4, 135.1, 132.4, 129.2, 128.9, 128.7, 127.3,
127.0, 125.7, 123.7, 120.6, 115.5, 114.8, 63.7, 49.8, 38.2, 14.6.
HRMS (ESI): Calcd for C25H21N3O3SNa, m/z [M+Na]+: 466.1201;
found 466.1196.
C
23H16FN3O2SNa, m/z [M+Na]+: 440.0845; found 440.0824.
4.2.39. 2-[2-Phenyl-1-(2-p-chlorophenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7k)
White solid (24% yield), mp 164–166 °C. 1H NMR (CDCl3): d 3.55
(dd, J = 12.8, 8.8 Hz, 1H), 3.80 (dd, J = 14.7, 7.0 Hz, 1H), 6.51 (dd,
J = 8.7, 7.0 Hz, 1H), 7.18–7.35 (m, 5H), 7.37–7.48 (m, 3H), 7.52–
7.57 (m, 1H), 7.59–7.66 (m, 1H), 7.89–7.96 (m, 2H), 7.99–8.04
(m, 1H). 13C NMR (CDCl3): d 165.2, 164.8, 164.1, 140.4, 138.4,
134.9, 132.5, 129.4, 129.2, 128.7, 128.4, 127.4, 127.1, 125.8,
123.6, 121.7, 120.6, 49.8, 38.2. HRMS (ESI): Calcd for C23H16
ClN3O2SNa, m/z [M+Na]+: 456.0549; found 456.0554.
4.2.44. 2-[2-Phenyl-1-(2-p-pyridyl-1,3,4-oxadiazol-5-yl)ethyl]-
1,2-benzisothiazol-3-one (7p)
White solid (20% yield), mp 176–177 °C. 1H NMR (CDCl3): d 3.57
(dd, J = 14.2, 8.7, 1H), 3.81 (dd, J = 14.3, 6.9 Hz, 1H), 6.53 (dd, J = 8.6,
7.1 Hz, 1H),7.19–7.35 (m, 5H), 7.39–7.44 (m, 1H), 7.53–7.57 (m,
1H), 7.61–7.66 (m, 1H), 7.82–7.87 (m, 2H), 8.00–8.05 (m, 1H),
8.75–8.82 (m, 2H). 13C NMR (CDCl3): d 165.3, 165.0, 163.8, 150.9,
140.3, 134.7, 132.6, 130.4, 129.2, 128.8, 127.5, 127.1, 125.9,
123.5, 120.6, 120.5, 49.9, 38.2. HRMS (ESI): Calcd for C22H16N4O2S-
Na, m/z [M+Na]+: 423.0892; found 423.0880.
4.2.40. 2-[2-Phenyl-1-(2-p-cyanophenyl-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7l)
White solid (20% yield), mp 182–184 °C. 1H NMR (CDCl3): d 3.57
(dd, J = 14.4, 8.7 Hz, 1H), 3.81 (dd, J = 14.3, 7.1 Hz, 1H), 6.52 (dd,
J = 8.7, 7.1 Hz, 1H), 7.18–7.35 (m, 5H), 7.38–7.44 (m, 1H), 7.53–
7.57 (m, 1H), 7.60–7.66 (m, 1H), 7.74–7.80 (m, 2H), 7.98–8.04
(m, 1H), 8.08–8.14 (m, 2H). 13C NMR (CDCl3): d 165.3, 164.8,
164.1, 140.3, 134.7, 132.8, 132.6, 130.2, 129.4, 129.1, 128.8,
127.6, 127.1, 125.9, 123.5, 120.6, 117.8, 115.5, 49.8, 38.2. HRMS
(ESI): Calcd for C24H16N4O2SNa, m/z [M+Na]+: 447.0892; found
447.0883.
4.2.45. 2-[2-Phenyl-1-(2-m-pyridyl-1,3,4-oxadiazol-5-yl)ethyl]-
1,2-benzisothiazol-3(2H)-one (7q)
White solid (19% yield), mp 148–149 °C. 1H NMR (CDCl3): d 3.57
(dd, J = 14.1, 8.7 Hz, 1H), 3.81 (dd, J = 13.8, 7.1 Hz, 1H), 6.53 (dd,
J = 8.9, 7.1 Hz, 1H), 7.18–7.35 (m, 5H), 7.38–7.45 (m, 2H), 7.53–
7.56 (m, 1H), 7.61–7.66 (m, 1H), 8.00–8.04 (m, 1H), 8.25–8.30
(m, 1H), 8.73–8.78 (m, 1H), 9.19–9.23 (m, 1H). 13C NMR (CDCl3):
d 165.3, 164.5, 163.5, 152.7, 148.0, 140.4, 134.8, 134.4, 132.5,
129.2, 128.8, 127.5, 127.1, 125.8, 123.8, 123.6, 120.6, 49.9, 38.3
HRMS (ESI): Calcd for C22H16N4O2SNa, m/z [M+Na]+: 423.0892;
found 423.0884.
4.2.41. 2-[2-Phenyl-1-(2-phenyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-one (7m)
To a stirred solution of compound 5b (1.20 g; 4 mmol) in dry
CH3CN (40 mL) was added phenyl hydrazide (0.55 g; 4 mmol) fol-
lowed by diisopropylethylamine (1.55 g; 12 mmol), and then TBTU
(1.42 g; 4.4 mmol). The resulting reaction mixture was stirred at
room temperature for 24 h. TLC of the reaction mixture showed
formation of the intermediate. Diisopropylethyl amine (1.03 g;
8 mmol) and p-toluenesulfonyl chloride (2.29 g; 12 mmol) were
4.2.46. 2-[2-Phenyl-1-(2-(2-furyl)-1,3,4-oxadiazol-5-yl)ethyl]-
1,2-benzisothiazol-3(2H)-one (7r)
White solid (32% yield), mp 142–144 °C. 1H NMR (DMSO-d6): d
3.56 (dd, J = 14.0, 10.8 Hz, 1H), 3.73 (dd, J = 14.3, 5.1 Hz, 1H), 6.42
(dd, J = 10.6, 5.1 Hz, 1H), 6.77–6.80 (m, 1H), 7.14–7.27 (m, 3H),
7.28–7.33 (m, 2H), 7.36–7.45 (m, 2H), 7.65–7.72 (m, 1H), 7.80–

H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
111
7.84 (m, 1H), 7.94–7.99 (m, 1H), 8.04–8.07 (m, 1H). 13C NMR
(CDCl3): d 165.4, 163.4, 158.6, 146.2, 140.5, 139.0, 135.1, 132.6,
129.4, 128.9, 127.6, 127.3, 126.0, 123.8, 120.8, 115.1, 112.4, 49.9,
164.7, 164.6, 140.7, 139.7, 135.3, 132.8, 132.3, 130.6, 128.8,
128.7, 127.3, 127.2, 126.7, 126.1, 125.4, 125.1, 123.9, 120.9, 48.4,
34.1, 32.0. HRMS (ESI): Calcd for C24H19N3O2SCl, m/z [M+H]+:
448.0887. Found 448.0875.
38.4.
HRMS
(ESI):
Calcd
for
C
21H15N3O3SNa,
m/z
[M+Na]+:412.0732; found 412.0746.
4.3. X-ray crystal analysis
4.2.47. 2-[2-Phenyl-1-(2-(2-thiophenyl)-1,3,4-oxadiazol-5-
yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7s)
Compound 7p crystallizes as colorless needles. The crystal was
affixed to a nylon cryoloop using oil (Paratone-n, Exxon) and
mounted in the cold stream of a Bruker Kappa-Apex-II area-detec-
tor diffractometer. The temperature of the crystal was maintained
at 150 K using a Cryostream 700EX Cooler (Oxford Cryosystems).
Data were measured with a redundancy using a CCD detector at
a distance of 50 mm from the crystal with a combination of phi
and omega scans. A scan width of 0.5° and scan time of 10 s was
White solid (25% yield), mp 128–130 °C. 1H NMR (CDCl3): d 3.51
(dd, J = 14.6, 8.8 Hz, 1H), 3.76 (dd, J = 14.2, 7.0 Hz, 1H), 6.46 (dd,
J = 9.3, 7.0 Hz, 1H), 7.08–7.12 (m, 1H), 7.15–7.32 (m, 5H), 7.35–
7.40 (m, 1H), 7.49–7.54 (m, 2H), 7.57–7.63 (m, 1H), 7.65–7.69 (m,
1H), 7.96–8.00 (m, 1H). 13C NMR (CDCl3): d 165.2, 163.3, 161.8,
140.4, 134.9, 132.4, 130.7, 130.5, 129.2, 128.7, 128.2, 127.4, 127.0,
125.8, 124.5, 123.7, 120.6, 49.8, 38.3. HRMS (ESI): Calcd for
C
21H15N3O2S2Na, m/z [M+Na]+: 428.0503; found 428.0496.
employed using graphite monochromated Molybdenum Ka radia-
tion (k = 0.71073 Å) that was collimated to a 0.6 mm diameter.
Data collection, reduction, structure solution, and refinement were
performed using the Bruker Apex2 suite (v2.0–2).46 All available
reflections to 2hmax = 52° were harvested (35,207 reflections,
3812 unique) and corrected for Lorentz and polarization factors
with Bruker SAINT (v6.45).47 Reflections were then corrected for
4.2.48. 2-[2-Phenyl-1-(2-methyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-one (7t)
White solid (25% yield), mp 98–99 °C. 1H NMR (CDCl3): d 2.48 (s,
3H), 3.49 (dd, J = 14.1, 9.1 Hz, 1H), 3.75 (dd, J = 14.3, 6.6 Hz, 1H),
6.39 (dd, J = 9.1, 6.6 Hz, 1H), 7.17–7.31 (m, 5H), 7.37–7.42 (m,
1H), 7.52–7.55 (m, 1H), 7.59–7.64 (m, 1H), 7.98–8.01 (m, 1H). 13C
NMR (CDCl3): d 165.2, 164.8, 164.3, 140.3, 135.0, 132.4, 129.1,
128.7, 127.3, 127.0, 125.7, 123.7, 120.6, 49.6, 37.9, 11.0. HRMS
(ESI): Calcd for C18H15N3O2SNa, m/z [M+Na]+:360.0783; found
360.0779.
absorption (numerical correction,
l
= 0.222 mmꢂ1), interframe
scaling, and other systematic errors with SADABS 2004/1.48 The
structure was solved (direct methods) and refined (full-matrix
least-squares against F2) with the Bruker SHELXTL package
(v6.14–1).49 All non-hydrogen atoms were refined using aniso-
tropic thermal parameters. All hydrogen atoms were included at
idealized positions; hydrogen atoms were not refined. Absolute
stereochemistry was assigned based on the synthetic protocol
and confirmed by a Flack parameter of 0.02. Pertinent crystal, data
collection, and refinement parameters are given in Table 2.
4.2.49. 2-[2-Phenyl-1-(2-ethyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-one (7u)
White solid (21% yield), mp 78–80 °C. 1H NMR (CDCl3): d 1.32 (t,
J = 7.6 Hz, 3H), 2.84 (q, J = 7.6 Hz, 2H), 3.49 (dd, J = 14.1, 9.0 Hz, 1H),
3.73 (dd, J = 14.2, 6.8 Hz, 1H), 6.39 (dd, J = 8.9, 6.9 Hz, 1H), 7.16–
7.32 (m, 5H), 7.36–7.43 (m, 1H), 7.50–7.66 (m, 2H), 7.97–8.02
(m, 1H). 13C NMR (CDCl3): d 168.9, 165.2, 164.1, 140.4, 135.0,
132.4, 129.2, 128.7, 127.3, 127.0, 125.7, 123.7, 120.6, 49.7, 38.1,
19.1, 10.6. HRMS (ESI): Calcd for C19H17N3O2SNa, m/z [M+Na]+:
374.0939; found 374.0932.
4.4. Biochemistry
4.4.1. DENV2 NS2B/NS3pro expression and purification
The construction of the pQE30-NS2BH(QR)NS3pro expression
plasmid has been described previously.38 The protease was ex-
pressed from Escherichia coli strain Top 10 F0 (Invitrogen) trans-
formed by pQE30-NS2BH(QR)-NS3pro plasmid and purified as
described.14 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/S3 junction site).
4.2.50. 2-[2-Phenyl-1-(2-propyl-1,3,4-oxadiazol-5-yl)ethyl]-1,2-
benzisothiazol-3(2H)-one (7v)
Light yellow solid (25% yield), mp 83–84 °C. 1H NMR (CDCl3): d
0.95 (t, J = 7.4 Hz, 3H), 1.69–1.80 (m, 2H), 2.74–2.81 (m, 2H), 3.49
(dd, J = 14.1, 9.0 Hz, 1H), 3.73 (dd, J = 14.4, 6.8 Hz, 1H), 6.40 (dd,
J = 8.7, 6.8 Hz, 1H), 7.17–7.30 (m, 5H), 7.37–7.42 (m, 1H), 7.52–
7.56 (m, 1H), 7.59–7.64(m, 1H), 7.97–8.02 (m, 1H). 13C NMR (CDCl3):
d 168.0, 165.2, 164.1, 140.4, 135.0, 132.4, 129.1, 128.7, 127.3, 127.0,
125.7, 123.7, 120.6, 49.7, 38.2, 27.1, 19.9, 13.5. HRMS (ESI): Calcd for
4.4.2. WNV NS2B/NS3pro expression and purification
The expression and purification of the WNV NS2B/NS3pro con-
taining the 5 amino acid spacer between the NS2B and NS3pro do-
mains used in these experiments were previously described.39
C
20H19N3O2SNa, m/z [M+Na]+:388.1096; found 388.1111.
4.4.3. In vitro DENV2 and WNV NS2B/NS3pro assays and
inhibition studies
Selected compounds 7a–x were analyzed by in vitro protease
assays performed in opaque 96-well plates. Standard reaction mix-
4.2.51. 2-[2-m-Fluorophenyl-1-(2-m-chlorophenyl-1,3,4-
oxadiazol-5-yl)ethyl]-1,2-benzisothiazol-3(2H)-one (7w)
Yellow oil (29% yield). 1H NMR (CDCl3): d 3.56 (dd, J = 12.4,
9.1 Hz, 1H), 3.80 (dd, J = 12.4, 9.1 Hz, 1H), 6.51 (dd, J = 8.9, 6.8 Hz,
1H), 6.85–8.05 (m, 12H). 13C NMR (CDCl3): d 165.3, 164.6, 164.4,
164.0, 140.3, 137.3, 135.2, 132.2, 130.8, 130.4, 130.0, 127.1,
125.9, 125.2, 124.8, 123.5, 120.7, 116.4, 116.1, 114.6, 114.4, 49.6,
tures (100
30% glycerol, 25 nM DENV2 NS2B/NS3 protease, and 10
25 M inhibitor (dissolved in DMSO) in each assay were incubated
15 min at 37 °C. Reactions were started by the addition of 5.0
lL) containing 200 mM TrisꢀHCl (pH 9.5), 6 mM NaCl,
l
M or
l
lM
tetra-peptide substrate Bz-Nle-Lys-Arg-Arg-AMC. Conditions were
the same for WNV protease except 28 nM WNV NS2B/NS3pro was
used in the assay.
37.8. HRMS (ESI): Calcd for
452.0636. Found 452.0637.
C
23H16N3O2SClF, m/z [M+H]+:
Release of free AMC was measured using a spectrofluorometer
(Molecular Devices) at excitation and emission wavelengths of
380 and 460 nm, respectively. Control fluorescence values ob-
tained in the absence of inhibitor were taken as 100%, and those
in the presence of inhibitors were calculated as the percentage of
4.2.52. 2-[3-Phenyl-1-(2-m-chlorophenyl-1,3,4-oxadiazol-5-
yl)propyl]-1,2-benzisothiazol-3(2H)-one (7x)
Yellow oil (31% yield). 1H NMR (CDCl3): d 2.60–2.82 (m, 4H),
6.21–6.27 (m, 1H), 7.18–8.11 (m, 13H). 13C NMR (CDCl3): d 165.7,

112
H. Lai et al. / Bioorg. Med. Chem. 21 (2013) 102–113
inhibition of the control using Microsoft Excel and plotted using
SigmaPlot 2001 v7.1 (Systat Software Inc.). The background of
AMC in the absence of protease was subtracted before the data
analysis. All assays were performed in triplicate. Compounds with
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The generous financial support of this work by the National
Institutes of Health (AI082068) is gratefully acknowledged.

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