Design, synthesis and evaluation of 1,2-benzisothiazol-3-one derivatives as potent caspase-3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.03.075

A number of 1,2-benzisothiazol-3-one derivatives were prepared through structural modification of the original compound from high-throughput screening. Some analogues (e.g., 6b, 6r, 6s and 6w) were identified as novel and potent caspase inhibitors with IC₅₀ of nanomolar. Structure-activity relationship (SAR) studies for caspase-3 inhibition were evaluated in vitro. Molecular modeling studies provided further insight into the interaction of this class of compounds with activated caspase-3. The present small molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.

Full text of DOI:10.1016/j.bmc.2013.03.075

Bioorganic & Medicinal Chemistry 21 (2013) 2960–2967
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of 1,2-benzisothiazol-3-one
derivatives as potent caspase-3 inhibitors
c,d
Dazhi Liu b, Zhen Tian b, Zhihui Yan a, Lixin Wu a, Yan Ma b, Quan Wang b, Wei Liu a, , Honggang Zhou
,
⇑
Cheng Yang c,d
a College of Sciences, Tianjin University of Science and Technology, Tianjin 300457, China
b College of Life Sciences, NanKai University, Tianjin 300071, China
c College of Pharmacy, NanKai University, Tianjin 300071, China
d High Throughput Molecular Drug Discovery Center, Tianjin International Joint Academy of Biotechnology and Medicine, TEDA, Tianjin 300457, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 1,2-benzisothiazol-3-one derivatives were prepared through structural modification of the
original compound from high-throughput screening. Some analogues (e.g., 6b, 6r, 6s and 6w) were iden-
tified as novel and potent caspase inhibitors with IC50 of nanomolar. Structure–activity relationship (SAR)
studies for caspase-3 inhibition were evaluated in vitro. Molecular modeling studies provided further
insight into the interaction of this class of compounds with activated caspase-3. The present small mol-
ecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors
revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-
regulated apoptosis.
Received 1 February 2013
Revised 13 March 2013
Accepted 24 March 2013
Available online 6 April 2013
Keywords:
Caspase-3
Inhibitor
1,2-Benzisothiazol-3-one
Apoptosis
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
as novel therapeutic targets for central nervous diseases in which
cell death occurs mainly by an apoptosis mechanism.
Apoptosis, or programmed cell death, plays an essential role in
all multi-cellular organisms. However, deregulation of the apopto-
tic process can contribute to many human diseases, including neu-
rodegenerative diseases, ischemic damage, autoimmune disorders,
and several forms of cancer.1 Cysteine-dependant aspartyl prote-
ases (caspases) have been implicated in the signal transduction
cascade leading to apoptosis. The 11 known human caspases can
be divided into three sub-types based on their structure and func-
tion.2,3 Group I caspases (1, 4, 5 and 13) are primarily involved in
inflammation.4 Group II caspases (2, 8, 9 and 10) are primary in-
volved in apoptosis as upstream regulators and Group III caspases
(3, 6 and 7) are the dominant effector caspases in apoptosis.5
Although various apoptotic signaling pathways have been dis-
covered, caspase-3 is activated in nearly every model of apoptosis
and is believed to be the ‘central executioner’ of the apoptotic
pathway.6 In pathological conditions, excessive neuronal apoptosis
leads to a variety of diseases such as stroke, Alzheimer’s disease,
Huntington’s disease and Parkinson’s disease.7,8 In consequence,
main apoptotic caspases, particularly caspase-3, are recognized
Hence, the inhibitors of caspases have been expected to be ben-
eficial pharmaceuticals for neurodegeneration. Although many
peptide and peptide mimetic inhibitors of caspases have been re-
ported,9–13 they have intrinsic limitations for therapeutic use be-
cause of their poor selectivity, cell-permeability, in vivo stability
and bioavailability.
Recently, a number of different nonpeptide inhibitors of cas-
pase-3 have been reported. One compound with isatin 1,2,3-tria-
zoles, have an medium to low nM IC50-values.14 Compounds with
either a pyrrolo[3,4-c]quinolone-1,3-dione or an isoquinoline-
1,3,4-trione moiety have also been reported to inhibit caspase-3
with IC50-values at low l
M to nM range.15 To find small-molecular
caspase-3 inhibitors with a novel scaffold directed against diseases
involving abnormally upregulated apoptosis, our work was initially
based on high-throughput screening of a diverse small-molecule li-
brary of 4000 compounds using recombinant human active cas-
pase-3. 1,2-Benzisothiazol-3-one 1, with an IC50 of 46.74 lM, was
O
O
NH
NH
⇑
Corresponding author. Tel.: +86 22 65378878.
E-mail address: liuwei2006@tust.edu.cn (W. Liu).
S
O
2
S
O
1
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.075
Figure 1. Structures of the hit compound 1 and its analogue 2 for the current study.

D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
2961
identified as a lead like hit (Fig. 1). To further research whether sul-
fur is necessary, we also explored commercially available saccharin
2 which is 1,2-benzisothiazol-3-one analogue with a sulfone group.
We found that the inhibitory activity of 2 to caspase-3 decreased to
110.5 lM inferior to 1,2-benzisothiazol-3-one 1. These results indi-
cated sulfur might be a necessary group for inhibitory activity of
displayed IC50 values in the
l
M range against caspase-3 (Table 1).
Such as compounds 3a and 3b, the replacement of the hydrogen
atom at the amide group of 1 with an alkyl group did not increase
in inhibitory potency. The compound 3b decreased inhibitory
activity compared with that of the initial compound 1. The intro-
duction of an allyl group 3c showed a higher potency inhibitory
activity relative to that of the initial compound 1. Compounds with
aryl groups 3d–f, benzyl groups 3g–i and phenethyl group 3j en-
hanced inhibitory activity compared with compound 1. Among of
these compounds, 3d and 3e had a nanomolar potency against cas-
pase-3 in vitro. Interestingly, extending the distance of N point and
aryl group led the inhibitory activity to downtrend. In addition,
compounds with substituted aromatic group 3e and substituted
benzyl groups 3h–i changed the activity little compared with
non-substituted aromatic group 3d and benzyl group 3g.
To further improve the inhibitory potency and determine the ef-
fect of the other substituted group at the N position. We replaced
the hydrogen atom at the amide group of 1 with a diverse series
of urea groups, including aliphatic acylamino, aromatic acylamino,
benzyl acylamino and phenylethyl acylamino groups. The inhibi-
tory activities of these compounds toward caspase-3 were summa-
rized in Table 2. The results showed that the introduction of aryl
acylamino groups 6b–l and 6n enhanced inhibitory activities com-
pared with that of the initial compound 1, and compound 6b had
IC50 values of 76 nM, which means its potency of inhibition was in-
creased about 1000-fold relative to that of the original compound
1. The introduction of an aromatic ring with an electron-donating
6c–e or electron-withdrawing group 6f–n decreased potency com-
pared to the corresponding nonsubstituted aniline 6b.
Compounds with benzyl acylamino 6o–v had a higher potency
of inhibition relative to that 1,2-benzisothiazol-3-one analogues
with aromatic acylamino. The introduction of a benzyl acylamino
with an electron-donating 6q–s increased potency of inhibition
compared to the corresponding compounds with electron-with-
drawing group 6t–v, and the result was more obvious when the
substitution was at the para position of benzylamine. Interestingly,
a higher caspase-3 potency was achieved upon replacing the ben-
zyl acylamino with a phenylethyl acylamino groups (6w). The cal-
culated logP values of the most of 1,2-benzisothiazol-3-one
analogues 3a–j and 6a–w were greater than or close to 3. There-
fore, these compounds may not have the drug’s ability to penetrate
the cell in vitro and label the target. Next work, it is necessary to
1,2-benzisothiazol-3-one 1 to caspase-3.
In this paper, we reported our discovery of a series of 1,2-ben-
zisothiazol-3-one derivatives as novel and potent inhibitors of cas-
pase-3 through structural modification based on screening hit 1. A
series of 1,2-benzisothiazol-3-one derivatives showed nanomolar
potency against caspase-3 in vitro.
2. Results and discussion
2.1. Chemistry
1,2-Benzisothiazol-3-one derivatives 3 and 6 were prepared
according to Scheme 1. Firstly, N-alkyl-benzisothiazol-3-one ana-
logues
3 were synthesized. Commercially available 1,2-ben-
zisothiazol-3-one 1 was alkylated by treatment with potassium
carbonate in tetrahydrofuran (THF) for the appropriate time, and
then the resulting salt reacted with an alkyl halide at ambient tem-
perature to obtain N-alkyl-benzisothiazol-3-one 3.16
A general approach to the preparation of 3-oxo-3H-benzo[d]iso-
thiazole-2-carboxylic acid amide analogues 6 were shown in
Scheme 1. The analogues 6 were prepared via the reaction of isocy-
anates 5, obtained by the reaction of a primary amine 4 with tri-
phosgene and triethylamine, with 1,2-benzisothiazol-3-one 1.
Yields for conversion of primary arylamines to isocyanates were
quantitative and yields for reactions of the isocyanates with 1,2-
benzisothiazol-3-one 1 were typically over 90%.17
2.2. Biological activity
1,2-Benzisothiazol-3-one 1 was obtained to confirm the scaffold
compound, and a series of derivatives were synthesized to evaluate
their potential in terms of modification of the initial compound, to
clarify their structure–activity relationships. Inhibition of recombi-
nant human caspase-3 by 1,2-benzisothiazol-3-one analogues 3
and 6 was assessed using a fluorimetric assay by measuring the
accumulation of a fluorogenic product, 7-amino-4-methycoumarin
(7-AMC). The tetrapeptidyl aldehyde Ac-DEVD-CHO was used as
the positive control.
Table 1
The N-alkyl-benzisothiazol-3-one inhibitors 3 synthesized were
examined for their ability to inhibit the activity of recombinant
human effector caspases-3. All N-alkyl-benzisothiazol-3-ones 3
Inhibitory activity of compounds 3 against human recombinant caspases 3
O
N
R1
S
3
Compound
R1
IC50
(l
M)
clog Pa
Ac-DEVD-CHO
1
0.0002 0.00001
46.74 2.63
42.39 3.03
91.24 16.48
22.72 1.18
0.5328 0.125
0.4657 0.0268
ND
10.80 3.01
12.09 0.18
13.64 1.11
18.55 1.33
H
CH3–
1.77
1.54
2.07
2.36
3.27
3.73
3.85
3.31
3.23
3.33
3.73
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3CH2–
CH2@CHCH2–
C6H5–
p-CH3-C6H5–
m-CF3-C6H5–
C6H5CH2–
p-CH3O-C6H5CH2–
p-F-C6H5CH2–
C6H5CH2CH2–
3j
ND = Not done.
a
clogP-values calculated with ACD/Chemsketch laboratories 12.0.
Scheme 1. Synthesis of substituted 1,2-benzisothiazol-3-ones.

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D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
Table 2
O
O
O
Inhibitory activity of compounds 6 against human recombinant caspases 3
O
N
N
HN
HN
S
O
O
S
N
HN
S
R2
6W
6O
6
Compound
R2
IC50
(lM)
clogPa
Ac-DEVD-CHO
0.0002 0.00001
ND
O
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
CH3CH2–
C6H5–
2.04
2.73
2.62
2.98
2.67
3.41
3.93
3.89
2.69
3.21
3.17
2.98
4.15
5.21
2.75
3.10
2.66
2.66
2.66
2.80
2.80
2.80
3.17
0.076 0.055
4.167 0.483
1.167 0.115
10.71 1.085
8.093 0.925
9.628 1.049
3.071 0.314
16.27 2.460
0.438 0.111
0.593 0.049
9.823 1.204
>100
22.050 1.880
0.086 0.011
0.085 0.095
0.088 0.002
0.074 0.008
0.040 0.001
0.206 0.018
0.241 0.008
0.115 0.007
0.031 0.004
o-CH3O-C6H4–
m-CH3O-C6H4–
p-CH3O-C6H4–
o-Br-C6H4–
m-Br-C6H4–
p-Br-C6H4–
o-F-C6H4–
m-F-C6H4–
p-F-C6H4–
o-NO2-C6H4–
p-I-C6H4–
O
O
N
S
O
N
N
co-crystallized inhibitor
Figure 2. Structures of the compounds 6o, 6w and co-crystallized inhibitor for the
docking study.
4-Cl-3-CF3-C6H4–
C6H5CH2–
C6H4CH(CH3)–
o-CH3O-C6H4CH2–
m-CH3O-C6H4CH2–
p-CH3O-C6H4CH2–
o-F-C6H4CH2–
m-F-C6H4CH2–
p-F-C6H4CH2–
C6H5CH2CH2–
better binding affinity than 6o (total_score 1.35). Judging from
what has been argued above, we have come to recognize that the
activity of the compounds can be improved to extend the distance
between the nitrogen atom and an aromatic ring and a better com-
bination of the compound with the protein pocket was obtained.
6s
6t
6u
6v
6w
3. Conclusion
ND = Not done.
a
clogP-values calculated with ACD/Chemsketch laboratories 12.0.
In summary, 1,2-benzisothiazol-3-one 1 was identified as a no-
vel and potent caspase-3 inhibitor by high-throughput screening.
We have completed the synthesis and in vitro evaluation of a series
of 1,2-benzisothiazol-3-one analogues having a high potency for
inhibiting the executioner caspase-3. The results of this study have
extended the structure–activity relationships of this class of com-
pounds and provide further insight into the development of non-
peptide-based inhibitors of caspase-3. Urea group at the N position
of compound 1 resulted in a higher activity compared with an alkyl
group or an aromatic group optimization and the activity have
been improved significantly, from the micromolar range to nano-
molar range. It was found that the activity can further be enhanced
to extend the distance between the urea group and an aromatic
ring. The docking studies also indicated that the activity of the
compounds could be improved to extend the distance between
the nitrogen atom and an aromatic ring and a better combination
of the compound with the protein pocket was obtained. Within
the active compounds, the best activity compound 6w presented
an IC50 value of 31 nM which is expected to be the new caspase-
3 inhibitor. Further work is in progress to optimize the structures
of compounds 6w with the aim of increasing its potency in both
the in vitro enzyme assay and cell assay.
improve the hydrophilicity of these compounds for their
druggability.
2.3. Docking studies
To get some insight to binding mode of 1,2-benzisothiazol-3-
one, compounds 6o and 6w (Fig. 2) were docked into the active site
of caspase-3. The Surflex-Dock program was used to predict the
binding modes of 6o and 6w to caspase-3. As shown in Figure 3,
the active site consists of S1, S2, S3, S4 four parts. Modelling of
co-crystallized inhibitor18 (IC50 = 7 nM) into the active site of cas-
pase-3 showed that the pyrimidine could be interacting with
H121, G122 amino acid residues in the S1 pocket and formed
two hydrogen bonds. The carbonyl and sulfuryl also could be inter-
acting with C163, R207 in the S2 pocket and developed two hydro-
gen bonds, respectively. The pyrrolyl and phenoxy of co-
crystallized inhibitor generated the hydrophobic interactions with
S3, S4 pockets of caspase-3. Similarly to co-crystallized inhibitor,
the carbonyl of benzoisothiazol-3-one part of 6w (IC50 = 31 nM)
molecule that could be generating two hydrogen bonds with
H121 and G122 in S1 pocket. The carbonyl of urea group also inter-
acted with H121 and formed one hydrogen bond in S2 pocket. The
benzene ring of 6w plunges into the hydrophobic S3 part that was
made of F256, Y204 and W206 and generates the hydrophobic
interactions, but there is no hydrophobic interactions between
6w and S4 part. Overall, co-crystallized inhibitor fitted well within
S1, S2, S3 and S4, and formed four hydrogen bonds, which could
provide higher potent inhibitory activity than 6w that can only
generat three hydrogen bonds and interactions with S1, S2, S3
pockets of caspase-3. Furthermore, compared with 6w, the 6o mol-
ecule is not enough fitted with the active pocket, especially in S1
and S3 part, which only forms a hydrogen bond with H121, mean-
while, the 6w molecule (total_score 2.82) is predicted to show the
4. Experimental
4.1. Chemistry: general procedures
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Melting points
were determined using an SGW-X4B digital melting point appara-
tus and were uncorrected. SHANGHAI SANPONT GF254 plates were
used as analytical TLC; flash column chromatography was per-
formed on SHANGHAI SANPONT Gel (200–300 mesh). 1H and 13C
NMR spectra were recorded on a 400 MHz or 600 MHz Bruker
Avance DPX spectrometers. All 13C NMR spectra were recorded

D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
2963
Figure 3. Caspase3-binding electrostatic potential groove (Left) and interactive map (Right) displaying the co-crystallized inhibitor (gray) and synthesized inhibitors 6o
(green) and 6w (violet). Hydrogen bonds are shown as yellow lines.
proton-decoupled. Chemical shifts for 1H and 13C spectra are
quoted in ppm downfield from TMS. Coupling constants are re-
ferred to as J values in hertz. ESI mass spectra were acquired using
a Bruker ESQUIRELC™ ESI ion trap spectrometer. FT-IR spectra
were recorded at room temperature in the region of 4000–
400 cmÀ1 with a PerkinElmer spectrum 65 FT-IR spectrometer
using KBr pellets.
1H), 7.34 (t, J = 8.0 Hz, 1H), 6.20–6.11 (m, 1H), 5.46 (d,
J = 16.0 Hz, 1H) 5.31 (d, J = 16.0 Hz, 1H), 5.05 (d, J = 4.0 Hz, 2H);
ESI-MS: m/z 192.12 ([M+H+]).
4.2.4. 2-Benzyl-2-hydrobenzo[d]isothiazol-3-one (3g)
Compound 3g was prepared according to standard procedure A
by using compound 1 and benzyl bromide, obtained a light yellow
solid in 60% yield. Mp 80.4–81.9 °C. 1H NMR (400 MHz, CDCl3, d
ppm) 8.03 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.44 (d,
J = 8.0 Hz, 1H), 7.42–7.31 (m, 6H), 5.01 (s, 2H); ESI-MS: m/z
241.97 ([M+H+]).
4.2. General procedure A: preparation of 2-alkyl-2-
hydrobenzo[d]isothiazol-3-one 3a–c, 3g–j
To a solution of 1,2-benzisothiazol-3-one 1 (755 mg, 5 mmol) in
10 ml of THF at 25 °C was added K2CO3 (1.75 g, 12.5 mmol), and
then R-X (10 mmol) was added. The reaction mixture was stirred
at room temperature for 2 h, removed of THF under vacuum, and
then diluted with 10 ml of ethyl acetate and 10 ml of H2O, and
the aqueous phase was extracted with 10 ml of ethyl acetate. The
combined organic phase was dried (MgSO4) and removed by rotary
evaporation, and the product was isolated by column chromatog-
raphy (ethyl acetate/petroleum ether, 1:1) to yield the final
compound.
4.2.5. 2-(4-Methoxybenzyl)-2-hydrobenzo[d]isothiazol-3-one
(3h)
Compound 3h was prepared according to standard procedure A
by using compound 1 and 4-methoxybenzyl chloride, obtained a
white solid in 42% yield. Mp 67.5–68.2 °C. 1H NMR (400 MHz,
CDCl3, d ppm) 8.06 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.47
(d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H),
6.88 (d, J = 8.0 Hz, 2H), 5.99 (s, 2H), 3.80 (s, 3H); ESI-MS: m/z
271.81 ([M+H+]).
4.2.1. 2-Methyl-2-hydrobenzo[d]isothiazol-3-one (3a)
Compound 3a was prepared according to standard procedure A
by using compound 1 and methyl iodide, obtained a dark green so-
lid in 50% yield. Mp 52.2–52.6 °C. 1H NMR (400 MHz, CDCl3, d ppm)
8.03 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz,
1H), 7.40 (t, J = 8.0 Hz, 1H), 3.44 (s, 3H); ESI-MS: m/z 166.11
([M+H+]).
4.2.6. 2-(4-Fluorobenzyl)-2-hydrobenzo[d]isothiazol-3-one (3i)
Compound 3i was prepared according to standard procedure A
by using compound 1 and 4-fluorobenzyl chloride, obtained a yel-
low solid in 35% yield. Mp 78.2–79.3 °C. 1H NMR (400 MHz, CDCl3,
d ppm) 8.07 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.51 (d,
J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 2H), 7.04
(t, J = 8.0 Hz, 2H), 5.03 (s, 2H); ESI-MS: m/z 260.18 ([M+H+]).
4.2.2. 2-Ethyl-2-hydrobenzo[d]isothiazol-3-one (3b)
Compound 3b was prepared according to standard procedure A
by using compound 1 and ethyl bromide, obtained a white solid in
45% yield. Mp 118.1–120.5 °C. 1H NMR (400 MHz, CDCl3, d ppm)
8.03 (d, J = 8.0 Hz, 1H), 7.62–7.54 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H),
3.95 (q, J = 8.0 Hz, 2H), 1.38 (t, J = 8.0 Hz, 3H); ESI-MS: m/z 180.22
([M+H+]).
4.2.7. 2-Phenethyl-2-hydrobenzo[d]isothiazol-3-one (3j)
Compound 3j was prepared according to standard procedure A
by using compound 1 and phenethyl bromide, obtained a light yel-
low solid in 55% yield. Mp 85.0–86.5 °C. 1H NMR (400 MHz, CDCl3,
d ppm) 8.02 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 8.0 Hz, 2H), 7.49 (d,
J = 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.29–7.22 (m, 5H), 4.12 (t,
J = 7.6 Hz, 2H), 3.06 (t, J = 7.6 Hz, 2H); ESI-MS: m/z 256.38 ([M+H+]).
4.2.3. 2-Allyl-2-hydrobenzo[d]isothiazol-3-one (3c)
4.3. General procedure B: preparation of 2-aryl-2-hydrobenzo[d
Compound 3c was prepared according to standard procedure A
by using compound 1 and allyl chloride, obtained a light yellow so-
lid in 51% yield. Mp 46.1–47.2 °C. 1H NMR (400 MHz, CDCl3, d ppm)
7.91 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.47 (t, J = 8.0 Hz,
]isothiazol-3-one 3d–f
To a crimp top microwave vial was added the benzoisothiazo-
lone 1 (76 mg, 0.5 mmol), aryl-X (1.05 mmol), K2CO3 (138 mg,

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D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
1.0 mmol), CuI (20 mol %), and DMEDA (20 mol %) in dioxane
(5 ml). The reaction mixture was heated in the microwave at
195 °C for 7 min. Following filtration and evaporation of solvents,
the products were isolated by column chromatography (ethyl ace-
tate/petroleum ether 1:5) to yield the final compounds.
120.6, 120.3. ESI-MS: m/z 271.40 ([M+H+]). IR (KBr, cmÀ1) 3087,
1725, 1651, 1615, 1594, 1295, 1206, 747.
4.4.3. N-(2-Methoxyphenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6c)
Compound 6c was prepared according to standard procedure C
by using compound 1 and o-anisidine, obtained a white solid in
98% yield. Mp 173.5–174.7 °C. 1H NMR (400 MHz, CDCl3, d, ppm):
11.20 (s, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.75
(t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 2H),
7.14 (t, J = 8.0 Hz, 1H), 7.04–6.96 (m, 1H), 4.02 (s, 3H). 13C NMR
(100 MHz, DMSO-d6, d ppm): 165.2, 148.7, 148.6, 141.2, 134.7,
127.2, 126.8, 126.7, 125.1, 124.9, 122.7, 121.2, 119.6, 111.7, 56.6.
ESI-MS: m/z 301.05 ([M+H+]). IR (KBr, cmÀ1) 3225, 1710, 1665,
1543, 1460, 1241, 1207, 736.
4.3.1. 2-Phenyl-2-hydrobenzo[d]isothiazol-3-one (3d)
Compound 3d was prepared according to standard procedure B
by using compound 1 and iodobenzene, obtained a white solid in
44% yield. Mp 138.5–140.3 °C. 1H NMR (400 MHz, DMSO-d6, d
ppm): 8.06 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.77 (t,
J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.56–7.50 (m, 3H), 7.38 (t,
J = 8.0 Hz, 1H); ESI-MS: m/z 227.82 ([M+H+]).
4.3.2. 2-(p-Tolyl)-2-hydrobenzo[d]isothiazol-3-one (3e)
Compound 3e was prepared according to standard procedure B
by using compound 1 and 4-iodotoluene, obtained a white solid in
43% yield. Mp 136.1–136.7 °C. 1H NMR (400 MHz, DMSO-d6, d
ppm): 8.05 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.76 (t,
J = 8.0 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.51 (t, J = 8.0 Hz, 1H), 7.34
(d, J = 8.0 Hz, 2H), 2.36 (s, 3H); ESI-MS: m/z 242.30 ([M+H+]).
4.4.4. N-(3-Methoxyphenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6d)
Compound 6d was prepared according to standard procedure
C by using compound 1 and m-anisidine, obtained a white solid
in 97% yield. Mp 165.7–167.3 °C. 1H NMR (400 MHz, CDCl3, d,
ppm): 11.08 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.74 (m, 1H), 7.63
(d, J = 8.0 Hz, 1H), 7.47 (m, 1H), 7.29 (m, 2H), 7.13 (m, 1H), 6.73
(m, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, DMSO-d6, d ppm):
165.3, 160.3, 148.8, 141.2, 138.4, 134.8, 130.5, 127.1, 126.7,
125.0, 122.7, 122.6, 110.6, 106.1, 55.7; ESI-MS: m/z 301.20
([M+H+]). IR (KBr, cmÀ1) 3182, 1714, 1601, 1555, 1214, 1156,
1050, 745.
4.3.3. 2-(3-(Trifluoromethyl)phenyl)-2-
hydrobenzo[d]isothiazol-3-one (3f)
Compound 3f was prepared according to standard procedure B
by using compound 1 and 3-Iodobenzotrifluoride, obtained a white
solid in 46% yield. Mp 134.5–135.7 °C. 1H NMR (400 MHz, DMSO-
d6, d ppm): 8.21 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.99–7.95 (m,
2H), 7.79 (t, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 1H), 7.52 (t,
J = 7.2 Hz, 1H). ESI-MS: m/z 296.17 ([M+H+]).
4.4.5. N-(4-Methoxyphenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6e)
4.4. General procedure C: preparation of N-alkyl-3-oxobenzo[d
]isothiazole-2(3H)-carboxamide analogues 6a–w
Compound 6e was prepared according to standard procedure C
by using compound 1 and p-anisidine, obtained a white solid in
99% yield. Mp 185.4–186.5 °C. 1H NMR (400 MHz, CDCl3, d ppm):
10.88 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 8.0 Hz, 1H), 7.62
(d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 8.0 Hz, 1H),
6.91 (d, J = 8.0 Hz, 2H), 3.82 (s, 3H); 13C NMR (100 MHz, DMSO-
d6, d ppm): 165.2, 156.7, 148.9, 141.2, 134.7, 130.1, 127.1, 126.7,
125.0, 122.7, 122.3, 114.8, 55.7. ESI-MS: m/z 301.22 ([M+H+]). IR
(KBr, cmÀ1) 3078, 1716, 1604, 1560, 1447, 1209, 1024, 746.
To a solution of triphosgene (2.96 g, 10 mmol) in DCM (20 ml)
was added dropwise to primary amine 4 (10 mmol) in DCM
(20 ml) followed by the dropwise addition of triethylamine
(3 ml) in DCM (10 ml). The solvent was removed on a rotary evap-
orator. The resulting residue was dissolved in DCM (20 ml), and
1,2-benzisothiazol-3-one (1.51 g, 10 mmol) in THF (20 ml) was
added. After the mixture was refluxed for 30 min, the solvent
was removed on a rotary evaporator. The residue was dissolved
in acetone (30 ml) and mixed with water (30 ml). The precipitate
was collected on a funnel by vacuum filtration and washed with
water–acetone (1:1, 4 Â 5 ml) to afford the final compound.
4.4.6. N-(2-Bromophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6f)
Compound 6f was prepared according to standard procedure C
by using compound 1 and o-bromaniline, obtained a white solid in
92% yield. Mp 197.3–197.8 °C. 1H NMR (400 MHz, DMSO-d6, d
ppm): 11.43 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.10–8.03 (m, 2H),
7.84 (m, 1H), 7.74 (dd, J = 8.0, 1.2 Hz, 1H), 7.53 (m, 1H), 7.44 (d,
J = 8.0 Hz, 1H), 7.15 (dd, J = 8.0, 1.2 Hz, 1H); 13C NMR (100 MHz,
CDCl3, d ppm): 165.2, 148.5, 140.9, 135.6, 134.3, 132.8, 128.3,
127.6, 126.1, 125.7, 124.7, 122.2, 120.5, 113.9. ESI-MS: m/z
348.82 ([M+H+]). IR (KBr, cmÀ1) 3104, 1705, 1543, 1440, 1211,
1179, 749, 730.
4.4.1. N-Ethyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide
(6a)
Compound 6a was prepared according to standard procedure C
by using compound 1 and ethylamine, obtained a white solid in
75% yield. Mp 123.1–124.5 °C. 1H NMR (400 MHz, CDCl3, d ppm)
8.84 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.58
(d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 3.51–3.48 (m, 2H), 1.28
(t, 3H); 13C NMR (100 MHz, CDCl3, d ppm): 165.1, 151.0, 140.8,
133.8, 127.2, 125.8, 125.1, 120.5, 35.6, 14.9. ESI-MS: m/z 223.12
([M+H+]). IR (KBr, cmÀ1) 3319, 2961, 1709, 1664, 1521, 1232, 745.
4.4.7. N-(3-Bromophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6g)
Compound 6g was prepared according to standard procedure C
by using compound 1 and m-bromaniline, obtained a white solid in
94% yield. Mp 217.9–220.5 °C. 1H NMR (400 MHz, CDCl3, d ppm):
11.10 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.74 (d,
J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.50 (dd, J = 14.4, 6.8 Hz,
2H), 7.30–7.23 (m, 2H); 13C NMR (100 MHz, CDCl3, d ppm):
165.4, 148.3, 140.8, 138.1, 134.3, 130.4, 127.8, 127.5, 126.1,
124.7, 123.2, 122.8, 120.6, 118.7. ESI-MS: m/z 349.02 ([M+H+]). IR
(KBr, cmÀ1) 3108, 3070, 1716, 1655, 1595, 1543, 1423, 1207,
1181, 769, 717.
4.4.2. N-Phenyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide
(6b)
Compound 6b was prepared according to standard procedure C
by using compound 1 and aniline, obtained a white solid in 95%
yield. Mp 186.3–188.5 °C. 1H NMR (400 MHz, CDCl3, d ppm)
11.04 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.64
(d, J = 8.0 Hz, 3H), 7.49 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 2H),
7.19 (t, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3, d ppm): 165.3,
148.4, 140.8, 136.8, 134.1, 129.2, 127.4, 126.0, 124.9, 124.8,

D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
2965
4.4.8. N-(4-Bromophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6h)
solid in 92% yield. Mp 222.9–225.1 °C. 1H NMR (400 MHz, CDCl3,
d ppm): 11.08 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz,
1H), 7.68 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.0 Hz, 1H), 7.47 (t,
J = 8.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, CDCl3,
d ppm): 165.3, 148.3, 140.8, 138.1 (C Â 2), 136.6, 134.3, 127.5,
126.1, 124.7, 122.0, 120.6, 88.1. ESI-MS: m/z 397.22 ([M+H+]). IR
(KBr, cmÀ1) 3062, 1710, 1545, 1298, 1000, 822, 737.
Compound 6h was prepared according to standard procedure C
by using compound 1 and p-bromaniline, obtained a white solid in
93% yield. Mp 232.6–234.8 °C. 1H NMR (400 MHz, CDCl3, d ppm):
11.09 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.62
(d, J = 8.0 Hz, 1H), 7.53–7.48 (m, 5H); 13C NMR (100 MHz, CDCl3,
d ppm): 165.4, 148.4, 140.8, 135.9, 134.3, 132.2, 127.5, 126.1,
124.7, 121.7, 120.6, 117.5. ESI-MS: m/z 348.94 ([M+H+]). IR (KBr,
cmÀ1) 3178, 3121, 3063, 1704, 1657, 1548, 1397, 1216, 824, 734.
4.4.14. N-(4-Chloro-3-(trifluoromethyl)phenyl)-3-
oxobenzo[d]isothiazole-2(3H)-carboxamide (6n)
Compound 6n was prepared according to standard procedure C
by using compound 1 and 4-chloro-3-(trifluoromethyl)aniline, ob-
tained a white solid in 97% yield. Mp 189.5–190.5 °C. 1H NMR
(400 MHz, CDCl3, d ppm): 11.27 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H),
7.99 (s, 1H), 7.79–7.75 (m, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.52–7.47
(m, 2H); 13C NMR (150 MHz, DMSO-d6, d ppm): 164.7, 152.3,
138.9, 131.9, 130.0, 126.6 (q, J = 30.0 Hz), 124.8, 124.6, 124.1,
124.0, 123.5, 122.8, 122.7 (q, J = 270.0 Hz), 121.4, 117.1 (q,
J = 6.0 Hz). ESI-MS: m/z 373.44 ([M+H+]). IR (KBr, cmÀ1) 3126,
1703, 1652, 1542, 1422, 1300, 1139, 744.
4.4.9. N-(2-Fluorophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6i)
Compound 6i was prepared according to standard procedure C
by using compound 1 and o-fluoroaniline, obtained a white solid in
98% yield. Mp 213.2–215.1 °C. 1H NMR (400 MHz, CDCl3, d ppm):
11.33 (s, 1H), 8.26 (t, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.74
(d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H),
7.18–7.12 (m, 3H). 13C NMR (100 MHz, CDCl3, d ppm): 165.4,
152.9 (J = 245 Hz), 148.3, 140.1, 134.3, 127.6, 126.1, 125.5 (d,
J = 11 Hz), 124.9 (d, J = 7 Hz), 124.7, 124.6 (t, J = 3 Hz), 121.7,
120.5, 115.2 (d, J = 19 Hz). ESI-MS: m/z 289.22 ([M+H+]). IR (KBr,
cmÀ1) 3069, 1721, 1556, 1447, 1300, 1214, 749.
4.4.15. N-Benzyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide
(6o)
Compound 6o was prepared according to standard procedure C
by using compound 1 and benzylamine, obtained a white solid in
98% yield. Mp 167.2–168.5 °C. 1H NMR (400 MHz, CDCl3, d ppm):
9.26 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.58
(d, J = 8.4 Hz, 1H), 7.42–7.36 (m, 6H), 4.64 (d, J = 5.6 Hz, 2H); 13C
NMR (100 MHz, CDCl3, d ppm): 165.2, 151.2, 140.9, 137.5, 133.9,
128.8, 127.7 (C Â 2), 127.3, 125.8, 125.0, 120.5, 44.6. ESI-MS: m/z
285.21 ([M+H+]). IR (KBr, cmÀ1) 3257, 3063, 1711, 1652, 1534,
1447, 1302, 741.
4.4.10. N-(3-Fluorophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6j)
Compound 6j was prepared according to standard procedure C
by using compound 1 and m-fluoroaniline, obtained a white solid
in 95% yield. Mp 189.5–190.3 °C. 1H NMR (400 MHz, CDCl3, d
ppm): 11.14 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 8.0 Hz,
1H), 7.63 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 10.8 Hz, 1H), 7.50 (t,
J = 8.0 Hz, 1H), 7.33–7.28 (m, 2H), 6.87 (t, J = 8.0 Hz, 1H). 13C
NMR (100 MHz, CDCl3, d ppm): 165.4, 163.1 (d, J = 244 Hz),
148.3, 140.8, 138.3 (d, J = 11 Hz), 134.3, 130.3 (d, J = 9 Hz), 127.5,
126.1, 124.7, 120.6, 115.5 (d, J = 3 Hz), 111.5 (d, J = 21 Hz), 107.7
(d, J = 27 Hz). ESI-MS: m/z 289.40 ([M+H+]). IR (KBr, cmÀ1) 3084,
1713, 1611, 1559, 1448, 1209, 745.
4.4.16. N-(a-Methylbenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6p)
Compound 6p was prepared according to standard procedure C
by using compound 1 and a-methylbenzylamine, obtained a white
solid in 99% yield. Mp 129.8–130.9 °C. 1H NMR (400 MHz, CDCl3, d
ppm): 9.29 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H),
7.57 (d, J = 8.0 Hz, 1H), 7.44–7.34 (m, 5H), 7.29 (d, J = 8.0 Hz, 1H),
5.19 (m, 1H), 1.63 (d, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3, d
ppm): 165.2, 150.3, 142.8, 140.9, 133.8, 128.7, 127.6, 127.2,
126.1, 125.8, 125.1, 120.5, 50.7, 22.8. ESI-MS: m/z 299.40
([M+H+]). IR (KBr, cmÀ1) 3289, 2980, 1705, 1662, 1517, 1298,
1184, 1099, 737.
4.4.11. N-(4-Fluorophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6k)
Compound 6k was prepared according to standard procedure C
by using compound 1 and p-fluoroaniline, obtained a white solid in
97% yield. Mp 187.7–188.8 °C. 1H NMR (400 MHz, CDCl3, d ppm):
11.01 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.63–
7.47 (m, 3H), 7.47 (t, J = 8.0 Hz, 1H), 7.07 (t, J = 8.0 Hz, 2H). 13C
NMR (100 MHz, CDCl3, d ppm): 165.3, 159.7 (d, J = 243 Hz),
148.5, 140.7, 134.2, 132.8 (d, J = 2 Hz), 127.4, 126.1, 124.7, 121.9
(d, J = 8 Hz), 120.5, 115.9 (d, J = 22 Hz). ESI-MS: m/z 289.25
([M+H+]). IR (KBr, cmÀ1) 3075, 1712, 1612, 1504, 1208, 839, 739.
4.4.17. N-(2-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6q)
Compound 6q was prepared according to standard procedure C
by using compound 1 and o-methoxybenzylamine, obtained a
white solid in 96% yield. Mp 126.5–128.3 °C. 1H NMR (400 MHz,
CDCl3, d ppm): 9.33 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.67 (t,
J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.34
(d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.92 (q, J = 8.0 Hz, 2H),
4.62 (d, J = 6.0 Hz, 2H), 3.92 (s, 3H); 13C NMR (100 MHz, CDCl3, d
ppm): 164.9, 157.6, 151.0, 140.9, 133.7, 129.6, 129.1, 127.2, 125.7
(C Â 2), 125.2, 120.6, 120.5, 110.4, 55.4, 44.5. ESI-MS: m/z 315.22
([M+H+]). IR (KBr, cmÀ1) 3307, 1707, 1658, 1520, 1249, 1028, 736.
4.4.12. N-(2-Nitrophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6l)
Compound 6l was prepared according to standard procedure C by
using compound 1 and o-nitroaniline, obtained a white solid in 85%
yield. Mp 227.3–228.9 °C. 1H NMR (400 MHz, CDCl3, d ppm): 12.84
(s, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.15 (d,
J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.61 (d,
J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H); 13C
NMR (100 MHz, CDCl3, d ppm): 165.0, 148.8, 140.8, 138.4, 135.2,
134.4, 133.0, 127.9, 126.2, 125.9, 124.6, 124.2, 123.4, 120.5. ESI-MS:
m/z316.25([M+H+]).IR(KBr,cmÀ1)3085,1715,1582,1504,1300,740.
4.4.18. N-(3-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6r)
Compound 6r was prepared according to standard procedure C
by using compound 1 and m-methoxybenzylamine, obtained a
white solid in 95% yield. Mp 137.5–139.1 °C. 1H NMR (400 MHz,
CDCl3, d ppm): 9.25 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.70 (t,
J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.27
4.4.13. N-(4-Iodophenyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6m)
Compound 6m was prepared according to standard procedure C
by using compound 1 and p-iodoaniline, obtained a light purple

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D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
(t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.91 (s, 1H), 6.84 (q,
J = 2.4 Hz, 1H), 4.62 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H); 13C NMR
(100 MHz, CDCl3, d ppm): 165.1, 159.9, 151.2, 140.9, 139.1, 133.9,
129.8, 127.3, 125.8, 125.0, 120.5, 119.9, 113.3, 113.2, 55.3, 44.5.
ESI-MS: m/z 315.15 ([M+H+]). IR (KBr, cmÀ1) 3247, 3011, 2934,
1705, 1654, 1547, 1272, 1152, 743.
solid in 95% yield. Mp 133.5–134.9 °C. 1H NMR (400 MHz, CDCl3,
d ppm): 8.93 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz,
1H), 7.57 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.33 (t,
J = 8.0 Hz, 2H), 7.27–7.23 (m, 3H), 3.71 (dd, J = 13.2, 7.2 Hz, 2H),
2.96 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3, d ppm): 165.0,
151.1, 140.9, 138.5, 133.8, 128.8, 128.7, 127.3, 126.7, 125.8,
125.1, 120.5, 42.1, 35.9. ESI-MS: m/z 299.10 ([M+H+]). IR (KBr,
cmÀ1) 3284, 2937, 1686, 1513, 1491, 1298, 1186, 738, 697.
4.4.19. N-(4-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6s)
Compound 6s was prepared according to standard procedure C
by using compound 1 and p-methoxybenzylamine, obtained a
white solid in 98% yield. Mp 153.1–154.7 °C. 1H NMR (400 MHz,
CDCl3, d ppm): 9.18 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.69 (t,
J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.30
(d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 4.57 (d, J = 5.6 Hz, 2H),
3.80 (s, 3H); 13C NMR (100 MHz, CDCl3, d ppm): 165.1, 159.2,
151.1, 140.9, 133.8, 129.6, 129.1, 127.2, 125.8, 125.0, 120.5,
4.5. Preparation of active caspase-3
Recombinant human caspase-3 was produced according to a
procedure published previously with minor modification.19,20
Briefly, caspase-3 was expressed and processed in Transetta
(DE3) cells harboring a pET-21d plasmid that contained the cloned
human caspase-3 cDNA. Fermentation was performed at 37 °C for
4–5 h in 800 ml LB media containing 100 lg/ml ampicillin until the
OD600 reached 0.6–0.8. Then, the culture was transferred to 25 °C,
114.2, 55.3, 44.1. ESI-MS: m/z 315.17 ([M+H+]). IR (KBr, cmÀ1
)
3266, 2942, 1701, 1510, 1304, 1241, 736.
and protein expression was induced for 3 h with 1.0 M isopropyl-
b-D-1-thiogalactopyranoside (IPTG). Harvested cells were resus-
4.4.20. N-(2-Fluorobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6t)
pended in lysis buffer containing 20 mM Tris–HCl (pH 8.0),
500 mM NaCl, and 5 mM imidazole and homogenized with a JN-
3000 PLUS low temperature ultra-high pressure cell disrupter
(JNBIO, Guangzhou). The lysate was centrifuged at 25,000g for
25 min at 4 °C to remove cell debris. The supernatant was then
loaded twice onto a Nickle-NTA agarose column pre-equilibrated
with lysis buffer. The caspase-3 was eluted with a gradient of 5–
300 mM imidazole in the above buffer. Fractions containing cas-
pase-3 were pooled and concentrated with a Millipore Ultrafree fil-
tration device, and then the caspase-3 was exchanged into a buffer
of 25 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM EDTA, 2 mM DTT.
The concentrated caspase-3 was loaded onto a Resource Q anion
exchange column (GE Healthcare) equilibrated with above buffer.
Fractions containing caspase-3 were pooled and concentrated.
The caspase-3 obtained with this method showed two subunits
of 17 and 12 kDa on 15% SDS–PAGE and aliquots stored at À80 °C.
Compound 6t was prepared according to standard procedure C
by using compound 1 and o-fluorobenzylamine, obtained a white
solid in 96% yield. Mp 184.2–185.1 °C. 1H NMR (400 MHz, CDCl3,
d ppm): 9.29 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz,
1H), 7.58 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 2H), 7.29 (m, 1H),
7.15–7.05 (m, 2H), 4.69 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3, d ppm): 165.2, 161.0 (d, J = 245 Hz), 151.2, 140.9, 133.9,
130.0 (d, J = 4 Hz), 129.6 (d, J = 8 Hz), 127.3, 125.9, 124.9, 124.6
(d, J = 15 Hz), 124.3 (d, J = 3 Hz), 120.5, 115.5 (d, J = 21 Hz), 38.6
(d, J = 5 Hz). ESI-MS: m/z 303.28 ([M+H+]). IR (KBr, cmÀ1) 3254,
1709, 1654, 1530, 1446, 1234, 1179, 743.
4.4.21. N-(3-Fluorobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6u)
Compound 6u was prepared according to standard procedure C
by using compound 1 and m-fluorobenzylamine, obtained a white
solid in 99% yield. Mp 157.2–158.3 °C. 1H NMR (400 MHz, CDCl3, d
ppm): 9.31 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H),
7.59 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.31 (dd, J = 14,
8 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.98 (td,
J = 8.0, 2.4 Hz, 1H), 4.63 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3, d ppm): 165.2, 163.0 (d, J = 245 Hz), 151.3, 140.9, 140.1 (d,
J = 7 Hz), 134.0, 130.3 (d, J = 8 Hz), 127.3, 125.9, 124.9, 123.2 (d,
J = 2 Hz), 120.5, 114.7 (d, J = 2 Hz), 114.5 (d, J = 3 Hz), 44.0 (d,
J = 2 Hz). ESI-MS: m/z 302.95 ([M+H+]). IR (KBr, cmÀ1) 3261,
3058, 2943, 1702, 1652, 1535, 1448, 1240, 740.
4.6. Caspase-3 inhibition assay
The test procedure to assess the inhibition of recombinant cas-
pase-3 activity of selected compounds was adapted from previ-
ously reported procedures.21,22 The typical assay of caspase-3
was carried out in a 100
7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% CHAPS, 20
Ac-LDEVD-AMC (GL Biochem, Shanghai) and 50 nM caspase-3 in
the presence of 1 l DMSO or 1 l inhibitor in DMSO. The proce-
ll system including 50 mM HEPES pH
l
M
l
l
dure was briefly described below. In room temperature, the cas-
pase-3 was incubated with inhibitors in 96-well plates for
10 min. Once added the substrate, enzymic cleavage between the
aspartate and the AMC fluorophore could liberate 7-amino-4-
tmethyl coumarin which was detected using an excitation wave-
length of 355 nm and an emission wavelength of 460 nm in Fluo-
roskan Ascent (Thermo). The AMC production was monitored
continuously for 10 min and the initial rate of hydrolysis was
determined using the early linear region of the enzymatic reaction
curve. For IC50 determination, about 15 concentrations of inhibitor
were freshly prepared by twofold gradient dilution with 95%
DMSO. After got the different initial rate according to this method,
we used the GraphPad Prism 5 software to calculate the IC50 value
of different inhibitors.
4.4.22. N-(4-Fluorobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6v)
Compound6vwaspreparedaccordingtostandardprocedureCby
using compound 1 and p-fluorobenzylamine, obtained a white solid
in 97% yield. Mp 171.0–172.9 °C. 1H NMR (400 MHz, CDCl3, d ppm)
9.26 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 7.59 (d,
J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.35 (dd, J = 8.0, 5.6 Hz, 2H),
7.04 (t, J = 8.0 Hz, 2H), 4.60 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz,
CDCl3, d ppm): 165.2, 162.3 (d, J = 244 Hz), 151.2, 140.9, 133.9,
133.9, 133.3 (d, J = 3 Hz), 129.5 (d, J = 8 Hz), 127.3, 125.9, 124.9,
120.5, 115.6 (d, J = 22 Hz), 43.8. ESI-MS: m/z 303.07 ([M+H+]). IR
(KBr, cmÀ1) 3267, 2942, 1704, 1638, 1531, 1308, 1218, 742.
4.7. Computational studies
4.4.23. N-Phenethyl-3-oxobenzo[d]isothiazole-2(3H)-
carboxamide (6w)
Compound 6w was prepared according to standard procedure C
by using compound 1 and 2-phenylethanamine, obtained a white
In order to predict the interactions and binding modes of 6o and
6w synthesized inhibitors in the caspase-3 active site, as well as
evaluate their relative binding affinities, the SYBYL-X 1.123

D. Liu et al. / Bioorg. Med. Chem. 21 (2013) 2960–2967
2967
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interface was used to perform the molecular docking studies. The
crystal structure of caspase-3 protein with small molecule was ac-
quired from the Protein Data Bank (PDB code 3H0E).18 As for the
preparation of the protein, the small molecule included in 3H0E
protein was extracted, hydrogen atoms were added and the dele-
tion of crystal waters using Prepare Protein Structure module with-
in Biopolymer package. To enhance the accuracy of docking, the co-
crystallized inhibitor was regarded as the reference molecule dur-
ing performing the docking.
Acknowledgments
This work was supported by Tianjin SME Technology Innovation
Fund (10ZCKFSY08300, 11ZXCXSY03500) and National Biomedical
Special Project of International Innovation Park (11ZCKFSY06800).
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2004, 47, 6455.
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M.; Ivachtchenko, A. V. Eur. J. Med. Chem. 2005, 40, 1377; (b) Kravchenko, D. V.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.075.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
16. Von Tacconi, G.; Righetti, P. P.; Desimoni, G. J. Prakt. Chem. (Leipzig) 1973, 315,
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18. Coordinates for the caspase-3 complex with co-crystallized inhibitor, have
been deposited in the Protein Data Bank under PDB ID code 3H0E, together
with the corresponding structure.
19. Ni, C.-Z.; Li, C. L.; Wu, J. C.; Spada, A. P.; Ely, K. R. J. Mol. Recognit. 2003, 16, 121.
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