Synthesis and bradykinin inhibitory activity of novel non-peptide compounds, and evaluation of in vivo analgesic activity

Article abstract of DOI:10.1016/j.bmc.2010.01.050

A series of novel non-peptide diamide compounds was synthesized and evaluated as antibradykinin agents by utilizing guinea-pig ileum smooth muscle. Among the final compounds, (Z)-4-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)-4-oxo-N-(4-phenylbutan-2-yl)

Full text of DOI:10.1016/j.bmc.2010.01.050

Bioorganic & Medicinal Chemistry 18 (2010) 2327–2336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bradykinin inhibitory activity of novel non-peptide
compounds, and evaluation of in vivo analgesic activity
Yoo Lim Kam a, Hee-Kyung Rhee b, Hwa-Jung Kim a, Seung Keun Back c, Heung Sik Na c,
,
*
Hea-Young Park Choo a,
*
a School of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b Department of Biological Science, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
c Department of Physiology, Korea University College of Medicine, Seoul 136-705, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel non-peptide diamide compounds was synthesized and evaluated as antibradykinin
agents by utilizing guinea-pig ileum smooth muscle. Among the final compounds, (Z)-4-(4-(bis(4-fluoro-
phenyl)methyl)piperazin-1-yl)-4-oxo-N-(4-phenylbutan-2-yl)but-2-enamide showed most favorable
bradykinin inhibitory activity and demonstrated analgesic efficacies in the rat models of inflammatory
and neuropathic pain.
Received 13 November 2009
Revised 18 January 2010
Accepted 20 January 2010
Available online 4 February 2010
Ó 2010 Published by Elsevier Ltd.
Keywords:
Antibradykinin agents
Formalin test
Allodynia
1. Introduction
DRG neurons with a more sustained effect than the B2 receptor.13
Taken together, these results suggested that the B2 receptors played
Bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH), an
endogenous nonapeptide generated by limited proteolysis of
kininogen in tissues and body fluids, elicits numerous responses
including vasodilation, edema, smooth muscle contraction, inflam-
mation, trauma, burns, shock, and allergy.1–3 It induces pain by di-
rect activation of nociceptive nerve terminals, by sensitizing other
fibers to become nociceptors, and by stimulating the release of
other substances such as neurokinin A, substance P, prostanoids,
and cytokines involved in nociception.4–7 Compelling evidence
has shown that bradykinin participates in a number of hyperalge-
sic and inflammatory preclinical models.8,9 The growing knowl-
edge of the biological role of kinins, in particular in pain and
inflammation, has sustained the development of potent and selec-
tive bradykinin receptor modulators as potential therapeutics.10
Two types of bradykinin receptors B1 and B2 have been identi-
fied: the B1 receptors are not present in tissues under normal condi-
tions but are induced during prolonged insults such as chronic
inflammation, and the B2 receptors constitutively expressed in the
nervous system and other tissues involve in the transmission of
nociceptive and acute inflammatory pain.11,12 In addition, the acti-
vation of the B1 receptor showing a small portion of normal dorsal
root ganglion (DRG) neurons exaggerated heat-evoked current in
a key role in nociceptive and acute types of pain, whereas the B1
receptors might be involved in the chronic types of pain.14–16
The formalin test, a commonly used method of inflammatory
pain, produces a biphasic behavioral response. The short-lived first
response is thought to be produced by direct activation of nocicep-
tive neurons by formalin and the second phase is thought to be a
part of an inflammatory response to tissue injury. Several studies
in mice and in rats have shown that bradykinin receptor antago-
nists greatly inhibit the second phase and at higher doses reduce
the first phase of pain behavior.17–27
Injury to a nociceptive pathway can also result in an exaggera-
tion of pain. Peripheral and central nerve injury leads to chronic
neuropathic pain characterized by spontaneous burning pain, allo-
dynia and hyperalgesia. The B1 and B2 receptor gene expression is
up-regulated in animal models of neuropathic pain.28,29 In addi-
tion, the novel induction of B1 receptor expression after peripheral
nerve injury is particularly meaningful due to that the B1 receptor
is not readily desensitized after agonist application, and thus these
changes can be maintained chronic types of pain.30,31
Icatibant (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH),
used as a positive control, is a peptide B2 receptor selective antago-
nist. Although icatibant has shown highly potent and long-acting
bradykinin antagonism both in vitro and in vivo studies,32,33 it has
a limited therapeutic use due to its poor oral bioavailability. In our
search for the new bradykinin antagonists, we previously reported
the bradykinin antagonistic effects of a series of non-peptide
* Corresponding authors.
E-mail addresses: hsna@korea.ac.kr (H.S. Na), hypark@ewha.ac.kr (H.-Y.P. Choo).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.01.050

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Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
compounds with piperazine, three amide bonds and a lipophilic ring
system in each molecule.34 In this study, we have designed and syn-
thesized new compounds modified the linker from previous series
having two amide bonds with various substituents in each side. Four
amines (b1–3 and b6) were selected from the previous series of
iminodiacetic acid triamide derivatives for their relatively high bra-
dykinin inhibitory activities.34 Compound 33, which has shown the
most potent inhibitory activity (87% inhibition at 1 lM), was se-
lected for further studies of its in vivo analgesic capabilities using
the rat models of inflammatory and neuropathic pain.
tion. As shown in Table 2, the inhibitory activity of synthesized
compounds to maximum contractility of bradykinin was 28–87%
(n = 4–13). By the modification of the linkers (a), compounds with
succinamide (a1 series), 3-methylpentanediamide (a2 series), or
maleamide linker (a4 series) showed higher bradykinin inhibitory
activity than compounds with 3,3-dimethylpentanediamide linker
(a3 series). In terms of amines (b), substituted benzyl or cyclo-
hexyl groups showed comparable inhibitory activity, while the di-
methyl amine group (13) showed decreased inhibitory activity.
Amines c1–3 exhibited similar inhibitory activity except for 1-
((4-chlorophenyl)(phenyl)methyl)-1,4-diazepan (c2) in com-
pounds with 3-methylpentanediamide linker (a2) that showed
an increased inhibitory activity compared with 1-(4-chloro-
benzhydryl)piperazine (c1) congeners. Among the synthesized
compounds, five compounds 6, 14, 17, 30, and 33 showed potent
inhibitory activity (>80%). The effect of compound 33 as analgesics
was tested in rodent models of inflammatory and neuropathic
pain.
2. Results and discussion
2.1. Chemistry
The combinatorial synthesis of the 33 diamide compounds was
accomplished by coupling reactions (Schemes 1 and 2). The anhy-
drides such as succinic anhydride (a1), 3-methylglutaric anhydride
(a2), and 3,3-dimethylglutaric anhydride (a3), or maleic acid (a4),
followed by treatment with various amines (b1–6) afforded the
monoamides axby. Each product was treated with 1-(4-chloro-
benzhydryl)piperazine (c1), 1-((4-chlorophenyl)(phenyl)methyl)-
1,4-diazepan (c2),35 or 1-(bis(4-fluorophenyl)methyl)piperazine
(c3) and PyBOP to afford diamides axbycz which were effectively
purified by acid and base extractions to remove reaction by-prod-
ucts, the unreacted starting materials and reagents. Each individual
compound synthesized was characterized by 1H NMR, 13C NMR,
and high resolution MS. The structures of different linkers and
amines in both ends are illustrated in Table 1.
2.3. Effect of compound 33 on formalin-induced pain
As illustrated in Figure 1, the rats treated with the mixture of
formalin and vehicle showed significant increase of the time spent
in licking, shaking or biting the affected paw during both the early
and late phases. However, 33 (60
lg, sc) injected with formalin sig-
nificantly reduced the response duration in the both phases
[*P <0.05 vs vehicle group (Student’s unpaired t-test)]. The re-
sponse duration of pain behaviors in the vehicle and 33 groups
in the early phase was 47.05 8.53 and 14.82 7.23 s, respectively.
In the late phase, the time spent in licking, shaking or biting the
paw in each group was 334.21 51.10 and 129.31 48.11 s,
respectively. These results indicate that compound 33 is effective
in relieving both nociceptive and inflammatory pain when treated
locally.
2.2. Bradykinin inhibitory activity
Each compound was tested for their bradykinin-induced con-
tractilities on guinea-pig ileum smooth muscle at 1
l
M concentra-
O
O
H
O
H
N
O
O
O
ii
i
N
+
RNH2
RNH2
RNH2
R
OH
R
R
R'
O
O
O
a1by
a2by
a1
a1bycz
O
O
O
O
O
ii
i
+
R
R
N
H
R'
R'
N
OH
OH
H
a2bycz
a2
O
O
O
O
O
O
O
ii
i
+
R
N
H
N
H
a3bycz
a3by
R= b1. 4-Methylbenzyl-
a3
b2. 4-Methoxylbenzyl-
R'= c1. 1-(4-chlorobenzhydryl)piperazine
c2. 1-((4-chlorophenyl)(phenyl)methyl)-
1,4-diazepan
b3. 3,4,5-Trimethoxybenzyl-
b4. Cyclohexyl-
b5. Dimethyl-
Scheme 1. Reagents and conditions: (i) DMF, rt, 20 h; (ii) c1 or c2, PyBOP, DIEA, DMF, rt, 16 h.
O
O
O
O
ii
O
O
i
R
R
+
OH
RNH2
N
H
N
H
OH
R'
HO
a4bycz
a4by
a4
R'= c1. 1-(4-chlorobenzhydryl)piperazine
R= b1. 4-Methylbenzyl-
c3.
1-(bis(4-fluorophenyl)methyl)-
piperazine
b2. 4-Methoxylbenzyl-
b4. Cyclohexyl-
b6
. 1-Methyl-3-phenylpropyl-
Scheme 2. Reagents and conditions: (i) EDCI, DMF, rt, 17 h; (ii) c1 or c3, PyBOP, DIEA, DMF, rt, 16 h.

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
2329
Table 1
Table 2
The structures of synthesized compounds
Bradykinin inhibitory activity of synthesized compounds at 1
guinea-pig ileum
l
M concentration in the
% Inhibition
a
b
c
Compd #
Structure
O
O
R'
R
1
2
3
4
5
6
7
8
a1b1c1
a1b2c1
a1b3c1
a1b4c1
a1b1c2
a1b2c2
a1b3c2
a1b4c2
a2b1c1
a2b2c1
a2b3c1
a2b4c1
a2b5c1
a2b1c2
a2b2c2
a2b3c2
a2b4c2
a3b1c1
a3b2c1
a3b3c1
a3b4c1
a4b1c1
a4b2c1
a4b4c1
a4b6c1
a4b1c2
a4b2c2
a4b4c2
a4b6c2
a4b1c3
a4b2c3
a4b4c3
a4b6c3
67.91 2.51
57.25 2.19
65.19 3.68
79.03 2.39
67.65 9.64
80.86 1.79
72.62 1.68
71.68 3.32
64.80 3.62
64.88 4.15
53.10 4.57
60.84 5.70
35.97 2.24
81.35 3.62
78.28 2.34
69.70 2.84
80.27 2.93
32.92 5.58
31.59 2.73
49.15 3.01
28.01 6.11
78.53 2.96
74.09 5.16
57.37 5.38
77.54 2.92
79.44 8.83
75.01 6.28
66.01 3.12
79.73 5.81
84.55 1.28
79.59 2.07
65.43 6.23
86.61 1.20
100.00 0.00
NH
N
N
linker
R''
a
O
O
O
9
O
O
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Icatibant
O
a1
a2
a3
a4
b
OCH3
OCH3
N
N
H
N
H
H
OCH3
OCH3
b2
b1
b4
b3
H
N
N
N
H
b5
b6
c
F
N
N
N
N
N
N
Cl
Cl
c1
c2
c3
with mild conditions. Most of the compounds synthesized showed
inhibitory activity on bradykinin-induced contraction at 1 M con-
F
l
centration in the guinea-pig ileum. The most potent compound 33
among the series demonstrated analgesic efficacies to formalin-in-
duced nociceptive and inflammatory pain, and cold and warm allo-
dynia following peripheral nerve injury.
2.4. Effect of compound 33 on neuropathic pain
Before nerve injury, animals rarely exhibited tail withdrawal re-
sponses to von Frey filaments, cold (4 °C) and warm (40 °C) water
stimuli (Fig. 2). However, 2 weeks after nerve injury, rats showed
significant decrease of tail withdrawal threshold and latencies in
response to mechanical and, cold and warm water stimuli, respec-
tively [P <0.05 vs presurgical value (Pre), Paired t-test]. We re-
garded these increased sensitivities as the signs of mechanical,
cold and warm allodynia, and these neuropathic animals were sub-
jected to the treatment of 33. As shown in Figure 2, compound 33
(60 mg, ip) administered significantly suppressed the cold and
warm, but not mechanical, allodynia until 5 hr after the injection
unlike vehicle group (*P <0.05 vs baseline, one-way repeated mea-
sured ANOVA followed by Bonferroni’s t-test). This discrepancy be-
tween the two efficacies may be due to that the spinal ascending
pathways involved in the processing of tactile allodynia is segre-
gated from that of cold and warm allodynia.36–38 These results
indicate that compound 33 has an analgesic effect against cold
and warm allodynia induced by peripheral nerve injury.
4. Experimental
4.1. Materials and methods
Melting points were measured on an electrothermal digital
melting point (Buchi, Germany) without calibration. 1H NMR and
13C NMR spectra were recorded on Varian NMR AS and Varian
Unity Inova 400 and 500 MHz NMR spectrometers. Chemical shifts
were reported in parts per million (d) units relative to the solvent
peak. The 1H NMR data were reported as peak multiplicities: s for
singlet; d for doublet; t for triplet; and m for multiplet. Coupling
constants were recorded in hertz. Mass spectral data were ob-
tained from the Korea Basic Science Institute (Daegu) on a Jeol
JMS 700 high resolution mass spectrometer. Reagents were of com-
mercial grade and were purchased from Sigma–Aldrich Co., Merck,
Ducksan Pure Chemical Co.
4.2. General procedure for substituted monoamide derivatives
(a1by–a3by)
3. Conclusion
A series of non-peptide diamide compounds were designed and
synthesized with different linkers and evaluated their bradykinin
inhibitory activity using guinea-pig smooth muscle. The synthetic
method for the preparation of the diamide compounds was simple
Anhydrides a1–3 (2 mmol) were dissolved in 3 mL of dimethyl-
formamide (DMF). Amines b1–5 (2 mmol) were added and stirred
at room temperature for 20 h. Twenty millilitres of 10% HCl was
put into the reaction solution, and extracted with 30 mL of EtOAc.

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Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
80
70
60
50
40
30
20
10
0
450
400
350
300
250
200
150
100
50
μ
Comp 33 (60 g, n=6)
μ
Comp 33 (60 g, n=6)
vehicle (n=12)
vehicle (n=12)
*
*
*
*
*
0
0
5
0
Early
Late
10 15 20 25 30 35 40 45 50 55
6
Post-injection time (min)
Phase
Figure 1. The effect of subcutaneous injection of compound 33 (60
interval following subcutaneous injection of formalin (5%, 50 l). In the bar graph, values represent licking or biting during the early (0–10 min) and late (10–60 min) phases
after formalin injection. Data represent means SEM. Significance of differences was analyzed by Student’s unpaired t-test. *P <0.05.
lg) on formalin-induced pain response. Values represent time (s) spent paw licking or biting in each 5 min
l
Cold (4 οC) water
A
von Frey filaments
B
15
15
*
*
*
12
9
12
9
Comp 33 (60 mg, n=8)
vehicle (n=7)
6
6
Comp 33 (60 mg, n=7)
vehicle (n=7)
3
3
0
0
PRE BL
1
3
5
24
PRE BL
1
3
5
24
Experimental time (hour)
Experimental time (hour)
C
Warm (40 oC) water
15
12
9
*
*
*
6
Comp 33 (60 mg, n=8)
vehicle (n=8)
3
0
PRE BL
1
3
5
24
Experimental time (hour)
Figure 2. Effect of compound 33 on mechanical (A), cold (B), and warm (C) allodynia induced by rat tail nerve injury (TNI). ‘PRE’ represents the 1 day prior to nerve injury. At
2 weeks after nerve injury, rats were given baseline (BL) test and then were subjected to the injection of compound 33 (60 mg, ip) or vehicle (Methyl pyrrolidone/Tween80/
Saline = 1:1:8). Behavioral tests were re-performed 1, 3, 5, and 24 h after the injection. *P <0.05 versus BL (one-way repeated measured ANOVA followed by Bonferroni’s
t-test).
The organic layer was washed with 20 mL of 10% HCl, and then
washed with 20 mL of a saturated NaCl solution twice. The organic
layer was collected, dried over anhydrous MgSO4, and filtered un-
der reduced pressure. The organic solvent in the filtrate was re-
moved under reduced pressure. The residue was recrystallized
from EtOAc and methanol to afford desired compounds.
4.2.1. 3-(4-Methylbenzylcarbamoyl)propanoic acid (a1b1)
White solid, yield: 77%; mp 147–149 °C; 1H NMR (CDCl3,
400 MHz) d 7.18–7.14 (m, 4CH), 5.90 (br s, NH), 4.42 (s, CH2),
2.74 (t, J = 6.0 Hz, CH2), 2.55 (t, J = 6.0 Hz, CH2) 2.34 (s, CH3);
HR-FABMS calcd for C12H16NO3 (M++1): 222.1130, found:
222.1127.

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
2331
4.2.2. 3-(4-Methoxybenzylcarbamoyl)propanoic acid (a1b2)
Light yellow solid, yield: 56%; mp 158–160 °C; 1H NMR (CDCl3,
400 MHz) d 7.24–7.19 (m, 2CH), 6.99–6.89 (m, 2CH), 5.87 (br s,
NH), 4.40 (s, CH2), 3.80 (s, OCH3), 2.73 (t, J = 6.4 Hz, CH2), 2.56 (t,
J = 6.4 Hz, CH2); HR-FABMS calcd for C12H16NO4 (M++1):
238.1079, found: 238.1076.
4CH), 4.21 (d, J = 6.0 Hz, CH2), 2.28 (s, CH2), 2.27 (s, CH3), 2.19 (s,
CH2), 1.02 (s, 2CH3); HR-FABMS calcd for C15H22NO3: (M++1):
264.1600, found: 264.1602.
4.2.11. 4-(4-Methoxybenzylcarbamoyl)-3,3-dimethylbutanoic
acid (a3b2)
White solid, yield: 85%; mp 102–103 °C; 1H NMR (CDCl3,
4.2.3. 3-(3,4,5-Trimethoxybenzylcarbamoyl)propanoic acid
(a1b3)
400 MHz)
d 12.05 (s, OH), 8.32 (t, J = 5.8 Hz, NH), 7.17 (d,
J = 8.0 Hz, 2CH), 6.87 (d, J = 8.0 Hz, 2CH), 4.18 (d, J = 5.6 Hz, CH2),
3.72 (s, OCH3), 2.28 (s, 2CH2), 1.04 (s, CH3), 1.02 (s, CH3); HR-FAB-
MS calcd for C15H22NO4 (M++1): 280.1549, found: 280.1542.
Light yellow solid, yield: 18%; mp 122–125 °C; 1H NMR (CDCl3,
400 MHz) d 6.49 (s, 2CH), 4.38 (s, CH2), 3.85 (s, 3OCH3), 2.74 (t,
J = 6.8 Hz, CH2), 2.55 (t, J = 6.8 Hz, CH2); HR-FABMS calcd for
C14H20NO6 (M++1): 298.1291, found: 298.1286.
4.2.12. 4-(3,4,5-Trimethoxybenzylcarbamoyl)-3,3-dimethylbutanoic
acid (a3b3)
4.2.4. 3-(Cyclohexylcarbamoyl)propanoic acid (a1b4)
White solid, yield: 60%; mp 170–173 °C; 1H NMR (CDCl3,
400 MHz) d 5.58 (s, NH), 3.81–3.74 (m, CH), 2.69 (t, J = 6.4 Hz,
CH2), 2.51 (t, J = 6.4 Hz, CH2), 1.95–1.90 (m, CH2), 1.75–1.60 (m,
2CH2), 1.42–1.13 (m, 2CH2); HR-FABMS calcd for C10H17NO3Na
(M++Na): 222.1106, found: 222.1103.
Light yellow solid, yield: 54%; mp 118–119 °C; 1H NMR (CDCl3,
400 MHz) d 12.07 (s, OH), 8.33 (t, J = 5.8 Hz, NH), 6.56 (d, J = 8.0 Hz,
2CH), 4.21 (d, J = 6.0 Hz, CH2), 3.74 (s, 2OCH3), 3.62 (s, OCH3), 2.29
(s, CH2), 2.21 (s, CH2), 1.05 (s, 2CH3); HR-FABMS calcd for
C17H26NO6: (M++1): 340.1760, found: 340.1757.
4.2.13. 4-(Cyclohexylcarbamoyl)-3,3-dimethylbutanoic acid
(a3b4)
4.2.5. 4-(4-Methylbenzylcarbamoyl)-3-methylbutanoic acid
(a2b1)
White solid, yield: 67%; mp 77–78 °C; 1H NMR (CDCl3,
400 MHz) d 12.07 (s, OH), 7.76 (d, J = 8.0 Hz, NH), 3.53 (s, CH),
2.25 (s, CH2), 2.09 (s, CH2), 1.52–1.72 (m, 2CH2), 1.04–1.30 (m,
3CH2), 1.05 (s, CH3), 1.01 (s, CH3); HR-FABMS calcd for
C13H24NO3 (M++1): 242.1756, found: 242.1752.
White solid, yield: 96%; mp 133–134 °C; 1H NMR (CDCl3,
400 MHz) d 7.16 (s, 4CH), 5.90 (s, NH), 4.41 (d, J = 6.0 Hz, CH2),
2.43–2.46 (m, CH), 2.40 (t, J = 7.2 Hz, CH2), 2.34 (s, CH3), 2.28 (t,
J = 6.4 Hz, CH2), 1.08 (d, J = 6.4 Hz, CH3); HR-FABMS calcd for
C14H20NO3 (M++1): 250.1443, found: 250.1444.
4.3. General procedure for (Z)-3-(substituted)acrylic acid
derivatives (a4by)
4.2.6. 4-(4-Methoxybenzylcarbamoyl)-3-methylbutanoic acid
(a2b2)
White solid, yield: 74%; mp 89–90 °C; 1H NMR (CDCl3,
400 MHz) d 7.20 (d, J = 9.2 Hz, 2CH), 6.87 (d, J = 9.2 Hz, 2CH), 5.94
(s, NH), 4.38 (d, J = 5.6 Hz, CH2), 3.80 (s, OCH3), 2.36–2.48 (m,
CH), 2.32 (dd, J = 6.4 Hz, CH2), 2.23 (dd, J = 6.4 Hz, CH2), 1.06 (d,
J = 6.4 Hz, CH3); HR-FABMS calcd for C14H20NO4 (M++1):
266.1392, found: 266.1382.
Maleic acid (1 mmol) was dissolved in 3 mL of DMF. Then,
1 mmol of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiamide HCl
(EDCI) was added and stirred at room temperature for 1 hour.
After 1 hour, amine (b1–2, b4 or b6) was added and stirred at
room temperature for 16 h. Thirty millilitres of 10% HCl was
put into the reaction solution, and extracted with 50 mL of
EtOAc. The organic layer was washed with 30 mL of 10% HCl,
and then washed with 20 mL of a saturated NaCl solution twice.
The organic layer was collected, dried over anhydrous MgSO4,
and filtered under reduced pressure. The organic solvent in the
filtrate was removed under reduced pressure. The residue was
recrystallized from EtOAc and methanol to obtain desired
compounds.
4.2.7. 4-(3,4,5-Trimethoxybenzylcarbamoyl)-3-methylbutanoic
acid (a2b3)
White solid, yield: 28%; mp 118–119 °C; 1H NMR (CDCl3,
400 MHz) d 6.50 (s, 2CH), 4.39 (d, J = 5.6 Hz, CH2), 3.85 (s,
3OCH3), 2.41–2.48 (m, CH), 2.45 (d, J = 4.4 Hz, CH2), 2.31 (d,
J = 5.6 Hz, CH2), 1.10 (d, J = 6.8 Hz, CH3); HR-FABMS calcd for
C16H24NO6 (M++1): 326.1604, found: 326.1606.
4.3.1. (Z)-3-(4-Methylbenzylcarbamoyl)acrylic acid (a4b1)
White solid, yield: 73%; mp 143–144 °C; 1H NMR (CDCl3,
400 MHz) d 6.923–6.945 (m, 4CH), 6.325 (d, J = 12.8 Hz, CH),
6.077 (d, J = 12.8 Hz, CH), 4.225 (s, CH2), 2.350 (s, CH3); HR-EIMS
calcd for C12H13NO3: (M+): 219.0895, found: 219.0895.
4.2.8. 4-(Cyclohexylcarbamoyl)-3-methylbutanoic acid (a2b4)
White solid, yield: 69%; mp 133–134 °C; 1H NMR (CDCl3,
400 MHz) d 5.56 (s, NH), 3.73–3.84 (m, CH), 2.35–2.45 (m, CH),
2.30; 2.27 (dd, J = 5.6; 6.0 Hz, CH2), 2.24; 2.21 (dd, J = 7.2 Hz,
CH2), 1.93 (d, J = 12.0 Hz, CH2), 1.72 (d, J = 12.4 Hz, CH2), 1.63 (d,
J = 12.8 Hz, CH2), 1.41; 1.34 (dd, J = 12.0 Hz, CH2), 1.16 (t,
J = 11.6 Hz, CH2), 1.08 (d, J = 6.4 Hz, CH3); HR-FABMS calcd for
C12H22NO3 (M++1): 228.1600, found: 228.1601.
4.3.2. (Z)-3-(4-Methoxybenzylcarbamoyl)acrylic acid (a4b2)
White solid, yield: 72%; mp 140–142 °C; 1H NMR (CDCl3.
400 MHz) d 6.766–6.800 (m, 2CH), 6.553–6.588(m, 2CH), 6.402
(d, J = 12.8 Hz, CH), 6.051 (d, J = 12.8 Hz, CH), 4.395 (s, CH2),
3.810 (s, CH3); HR-EIMS calcd for C12H13NO4: (M+): 235.0845,
found: 235.0843.
4.2.9. 4-(Dimethylcarbamoyl)-3-methylbutanoic acid (a2b5)
White solid, yield: 2%; mp 116–117 °C; 1H NMR (CDCl3,
400 MHz) d 3.05 (s, CH3), 2.98 (s, CH3), 2.35–2.49 (m, CH), 2.33
2.31 (dd, J = 5.6 Hz, CH2), 2.27; 2.23 (dd, J = 7.6; 7.2 Hz, CH2), 1.08
(d, J = 6.0 Hz, CH3); HR-FABMS calcd for C8H15NO3Na (M++Na):
196.0950, found: 196.0949.
4.3.3. (Z)-3-(Cyclohexylcarbamoyl)acrylic acid (a4b4)
White solid, yield: 34%; mp 150–151 °C; 1H NMR (CDCl3.
400 MHz) d 6.357 (d, J = 12.8 Hz, CH), 6.197 (d, J = 12.8 Hz, CH),
3.812–3.906 (m, CH), 2.046–1.959 (m, CH2), 1.800–1.747 (m,
CH2), 1.705–1.635 (m, 1/2CH2), 1.458–1.348 (m, CH2), 1.292–
1.156 (m, CH2, 1/2CH2); HR-EIMS calcd for C10H15NO3: (M+):
197.1052, found: 197.1055.
4.2.10. 4-(4-Methylbenzylcarbamoyl)-3,3-dimethylbutanoic
acid (a3b1)
White solid, yield: 79%; mp 85–86 °C; 1H NMR (CDCl3,
400 MHz) d 12.09 (s, OH), 8.32 (t, J = 5.8 Hz, NH), 7.10–7.15 (m,

2332
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
4.3.4. (Z)-3-(4-Phenylbutan-2-yl-carbamoyl)acrylic acid (a4b6)
White solid, yield: 91%; mp 113–115 °C; 1H NMR (CDCl3.
400 MHz) d 7.08–7.21 (m, 5CH), 6.75 (d, J = 12.8 Hz, CH), 6.510
(d, J = 12.8 Hz, CH), 3.762–3.796 (m, CH), 2.602–2.630 (m, CH2),
1.838–1.402 (m, CH2), 1.302 (d, J = 6.8 Hz, CH3); HR-EIMS calcd
for C14H17NO3: (M+): 247.1208, found: 247.1205.
NMR (CDCl3, 100 MHz) d 171.29, 170.31, 141.38, 140.60, 131.33,
129.79, 129.00, 128.68, 128.62, 127.64, 127.29, 75.04, 51.71,
51.35, 48.00, 45.34, 32.86, 31.52, 28.58, 25.44, 24.68; HR-FABMS
calcd for C27H35ClN3O2: (M++1): 468.2418, found: 468.2407.
4.4.5. 4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-(4-methylbenzyl)-4-oxobutanamide (5)
4.4. General procedure for substituted diamide derivatives (1–27)
Light yellow solid, yield: 58%; mp 72–74 °C; 1H NMR (CDCl3,
400 MHz); d 7.36–7.34 (m,4CH), 7.23–7.18 (m, 5CH), 7.17–7.11
(m, 4CH), 6.34 (s, NH), 4.55 (s, CH), 4.39 (s, CH2), 3.66–3.45 (m,
2CH2), 2.72–2.66 (m, 2CH2), 2.67–2.53 (m, 4CH), 2.32 (s, CH3),
1.82–1.73 (m, CH2); 13C NMR (CDCl3, 100 MHz) d 172.33, 171.55,
171.45, 142.48, 141.60, 136.77, 135.45, 132.63, 132.55, 129.17,
129.03, 128.95, 128.71, 128.65, 128.60, 127.67, 127.57, 127.26,
127.19, 74.21, 74.02, 54.27, 53.63, 53.24, 52.64, 48.65, 46.41,
46.37, 44.58, 43.19, 31.43, 28.92, 28.75, 28.50, 27.50, 21.02; HR-
FABMS calcd for C30H35ClN3O2: (M++1): 504.2418, found: 504.2393.
Monoamide derivatives axby (0.5 mmol), 0.55 mmol of
amines c1–3, and benzotriazol-1-yloxytripyrrolidino phospho-
nium hexafluorophosphate (PyBOP, 0.55 mmol) were dissolved
in 3 mL of DMF. Then, 1.0 mmol of N,N-diisopropylethylamine
(DIEA) was added and stirred at room temperature for 16 h.
Twenty millilitres of 10% HCl was put into the reaction solution,
and extracted with 30 mL of EtOAc. The organic layer was
washed with 20 mL of 10% HCl, 20 mL of a saturated NaHCO3
solution twice and with 20 mL of a saturated NaCl solution
twice. The organic layer was collected, dried over anhydrous
MgSO4, and filtered. The organic solvent in the filtrate was re-
moved under reduced pressure. The residue was purified by col-
umn chromatography with EtOAc and methanol (20:1) to afford
desired compounds.
4.4.6. 4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-(4-methoxybenzyl)-4-oxobutanamide (6)
Light yellow solid, yield: 21%; mp 57–60 °C; 1H NMR (CDCl3,
400 MHz) d 7.36–7.31 (m, 4CH), 7.29–7.20 (m, 5CH), 7.19–7.17
(m, 2CH), 6.84–6.81 (m, 2CH), 6.85 (s, NH), 4.66 (d, J = 6.8 Hz,
CH), 4.35 (d, J = 5.6 Hz, CH2), 3.77 (s, OCH3), 3.63–3.55 (m, CH2),
3.54–3.41 (m, CH2), 2.70–2.52 (m, 4CH2), 1.81–1.71 (m, CH2); 13C
NMR (CDCl3, 100 MHz) d 172.31, 171.52, 171.44, 158.80, 142.41,
141.63, 132.64, 132.55, 129.04, 128.95, 128.72, 128.66, 128.60,
127.68, 127.60, 127.27, 127.18, 113.93, 74.22, 74.02, 55.23, 54.29,
53.66, 53.26, 52.66, 48. 68, 46.43, 44.61, 42.93, 31.47, 28.96,
28.80, 28.54, 27.52; HR-FABMS calcd for C30H35ClN3O3: (M++1):
520.2367, found: 520.2346.
4.4.1. 4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-N-
(4-methylbenzyl)-4-oxobutanamide (1)
Light yellow solid, yield: 60%; mp 75–78 °C; 1H NMR (CDCl3,
400 MHz) d 7.37–7.34 (m, 4CH), 7.32–7.13 (m, 5CH), 7.15–7.11
(t, J = 4.4 Hz, CH2), 3.46 (t, J = 4.4 Hz, CH2), 2.64 (t, J = 6.4 Hz, CH2),
2.53 (t, J = 6.4 Hz, CH2), 2.36–2.32 (m, 2CH2), 2.31(s, CH3); 13C
NMR (CDCl3, 100 MHz) d 172.17, 170.26, 141.39, 140.61, 136.63,
135.38, 132.68, 129.05, 128.98, 128.68, 128.62, 127.62, 127.50,
127.30, 74.99, 51.61, 51.25, 45.23, 43.07, 31.10, 28.37, 20.92; HR-
FABMS calcd for C29H33ClN3O2: (M++1): 490.2261, found:
490.2249.
4.4.7. 4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-4-oxo-N-(3,4,5-trimethoxybenzyl)butanamide (7)
Light yellow solid, yield: 41%; mp 83–85 °C; 1H NMR (CDCl3,
400 MHz) d 7.37–7.33 (m, 4CH), 7.29–7.17 (m, 5CH), 6.52 (s,
2CH), 4.55 (d, J = 6.4 Hz, CH), 4.37 (d, J = 5.6 Hz, CH2), 3.84 (s,
3OCH3), 3.65–3.62 (m, 1/2CH2), 3.59–3.55 (m, CH2), 3.48–3.45
(m, 1/2CH2), 2.74–2.53 (m, 4CH2), 1.83–1.72 (m, CH2); 13C NMR
4.4.2. 4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-N-
(4-methoxybenzyl)-4-oxobutanamide (2)
Light yellow solid, yield: 44%; mp 119–121 °C; 1H NMR (CDCl3,
400 MHz) d 7.36–7.32 (m, 4CH), 7.29–7.19 (m, 5CH), 7.17–7.15 (m,
2CH2), 6.82–6.78 (m, 2CH), 4.31(s, CH2), 4.19 (s, CH), 3.74 (s, OCH3),
3.59–3.50 (m, CH2), 3.44–3.31 (m, CH2), 2.61 (t, J = 6.4 Hz, CH2),
2.94 (t, J = 6.4 Hz, CH2), 2.34–2.33 (m, CH2), 2.30–2.28 (m, CH2);
13C NMR (CDCl3, 100 MHz) d 172.15, 170.21, 158.85, 141.50,
140.70, 130.55, 129.08, 128.98, 128.80, 128.73, 127.74, 127.40,
113.97, 75.14, 55.25, 51.76, 51.42, 45.37, 42.97, 31.38, 28.58; HR-
FABMS calcd for C29H33ClN3O3: (M++1): 506.2210, found:
506.2196.
(CDCl3, 100 MHz)
d 172.44, 171.47, 171.39, 153.34, 142.41,
141.64, 137.12, 134.27, 134.24, 132.70, 132.60, 129.07, 129.00,
128.70, 128.64, 127.71, 127.63, 127.32, 127.22, 104.62, 74.23,
74.05, 60.80, 56.11, 54.24, 53.72, 53.34, 52.69, 48.68, 46.46,
44.58, 43.75, 31.55, 28.96, 28.81, 28.56, 27.59; HR-FABMS calcd
for C32H39ClN3O5: (M++1): 580.2578, found: 580.2588.
4.4.8. 4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-cyclohexyl-4-oxobutanamide (8)
White solid, yield: 55%; mp 68–70 °C; 1H NMR (CDCl3,
400 MHz) d 7.36–7.32 (m, 4CH), 7.29–7.18 (m, 5CH), 6.16 (s, NH),
4.56 (s, CH), 3.76–3.69 (m, CH), 3.65 (t, J = 5.6 Hz, 1/2CH2), 3.58
(t, J = 5.6 Hz, CH2), 3.45 (t, J = 5.6 Hz, 1/2CH2), 2.69–2.49 (m,
4CH2), 1.89–1.85 (m, CH2), 1.81–1.74 (m, CH2), 1.71–1.65 (m,
CH2), 1.61–1.56 (m, 1/2CH2), 1.38–1.30 (m, CH2), 1.19–1.06 (m,
4.4.3. 4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-4-
oxo-N-(3,4,5-trimethoxybenzyl)butanamide (3)
White solid, yield: 20%; mp 71–73 °C; 1H NMR (CDCl3,
400 MHz) d 7.37–7.31 (m, 4CH), 7.30–7.25 (m, 5CH), 6.50(s,
2CH), 4.35(s, CH2), 4.21 (s, CH), 3.84 (s, 3OCH3), 3.56–3.46 (m,
2CH2), 2.66 (t, J = 6.4 Hz, CH2), 2.54 (t, J = 6.4 Hz, CH2), 2.38–2.27
(m, 2CH2); 13C NMR (CDCl3, 125 MHz) d 172.62, 170.45, 153.56,
137.31, 134.48, 129.35, 129.01, 128.00, 106.97, 104.78, 75.40,
61.04, 56.52, 56.35, 51.99, 46.56, 43.96, 31.62, 26.67; HR-FABMS
calcd for C31H37ClN3O5: (M++1): 566.2422, found: 566.2415.
CH2); 13C NMR (CDCl3, 100 MHz)
d 171.62, 171.55, 142.44,
142.39, 141.66, 141.61, 132.68, 132.59, 129.06, 128.97, 128.74,
128.68, 128.61, 127.70, 127.62, 127.30, 127.21, 74.27, 74.13,
54.38, 53.75, 53.28, 52.73, 48.73, 48.04, 46.47, 46.41, 44.66,
33.00, 31.83, 29.20, 29.04, 28.64, 27.58, 25.54, 24.75; HR-FABMS
calcd for C28H37ClN3O2: (M++1): 482.2574, found: 482.2551.
4.4.4. 4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-N-
cyclohexyl-4-oxobutanamide (4)
4.4.9. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-N-
(4-methylbenzyl)-3-methyl-5-oxopentanamide (9)
Yellow solid, yield: 52%; mp 59–61 °C; 1H NMR (CDCl3,
400 MHz) d 7.36–7.34 (m, 4CH), 7.30–7.19 (m, 5CH), 5.34 (s, NH),
4.21 (s, CH), 3.60–3.55 (m, CH2), 3.49–3.46 (m, CH2), 2.61 (t,
J = 6.8 Hz, CH2), 2.46 (t, J = 6.8 Hz, CH2), 2.35–2.33 (m, 2CH2); 13C
Light yellow solid, yield: 65%; mp 62–63 °C; 1H NMR (CDCl3,
400 MHz) d 7.35 (d, J = 8.0 Hz, 2CH), 7.29 (d, J = 7.2 Hz, 2CH), 7.26
(t, J = 8.2 Hz, 2CH), 7.22 (t, J = 7.3 Hz, CH), 7.16 (d, J = 8.0 Hz,

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
2333
2CH), 7.11 (d, J = 7.2 Hz, 2CH), 6.29 (s, NH), 4.37 (d, J = 5.6 Hz, CH2),
4.21 (s, CH), 3.59 (t, J = 5.6 Hz, CH2), 3.48 (q, J = 5.6 Hz, CH2), 2.43
(d, J = 6.8 Hz, CH2), 2.34–2.39 (m, CH2), 2.32 (s, CH3), 2.28 (d,
J = 6.0 Hz, CH2), 2.25 (d, J = 6.4 Hz, CH2), 2.13–2.23 (m, CH), 1.03
(d, J = 6.4 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d 171.68, 170.49,
141.54, 135.44, 132.82, 129.28, 129.08, 128.80, 128.74, 127.73,
127.40, 75.19, 52.07, 51.58, 45.96, 43.20, 43.10, 41.65, 39.43,
28.49, 21.06, 20.28; HR-FABMS calcd for C31H37ClN3O2: (M++1):
518.2574, found: 518.2584.
CH3); 13C NMR (CDCl3, 100 MHz)
140.59, 132.83, 129.13, 128.79, 127.75, 75.11, 51.95, 51.52, 46.37,
46.32, 41.53, 40.13, 37.61, 27.58, 20.14; HR-FABMS calcd for
C25H33ClN3O2: (M++1): 442.2261, found: 442.2259.
d
170.77, 170.60, 141.44,
4.4.14. 5-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-(4-methylbenzyl)-3-methyl-5-oxopentanamide (14)
Light yellow solid, yield: 75%; mp 53–54 °C; 1H NMR (CDCl3,
400 MHz) d 7.30–7.36 (m, 4CH), 7.23–7.29 (m, 3CH), 7.19–7.23
(m, 2CH), 7.17 (d, J = 8.4 Hz, 2CH), 7.12 (d, J = 8.0 Hz, 2CH), 6.42
(s, NH), 4.55 (d, J = 4.8 Hz, CH), 4.39 (d, J = 6.0 Hz, CH2), 3.61–3.67
(m, 1/2CH2), 3.49–3.60 (m, CH2), 3.43–3.48 (m, 1/2CH2), 2.53–
2.68 (m, 2CH2), 2.22–2.47 (m, 2CH2), 2.13–2.22 (m, CH), 1.77 (q,
J = 5.7 Hz, CH2), 1.07 (d, J = 6.0 Hz, CH3) 2.43 (d, J = 6.8 Hz, CH2),
2.34–2.39 (m, CH2), 2.32 (s, CH3), 2.28 (d, J = 6.0 Hz, CH2), 2.25 (d,
J = 6.4 Hz, CH2), 2.13–2.23 (m, CH), 1.03 (d, J = 6.4 Hz, CH3); 13C
NMR (CDCl3, 100 MHz) d 171.88, 171.82, 171.61, 171.54, 142.26,
142.23, 141.50, 136.59, 135.49, 132.46, 132.37, 129.03, 128.89,
128.83, 128.54, 128.47, 127.50, 127.13, 127.05, 74.18, 74.01,
54.52, 53.48, 53.12, 52.36, 48.85, 46.55, 46.12, 44.40, 42.92,
39.45, 39.28, 28.69, 28.35, 27.31, 20.89, 20.18; HR-FABMS calcd
for C32H39ClN3O2: (M++1): 532.2731, found: 532.2720.
4.4.10. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-(4-methoxybenzyl)-3-methyl-5-oxopentanamide (10)
White solid, yield: 63%; mp 61–62 °C; 1H NMR (CDCl3,
400 MHz) d 7.35 (d, J = 8.4 Hz, 2CH), 7.29 (d, J = 7.6 Hz, 2CH), 7.26
(t, J = 8.2 Hz, 2CH), 7.23 (d, J = 7.6 Hz, CH), 7.20 (d, J = 8.8 Hz,
2CH), 6.84 (d, J = 8.0 Hz, 2CH), 6.31 (s, NH), 4.35 (d, J = 5.6 Hz,
CH2), 4.21 (s, CH), 3.79 (s, OCH3), 3.59 (t, J = 5.0 Hz, CH2), 3.48 (q,
J = 5.2 Hz, CH2), 2.42 (d, J = 7.2 Hz, CH2), 2.34–2.38 (m, CH2), 2.28
(d, J = 6.4 Hz, CH2), 2.25 (d, J = 6.0 Hz, CH2), 2.18–2.22 (m, CH),
1.03 (d, J = 6.8 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d 171.66,
170.49, 158.87, 141.53, 140.74, 132.80, 130.61, 129.07, 128.79,
128.72, 127.72, 127.39, 113.99, 75.17, 55.25, 52.05, 51.56, 45.95,
43.07, 42.89, 41.64, 39.40, 28.48, 20.28; HR-FABMS calcd for
C31H37ClN3O3: (M++1): 534.2523, found: 534.2528.
4.4.15. 5-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-(4-methoxybenzyl)-3-methyl-5-oxopentanamide (15)
Brown solid, yield: 66%; mp 52–53 °C; 1H NMR (CDCl3,
400 MHz) d 7.30–7.36 (m, 4CH), 7.24–7.29 (m, 3CH), 7.18–7.23
(m, 2CH), 6.84 (d, J = 6.8 Hz, 2CH), 6.43 (s, NH), 4.55 (d, J = 4.4 Hz,
CH), 4.36 (d, J = 5.6 Hz, CH2), 3.79 (s, OCH3), 3.61–3.67 (m, 1/
2CH2), 3.53–3.58 (m, CH2), 3.43–3.48 (m, 1/2CH2), 2.54–2.68 (m,
2CH2), 2.22–2.47 (m, 2CH2), 2.13–2.21 (m, CH), 1.77 (q, J = 6.1 Hz,
CH2), 1.07 (d, J = 6.0 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d
171.85, 171.80, 171.70, 171.65, 158.78, 142.39, 142.34, 141.59,
132.62, 132.53, 130.72, 129.00, 128.94, 128.69, 128.63, 128.58,
127.65, 127.58, 127.26, 127.17, 113.91, 74.34, 74.18, 55.20, 54.67,
53.66, 53.28, 52.50, 48.99, 46.69, 46.27, 44.53, 43.08, 42.80,
39.51, 39.32, 28.84, 28.55, 27.46, 20.35; HR-FABMS calcd for
C32H39ClN3O3: (M++1): 548.2680, found: 548.2690.
4.4.11. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-(3,4,5-trimethoxybenzyl)-3-methyl-5-oxopentanamide (11)
White solid, yield: 71%; mp 72–73 °C; 1H NMR (CDCl3,
400 MHz) d 7.35 (d, J = 8.4 Hz, 2CH), 7.29 (d, J = 6.8 Hz, 2CH), 7.27
(t, J = 8.4 Hz, 2CH), 7.24 (d, J = 7.2 Hz, CH), 7.22 (d, J = 7.2 Hz,
2CH), 6.56 (s, NH), 6.51 (s, 2CH), 4.35 (d, J = 5.6 Hz, CH2), 4.21 (s,
CH), 3.83 (d, J = 4.8 Hz, 3OCH3), 3.59 (t, J = 4.0 Hz, CH2), 3.49 (q,
J = 4.0 Hz, CH2), 2.41 (d, J = 7.2 Hz, CH2), 2.31–2.38 (m, CH2), 2.29
(d, J = 6.0 Hz, CH2), 2.25 (d, J = 8.4 Hz, CH2), 2.14–2.20 (m, CH),
1.05 (d, J = 6.4 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d 171.81,
170.45, 153.27, 140.45, 137.06, 134.36, 132.99, 129.12, 128.85,
128.79, 127.75, 127.54, 104.70, 75.28, 60.78, 56.05, 52.02, 51.55,
46.29, 46.24, 43.62, 43.00, 41.40, 39.30, 28.55, 26.41, 26.33,
20.36; HR-FABMS calcd for C33H41ClN3O5: (M++1): 594.2735,
found: 594.2757.
4.4.16. 5-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-(3,4,5-trimethoxybenzyl)-3-methyl-5-oxopentanamide
(16)
4.4.12. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-cyclohexyl-3-methyl-5-oxopentanamide (12)
White solid, yield: 46%; mp 60–61 °C; 1H NMR (CDCl3,
400 MHz) d 7.30–7.35 (m, 4CH), 7.25–7.29 (m, 3CH), 7.18–7.24
(m, 2CH), 6.66 (s, NH), 6.53 (s, 2CH), 4.55 (d, J = 4.0 Hz, CH), 4.37
(d, J=6.0 Hz, CH2), 3.83 (d, J = 5.2 Hz, 3OCH3), 3.61–3.67 (m, 1/
2CH2), 3.55–3.59 (m, CH2), 3.46 (t, J = 4.6 Hz, 1/2CH2), 2.53–2.69
(m, 2CH2), 2.26–2.47 (m, 2CH2), 2.16–2.22 (m, CH), 1.77 (q,
J = 5.7 Hz, CH2), 1.08 (d, J = 5.8 Hz, CH3); 13C NMR (CDCl3,
White solid, yield: 73%; mp 75–76 °C; 1H NMR (CDCl3,
400 MHz) d 7.35 (d, J = 7.2 Hz, 2CH), 7.29 (d, J = 7.6 Hz, 2CH), 7.26
(t, J = 8.0 Hz, 2CH), 7.23 (d, J = 7.6 Hz, CH), 7.22 (d, J = 7.2 Hz,
2CH), 5.97 (s, NH), 4.21 (s, CH), 3.69–3.79 (m, CH), 3.61 (t,
J = 4.8 Hz, CH2), 3.51 (q, J = 4.8 Hz, CH2), 2.43 (d, J = 6.8 Hz, CH2),
2.31–2.39 (m, CH2), 2.23 (d, J = 7.6 Hz, CH2), 2.16–2.20 (m, CH),
2.12 (d, J = 6.0 Hz, CH2), 1.88 (d, J = 8.8 Hz, CH2), 1.69 (d,
J = 12.0 Hz, CH2), 1.59 (d, J = 12.4 Hz, CH2), 1.35 (q, J = 12.7 Hz,
CH2), 1.14 (t, J = 11.4 Hz, CH2), 1.02 (d, J = 6.4 Hz, CH3); 13C NMR
100 MHz)
d 171.98, 171.94, 171.73, 171.69, 153.27, 142.39,
142.31, 141.60, 137.06, 134.43, 132.69, 132.61, 129.03, 128.95,
128.74, 128.67, 128.62, 127.67, 127.59, 127.31, 127.23, 104.69,
74.40, 74.26, 60.79, 56.04, 54.70, 53.73, 53.34, 52.51, 49.06,
46.76, 46.36, 44.56, 43.61, 43.08, 39.50, 39.33, 28.88, 28.74,
27.50, 20.46; HR-FABMS calcd for C34H43ClN3O5: (M++1):
608.2891, found: 608.2901.
(CDCl3, 100 MHz)
d 170.75, 170.60, 141.53, 140.73, 132.81,
129.08, 128.79, 128.72, 127.72, 127.39, 75.20, 52.10, 51.60, 47.98,
46.01, 41.66, 39.29, 33.16, 33.13, 28.59, 25.51, 24.81, 20.15; HR-
FABMS calcd for C29H39ClN3O2: (M++1): 496.2731, found:
496.2724.
4.4.17. 5-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepan-1-
yl)-N-cyclohexyl-3-methyl-5-oxopentanamide (17)
4.4.13. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N,N,3-trimethyl-5-oxopentanamide (13)
White solid, yield: 60%; mp 62–63 °C; 1H NMR (CDCl3,
400 MHz) d 7.31–7.35 (m, 4CH), 7.25–7.30 (m, 3CH), 7.18–7.24
(m, 2CH), 5.06 (s, NH), 4.55 (s, CH), 3.71–3.82 (m, CH), 3.63–3.70
(m, 1/2CH2), 3.59 (t, J = 6.0 Hz, CH2), 3.46–3.50 (m, 1/2CH2), 2.61–
2.68 (m, CH2), 2.55–2.60 (m, CH2), 2.35–2.47 (m, CH2), 2.22–2.33
(m, CH), 2.09–2.15 (m, CH2), 1.89 (d, J = 12.0 Hz, CH2), 1.78 (q,
J = 6.0 Hz, CH2), 1.70 (d, J = 13.6 Hz, CH2), 1.60 (d, J = 12.4 Hz,
Light yellow gel, yield: 8%; 1H NMR (CDCl3, 400 MHz) d 7.35 (d,
J = 7.2 Hz, 2CH), 7.29 (d, J = 7.6 Hz, 2CH), 7.26 (t, J = 8.2 Hz, 2CH),
7.23 (d, J = 8.0 Hz, CH), 7.21 (d, J = 7.2 Hz, 2CH), 4.21 (s, CH),
3.60–3.71 (m, CH2), 3.48–3.59 (m, CH2), 3.01 (s, CH3), 2.91 (s,
CH3), 2.57; 2.53 (dd, J = 5.2; 5.6 Hz, CH), 2.27–2.48 (m, 3CH2),
2.16–2.26 (m, 1/2CH2), 2.00–2.13 (m, 1/2CH2), 1.03 (d, J = 6.4 Hz,

2334
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
CH2), 1.36 (q, J = 12.5 Hz, CH2), 1.15 (t, J = 10.2 Hz, CH2), 1.05 (d,
J = 5.8 Hz, CH3); 13C NMR (CDCl3, 100 MHz) d 171.79, 171.74,
170.96, 170.89, 142.34, 141.58, 132.60, 132.51, 129.00, 128.92,
128.67, 128.61, 128.56, 127.63, 127.55, 127.23, 127.15, 74.35,
74.19, 54.70, 53.67, 53.27, 52.50, 49.01, 47.94, 46.73, 46.27,
44.51, 43.23, 43.17, 39.41, 39.25, 33.05, 28.86, 28.61, 27.49,
25.47, 24.78, 20.18; HR-FABMS calcd for C30H41ClN3O2: (M++1):
510.2887, found: 510.2864.
4.4.22. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-N-(4-methylbenzyl)-4-oxobut-2-enamide (22)
Light yellow solid, yield: 81%; mp 76–78 °C; 1H NMR (CDCl3.
400 MHz)
d 7.330–7.360 (m, 5CH), 7.290–7.308 (m, 2CH),
7.189–7.234 (m, 2CH), 7.119–7.169 (m, 4CH), 6.328 (d,
J = 12.8 Hz, CH), 6.076 (d, J = 12.8 Hz, CH), 4.425 (d, J = 5.6 Hz,
CH2), 4.204 (s, CH), 3.591–3.614 (m, CH2), 3.441–3.467 (m, CH2),
2.337 (s, CH3), 2.310–2.232 (m, 2CH2); 13C NMR (CDCl3,
100 MHz)
d 165.71, 164.41, 141.40, 140.61, 136.94, 135.01,
4.4.18. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-(4-methylbenzyl)-3,3-dimethyl-5-oxopentanamide (18)
Light yellow solid, yield: 70%; mp 63–64 °C; 1H NMR (CDCl3,
400 MHz) d 8.05 (t, J = 5.8 Hz, NH), 7.35 (d, J = 8.0 Hz, 4CH), 7.30
(d, J = 7.2 Hz, 3CH), 7.26 (t, J = 8.4 Hz, 2CH), 7.18 (d, J = 8.0 Hz,
2CH), 7.10 (d, J = 8.0 Hz, 2CH), 4.38 (d, J = 5.6 Hz, CH2), 4.20 (s,
CH), 3.56–3.66 (m, CH2), 3.51 (t, J = 5.0 Hz, CH2), 2.34–2.37 (m,
2CH2), 2.33 (s, CH3), 2.32 (s, 2CH2), 1.02 (s, 2CH3); 13C NMR (CDCl3,
100 MHz) d 171.31, 170.73, 141.44, 140.64, 136.52, 136.14, 132.88,
129.13, 129.03, 128.83, 128.76, 127.69, 127.66, 127.45, 75.16,
51.93, 51.56, 47.35, 46.77, 42.89, 41.65, 40.62, 34.42, 29.30,
21.07; HR-FABMS calcd for C32H39ClN3O2: (M++1): 532.2731,
found: 532.2719.
132.86, 130.21, 130.03, 129.25, 129.03, 128.81, 128.74, 127.72,
127.69, 127.44, 74.98, 51.60, 51.06, 46.51, 43.17, 41.50, 21.09;
HR-FABMS calcd for C29H31O2N3Cl: (M++1): 488.2105, found:
488.2108.
4.4.23. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-N-(4-methoxybenzyl)-4-oxobut-2-enamide (23)
Light yellow solid, yield: 80%; mp 77–78 °C; 1H NMR (CDCl3.
400 MHz) d 7.330–7.360 (m, 5CH), 7.284–7.305 (m, 2CH), 7.196–
7.250 (m, 4CH), 6.850–6.872 (m, 2CH), 6.322 (d, J = 12.8 Hz, CH),
6.072 (d, J = 12.8 Hz, CH), 4.396 (d, J = 6.0 Hz, CH2), 4.186 (s, CH),
3.801 (s, CH3), 3.578–3.619 (m, CH2), 3.429–3.454 (m, CH2),
2.290–2.329 (m, 2CH2); 13C NMR (CDCl3, 100 MHz) d 165.79,
164.35, 158.86, 141.39, 140.61, 132.81, 130.17, 129.99, 129.11,
129.01, 128.78, 128.72, 127.66, 127.40, 113.95, 74.92, 55.23,
51.55, 51.02, 46.47, 42.84, 41.46; HR-FABMS calcd for
C29H31N3O3Cl: (M++1): 504.2054, found: 504.2058.
4.4.19. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-(4-methoxybenzyl)-3,3-dimethyl-5-oxopentanamide (19)
White solid, yield: 63%; mp 64–65 °C; 1H NMR (CDCl3,
400 MHz) d 8.06 (t, J = 5.6 Hz, NH), 7.35 (d, J = 6.4 Hz, 4CH), 7.29
(t, J = 8.1 Hz, 2CH), 7.26 (d, J = 8.8 Hz, 2CH), 7.21 (t, J = 8.8 Hz,
CH), 6.83 (d, J = 8.8 Hz, 4CH), 4.35 (d, J = 6.0 Hz, CH2), 4.20 (s, CH),
3.79 (s, OCH3), 3.56–3.65 (m, CH2), 3.51 (t, J = 5.0 Hz, CH2), 2.33–
2.39 (m, 2CH2), 2.32 (s, CH2), 2.30 (s, CH2), 1.05 (s, 2CH3); 13C
NMR (CDCl3, 100 MHz) d 171.23, 170.62, 158.52, 141.35, 140.58,
132.73, 131.29, 128.95, 128.89, 128.72, 128.66, 127.60, 127.34,
113.76, 75.02, 55.11, 51.81, 51.44, 47.22, 46.66, 42.46, 41.56,
40.50, 34.31, 29.17; HR-FABMS calcd for C32H39ClN3O3: (M++1):
548.2680, found: 548.2684.
4.4.24. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-N-cyclohexyl-4-oxobut-2-enamide (24)
White solid, yield: 69%; mp 152–154 °C; 1H NMR (CDCl3.
400 MHz) d 7.208–7.351 (m, 9CH), 6.282 (d, J = 12.4 Hz, CH),
6.038 (d, J = 12.4 Hz, CH), 4.240 (s, CH), 3.775–3.796 (m, CH),
3.653–3.677 (m, CH2), 3.457–3.481 (m, CH2), 2.341–2.402 (m,
2CH2), 1.795–1.878 (m, CH2), 1.666–1.718 (m, CH2), 1.559–
1.600 (m, 1/2CH2), 1.310–1.407 (m, CH2), 1.142–1.248 (m, CH2,
1/2CH2); 13C NMR (CDCl3. 100 MHz) d 165.85, 163.69, 141.31,
140.52, 130.84, 129.39, 129.07, 128.81, 128.74, 127.73, 127.45,
74.98, 51.67, 51.16, 48.04, 46.57, 46.30, 32.76, 25.48, 24.55;
HR-EIMS calcd for C27H32N3O2Cl: (M+): 465.2183, found:
465.2184.
4.4.20. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-(3,4,5-trimethoxybenzyl)-3,3-dimethyl-5-oxopentanamide
(20)
White solid, yield: 46%; mp 67–68 °C; 1H NMR (CDCl3,
400 MHz) d 8.37 (t, J = 5.8 Hz, NH), 7.35 (d, J = 8.4 Hz, 4CH), 7.29
(t, J = 7.6 Hz, 2CH), 7.26 (d, J = 8.8 Hz, 2CH), 7.21 (t, J = 7.2 Hz,
CH), 6.83 (d, J = 8.8 Hz, 2CH), 4.37 (d, J = 6.0 Hz, CH2), 4.20 (s, CH),
3.82 (s, 3OCH3), 3.56–3.67 (m, CH2), 3.53 (t, J = 5.0 Hz, CH2),
2.34–2.41 (m, 2CH2), 2.34 (s, CH2), 2.30 (s, CH2), 1.06 (s, 2CH3);
13C NMR (CDCl3, 100 MHz) d 171.33, 170.75, 153.18, 141.40,
140.60, 136.83, 135.10, 132.90, 129.03, 128.84, 128.78, 127.68,
127.48, 104.62, 75.14, 60.80, 56.01, 51.95, 51.57, 47.33, 46.90,
43.33, 41.70, 40.45, 34.61, 29.33; HR-FABMS calcd for
C34H43ClN3O5: (M++1): 608.2891, found: 608.2861.
4.4.25. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-
yl)-N-(4-phenylbutan-2-yl)-4-oxobut-2-enamide (25)
Light yellow solid, yield: 89%; mp 154–157 °C; 1H NMR
(CDCl3. 400 MHz) d 7.210–7.324 (m, 12CH), 7.176–7.196 (m,
2CH), 6.305 (d, J = 12.8 Hz, CH), 6.059 (d, J = 12.8 Hz, CH), 4.139
(s, CH), 4.026–4.063 (m, CH), 3.620–3.694 (m, CH2), 3.463–
3.489 (m, CH2), 2.602–2.633 (m, CH2), 2 .286–2.386 (m, 2CH2),
1.741–1.824 (m, CH2), 1.184 (d, J = 6.8 Hz, CH3); 13C NMR (CDCl3,
100 MHz)
d 165.75, 164.09, 141.80, 141.31, 140.50, 131.35,
129.05, 128.81, 128.75, 128.40, 128.36, 127.69, 127.45, 125.87,
74.98, 51.75, 51.17, 46.60, 45.23, 41.60, 38.49, 32.37, 20.84;
HR-FABMS calcd for C31H35N3O2Cl: (M++1): 516.2418, found:
516.2418.
4.4.21. 5-(4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)-
N-cyclohexyl-3,3-dimethyl-5-oxopentanamide (21)
White solid, yield: 9%; mp 77–78 °C; 1H NMR (CDCl3, 400 MHz)
d 7.65 (d, J = 8.4 Hz, NH), 7.35 (d, J = 8.4 Hz, 2CH), 7.29 (t, J = 7.4 Hz,
2CH), 7.25 (t, J = 8.0 Hz, CH), 7.22 (d, J = 6.8 Hz, 2CH), 7.21 (d,
J = 6.4 Hz, 2CH), 4.21 (s, CH), 3.76 (s, CH), 3.60–3.70 (m, CH2),
3.55 (t, J = 5.0 Hz, CH2), 2.30–2.43 (m, 2CH2), 2.24 (s, CH2), 2.17
(s, CH2), 1.86 (d, J = 12.4 Hz, CH2), 1.67 (d, J = 12.0 Hz, CH2), 1.29–
1.40 (m, CH2), 1.10–1.28 (m, 2CH2), 1.05 (s, CH3), 1.01 (s, CH3);
13C NMR (CDCl3, 100 MHz) d 170.84, 170.26, 141.45, 140.65,
132.92, 129.08, 128.87, 128.79, 127.74, 127.49, 75.22, 51.99,
51.63, 47.62, 47.47, 46.87, 41.72, 40.67, 34.38, 33.19, 29.34,
25.62, 24.85; HR-FABMS calcd for C30H41ClN3O2: (M++1):
510.2887, found: 510.2874.
4.4.26. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-
diazepan-1-yl)-N-(4-methylbenzyl)-4-oxobut-2-enamide (26)
Yellow solid, yield: 71%; mp 73–74 °C; 1H NMR (CDCl3.
400 MHz) d 7.092–7.321 (m, 13CH), 6.350 (dd, J = 12.8 Hz, CH),
6.101 (dd, J = 13.2 Hz, CH), 4.535 (d, J = 4.4 Hz, CH), 4.437 (t,
J = 5.4 Hz, CH2), 3.443–3.579 (m, 2CH2), 2.510–2.642 (m, 2CH2),
2.303 (s, CH3), 1.729–1.802 (m, CH2); 13C NMR (CDCl3. 100 MHz)
d 166.91, 164.62, 142.3, 141.50, 131.02, 129.78, 129.24, 128.98,
128.74, 127.61, 127.34, 74.12, 53.33, 52.91, 47.04, 46.38, 43.24,
26.35, 21.09; HR-EIMS calcd for C30H32O2N3Cl: (M+): 501.2183,
found: 501.2178.

Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
2335
4.4.27. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-
3.779–3.799 (m, CH), 3.665 (t, J = 5.0 Hz, CH2), 3.470 (t, J = 5.0 Hz,
diazepan-1-yl)-N-(4-methoxylbenzyl)-4-oxobut-2-enamide (27)
Yellow solid, yield: 52%; mp 63–64 °C; 1H NMR (CDCl3.
400 MHz) d 7.176–7.389 (m, 10CH), 6.812–6.856 (m, 2CH), 6.417
(dd, J = 12.8, 14.0 Hz, CH), 6.104 (t, J = 13.2 Hz, CH), 4.525 (s, CH),
4.401 (t, J = 5.4 Hz, CH), 3.785 (s, CH3), 3.429–3.654 (m, 2CH2),
2.507–2.641 (m, 2CH2), 1.712–1.780 (m, CH2); 13C NMR (CDCl3.
CH2), 2.324–2.385 (m, 2CH2), 1.139–1.954 (m, 5CH2); 13C NMR
(CDCl3. 100 MHz)
d 165.90, 163.67, 163.15, 160.70, 137.41,
130.73, 129.47, 129.23, 129.15, 115.69, 115.48, 74.05, 51.61,
51.09, 48.05, 46.55, 41.54, 32.76, 25.47, 24.55; HR-EIMS calcd for
C27H31N3O2F2: (M+): 467.2384, found: 467.2383.
100 MHz)
d
166.84, 164.56, 158.83, 142.31, 141.53, 131.37,
4.4.33. (Z)-4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-
(4-phenylbutan-2-yl)-4-oxobut-2-enamide (33)
131.01, 129.73, 129.45, 129.07, 128.95, 128.70, 127.59, 127.28,
113.95, 74.03, 55.23, 54.17, 52.90, 47.29, 44.44, 42.90, 27.05; HR-
EIMS calcd for C30H32O3N3Cl: (M+): 517.2132, found: 517.2131.
Light yellow solid, yield: 68%; mp 149–152 °C; 1H NMR (CDCl3.
400 MHz)
d 7.248–7.303 (m, 5CH), 7.176–7.196 (m, 4CH),
6.937ꢀ6.996 (m, 4CH), 6.304 (d, J = 12.8 Hz, CH), 6.059 (d,
J = 12.8 Hz, CH), 4.143 (s, CH), 4.027–4.064 (m, CH), 3.604ꢀ3.686
(m, CH2), 3.461–3.488 (m, CH2), 2.601–2.661 (m, CH2), 2.258–
2.378 (m, 2CH2), 1.741–1.808 (m, CH2), 1.183 (d, CH3, J = 6.4 Hz);
13C NMR (CDCl3, 100 MHz) d 165.73, 164.06, 163.13, 160.69,
141.80, 137.42, 137.39, 131.34, 129.18, 129.10, 129.05, 128.36,
125.87, 115.68, 115.47, 74.05, 51.71, 51.11, 46.58, 45.23, 41.58,
38.50, 32.38, 20.54; HR-FABMS calcd for C31H34N3O2F2: (M++1):
518.2619, found: 518.2619.
4.4.28. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-
diazepan-1-yl)-N-cyclohexyl-4-oxobut-2-enamide (28)
Pale yellow solid, yield: 62%; mp 74–75 °C; 1H NMR (CDCl3.
400 MHz) d 7.173–7.532 (m, 9CH), 6.812–6.856 (m, 2CH), 6.417
(dd, J = 12.8, 10.8 Hz, CH), 6.104 (dd, J = 9.2, 4.0 Hz, CH), 4.576 (d,
J = 8.0 Hz, CH), 3.781–3.837 (m, CH), 3.438–3.718 (m, 2CH2),
2.553–2.697 (m, 2CH2), 1.191–1.948 (m, 6CH2); 13C NMR (CDCl3.
125 MHz)
d 167.34, 164.11, 142.53, 141.72, 132.99, 131.83,
129.37, 129.22, 129.03, 128.93, 127.93, 127.56, 74.49, 54.53,
53.07, 48.28, 47.49, 44.73, 31.82, 26.54, 25.77, 24.84; HR-EIMS
calcd for C28H34O2N3Cl: (M+): 479.2340, found: 479.2336.
4.5. Bradykinin-induced contractions of guinea-pig ileum
Bradykinin, captopril, indomethacin, dithiothreitol, atropine
and Icatibant were obtained from Sigma (St. Louis, MO, USA). The
composition of the Tyrode solution was as follows (in mM): NaCl
136.9, KCl 2.7, CaCl2 1.8, MgCl2 1.15, NaH2PO4 0.4, NaHCO3 11.9,
glucose 5.6. The final DMSO concentration in bath was less than
0.1% and had no effect on the tissue’s responsiveness to bradykinin.
Male Hartley guinea-pigs weighing 275–500 g (Jaeil, Korea) were
fasted overnight and decapitated. At a level 2 cm above the ileoce-
cal junction, a section of ileum approximately 40 cm in length was
removed and placed in warm (37 °C) Tyrode solution. Strips of
muscle, 1.5–2 cm in length, were then mounted in a 50 mL bath
containing Tyrode solution (37 °C) and aerated with 95%O2/
5%CO2. Tissue contractions were recorded isometrically on a Grass
model 76E polygraph. After an equilibration period of about
60 min a stable baseline tone was reached and two or three con-
4.4.29. (Z)-4-(4-((4-Chlorophenyl)(phenyl)methyl)-1,4-
diazepan-1-yl)-N-(4-phenylbutan-2-yl)-4-oxobut-2-enamide
(29)
Yellow oil, yield: 73%; 1H NMR (CDCl3. 400 MHz) d 7.411–7.149
(m, 14CH), 6.342 (dd, J = 11.2, 13.2 Hz, CH), 6.100 (t, J = 13.0 Hz,
CH), 4.505 (d, J = 8.8 Hz, CH), 4.040–4.057 (m, CH), 3.568–3.725
(m, 2CH2), 2.545–2.679 (m, 3CH2), 1.748–1.828 (m, 2CH2), 1.195
(dd, J = 1.6, 5.2 Hz, CH3); 13C NMR (CDCl3. 125 MHz) d 167.23,
164.47, 142.48, 142.19, 141.68, 132.93, 132.20, 129.22, 129.03,
128.61, 128.55, 127.85, 126.07, 74.44, 54.55, 52.99, 47.58, 45.46,
44.82, 38.76, 32.65, 27.43, 21.07; HR-EIMS calcd for C32H36N3O2Cl:
(M+): 529.2496, found: 529.2499.
4.4.30. (Z)-4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-
(4-methylbenzyl)-4-oxobut-2-enamide (30)
tractions were obtained in response to BK (0.1 lM), every 20 min
Light red solid, yield: 67%; mp 73–75 °C; 1H NMR (CDCl3.
400 MHz) d 7.308–7.344 (m, 4CH), 7.114–7.184 (m, 4CH), 6.963–
7.012 (m, 4CH), 6.319 (d, J = 12.8 Hz, CH), 6.070 (d, J = 12.8 Hz,
CH), 4.421 (d, J = 6.0 Hz, CH2), 4.213(s, CH), 3.589–3.614 (m, CH2),
3.438–3.487 (m, CH2), 2.332 (s, CH3), 2.296–2.305 (m, 2 CH2); 13C
NMR (CDCl3, 100 MHz) d 165.70, 164.46, 163.17, 160.73, 137.52,
137.01, 130.38, 129.96, 129.30, 129.21, 129.13, 127.77, 115.73,
115.52, 74.11, 51.61, 51.07, 46.54, 43.24, 41.54, 21.12; HR-FABMS
calcd for C29H30O2N3F2: (M++1): 490.2306, found: 490.2304.
interval, to assay sensitivity and reproducibility of the contractile
response.39,40 Only segments producing reproducible responses
were used. The last control response was taken as 100% and subse-
quent results obtained with bradykinin antagonists expressed as a
percentage of this. The segments were incubated with the bradyki-
nin antagonists (1 lM) for 15 min before BK was added. To mini-
mize degradation of bradykinin and to prevent responses due to
neuronal activation or prostaglandin production, Tyrode solutions
contained 1
indomethacin. Moreover, to prevent histaminergic responses,
M of dibenamine, which is an irreversible histamine H1 blocker,
lM each of captopril, atropine, dithiothreitol, and
4.4.31. (Z)-4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-
(4-methoxybenzyl)-4-oxobut-2-enamide (31)
1 l
was also added to the Tyrode solution.
Light yellow solid, yield: 73%; mp 73–75 °C; 1H NMR (CDCl3.
400 MHz) d 7.307–7.340 (m, 4CH), 7.176–7.220 (m, 2CH), 6.957–
6.999 (m, 4CH), 6.837–6.858 (m, 2CH), 6.300 (d, J = 12.8 Hz, CH),
6.058 (d, J = 12.8 Hz, CH), 4.381 (d, J = 5.6 Hz, CH2), 4.192 (s, CH),
3.787 (s, CH3), 3.587 (br s, CH2), 3.342 (br s, CH2), 2.292 (br s,
2CH2); 13C NMR (CDCl3, 125 MHz) d 166.01, 164.66, 163.15,
161.20, 159.18, 137.77, 130.51, 130.29, 129.43, 129.37, 115.95,
114.27, 74.31, 55.53, 51.84, 51.28, 46.77, 43.16, 41.75; HR-FABMS
calcd for C29H30N3O3F2: (M++1): 506.2255, found: 506.2258.
4.6. Nociceptive and inflammatory pain
4.6.1. Formalin and compound 33 injection
To investigate the effect of 33 on both nociceptive and inflam-
matory pain, the formalin test was carried out. Each rat was placed
in the observation chamber (20 Â 20 Â 20 cm) equipped with a
mirror placed behind the chamber to allow an unobstructed view
of the paw for a 30 min habituation period. Thereafter, animals
were assigned to the two groups; the first group was subjected to
4.4.32. (Z)-4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-N-
cyclohexyl-4-oxobut-2-enamide (32)
the subcutaneous injection of the mixture of formalin (5%, 50
and vehicle (Methyl pyrrolidone/Tween80/Saline = 1:1:8, 10
under the plantar surface of the right hind paw using a 30-gauge
syringe, the second group was treated with the mixture of formalin
l
l
l)
l)
Pale yellow solid, yield: 54%; mp 92–94 °C; 1H NMR (CDCl3.
400 MHz) d 7.307–7.350 (m, 4CH), 6.961–7.009 (m, 4CH), 6.283
(d, J = 12.4 Hz, CH), 6.035 (d, J = 12.8 Hz, CH), 4.251 (s, CH),
(5%, 50 ll) plus 33 (60 lg/10 ll) by the same way described above.

2336
Y. L. Kam et al. / Bioorg. Med. Chem. 18 (2010) 2327–2336
4.6.2. Behavioral testing for Formalin test
were used wherever appropriate. A difference of P <0.05 was con-
After the injection, animals were returned to the chamber
immediately and the time spent in licking, shaking or biting the in-
jected paw was recorded for every 5 min over a 60 min period. The
early phase was defined as the first 10 min after formalin injection,
and the late phase was the following 50 min.41,42 The observations
in this test were carried out by three investigators who were una-
ware of the treatment status.
sidered to be statistically significant.
Acknowledgments
This study was supported in part by the Korea Research founda-
tion (Grants KRF-2004-E00406 and KRF-2005-041-E20291) and
Grant (M103KV010015-08K2201-01510) from Brain Research Cen-
ter of the 21st Century Frontier Research Program funded by the
Ministry of Education, Science and Technology, the Republic of
Korea.
4.7. Neuropathic pain
4.7.1. Tail nerve injury
Tail nerve injury (TNI) was based on the procedure previously
described by Na et al.43 Briefly, under enflurane anesthesia, ani-
mals were subjected to unilateral transection of the superior and
inferior caudal trunks at the level between the S1 and the S2 spinal
nerves. To prevent possible rejoining of the proximal and distal
ends of the severed trunks, pieces of nerve about 2 mm in length
were removed from the distal nerve ends. This surgery injured
the S1 spinal nerve which innervates the tail.
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At 2 weeks after TNI, rats were given a baseline test and there-
after compound 33 (60 mg/kg) or vehicle (Methyl pyrrolidone/
Tween80/Saline = 1:1:8) was administered intraperitoneally.
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