Synthesis and pharmacological characterization of 2-aminobenzaldehyde oxime analogs as dual inhibitors of neutrophil elastase and proteinase 3

Article abstract of DOI:10.1016/j.bmc.2014.12.056

Proteinase 3 (Pr3), and human neutrophil elastase (HNE) are two major neutrophilic serine proteases (NsPs) expressed in neutrophil azurophil granules. Emerging data suggest that excessive release of proteases mediates tissue damage, and therefore prolonged neutrophil accumulation has an important role in the pathogenesis of many diseases. Thus, HNE and Pr3 inhibitors may prove to be targets for the generation of agents in the treatment of neutrophilic inflammatory disease. Sivelestat is the only commercially available selective HNE inhibitor. Therefore, sivelestat was chosen as the model structure in an attempt to obtain more potent anti-NsPs agents. In the present study, a series 2-aminobenzaldehyde oxime and 2-aminobenzoate analogs were synthesized and their inhibitory effects on NsPs (CatG, Pr3, and HNE) were determined, respectively. The results of structure-activity relationships studies concluded that a hydroxyl oxime moiety plays an important role in ligand-enzyme affinity through hydrogen bonding. As compound 6 had more potency and showed dual inhibitory effects on NE and Pr3, both in vitro and in vivo experiments were carried out to evaluate its selectivity, effects in cell-based assays, and efficacy in models of inflammation and damage. Compound 6 had the potential to reduce paw edema induced by LPS and HNE, as well as acute lung injury, and may be approved as a candidate for the development of new agents in the treatment of neutrophilic inflammatory diseases.

Full text of DOI:10.1016/j.bmc.2014.12.056

Bioorganic & Medicinal Chemistry 23 (2015) 1123–1134
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological characterization
of 2-aminobenzaldehyde oxime analogs as dual inhibitors
of neutrophil elastase and proteinase 3
Tsong-Long Hwang a,d,e, , Wen-Hui Wang a, , Ting-Yi Wang a, , Huang-Ping Yu b,c, Pei-Wen Hsieh a,d,e,
⇑
a Graduate Institute of Natural Products, School of Traditional Chinese Medicine, and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University,
Taoyuan 333, Taiwan
b Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
c School Medicine, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
d Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan, Taiwan
e Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteinase 3 (Pr3), and human neutrophil elastase (HNE) are two major neutrophilic serine proteases
(NsPs) expressed in neutrophil azurophil granules. Emerging data suggest that excessive release of pro-
teases mediates tissue damage, and therefore prolonged neutrophil accumulation has an important role
in the pathogenesis of many diseases. Thus, HNE and Pr3 inhibitors may prove to be targets for the gen-
eration of agents in the treatment of neutrophilic inflammatory disease. Sivelestat is the only commer-
cially available selective HNE inhibitor. Therefore, sivelestat was chosen as the model structure in an
attempt to obtain more potent anti-NsPs agents. In the present study, a series 2-aminobenzaldehyde
oxime and 2-aminobenzoate analogs were synthesized and their inhibitory effects on NsPs (CatG, Pr3,
and HNE) were determined, respectively. The results of structure–activity relationships studies con-
cluded that a hydroxyl oxime moiety plays an important role in ligand–enzyme affinity through hydro-
gen bonding. As compound 6 had more potency and showed dual inhibitory effects on NE and Pr3, both
in vitro and in vivo experiments were carried out to evaluate its selectivity, effects in cell-based assays,
and efficacy in models of inflammation and damage. Compound 6 had the potential to reduce paw edema
induced by LPS and HNE, as well as acute lung injury, and may be approved as a candidate for the devel-
opment of new agents in the treatment of neutrophilic inflammatory diseases.
Received 27 November 2014
Revised 22 December 2014
Accepted 23 December 2014
Available online 16 January 2015
Keywords:
2-Aminobenzaldehyde oxime analogs
Acute lung injury
Neutrophil elastase
Proteinase 3
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Serine proteases are the largest known family of protein-cleav-
ing enzymes, and modulate blood coagulation, apoptosis and
Neutrophils are a major type of blood leukocyte, and are essen-
tial for host defense against invading microorganisms.1 In addition,
neutrophils are involved in the activation, regulation and effector
function of innate and adaptive immune cells.2 In response to
microbial attack, neutrophils are activated and recruited to the
infection site, thereby releasing reactive oxygen species (ROS)
and serine proteases into the extracellular space.1,3 However,
emerging data suggest that oxidant stress and excessive release
of proteases mediate tissue damage. Thus, prolonged neutrophil
accumulation has an important role in the pathogenesis of chronic
inflammation, autoimmunity, and cancer.1–4
inflammation.5 Of these, cathepsin G (CatG), proteinase 3 (Pr3),
and human neutrophil elastase (HNE) are three major neutrophilic
serine proteases (NsPs) expressed in neutrophil azurophil gran-
ules.6,7 In particular, HNE and Pr3 are two major NsPs which play
crucial roles in antimicrobial defense with overlapping and poten-
tially redundant substrate specificity.6 However, recent reports
suggest that excess proteolysis which results from an influx of neu-
trophils into the airways is pivotal in the progressive destruction of
lung parenchyma, for example, acute lung injury (ALI), acute respi-
ratory distress syndrome (ARDS), and chronic obstructive pulmon-
ary disease (COPD).7–10 Therefore, HNE and Pr3 may prove to be
targets for the generation of agents in the treatment of neutrophilic
inflammatory disease.6,11
⇑
Corresponding author. Fax: +886 3 211 8643.
E-mail address: pewehs@mail.cgu.edu.tw (P.-W. Hsieh).
In 2000, an acetylating inhibitor, sivelestat, was developed for
the treatment of ALI in Japan, and is the only nonpeptidic inhibitor
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.12.056
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
hydrophobic and hydrogen-bonding interactions with HNE
through pivalate ester, sulfonamide, and glycine moieties,16 struc-
tural modification was carried out by changing the glycine moiety
to hydrogen, aliphatic chains, or esters, and replacing the anthrani-
late moiety with 2-aminobenzaldehyde oximes, as well as
exchanging pivalate ester with isostere. In the present study, we
report the synthesis of target components (Schemes 1–7), as well
as their structure–activity relationships (SARs) and both in vitro
and in vivo pharmacologic data.
2. Results and discussion
2.1. Effects of synthetics on neutrophil serine proteases
In attempt to investigate the SARs of the synthetics, the effects
of all the synthetics on neutrophil serine proteases (HNE, Pr3 and
CatG) were determined.9,12,17 Most of these compounds had a
potent and dual inhibitory effect on HNE and Pr3. Of these synthet-
ics, a new 2-aminobenzaldehyde oxime analog, compound 6,
exhibited the most potent inhibitory effects on the activity of
HNE and Pr3 with IC50 values of 0.05 and 0.22 lM, respectively,
and was slightly potent than sivelestat (Tables 1 and 2). Further
analysis of the SARs of the synthetics indicated that the activities
of the compounds with O-methyl oxime (9 and 10) were markedly
decreased compared to the compound with hydroxyl oxime (6),
indicating that hydrogen bonding interactions play an important
role in ligand–enzyme affinity. In 2012, Feng and his co-worker
showed that sivelestat was able to bind to the S1 pocket of HNE
through hydrophobic and hydrogen bonding interactions.16 In par-
ticular, sivelestat was able to form hydrogen bonding interactions
between the sulfonyl moiety and residues Gly 193 and Ser 195 of
HNE, as well as between residue Ser 214 of HNE and the amine
and carboxylic acid moieties of glycine in sivelestat.16 In present
study, the potential mode of binding of 6 and sivelestat to the
Scheme 1. Synthesis of Intermediates 4-(chlorosulfonyl)phenyl pivalate and 4-
pivalamidobenzene-1-sulfonyl chloride. Reagents and conditions: (a) 3,3-dimeth-
ylbutyryl chloride, MeCN, 0 °C, 12 h; (b) pivaloyl chloride, Et3N, 0 °C to rt, 2 h; (c)
chlorosulfonic acid, MeCN, 0 °C to 75 °C, 2 h.
marketed.12–14 However, the use of sivelestat was limited by its
poor pharmacokinetics and serious risks of organ toxicity because
it irreversibly inhibits HNE.15 In addition, the clinical trials of siv-
elestat in the western world were terminated due to a lack in proof
of therapeutic effect.12 Therefore, sivelestat was chosen as the
model structure in the present study in an attempt to obtain more
potent anti-neutrophil serine protease agents. As sivelestat has
Scheme 2. The route for the preparation of compounds 1-8 and 11. Reagents and conditions: (a) 4-(chlorosulfonyl)phenyl pivalate, pyridine, 4 h; (b) hydroxylamine
hydrochloride, EtOH, reflux, 14 h.
Scheme 3. The route for the synthesis of compounds 9 and 10. Reagents and conditions: (a) methoxylamine hydrochloride, EtOH, reflux, 12 h; (b) 4-(chlorosulfonyl)phenyl
pivalate, pyridine, 4 h.

T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
1125
Scheme 4. The route for the preparation of 2-benzamidobenzoate esters (12–17) Reagents and conditions: (a) methyl anthranilate, 1 M NaOH, dioxane, reflux, 12 h; (b) DCC,
DMAP, anhydrous methanol, 12 h; (c) pivaloyl chloride, Et3N/DCM (1:3), 2 h; (d) 3,3,3-trifluoro-2,2-dimethylpropionic acid, DMAP, EDC, DCM, 2 h.
Scheme 7. The route for the preparation of compounds 20 and 21. Reagents and
conditions: (a) 2 M NaOHaq, 12 h; (b) t-butylsulfinyl chloride, pyridine, 0 °C, 1 h.
was modified to the O-methyl oxime moiety and the affinity to
HNE was suppressed by blocking the role of the hydrogen bond
donor. Moreover, those with extending carbon chains (e.g., com-
pounds 2–4, 7, and 8), or replacing pivalate ester with its isosteres
(e.g., compounds 15–21) had significantly reduced effects on pro-
teases activities. Interestingly, the E form of the O-methyl oxime
Scheme 5. The route for the preparation of compound 18. Reagents and conditions:
(a) 2 M NaOH, reflux, 6 h; (b) DCC, DMAP, anhydrous methanol, 12 h; (c) 3,3,3-
trifluoro-2,2-dimethylpropionic acid, DMAP, EDC, DCM, 16 h.
derivative (9) had a more favorable effect on Pr3 than the Z form
(10).
2.2. Effects of synthetics on OÅ2ꢀ generation and elastase release
in human neutrophils
Since neutrophil respiratory burst and degranulation are impor-
tant in many inflammatory disorders, thus investigating the influ-
ence of synthetics on OÅ2ꢀ generation and elastase release in fMLF/
CB-induced human neutrophils was determined.18,19 The results
showed that, with the exception of compounds 6 and 8, most of
the synthetics selectively inhibited elastase release in fMLF/CB-
induced human neutrophils (Tables 3 and 4). Moreover, com-
pounds 2, 3, 5, 6, and 11 showed equal potency to that of sivelestat
in inhibiting neutrophil elastase release. The potency of com-
pounds 3 and 11 in cell-based assays was approximately 5-fold
greater than in enzyme-based assays, suggesting that these two
compounds may not only suppress HNE directly, but also indirectly
modulate HNE release through the signaling pathway.
Accordingly, the selectivity of compound 6 for HNE was con-
firmed by the cell-associated HNE assay, which showed that inhi-
bition of HNE release was greater than superoxide generation in
fMLF-induced human neutrophils. Moreover, the selectivity of
Scheme 6. The route for the preparation of compound 19. Reagents and conditions:
(a) 4-pivalamidobenzene-1-sulfonyl chloride, pyridine, 4 h.
HNE were carried out docking studies.16 As Figure S1, sivelestat
and 6 are able to form hydrogen bonding interactions through their
pivalate ester moiety and residues Gly 193 and Ser 195 of HNE, as
well as between residues Pro 98 of HNE and oxime group of 6 or
carboxylic acid moieties of glycine in sivelestat, respectively
(Fig. S1). Accordingly, the hydroxyl oxime moiety in compound 6
consisted of a hydrogen bond, while the hydroxyl oxime moiety

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T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
Table 1
Table 3
Neutrophil serine proteases (HNE, Pr3 and CatG) inhibitory activities of synthetics (1–
11)
Effects of synthetics (1–11) on OÅ2ꢀ generation and elastase release in fMLF-induced
human neutrophils
Synthetics
R1
R2
HNE
Pr3
CatG
Synthetics
R1
R2
OÅ2ꢀ generation
Elastase release
a
a
IC50
(lM)
IC50
(
lM)
1
2
3
4
5
6
7
O
O
O
O
CH3
0.11 0.01
0.19 0.01
0.37 0.01
0.47 0.03
1.09 0.08
0.36 0.03
2.31 0.10
2.05 0.05
0.22 0.02
2.50 0.12
1.73 0.03
0.61 0.03
2.37 0.11
0.53 0.03
0.34 0.03
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1
2
3
4
5
6
7
O
O
O
O
CH3
>20
>20
>20
>20
>20
19.61 3.49
>20
13.37 0.89
>20
0.09 0.02
0.04 0.01
0.05 0.02
0.11 0.03
0.05 0.01
0.03 0.01
0.11 0.03
0.13 0.03
0.17 0.02
0.18 0.03
0.08 0.02
0.05 0.02
CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
H
CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
H
2.24 0.17
0.12 0.04
0.05 0.01
0.68 0.03
0.36 0.02
0.32 0.04
0.37 0.03
0.39 0.02
0.07 0.00
O
O
N-OH
N-OH
N-OH
N-OCH3
N-OCH3
N-OH
O
CH3
N-OH
N-OH
N-OH
N-OCH3
N-OCH3
N-OH
O
CH3
CH2CH3
CH2CH2CH3
CH3
CH3
H
CH2CH3
CH2CH2CH3
CH3
CH3
H
8
8
9b
9b
10b
10b
>20
>20
>20
11
11
Sivelestatc
NHCH2COOH
Sivelestatc
NHCH2COOH
a
b
c
a
b
c
The IC50 values are presented as mean SEM (n = 3).
Compounds 9 and 10 are E/Z form isomers, respectively.
Sivelestat was used as positive control.
The IC50 values are presented as mean SEM (n = 3).
Compounds 9 and 10 are E/Z form isomers, respectively.
Sivelestat was used as positive control.
Table 2
Table 4
Neutrophil serine proteases (HNE, Pr3 and CatG) inhibitory activities of synthetics
(12–23)
Effects of synthetics (12–23) on OÅ2ꢀ generation and elastase release in fMLF-induced
human neutrophils
Synthetics R3
R4
X
Y
HNE
Pr3
CatG
Synthetics R3
R4
X
Y
OÅ2ꢀ
generation
Elastase
release
a
IC50 (lM)
a
IC50
(
l
M)
12
13
14
15
16
17
18
19
20
21
22
23
OCH3
OCH2CH3
OCH2CH2CH3 CH3 CO
OCH3
OCH2CH3
CH3 CO
CH3 CO
C
C
C
C
C
C
C
N
S
0.82 0.03 2.05 0.13 >10
0.48 0.03 3.02 0.07 >10
1.23 0.09 3.91 0.29 >10
12
13
14
15
16
17
18
19
20
21
22
23
OCH3
OCH2CH3
OCH2CH2CH3 CH3 CO
OCH3
OCH2CH3
CH3 CO
CH3 CO
C
C
C
C
C
C
C
N
S
>20
>20
>20
>20
>20
>20
3.69 0.12
>20
>20
>20
>20
>20
>20
0.55 0.11
0.30 0.02
0.62 0.16
>20
13.84 4.16
>20
CF3
CF3
CO
CO
CO
SO2
>10
>10
>10
>10
>10
>10
>10
>10
>10
CF3
CF3
CO
CO
CO
SO2
OCH2CH2CH3 CF3
OCH3
OCH3
CH3
H
CF3
3.69 0.12 4.94 0.36 >10
OCH2CH2CH3 CF3
CH3 SO2
CH3 SO2
CH3 SO2
>10
>10
>10
>10
>10
>10
>10
>10
>10
OCH3
OCH3
CH3
H
CF3
4.94 0.36
13.59 3.16
3.44 0.90
9.41 2.13
0.05 0.02
0.05 0.01
0.05 0.02
CH3 SO2
CH3 SO2
CH3 SO2
S
NH(CH2)2OH CH3 SO2
O(CH2)2OH CH3 SO2
C
C
C
0.13 0.01 0.31 0.02 >10
0.29 0.01 0.61 0.06 >10
0.07 0.00 0.34 0.03 >10
S
NH(CH2)2OH CH3 SO2
O(CH2)2OH CH3 SO2
C
C
C
Sivelestatb NHCH2COOH CH3 SO2
Sivelestatb NHCH2COOH CH3 SO2
a
The IC50 values are presented as mean SEM (n = 3).
b
a
Sivelestat was used as positive control.
The IC50 values are presented as mean SEM (n = 3).
Sivelestat was used as positive control.
b
sivelestat for Pr3 was better than compound 6, but the inhibitory
effect of compound 6 was more potent than sivelestat. To our
knowledge, Pr3 and CatG are the other serine proteases in neutro-
phils, and have an important role in neutrophilic inflammation by
2.3. Specificity and species crossover evaluations of compound 6
on serine proteases
multiple mechanisms often with significant overlap to HNE.17,20
A
To evaluate inhibitor specificity, we analyzed the effects of the
most potent compound, 6, on three other serine proteases, bovine
pancreatic trypsin, bovine plasmatic thrombin, and bovine pancre-
atic chemotrypsin. The results showed that compound 6 was at
least 11-fold more selective for HNE compared with the other ser-
ine proteases (Table 5). Compared with the positive HNE inhibitor,
compound 6 showed greater specificity than sivelestat for HNE
recent study indicated that active NsPs collectively caused more
severe lung damage and enhanced the activity of tissue destructive
proteases than HNE alone.21 Although HNE is a traditional thera-
peutic target in neutrophilic inflammation, these findings indicate
that this doctrine may no longer be valid; CatG and Pr3 are patho-
genic proteases that are associated with the initiation and/or chro-
nicity of lung inflammation and tissue injury.

T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
1127
Table 5
Potency and Specificity of compound 6 and sivelestat for HNE and other proteases
Compounds
6
Sivelestat
Serine proteases
a
IC50 (nM)/selective index (SI)
HNE
51.26 7.88/1
65.04 4.26/1
Pr3
CatG
Trypsin
Chemotrypsin
Thrombin
211.56 17.31/4.32
>10000/>195.08
>10000/>195.08
593.56 20.38/11.58
>10000/>195.08
338.22 28.35/5.20
>10000/>153.75
>10000/>153.75
1947.06 86.60/29.94
>10000/>153.75
a
The IC50 values are presented as mean SEM (n = 3).
Table 6
Potency of 6 for NE species crossover
Compounds
HNE
Mouse NE
Procine Pancreas NE
a
IC50 (nM)
6
51.26 7.88
65.04 4.26
190.75 11.69
145.39 17.41
1881.98 35.83
2185.17 34.02
Sivelestatb
a
The IC50 values are presented as mean SEM (n = 3).
Sivelestat was used as positive control.
b
over the other serine proteases, CatG, trypsin, and thrombin, and
exhibited more inhibitory effects on Pr3 and chymotrypsin than
sivelestat. Furthermore, compound 6 showed good crossover
potency for HNE from other species (Table 6).
2.4. Compound 6 attenuated HNE-induced paw edema in mice
To evaluate the in vivo activity of compound 6 against HNE, we
determined the effect of this compound on HNE-induced paw
edema in mice via intraperitoneal administration.22 HNE (5
lg/
site) was injected into the plantar region of the right hind paw
thereby causing notable foot pad swelling, which reached a maxi-
mum at 10 min, and then slowly decreased, but was still significant
at 4 h (Fig. 1A–C). Compared with the control group, intraperito-
neal administration of compound 6 (100 or 50 mg/kg) resulted in
a significant reduction in paw edema in a time- and dose-depen-
dent manner. It should be noted that a reduction in paw edema
was also observed in the sivelestat (100 mg/kg) treatment group;
however, this reduction was less than that with the same dosage
of compound 6.
Figure 1. Effects of 6 on human neutrophil elastase-induced paw edema in mice.
(A) Compound 6 and sivelestat (each 100 mg/kg body wt, ip), respectively; (B)
compound
6 (50 mg/kg body wt, ip) and sivelestat (100 mg/kg body wt, ip),
respectively; (C) compound 6 (50 mg/kg or 100 mg/kg body wt, ip, respectively).
Values are shown as the means SEM (n = 6). ⁄⁄⁄p <0.001, ⁄⁄p <0.01, ⁄p <0.05, ns:
p >0.05 compared to corresponding data.
2.5. Compound 6 diminished LPS-induced paw edema in mice
significantly augmented compared with the sham group after
intratracheal LPS administration (Fig. 3). In addition, both sivele-
stat and compound 6 significantly suppressed lung MPO activity
in ALI mice compared with the LPS-induced group (Fig. 3).
As compound 6 had a potent effect on HNE-induced paw edema
in mice, the effect of compound 6 on LPS-induced paw edema in
mice was also determined.23 Paw edema was induced by an
intraplantar injection of 25 ll (20 lg/site) LPS into the right hind
Neutrophils form the first line of defense during infection and
are essential in this function.5 In pathological conditions, neutro-
phils upregulate the release of proteases into the extracellular
space and cause host tissue damage.20,25 Many clinical studies
and animal models of ALI have shown that MPO overexpression,
leukocyte infiltration, lung edema, endothelial and epithelial injury
are accompanied by the invasion of neutrophils in the extracellular
and bronchoalveolar space, as well as by overexpression of neutro-
phil proteases. Thus, neutrophil proteases are considered a target
in the treatment of ALI.9,20,25 Herein, the protective effects of com-
pound 6 in lung injury following LPS treatment were examined.
The results showed no significant differences in MPO activity and
histological analysis between the compound 6-treated group and
the sham-treated animals. The administration of compound 6 or
paw, and paw swelling was measured at 10, 20, 40, 60, 120, and
240 min after LPS administration. The results indicated that edema
formation reached a maximum at 10 min, and decreased from 20
to 240 min (Fig. 2). Pretreatment with compound 6 (100 mg/kg)
significantly suppressed the edema induced by LPS, and the reduc-
tion in paw edema was less, but not significantly different to that
in mice pre-treated with sivelestat (100 mg/kg).
2.6. Compound 6 suppressed the increase of MPO activity in
lung tissue from mice with LPS-induced ALI
Myeloperoxidase (MPO) is an index of neutrophil infiltration,
and plays an important role in the development of ALI.24 The result
of this experiment showed that MPO level in lung tissue was

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T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
Figure 2. Effects of 6 on LPS-induced paw edema in mice. Compound 6 and sivelestat (each 100 mg/kg body wt, ip) were administered intraperitoneally, respectively. Values
are shown as the means SEM (n = 6). ⁄⁄⁄p <0.001, ⁄⁄p <0.01, ⁄p <0.05, ns: p >0.05 compared to corresponding data.
3. Conclusion
In conclusion, we synthesized twenty-three 2-aminobenzalde-
hyde oxime analogs and compared their inhibitory effects on NsPs
(CatG, Pr3, and HNE), respectively. The results of SARs studies con-
cluded that the hydroxyl oxime moiety was favorable for HNE and
Pr3 activity, and indicated that hydrogen bonding interactions play
an important role in ligand–enzyme affinity. As compound 6 had
significant potency and showed dual inhibitory effects on HNE
and Pr3, both in vitro and in vivo experiments were carried out
to evaluate its selectivity, effects in cell-based assays, and efficacy
in models of inflammation and damage. Particularly, compound 6
synthesized easier than sivelestat.26 In summary, compound 6
has the potential to reduce ALI and associated inflammatory disor-
ders, and may be approved as a candidate for the development of
new agents in the treatment of neutrophilic inflammatory
diseases.
Figure 3. Effects of 6 and sivelestat (each 100 mg/ kg) on MPO activity in lung
tissue from mice with LPS-induced ALI. Values are shown as the means SEM
(n = 6). ⁄⁄p <0.01 compared with the LPS-treated group.
4. Materials and methods
4.1. Chemicals and reagents
sivelestat (each 100 mg/kg of body weight) significantly attenu-
ated the increase in MPO activity, leukocyte infiltration, and lung
tissue destruction after administration of LPS (Figs. 3 and 4). These
data indicated that compound 6 had similar potency to sivelestat
and attenuated lung injury in LPS-induced ALI.
a
-Chymotrypsin from bovine pancreas, trypsin from bovine
pancreas, thrombin from bovine plasma, elastase from human leu-
kocytes, N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-methylcoumarin,
N -benzoyl-L-arginine-7-amido-4-methylcoumarin, porcine pan-
a
creas elastase, lipopolysaccharides (Escherichia coli 055:B5),
sodium azide, o-dianisidine dihydrochloride, phenol, and 2-amino-
acetophenone were purchased from Sigma–Aldrich Chemicals (St.
Louis, MO, USA). Human neutrophil elastase and Cathepsin G,
and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were provided by
Enzo (Farmingdale, NY, USA). Proteinase 3 (human neutrophils),
t-butyloxycarbonyl-Ala-Ala-Nva-thiobenzyl ester, benzoyl-Phe-
Val-Arg-7-amido-4-methylcoumarin hydrochloride, and meth-
oxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide were purchased from
Calbiochem (Darmstadt, Germany). Zoletil 50 was provided by Vir-
bac Laboratories (France). Reactions were detected by thin-layer
chromatography (TLC) using Merck 60 F254 silica gel aluminum
backed plates; spots were recorded under ultraviolet irradiation
(254 and 365 nm). Flash column chromatography was performed
using silica gel (Silicycle, 70–230 mesh or 230–400 mesh). The
nuclear magnetic resonance (NMR) spectra using CDCl3 as the
2.7. Compound 6 significantly attenuated histological changes
in lung tissue from mice with LPS-induced ALI
To evaluate histological changes in lung tissues after pretreat-
ment with sivelestat or compound 6 in LPS-treated mice, the ani-
mals were sacrificed 6 h after injection of LPS, and lung tissue
was subjected to H&E staining. As shown in Figure 4, lung sections
from the control and compound 6 (100 mg/kg) alone groups,
showed intact structure and clear pulmonary alveoli, suggested
compound 6 was safe at dosage of 100 mg/kg. Administration of
LPS resulted in diffuse edematous changes, alveolar thickening,
leukocyte infiltration, and lung tissue destruction (Fig. 4B). In con-
trast, the LPS-induced histological changes in lung were markedly
attenuated by treatment with sivelestat or compound 6 (each
100 mg/kg) (Fig. 4C, D and E).

T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
1129
Figure 4. Histological effects of compound 6 and sivelestat on lungs of rats after a sham operation (Sham) or LPS-treatment. Representative photomicrographs of lung of (A)
sham animals receiving vehicle; (B) LPS-treated animals receiving vehicle; (C and D) LPS-treated animals receiving 6 or sivelestat (each 100 mg/kg body wt, ip), respectively;
(E) sham animals receiving 6 (100 mg/kg body wt, ip) Tissue sections were stained with hematoxylin–eosin, examined at an original magnification of ꢁ400, and
photographed.
solvent were obtained on a Bruker AVANCE-400 MHz FT-NMR
spectrometer. Chemical shifts were internally referenced to the
solvent signals in tetramethylsilane (TMS). Low-resolution EI-MS
were recorded on a Quattro GC/MS spectrometer having a direct
inlet system, low-resolution and high-resolution ESI-MS spectra
on a Bruker Daltonics APEX II 30e spectrometer.
(9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d 202.5 (s, –COCH3–),
176.2 (s, –OCO–), 154.6 (s, C-10), 139.7 (s, C-1), 136.5 (s, C-40),
134.9 (d, C-5), 131.9 (d, C-3), 128.8 (d, C-30, 50), 123.0 (d, C-4),
122.7 (s, C-2), 122.2 (d, C-20, 60), 119.5 (d, C-6), 39.2 (s, C(CH3)3),
28.1 (q, COCH3), 27.0 (q, (CH3)3). ESI-MS (m/z, %): 373.8 [MꢀH]ꢀ
(100). HRESI-MS m/z 374.1057 [MꢀH]ꢀ (calcd for C19H20O5NS
374.1066).
4.1.1. General procedure for synthesis of 2-aminobenzaldehyde
analogs (1–5)
4.1.1.2. 4-(N-(2-Propionylphenyl)sulfamoyl)phenyl pivalate
(2).
Initially, phenol (10 mmol) was dissolved in acetonitrile. The
mixture solution was slowly added 3,3-dimethylbutyryl chloride
(1.5 equiv) at 0 °C, and then stirred at room temperature for 12 h.
The solvent was evaporated at reduced pressure. The residue was
purified by silica gel column chromatography using a mixture of
n-hexane and acetone to afford the phenyl pivalate. Subsequently,
phenyl pivalate was dissolved in acetonitrile. The mixture solution
was slowly added chlorosulfonic acid (1.5 equiv) at 0 °C and
reacted at 0 °C for 15 min, and then refluxed at 75 °C for 2 h. The
solutions were quenched by ice; the resulting mixtures were fil-
trated; and the residues was purified by silica gel column chroma-
tography using a mixture of n-hexane/ethyl acetate to afford
4-(chlorosulfonyl)phenyl pivalate (Scheme 1). To a mixture solu-
tion of 2-aminoacetophenone, 1-(2-aminophenyl)propan-1-one,
1-(2-aminophenyl)butan-1-one, 1-(2-aminophenyl)pentan-1-one,
or 2-aminobenzaldehyde (each 1.0 equiv) in pyridine, respectively,
was added 4-(chlorosulfonyl)phenyl pivalate, and then stirred at
room temperature for 4 h. The reaction mixture was concentrated
and purified by silica gel column chromatography using a mixture
of n-hexane/acetone solutions, to afford the products (Scheme 2,
1–5).
68% yield. White powder, mp 129–131 °C. 1H NMR
(400 MHz, CDCl3) d 11.45 (1H, s, NH), 7.80–7.84 (3H, m, H-30, 50,
3), 7.70 (1H, br dd, J = 1.6, 8.4 Hz, H-6), 7.44 (1H, td, J = 1.6,
8.4 Hz, H-5), 7.11 (2H, d, J = 8.8 Hz, H-20, 60), 7.08 (1H, td, J = 1.6,
8.4 Hz, H-4), 2.92 (2H, q, J = 7.2 Hz, CH2CH3), 1.31 (9H, s, (CH3)3),
1.12 (3H, t, J = 7.2 Hz, CH2CH3). 13C NMR (100 MHz, CDCl3) d
205.1 (s, –COCH2CH3–), 176.2 (s, –OCO–), 154.5 (s, C-10), 139.4 (s,
C-1), 136.5 (s, C-40), 134.5 (d, C-5), 130.9 (d, C-3), 128.7 (d, C-30,
50), 123.1 (d, C-4), 122.6 (s, C-2), 122.1 (d, C-20, 60), 119.7 (d, C-6),
39.1 (s, C(CH3)3), 32.8 (t, CH2CH3), 26.9 (q, (CH3)3), 8.1 (q, CH2CH3).
ESI-MS (m/z, %): 387.9 [MꢀH]ꢀ (100). HRESI-MS m/z 388.1213
[MꢀH]ꢀ (calcd for C20H22O5NS 388.1223).
4.1.1.3.
(3).
4-(N-(2-Butyrylphenyl)sulfamoyl)phenyl
pivalate
73% yield. White powder, mp 82–84 °C. 1H NMR
(400 MHz, CDCl3) d 11.46 (1H, s, NH), 7.82 (2H, d, J = 8.8 Hz, H-30,
50), 7.81 (1H, dd, J = 1.2, 8.4 Hz, H-3), 7.71 (1H, dd, J = 1.2, 8.4 Hz,
H-6), 7.45 (1H, td, J = 1.2, 8.4 Hz, H-5), 7.11 (2H, d, J = 8.8 Hz, H-
20, 60), 7.08 (1H, td, J = 1.2, 8.4 Hz, H-4), 2.86 (2H, t, J = 7.6 Hz,
CH2CH2CH3), 1.65 (2H, sext., J = 7.6 Hz, CH2CH2CH3), 1.32 (9H, s,
(CH3)3), 0.94 (3H, t, J = 7.6 Hz, CH2CH2CH3). 13C NMR (100 MHz,
CDCl3) d 204.7 (s, –COCH2CH3–), 176.2 (s, –OCO–), 154.5 (s, C-10),
139.6 (s, C-1), 136.6 (s, C-40), 134.6 (d, C-5), 131.0 (d, C-3), 128.8
(d, C-30, 50), 123.1 (d, C-4), 122.8 (s, C-2), 122.1 (d, C-20, 60), 119.9
(d, C-6), 41.5 (t, CH2CH2CH3), 39.1 (s, C(CH3)3), 26.9 (q, (CH3)3),
17.8 (t, CH2CH2CH3), 13.6 (q, CH2CH2CH3). HRESI-MS m/z
404.1526 [M+H]+ (calcd for C21H26O5NS 404.1533).
4.1.1.1.
(1).
4-(N-(2-Acetylphenyl)sulfamoyl)phenyl
pivalate
89% yield. White powder, mp 157–159 °C. 1H NMR
(400 MHz, CDCl3) d 11.45 (1H, s, NH), 7.86 (2H, d, J = 8.8 Hz, H-30,
50), 7.80 (1H, dd, J = 1.2, 8.4 Hz, H-3), 7.72 (1H, dd, J = 1.2, 8.4 Hz,
H-6), 7.48 (1H, td, J = 1.2, 8.4 Hz, H-5), 7.14 (2H, d, J = 8.4 Hz, H-
20, 60), 7.10 (1H, td, J = 1.2, 8.4 Hz, H-4), 2.56 (3H, s, CH3), 1.33

1130
T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
4.1.1.4. 4-(N-(2-Pentanoyl phenyl)sulfamoyl)phenyl pivalate
(4).
62% yield. White powder, mp 69–71 °C. 1H NMR
(1H, s, OH), 7.76 (2H, d, J = 8.8 Hz, H-20, 60), 7.67 (1H, dd, J = 1.2,
8.0 Hz, H-3), 7.37 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.27 (1H, td,
J = 1.2, 8.0 Hz, H-4), 7.10 (1H, td, J = 1.2, 8.0 Hz, H-5), 7.09 (2H, d,
J = 8.8 Hz, H-30, 50), 2.64 (2H, q, J = 7.6 Hz, CH2CH3), 1.32 (9H, s,
(CH3)3), 0.99 (3H, t, J = 7.6 Hz, CH2CH3). 13C NMR (100 MHz, CDCl3)
d 176.4 (s, –OCO–), 161.8 (s, C@N), 154.4 (s, C-40), 136.3 (s, C-10),
135.9 (s, C-2), 129.6 (d, C-4), 128.8 (d, C-20, 60), 128.3 (d, C-6),
124.4 (s, C-1), 123.6 (d, C-5), 122.0 (d, C-3, 30, 50), 39.2 (s,
C(CH3)3), 27.0 (q, (CH3)3), 19.5 (t, CH2CH3), 10.8 (q, CH2CH3). ESI-
MS (m/z, %): 405.2 [M+H]+ (100). HRESI-MS m/z 405.1500 [M+H]+
(calcd for C20H25O5N2S 405.1500).
(400 MHz, CDCl3) d 11.46 (1H, s, NH), 7.83 (2H, d, J = 8.8 Hz, H-30,
50), 7.82 (1H, dd, J = 1.2, 8.0 Hz, H-3), 7.72 (1H, dd, J = 1.6, 8.0 Hz,
H-6), 7.46 (1H, td, J = 1.2, 8.0 Hz, H-5), 7.12 (2H, d, J = 8.8 Hz, H-
20, 60), 7.09 (1H, td, J = 1.2, 8.0 Hz, H-4), 2.88 (2H, t, J = 7.2 Hz,
CH2CH2CH2CH3), 1.60 (2H, Quint., J = 7.6 Hz, CH2CH2CH2CH3),
1.34 (2H, sext., J = 7.6 Hz, CH2CH2CH2CH3), 1.33 (9H, s, (CH3)3),
0.93 (3H, t, J = 7.2 Hz, CH2CH2CH2CH3). 13C NMR (100 MHz, CDCl3)
d 204.9 (s, –COC–), 176.2 (s, –OCO–), 154.5 (s, C-10), 139.6 (s, C-1),
136.6 (s, C-40), 134.6 (d, C-5), 131.0 (d, C-36), 128.8 (d, C-30, 50),
123.1 (d, C-4), 122.8 (s, C-2), 122.1 (d, C-20, 60), 119.9 (d, C-6),
39.3 (t, CH2CH2 CH2CH3), 39.1 (s, C(CH3)3), 26.9 (q, (CH3)3), 26.5
(t, CH2CH2 CH2CH3), 22.2 (q, CH2 CH2CH2CH3), 13.8 (q, CH2CH2CH2CH3).
ESI-MS (m/z, %): 440.2 [M+Na]+ (100). HRESI-MS m/z 440.1502
[M+Na]+ (calcd for C22H27O5NNaS 440.1511).
4.1.2.3.
moyl)phenyl pivalate (8).
(E)-4-(N-(2-(1-(Hydroxyimino)butyl)phenyl)sulfa-
70% yield. White powder, mp
168–170 °C. 1H NMR (400 MHz, CDCl3) d 11.07 (1H, s, NH), 8.34
(1H, s, OH), 7.77 (2H, d, J = 8.8 Hz, H-20, 60), 7.68 (1H, dd, J = 1.2,
8.0 Hz, H-3), 7.36 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.27 (1H, td,
J = 1.2, 8.0 Hz, H-4), 7.10 (2H, d, J = 8.8 Hz, H-30, 50), 7.09 (1H, td,
J = 1.2, 8.0 Hz, H-5), 2.61 (2H, t, J = 7.6 Hz, CH2CH2CH3), 1.35 (2H,
sext., J = 7.6 Hz, CH2CH2CH3), 1.32 (9H, s, (CH3)3), 0.89 (3H, t,
J = 7.6 Hz, CH2CH2CH3). 13C NMR (100 MHz, CDCl3) d 176.3 (s, –
OCO–), 160.8 (s, C@N), 154.4 (s, C-40), 136.4 (s, C-10), 136.0 (s, C-
2), 129.6 (d, C-4), 128.7 (d, C-20, 60), 128.4 (d, C-6), 124.3 (s, C-1),
123.7 (d, C-5), 122.0 (d, C-30, 50), 121.8 (d, C-3), 39.2 (s, C(CH3)3),
27.9 (t, CH2CH2CH3), 27.0 (q, (CH3)3), 19.9 (t, CH2CH2CH3), 14.2
(q, CH2CH2CH3). ESI-MS (m/z, %): 419.2 [M+H]+ (100). HRESI-MS
m/z 419.1635 [M+H]+ (calcd for C21H27O5N2S 419.1643).
4.1.1.5.
(5).
4-(N-(2-Formylphenyl)sulfamoyl)phenyl
pivalate
64% yield. White powder, mp 121–123 °C. 1H NMR
(400 MHz, CDCl3) d 10.81 (1H, s, NH), 9.81 (1H, s, H), 7.90 (2H, d,
J = 8.8 Hz, H-20, 60), 7.69 (1H, br dd, J = 8.4 Hz, H-3), 7.60 (1H, dd,
J = 1.2, 8.4 Hz, H-6), 7.51 (1H, td, J = 1.2, 8.4 Hz, H-4), 7.17 (1H, td,
J = 1.2, 8.4 Hz, H-5), 7.16 (2H, d, J = 8.8 Hz, H-30, 50), 1.33 (9H, s,
(CH3)3). 13C NMR (100 MHz, CDCl3) d 195.0 (s, C@O), 176.1 (s, –
OCO–), 154.7 (s, C-40), 139.5 (s, C-2), 136.2 (s, C-10), 136.1 (d, C-
6), 135.8 (d, C-4), 128.8 (d, C-20, 60), 123.2 (d, C-5), 122.3 (s, C-30,
50), 121.9 (d, C-1), 117.7 (d, C-3), 39.1 (s, C(CH3)3), 26.9 (q,
(CH3)3). ESI-MS (m/z, %): 360.3 [MꢀH]ꢀ (100). HRESI-MS m/z
360.0900 [MꢀH]ꢀ (calcd for C18H18O5NS 360.0909).
4.1.2.4.
moyl)phenyl pivalate (9).
(E)-4-(N-(2-(1-(Methoxyimino)ethyl)phenyl)sulfa-
60% yield. White powder, mp 88–
4.1.2. General procedure for synthesis of 2-aminobenzaldehyde
oxime analogs (6–11)
90 °C. 1H NMR (400 MHz, CDCl3) d 10.77 (1H, s, NH), 7.69 (2H, d,
J = 8.8 Hz, H-20, 60), 7.65 (1H, br dd, J = 1.2, 8.4 Hz, H-3), 7.30 (1H,
br td, J = 1.2, 8.4 Hz, H-4), 7.27 (1H, d, J = 8.4 Hz, H-6), 7.11 (1H,
td, J = 1.2, 8.4 Hz, H-5), 7.08 (2H, d, J = 8.8 Hz, H-30, 50), 4.06 (3H,
s, OCH3), 2.02 (3H, s, CH3), 1.33 (9H, s, (CH3)3). 13C NMR
(100 MHz, CDCl3) d 176.2 (s, –OCO–), 156.1 (s, C@N), 154.4 (s, C-
40), 136.4 (s, C-10), 135.4 (s, C-2), 129.7 (d, C-6), 128.7 (d, C-20, 60),
128.5 (d, C-4), 125.0 (s, C-1), 124.5 (d, C-5), 122.3 (d, C-30, 50),
121.9 (d, C-3), 62.6 (q, OCH3), 39.2 (s, C(CH3)3), 27.0 (q, (CH3)3),
13.2 (q, CH3). ESI-MS (m/z, %): 403.4 [MꢀH]ꢀ (100). HRESI-MS m/
z 403.1322 [MꢀH]ꢀ (calcd for C20H23O5N2S 403.1331).
To a mixture solution of compounds 1–3 or 5 (each 1 equiv) in
ethanol, respectively, was added hydroxylamine hydrochloride
(1.5 equiv), and refluxed for 14 h.27 The reaction mixture was con-
centrated and purified by silica gel column chromatography using
a mixture of n-hexane/acetone (4:1 or 5:1) solutions, to afford
compounds 6–8, and 11 (Scheme 2).
To a mixture solution of 2-aminoacetophenone (1 equiv) in eth-
anol was added methoxylamine hydrochloride (1.5 equiv), and
refluxed for 12 h.27 The reaction mixture was concentrated, parti-
tioned in dichloromethane and water, and gave the 1-(2-amino-
phenyl)ethanone
O-methyl
oxime.
Subsequently,
1-(2-
4.1.2.5.
moyl)phenyl pivalate (10).
(Z)-4-(N-(2-(1-(Methoxyimino)ethyl)phenyl)sulfa-
2.5% yield. White powder, mp
aminophenyl)ethanone O-methyl oxime was dissolved in pyridine,
and added 4-(chlorosulfonyl)phenyl pivalate. The mixture solution
was stirred for 4 h at room temperature, and then purified by silica
gel column chromatography using a mixture of n-hexane/acetone
(4:1) solutions, to afford compounds 9 and 10 (Scheme 3).
146–148 °C. 1H NMR (400 MHz, CDCl3) d 7.66 (2H, d, J = 8.8 Hz,
H-20, 60), 7.64 (1H, br dd, J = 1.2, 7.6 Hz, H-3), 7.40 (1H, td, J = 1.2,
7.6 Hz, H-4), 7.23 (1H, td, J = 1.2, 7.6 Hz, H-5), 7.10 (2H, d,
J = 8.8 Hz, H-30, 50), 7.05 (1H, d, J = 1.2, 7.6 Hz, H-6), 3.91 (3H, s,
OCH3), 1.57 (3H, s, CH3), 1.36 (9H, s, (CH3)3). 13C NMR (100 MHz,
CDCl3) d 176.3 (s, –OCO–), 154.6 (s, C@N), 153.6 (s, C-40), 136.7
(s, C-10), 132.7 (s, C-2), 130.3 (d, C-1), 130.1 (s, C-6), 128.8 (d, C-
20, 60), 127.3 (d, C-4), 126.5 (d, C-3), 126.3 (d, C-3), 122.3 (d, C-30,
50), 62.1 (q, OCH3), 39.2 (s, C(CH3)3), 27.0 (q, (CH3)3), 21.3 (q,
CH3). ESI-MS (m/z, %): 427.1 [M+Na]+ (100). HRESI-MS m/z
427.1298 [M+Na]+ (calcd for C20H24O5N2SNa 427.1303).
4.1.2.1.
moyl)phenyl pivalate (6).
(E)-4-(N-(2-(1-(Hydroxyimino)ethyl)phenyl)sulfa-
27% yield. White powder, mp
166–168 °C. 1H NMR (400 MHz, CDCl3) d 10.86 (1H, s, NH), 8.51
(1H, s, OH), 7.73 (2H, d, J = 8.8 Hz, H-20, 60), 7.65 (1H, dd, J = 1.2,
8.0 Hz, H-3), 7.33 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.28 (1H, td,
J = 1.2, 8.0 Hz, H-4), 7.11 (1H, td, J = 1.2, 8.0 Hz, H-5), 7.09 (2H, d,
J = 8.8 Hz, H-30, 50), 2.05 (3H, s, CH3), 1.33 (9H, s, (CH3)3). 13C
NMR (100 MHz, CDCl3) d 176.9 (s, –OCO–), 157.3 (s, C@N), 154.9
(s, C-40), 136.5 (s, C-10), 135.7 (s, C-2), 130.0 (d, C-4), 129.2 (d, C-
20, 60), 129.0 (d, C-6), 125.9 (s, C-1), 125.1 (d, C-5), 122.9 (d, C-3),
122.5 (d, C-30, 50), 39.6 (s, C(CH3)3), 27.4 (q, (CH3)3), 12.8 (q, CH3).
ESI-MS (m/z, %): 390.9 [M+H]+ (100). HRESI-MS m/z 391.1322
[M+H]+ (calcd for C19H23O5N2S 391.1330).
4.1.2.6.
moyl)phenyl pivalate (11).
(E)-4-(N-(2-(1-(Hydroxyimino)methyl)phenyl)sulfa-
52% yield. White powder, mp
129–131 °C. 1H NMR (400 MHz, CDCl3) d 10.52 (1H, s, NH), 8.06
(1H, s, H), 7.87 (2H, d, J = 8.8 Hz, H-20, 60), 7.64 (1H, br dd,
J = 8.0 Hz, H-3), 7.26 (1H, td, J = 1.2, 8.0 Hz, H-4), 7.14 (2H, d,
J = 8.8 Hz, H-30, 50), 7.13 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.05 (1H, td,
J = 1.2, 8.0 Hz, H-5), 1.33 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3)
d 176.4 (s, –OCO–), 154.6 (s, C@N), 152.1 (s, C-40), 136.3 (s, C-10, 2),
132.1 (d, C-4), 130.5 (d, C-6), 128.9 (d, C-20, 60), 123.7 (d, C-5),
122.2 (d, C-30, 50), 119.5 (s, C-1), 119.1 (d, C-3), 39.2 (s, C(CH3)3),
4.1.2.2.
moyl)phenyl pivalate (7).
167–169 °C. 1H NMR (400 MHz, CDCl3) d 11.04 (1H, s, NH), 8.40
(E)-4-(N-(2-(1-(Hydroxyimino)propyl)phenyl)sulfa-
38% yield. White powder, mp

T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
1131
27.0 (q, (CH3)3). ESI-MS (m/z, %): 399.1 [M+Na]+ (100). HRESI-MS m/z
J = 7.2 Hz, OCH2CH2CH3), 1.37 (9H, s, (CH3)3), 1.06 (3H, t,
J = 7.2 Hz, OCH2CH2CH3). 13C NMR (100 MHz, CDCl3) d 176.6 (s, –
OCO–), 168.7 (s, –COO–), 164.8 (s, –CONH–), 154.0 (s, C-40), 141.8
(s, C-2), 134.7 (d, C-4), 132.2 (s, C-10), 130.8 (d, C-6), 128.8 (d, C-
20, 60), 122.6 (d, C-5), 121.9 (d, C-30, 50), 120.4 (d, C-3), 115.5 (s,
C-1), 67.1, (t, OCH2CH2CH3), 39.2 (s, C(CH3)3), 27.1 (q, (CH3)3),
21.9 (t, OCH2CH2CH3), 10.5 (q, OCH2CH2CH3). ESI-MS (m/z, %):
406 [M+Na]+ (100). HRESI-MS m/z 406.1625 [M+Na]+ (calcd for
399.0985 [M+Na]+ (calcd for C18H20N2O5SNa 399.0983).
4.1.3. General procedure for synthesis of 2-benzamidobenzoate
esters (12–17)
Initially, dissolved 4-(trifluoromethyl)phenol (1.0 equiv) and
methyl anthranilate (2.0 equiv) in dioxane (9 mL), and then 1 M
NaOH (4 equiv) was added. The reaction mixture was refluxed
for 12 h, and then removed dioxane in vacuum. The residue was
suspended in water, and adjusted pH value to 14, and then parti-
tioned in dichloromethane (DCM) and water. The water layer
was acidified to pH 1.0 with 1 M hydrochloric acid, and a precipi-
tate formed. The precipitate was collected by filtration affording
2-(4-hydroxybenzamido)benzoic acid.28 Secondarily, A dispersion
of 2-(4-hydroxybenzamido)benzoic acid (1 equiv) and N,N0-dicy-
clohexylcarbodiimide (DCC, 1 equiv) in anhydrous methanol
(10 ml) was stirred at room temperature for 1 h. 4-dimethylamino-
pyridine (DMAP, 0.5 equiv) was added and stirred at room temper-
ature for 12 h, and gave 2-(4-hydroxybenzamido)benzoate esters.
Finally, compounds 12–14 were synthesized through dissolving
corresponding 2-(4-hydroxybenzamido)benzoate esters, respec-
tively, in triethylamine/DCM (1:3) solution, and esterified through
reacting with pivaloyl chloride for 2 h, and then purified by silica
gel column chromatography using a mixture of n-hexane/ethyl
acetate (2:1) solutions (Scheme 4). On the other hand, compounds
15–17 were synthesized through dissolving 3,3,3-trifluoro-2,2-
dimethylpropionic acid and DMAP in DCM, and esterified through
reacting with N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC) and corresponding 2-(4-hydroxybenzamido)benzoate esters
for 2 h, and then purified by silica gel column chromatography
C22H25O5NNa 406.1633).
4.1.3.4. Methyl 2-(4-(3,3,3-trifluoro-2,2-dimethylpropanoyl-
oxy)benzamido)benzoate (15). 31% yield. White powder,
mp 63–65 °C. 1H NMR (400 MHz, CDCl3) d 12.08 (1H, s, NH), 8.91
(1H, d, J = 8.0 Hz, H-3), 8.11 (3H, m, H-6, 20, 60), 7.62 (1H, td,
J = 1.2, 8.0 Hz, H-4), 7.27 (2H, dd, J = 1.6, 7.2 Hz, H-30, 50), 7.14
(1H, td, J = 1.6, 8.0 Hz, H-5), 3.97 (1H, s, OCH3), 1.60 (6H, s,
(CH3)2). 13C NMR (100 MHz, CDCl3) d169.1 (s, –COO–), 168.6 (s, –
OCO–), 164.5 (s, –CONH–), 153.1 (s, C-40), 142.0 (s, C-2), 134.8 (d,
C-4), 133.0 (s, C-10), 130.9 (d, C-6), 129.0 (d, C-20, 60), 126.1 (s, JC–
F = 281 Hz, C-9), 122.0 (d, C-5), 121.6 (d, C-30, 50), 120.3 (d, C-3),
115.1 (s, C-1), 52.4 (q, OCH3), 49.0 (s, JC–F = 27 Hz, C-8), 19.6 (q,
JC–F = 2 Hz, C-10, 11). ESI-MS (m/z, %): 408 [MꢀH]ꢀ (100). HRESI-
MS m/z 432.1029 [M+Na]+ (calcd for C20H18O5NF3Na 432.1039).
4.1.3.5.
Ethyl
2-(4-(3,3,3-trifluoro-2,2-dimethylpropanoyl-
34% yield. White powder,
oxy)benzamido)benzoate (16).
mp 70–72 °C. 1H NMR (400 MHz, CDCl3) d 12.14 (1H, s, NH), 8.91
(1H, d, J = 8.0 Hz, H-3), 8.11 (3H, m, H-6, 20, 60), 7.61 (1H, td,
J = 1.6, 8.0 Hz, H-4), 7.26 (2H, br dd, J = 8.4 Hz, H-30, 50), 7.14 (1H,
td, J = 1.6, 8.0 Hz, H-5), 4.43 (2H, q, J = 7.2 Hz, OCH2CH3), 3.97
(1H, s, OCH3), 1.60 (6H, s, (CH3)2), 1.44 (3H, t, J = 7.2 Hz, OCH2CH3).
13C NMR (100 MHz, CDCl3) d 169.1 (2s, –COO–, –OCO–), 165.0 (s, –
CONH–), 153.5 (s, C-40), 142.2 (s, C-2), 135.1 (d, C-4), 133.4 (d, C-10),
131.4 (d, C-6), 129.4 (d, C-20, 60), 126.6 (s, JC–F = 281 Hz, C-9), 123.1
(d, C-5), 122.0 (d, C-30, 50), 120.8 (d, C-3), 115.1 (s, C-1), 62.0 (t,
OCH2CH3), 49.4 (s, JC–F = 53 Hz, C-8), 20.1 (q, JC–F = 2 Hz, C-10, 11),
14.6 (q, OCH2CH3). ESI-MS (m/z, %): 422 [MꢀH]ꢀ (100). HRESI-MS
m/z 422.1210 [MꢀH]ꢀ (calcd for C21H19O5NF3 422.1200).
using
a mixture of n-hexane/ethyl acetate (2:1) solutions
(Scheme 4).
4.1.3.1.
(12).
Methyl
2-(4-(pivaloyloxy)benzamido)benzoate
48% yield. White powder, mp 118–120 °C. 1H NMR
(400 MHz, CDCl3) d 12.04 (1H, s, NH), 8.91 (1H, d, J = 8.4 Hz, H-3),
8.09 (3H, m, H-6, 20, 60), 7.61 (1H, td, J = 1.2, 8.4 Hz, H-4), 7.22
(2H, dd, J = 1.6, 6.8 Hz, H-30, 50), 7.12 (1H, td, J = 1.2, 8.4 Hz, H-5),
3.96 (1H, s, CH3), 1.38 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3)
d 176.6 (s, –OCO–), 169.1 (s, –COO–), 164.8 (s, –CONH–), 154.1
(s, C-40), 141.8 (s, C-2), 134.8 (d, C-4), 132.2 (s, C-10), 130.9 (d, C-
6), 128.8 (d, C-20, 60), 122.6 (d, C-5), 121.9 (d, C-30, 50), 120.4 (d,
C-3), 115.2 (s, C-1), 52.5 (q, OCH3), 39.2 (s, C(CH3)3), 27.1 (q,
(CH3)3). ESI-MS (m/z, %): 378 [M+Na]+ (100). HRESI-MS m/z
378.1312 [M+Na]+ (calcd for C20H21O5NNa 378.1323).
4.1.3.6. Propyl 2-(4-(3,3,3-trifluoro-2,2-dimethylpropanoyl-
oxy)benzamido)benzoate (17).
41% yield. White powder,
mp 90–92 °C. 1H NMR (400 MHz, CDCl3) d 12.14 (1H, s, NH), 8.91
(1H, d, J = 7.6 Hz, H-3), 8.10–8.12 (3H, m, H-6, 20, 60), 7.61 (1H, td,
J = 1.6, 7.6 Hz, H-4), 7.25 (2H, dd, J = 8.8 Hz, H-30, 50), 7.14 (1H, td,
J = 1.6, 7.6 Hz, H-5), 4.32 (2H, t, J = 7.2 Hz, OCH2CH2CH3), 1.83
(3H, q, J = 7.2 Hz, OCH2CH2CH3), 1.60 (6H, (CH3)2), 1.06 (3H, t,
J = 7.2 Hz, OCH2CH2CH3). 13C NMR (100 MHz, CDCl3) d 168.7 (s, –
COO–), 168.6 (s, –OCO–), 164.5 (s, –CONH–), 153.0 (s, C-40), 141.7
(s, C-2), 134.7 (d, C-4), 133.0 (s, C-10), 130.8 (d, C-6), 129.0 (d, C-
20, 60), 126.1 (s, JC–F = 281 Hz, C-9), 122.7 (d, C-5), 121.6 (d, C-30,
50), 120.3 (d, C-3), 115.4 (s, C-1), 67.1 (t, OCH2CH2CH3), 49.0 (s,
JC–F = 26 Hz, C-8), 21.9 (t, OCH2CH2CH3), 19.6 (q, JC–F = 2 Hz, C-10,
11), 10.4 (q, OCH2CH2CH3). ESI-MS (m/z, %): 436 [MꢀH]ꢀ (100).
HRESI-MS m/z 436.1366 [MꢀH]ꢀ (calcd for C22H21O5NF3
436.1370).
4.1.3.2.
(13).
Ethyl
2-(4-(pivaloyloxy)benzamido)benzoate
61% yield. White powder, mp 121–123 °C. 1H NMR
(400 MHz, CDCl3) d 12.10 (1H, s, NH), 8.91 (1H, d, J = 8.4 Hz, H-3),
8.10 (3H, m, H-6, 20, 60), 7.60 (1H, td, J = 1.2, 8.4 Hz, H-4), 7.22
(2H, d, J = 8.4 Hz, H-30, 50), 7.12 (1H, t, J = 8.4 Hz, H-5), 4.42 (2H,
q, J = 7.2 Hz, OCH2CH3), 1.43 (3H, t, J = 7.2 Hz, OCH2CH3), 1.38
(9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d 176.6 (s, –OCO–),
168.6 (s, –COO–), 164.9 (s, –CONH–), 154.0 (s, C-40), 141.8 (s, C-
2), 134.7 (d, C-4), 132.3 (s, C-10), 130.9 (d, C-6), 128.9 (d, C-20, 60),
122.6 (d, C-5), 121.9 (d, C-30, 50), 120.4 (d, C-3), 115.5 (s, C-1),
61.54, (t, OCH2CH3), 39.2 (s, C(CH3)3), 27.1 (q, (CH3)3), 14.19 (q,
OCH2CH3). ESI-MS (m/z, %): 392 [M+Na]+ (100). HRESI-MS m/z
392.1468 [M+Na]+ (calcd for C21H23O5NNa 392.1472).
4.1.4. General procedure for synthesis of methyl 2-(4-(3,3,3-trifluoro-
2,2-dimethylpropanoyloxy)phenylsulfonamide)benzoate (18)
Initially, dissolved compound 13 (1.0 equiv) in 2 M NaOH solu-
tion (4 mL), and refluxed for 6 h, and then partitioned in chloro-
form and water. The organic layer was concentrated and purified
by silica gel column chromatography using a mixture of chloro-
form/methanol (5:1) solutions to yield 2-(4-hydroxyphenylsulfo-
namido)benzoate. 2-(4-Hydroxyphenylsulfonamido)benzoate was
further esterified by DMAP and DCC as described in Scheme 4,
and afford methyl 2-(4-hydroxyphenylsulfonamido)benzoate.
4.1.3.3.
(14).
Propyl
2-(4-(pivaloyloxy)benzamido)benzoate
60% yield. White powder, mp 84–86 °C. 1H NMR
(400 MHz, CDCl3) d 12.10 (1H, s, NH), 8.91 (1H, dd, J = 0.4, 8.0 Hz,
H-3), 8.09 (3H, m, H-6, 20, 60), 7.60 (1H, td, J = 1.2, 8.4 Hz, H-4),
7.22 (2H, dd, J = 2.0, 8.4 Hz, H-30, 50), 7.13 (1H, td, J = 1.2, 8.4 Hz,
H-5), 4.31 (2H, t, J = 7.2 Hz, OCH2CH2CH3), 1.84 (2H, sext.,

1132
T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
Finally, compound 18 were obtained through dissolving 3,3,3-tri-
fluoro-2,2-dimethylpropionic acid and DMAP in acetonitrile
(1 mL), and esterified through reacting with N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide (EDC) and methyl 2-(4-hydrox-
yphenylsulfonamido)benzoate for 16 h, and then purified by
silica gel column chromatography using a mixture of n-hexane/
acetone (4:1) solutions (Scheme 5).
4.1.6.1. 4-(N-(2-Acetylphenyl)sulfamoyl)phenyl 2-methylpro-
pane-2-sulfinate (20). 90% yield. White powder, mp 137–
139 °C. 1H NMR (400 MHz, CDCl3) d 11.48 (1H, s, NH), 7.83 (2H,
d, J = 8.8 Hz, H-20, 60), 7.81 (1H, dd, J = 1.2, 8.4 Hz, H-6), 7.70 (1H,
dd, J = 1.2, 8.4 Hz, H-3), 7.47 (1H, td, J = 1.2, 8.4 Hz, H-4), 7.19
(2H, d, J = 8.8 Hz, H-30, 50), 7.10 (1H, td, J = 1.2, 8.4 Hz, H-5), 2.57
(3H, s, CH3), 1.33 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d
202.5 (s, C@O), 158.1 (s, C-40), 139.7 (s, C-2), 135.8 (s, C-10), 135.0
(d, C-4), 132.0 (d, C-6), 129.4 (d, C-20, 60), 123.0 (d, C-5), 122.5 (s,
C-1), 120.0 (d, C-30, 50), 119.2 (d, C-3), 59.2 (s, C(CH3)3), 28.1 (q,
CH3), 21.6 (q, (CH3)3). ESI-MS (m/z, %): 418 [M+Na]+ (100). HRESI-
MS m/z 418.0753 [MꢀH]ꢀ (calcd for C18H2O5NS2Na 418.0753).
35% yield. White powder, mp <25 °C. 1H NMR (400 MHz, CDCl3)
d 10.66 (1H, s, NH), 7.93 (1H, dd, J = 1.6, 8.0 Hz, H-6), 7.88 (2H, d,
J = 8.8 Hz, H-20, 60), 7.70 (1H, br dd, J = 8.0 Hz, H-3), 7.47 (1H, td,
J = 1.6, 8.0 Hz, H-4), 7.17 (2H, d, J = 8.8 Hz, H-30, 50), 7.06 (1H, td,
J = 1.6, 8.0 Hz, H-5), 3.87 (3H, s, OCH3), 1.53 (6H, s, (CH3)2). 13C
NMR (100 MHz, CDCl3) d 168.7 (2s, –COO–, –OCO–), 154.0 (s, C-
4), 140.5 (s, C-2), 137.7 (s, C-10), 135.0 (d, C-4), 130.6 (d, C-6),
129.4 (d, C-20, 60), 127.8 (d, C-5), 126.4 (s, J = 281 Hz, C-9), 123.7
(d, C-5), 122.4 (d, C-30, 50), 119.7 (d, C-3), 116.6 (s, C-1), 52.9 (q,
OCH3), 49.4 (s, J = 27 Hz, C-8), 20.0 (q, C-10, 11). ESI-MS (m/z, %):
446 [M+H]+ (100). HRESI-MS m/z 446.0880 [M+H]+ (calcd for
C19H19O6NF3S 446.0887).
4.1.6.2. 4-(N-(2-Formylphenyl)sulfamoyl)phenyl 2-methylpro-
pane-2-sulfinate (21).
91% yield. White powder, mp 98–
100 °C. 1H NMR (400 MHz, CDCl3) d 10.81 (1H, s, NH), 9.84 (1H, s,
COH), 7.88 (2H, d, J = 8.8 Hz, H-30, 50), 7.69 (1H, br dd, J = 8.0 Hz,
H-3), 7.62 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.53 (1H, td, J = 1.2,
8.0 Hz, H-5), 7.21 (2H, d, J = 8.8 Hz, H-20, 60), 7.20 (1H, br t,
J = 8.8 Hz, H-4), 1.33 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d
195.1 (s, CHO), 158.3 (s, C-10), 139.6 (s, C-1), 136.2 (d, C-5), 135.9
(d, C-3), 135.5 (d, C-40), 129.4 (d, C-30, 50), 123.3 (d, C-4), 122.0 (s,
C-2), 120.0 (d, C-20, 60), 117.8 (d, C-6), 59.3 (s, C(CH3)3), 21.5 (q,
(CH3)3). ESI-MS (m/z, %): 404 [M+Na]+ (100). HRESI-MS m/z
404.0597 [M+Na]+ (calcd for C17H19O5NS2Na 404.0592).
4.1.5. General procedure for synthesis of methyl 2-(4-
pivalamidophenylsulfonamido)benzoate (19)
Initially, aniline (10 mmol) was dissolved in DCM. The mixture
solution was slowly added pivaloyl chloride (1.1 equiv) and trieth-
ylamine (2.0 equiv) at 0 °C, and then stirred at room temperature
for 2 h. The mixture was partitioned with DSM and water, and col-
lected organic layer to afford the N-phenylpivalamide. Subse-
quently, N-phenylpivalamide was dissolved in DCM (5 mL), and
chlorosulfonic acid (5 equiv) was slowly added at 0 °C, and then
refluxed for 2 h. The solutions were quenched by ice, the mixture
was partitioned with DSM and water, and collected organic layer
to afford 4-pivalamidobenzene-1-sulfonyl chloride (Scheme 1).
To a mixture solution of 4-pivalamidobenzene-1-sulfonyl chloride
(0.05 mmol) in pyridine (3 mL) was added methyl anthranilate
(0.45 mmol), and then stirred at room temperature for 4 h. The
reaction mixture was concentrated and purified by silica gel col-
umn chromatography using a mixture of n-hexane/acetone (4:1)
solutions, to afford compound 19 (35%, Scheme 6), white powder,
mp 125–127 °C. 1H NMR (400 MHz, CDCl3) d 10.67 (1H, s, NHSO2),
7.88 (1H, dd, J = 1.2, 8.0 Hz, H-6), 7.74 (1H, s, NHCO), 7.71 (2H, d,
J = 8.8 Hz, H-20, 60), 7.61 (3H, d, J = 8.8 Hz, H-3, 30, 50), 7.39 (1H,
td, J = 1.2, 8.0 Hz, H-4), 6.70 (1H, br td, J = 8.0 Hz, H-5), 3.85 (3H,
s, OCH3), 1.25 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d 177.6
(s, –COO–), 168.8 (s, –OCO–), 143.1 (s, C-40), 140.7 (s, C-2), 134.9
(d, C-4), 134.0 (s, C-10), 131.7 (d, C-6), 128.8 (d, C-30, 50), 123.4 (d,
C-5), 120.1 (d, C-20, 60), 119.2 (d, C-3), 116.2 (s, C-1), 52.9 (q,
OCH3), 40.3 (s, C(CH3)3), 27.8 (q, (CH3)3). ESI-MS (m/z, %): 403
[MꢀH]ꢀ (100). HRESI-MS m/z 403.1322 [MꢀH]ꢀ (calcd for
4.1.7. Synthesis of 4-(N-(2-(2-Hydroxyethylcarbamoyl)phenyl)
sulfamoyl)phenyl pivalate (22)
To a solution of isoatic anhydride (1 mmol) in terahydrofuran
(THF, 5 mL) were added ethylanolamine (1.5 mmol), and the mix-
ture was stirred at room temperature for 12 h. The resulting solu-
tion was concentrated in vacuum to yield a crude residue which
was purified by chromatography on silica gel (acetone/hex-
ane = 1:1) to provide intermediate 2-amino-N-(2-hydroxy-
ethyl)benzamide. The intermediate (0.2 mmol) was dissolved in
10% pyridine/DCM solution (5 mL), and 4-(chlorosulfonyl)phenyl
pivalate, which obtaining as Scheme 1 described, was added slowly
at room temperature, then; the mixture was stirred at same tem-
perature for 4 h. The resulting solution was concentrated and puri-
fied by chromatography on silica gel (acetone/hexane = 1:1) to
furnish 22 (yielding 72%, Scheme 8), white powder, mp 112–
114 °C. 1H NMR (400 MHz, CDCl3) d 10.52 (1H, s, NH), 7.70 (2H,
d, J = 8.8 Hz, H-30, 50), 7.69 (1H, br d, J = 8.0 Hz, H-6), 7.42 (1H, br
td, J = 8.0, 8.0 Hz, H-5), 7.39 (1H, br d, J = 8.0 Hz, H-6), 7.10 (1H,
br td, J = 8.0, 8.0 Hz, H-4), 7.05 (2H, d, J = 8.8 Hz, H-20, 60), 3.68
(2H, t, J = 4.8 Hz, CH2CH2), 3.40 (2H, q, J = 4.8 Hz, CH2CH2), 1.33
(9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d 177.6 (s, –OCO–),
168.73 (s, –COO–), 154.2 (s, C-10), 137.9 (s, C-1), 136.6 (s, C-40),
132.6 (d, C-5), 128.8 (d, C-30, 50), 126.9 (d, C-3), 125.0 (d, C-4),
124.3 (d, C-6), 124.0 (s, C-2), 122.0 (d, C-20, 60), 61.4 (t, CH2CH2),
42.2 (t, CH2CH2), 39.3 (s, C(CH3)3), 27.0 (q, (CH3)3). ESI-MS (m/z,
%): 421 [M+H]+ (100). HRESI-MS m/z 421.1425 [M+H]+ (calcd for
C20H23O5N2S 403.1335).
4.1.6. General procedure for synthesis of compounds 20 and 21
Sodium hydroxide aqueous solution (2 M, 3 mL) was added to a
suspension of 1 or 5 (1 mmol), respectively. The reaction mixture
was stirred for 12 h at room temperature; then, the mixture was
diluted with sodium bicarbonate aqueous solution and extracted
with ethyl acetate. The organic layer was washed with brine, dried,
and filtered. Removal of the solvent gave intermediates N-(2-acet-
ylphenyl)4-hydroxybenzenesulfonamide or N-(2-formylphenyl)4-
hydroxybenzenesulfonamide, respectively. Each intermediate was
dissolved in pyridine (2 mL), respectively, and t-butylsulfinyl chlo-
ride (5 equiv) was added slowly at 0 °C, then; the mixture was stir-
red at same temperature for 1 h. The mixture was diluted with
sodium bicarbonate aqueous solution and extracted with ethyl
acetate. Removal of the solvent of organic layer gave a residue that
was purified by column chromatography (silica gel, n-hexane/ace-
tone = 4:1 as eluent) to furnish 20 and 21, respectively (Scheme 7).
C20H25N2O6S 421.1433).
4.1.8. Synthesis of 2-hydroxyethyl 2-(4-(pivaloyloxy)phenylsulfo-
namido)benzoate (23)
To a solution of isoatic anhydride (1 mmol) in terahydrofuran
(THF, 5 mL) were added ethylene glycol (1.5 mmol) and DMAP
(1.0 mmol), the mixture was stirred at 75 °C for 12 h. The resulting
solution was concentrated in vacuum to yield a crude residue
which was purified by chromatography on silica gel (acetone/hex-
ane = 1:4) to provide intermediate 2-hydroxyethyl-2-aminobenzo-
ate. The intermediate was reacted with 4-(chlorosulfonyl)phenyl
pivalate (2 mmol), which obtaining as Scheme 1 described, was
added slowly at room temperature, then; the mixture was stirred
at same temperature for 4 h. The resulting solution was concen-

T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
1133
described.13,17 Species crossover was evaluated in the similar
way using NE from pig and mouse.17 The final concentrations of
serine proteases and their corresponding substrates as follows:
chymotrypsin
methylcoumarin (5
(5 nM)/N-succinyl-Ala-Ala-Pro-Phe-7-amido-4-
M), trypsin (50 nM)/N -benzoyl-L-arginine-
l
a
7-amido-4-methylcoumarin (50
zoyl-Phe-Val-Arg-7-amido-4-methylcoumarin
(100 M), porcine pancreas elastase (400 nM)/methoxysuccinyl-
Ala-Ala-Pro-Val-p-nitroanilide (250 M), and recombinant mouse
neutrophil elastase (50 nM)/methoxysuccinyl-Ala-Ala-Pro-Val-p-
nitroanilide (125 M).
lM), thrombin (100 nM)/Ben-
hydrochloride
l
l
l
4.2.1.3. Preparation of human neutrophils18
.
Blood was
taken from healthy human donors (20–34 years old) by venipunc-
ture, following a protocol approved by the Institutional Review
Board at Chang Gung Memorial Hospital. Neutrophils were isolated
with a standard method of dextran sedimentation prior to centri-
fugation in a Ficoll Hypaque gradient and the hypotonic lysis of
erythrocytes. Purified neutrophils that contained >98% viable cells,
as determined by the trypan blue exclusion method, were resus-
pended in Ca2+-free HBSS buffer at pH 7.4 and maintained at 4 °C
before use.
Scheme 8. The route for the preparation of compounds 22 and 23. Reagents and
conditions: (a) ethylanolamine, THF, 12 h; (b) 4-(chlorosulfonyl)phenyl pivalate,
10% pyridine/DCM, 2 h; (c) ethylene glycol, DMAP, THF, 75 °C, 12 h.
trated and purified by chromatography on silica gel (acetone/hex-
ane = 1:1) to furnish 23 (38%, Scheme 8), white powder, mp 106–
108 °C. 1H NMR (400 MHz, CDCl3) d 10.43 (1H, s, NH), 7.94 (1H,
dd, J = 1.2, 8.0 Hz, H-6), 7.80 (2H, d, J = 8.8 Hz, H-20, 60), 7.69 (1H,
br d, J = 8.0 Hz, H-3), 7.46 (1H, td, J = 1.2, 8.0 Hz, H-4), 7.10 (2H,
d, J = 8.8 Hz, H-30, 50), 7.06 (1H, t, J = 8.0 Hz, H-5), 4.36 (2H, t,
J = 4.4 Hz, CH2CH2), 3.87 (2H, t, J = 4.4 Hz, CH2CH2), 2.36 (1H, s,
OH), 1.31 (9H, s, (CH3)3). 13C NMR (100 MHz, CDCl3) d 176.5 (s, –
OCO–), 167.8 (s, –COO–), 154.5 (s, C-40), 140.0 (s, C-2), 136.3 (s,
C-10), 134.6 (d, C-4), 131.3 (d, C-6), 128.8 (d, C-20, 60), 123.5 (d, C-
5), 122.1 (d, C-30, 50), 120.2 (d, C-3), 116.7 (s, C-1), 66.9 (t, CH2CH2),
60.7 (t, CH2CH2), 39.2 (s, C(CH3)3), 26.9 (q, (CH3)3). ESI-MS (m/z, %):
444 [M+Na]+ (100). HRESI-MS m/z 422.1268 [M+H]+ (calcd for C22-
H24NO7S 422.1273).
4.2.1.4. Measurement of OÅ2ꢀ generation.
The assay of OÅ2ꢀ
generation was described as literatures.18 Briefly, after supplemen-
tation with 0.5 mg/ml ferricytochrome c and 1 mM Ca2+, neutro-
phils were equilibrated at 37 °C for 2 min and incubated with
drugs for 5 min. Cells were activated with formyl-
leucyl- -phenylalanine (fMLF, 30 nM) for 10 min in the pretreat-
ment of cytochalasin B (CB, 1 g/ml) for 3 min. Changes in absor-
L-methionyl-L-
L
l
bance with the reduction of ferricytochrome c at 550 nm were
continuously monitored in a double-beam, six-cell positioner spec-
trophotometer with constant stirring (Hitachi U-3010, Tokyo,
Japan). Calculations were based on differences in the reactions
with and without SOD (100 U/ml) divided by the extinction coeffi-
4.2. Biological assays
cient for the reduction of ferricytochrome c (
e
= 21.1/mM/10 mm).
4.2.1. In vitro tests
4.2.1.1. Neutrophil serine proteases (HNE, Pr3 and CatG) inhi-
4.2.1.5. Measurement of elastase release.
The method to
determine of elastase release of degranulation of azurophilic gran-
ules in human neutrophil as described previously.18,19 Experiments
were performed using methoxysuccinyl-Ala-Ala-Pro-Val-p-nitro-
anilide as the elastase substrate. Briefly, after supplementation
bition assay.
The selectivity and potency of synthetics were
modified and determined by observing the hydrolysis of the cleav-
age of peptide substrates to products by serine protease.9,12,17 HNE
(50 nM in 20 mM Tris–HCl, pH 7.4 containing 0.1% sodium azide
buffer solution) and different concentrations of synthetics
(10 nM–10 lM in 0.2% DMSO in 20 mM Tris–HCl, pH 7.4 contain-
ing 0.1% sodium azide and 5 mM calcium chloride buffer solution)
were incubated in assay buffer for 5 min at 30 °C. The substrate
(125 lM in 0.2% DMSO in 20 mM Tris–HCl, pH 7.4 containing
0.1% sodium azide and 5 mM calcium chloride buffer solution)
was added to the wells (assays were run in triplicates in a 96-well
with methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (100 lM),
neutrophils (5 ꢁ 105/ml) were equilibrated at 37 °C for 2 min and
incubated with drugs for 5 min. Cells were activated by 30 nM
FMLP for 10 min in the pretreatment of 0.5 lg/ml CB for 3 min,
and changes in absorbance at 405 nm were continuously moni-
tored to assay elastase release. Results are expressed as a percent
of elastase release in the drug-free control group.
plate in a total volume of 100 ll), and the rate of hydrolysis was
4.2.2. In vitro tests
4.2.2.1. Animals.
determined by monitoring the absorbance at 405 nm at 30 °C for
30 min. In addition, all synthetics and inhibitors were also tested
their effects on inhibiting Pr 3 (50 nM in 100 mM HEPES buffer,
pH 7.5 containing 500 mM NaCl, 10% DMSO, 170 lM DTNB) and
CatG (100 nM in 100 mM Tris–HCl buffer containing 1.6 M NaCl,
Male C57BL/6 mice (6–8 weeks old) weigh-
ing 20–25 g were used in this study. The animal protocol was
approved by the Institutional Animal Care and Use Committee of
Chang Gung University (Taoyuan, Taiwan, IACUC Approval no.:
CGU12-011, period of protocol: valid from Jun. 01, 2012 to May.
31, 2015). Animals were anesthetized with pentobarbital (50 mg/
kg) before intraperitoneal or intranasal administration.
pH 7.5) activities with corresponding substrates t-butyloxycar-
bonyl-Ala-Ala-Nva-thiobenzyl ester (100
lM) and N-succinyl-Ala-
Ala-Pro-Phe-p-nitroanilide (200 M), and the rate of hydrolysis
l
was determined by monitoring the absorbance at 405 nm at
30 °C for 180 min.
4.2.2.2. LPS- and HNE-induced paw edema.
Lipopolysaccha-
rides (LPS) from Escherichia coli 055:B5 and human neutrophil elas-
tase (HNE) were purchased from Sigma–Aldrich and dissolve in
saline. Acute inflammation of mice hind paws was induced as liter-
4.2.1.2. Selectivity and species crossover of lead compo-
nents.
The selectivity of lead components were also deter-
ature.22,23 Briefly, LPS (20
25
region of the right hind paw. The thickness of the LPS-/or
lg in 25
ll of saline) or HNE (5 lg in
mined by measuring the cleavage of substrates to products by
serine proteases: chymotrypsin, trypsin, and thrombin as
ll of saline) were injected subcutaneously into the plantar

1134
T.-L. Hwang et al. / Bioorg. Med. Chem. 23 (2015) 1123–1134
HNE-treated paws was measured before and after LPS/or HNE
injection at the time points, using digital calipers (Insize, Austria).
Each measurement was repeated three times, and the mean values
were calculated and expressed to the nearest 0.01 mm.6 The
inflamed animals were pretreated intraperitoneally with 6 (100
and 50 mg/kg) or sivelestat (100 mg/kg), respectively, 1 h before
induced by LPS- or HNE-injected.
Acknowledgements
The investigation was supported by research grants to P. W.
Hsieh from the National Science Council of the Republic of China
(NSC101-2320-B-182-008) and Chang Gung Medical Research
Foundation (CMRPD1B0301–CMRPD1B0303) in Taiwan.
Supplementary data
4.2.2.3. LPS-induced acute lung injury.
As described LPS
from Escherichia coli 055:B5 was dissolve in saline. Acute inflam-
mation of mice hind paws was induced as literature.24,29,30 Thirty
male C57BL/6 mice were randomly divided into five groups
(n = 6): sham group (without LPS treated); LPS treated; LPS + 6
(100 mg/kg); LPS + sivelestat (100 mg/kg); and 6 alone. Mice were
given intraperitoneal injection of 6 or sivelestat, respectively. Mice
from the sham group and LPS groups were treated with an equal
volume of vehicles instead. Except for sham group, all test animals
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.12.056.
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