Anthranilic acid-based inhibitors of phosphodiesterase: Design, synthesis, and bioactive evaluation

Article abstract of DOI:10.1039/c1ob05714f

Our previous studies identified two 2-benzoylaminobenzoate derivatives 1, which potently inhibited superoxide (O₂^-) generation induced by formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) in human neutrophils. In an attempt to improve their activities, a series of anthranilic acid derivatives were synthesized and their anti-inflammatory effects and underlying mechanisms were investigated in human neutrophils. Of these, compounds 17, 18, 46, 49, and 50 showed the most potent inhibitory effect on FMLP-induced release of O₂^- in human neutrophils with IC₅₀ values of 0.20, 0.16, 0.15, 0.06, and 0.29 μM, respectively. SAR analysis showed that the activities of most compounds were dependent on the ester chain length in the A ring. Conversely, a change in the linker between the A and B ring from amide to sulfonamide or N-methyl amide, as well as exchanges in the benzene rings (A or B rings) by isosteric replacements were unfavorable. Further studies indicated that inhibition of O₂^- production in human neutrophils by these anthranilic acids was associated with an elevation in cellular cAMP levels through the selective inhibition of phosphodiesterase 4. Compound 49 could be approved as a lead for the development of new drugs in the treatment of neutrophilic inflammatory diseases.

Full text of DOI:10.1039/c1ob05714f

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PAPER
Anthranilic acid-based inhibitors of phosphodiesterase: Design, synthesis, and
bioactive evaluation†
Yih-Dih Cheng,‡a,b Tsong-Long Hwang,‡a Han-Hsiang Wang,a Tai-Long Pan,c Chin-Chung Wu,d
Wen-Yi Chang,a Yi-Ting Liu,a Tzu-Chi Chua and Pei-Wen Hsieh*a
Received 6th May 2011, Accepted 30th June 2011
DOI: 10.1039/c1ob05714f
Our previous studies identified two 2-benzoylaminobenzoate derivatives 1, which potently inhibited
∑
superoxide (O2 -) generation induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in
human neutrophils. In an attempt to improve their activities, a series of anthranilic acid derivatives were
synthesized and their anti-inflammatory effects and underlying mechanisms were investigated in human
neutrophils. Of these, compounds 17, 18, 46, 49, and 50 showed the most potent inhibitory effect on
∑
-
FMLP-induced release of O2 in human neutrophils with IC50 values of 0.20, 0.16, 0.15, 0.06, and
0.29 mM, respectively. SAR analysis showed that the activities of most compounds were dependent on
the ester chain length in the A ring. Conversely, a change in the linker between the A and B ring from
amide to sulfonamide or N-methyl amide, as well as exchanges in the benzene rings (A or B rings) by
∑
-
isosteric replacements were unfavorable. Further studies indicated that inhibition of O2 production in
human neutrophils by these anthranilic acids was associated with an elevation in cellular cAMP levels
through the selective inhibition of phosphodiesterase 4. Compound 49 could be approved as a lead for
the development of new drugs in the treatment of neutrophilic inflammatory diseases.
of neutrophils.6,7 Phosphodiesterases (PDEs) are important in
1. Introduction
regulating intracellular concentrations of cAMP by catalyzing
Inflammatory lung diseases, including asthma, chronic obstructive
its hydrolysis and inactivating cAMP. Thus, PDE inhibitors are
pulmonary disease (COPD), cystic fibrosis, and lung injury are
currently being investigated as anti-inflammatory drugs because
of their suppressive effects on neutrophil function.6,8–10 PDEs have
been classified into 11 families (PDE1–PDE11) based on protein
sequence, structure, substrate specificity, enzymatic properties, and
tissue distribution.11 Among these, PDE3 and PDE4 were found
in neutrophils and were observed to regulate intracellular cAMP
levels and modulate the activation of neutrophils.12,13 However,
on the basis of our unpublished data, only PDE4 inhibitors
caused by activated neutrophils recruiting and releasing chemoat-
tractants into the airway.1,2 Although neutrophils play a critical
role in the defense of the human body against infections, abnormal
activation of neutrophils releases high concentrations of reactive
oxygen species (ROS) and elastase, and mediates immunological
insults and oxidative damage.2 The formation of superoxide anion
∑
(O2 -), a precursor of other ROS, by NADPH oxidase in human
∑
-
neutrophils is directly or indirectly linked to damage or destruction
of surrounding tissues.3,4 Several experimental studies showed that
oxidant stress enhances many pathological processes in various
inflammatory diseases and leads to harmful tissue damage.5 There-
fore, it is crucial to prevent neutrophil oxidative burst in inflamma-
tory tissues and organs. To our knowledge, the cyclic nucleotide
cAMP is an important second messenger in preventing activation
exhibited an inhibitory effect on O2 generation in neutrophils.
Therefore, we hypothesize that evaluating the inhibitory effect on
∑
O2 - generation in neutrophils is a new way of investigating specific
PDE4 inhibitors, and may be the target for a new generation of
agents in the treatment of neutrophilic inflammatory disease.
Anthranilic acid derivatives have been recognized to have
diverse biological activities,14–31 in particular, they are enzymatic
inhibitors of VEGF receptor kinase,14 matrix metalloproteinase
(MMP),15 acyl carrier protein synthase (AcpS),16 methionine
aminopeptidase-2,17,18 phospholipase A2,19 human hydroxysteroid
dehydrogenase AKR1C1,20 plasminogen activator inhibitor-1,21
and cholecystokinin (CCK).22–24 In addition, they also exhibit
anti-cancer, anti-hepatitis C virus (anti-HCV), anti-Alzheimer,
anti-inflammatory, anti-platelet, and anti-bacterial activities.25–35
Of these, the N-phenylanthranilic acids, e.g. mefenamic acid
analogs, have beenusedas anti-inflammatoryagents intherapy.29–31
Our previous studies identified two 2-benzoylaminobenzoate
aGraduate Institute of Natural Products, Chang Gung University, Taoyuan,
33302, Taiwan. E-mail: pewehs@mail.cgu.edu.tw; Fax: +886-3-211-8643;
Tel: +886-3-211-8800, ext. 3105
bChang Gung Memorial Hospital, Chia-Yi, 613, Taiwan
cSchool of Traditional Chinese Medicine, Chang Gung University, Taoyuan,
33302, Taiwan
dGraduate Institute of Natural Products, Kaohsiung Medical University,
Kaohsiung, 807, Taiwan
† Electronic supplementary information (ESI) available: HPLC analysis of
the target compounds. See DOI: 10.1039/c1ob05714f
‡ These authors contributed equally to this work.
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Table 1 Inhibitory effects of 2-benzoylaminobenzoic esters and N-methyl 2-benzoylaminobenzoic esters on superoxide anion generation and elastase
release by human neutrophils in response to fMLP/CB
Anti-inflammation (mM)a
∑
-
R1
H
R2
R3/R4
F/H
O2 generation
NE release
1b
CH3
0.64 0.01
> 20
> 20
Sivelestatb
0.050 0.0002
8
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH2CH3
CH2CH3
Cl/H
Br/H
F/H
Cl/H
Br/H
F/H
Cl/H
Br/H
F/F
F/F
F/F
F/F
F/F
0.76 0.26
6.92 2.54
> 20
> 20
> 20
> 20
> 20
> 20
0.62 0.07
0.20 0.03
0.16 0.03
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
9
10
11
12
13
14
15
16
17
18
19
20
21
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH3
CH2CH3
CH2CH2CH3
CH3
CH2CH3
CH2CH2CH3
F/F
a Concentration necessary for 50% inhibition (IC50). b Compound 1 and sivelestat were used as positive controls.
∑
derivatives, 1, which potently inhibited O2 - generation induced by
formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in human
neutrophils.32,36 Furthermore, derivative 1 was able to elevate
cAMP levels by inhibiting phosphodiesterase, and attenuate
hemorrhagic shock-induced lung injury in rats.36 Accordingly, the
leading agent of phosphodiesterase inhibitors, 1, was modified in
an attempt to improve anti-inflammatory activity by increasing
the ester chain length, changing the amide to sulfonamide or N-
methyl amide, as well as extending the space between the two
aromatic rings. Furthermore, replacing the aromatic rings with
isosteres is also discussed. All synthetic compounds (Tables 1–
carboxylic acid, 4-aminonicotinic acid, and 3-amino-2-pyrazine
carboxylate were esterified and then coupled with the cor-
responding substituted benzoyl chlorides to afford 22–27
(Table 2 and Scheme 1). Furthermore, the anthranilic esters
were transformed to 2-phenylsulfonamidobenzoic esters (29–
37, Table 3) by reacting with the corresponding benzene-
sulfonyl chloride (Scheme 2). 2-Phenylacetamidobenzoic esters
(40–51, Table 4) were prepared by a coupling reaction with
2-benzoylaminobenzoic esters and the corresponding pheny-
lacetic acids in HBTU/DIEA/DCM solution (Scheme 3). In
addition, conversion of 2-benzoylaminobenzoic esters to 3-
phenylpropanamidobenzoic esters (52–54, Table 4, Scheme 3)
and 2-cinnamamidobenzoic esters were described using the above
protocol (56 and 58, Scheme 4). Accordingly, all synthetics
(Tables 1–4) were fully characterized using spectroscopic data. The
spectroscopic data of the new compounds (10–15, 18–26, 34–37,
42–48, 49–54, and 58) are shown in the experimental section.
∑
-
4) were evaluated for their in vitro inhibitory effects on O2
generation induced by FMLP in human neutrophils. Herein, we
report on the synthesis of anthranilic acid derivatives, as well as
their pharmacological data and structure–activity relationships
(SARs).
2. Chemistry
3. Results and discussion
Initially, corresponding anthranilic and N-methyl anthranilic
esters were synthesized by refluxing anthranilic or N-
methyl anthranilic acids, respectively, in alcohols/c-H2SO4 so-
lutions to yield intermediates, which were subsequently re-
acted with corresponding substituted benzoyl chlorides to ob-
tain 2-benzoylaminobenzoic esters (8–18) and N-methyl 2-
benzoylaminobenzoic esters (19–21) (Table 1 and Scheme 1).
To study the isosteres of the aromatic rings, 2-aminopyridine-3-
Neutrophil function assay
All synthetics (8–27, 29–37, 40–54, 56, and 58) and positive
∑
-
controls (1 and sivelestat) were subjected to O2 generation
and neutrophil elastase release with FMLP as the inducer in
neutrophils. Of these, five compounds 17, 18, 46, 49, and 50
exhibited the most potent and selective inhibitory effects on
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Table 2 Inhibitory effects of the isosteres of 2-benzoylaminobenzoic esters on superoxide anion generation and elastase release by human neutrophils
in response to fMLP/CB
Anti-inflammation (mM)a
∑
-
R2
R3
F
W/X/Y/Z
O2 generation
NE release
1b
CH3
CH/CH/CH/CH
0.64 0.01
>20
>20
0.050 0.0002
>20
>20
>20
>20
>20
>20
>20
Sivelestatb
8
9
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
Cl
Br
F
CH/CH/CH/CH
CH/CH/CH/CH
CH/N/CH/CH
N/CH/CH/CH
N/CH/CH/CH
N/CH/CH/CH
N/CH/N/CH
0.76 0.26
6.92 2.54
1.00 0.20
0.45 0.17
12.20 6.97
11.32 5.92
>20
22
23
24
25
26
27
F
Cl
Br
F
CH2CH3
Cl
CH/CH/CH/N
10.92 2.86
>20
a Concentration necessary for 50% inhibition (IC50). b Compound 1 and sivelestat were used as positive controls.
Table 3 Inhibitory effects of the isosteres of 2-phenylsulfonamidobenzoic esters on superoxide anion generation and elastase release by human neutrophils
in response to fMLP/CB
Anti-inflammation (mM)a
∑
-
R2
R4
F
O2 generation
NE release
1b
CH3
0.64 0.01
> 20
> 20
Sivelestatb
0.050 0.0002
29
30
31
32
33
34
35
36
37
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
F
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
Cl
Br
F
Cl
Br
F
6.10 2.14
2.42 0.15
2.43 0.16
2.02 0.56
1.05 0.40
0.74 0.0007
Cl
Br
11.39 2.23
> 20
a Concentration necessary for 50% inhibition (IC50). b Compound 1 and sivelestat were used as positive controls.
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Table 4 Inhibitory effects of the isosteres of 2-phenylacetamidobenzoic esters and 3-phenylpropanamidobenzoic esters on superoxide anion generation
and elastase release by human neutrophils in response to fMLP/CB
Anti-inflammation (mM)a
∑
-
R2
R5
F
n
O2 generation
NE release
1b
CH3
0
0.64 0.01
> 20
> 20
Sivelestatb
40
0.050 0.0002
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
F
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
—
1.18 0.10
1.35 0.01
3.42 1.04
0.47 0.33
0.57 0.38
0.66 0.11
0.15 0.03
0.39 0.004
0.48 0.21
0.06 0.01
0.29 0.09
0.67 0.41
0.95 0.19
1.57 0.42
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
41
Cl
Br
F
Cl
Br
F
Cl
Br
F
Cl
Br
F
42
43
44
45
46
47
48
17.60 7.76
49
> 20
> 20
> 20
> 20
> 20
> 20
> 20
50
51
52
53
Cl
Br
F
54
56
> 20
58
CH2CH3
Cl
—
> 20
> 20
a Concentration necessary for 50% inhibition (IC50). b Compound 1 and sivelestat were used as positive controls.
∑
-
FMLP-induced O2 generation in neutrophils with IC50 values
of 0.20, 0.16, 0.15, 0.06, and 0.29 mM, respectively.
was increased by one carbon atom, whereas the activities were
reversed when the spacer length was increased by two carbon
atoms (52–54). Moreover, the activities were attenuated by the
replacement of a 3-phenylpropanoyl moiety in anthranilic acid
(52–54) with a cinnamoyl moiety (56 and 58).
The neutrophil function assay data and SAR analysis showed
the enhancement of activity in most synthetics, e.g. compounds
16–18 and 29–51, where the ester chain length in the A ring was
increased. A change in the linker between the A and B rings from
amide to sulfonamide drastically reduced the inhibitory effect, as
seen for 29–34. The activities of N-methyl amide derivatives (19–
21) vanished due to removal of the intramolecular hydrogen bond,
which results in a drastic loss of binding affinity.37 Furthermore,
except for 23, exchanges in the benzene rings (A or B rings) of 1
by different isosteric replacements (22–27) were unfavorable. There
are many reports that demonstrate that replacing the CH with an N
at each of the aromatic sites leads to improved aqueous solubility,
preserves hydrogen bonding, or reduces metabolism.38 However,
the N atom in aromatic rings acts as an electron-withdrawing
substitution changing the inductive effect or resonance, and
modifies the hydrogen-bonding intensity of the amide between
the A and B rings, thus the activities of compounds 22–27 were
decreased.
cAMP level determination
Compound 1 was able to enhance cAMP levels and PKA
activity by inhibiting phosphodiesterase. To gain further insight
into the mechanism of action of active synthetics, cAMP levels
in the presence of these active compounds were determined.
Neutrophils were incubated with compounds 17, 18, 21, and 49
(1 mM) as described above, and an enzyme immunoassay kit was
used to determine cAMP concentrations.36 The results showed
that compound 49 exhibited the most potent inhibitory effects
on cAMP degradation, however, 21, a non-active component
∑
-
for inhibiting O2 generation in neutrophils, had no effect on
modulating cAMP levels (Fig. 1). These results suggested and
∑
-
confirmed that cAMP is involved in the inhibitory effects on O2
generation.
To investigate the bioactive relationship of the spacer between
the two aromatic rings, compounds 43–51 were synthesized and
they showed an increase in inhibitory effects as the spacer length
Additionally, in an attempt to examine whether cAMP was
∑
-
involved in the inhibitory effect on O2 generation of 49, a PKA
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Scheme 1 The route for the preparation of compounds 8–27.
Scheme 2 The route for the preparation of compounds 29–37.
inhibitor, H89 (3 mM), was used to elucidate the mechanism.
The result showed negative regulation of FMLP-caused human
∑
-
neutrophil O2 generation mediated by 49 was reversed by H89
Scheme 3 The route for the preparation of compounds 40–54.
(Fig. 2). This suggests that cAMP/PKA-dependent pathway
∑
-
mediates the inhibition of FMLP/CB-activated O2 production
evaluate their inhibitory effects on PDE3, PDE4, and PDE5,
respectively.39,40 In the present study, 49 exhibited selective activity
on PDE4 (Selective Index; SI = 15.2). Interestingly, compound 49
showed minor inhibitory effects on PDE3 (Table 5), and no effect
on PDE5 (the IC50 value of zaprinast was 2.5 mM).
cAMP is an important second messenger with a variety of
physiological and pathophysiological manifestations. Elevation of
intracellular cAMP levels is believed to suppress the activation of
neutrophils.36 Cellular cAMP concentrations are modulated either
by synthesis via adenylate cyclase or by degradation via PDEs.
caused by 49.
Inhibitory effects of compounds on PDEs
Phosphodiesterases, e.g. PDE3, PDE4, and PDE5, regulate intra-
∑
-
cellular cAMP and/or cGMP levels to modulate O2 generation
in neutrophils. To investigate whether bioactive synthetics regulate
cAMP levels by specifically inhibiting subtype phosphodiesterases,
the most potent compound (49), and three positive controls
(Ro20-1724, cilostamide, and zaprinast) were chosen to further
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Scheme 4 The route for the preparation of compounds 56 and 58.
Fig. 1 Effects of compounds 2, 17, 18, 21, and 49 on cAMP levels. Human
neutrophils were incubated with DMSO (control) or compounds 2, 17, 18,
21, and 49 (1.0 mM) for 5 min before stimulation with or without FMLP
(0.1 mM) for another 1 min. cAMP levels were measured by enzyme
immunoassay kits. All data are expressed as the mean S.E.M. (n = 3).
*p < 0.05; **p < 0.01; ***p < 0.001 compared to the control.
∑
-
Fig. 2 Effects of the cAMP pathway on 49 caused inhibition of O2
∑
-
release. O2 generation was induced by FMLP/CB and measured using
SOD-inhibitable cytochrome c reduction. H89 (3 mM). All data are
expressed as the mean S.E.M. (n = 3). * p < 0.05; ** p < 0.01; ***
p < 0.001 compared to the control. # p < 0.01; ## p < 0.01; ### p < 0.001
compared to the corresponding control.
Table 5 Inhibitory effects of 49 on PDE3 and PDE4
IC50 (mM)a
PDE3
PDE4
∑
-
O2 generation than PDE4, indicating that 49 may exhibit an
additional cAMP-independent mechanism of action.
49
64.0
0.0312
—
4.20
—
1.58
Cilostamideb
Ro20-1724b
Although a few selective PDE4 inhibitors have exhibited broad
spectrum anti-inflammatory effects, and reached clinical effects in
asthma and COPD, the side effects of these inhibitors limit their
clinical use.13,43 Therefore, the development of a new generation
of PDE inhibitors combining PDE4 inhibition may be another
way forward.42 The dual PDE3–PDE4 compounds, zardaverine
and its related compounds, provide additive or synergistic effects
to suppress the activation/function of cells playing a role in
inflammatory lung diseases, and provide more bronchodilator
and bronchoprotective effects in addition to the beneficial PDE4
effects.13,41,42 Accordingly, compound 49 showed dual inhibitory
effects on PDE3 and PDE4, and no significantly cytotoxic or anti-
platelet effects, suggesting that this compound may be approved as
a new candidate in the treatment of airway inflammatory diseases.
a Concentration necessary for 50% inhibition (IC50). b Cilostamide and
Ro20-1724 were used as positive controls.
The predominant cAMP-specific PDE in most inflammatory cells
belongs to the PDE4 family,41 and inhibitors of PDE4 are currently
being developed clinically as potential anti-inflammatory agents.42
Human neutrophils contain abundant amounts of PDE4, and the
activity of PDE4 plays a significant function in regulating cellular
cAMP level. In this study, our results demonstrate that inhibition
∑
-
of O2 production in human neutrophils by 49 is associated
with an elevation of cellular cAMP concentration through its
inhibition of PDE4. 49 was shown to be more effective at inhibiting
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(d, C-4), 133.9 (d, C-4¢, JC-F = 9 Hz), 132.1 (d, C-6¢), 131.3 (d,
C-6), 125.1 (d, C-5¢, JC-F = 3 Hz), 123.4 (d, C-5), 123.2 (s, C-1¢,
JC-F = 12 Hz), 121.7 (d, C-3), 117.0 (d, C-3¢, JC-F = 23 Hz), 116.7 (s,
C-1), 67.4 (t, -COOCH2CH2CH3), 22.4 (t, -COOCH2CH2CH3),
10.9 (q, -COOCH2CH2CH3). ESI-MS (m/z,%): 302 [M + H]+
(100). HRESI-MS m/z 302.1191 [M + H]+ (calcd for C17H17FNO3
302.1192).
4. Conclusion
In this study, we synthesized forty-six anthranilic acid derivatives
∑
-
and compared their inhibitory effects on O2 generation in neu-
trophils. The analysis of structure–activity relationships revealed
that the ester chain length in the A ring, as well as an amide
linker between the A and B rings, played important roles in
∑
-
O2 generation. Furthermore, our study demonstrated that 49
5.1.1.2 Propyl 2-(2-chlorobenzamido)benzoate (11). 83%
yield. Pale yellow oil. 1H NMR (CDCl3) d 11.59 (1H, br. s, NH),
8.91 (1H, d, J = 8.4 Hz, H-3), 8.07 (1H, dd, J = 8.0, 1.2 Hz, H-
6), 7.65 (1H, dd, J = 7.2, 2.0 Hz, H-6¢), 7.57 (1H, td, J = 8.4,
1.2 Hz, H-4), 7.42 (1H, dd, J = 8.8, 1.2 Hz, H-3¢), 7.34 (2H, m,
H-4¢, 5¢), 7.12 (1H, t, J = 8.0 Hz, H-5), 4.22 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 1.74 (2H, qt, J = 7.2, 6.8 Hz, OCH2CH2CH3),
0.98 (3H, t, J = 7.2 Hz, OCH2CH2CH3). 13C NMR (CDCl3) d
168.5 (s, -COOCH2CH2CH3), 165.8 (s, -CONH-), 141.6 (s, C-2),
136.6 (s, C-1¢), 135.0 (d, C-4), 131.8 (d, C-6), 131.6 (s, C-2¢), 131.3
(d, C-4¢), 131.0 (d, C-3¢), 129.6 (d, C-6¢), 127.5 (d, C-5¢), 123.5 (d,
C-5), 120.9 (d, C-3), 116.1 (s, C-1), 67.4 (t, -COOCH2CH2CH3),
22.3 (t, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3). ESI-
MS (m/z,%): 318[M + H]+, 320 [M + 2 + H]+. HRESI-MS m/z
318.0899 [M + H]+ (calcd for C17H17ClNO3 318.0897).
∑
-
inhibited O2 generation via cAMP-dependent pathways. This
compound could be approved as a lead for the development of
new agents in the treatment of inflammatory lung diseases.
5. Experimental section
5.1. Materials and methods
All commercial chemicals and solvents are reagent grade and
were used without further treatment unless otherwise noted.
Reactions were detected by TLC using Merck 60 F254 silica gel
aluminum packed plates; spots were recorded under ultraviolet
irradiation (254 and 365 nm). The flash column chromatography
was performed with silica gel (Silicycle, 70–230 mesh or 230–400
mesh). The purities of synthetics were determined by HPLC using
a Jasco PU-1580 intelligent HPLC pump and a Jasco AS 1555-
5.1.1.3. Propyl 2-(2-bromobenzamido)benzoate (12). 63%
yield. Brown oil. 1H NMR (CDCl3) d 11.53 (1H, br. s, NH), 8.91
(1H, d, J = 8.4 Hz, H-3), 8.07 (1H, d, J = 8.0 Hz, H-6), 7.59 (3H,
m, H-4, 3¢, 6¢), 7.37 (1H, t, J = 7.6 Hz, H-5¢), 7.27 (1H, t, J =
7.6 Hz, H-4¢), 7.12 (1H, t, J = 7.6 Hz, H-5), 4.22 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 1.74 (2H, qt, J = 7.6, 6.8 Hz, OCH2CH2CH3),
0.98 (3H, t, J = 7.6 Hz, OCH2CH2CH3). 13C NMR (CDCl3) d
168.5 (s, -COOCH2CH2CH3), 166.6 (s, -CONH-), 141.6 (s, C-2),
138.8 (s, C-1¢), 135.0 (d, C-4), 134.1 (d, C-3¢), 131.9 (d, C-6), 131.3
(d, C-4¢), 129.3 (d, C-6¢), 128.1 (d, C-5¢), 123.5 (d, C-5), 120.9 (d,
C-3), 120.1 (s, C-2¢), 116.1 (s, C-1), 67.4 (t, -COOCH2CH2CH3),
22.3 (t, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3). ESI-
MS (m/z,%): 362 [M + H]+, 364 [M + 2 + H]+. HRESI-MS m/z
362.0393 [M + H]+ (calcd for C17H17BrNO3 362.0392).
R
10 intelligent sampler with an Ascentisꢀ C-18 analytical column
(Superlcu, 5 mm, 4.6 mm ¥ 250 mm). Detection was conducted
at 248 nm in a Jassco UV-1575 UV-Vis detector. Purities of all
the tested components were found to be more than 95% unless
otherwise stated. The NMR spectra using CDCl3 as solvents were
obtained ona Bruker AVANCE-400MHzFT-NMRspectrometer.
Chemical shifts were internally referenced to the solvent signals
in 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.
5.1.1. General
procedure
for
synthesis
of
2-
benzoylaminobenzoic esters (8–18) and N-methyl 2-benzoyl-
aminobenzoic esters (19–21). Compounds 3a–3d and 4a–4c
were synthesized through refluxing 2a or 2b, respectively, in
corresponding alcohols (50 ml)/c-H2SO4 (1.0 ml) for 2 h, the
solutions were subsequently neutralized by ammonium water; the
resulting mixtures were partitioned between ice water (100 ml)
and chloroform; and the organic layers were concentrated. To a
mixture solution of 3a–3d and 4a–4c (1.0 mmole) in DCM was
added the suitable benzoyl chlorides (2.0 mmole). The reaction
mixture was 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–ethyl
acetate to afford the products.
5.1.1.1. Propyl 2-(2-fluorobenzamido)benzoate (10). 51%
yield. Colorless oil. 1H NMR (CDCl3) d 11.89 (1H, d, J = 6.8 Hz,
NH), 8.91 (1H, d, J = 8.4 Hz, H-3), 8.07 (1H, dd, J = 8.0, 1.2 Hz, H-
6¢), 8.05 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.58 (1H, t, J = 8.4, 1.2 Hz,
H-4), 7.48 (1H, td, J = 7.2, 1.2 Hz, H-4¢), 7.27 (1H, t, J = 7.6 Hz,
H-5¢), 7.15 (1H, dd, J = 11.6, 8.4 Hz, H-3¢), 7.13 (1H, t, J = 8.0 Hz,
H-5), 4.29 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 1.79 (2H, qt, J = 7.6,
6.8 Hz, OCH2CH2CH3), 1.02 (3H, t, J = 7.6 Hz, OCH2CH2CH3).
13C NMR (CDCl3) d 168.3 (s, -COOCH2CH2CH3), 162.7 (s,
-CONH-), 160.6 (s, C-2¢, JC-F = 249 Hz), 141.5 (s, C-2), 134.7
5.1.1.4. Butyl 2-(2-fluorobenzamido)benzoate (13). 48%
yield. Colorless oil. 1H NMR (CDCl3) d 11.90 (1H, br. s,
NH), 8.91 (1H, d, J = 8.8 Hz, H-3), 8.06 (2H, m, H-6, 6¢),
7.57 (1H, td, J = 8.8, 1.6 Hz, H-4), 7.47 (1H, ddd, J = 11.6,
8.0, 2.0 Hz, H-4¢), 7.26 (1H, t, J = 8.0 Hz, H-5¢), 7.18 (1H,
dd, J = 11.6, 8.0 Hz, H-3¢), 7.12 (1H, t, J = 7.6 Hz, H-5),
4.33 (2H, t, J = 6.4 Hz, OCH2CH2CH2CH3), 1.74 (2H, tt, J =
7.2, 6.4 Hz, OCH2CH2CH2CH3), 1.46 (2H, qt, J = 7.6, 7.2 Hz,
OCH2CH2CH2CH3), 0.96 (3H, t, J = 7.6 Hz, OCH2CH2CH2CH3).
13C NMR (CDCl3) d 168.3 (s, -COOCH2CH2CH2CH3), 162.6 (s,
JC-F = 3 Hz, -CONH-), 160.6 (s, JC-F = 250 Hz, C-2¢), 141.5 (s,
C-2), 134.7 (d, C-4), 133.8 (d, JC-F = 9 Hz, C-4¢), 132.0 (d, JC-F
2 Hz, C-6¢), 131.2 (d, C-6), 125.0 (d, JC-F = 4 Hz, C-5¢), 123.4 (d,
C-5), 123.2 (s, JC-F = 12 Hz, C-1¢), 121.8 (d,C-3), 116.8 (d, JC-F
=
=
23 Hz, C-3¢), 116.7 (s, C-1), 65.6 (t, -COOCH2CH2CH2CH3), 31.0
(t, -COOCH2CH2CH2CH3), 19.6 (t, -COOCH2CH2CH2CH3),
14.1 (q, -COOCH2CH2CH2CH3). ESI-MS (m/z,%): 316 [M +
H]+. HRESI-MS m/z 316.1348 [M + H]+ (calcd for C18H19FNO3
316.1349).
5.1.1.5. Butyl 2-(2-chlorobenzamido)benzoate (14). 55%
1
yield. Colorless oil. H NMR (CDCl3) d 11.58 (1H, br. s, NH),
8.91 (1H, d, J = 8.4 Hz, H-3), 8.06 (1H, dd, J = 8.0, 1.2 Hz, H-6),
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7.65 (1H, dd, J = 7.2, 2.0 Hz, H-6¢), 7.58 (1H, td, J = 8.4, 1.2 Hz,
H-4), 7.44 (1H, dd, J = 7.6, 1.2 Hz, H-3¢), 7.38 (1H, td, J = 7.6,
1.2 Hz, H-5¢), 7.37 (1H, td, J = 7.6, 2.0 Hz, H-4¢), 7.12 (1H, t,
J = 8.0 Hz, H-5), 4.28 (2H, t, J = 6.8 Hz, OCH2CH2CH2CH3),
1.71 (2H, tt, J = 7.2, 6.8 Hz, OCH2CH2CH2CH3), 1.43 (2H,
qt, J = 7.6, 7.2 Hz, OCH2CH2CH2CH3), 0.94 (3H, q, J =
7.6 Hz, OCH2CH2CH2CH3). 13C NMR (CDCl3) d 168.5 (s,
-COOCH2CH2CH2CH3), 165.8 (s, -CONH-), 141.6 (s, C-2),
136.7 (s, C-1¢), 135.0 (d, C-4), 131.8 (s, C-2¢), 131.7 (d, C-6),
131.3 (d, C-4¢), 131.0 (d, C-3¢), 129.6 (d, C-6¢), 127.5 (d,
C-5¢), 123.5 (d, C-5), 121.0 (d, C-3), 116.2 (s, C-1), 65.7 (t,
-COOCH2CH2CH2CH3), 30.9 (t, -COOCH2CH2CH2CH3), 19.6
(t, -COOCH2CH2CH2CH3), 14.1 (q, -COOCH2CH2CH2CH3).
ESI-MS (m/z,%): 332 [M + H]+, 334 [M + 2 + H]+. HRESI-MS
m/z 332.1052 [M + H]+ (calcd for C18H19ClNO3 332.1053).
5.1.1.9. Ethyl 2-(2-fluoro-N-methylbenzamido)benzoate (20).
48% yield. White powder, mp 84–86 ◦C. 1H NMR (CDCl3) d 7.81
(1H, d, J = 7.6 Hz, H-6), 7.45 (1H, t, J = 7.6 Hz, H-4, 7.36 (1H,
d, J = 7.6 Hz, H-3), 7.29 (1H, t, J = 7.6 Hz, H-5), 7.10 (1H, tt,
J = 8.0, 7.6 Hz, H-4¢), 6.65 (2H, m, H-3¢, 5¢), 4.87 (2H, q, J =
6.8 Hz, OCH2CH3), 3.48 (3H, s, NCH3), 1.40 (3H, t, J = 6.8 Hz,
OCH2CH3). 13C NMR (CDCl3) d 165.6 (s, -COOCH2CH3), 161.5
(s, -CONCH3), 142.2 (s, C-2), 133.7 (d, C-4), 133.2 (d, C-6), 131.3
(d, JC-F = 10 Hz C-4¢), 129.9 (d, C-5), 129.7 (s, C-1), 128.9 (d, C-3),
115.2 (s, JC-F = 23 Hz, C-1¢), 111.6 (d, JC-F = 21 Hz, C-3¢, 5¢), 62.1
(t, -COOCH2CH3), 37.9 (q, -CONCH3), 14.5 (q, -COOCH2CH3).
ESI-MS (m/z,%): 320 [M + H]+. HRESI-MS m/z 320.1097 [M +
H]+ (calcd for C17H16F2NO3 320.1098).
5.1.1.10. Propyl 2-(2,6-difluoro-N-methylbenzamido)benzoate
(21). 52% yield. White powder, mp 72–74 ◦C. 1H NMR (CDCl3) d
7.82 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.45 (1H, td, J = 7.6, 1.2 Hz, H-
4), 7.36 (1H, d, J = 7.6 Hz, H-3), 7.29 (1H, t, J = 8.0 Hz, H-5), 7.09
(1H, tt, J = 8.4, 7.6 Hz, H-4¢), 6.64 (2H, m, H-3¢, 5¢), 4.30 (2H, t, J =
6.8 Hz, OCH2CH2CH3), 3.52 (3H, s, NCH3), 1.82 (2H, qt, J = 7.2,
6.8 Hz, OCH2CH2CH3), 1.02 (3H, t, J = 7.2 Hz, OCH2CH2CH3).
13C NMR (CDCl3) d 165.5 (s, -COOCH2CH2CH3), 161.6 (s,
-CONCH3), 158.9 (s, JC-F = 250 Hz, C-2¢, 6¢), 142.0 (s, C-2), 133.2
(d, C-4), 131.9 (d, C-6), 131.5 (d, JC-F = 10 Hz, C-4¢), 129.8 (d,
C-5), 129.5 (s, C-1), 129.0 (d, C-3), 115.0 (d, JC-F = 23 Hz, C-1¢),
111.7 (d, JC-F = 21 Hz, C-3¢, 5¢), 67.7 (t, -COOCH2CH2CH3),
37.8 (q, -CONCH3), 22.15 (t, -COOCH2CH2CH3), 10.77 (q, -
COOCH2CH2CH3). ESI-MS (m/z,%): 334 [M + H]+. HRESI-MS
m/z 334.1257 [M + H]+ (calcd for C18H18F2NO3 334.1255).
5.1.1.6. Butyl 2-(2-bromobenzamido)benzoate (15). 60%
yield. Pale yellow oil. 1H NMR (CDCl3) d 11.50 (1H, br. s, NH),
8.90 (1H, d, J = 8.0 Hz, H-3), 8.07 (1H, dd, J = 8.0, 2.0 Hz,
H-6), 7.60 (3H, m, H-4, 3¢, 6¢), 7.38 (1H, td, J = 8.0, 0.8 Hz, H-
5¢), 7.28 (1H, td, J = 8.0, 2.0 Hz, H-4¢), 7.13 (1H, t, J = 8.0 Hz,
H-5), 4.27 (2H, t, J = 6.8 Hz, OCH2CH2CH2CH3), 1.71 (2H, tt,
J = 7.6, 6.8 Hz, OCH2CH2CH2CH3), 1.43 (2H, qt, J = 7.6, 7.2 Hz,
OCH2CH2CH2CH3), 0.94 (3H, t, J = 7.2 Hz, OCH2CH2CH2CH3).
13C NMR (CDCl3) d 168.4 (s, -COOCH2CH2CH2CH3), 166.5 (s,
-CONH-), 141.4 (s, C-2), 138.7 (s, C-1¢), 134.9 (d, C-4), 134.0 (d,
C-3¢), 131.7 (d, C-6), 131.2 (d, C-4¢), 129.2 (d, C-6¢), 127.9 (d, C-5¢),
123.4 (d, C-5), 120.8 (d, C-3), 120.0 (s, C-2¢), 116.0 (s, C-1), 65.6 (t,
-COOCH2CH2CH2CH3), 30.8 (t, -COOCH2CH2CH2CH3), 19.5
(t, -COOCH2CH2CH2CH3), 14.0 (q, -COOCH2CH2CH2CH3).
ESI-MS (m/z,%): 376 [M + H]+, 378 [M + 2 + H]+. HRESI-MS
m/z 376.0548 [M + H]+ (calcd for C18H19BrNO3 376.0547).
5.1.1.11. Ethyl 2-(2-fluorobenzamido)nicotinate (22). Com-
pound 22 was synthesized in 45% yield from 4-amino-nicotinic
acid (2c) i◦n similar procedure as described. Pale yellow powder,
mp 72–74 C. 1H NMR (CDCl3) d 11.55 (1H, d, J = 8.4 Hz, NH),
8.69 (1H, dd, J = 4.8, 1.6 Hz, H-6), 8.36 (1H, dd, J = 7.6, 1.6 Hz,
H-4), 8.12 (1H, dd, J = 7.6, 1.2 Hz, H-6¢), 7.52 (1H, ddd, J = 10.2,
7.6, 1.2 Hz, H-4¢), 7.28 (1H, t, J = 7.6 Hz, H-5¢), 7.19 (1H, dd,
J = 10.2, 8.4 Hz, H-3 ¢), 7.15 (1H, dd, J = 7.6, 4.8 Hz, H-5), 4.41
(2H, q, J = 6.8 Hz, CH2CH3), 1.39 (3H, t, J = 6.8 Hz, CH2CH3).
13C NMR (CDCl3) d 166.7 (s, -COOCH2CH3), 161.6 (s, -CONH-),
160.1 (s, JC-F = 249 Hz, C-2¢), 153.1 (d, C-6), 152.4 (s, C-2), 140.2
5.1.1.7. Propyl 2-(2,6-difluorobenzamido)benzoate (18). 32%
1
yield. Colorless oil. H NMR (CDCl3) d 11.64 (1H, br. s, NH),
8.90 (1H, d, J = 8.0 Hz, H-3), 8.08 (1H, dd, J = 7.6, 1.6 Hz, H-6),
7.60 (1H, td, J = 8.0, 1.2 Hz, H-4), 7.39 (1H, tt, J = 12.4, 8.0 Hz,
H-4¢), 7.15 (1H, t, J = 7.6 Hz, H-5), 6.99 (2H, t, J = 8.0 Hz, H-3¢,
5¢), 4.25 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 2.77 (2H, qt, J = 7.2,
6.8 Hz, OCH2CH2CH3), 1.00 (3H, t, J = 7.2 Hz, OCH2CH2CH3).
13C NMR (CDCl3) d 168.5 (s, -COOCH2CH2CH3), 161.6 (s,
-CONH-), 159.1 (s, JC-F = 252, 7 Hz, C-2¢, 5¢), 141.3 (s, C-2),
135.0 (d, C-4), 132.7 (d, JC-F = 10 Hz, C-4¢), 131.3 (d, C-6), 123.7
(d, C-5), 121.0 (d, C-3), 116.1 (s, C-1), 115.5 (s, JC-F = 20 Hz, C-
1¢), 112.5 (d, JC-F = 25 Hz, C-3¢, 5¢), 67.4 (t, -COOCH2CH2CH3),
22.3 (t, -COOCH2CH2CH3), 10.8 (q, -COOCH2CH2CH3). ESI-
MS (m/z,%): 320 [M + H]+. HRESI-MS m/z 320.1099 [M + H]+
(calcd for C17H16F2NO3 320.1098).
(d, C-4), 134.1 (d, JC-F = 9 Hz, C-4¢), 132.5 (d, C-5¢), 125.1 (d, JC-F
=
3 Hz, C-6¢), 122.7 (s, JC-F = 12 Hz, C-1¢), 119.3 (d, C-5), 116.6 (d,
JC-F = 24 Hz, C-3¢), 113.5 (s, C-3), 62.3 (t, -COOCH2CH3), 14.4
(q, -COOCH2CH3). ESI-MS (m/z,%): 289 [M + H]+. HRESI-MS
m/z 289.0986 [M + Na]+ (calcd for C15H14FN2O3 289.0986).
5.1.1.12. Ethyl 4-(2-fluorobenzamido)nicotinate (23). Com-
pound 23 was synthesized in 49% yield from 2-aminonicotinic
acid (2d) in a procedure similar to 22. White powder, mp 74–
5.1.1.8. Methyl 2-(2,6-difluoro-N-methylbenzamido)benzoate
◦
1
1
(19). 55% yield. Colorless oil. H NMR (CDCl3) d 7.78 (1H,
76 C. H NMR (CDCl3) d 12.00 (1H, d, J = 8.0 Hz, NH), 9.21
(1H, s, H-2), 8.81 (1H, d, J = 6.0 Hz, H-6), 8.66 (1H, d, J = 6.0 Hz,
H-5), 8.07 (1H, td, J = 8.4, 1.6 Hz, H-6¢), 7.54 (1H, ddd, J = 13.2,
8.4, 1.6 Hz, H-4¢), 7.30 (1H, t, J = 8.4 Hz, H-5¢), 7.22 (1H, dd,
J = 11.2, 8.4 Hz, H-3¢), 4.46 (2H, q, J = 6.8 Hz, OCH2CH3), 1.44
(3H, t, J = 6.8 Hz, OCH2CH3). 13C NMR (CDCl3) d 167.5 (s,
-COOCH2CH3), 163.2 (s, -CONH-),160.2 (s, JC-F = 250 Hz, C-2¢),
155.0 (d, C-2), 152.9 (d, C-6), 147.7 (s, C-4), 134.6 (d, JC-F = 9 Hz,
dd, J = 8.0, 1.2 Hz, H-6), 7.45 (1H, td, J = 8.0, 1.2 Hz, H-4),
7.36 (1H, d, J = 8.0 Hz, H-3), 7.27 (1H, td, J = 8.0, 1.2 Hz, H-5),
7.08 (1H, tt, J = 8.0, 6.8 Hz, H-4¢), 6.63 (2H, m, H-3¢, 5¢), 3.90
(3H, OCH3), 3.89 (3H, NCH3). 13C NMR (CDCl3) d 166.3 (s,
-COOCH3), 165.8 (s, -CONCH3), 158.7(s, JC-F = 250 Hz, C-2¢, 6¢),
142.1 (s, C-2), 133.4 (d, C-4), 131.9 (d, C-6), 131.4 (d, JC-F = 10 Hz,
C-4¢), 129.9 (d, C-5), 29.1 (s, C-1), 129.0 (d, C-3), 125.0 (s, JC-F
=
22 Hz, C-1¢), 111.6 (d, JC-F = 22 Hz, C-3¢, 5¢), 52.8 (q, -COOCH3),
37.7 (q, -CONCH3). ESI-MS (m/z,%): 306 [M + H]+. HRESI-MS
m/z 306.0941 [M + H]+ (calcd for C16H14F2NO3 306.0942).
C-6¢), 132.3 (d, C-5¢), 125.4 (d, JC-F = 3 Hz, C-4¢), 122.1 (d, JC-F =
12 Hz, C-3¢), 116.9 (s, JC-F = 23 Hz, C-1¢), 114.7 (d, C-5), 112.0 (s,
C-3), 62.2 (t, -COOCH2CH3), 14.5 (q, -COOCH2CH3). ESI-MS
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(m/z,%): 289 [M + H]+. HRESI-MS m/z 289.0989 [M + H]+ (calcd
for C15H14FN2O3 289.0988).
5.1.2.1. Ethyl 2-(2-bromophenylsulfonamido)benzoate (34).
23% yield. Pale yellow powder, mp 88–90 ◦C. 1H NMR (CDCl3)
d 11.33 (1H, br. s, NH), 8.26 (1H, dd, J = 7.6, 1.2 Hz, H-3), 7.96
(1H, dd, J = 8.0, 1.6 Hz, H-6), 7.63 (1H, d, J = 8.4 Hz, H-3¢),
7.49 (1H, d, J = 8.4 Hz, H-6¢), 7.44 (1H, td, J = 7.2, 1.2 Hz, H-4),
7.35 (2H, m, H-4¢, 5¢), 6.97 (1H, t, J = 7.6 Hz, H-5), 4.38 (2H,
q, J = 7.2 Hz, OCH2CH3), 1.39 (3H, t, J = 7.2 Hz, OCH2CH3).
13C NMR (CDCl3) d 168.2 (s, -COOCH2CH3), 140.2 (s, C-2),
138.8 (s, C-1¢), 135.9 (d, C-4), 134.7 (d, C-3¢), 134.5 (d, C-4¢),
132.7 (d, C-6), 131.8 (d, C-6¢), 128.0 (d, C-5¢), 122.7 (s, C-1), 120.7
(s, C-2¢), 117.2 (d, C-5), 115.9 (d, C-3), 62.1 (t, -COOCH2CH3),
14.6 (q, -COOCH2CH3). ESI-MS (m/z,%): 406 [M + Na]+, 408
[M + 2 + Na]+. HRESI-MS m/z 405.9725 [M + Na]+ (calcd for
C15H14BrNO4SNa 405.9728).
5.1.2.2. Propyl 2-(2-fluorophenylsulfonamido)benzoate (35).
29% yield. Pale yellow powder, mp 79–81 ◦C. 1H NMR (CDCl3)
d 11.13 (1H, br. s, NH), 7.98 (2H, m, H-6, 6¢), 7.63 (1H, d, J =
8.4 Hz, H-3), 7.54 (1H, m, H-4¢), 7.40 (1H, td, J = 8.4, 1.2 Hz,
H-4), 7.27 (1H, t, J = 7.6 Hz, H-5¢), 7.11 (1H, t, J = 9.6 Hz,
H-3¢), 7.03 (1H, t, J = 7.6 Hz, H-5), 4.30 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 1.79 (2H, qt, J = 7.2, 6.8 Hz, OCH2CH2CH3),
1.02 (3H, t, J = 7.2 Hz, OCH2CH2CH3). 13C NMR (CDCl3) d
168.2 (s, -COOCH2CH2CH3), 159.3 (s, C-2¢, JC-F = 255 Hz), 140.3
(s, C-2), 136.1 (d, C-4¢, JC-F = 9 Hz), 134.8 (d, C-4), 131.7 (d,
C-6¢), 131.3 (d, C-6), 127.5 (s, C-1¢, JC-F = 14 Hz), 124.8 (d, C-
5¢, JC-F = 4 Hz), 123.2 (d, C-5), 118.1 (d, C-3), 117.6 (d, C-3¢,
JC-F = 21 Hz), 116.2 (s, C-1), 67.7 (t, -COOCH2CH2CH3), 22.3
(t, -COOCH2CH2CH3), 10.9(q, t, -COOCH2CH2CH3). ESI-MS
(m/z,%): 360 [M + Na]+. HRESI-MS m/z 360.0682 [M + Na]+
(calcd for C16H16FNO4SNa 360.0684).
5.1.1.13. Ethyl 2-(2-chlorobenzamido)nicotinate (24). Com-
pound 24 was synthesized in 58% yield from 2-aminonicotinic
acid◦(2d) in a procedure similar to 8. Pale yellow powder, mp 108–
110 C. 1H NMR (CDCl3) d 11.25 (1H, br. s, NH), 8.63 (1H, dd,
J = 4.8, 1.6 Hz, H-6), 8.35 (1H, dd, J = 8.0, 2.0 Hz, H-4), 7.67
(1H, dd, J = 8.0, 2.0 Hz, H-3 ¢), 7.43 (1H, td, J = 8.0, 2.0 Hz,
H-5 ¢), 7.38 (1H, dd, J = 8.0, 2.0 Hz, H-6 ¢), 7.35 (1H, td, J = 8.0,
2.0 Hz, H-4 ¢), 7.13 (1H, dd, J = 8.0, 4.8 Hz, H-5), 4.33 (1H, q,
J = 6.8 Hz, OCH2CH3), 1.38 (1H, t, J = 6.8 Hz, OCH2CH3). 13
C
NMR (CDCl3) d 166.3 (s, -COOCH2CH3), 164.3 (s, -CONH-),
152.7 (d, C-6), 151.8 (s, C-2), 139.6 (s, C-1¢), 135.9 (d, C-4), 131.2
(s, C-2¢), 130.7 (d, C-4¢), 130.1 (d, C-3¢), 129.2 (d, C-6¢), 126.8 (d,
C-5¢), 118.6 (d, C-5), 112.1 (s, C-3), 61.8 (t, -COOCH2CH3), 13.8
(q, -COOCH2CH3). ESI-MS (m/z,%): 305 [M + H]+, 307 [M + 2 +
H]+. HRESI-MS m/z 305.0691 [M + H]+ (calcd for C15H14ClN2O3
305.0693).
5.1.1.14. Ethyl 2-(2-bromobenzamido)nicotinate (25). Com-
pound 25 was synthesized in 62% yield from 2-aminonicotinic acid
(3d) in a procedure similar to 9. Pale yellow powder, mp 98–100 ◦C.
1H NMR (CDCl3) d 11.21 (1H, br. s, NH), 8.61 (1H, dd, J = 4.8,
2.0 Hz, H-6), 8.34 (1H, dd, J = 8.0, 2.0 Hz, H-4), 7.61 (2H, dd,
J = 8.0, 0.8 Hz, H-3¢, 6¢), 7.39 (1H, td, J = 8.0, 0.8 Hz, H-5¢),
7.30 (1H, td, J = 8.0, 0.8 Hz, H-4¢), 7.13 (1H, dd, J = 8.0, 4.8 Hz,
H-5), 4.37 (2H, q, J = 6.4 Hz, OCH2CH3), 1.37 (3H, t, J = 6.4 Hz,
OCH2CH3). 13C NMR (CDCl3) d 166.9 (s, -COOCH2CH3), 165.7
(s, -CONH-), 153.2 (d, C-6), 152.3 (s, C-2), 140.3 (d, C-4), 138.7
(s, C-1¢), 133.9 (d, C-3¢), 131.8 (d, C-4¢), 129.4 (d, C-6¢), 128.0
(d, C-5¢), 119.7 (d, C-5), 119.3 (s, C-2¢), 112.8 (s, C-3), 62.4 (t,
-COOCH2CH3), 14.48 (q, -COOCH2CH3). ESI-MS (m/z,%): 349
[M + H]+, 357 [M + 2 + H]+. HRESI-MS m/z 349.0185 [M + H]+
(calcd for C15H14BrN2O3 349.0188).
5.1.2.3. Propyl 2-(2-chlorophenylsulfonamido)benzoate (36).
◦
1
39% yield. Yellow powder, mp 83–85 C. H NMR (CDCl3) d
11.33 (1H, br. s, NH), 8.22 (1H, dd, J = 7.6, 1.6 Hz, H-3), 7.98
(1H, dd, J = 8.0, 1.6 Hz, H-6), 7.54 (1H, d, J = 8.4 Hz, H-6¢),
7.41 (4H, m, H-4, 3¢, 4¢, 5¢), 7.00 (1H, t, J = 8.0 Hz, H-5), 4.30
(2H, t, J = 6.8 Hz OCH2CH2CH3), 1.80 (2H, qt, J = 7.2, 6.8 Hz,
OCH2CH2CH3), 1.03 (3H, t, J = 7.2 Hz OCH2CH2CH3). 13C NMR
(CDCl3) d 168.2 (s, -COOCH2CH2CH3), 140.23 (s, C-2), 136.9 (s,
C-1¢), 134.8 (d, C-2¢), 134.7 (d, C-6), 132.5 (d, C-4¢), 134.4 (d,
C-3¢), 132.4 (d, C-4), 131.8 (d, C-6¢), 127.4 (d, C-5¢), 122.9 (d,
C-5), 117.2 (d, C-3), 115.8 (s, C-1), 67.6 (d, -COOCH2CH2CH3),
22.3 (d, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3). ESI-
MS (m/z,%): 376 [M + Na]+, 378 [M + 2 + Na]+. HRESI-MS m/z
376.0386 [M + Na]+ (calcd for C16H16ClNO4S Na 376.0384).
5.1.2.4. Propyl 2-(2-bromophenylsulfonamido)benzoate (37).
5.1.1.15. Methyl
3-(2-fluorobenzamido)pyrazine-2-carbo-
xylate (26). Compound 26 was synthesized in 14% yield from
3-aminopyrazine-2-carboxylic acid (3e) ◦in a procedure similar to
1
described. White powder, mp 123–125 C. H NMR (CDCl3) d
11.58 (1H, d, J = 9.6 Hz, NH), 8.70 (1H, d, J = 2.0 Hz, H-6),
8.47 (1H, d, J = 2.0 Hz, H-5), 8.16 (1H, t, J = 7.6 Hz, H-6¢), 7.57
(1H, ddd, J = 7.6, 5.2, 2.0 Hz, H-4¢), 7.33 (1H, t, J = 7.6 Hz,
H-5¢), 7.23 (1H, dd, J = 11.6, 7.6 Hz, H-3¢), 4.08 (3H, s, OCH3).
13C NMR (CDCl3) d 166.5 (s, -COOCH3), 161.6 (s, -CONH-),
159.6 (s, JC-F = 248 Hz, C-2¢), 149.8 (s, C-3), 147.0 (d, C-6), 139.2
(d, C-5), 134.8 (d, JC-F = 9 Hz, C-6¢), 132.8 (d, C-5¢), 130.4 (s,
◦
1
21% yield. Yellow powder, mp 101–103 C. H NMR (CDCl3) d
11.37 (1H, br. s, NH), 8.28 (1H, dd, J = 8.0, 1.6 Hz, H-3), 7.79
(1H, dd, J = 8.0, 1.2 Hz, H-6), 7.64 (1H, d, J = 7.2 Hz, H-3¢),
7.51 (1H, d, J = 8.8 Hz, H-6¢), 7.46 (1H, t, J = 7.2 Hz, H-4),
7.36 (1H, t, J = 7.2 Hz, H-4¢, 5¢), 6.99 (1H, t, J = 7.6 Hz, H-5),
4.30 (1H, t, J = 6.8 Hz, OCH2CH2CH3), 1.80 (2H, qt, J = 7.2,
6.8 Hz, OCH2CH2CH3), 1.08 (3H, t, J = 7.2 Hz, OCH2CH2CH3).
13C NMR (CDCl3) d 168.2 (s, -CO), 140.2 (s, C-2), 138.7 (s, C-
1¢), 135.9 (d, C-4), 134.8 (d, C-3¢), 134.6 (d, C-4), 132.7 (d, C-
6), 131.8 (d, C-6¢), 128.0 (d, C-5¢), 122.7 (s, C-1), 120.7 (s, C-
2¢), 117.1 (d, C-5), 115.8 (d, C-3), 67.6 (t, -COOCH2CH2CH3),
22.4 (t, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3). ESI-
MS (m/z,%): 420 [M + Na]+, 422 [M + 2 + Na]+. HRESI-MS m/z
419.9880 [M + Na]+ (calcd for C16H16BrNO3SNa 419.9880).
C-2), 125.4 (d, C-4¢), 121.8 (d, JC-F = 11 Hz, C-3¢), 116.8 (s, JC-F
=
23 Hz, C-1¢), 54.0 (q, -COOCH3). ESI-MS (m/z,%): 276 [M +
H]+. HRESI-MS m/z 276.0786 [M + H]+ (calcd for C13H11FN3O3
276.0784).
5.1.2 General procedure for synthesis of 2-phenyl-
sulfonamidobenzoic esters (29–37). Compounds 3a–3c were
synthesized in a procedure similar to described. To a mixture
solution of 3a–3c (1.0 mmole) in DCM (20.0 ml) was added the
corresponding benzenesulfonyl chloride (28a–28c, 1.5 mmole).
The reaction mixture was 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–acetone to afford the products.
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5.1.3. General procedure for synthesis of 2-phenyl-
acetamidobenzoic esters (40–51) and 3-phenylpropanamido-
benzoic esters (52–54). Compounds 3a–3d were synthesized
in a procedure similar to described. To a solution of 3a–3d
(1.0 mmole) with the corresponding phenylacetic acids (38a–38c)
or phenylpropionic acids (39a–39c) (each 1.5 mmole) in CH2Cl2
(20 mL), respectively, were added successively coupling agents
HBTU (1.5 mmol) and DIEA (3.0 mmol). The reaction mixture
was stirred at room temperature for 24 h. The solvent was
evaporated at reduced pressure. The residue was purified by silica
gel column chromatography using a mixture of n-hexane–acetone
to afford the products.
8.02 (1H, dd, J = 7.6, 1.2 Hz, H-6), 7.62 (1H, d, J = 8.0 Hz, H-3¢),
7.53 (1H, td, J = 7.6, 1.2 Hz, H-4), 7.45 (1H, dd, J = 8.0, 1.6 Hz,
H-6 ¢), 7.36 (1H, t, J = 8.0 Hz, H-5 ¢), 7.20 (1H, td, J = 8.0, 1.6 Hz,
H-4¢), 7.08 (1H, t, J = 7.6 Hz, H-5), 4.32 (2H, q, J = 7.2 Hz
OCH2CH3), 3.96 (2H, s, Ar-CH2-NCO), 1.39 (3H, t, J = 7.2 Hz,
OCH2CH3). 13C NMR (CDCl3) d 169.0 (s, -COOCH2CH3), 168.3
(s, -CONH-), 141.7 (s, C-2), 134.8 (s, C-1¢), 133.4 (s, C-4), 132.4
(s, C-3¢), 131.1 (s, C-4¢), 129.5 (s, C-6¢), 128.2 (s, C-5¢), 125.8
(s, C-5), 123.0 (s, C-3), 123.9 (s, C-2), 116.0 (s, C-1), 61.7 (s,
-COOCH2CH3), 46.1 (s, CH2), 14.6 (s, -COOCH2CH3). ESI-MS
(m/z,%): 384 [M + Na]+, 386 [M + 2 + Na]+. HRESI-MS m/z
384.0211 [M + Na]+ (calcd for C17H16BrNO3Na 384.0214).
5.1.3.1. Methyl
2-(2-(2-bromophenyl)acetamido)benzoate
5.1.3.5. Propyl
2-(2-(2-fluorophenyl)acetamido)benzoate
◦
1
(42). 24% yield. Pale yellow powder, Mp: 88–90 C. H NMR
(CDCl3) d 11.03 (1H, br. s, NH), 8.73 (1H, d, J = 8.4 Hz, H-3),
7.98 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.61 (1H, d, J = 8.0 Hz, H-6¢),
7.51 (1H, td, J = 8.4, 1.2 Hz, H-4), 7.43 (1H, dd, J = 7.2, 1.2 Hz,
H-5 ¢), 7.35 (1H, t, J = 7.2 Hz, H-4 ¢), 7.19 (1H, td, J = 8.0, 1.2 Hz,
H-3¢), 7.06 (1H, t, J = 8.0 Hz, H-5), 3.94 (2H, s, Ar-CH2-NCO),
3.84 (3H, s, OCH3). 13C NMR (CDCl3) d 169.1 (s, -COOCH3),
168.7(s, -CONH-), 141.6(s, C-2), 134.9(s, C-1¢), 134.7 (d, C-4),
133.5 (d, C-3¢), 131.2 (d, C-4¢), 129.5 (d, C-6¢), 128.3 (d, C-5¢),
125.8 (d, C-5), 123.1 (d, C-3), 120.9 (s, C-2¢), 115.7 (s, C-1), 52.6
(q, -COOCH3), 46.12 (t, CH2). ESI-MS (m/z,%): 370 [M + Na]+,
372 [M + 2 + Na]+. HRESI-MS m/z 370.0055 [M + Na]+ (calcd
for C16H14BrNO3Na 370.0057).
5.1.3.2. Ethyl 2-(2-(2-fluorophenyl)acetamido)benzoate (43).
25% yield. Yellow oil. 1H NMR (CDCl3) d 11.18 (1H, br. s, NH),
8.71 (1H, d, J = 8.0 Hz, H-3), 8.01 (1H, dd, J = 8.0, 1.6 Hz, H-
6), 7.51 (1H, td, J = 8.0, 1.6 Hz, H-6¢), 7.39 (1H, dd, J = 8.0,
1.6 Hz, H-4), 7.29 (1H, m, H-4¢), 7.15 (1H, t, J = 8.0 Hz, H-5¢),
7.10 (1H, t, J = 9.6 Hz, H-3¢), 7.06 (1H, t, J = 8.0 Hz, H-5), 4.36
(2H, q, J = 7.2 Hz, OCH2CH3), 3.84 (2H, s, Ar-CH2-NCO), 1.40
(3H, t, J = 7.2 Hz, OCH2CH3). 13C NMR (CDCl3) d 169.3 (s,
-COOCH2CH3), 168.4 (s, -CONH-), 162.8 (s, C-2¢, JC-F = 245 Hz),
141.78 (s, C-2), 134.8 (d, C-4), 132.2 (d, C-4¢, JC-F = 4 Hz), 131.1
(d, C-6), 129.7 (d, C-6¢, JC-F = 8 Hz), 124,8 (d, C-5¢, JC-F = 4 Hz),
123.0 (d, C-5), 122.1 (s, C-1¢, JC-F = 16 Hz), 120.8 (d, C-3), 116.0
(s, C-1), 115.9 (d, C-3¢, JC-F = 4 Hz), 61.7 (t, -COOCH2CH3), 39.1
(t, CH2, J = 3 Hz), 14.6 (q, -COOCH2CH3). ESI-MS (m/z,%):
324 [M + Na]+. HRESI-MS m/z 324.1012 [M + Na]+ (calcd for
C17H16FNO3Na 324.1010).
(46). 38% yield. Pale yellow oil. 1H NMR (CDCl3) d 11.23 (1H,
br. s, NH), 8.74 (1H, d, J = 8.8 Hz, H-3), 8.03 (1H, dd, J = 8.0,
1.6 Hz, H-6), 7.53 (1H, td, J = 8.8, 1.6 Hz, H-4), 7.42 (1H, td,
J = 8.0, 1.2 Hz, H-6¢), 7.31 (1H, m, H-4¢), 7.17 (1H, td, J = 8.0,
1.2 Hz, H-5¢), 7.12 (1H, dd, J = 8.8, 1.2 Hz, H-3¢), 7.08 (1H, t, J =
8.0 Hz, H-5), 4.26 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 3.84 (2H,
s, Ar-CH2-NCO), 1.80 (2H, qt, J = 7.6, 6.8 Hz, OCH2CH2CH3),
1.05 (3H, t, J = 7.6 Hz, OCH2CH2CH3). 13C NMR (CDCl3)
d 169.3 (s, -COOCH2CH2CH3), 168.5 (s, -CONH-), 162.8 (s,
C-2¢, JC-F = 244 Hz), 141.8 (s, C-2), 134.8 (d, C-4), 132.2 (d,
C-4¢, JC-F = 4 Hz), 131.1 (d, C-6), 129.7 (d, C-6¢, JC-F = 8 Hz),
124.9 (d, C-5¢, JC-F = 4 Hz), 123.0 (d, C-5), 122.2 (d, C-1¢, JC-F
=
15 Hz), 120.8 (d, C-3), 116.1 (d, C-3¢, JC-F = 16 Hz), 115.9 (s,
C-1), 67.3 (t, -COOCH2CH2CH3), 39.1 (t, JC-F = 2 Hz, CH2), 22.4
(t, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3). ESI-MS
(m/z,%): 338 [M + Na]+. HRESI-MS m/z 338.1168 [M + Na]+
(calcd for C18H18FNO3Na 338.1165).
5.1.3.6. Propyl
2-(2-(2-chlorophenyl)acetamido)benzoate
◦
1
(47). 40% yield. pale yellow powder, mp 101–103 C. H NMR
(CDCl3) d 11.16 (1H, br. s, NH), 8.75 (1H, d, J = 8.0 Hz, H-3), 8.02
(1H, dd, J = 8.0, 1.2 Hz, H-6, H-6), 7.53 (1H, td, J = 8.0, 1.2 Hz,
H-4), 7.44 (2H, m, H-3¢, 6¢), 7.29 (2H, m, H-4¢, 5¢), 7.08 (1H, t, J =
8.0 Hz, H-5), 4.32 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 3.94 (2H, s,
Ar-CH2-NCO), 1.78 (2H, qt, J = 7.2, 6.8 Hz, OCH2CH2CH3), 1.04
(3H, t, J = 7.2 Hz, OCH2CH2CH3). 13C NMR (CDCl3) d 169.2 (s,
-COOCH2CH2CH3), 168.4 (s, -CONH-), 141.7 (s, C-2), 135.2 (s,
C-1¢), 134.8 (d,C-4), 133.0 (s, C-2¢), 132.3 (d, C-6), 131.0 (d, C-4¢),
130.1(d, C-3¢), 129.3 (d, C-6¢), 127.6 (d, C-5¢), 123.0 (d, C-5),
120.9 (d, C-3), 116.0 (s, C-1), 67.2 (t, -COOCH2CH2CH3), 43.6 (t,
CH2), 22.3 (t, -COOCH2CH2CH3), 10.9 (q, -COOCH2CH2CH3).
ESI-MS (m/z,%): 354 [M + Na]+, 356 [M + 2 + Na]+. HRESI-MS
m/z 354.0873 [M + Na]+ (calcd for C18H18ClNO3Na 354.0870).
5.1.3.3. Ethyl 2-(2-(2-chlorophenyl)acetamido)benzoate (44).
◦
1
22% yield. pale yellow powder, mp 59–61 C. H NMR (CDCl3)
d 11.12 (1H, br. s, NH), 8.72 (1H, d, J = 8.0 Hz, H-3), 8.03 (1H,
dd, J = 8.0, 1.2 Hz, H-6), 7.51 (1H, td, J = 8.0, 1.2 Hz, H-4), 7.42
(2H, m, H-3¢, 6¢), 7.28 (2H, m, H-4¢, 5¢), 7.07 (1H, t, J = 8.0 Hz,
H-5), 4.31 (2H, q, J = 6.8 Hz, OCH2CH3), 3.92 (2H, s, Ar-CH2-
NCO), 1.37 (3H, t, J = 6.8 Hz, OCH2CH3). 13C NMR (CDCl3) d
169.2 (s, -COOCH2CH3), 168.4 (s, -CONH-), 141.7 (s, C-2), 135.2
(s, C-1¢), 134.8 (s, C-4), 133.0 (d, C-6), 132.3 (d, C-4¢), 131.1 (s,
C-2¢), 130.1 (d, C-3¢), 129.3 (d, C-6¢), 127.6 (d, C-5¢), 123.0 (d, C-
5), 120.9 (d, C-3), 116.0 (s, C-1), 61.7 (t, -COOCH2CH3), 43.6 (t,
CH2), 14.6 (q, -COOCH2CH3). ESI-MS (m/z,%): 340 [M + Na]+,
342 [M + 2 + Na]+. HRESI-MS m/z 340.0716 [M + Na]+ (calcd
for C17H16ClNO3Na 340.0713).
5.1.3.7. Propyl
2-(2-(2-bromophenyl)acetamido)benzoate
◦
1
(48). 40% yield. Pale yellow needles, mp 57–59 C. H NMR
(CDCl3) d 11.14 (1H, br. s, NH), 8.75 (1H, d, J = 8.0 Hz, H-3),
8.03 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.63 (1H, d, J = 8.0 Hz, H-6¢),
7.54 (1H, td, J = 8.4, 1.2 Hz, H-4), 7.45 (1H, dd, J = 7.6, 1.2 Hz,
H-3¢), 7.36 (1H, td, J = 8.4, 1.2 Hz, H-5¢), 7.20 (1H, td, J = 7.6,
1.2 Hz,H-4¢), 7.09 (1H, t, J = 7.6 Hz, H-5), 4.23 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 3.96 (2H, s, Ar-CH2-NCO), 1.80 (2H, qt, J = 7.2,
6.8 Hz, OCH2CH2CH3), 1.05 (3H, t, J = 7.2 Hz, OCH2CH2CH3).
13C NMR (CDCl3) d 169.1 (s, -COOCH2CH2CH3), 168.4 (s,
-CONH-), 141.7 (s, C-2), 134.8 (d, C-4), 134.8 (s, C-1¢), 133.4 (d,
C-3¢), 132.4 (d, C-6), 131.1 (d, C-4¢), 129.5 (d, C-6¢), 128.2 (d,
C-5¢), 125.8 (d, C-5), 123.0 (d, C-3), 120.1 (s, C-2¢), 116.0 (s, C-1),
5.1.3.4. Ethyl
2-(2-(2-bromophenyl)acetamido)benzoate
◦
1
(45). 24% yield. pale yellow powder, mp 83–85 C. H NMR
(CDCl3) d 11.13 (1H, br. s, NH), 8.75 (1H, d, J = 7.6 Hz, H-3),
7122 | Org. Biomol. Chem., 2011, 9, 7113–7125
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67.2 (t, -COOCH2CH2CH3), 46.1 (t, -COOCH2CH2CH3), 22.4 (t,
CH2), 10.9 (q, -COOCH2CH2CH3). ESI-MS (m/z,%): 398 [M +
Na]+, 400 [M + 2 + Na]+. HRESI-MS m/z 398.0368 [M + Na]+
(calcd for C18H18BrNO3Na 398.0369).
ESI-MS (m/z,%): 390 [M + H]+, 392 [M + 2 + H]+. HRESI-MS
m/z 390.0706 [M + H]+ (calcd for C19H21ClNO3 390.0705).
5.1.3.11. Ethyl 2-(3-(2-fluorophenyl)propanamido)benzoate
◦
1
(52). 39% yield. Pale yellow powder, mp 58–60 C. H NMR
(CDCl3) d 11.12 (1H, br. s, NH), 8.71 (1H, d, J = 8.0 Hz, H-3),
8.02 (1H, dd, J = 8.0, 1.2 Hz, H-6), 7.52 (1H, td, J = 8.8, 1.6 Hz,
H-6¢), 7.26 (1H, td, J = 8.0, 1.2 Hz, H-4), 7.17 (1H, m, H-4¢), 7.03
(3H, m, H-5, 3¢, 5¢), 4.35 (2H, q, J = 6.8 Hz, OCH2CH3), 3.10
(2H, t, J = 8.0 Hz, CH2CH2), 2.76 (2H, t, J = 8.0 Hz, CH2CH2),
1.40 (3H, t, J = 6.8 Hz, OCH2CH3). 13C NMR (CDCl3) d 171.2 (s,
-COOCH2CH3), 168.6 (s, -CONH-), 161.6 (s, C-2 ¢, JC-F = 244 Hz),
141.9 (s, C-2), 134.9 (s, C-4), 131.2 (d, C-6 ¢, JC-F = 6 Hz), 131.1 (d,
C-6), 128.4 (d, C-4 ¢, JC-F = 8 Hz), 127.9 (s, C-1 ¢, JC-F = 15 Hz),
124.5 (d, C-5 ¢, JC-F = 4 Hz), 122.7 (d, C-5), 120.8 (d, C-3), 115.8 (s,
C-3 ¢, JC-F = 22 Hz), 115.6 (d, C-1), 61.8 (t, -COOCH2CH3), 38.9
(t, CH2CH2), 25.3 (t, CH2CH2, J = 3 Hz), 14.6 (q, -COOCH2CH3).
ESI-MS (m/z,%): 316 [M + H]+. HRESI-MS m/z 316.1346 [M +
H]+ (calcd for C18H19FNO3 316.1349).
5.1.3.8. Butyl 2-(2-(2-fluorophenyl)acetamido)benzoate (49).
62% yield. Pale yellow powder, mp 48–50 ◦C. 1H NMR (CDCl3) d
11.20 (1H, br. s, NH), 8.72 (1H, d, J = 8.4 Hz, H-3), 7.95 (1H, d, J =
7.6 Hz, H-6), 7.42 (1H, t, J = 8.4 Hz, H-4), 7.36 (1H, t, J = 7.6 Hz,
H-6¢), 7.24 (1H, ddd, J = 7.2, 6.4, 1.6 Hz, H-4¢), 7.10 (1H, t, J =
7.6 Hz, H-5¢), 7.07 (1H, t, J = 9.2 Hz, H-3¢), 7.00 (1H, t, J = 7.6 Hz,
H-5), 4.23 (2H, t, J = 6.8 Hz, OCH2CH2CH2CH3), 3.78 (2H, s,
Ar-CH2-NCO), 2.67 (2H, q, J = 8.0, 6.8 Hz, OCH2CH2CH2CH3),
1.42 (2H, qt, J = 8.0, 7.2 Hz, OCH2CH2CH2CH3), 0.95 (3H, t,
J = 7.2 Hz, OCH2CH2CH2CH3). 13C NMR (CDCl3) d 169.1 (s,
-CONH-), 168.4 (s, -COOCH2CH2CH2CH3), 161.6 (s, C-2¢, JC-F
245 Hz), 141.1 (s, C-2), 134.0 (d, C-4), 131.5 (d, C-6¢, JC-F = 3 Hz),
130.3 (d, C-6), 128.9 (s, C-4¢, JC-F = 8 Hz), 124.0 (d, C-5¢, JC-F
=
=
3 Hz), 122.2 (d, C-5), 121.4 (d, C-1¢, JC-F = 16 Hz), 120.0 (d, C-3),
115.3 (d, C-3¢), 115.0 (d, C-1), 65.5 (t, -COOCH2CH2CH2CH3),
40.0 (t, CH2, J = 2 Hz), 31.0 (t, -COOCH2CH2CH2CH3), 19.6 (t,
-COOCH2CH2CH2CH3), 14.1 (q, -COOCH2CH2CH2CH3). ESI-
MS (m/z,%): 330 [M + H]+. HRESI-MS m/z 330.1504 [M + H]+
(calcd for C19H21FNO3 330.1505).
5.1.3.12. Ethyl 2-(3-(2-chlorophenyl)propanamido)benzoate
(53). 37% yield. White powder, mp 48–50 ◦C. 1H NMR (CDCl3)
d 11.13 (1H, br. s, NH), 8.72 (1H, d, J = 8.4 Hz, H-3), 8.06 (1H, dd,
J = 8.0, 1.2 Hz, H-6), 7.51 (1H, td, J = 8.4, 1.2 Hz, H-4), 7.38 (1H,
dd, J = 7.2, 1.2 Hz, H-3¢), 7.29 (1H, dd, J = 7.2, 1.6 Hz, H-6¢), 7.15
(1H, td, J = 7.2, 1.2 Hz, H-5¢), 7.12 (1H, td, J = 7.2, 1.6 Hz, H-4¢),
7.04 (1H, t, J = 8.0 Hz, H-5), 4.39 (2H, q, J = 7.2 Hz, OCH2CH3),
3.18 (2H, t, J = 8.0 Hz, CH2CH2), 2.76 (2H, t, J = 8.0 Hz, CH2CH2),
1.38 (3H, t, J = 7.2 Hz, OCH2CH3). 13C NMR (CDCl3) d 171.2 (s,
-COOCH2CH3), 168.6 (s, -CONH-), 141.9 (s, C-2), 138.6 (s, C-4),
134.9 (s, C-1¢), 134.4 (d, C-6), 131.2 (s, C-2¢), 131.0 (d, C-4¢), 130.0
(d, C-3¢), 128.2 (d, C-6¢), 127.3 (d, C-5¢), 122.7 (d, C-5), 120.7 (d,
C-3), 115.5 (s, C-1), 61.8 (t, -COOCH2CH3), 38.5 (t, CH2CH2),
29.7 (t, CH2CH2), 14.6 (q, -COOCH2CH3). ESI-MS (m/z,%): 332
[M + H]+, 334[M + 2 + H]+. HRESI-MS m/z 332.1054 [M + H]+
(calcd for C18H19ClNO3 332.1053).
5.1.3.9. Butyl
2-(2-(2-chlorophenyl)acetamido)benzoate
◦
1
(50). 61% yield. pale yellow powder, mp 49–51 C. H NMR
(CDCl3) d 11.14 (1H, br. s, NH), 8.74 (1H, d, J = 8.0 Hz, H-3),
7.94 (1H, d, J = 8.0 Hz, H-6), 7.43 (1H, td, J = 8.0 Hz, 1.2 Hz,
H-4), 7.38 (1H, d, J = 7.6 Hz, H-6¢), 7.36 (1H, dd, J = 8.0,
1.2 Hz, H-3¢), 7.23 (1H, td, J = 7.6, 1.6 Hz, H-5¢), 7.69 (1H, td,
J = 7.6, 2.0 Hz, H-4¢), 6.99 (1H, t, J = 8.0 Hz, H-5), 4.20 (2H,
t, J = 6.8 Hz, OCH2CH2CH2CH3), 3.89 (2H, s, Ar-CH2-NCO),
1.66 (2H, tt, J = 7.2, 6.8 Hz, OCH2CH2CH2CH3), 1.41 (2H, qt,
J = 7.2, 6.8 Hz, OCH2CH2CH2CH3), 0.95 (3H, t, J = 6.8 Hz,
OCH2CH2CH2CH3). 13C NMR (CDCl3) d 169.0 (s, -CONH-),
168.3 (s, -COOCH2CH2CH2CH3), 141.8 (s, C-2), 135.1 (d, C-4),
134.7 (s, C-1¢), 133.0 (s, C-2¢), 132.3 (d, C-6), 131.0 (d, C-4¢),
130.0 (d, C-3¢), 129.2 (d, C-6¢), 127.6 (d, C-5¢), 122.9 (d, C-5),
120.7 (d, C-3), 115.8 (s, C-1), 65.4 (t, -COOCH2CH2CH2CH3),
43.5 (t, CH3), 31.0 (t, -COOCH2CH2CH2CH3), 19.6 (t, -
COOCH2CH2CH2CH3), 14.1 (q, -COOCH2CH2CH2CH3).
ESI-MS (m/z,%): 346 [M + H]+, 348 [M + 2 + H]+. HRESI-MS
m/z 346.1207 [M + Na]+ (calcd for C19H21ClNO3 346.1209).
5.1.3.13. Ethyl 2-(3-(2-bromophenyl)propanamido)benzoate
(54). 40% yield. White powder, mp 49–51 ◦C. 1H NMR (CDCl3)
d 11.13 (1H, br. s, NH), 8.72 (1H, d, J = 7.6 Hz, H-3), 8.00 (1H,
d, J = 7.6 Hz, H-6), 7.51 (1H, d, J = 8.0 Hz, H-3¢), 7.50 (1H, d,
J = 7.6 Hz, H-6¢), 7.29 (1H, d, J = 7.6 Hz, H-5¢), 7.20 (1H, t, J =
8.0 Hz, H-4¢), 7.04 (2H, t, J = 7.6 Hz, H-4, 5), 4.34 (2H, q, J =
7.2 Hz, OCH2CH3), 3.18 (2H, t, J = 8.0 Hz, CH2CH2), 2.76 (2H,
t, J = 8.0 Hz, CH2CH2), 1.38 (3H, t, J = 7.2 Hz, OCH2CH3).
13C NMR (CDCl3) d 171.1 (s, -COOCH2CH3), 168.6 (s,
-CONH-), 141.9 (s, C-2), 140.4 (s, C-1¢), 134.9 (d, C-4), 133.3
(d, C-3¢), 131.2 (d, C-6), 131.0 (d, C-4¢), 128.4 (d, C-6¢), 128.0 (d,
C-5¢), 124.8 (d, C-5), 122.8 (d, C-3), 120.7 (s, C-2¢), 115.5 (s, C-1),
61.8 (t, -COOCH2CH3), 38.6 (t, CH2CH2), 32.3 (t, CH2CH2), 14.6
(q, -COOCH2CH3). ESI-MS (m/z,%): 376 [M + H]+, 378 [M + 2 +
H]+. HRESI-MS m/z 376.0546 [M + H]+ (calcd for C18H19BrNO3
376.0548).
5.1.3.10. Butyl
2-(2-(2-bromophenyl)acetamido)benzoate
(51). 74% yield. yellow powder, mp 41–43 ◦C. 1H NMR (CDCl3)
d 11.12 (1H, br. s, NH), 8.74 (1H, d, J = 8.0 Hz, H-3), 7.94 (1H,
d, J = 8.0 Hz, H-6), 7.54 (1H, d, J = 8.0 HzH-3¢), 7.44 (1H, t, J =
7.6 Hz, H-4), 7.39 (1H, d, J = 8.0 Hz, H-6¢), 7.28 (1H, t, J = 7.6 Hz,
H-5¢), 7.11 (1H, t, J = 7.6 Hz, H-4¢), 7.00 (1H, t, J = 7.6 Hz,
H-5), 4.20 (2H, t, J = 6.8 Hz, OCHCH2CH2CH3), 3.90 (2H, s,
Ar-CH2-NCO), 1.67 (2H, tt, J = 7.6, 6.8 Hz, OCHCH2CH2CH3),
1.42 (2H, qt, J = 7.6, 6.8 Hz, OCHCH2CH2CH3), 0.94 (3H, t,
J = 6.8 Hz, OCHCH2CH2CH3). 13C NMR (CDCl3) d 168.9 (s,
-CONH-), 168.3 (s, -COOCH2CH2CH2CH3), 141.8 (s, C-2), 134.8
(d, C-4), 134.7 (d, C-1¢), 133.3 (d, C-3¢), 132.4 (d, C-6), 131.0 (d,
C-4¢), 129.5 (d, C-6¢), 128.2 (d, C-5¢), 125.7 (d, C-5), 122.9 (d, C-3),
120.7 (s, C-2¢), 115.9 (s, C-1), 65.4 (t, -COOCH2CH2CH2CH3),
46.0 (t, CH2), 31.0 (t, -COOCH2CH2CH2CH3), 19.6 (t,
-COOCH2CH2CH2CH3), 14.2 (q, -COOCH2CH2CH2CH3).
5.1.4. (E)-Ethyl
2-(3-(2-fluorophenyl)acrylamido)benzoate
(56). Compound 56 was synthesized in 5% yield from 2-
fluorocinnamic acid (55, 1.5 ◦mmole) in a procedure similar to 54.
1
White powder, mp 108–110 C. H NMR (CDCl3) d 11.43 (1H,
br. s, NH), 8.87 (1H, d, J = 8.8 Hz, H-3), 8.08 (1H, dd, J = 8.0,
1.2 Hz, H-6), 7.88 (1H, d, J = 16.0 Hz, H-a), 7.59 (2H, m, H-4,
6¢), 7.35 (1H, ddd, J = 8.0, 5.8, 1.6 Hz, H-4¢), 7.53 (1H, t, J =
8.0 Hz, H-5¢), 7.13 (1H, dd, J = 10.0, 8.0 Hz, H-3¢), 7.11 (1H, t,
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J = 8.0 Hz, H-5), 6.74 (1H, d, J = 16.0 Hz, H-b), 4.41 (2H, q,
J = 7.2 Hz, OCH2CH3), 1.44 (3H, t, J = 7.2 Hz, OCH2CH3). 13
supernatants were used as a source for the cAMP samples. The
assay was performed according to the manufacturer’s instructions.
C
NMR (CDCl3) d 168.9 (s, -COOCH2CH3), 164.7 (s, -CONH-),
161.8 (s, JC-F = 253 Hz, C-2¢), 142.3 (s, C-2), 135.4 (d, C-a), 135.0
(d, C-4), 131.7 (d, JC-F = 9 Hz, C-4¢), 131.3 (d, C-6), 129.8 (d, C-5¢),
125.1 (d, JC-F = 6 Hz, C-3¢), 124.8 (d, JC-F = 3 Hz, C-6¢), 123.2 (s,
JC-F = 11 Hz, C-1¢), 123.0 (d, C-5), 121.0 (d, C-3), 116.6 (d, C-b),
115.7 (s, C-1), 61.9 (t, -COOCH2CH3), 14.6 (q, -COOCH2CH3).
ESI-MS (m/z,%): 314 [M + H]+. HRESI-MS m/z 314.1194 [M +
H]+ (calcd for C18H17FNO3 314.1192).
5.2.5. Phosphodiesterase assay39,40
. PDE activity was modi-
fied and determined as the method.39,40 Washed human platelets
were used for both PDE3 and PDE5 analyses and human U937
cells for PDE4. Purified protein containing PDE3, 4 or 5 enzyme
and concentration of each test compounds were resuspended in 50
mM Tris–HCl containing 5 mM MgCl2 (pH 7.5). Subsequently,
the enzyme (11.5 mg mL-1, 10 mL) was incubated with Tris–HCl
(80 mL) and 10 mM cyclic GMP or cyclic AMP substrate (final
concentration 1.01 mM containing 0.01 mM [3H]-cyclic GMP or
[3H]-cyclic AMP) was added. After 15 min at 25 ◦C, the samples
were heated to 100 ◦C for 2 min. The reaction was terminated
by boiling for 2 min and the resulting AMP was converted to
adenosine by the addition of 10 mg mL-1 (10 mL) snake venom
nucleotidase and further incubation at 25 ◦C for 20 min. An AGI-
X2 resin (200 ml) was added to bind all unconverted cyclic GMP
or cyclic AMP. After centrifugation, the supernatant was removed
for determination of radiolabelled guanosine or adenosine by a
liquid scintillation counter. The test compounds were dissolved
separately in DMSO and tested at concentrations of 300, 100, 30,
10, 3, 1, and 0.3 mM, respectively.
5.2. Biological assays
5.2.1. Preparation of human neutrophils36. Blood was taken
from healthy human donors (20–32 years old) by venipuncture,
using 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
centrifugation in a Ficoll Hypaque gradient and hypotonic lysis
of erythrocytes.36 Purified neutrophils that contained >98% viable
cells, as determined by the trypan blue exclusion method, were
resuspended in calcium (Ca2+)-free HBSS buffer at pH 7.4, and
were maintained at 4 ◦C before use.
∑
∑
-
-
5.2.2. Measurement of O2 generation36. The assay of O2
5.2.6. Statistical analysis. Results are expressed as the mean
S.E.M. Data were analysed using the GraphPad Prism software
(GraphPad Software, San Diego, CA). Statistical analysis was
performed using Student’s t-test or one-way analysis of variance
(ANOVA) followed by Tukey’s multiple comparison test. A value
of p < 0.05 was considered statistically significant.
generation was based on the SOD-inhibitable reduction of ferri-
cytochrome c.36 In brief, after supplementation with 0.5 mg ml-1
ferricytochrome c and 1 mM Ca2+, neutrophils were equilibrated
at 37 ◦C for 2 min and incubated with drugs for 5 min. Cells
were activated with 100 nM FMLP for 10 min. When FMLP
was used as a stimulant, CB (1 mg ml-1) was incubated for
3 min before activation by the peptide (FMLP/CB). Changes
in absorbance with the reduction of ferricytochrome c at 550
nm were continuously monitored in a double-beam, six-cell
positioner spectrophotometer with constant stirring (Hitachi U-
3010, Tokyo, Japan). Calculations were based on the differences in
the reactions with and without SOD (100 U ml-1) divided by the
extinction coefficient for the reduction of ferricytochrome c (e =
21.1/mM/10 mm).
5.2.7. Cytotoxic assay. Compounds were tested against
HepG2 cells using the MTT method as described before.44 A total
of 1 ¥ 105 HepG2 cells were seeded in 24-well plates for 24 h and
made quiescent by incubating in DMEM containing 0.2% FBS
for 24 h before use and cells were incubated with control and 49
(100 mM) followed by incubating for another 24 h. For the growth
rate determination, isopropanol solution mixed with tetrazolium
salt was added to the wells and incubated for an additional 4 h at
37 ◦C.45 The optical density of the dissolved material was measured
spectrophotometrically at 570 nm, and assays were performed in
triplicate.
5.2.3. Measurement of elastase release. Degranulation of
azurophilic granules was determined by elastase release as de-
scribed previously.36 Experiments were performed using MeO-
Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate.
Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-
nitroanilide (100 mM), neutrophils (5 ¥ 105 ml-1) were equilibrated
at 37 ◦C for 2 min and incubated with drugs for 5 min. Cells
were activated by100 nM FMLP and 0.5 mg ml-1 CB, and changes
in absorbance at 405 nm were continuously monitored to assay
elastase release. The results are expressed as the percent of the
initial rate of elastase release in the FMLP/CB-activated, drug-
free control system.
5.2.8. Preparation of washed human platelets. Human blood
anticoagulated with acid citrate dextrose (ACD) was obtained
from healthy human volunteers who had not taken any
drugs within the last two weeks. The platelet suspension was
then prepared according to the washing procedure previously
described.32,33 Platelets were finally suspended in Tyrode’s solution
containing Ca2+ (2 mM), glucose (11.1 mM) and bovine serum
albumin (3.5 mg ml-1) at a concentration of 3 ¥ 108 platelets ml-1.
5.2.9. Measurement of platelet aggregation. Platelet aggre-
gation was measured turbidimetrically with a light-transmission
aggregometer (Chrono-Log Co., USA). The platelet suspension
was incubated with dimethyl sulfoxide (DMSO, vehicle) or test
compounds (10 mM) at 37 ◦C for 3 min under a stirring condition
(1200 rpm) prior to the addition of thrombin as inducer. The extent
of platelet aggregation was measured as the maximal increase of
light transmission within 5 min after the treatment of thrombin.32,33
5.2.4. Determination of cAMP concentrations36,43
.
cAMP
levels were assayed using an enzyme immunoassay kit (Amersham
Biosciences, Buckinghamshire, UK). Human neutrophils were
incubated with drugs for the indicated time before stimulation
with or without FMLP for another 1 min, and the reaction was
terminated by adding 0.5% dodecyltrimethylammonium bromide.
Samples were then centrifuged at 3000 ¥ g for 5 min at 4 ◦C. The
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The Royal Society of Chemistry 2011
©

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21 N. Yamaoka, H. Kodama, Y. Izuhara, T. Miyata and K. Meguro, Chem.
Pharm. Bull., 2011, 59, 215–224.
Acknowledgements
22 L. Lassiani, M. V. Pavan, F. Berti, G. Kokotos, T. Markidis, L. Mennuni,
F. Makovec and A. Varnavas, Bioorg. Med. Chem., 2009, 17, 2336–2350.
23 D. Hadjipavlou-Litina, P. Braiuca, L. Lassiani, M. Pavan and A.
Varnavas, Bioorg. Med. Chem., 2009, 17, 5198–5206.
24 D. De Luca, M. Saviano, L. Lassiani, K. Yannakopoulou, P. Ste-
fanidou, L. Aloj, G. Morelli and A. Varnavas, J. Med. Chem., 2006, 49,
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The investigation was supported by research grants to P. W. Hsieh
from the National Science Council of the Republic of China
(NSC95-2320-B-037-002) and Chang Gung Medical Research
Foundation (CMRPG-690031) in Taiwan.
25 C. Congiu, M. T. Cocco, V. Lilliu and V. Onnis, J. Med. Chem., 2005,
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