Inhibition of dengue virus and west Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives

Article abstract of DOI:10.1016/j.bmc.2011.12.047

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j-n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Full text of DOI:10.1016/j.bmc.2011.12.047

Bioorganic & Medicinal Chemistry 20 (2012) 1213–1221
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of Dengue virus and West Nile virus proteases by click
chemistry-derived benz[d]isothiazol-3(2H)-one derivatives
Kok-Chuan Tiew a, Dengfeng Dou a, Tadahisa Teramoto b, Huiguo Lai b, Kevin R. Alliston a,
Gerald H. Lushington c, 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:
Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were
synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several com-
pounds (4l, 7j–n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in
the absence and presence of added detergent. These compounds could potentially serve as a launching
pad for a hit-to-lead optimization campaign.
Received 10 October 2011
Revised 14 December 2011
Accepted 21 December 2011
Available online 30 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dengue virus
West Nile virus
Protease
Inhibitor
Click chemistry
1. Introduction
as an attractive target for the discovery and development of ther-
apeutics for DENV infection.6,7 NS3 is a multifunctional protein
The genus Flavivirus in the family Flaviviridae comprises an array
of major viral pathogens, including West Nile virus (WNV), Dengue
virus (DENV), Hepatitis C virus (HCV), Yellow Fever virus (YFV), and
Japanese encephalitis virus (JEV).1 DENV is a mosquito-borne virus
that has four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4.
Worldwide, DENV is the cause of 50–100 million infections annu-
ally, with approximately 500,000 cases progressing to dengue
hemorrhagic fever/dengue shock syndrome, resulting in ꢀ25,000
deaths.2,3 There are no vaccines or small molecule therapeutics for
the treatment of DENV infection; consequently, there is currently
an urgent and unmet need for the discovery and development of
therapeutic agents for DENV infection.
DENV is a small, enveloped virus with a single-stranded, posi-
tive sense 11-kb RNA genome, which encodes a polyprotein
precursor. Co- and post-translational cleavage of the polyprotein
produces three structural proteins (C, prM, and E) and seven
nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and
NS5) by the concerted action of host proteases (furin, signalase)
and the two-component trypsin-like protease NS2B-NS3 (NS2B-
NS3pro).4,5 Processing of the polyprotein by NS2B-NS3pro is essen-
tial for viral replication; consequently, NS2B-NS3pro has emerged
that comprises a protease (NS3pro), an RNA helicase, a nucleoside
triphosphatase, and 50-RNA triphosphatase activities.7 NS3pro is a
serine endoprotease with a His51-Asp75-Ser135 catalytic triad
and a strongly-preferred substrate specificity for a -X-K-R-R-G/
8
0
S- sequence corresponding to the subsites -S4-S3-S2-S1-S1 -. Cleav-
0
age is at the P1–P1 scissile bond (R–G/S). The hydrophilic core of
the NS2B protein cofactor is required for optimal catalytic effi-
ciency.9,10 X-ray crystal structures of NS2B-NS3pro of WNV and
DENV-2 and DENV-1 have been reported.10–13 Inhibitors of NS2B-
NS3 protease containing a highly charged peptidyl recognition
element with a dibasic motif at P1–P2 coupled to an array of
electrophilic warheads (aldehydes, trifluoromethyl ketones, and
boronic acids)14–16 or non-peptidyl
have been described.
a
-ketoamides,17 and others18,19
We describe herein the results of synthetic and biochemical
studies related to the inhibition of DENV NS2B-NS3 protease by
click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives
(I–II) (Fig. 1).
1.1. Chemistry
The synthesis of compounds 4a–p and 7a–p is summarized in
Scheme 1. Thus, starting acid 120 was activated with 1,10-carbonyl-
diimidazole (CDI) and then coupled to propargylamine to yield
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.047

1214
K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
O
assays and inhibition studies were carried out as previously de-
O
scribed25–28 and the results are summarized in Figures 2 and 3.
N
O
N
N
N
N
N
N
N
S
NH
S
R
NH
R
2. Results and discussion
O
O
(I)
(II)
Diseases caused by Dengue and related flaviviruses constitute a
world-wide health problem for which there are currently no effec-
tive vaccines or small-molecule therapeutics. Consequently, there
has been an increasing interest in the discovery and development
of agents for DENV infection.18
Figure 1. General structures of DENV2 NS2B-NS3pro inhibitors (I–II).
intermediate 2. Click chemistry methodology was employed to
generate the desired triazole derivatives 4a–p by coupling com-
pound 2 to an array of structurally diverse azides 3a–p. The latter
were readily synthesized from the corresponding halides and so-
dium azide in dimethyl sulfoxide.21 Intermediate 1 was also cou-
pled to aniline derivative 522 to yield compound 6, which was
subsequently converted to compounds 7a–p via reaction with a
series of azides 3a–p under click chemistry conditions. Compounds
4p and 7p were obtained by hydrolyzing compounds 4o and 7o,
respectively.
As part of
a program related to the discovery of DENV
NS2B-NS3pro inhibitors, screening of a representative subset of
compounds from a pharmacologically-rich in-house library of com-
pounds lead to the identification of a benz[d]isothiazol-3(2H)-one
derivative that exhibited inhibitory activity against DENV NS2B-
NS3pro. This finding provided the impetus for the synthesis of two
focused libraries based on general structures (I) and (II) ( Fig. 1).
The high synthetic tractability of (I) and (II) made possible the facile
synthesis of the desired compounds (Scheme 1). Considerations
associated with the design strategy employed in the construction
of the libraries included using click chemistry to generate an
electron-rich ring with hydrogen acceptor capabilities and enhanc-
ing binding by probing the nature of R (Scheme 1, compounds
4a–p). The active site of DENV NS2B-NS3pro is rather shallow;
1.2. Biochemistry
The expression and purification of DENV NS2B-NS3pro and
WNV NS2B-NS3pro have been previously described.23,24 Enzyme
ii
R
X
R
N3
3a-o
O
N
R
iv
N
N
O
O
S
NH
N
O
N
i
N
S
NH
4a-o
S
OH
O
O
1
2
iii
O
i
OH
H2N
H2N
iv
5
R
O
N3
3a-o
O
N
N
O
N
N
R
N
O
S
NH
S
NH
O
O
7a-o
6
R =
S
O
O
F
OCH3
F
O
a
b
c
d
e
f
g
h
i
O
HN
O
HN
O
O
O
O
HN
O
HN
O
O
O
OH
O
v
j
k
l
m
n
o
p(4p,7p)
Scheme 1. Reagents and conditions: (i) CDI/THF/reflux then propargylamine or compound 5; (ii) NaN3/DMSO; (iii) NaOH/CH3OH, removal of CH3OH, then propargyl bromide/
CH3CN; (iv) sodium ascorbate/CuSO4/BuOH/H2O; (v) TFA.

K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
1215
100
80
60
40
20
0
Compound
Figure 2. Inhibition of DENV2 NS2B-NS3pro by derivatives of compounds (I–II). The concentrations of the tested compounds and DENV2 NS2B-NS3 protease were 25
l
M and
25 nM, respectively. The buffer used contained 200 mM Tris hydrochloride, 6 mM NaCl, and 30% glycerol, pH 9.5. The percent values were calculated from the relative
fluorescence units obtained in the presence and absence of tested compound. All assays were performed in triplicate and the average values are shown. The assays were
performed as described in Section 1.2.
100
80
60
40
20
0
100
80
60
40
20
DENV2
WNV
-2
-1
0
1
4l
7j
7k
7l
7m
7n Aprotinin
Compound
Log [Inhibitor (μΜ)]
Figure 3. Inhibition of DENV2 and WNV NS2B-NS3pro by selected derivatives of (I–
II) at 25 M. The concentrations of DENV2 and WNV NS2B-NS3 proteases were 25
Figure 4. Determination of IC50 values of selected benz[d]isothiazol-3(2H)-one
derivatives for inhibition of DENV2 NS2B-NS3 protease. The inhibitors were
incubated with DENV2 NS2B-NS3 protease (50 nM) in buffer (200 mM Tris–HCl,
l
and 28 nM, respectively. The buffer used contained 200 mM Tris–HCl, 6 mM NaCl,
and 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. All assays were performed in triplicate and the average values are
shown. The assays were performed as described in Section 1.2.
6 mM NaCl and 30% glycerol, pH 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
l
was measured at 460 nm with excitation at 380 nm and converted to the
percentage of protease activity in the absence and the presence of inhibitors. The
inhibitors analyzed were as follows: (d) 7 l; (s) 7n. The solid line is the theoretical
fitting curve based on the Sigmoidal Equation. All the spectra were recorded at
37 °C and shown after subtraction of the buffer spectrum. The apparent IC50 for
compounds 7 l and 7n were 4.45 0.06 and 3.48 0.05, respectively.
consequently, we envisaged tethering the benz[d]isothiazol-3(2H)-
one ring to the triazole ring via a m-aminophenol linker to provide
additional favorable binding interactions with the enzyme (Scheme
1, compounds 7a–p).
The results related to the interaction of the synthesized com-
pounds with DENV NS2B-NS3pro are shown in Figure 2. With the
exception of compounds 4l and 4m, the rest of the compounds
based on structure (I) were inactive. However, several derivatives
based on structure (II) showed noteworthy inhibitory activity
(compounds 7e and 7j–n, Fig. 2). The IC50 values of compounds
4l and 7j–n were subsequently determined (Fig. 4) and are listed
in Table 2. The kinetics of the interaction of a representative
inhibitor 7n against DENV2 NS2B-NS3pro was investigated in
greater detail by determining Km and Vmax (Figs. 5 and 6, Table
2), as well as the Ki (determined to be 4.77 0.05 lM). Because
at micromolar concentrations many small molecules self–associate
and the resulting colloid-like aggregates can lead to non-specific
inhibition,29,30 the synthesized compounds were re-screened in
the presence of 0.1% CHAPS to confirm the activity of the com-
pounds and to eliminate the possibility that the compounds acted
as promiscuous inhibitors.31,32 Indeed, the inhibitory activity of all

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K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
Table 2
0.08
0.06
0.04
0.02
0.00
Kinetics of 7n against DENV2 NS2B-NS3pro
Inhibitor [7n] (
lM)
Km
(
l
M)
Vmax (lM/min)
0
1
2
5
12.8732 0.4064
17.5003 1.1541
19.5461 0.8851
27.0101 0.5086
0.0999 0.0012
0.1006 0.0029
0.0978 0.0020
0.0994 0.0010
active compounds remained essentially the same in the presence
of detergent. It is evident from the results shown in Table 1 that
the presence of a (phenoxy)phenyl moiety enhances inhibitory
activity, however, the gain in potency is accompanied by a signifi-
cant increase in molecular weight and hydrophobicity which are
likely to impact adversely ADMET and drug-like characteris-
tics.33–35
The closely related West Nile virus (WNV), a member of the Fla-
vivirus genus of the Flaviviridae family, is an emergent viral patho-
gen, for which there are no effective vaccines or antiviral
agents.18,36,37 Consequently, the synthesized compounds were also
screened against WNV NS2B-NS3pro26 and the results are summa-
rized in Figure 3. Compounds 4l and 7j–n displayed weak inhibi-
tory activity toward the enzyme.
0
10
20
30
40
50
[Substrate] (μM)
Figure 5. DENV2 NS2B-NS3pro activity in the absence and presence of inhibitor 7n.
The in vitro protease assays were performed as described in Section 3. The initial
reaction rates of the tetra-peptide substrate cleavage catalyzed by 0.025
NS2B-NS3 protease in the absence (d–d) and the presence of 1.0 M (o-o), 2.0
(.–.) and 5.0
M (5–5) were determined by varying concentrations of tetra-
peptide substrate (0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40 and 50 M range). The reactions
were initiated by the addition of DENV2 NS2B-NS3pro 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 calculated Km
lM DENV2
l
lM
l
l
Inspection of the structures of the six most active derivatives of
compound (II) toward DENV NS2B-NS3pro suggests that they have
in common an R group (keto, amide or ester carbonyl) capable of
app
and Vmax values are shown in Table 2.
engaging in hydrogen bonding, as well as hydrophobic or p–p inter-
actions. A plausible mode of binding of energy-minimized inhibitor
7j to the active site of DENV2 NS2B-NS3pro (homology model de-
rived by Wichapong et al. from the corresponding WNV homolog10
)
30
28
26
24
22
20
18
16
14
12
is shown in Figure 7. This bound conformation, derived from molec-
ular docking simulations, predicts that the ligand will occupy space
adjacent to catalytic triad residues Ser135 and His51, with the core
benz[d]isothiazol-3(2H)-one engaging in lipophilic interactions
with the His51 and Trp50 side chains. The ligand triazole also
accepts H-bonds from the backbone amide proton of Gly153 and
the hydroxyl proton of Tyr161, donates a H-bond to the hydroxyl
oxygen of Ser83, and engages in favorable lipophilic interactions
with Pro132, Tyr150 and Tyr161. The terminal phenyl group on
the ligand appears to be somewhat sterically limited by its presence
in a relatively small lipophilic pocket, which might limit the
prospects for lead optimization via aryl substituents, however the
torsional flexibility availed from an aliphatic carbon immediately
adjacent to the triazole (as per compounds 7k,l,m,n) may permit
the ligand to bending downwards to engage with other lipophiles
in the more spacious hydrophobic pocket marked by the nearby
Tyr161.
10
0
1
2
3
4
5
[Inhibitors] (μM)
In conclusion, the synthesis and in vitro evaluation of two fo-
cused libraries of compounds have resulted in the identification
of several inhibitors of DENV2 NS2B-NS3pro. The novelty of the
structures and high synthetic tractability provide a reasonable ba-
sis for a hit-to-lead optimization campaign.
Figure 6. Plot of Km values versus concentration of compound 7n. Km values with no
inhibitor and at different concentrations of inhibitor were calculated and plotted
versus different concentrations of inhibitor. From these data, the slope = 2.6988
0.2712 lM and the intercept = 12.8732 0.7427 lM were obtained and the Ki value
calculated from the equation intercept/slope is 4.77 0.05 lM.
3. Experimental section
3.1. General
Table 1
IC50 values against DENV-2 proteasea
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.
Compound
IC50 (lM)
4l
7j
7k
7l
7m
7n
4.87 0.07
6.22 0.09
13.36 0.21
4.45 0.06
4.59 0.07
3.48 0.05
a
The IC50 values were determined as described
under Section 3.

K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
1217
Figure 7. Conformation for inhibitor 7j bound to the catalytic site of DENV2 NS2B-NS3 protease, as predicted from molecular docking simulations. The receptor surface is
colored as follows: red = polar O, blue = polar N, cyan = polar (donatable) H, white = weakly polar aliphatic or aryl CH groups, yellow = nonpolar lipophiles. The ligand is
rendered in stick form according to standard CPK coloring.
The TLC plates for the final compounds were eluted using two dif-
ferent solvent systems and were visualized using iodine chamber
and/or UV light. Each individual compound was identified as a
single spot on TLC plate (purity greater than 95%). DENV2 NS2B-
NS3 protease (or WNV NS2B-NS3pro) substrate Bz-Nle-Lys-Arg-
Arg-AMC was purchased from Bachem, Torrance, CA. or custom
synthesized by NeoBioScience, Cambridge, MA.
sodium sulfate. The drying agent was filtered off and the filtrate
was concentrated, leaving compound 3a as a yellow oil (6.31 g;
79% yield). 1H NMR (CDCl3): d 4.38 (s, 2H), 7.27–7.44 (m, 5H).
3.3.2. p-Fluorobenzyl azide (3b)
Yellow oil (99% yield). 1H NMR (CDCl3): d 4.31 (s, 2H), 7.00–
7.12(m, 2H), 7.24–7.35 (m, 2H).
3.3.3. m-Fluorobenzyl azide (3c)
3.2. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(prop-2-ynyl)ace-
tamide (2)
Yellow oil (90% yield). 1H NMR (CDCl3): d 4.38 (s, 2H), 6.98–7.13
(m, 3H), 7.33–7.40 (m, 1H).
To a solution of compound 1 (10.45 g; 50 mmol) in dry THF
(100 mL) was added portionwise a suspension of 1,10-carbonyldi-
imidazole (8.10 g; 50 mmol) in THF (25 mL). The resulting solution
was stirred at room temperature for 20 min and refluxed for
10 min. A solution of propargylamine (2.75 g; 50 mmol) in THF
(25 mL) was added and the solution was allowed to stir at room
temperature overnight. A precipitate formed which was collected
by vacuum filtration, leaving compound 2 as a white solid
(5.71 g; 46% yield), mp 156–158 °C. 1H NMR (CDCl3): d 2.20 (t,
J = 4.2 Hz, 1H), 4.05 (dd, J = 7.9, 7.9 Hz, 2H), 4.50 (s, 2H), 6.62 (s,
1H), 7.45 (t, J = 11.1 Hz, 1H), 7.60 (d, J = 4.3 Hz, 1H), 7.68 (t,
J = 11.1 Hz, 1H), 8.06 (d, J = 4.3 Hz, 1H).
3.3.4. p-Methoxybenzyl azide (3d)
Colorless oil (98% yield). 1H NMR (CDCl3): d 3.80 (s, 3H), 4.23 (s,
2H), 6.90 (d, J = 6.9 Hz, 2H), 7.25 (d, J = 6.9 Hz, 2H).
3.3.5. Methyl 3-(azidomethyl)benzoate (3e)
Colorless oil (71% yield). 1H NMR (CDCl3): d 3.98 (s, 3H), 4.41 (s,
2H), 7.42–7.59 (m, 3H), 8.00–8.15 (m, 1H).
3.3.6. (Azidomethyl)(phenyl)sulfane (3f)
Colorless oil (98% yield). 1H NMR (CDCl3): d 4.58 (s, 2H), 7.25–
7.39 (m, 3H), 7.50 (d, J = 8.3 Hz, 2H).
3.3.7. (3-Azidopropyl)benzene (3g)
Yellow oil (79% yield). 1H NMR (CDCl3): d 1.85–2.00 (m, 2H),
2.72 (t, J = 25.0 Hz, 2H), 3.28 (t, J = 25.0 Hz, 2H), 7.18–7.40 (m, 5H).
3.3. Azides 3a–j were synthesized as described below using sta-
ndard literature procedures21
3.3.1. Benzyl azide (3a)
3.3.8. (2-Azidoethyl)benzene (3h)
Yellow oil (41% yield). 1H NMR (CDCl3): d 2.90 (t, J = 8.3 Hz, 2H),
3.50 (t, J = 8.3 Hz, 2H), 7.20–7.38 (m, 5H).
To a solution of sodium azide (4.32 g; 66 mmol) in dry dimethyl
sulfoxide (120 mL) was added benzyl chloride (7.62 g; 60 mmol),
and the reaction mixture was stirred at room temperature over-
night. Water (80 mL) was carefully added to the reaction mixture
and the aqueous layer was extracted with diethyl ether
(2 ꢁ 150 mL). The combined organic extracts were washed with
water (50 mL) and the organic layer was dried over anhydrous
3.3.9. (2-Azidoethoxy)benzene (3i)
Yellow oil (69% yield). 1H NMR (CDCl3): d 3.89 (t, J = 20.3 Hz,
2H), 4.29 (t, J = 20.3 Hz, 2H), 6.90–7.03 (m, 3H), 7.25–7.39 (m,
2H).

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K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
3.3.10. 2-Azido-1-phenylethanone (3j)
3.5.2. N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-
(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (4b)
Reddish oil (85% yield). 1H NMR (CDCl3): d 4.59 (s, 2H), 7.43–
7.70 (m, 3H), 7.95 (d, J = 8.3 Hz, 2H).
White solid (95% yield), mp 187–189 °C. 1H NMR (CDCl3): d 4.49
(s, 2H), 4.51 (d, J = 5.2 Hz, 2H), 5.44 (s, 2H), 6.80 (s, 1H), 7.05 (t,
J = 6.9 Hz, 2H), 7.26 (m, 1H), 7.43 (m, 2H), 7.59 (d, J = 5.2 Hz, 1H),
7.68 (d, J = 5.2 Hz, 1H), 8.02 (d, J = 5.2 Hz, 1H). HRMS (ESI): Calcu-
lated for C19H16FN5O2S [M+Na]+ 420.0906; found 402.0903.
3.4. Representative synthesis of compound 3k–o
3.4.1. 2-Azido-N-phenylacetamide (3k)
To a solution of aniline (9.3 mL; 100 mmol) in acetone (20 mL)
kept in an ice bath was added 2-chloroacetyl chloride (4.1 mL;
50 mmol) dropwise with stirring. After the addition the ice bath
was removed and the reaction mixture was stirred at room tem-
perature for 4 h. Aqueous hydrochloric acid (10% v/v; 40 mL) was
added to the reaction mixture, whereupon a white solid formed.
The white precipitate was collected by vacuum filtration and
washed with 5% hydrochloric acid (3 ꢁ 40 mL) and water
(2 ꢁ 40 mL). The solid was allowed to air dry overnight (7.8 g;
92% yield), mp 122–124 °C. The dry solid (7.6 g; 45 mmol) was
added to a solution of sodium azide (3.24 g; 49.5 mmol) in dry di-
methyl sulfoxide (100 mL) and the reaction mixture was stirred at
room temperature overnight. Water (50 mL) was added to the
reaction mixture and the aqueous layer was extracted with diethyl
ether (3 ꢁ 100 mL). The combined extracts were washed with
water (50 mL) and dried by anhydrous sodium sulfate. The drying
agent was filtered off and the filtrate concentrated, leaving com-
pound 3k as a white solid (8.7 g; 98% yield), mp 57–59 °C. 1H
NMR (CDCl3): d 4.18 (s, 2H), 7.18 (t, J = 6.9 Hz, 1H), 7.35 (t,
J = 8.6 Hz, 2H), 7.57 (d, J = 5.2 Hz, 2H), 8.00 (s, 1H).
3.5.3. N-((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-
(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (4c)
White solid (78% yield), mp 191–193 °C. 1H NMR (CDCl3): d 4.49
(s, 2H), 4.51 (d, J = 5.2 Hz, 2H), 5.44 (s, 2H), 6.80 (s, 1H), 6.98 (d,
J = 6.9 Hz, 1H), 7.05 (d, J = 6.9 Hz, 1H), 7.36 (m, 1H), 7.43 (m, 2H),
7.59 (d, J = 5.2 Hz, 1H), 7.68(d, J = 5.2 Hz, 1H), 8.02 (d, J = 5.2 Hz,
1H). HRMS (ESI): calcd for C19H16FN5O2S [M+H]+ 398.1087; found
398.1068.
3.5.4. N-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-
(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (4d)
White solid (92% yield), mp 188–190 °C. 1H NMR (CDCl3): d 3.81
(s, 3H), 4.49 (s, 4H), 5.40 (s, 2H), 6.89 (d, J = 8.6 Hz, 2H), 7.22 (d,
J = 8.6 Hz, 2H), 7.41 (m, 2H), 7.58 (d, J = 6.9 Hz, 1H), 7.63 (d,
J = 6.9 Hz, 1H), 8.02 (d, J = 6.9 Hz, 1H). HRMS (ESI): calcd for
C
20H19N5O3S [M+H]+ 410.1287; found 410.1297.
3.5.5. Methyl 4-((4-((2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamido)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzoate (4e)
White solid (10% yield), mp 138–140 °C. 1H NMR (CDCl3): d 3.96
(s, 3H), 4.45 (s, 2H), 4.47 (d, J = 5.2 Hz, 2H), 5.55 (s, 2H), 6.80 (s, 1H),
7.40–7.49 (m, 8H), 8.01 (d, J = 5.2 Hz, 1H). HRMS (ESI): calcd for
3.4.2. 2-Azido-N-(4-phenoxyphenyl)acetamide (3l)
Reddish oil (80% total yield). 1H NMR (CDCl3): d 4.18 (s, 2H),
7.00 (m, 4H), 7.09 (t, J = 6.7 Hz, 1H), 7.35 (t, J = 6.7 Hz, 2H), 7.50
(d, J = 4.9 Hz, 2H), 8.03 (s, 1H).
C
21H19N5O4S [M+Na]+ 460.1055; found 460.1044.
3.5.6. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-((1-(phenylthio-
methyl)-1H-1,2,3-triazol-4-yl)methyl)acetamide (4f)
White solid (49% yield), mp 176–178 °C. 1H NMR (CDCl3): d 4.49
(s, 2H), 4.51 (s, 2H), 5.58 (s, 2H), 6.79 (s, 1H), 7.31(s, 3H), 7.41 (t,
J = 3.4 Hz, 2H), 7.58 (d, J = 3.4 Hz, 1H), 7.65 (t, J = 3.4 Hz, 2H), 8.03
(d, J = 3.4 Hz, 1H). HRMS (ESI): calcd for C19H17N5O2S2 [M+H]+
412.0902; found 412.0909.
3.4.3. 2-Azido-N-(3-phenoxyphenyl)acetamide (3m)
Reddish oil (56% total yield). 1H NMR (CDCl3): d 4.17 (s, 2H),
6.80 (m, 1H), 7.02 (d, J = 6.9 Hz, 2H), 7.15 (t, J = 8.6 Hz, 1H), 7.24–
7.40 (m, 5H), 8.00 (s, 1H).
3.4.4. 2-Azido-N-(2-phenoxyphenyl)acetamide (3n)
Brown oil (93% total yield). 1H NMR (CDCl3): d 4.18 (s, 2H), 6.90
(d, J = 5.5 Hz, 1H), 7.00–7.20 (m, 6H), 7.39 (t, J = 9.1 Hz, 2H), 8.62 (s,
1H).
3.5.7. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-((1-(3-phenyl-
propyl)-1H-1,2,3-triazol-4-yl)methyl)acetamide (4g)
Brown solid (75% yield), mp 143–145 °C. 1H NMR (CDCl3): d
2.19–2.25 (m, 2H), 2.65 (t, J = 12.1 Hz, 2H), 4.34 (t, J = 8.6 Hz, 2H),
4.51 (s, 2H), 4.53 (d, 2H), 6.80 (s, 1H), 7.18 (d, J = 5.2 Hz, 2H),
7.20–7.35 (m, 2H), 7.42 (t, J = 8.6 Hz, 1H), 7.49–7.60 (m, 2H), 7.63
(t, J = 8.6 Hz, 1H), 8.01 (s, 1H). HRMS (ESI): calcd for C21H21N5O2S
[M+H]+ 408.1494; found 408.1475.
3.4.5. tert-Butyl 2-azidoacetate (3o)
Colorless oil (99% total yield). 1H NMR (CDCl3): d 1.50 (s, 9H),
3.79 (s, 2H).
3.5. Representative synthesis of compounds 4a–p
3.5.8. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-((1-phenethyl-1H-1,
2,3-triazol-4-yl)methyl)acetamide (4h)
Grey solid (78% yield), mp 173–175 °C. 1H NMR (CDCl3): d 3.20
(t, J = 8.6 Hz, 2H), 4.46–4.59 (m, 6H), 6.81 (s, 1H), 7.10 (d, J = 6.9 Hz,
2H), 7.22–7.35 (m, 3H), 7.43 (t, J = 8.6 Hz, 2H), 7.59 (d, J = 5.2 Hz,
1H), 7.66 (t, J = 8.6 Hz, 2H), 8.02 (d, J = 5.2 Hz, 1H). HRMS (ESI):
calcd for C20H19N5O2S [M+H]+ 394.1338; found 394.1333.
3.5.1. N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-(3-oxobenzo-
[d]isothiazol-2(3H)-yl)acetamide (4a)
To a solution of compound 2 (0.25 g; 1.0 mmol) and compound
3a (0.33 g; 2.5 mmol) in tert-butyl alcohol and water (1:1) (15 mL)
were added sodium ascorbate (0.04 g; 0.2 mmol) and copper(II)
sulfate pentahydrate (0.005 g; 0.02 mmol) and the reacting mix-
ture was stirred at room temperature overnight. The disappearance
of compound 2 was monitored by TLC. Water (25 mL) was added
and the reaction mixture was stirred for 5 min. The precipitate
was collected by vacuum filtration and washed with water
(25 mL) and diethyl ether (30 mL), leaving compound 4a as a white
solid (0.30 g; 79% yield), mp 193–195 °C. 1H NMR (CDCl3): 4.50 (s,
2H), 4.51 (s, 2H), 5.48 (s, 2H), 6.82 (s, 1H), 7.32–7.44 (m, 6H), 7.59
(d, J = 5.1 Hz, 1H), 7.64 (t, J = 6.9 Hz, 1H), 8.02 (s, 1H). HRMS (ESI):
calcd for C19H17N5O2S [M+Na]+ 402.1001; found 402.0992.
3.5.9. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-((1-(2-phenoxye-
thyl)-1H-1,2,3-triazol-4-yl)methyl)acetamide (4i)
Gray solid (82% yield), mp 168–170 °C. 1H NMR (CDCl3): d 4.33
(t, J = 6.9 Hz, 2H), 4.51 (s, 2H), 4.53 (d, J = 5.2 Hz, 2H), 4.75 (t,
J = 6.9 Hz, 2H), 6.80 (s, 1H), 6.88 (d, J = 6.9 Hz, 2H), 7.00 (t,
J = 8.6 Hz, 1H), 7.25–7.32 (m, 1H), 7.42 (t, J = 8.6 Hz, 2H), 7.53 (d,
J = 6.9 Hz, 1H), 7.63 (t, J = 8.6 Hz, 2H), 7.75 (s, 1H), 8.02 (d,
J = 5.2 Hz, 1H). HRMS (ESI): calcd for
C
20H19N5O3S [M+Na]+
432.1106; found 410.1120.

K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
1219
3.5.10. N-((1-(2-Oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)methyl)-
3.6. 3-(Prop-2-ynyloxy)aniline (5)
2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (4j)
Brown solid (90% yield), mp 180–182 °C. 1H NMR (DMSO): d
4.40 (d, J = 3.7 Hz, 2H), 4.50 (s, 2H), 6.18 (s, 2H), 7.42 (t,
J = 9.1 Hz, 1H), 7.60–7.79 (m, 5H), 7.85–8.10 (m, 4H), 8.80 (t,
J = 9.1 Hz, 1H). HRMS (ESI): calcd for
408.1130; found 408.1150.
Sodium hydroxide (4.30 g; 107.5 mmol) and 3-aminophenol
(11.0 g; 100.9 mmol) were dissolved in methanol (200 mL) at
gentle heating. The volatiles were evaporated off and the residual
water was removed by four successive co-distillations with abso-
lute ethanol (80 mL). The residue was dissolved in dry acetonitrile
(200 mL) and propargyl bromide (14.24 g; 119.7 mmol) was added
in three portions over 1 h period. The reaction mixture was stirred
overnight, concentrated, and partitioned between 0.1 M sodium
hydroxide (200 mL) and diethyl ether (200 mL). The layers were
separated and the aqueous layer was extracted with diethyl ether
(2 ꢁ 200 mL). The combined organic extracts were washed with
brine (100 mL) and dried over anhydrous sodium sulfate. Evapora-
tion of the solvent left a brown oily residue which was dissolved in
methanol (160 mL) and treated with 4 M hydrochloric acid in diox-
ane (54 mL; 215.3 mmol). The volatiles were removed and the
resulting brownish solid was suspended in boiling ethyl acetate
(140 mL). Cooling the suspension to room temperature yielded a
white crystalline solid which was collected by suction filtration
and dried. The solid was dissolved in water (50 mL) and the pH
was adjusted to ꢀ11 using 10% aqueous sodium hydroxide
(35 mL). The aqueous layer was extracted with ethyl acetate
(2 ꢁ 100 mL), dried over sodium sulfate, filtered, and concentrated,
leaving a yellow oil (10.3 g; 70% yield). 1H NMR (CDCl3): d 2.50 (t,
J = 3.1 Hz, 1H), 3.65 (br s, 2H), 4.63 (d, J = 3.1 Hz, 2H), 6.29–6.40 (m,
3H), 7.05 (t, J = 7.8 Hz, 1H).
C
20H17N5O3S [M+H]+
3.5.11. N-((1-(2-Oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)-
methyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (4k)
White solid (74% yield), mp 176–178 °C. 1H NMR (DMSO): d
4.40 (d, J = 4.2 Hz, 2H), 4.51 (s, 2H), 5.38 (s, 2H), 7.10 (t,
J = 8.3 Hz, 1H), 7.39 (t, J = 8.3 Hz, 2H), 7.45 (t, J = 8.3 Hz, 1H), 7.60
(d, J = 6.5 Hz, 1H), 7.70 (t, J = 8.3 Hz, 1H), 7.91 (d, J = 6.5 Hz, 1H),
8.00 (d, J = 6.5 Hz, 2H), 8.82 (s, 1H), 10.51 (s, 1H). HRMS (ESI): calcd
for C20H18N6O3S [M+Na]+ 445.1059; found 445.1068.
3.5.12. N-((1-(2-Oxo-2-(4-phenoxyphenylamino)ethyl)-1H-1,2,
3-triazol-4-yl)methyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamide (4l)
Gray solid (65% yield), mp 240–242 °C. 1H NMR (DMSO): d 4.39
(d, J = 4.3 Hz, 2H), 4.50 (s, 2H), 5.30 (s, 2H), 6.95–7.05 (m, 3H), 7.10
(t, J = 6.5 Hz, 2H), 7.38–7.45 (m, 3H), 7.60 (d, J = 6.5 Hz, 1H), 7.69 (t,
J = 6.5 Hz, 2H), 7.90 (d, J = 4.3 Hz, 1H), 8.00 (d, J = 4.3 Hz, 2H), 8.78
(t, J = 6.5 Hz, 1H), 10.51 (s, 1H). HRMS (ESI): calcd for C26H22N6O4S
[M+Na]+ 537.1321; found 537.1338.
3.5.13. N-((1-(2-Oxo-2-(3-phenoxyphenylamino)ethyl)-1H-1,2,
3-triazol-4-yl)methyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamide (4m)
3.7. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(3-(prop-2-ynyloxy)-
phenyl)acetamide (6)
Gray solid (66% yield), mp 220–222 °C. 1H NMR (DMSO): d 4.39
(d, J = 4.3 Hz, 2H), 4.50 (s, 2H), 5.31 (s, 2H), 6.78 (d, J = 4.3 Hz, 2H),
7.03 (d, J = 4.3 Hz, 2H), 7.39 (t, J = 6.5 Hz, 2H), 7.29–7.48 (m, 4H),
7.70 (t, J = 6.5 Hz, 1H), 7.89 (d, J = 4.3 Hz, 1H), 7.98 (d, J = 4.3 Hz,
2H), 8.79 (t, J = 6.5 Hz, 1H), 10.56 (s, 1H). HRMS (ESI): calcd for
To a solution of compound 1 (6.27 g; 30 mmol) in dry THF
(60 mL) was added portion wise a suspension of carbonyldiimidaz-
ole (4.86 g; 30 mmol) in THF (15 mL) and the mixture was stirred
at rt for 20 min and then refluxed for 10 min. A solution of com-
pound 5 (4.41 g; 30 mmol) in THF (15 mL) was added and the reac-
tion mixture was stirred at rt overnight. A white precipitate formed
which was collected by suction filtration (4.46 g; 44% yield), mp
173–175 °C. 1H NMR (CDCl3): d 2.40–2.60 (m, 1H), 4.60 (s, 2H),
4.65 (s, 2H), 6.30–6.42 (m, 1H), 6.75 (d, J = 5.5 Hz, 1H), 7.00–7.30
(m, 2H), 7.45 (t, J = 7.3 Hz, 2H), 7.60 (d, J = 3.6 Hz, 1H), 7.70 (d,
J = 3.6 Hz, 1H), 8.10 (d, J = 3.6 Hz, 1H), 8.63 (s, 1H).
C
26H22N6O4S [M+Na]+ 537.1321; found 537.1355.
3.5.14. N-((1-(2-Oxo-2-(2-phenoxyphenylamino)ethyl)-1H-1,2,
3-triazol-4-yl)methyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamide (4n)
Gray solid (77% yield), mp 213–215 °C. 1H NMR (DMSO): d 4.38
(d, J = 4.3 Hz, 2H), 4.50 (s, 2H), 5.39 (s, 2H), 6.87 (m, 1H), 7.02–7.21
(m, 6H), 7.40–7.46 (m, 3H), 7.70 (t, J = 6.9 Hz, 1H), 7.85–8.05 (m,
3H), 8.79 (t, J = 5.2 Hz, 1H), 10.09 (s, 1H). HRMS (ESI): calcd for
26H22N6O4S [M+Na]+ 537.1321; found 537.1339.
3.8. Representative synthesis of compounds 7a–p
C
3.8.1. N-(3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-
(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (7a)
3.5.15. tert-Butyl 2-(4-((2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamido)methyl)-1H-1,2,3-triazol-1-yl)acetate (4o)
White solid (80% yield), mp 177–179 °C. 1H NMR (CDCl3): d 4.52
(s, 2H), 4.59 (d, J = 3.4 Hz, 2H), 5.01 (s, 2H), 6.79 (s, 1H), 7.43 (t,
J = 6.8 Hz, 1H), 7.56–7.70 (m, 3H), 8.04 (d, J = 5.2 Hz, 1H). HRMS
(ESI): calcd for C18H21N5O4S [M+H]+ 404.1393; found 404.1391.
A solution of compound 6 (0.34 g; 1.0 mmol) and compound 3a
(0.33 g; 2.5 mmol) in tert-butyl alcohol and water (1:1) (20 mL
was treated with sodium ascorbate (0.20 g; 1.0 mmol) and cop-
per(II) sulfate pentahydrate (0.02 g; 0.10 mmol) and the reacting
mixture was stirred at room temperature overnight. TLC analysis
showed the presence of unreacted compound 6. An extra portion
of compound 3a (0.33 g; 2.5 mmol) was added and the reaction
mixture was stirred at 40 °C overnight. Water (35 mL) was added
and the resulting mixture was stirred for 5 min while kept in an
ice bath. A precipitate formed which was collected by vacuum
filtration and washed with water (25 mL) and diethyl ether
(100 mL), leaving compound 7a as a gray solid (0.33 g; 70% yield),
mp 185–187 °C. 1H NMR (CDCl3): d 4.60 (s, 2H), 5.18 (s, 2H), 5.50
(s, 2H), 6.70 (d, J = 5.5 Hz, 1H), 7.01 (d, J = 5.5 Hz, 1H), 7.15–7.40
(m, 5H), 7.41–7.72 (m, 6H), 8.09 (d, J = 5.5 Hz, 1H), 8.75 (s, 1H).
HRMS (ESI): calcd for C25H21N5O3S [M+Na]+ 494.1263; found
494.1258.
3.5.16. 2-(4-((2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)acetamido)-
methyl)-1H-1,2,3-triazol-1-yl)acetic acid (4p)
A solution of compound 4o (0.21 g; 0.5 mmol) in TFA (15 mL)
was allowed to stir at rt for 5 h while monitoring the disappear-
ance of the starting material by TLC. TFA was evaporated off and
the residue was treated with diethyl ether (75 mL) and stirred
for 30 min at rt. A white solid formed which was collected by suc-
tion filtration (0.13 g; 75% yield), mp 230–232 °C. 1H NMR (DMSO):
d 4.38 (d, J = 5.6 Hz, 2H), 4.50 (s, 2H), 5.25 (s, 2H), 7.43 (t, J = 5.4 Hz,
1H), 7.71 (t, J = 5.4 Hz, 1H), 7.83–8.00 (m, 3H), 8.79 (t, J = 5.4 Hz,
1H), 13.38 (s, 1H). HRMS (ESI): calcd for C14H14N5O4S [M+Na]+
348.0767; found 348.0779.

1220
K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
3.8.2. N-(3-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-
phenyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (7b)
Gray solid (94% yield), mp 185–187 °C. 1H NMR (CDCl3): d 4.60
(s, 2H), 5.18 (s, 2H), 5.49 (s, 2H), 6.71 (d, J = 6.3 Hz, 1H), 6.98–7.10
(m, 3H), 7.18–7.37 (m, 4H), 7.44 (t, J = 5. 6 Hz, 1H), 7.55–7.74 (m,
4H), 8.10 (d, J = 4.3 Hz, 1H), 8.63 (s, 1H). HRMS (ESI): calcd for
2H), 6.78 (d, J = 4.3 Hz, 1H), 6.89–7.00 (m, 3H), 7.12–7.38 (m,
5H), 7.42 (t, J = 6.5 Hz, 1H), 7.70 (t, J = 6.5 Hz, 1H), 7.90 (d,
J = 4.3 Hz, 1H), 8.00 (d, J = 4.3 Hz, 1H), 8.29 (s, 1H), 10.28 (s, 1H).
HRMS (ESI): calcd for
502.1547.
C
26H23N5O4S [M+H]+ 502.1549; found
C
25H20FN5O3S [M+Na]+ 512.1169; found 512.1190.
3.8.10. N-(3-((1-(2-Oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)-
methoxy)phenyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamide (7j)
3.8.3. N-(3-((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-
phenyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (7c)
Gray solid (93% yield), mp 198–200 °C. 1H NMR (CDCl3): d 4.60
(s, 2H), 5.20 (s, 2H), 5.52 (s, 2H), 6.72 (d, J = 5.5 Hz, 1H), 6.95–7.10
(m, 4H), 7.19–7.40 (m, 3H), 7.45 (t, J = 7.4 Hz, 1H), 7.60 (d,
J = 5.5 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 8.10 (d, J = 5.5 Hz, 1H), 8.62
(s, 1H). HRMS (ESI): calcd for C25H20FN5O3S [M+H]+ 490.1349;
found 490.1345.
Gray solid (76% yield), mp 165–167 °C. 1H NMR (CDCl3): d 4.60
(s, 2H), 5.22 (s, 2H), 5.82 (s, 2H), 6.77 (d, J = 4.3 Hz, 1H), 7.02 (d,
J = 4.3 Hz, 1H), 7.15–7.35 (m, 3H), 7.40–7.70 (m, 6H), 7.80 (s, 1H),
8.00 (d, J = 4.3 Hz, 1H), 8.09 (d, J = 4.3 Hz, 1H), 8.60 (s, 1H). HRMS
(ESI): calcd for C26H21N5O4S [M+H]+ 500.1393; found 500.1377.
3.8.11. N-(3-((1-(2-Oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-
4-yl)methoxy)phenyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamide (7k)
3.8.4. N-(3-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-
phenyl)-2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetamide (7d)
White solid (68% yield), mp 163–165 °C. 1H NMR (CDCl3): d 3.80
(s, 3H), 4.59 (s, 2H), 5.17 (s, 2H), 5.45 (s, 2H), 6.70 (d, J = 5.4 Hz, 1H),
6.92 (d, J = 5.4 Hz, 2H), 7.00 (d, J = 3.6 Hz, 1H), 7.18–7.35 (m, 4H),
7.45–7.55 (m, 2H), 7.60 (d, J = 3.6 Hz, 1H), 7.68 (d, J = 3.6 Hz, 1H),
8.10 (d, J = 5.4 Hz, 1H), 8.60 (s, 1H). HRMS (ESI): calcd for
White solid (74% yield), mp 205–207 °C. 1H NMR (DMSO): d
4.66 (s, 2H), 5.12 (s, 2H), 5.38 (s, 2H), 6.80 (d, J = 4.2 Hz, 1H),
7.02–7.39 (m, 7H), 7.41 (t, J = 6.5 Hz, 1H), 7.59 (d, J = 4.2 Hz, 2H),
7.69 (t, J = 6.5 Hz, 1H), 7.90 (d, J = 4.2 Hz, 1 h), 8.00 (d, J = 4.2 Hz,
1H), 8.22 (s, 1H), 10.38 (s, 1H). HRMS (ESI): calcd for C26H22N6O4S
[M+H]+ 515.1502; found 515.1505.
C
26H23N5O4S [M+H]+ 502.1549; found 502.1526.
3.8.12. N-(3-((1-(2-Oxo-2-(4-phenoxyphenylamino)ethyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)-2-(3-oxobenzo[d]isothiazol-2-
(3H)-yl)acetamide (7l)
3.8.5. Methyl 4-((4-((3-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-
benzoate (7e)
Brown solid (90% yield), mp 198–200 °C. 1H NMR (DMSO): d
4.66 (s, 2H), 5.13 (s, 2H), 5.36 (s, 2H), 6.80 (d, J = 4.3 Hz, 1H),
6.92–7.03 (m, 2H), 7.08–7.30 (m, 4H), 7.32–7.49 (m, 5H), 7.60 (d,
J = 6.3 Hz, 1H), 7.70 (t, J = 6.5 Hz, 1H), 7.91 (d, J = 4.3 Hz, 1H), 8.00
(d, J = 4.3 Hz, 1H), 8.24 (s, 1H), 10.39 (s, 1H). HRMS (ESI): calcd
for C32H26N6O5S [M+Na]+ 629.1583; found 629.1583.
White solid (15% yield), mp 98–100 °C. 1H NMR (CDCl3): d 3.95
(s, 3H), 4.60 (s, 2H), 5.19 (s, 2H), 5.59 (s, 2H), 6.70 (d, J = 5.6 Hz, 1H),
7.00 (d, J = 5.6 Hz, 1H), 7.10–7.40 (m, 3H), 7.40–7.72 (m, 5H), 8.00–
8.20 (m, 3H), 8.61 (s, 1H). HRMS (ESI): calcd for C27H23N5O5S
[M+Na]+ 552.1318; found 552.1302.
3.8.6. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(3-((1-(phenylthio-
methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetamide (7f)
White solid (60% yield), mp 158–160 °C. 1H NMR (CDCl3): d
4.60 (s, 2H), 5.18 (s, 2H), 5.60 (s, 2H), 6.71 (d, J = 5.6 Hz, 1H),
7.01 (d, J = 5.6 Hz, 1H), 7.20–7.39 (m, 5H), 7.48 (t, J = 7.7 Hz,
1H), 7.60–7.75 (m, 4H), 8.11 (d, J = 3.7 Hz, 1H), 8.62 (s, 2H).
HRMS (ESI): calcd for C25H21N5O3S2 [M+Na]+ 526.0984; found
526.0969.
3.8.13. N-(3-((1-(2-Oxo-2-(3-phenoxyphenylamino)ethyl)-1H-1,
2,3-triazol-4-yl)methoxy)phenyl)-2-(3-oxobenzo[d]isothiazol-
2(3H)-yl)acetamide (7m)
Gray solid (22% yield), mp 230–232 °C. 1H NMR (DMSO): d 4.65
(s, 2H), 5.11 (s, 2H), 5.30 (s, 2H), 6.78 (t, J = 10.6 Hz, 2H), 7.02 (d,
J = 6.7 Hz, 1H), 7.17 (d, J = 6.7 Hz, 1H), 7.20–7.44 (m, 9H), 7.70 (t,
J = 8.5 Hz, 1H), 7.91 (d, J = 4.3 Hz, 1H), 8.00 (d, J = 4.3 Hz, 1H),
8.20 (s, 1H), 10.33 (s, 1H), 10.52 (s, 1H). HRMS (ESI): calcd for
C
32H26N6O5S [M+Na]+ 629.1583; found 629.1586.
3.8.7. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(3-((1-(3-phenyl-
propyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetamide (7g)
White solid (40% yield), mp 169–171 °C. 1H NMR (CDCl3): d
2.20–2.31(m, 2H), 2.63 (t, J = 8.9 Hz, 2H), 4.35 (t, J = 7.1 Hz, 2H),
4.59 (s, 2H), 5.20 (s, 2H), 6.75 (d, J = 3.6 Hz, 1H), 7.00 (d,
J = 3.6 Hz, 1H), 7.14–7.40 (m, 7H), 7.45 (t, J = 5.4 Hz, 1H), 7.58–
7.73 (m, 3H), 8.07 (d, J = 3.6 Hz, 1H), 8.60 (s, 1H). HRMS (ESI): calcd
for C27H25N5O3S [M+H]+ 500.1756; found 500.1748.
3.8.14. N-(3-((1-(2-Oxo-2-(2-phenoxyphenylamino)ethyl)-1H-1,
2,3-triazol-4-yl)methoxy)phenyl)-2-(3-oxobenzo[d]isothiazol-2-
(3H)-yl)acetamide (7n)
White solid (94% yield), mp 185–187 °C. 1H NMR (CDCl3): d 4.59
(s, 2H), 5.13 (s, 2H), 5.19 (s, 2H), 6.70 (d, J = 5.4 Hz, 1H), 6.80–6.95
(m, 3H), 6.95–7.38 (m, 7H), 7.45 (t, J = 5.4 Hz, 1H), 7.59–7.75 (m,
3H), 8.10 (m, 2H), 8.32 (d, J = 5.4 Hz, 1H), 8.61 (s, 1H). HRMS
(ESI): calcd for C32H26N6O5S [M+Na]+ 629.1583; found 629.1573.
3.8.8. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(3-((1-phenethyl-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetamide (7h)
3.8.15. tert-Butyl 2-(4-((3-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)-
acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetate (7o)
White solid (82% yield), mp 188–190 °C. 1H NMR (CDCl3): d 1.46
(s, 9H), 4.60 (s, 2H), 5.03 (s, 2H), 5.20 (s, 2H), 6.75 (d, J = 5.4 Hz, 1H),
7.00 (d, J = 5.4 Hz, 1H), 7.18–7.38 (m, 3H), 7.44 (t, J = 7.3 Hz, 1H),
7.58–7.75 (m, 2H), 8.10 (d, J = 5.4 Hz, 1H), 8.60 (s, 1H). HRMS
(ESI): calcd for C24H25N5O5S [M+H]+ 496.1655; found 496.1641.
White solid (52% yield), mp 175–177 °C. 1H NMR (CDCl3): d 3.20
(t, J = 8.9 Hz, 2H), 4.59 (t, J = 8.9 Hz, 2H), 4.60 (s, 2H), 5.16 (s, 2H),
6.69 (d, J = 5.4 Hz, 2H), 7.00 (d, J = 5.4 Hz, 1H), 7.03–7.40 (m,8H),
7.45 (t, J = 7.1 Hz, 1H), 7.60 (d, J = 3.6 Hz, 1H), 7.65 (d, J = 3.6 Hz,
1H), 8.09 (d, J = 3.6 Hz, 1H), 8.62 (s, 1H). HRMS (ESI): calcd for
C
26H23N5O3S [M+H]+ 486.1600; found 486.1597.
3.8.9. 2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)-N-(3-((1-(2-pheno-
xyethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetamide (7i)
Gray solid (96% yield), mp 163–165 °C. 1H NMR (DMSO-d6): d
4.39 (t, J = 4.4 Hz, 2H), 4.63 (s, 2H), 4.79 (t, J = 4.4 Hz, 2H), 5.10 (s,
3.8.16. 2-(4-((3-(2-(3-Oxobenzo[d]isothiazol-2(3H)-yl)acetamido)-
phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (7p)
Compound 7o (0.25 g; 0.5 mmol) was treated with TFA (15 mL)
and the solution was stirred at RT for 5 h. TFA was evaporated off

K.-C. Tiew et al. / Bioorg. Med. Chem. 20 (2012) 1213–1221
1221
and the residue was treated with diethyl ether (100 mL) and stir-
red at RT for 30 min. The solid was collected by suction filtration,
leaving a white powder (0.11 g; 50% yield), mp 115–117 °C. 1H
NMR (DMSO): d 4.65 (s, 2H), 5.11 (s, 2H), 5.30 (s, 2H), 6.79 (d,
J = 5.6 Hz, 1H), 7.15–7.28 (m, 2H), 7.35 (s, 1H), 7.44 (t, J = 5.6 Hz,
1H), 7.76 (t, J = 5.6 Hz, 1H), 7.90 (d, J = 4.2 Hz, 1H), 8.00 (d,
J = 4.2 Hz, 1H), 8.20 (s, 1H), 10.39 (s, 1H). HRMS (ESI): calcd for
presumed anionic aspartate and glutamate groups, and cationic
arginines and lysines).
Acknowledgment
The generous financial support of this work by the National
Institutes of Health (AI082068) is gratefully acknowledged.
C
20H17N5O5S [M+H]+ 440.1029; found 440.1024.
References and notes
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3.9. DENV2 NS2B-NS3pro expression and purification
The construction of the pQE30-NS2BH(QR)NS3pro expression
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expressed from Eschemrichia coli strain Top 10 F0 (Invitrogen)
transformed by pQE30-NS2BH(QR)-NS3pro plasmid and purified
as described.25 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).
6. Sampath, A.; Padmanabhan, R. Antiviral Res. 2009, 81, 6.
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3.10. WNV NS2B-NS3pro expression and purification
The expression and purification of the WNV NS2BH-NS3pro
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13. Aleshin, A. E.; Shiryaev, S. A.; Strongin, A. Y.; Liddington, R. C. Protein Sci. 2007,
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3.11. In vitro DENV2 and WNV NS2B-NS3pro assays and
inhibition studies
The compounds were dissolved in dimethylsulfoxide (DMSO) to
make 50 mM stock solutions. The compounds were screened at
25
says were performed in triplicates in Greiner Black 96 well plates.
Each assay consisted of the reaction mixture of 100 L containing
lM in 1% v/v DMSO in the final reaction mixture. Protease as-
l
200 mM Tris–HCl buffer, pH 9.5, 30% glycerol, 25 nM DENV2
NS2B-NS3 protease (or 28 nM WNV NS2BH-NS3pro) and the com-
pound. The enzyme and the compound were pre-incubated at
room temperature 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
was first determined. For determining IC50 values, the range of
10 nM, 50 nM, 0.1, 1, 2, 5, 10, and 25 M concentrations of selected
lM
l
compounds were used. IC50 values were calculated using the Sig-
maPlot software.
3.12. Molecular modeling
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2009), and the receptor was modeled based on the homology mod-
el generated for DENV NS3/NS2Bpro10 from the corresponding
WNV homolog (the protein protonation state used herein
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