Solution-phase combinatorial synthesis of nonpeptide bradykinin antagonists

Article abstract of DOI:10.1016/j.bmc.2004.04.031

We describe the solution-phase combinatorial synthesis and pharmacological effect of fifty N,N^′-substituted-N″-1-(4- chlorobenzhydryl)piperazine iminodiacetic acid triamide derivatives as nonpeptide B2 antagonists. The synthesized compounds wer

Full text of DOI:10.1016/j.bmc.2004.04.031

Bioorganic & Medicinal Chemistry 12 (2004) 3543–3552
Solution-phase combinatorial synthesis of nonpeptide
bradykinin antagonists
Yoo Lim Kam, Soo-Jin Rhee and Hea-Young P. Choo*
School of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea
Received 17 February 2004; revised 26 April 2004; accepted 26 April 2004
Available online
Abstract—We describe the solution-phase combinatorial synthesis and pharmacological effect of fifty N,N0-substituted-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide derivatives as nonpeptide B2 antagonists. The synthesized compounds
were tested for their antibradykinin activity by utilizing guinea-pig ileum smooth muscle. Most of the compounds showed antag-
onistic effects on bradykinin induced contraction. N-acetyl-N0-(4-methylbenzyl)-N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic
acid triamide (A3B1C1) showed the 46% inhibition at 100 nM.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
which is structurally related to the Fujisawa compounds
includes the basic phenylamidine moiety on one termi-
nus and a central sulfonamide linkage.11 Bradyzide,
which evolved from high-throughput screening, has the
prolylamide section of the Fournier compound and
lipophilic benzodiazepine portion.12
Bradykinin is an autocoid related to acute and chronic
pain and inflammation. Two bradykinin receptors B1
and B2 are known and the B2 receptor is constitutively
expressed on most cell types, whereas the B1 receptors
are induced during inflammatory insults.1–3 Most of the
antagonists for bradykinin B2 receptors reported are
bradykinin derived peptides such as HOE 140.4
However the structural features required for the bra-
dykinin antagonist is not clear. From the bradykinin
structure, it was generally accepted that the antagonist
ꢀ
The nonpeptide bradykinin antagonists were of interest
as novel anti-inflammatory therapeutics. The first non-
peptide antagonist Win 64338 was reported in 1993 by
Sterling Winthrop researchers,5 but it showed low
specificity and a species-dependent variable affinity for
B2 receptors. Some active compounds such as FR
173657, LF 16-0687, and bradyzide have been reported
recently (Fig. 1).6;7 The derivatives synthesized by Fu-
jisawa showed antagonist activity at the bradykinin B2
receptor and as in FR 173657, they have the structural
feature that a dichlorobenzene-linked terminal quinoline
stands one side, while the other side varies from basic
heterocycles to amidinobenzene. Especially the FR
173657 is a potent, orally available B2 antagonist, and
its biological activity has been studied extensively.8–10
On the other hand, Fournier compound LF 16-0687,
should have two basic groups separated by 10 A, with a
hydrophobic aromatic group;5 even though the need for
basic groups seems not to be absolute.13
In our search for the new bradykinin antagonists, we
reported the bradykinin antagonistic effects of pipera-
zine compounds derived from the anti-histamine drug,
cetirizine.14 Now we have designed and synthesized new
compounds with piperazine, three amide bonds and a
lipophilic ring system in each molecule. The solution-
phase combinatorial synthesis route with iminodiacetic
anhydride as template was employed for our discovery
of nonpeptide bradykinin antagonists.15;16
1.1. Solution-phase combinatorial synthesis
To produce large numbers of diverse compounds rapidly
and efficiently, a combinatorial synthesis using the
iminodiacetic anhydride template was employed. The
solution-phase, parallel synthesis of chemical libraries
allowed the preparation of multi-milligram quantities of
Keywords: Solution-phase combinatorial synthesis; Nonpeptide
bradykinin; Antagonists.
* Corresponding author. Tel.: +82-2-32773042; fax: +82-2-32772851;
e-mail: hypark@ewha.ac.kr
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.031

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Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
H+
N
N
H
N
O
O
Cl
O
HN
NH
N
P+(C4H9)3
N
H
O
O
Cl
N
N
H
b
a
CH3
H
CH3
CH3
NO2
O
NH
N
S
N
N
NH
NH2
NH
N
CH3
NH
CH3
NH
O
N
H
N
S
O
Cl
Cl
S
O
O
O
N
O
O
c
d
Figure 1. The structure of Win 64338 (a), FR 173657 (b), LF 16-0687 (c), and Bradyzide (d).
each individual member. The template contained three
positions that could be sequentially functionalized en-
abling the synthesis of libraries with up to three variable
units. Also in each step simple liquid/liquid extraction
was used for both the isolation and purification of each
intermediate and final product from the starting mate-
rial, reactants, reagents, and their reaction by-products.
Boc groups were removed using HCl in dioxane and the
resulting secondary amines were coupled to carboxylic
acids (R3COOH) affording triamides AXB1CX using
PyBrOP. The library consisted of individual compounds
as a 10 ꢀ 1 ꢀ 5 matrix affording 50 compounds. Each
individual compound synthesized (intermediate and
final product) was traditionally characterized by 1H
NMR and low-resolution FABMS (high-resolution
FABMS for several compounds). Most of the final
library products were suitable for direct use in screening
efforts without further purification (Scheme 2).
Each of the expected library members was obtained in a
purified form in amounts ranging from 1 to 100 mg. In
situ closure of N-Boc-iminodiacetic acid to the anhy-
dride 1 followed by treatment with R1NH2 afforded the
monoamides AX, which were purified by simple acid
extraction to remove unreacted R1NH2, EDCI, and its
reaction by-products. Each product was treated with 1-
(4-chlorobenzhydryl)piperazine (P) and PyBOP to af-
ford diamides AXB1, which were effectively purified by
acid and base extractions to remove reaction by-prod-
ucts, the unreacted starting materials, and reagents. The
2. Results and discussion
The structures of amines and acids used are illustrated in
Table 1. The percent reaction yields are given in Table 2.
The purity of selected final compounds was 80–90% in
HPLC analysis using reported conditions.16
Each compound was tested for their bradykinin-induced
contractilities on guinea-pig ileum smooth muscle in
0.1 lM concentration. As shown in Table 3, inhibitory
activity of synthesized compounds to maximum con-
tractility of bradykinin was varied from 0.4% to 46.3%
(n ¼ 6).
N
O
Cl
O
N
N
H
O
O
Cl
N
N
H
FR 173657
2.1. Antibradykinin effects
B
As shown in Scheme 1, FR 173657 and synthesized
compounds can be considered as composed of three
parts: terminal ring system (A), the aromatic rings (B),
and amide bonds (C).
C
A
CH
R''
O
N
H
N
N
N
R'
O
O
For the terminal (A) of the compounds, 10 amines (M1–
M10) were employed. Because there was no structural
feature for antibradykinin antagonist, some alkyl, or
substituted aromatic amines were used. The compounds
Cl
Scheme 1. Design of the iminodiacetic acid triamide derivatives based
on FR 173657.

Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
3545
O
O
O
O
BOC O
N
O
BOC O
N
R1NH2
EDCI
R1HN
OH
HO
OH
N
BOC
1
R' = M1-M10
AX
(1)HCl-dioxane
R3
O
O
BOC O
N
PyBOP
(2)R3COOH,PyBrOP
R1HNOC
N
COR2
R1HN
R2
R2=
R3 = S1-S5
AXB1CX
CH N NH
P
AXB1
Cl
Scheme 2. Solution-phase combinatorial synthesis route using the iminodiacetic anhydride template.
Table 1. The structures of amines and acids
Table 2. The yield (%) of synthesized compounds
C1
C2
C3
C4
C5
R3
O
O
O
A1B1
A2B1
A3B1
A4B1
A5B1
A6B1
A7B1
A8B1
A9B1
A10B1
NRa
2
40
14
66
53
77
21
53
27
21
95
64
17
74
77
34
9
45
19
85
77
97
14
61
25
34
13
26
25
82
84
89
39
47
32
22
13
N
R1HN
R2
64
71
76
33
13
17
20
30
R1NH2
H3C
H3C
H3C
CH2NH2
CH2NH2
NH2
99
32
23
26
M1
M2
M3
H3CO
H3CO
H3CO
CH3
CH2NH2
CH2CH2CH NH2
H3CO
CH2NH2
a No reaction.
M4
M5
M6
NH2
N
S
N
Br
Cl
NH2
(H3C)3C
N
O
NH2
S
Table 3. Antibradykinin activity of synthesized compounds at 0.1 lM
concentration in the guinea-pig ileum
M7
M9
M8
Sample no
%Inhibition
Sample no
%Inhibition
NH2
A1B1C1
A1B1C2
A1B1C2
A1B1C4
A1B1C5
NR
A2B1C1
A2B1C2
A2B1C3
A2B1C4
A2B1C5
12.90 ꢁ 2.04
19.46 ꢁ 2.22
18.29 ꢁ 4.78
19.64 ꢁ 2.98
16.67 ꢁ 2.95
M10
7.67 ꢁ 3.48
13.26 ꢁ 1.72
23.67 ꢁ 5.78
7.12 ꢁ 3.44
R2NH
CH
N
NH
A3B1C1
A3B1C2
A3B1C3
A3B1C4
A3B1C5
46.32 ꢁ 6.32
24.52 ꢁ 2.49
42.58 ꢁ 0.28
31.19 ꢁ 6.78
23.70 ꢁ 3.15
A4B1C1
A4B1C2
A4B1C3
A4B1C4
A4B1C5
)0.10 ꢁ 2.75
5.96 ꢁ 3.05
39.16 ꢁ 5.69
18.21 ꢁ 3.40
18.55 ꢁ 2.30
P
Cl
R3COOH
O
O
A5B1C1
A5B1C2
A5B1C3
A5B1C4
A5B1C5
19.95 ꢁ 3.58
27.68 ꢁ 4.72
24.00 ꢁ 1.98
25.96 ꢁ 1.72
33.60 ꢁ 9.60
A6B1C1
A6B1C2
A6B1C3
A6B1C4
A6B1C5
20.76 ꢁ 5.39
18.45 ꢁ 2.47
32.63 ꢁ 7.77
22.50 ꢁ 7.04
21.90 ꢁ 3.89
H3C
OH
OH
OH
O
OH
OH
O
O
S1
S2
S3
S4
S5
A7B1C1
A7B1C2
A7B1C3
A7B1C4
A7B1C5
17.96 ꢁ 7.96
13.38 ꢁ 6.31
7.30 ꢁ 5.90
10.86 ꢁ 7.61
7.78 ꢁ 9.87
A8B1C1
A8B1C2
A8B1C3
A8B1C4
A8B1C5
16.57 ꢁ 4.56
20.82 ꢁ 3.04
12.90 ꢁ 3.85
26.02 ꢁ 4.54
)6.02 ꢁ 7.26
with substituted benzyl groups (A3- and A6-series)
showed good antibradykinin activities. But the com-
pounds with simple alkyl groups, such as isopropyl (A1-
series) and cyclohexyl (A10-series), exhibited poor
activities. Compounds with heterocycles, such as isox-
azole and thiazoles, showed poor activities, too.
A9B1C1
A9B1C2
A9B1C3
A9B1C4
A9B1C5
12.76 ꢁ 5.84
8.50 ꢁ 3.91
9.19 ꢁ 5.13
2.16 ꢁ 4.00
9.90 ꢁ 6.73
A10B1C1
A10B1C2
A10B1C3
A10B1C4
A10B1C5
0.42 ꢁ 1.38
13.33 ꢁ 4.82
8.75 ꢁ 3.86
4.71 ꢁ 2.73
19.15 ꢁ 9.37
To mimic the dichlorobenzene-linked terminal quinoline
(B), 1-(4-chlorobenzhydryl)piperazine (P) was intro-
duced in our synthesized compounds. In our previous
HOE 140
82.22 ꢁ 2.31

3546
Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
report14 on the second-generation antihistamine agent
cetirizine, the antibradykinin effects were observed from
the compounds with 1-(4-chlorobenzhydryl)piperazine
moiety. However the dual effect of antihistamine and
antibradykinin could be expected.
4. Experimental
4.1. General procedure for the first derivatization of
N-Boc iminodiacetic acids
A mixture of N-((tert-butyloxy)carbonyl)iminodiacetic
acid (2.13 mmol) and EDCI (2.13 mmol) in 6.4 mL of
DMF was stirred for 1 h at room temperature (A2–A10)
or at 50 °C (A1). Amine (M1–M10, 2.13 mmol) was
added to the resulting solution via syringe while stirring
(slightly exothermic). In reactions with aromatic amines
(M7–M9) the catalyst DMAP (4.26 mmol, 2 equiv) were
added to the resulting solution. The reaction mixture
was stirred for 20 h before it was poured into a 250 mL
separatory funnel. The product was diluted with EtOAc
(60 mL), washed sequentially with 10% aqueous HCl
(2 ꢀ 40 mL), and saturated aqueous NaCl (40 mL), dried
over Na2SO4 and the solvent removed in vacuo to afford
the products A1–A10.
For the substitution on the nitrogen of the middle amide
bond (C) in our molecules, five acids (S1–S5) including
naphthyl carboxylic acid adopted from structure of Win
64338 were employed. As the 2-naphthyl of Win 64338,
this part was introduced for a distinct hydrophobic
binding site in the B2 receptor since the aromatic residue
in position 8 of bradykinin is known as an absolute
requirement for high affinity binding. However when
five different acids were introduced, little difference in
antibradykinin activity was observed. Only the benzyl
(S3) substitution showed slightly increased activity than
the other compounds derived from different acids.
Among the 50 synthesized compounds, five compounds
A3B1C1, A3B1C3, A4B1C3, A5B1C5, and A6B1C3
showed high activity. The IC50 value of A4B1C3 was ca.
10ꢂ7 M while that of the reference compound HOE 140
was 10ꢂ8 M.17 The potency of our compounds was less
than the peptide antagonist HOE 140 but the synthetic
compounds has the merit of being nonpeptide. The
potency (IC50) of nonpeptide antagonists Win 64338 and
FR 173657 has been reported as 3.9 lM and 0.56 nM,
respectively, in a guinea-pig ileum binding affinity
assay.6 However the synthetic route for our compounds
was very simple and conditions were very mild com-
pared to Fujisawa compounds. The further optimization
of these series of compounds by changing 1-(4-chloro-
benzhydryl)piperazine (part B in Scheme 1) to various
amines are under study.
4.1.1. N-((tert-Butyloxy)carbonyl)-N0-isopropyl iminodi-
acetic acid monoamide (A1). 0.31 g (53%); 1H NMR
(acetone-d6, 400 MHz) d 3.9 (4H, m), 2.6 (1H, s), 1.3
(9H, s), 1.0 (6H, dd, J ¼ 6:4 and 5.6 Hz); FABMS m=z
177.83 (100), 274.92 (34).
4.1.2. N-((tert-Butyloxy)carbonyl)-N0-benzyl iminodiace-
tic acid monoamide (A2). 0.52 g (75%); H NMR (ace-
1
tone-d6, 400 MHz) d 7.2 (5H, m), 4.4 (2H, m), 3.9–4.0
(4H, m), 1.3 and 1.4 (9H, two s); FABMS m=z 266.9
(100), 322.96 (62).
4.1.3. N-((tert-Butyloxy)carbonyl)-N0-(4-methylbenzyl)-
iminodiacetic acid monoamide (A3). 0.49 g (68%); 1H
NMR (acetone-d6, 400 MHz) d 7.2 (4H, m), 4.4 (2H, m),
3.9–4.0 (4H, m), 2.3 (3H, s), 1.3 and 1.5 (9H, two s);
FABMS m=z 57.5 (100), 336.95 (42).
H2
C
O
O
N
O
N
N
CH
Cl
NH
H2C
4.1.4. N-((tert-Butyloxy)carbonyl)-N0-(4-methoxybenzyl)-
iminodiacetic acid monoamide (A4). 0.43 g (55%); 1H
NMR (acetone-d6, 400 MHz) d 7.1 (2H, m), 6.8 (2H, m),
4.3 (2H, d, J ¼ 5:6 Hz), 3.9–4.0 (4H, m), 3.6 (3H, s), 1.4
(9H, two s); FABMS m=z 153.9 (100), 352.93 (24).
OCH3
A4B1C3
4.1.5. N-((tert-Butyloxy)carbonyl)-N0-(1-(methyl)-3-(phen-
yl)propyl)iminodiacetic acid monoamide (A5). 0.62 g
(80%); H NMR (acetone-d6, 400 MHz) d 7.1–7.4 (5H,
3. Conclusions
1
Fifty nonpeptide bradykinin B2 receptor antagonists
were designed and synthesized by solution-phase com-
binatorial synthesis using the iminodiacetic anhydride
template. The synthetic route for the preparation of the
compounds was very simple and conditions were very
mild. Most of the compounds synthesized showed
inhibitory activity on bradykinin induced contraction at
0.1 lM concentration in the guinea-pig ileum. The po-
tency was less than HOE 140, the peptide antagonist,
but the synthetic compounds have the merit of being
nonpeptide.
m), 4.1 (1H, m), 3.9–4.0 (4H, m), 2.6 (2H, m), 1.8 (2H,
m), 1.4 (9H, two s), 1.2 (3H, d, J ¼ 7:2 Hz); FABMS
m=z 308.9 (100), 364.95 (58).
4.1.6. N-((tert-Butyloxy)carbonyl)-N0-(3,4,5-trimethoxy-
benzyl)iminodiacetic acid monoamide (A6). 0.23 g
1
(26%); H NMR (acetone-d6, 400 MHz) d 6.5 (2H, m),
4.4 (2H, d, J ¼ 5:6 Hz), 4.0 (4H, m), 3.8 (9H, s), 1.4 (9H,
s); FABMS m=z 181.0 (100), 413.06 (22).

Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
3547
4.1.7. N-((tert-Butyloxy)carbonyl)-N0-(2-(4-(4-chlorophen-
yl)thiazole))iminodiacetic acid monoamide (A7). 0.29 g
(32%); H NMR (acetone-d6, 400 MHz) d 7.2–7.9 (5H,
m), 3.7–3.9 (4H, m), 1.2 and 1.3 (9H, two s); FABMS
400 MHz) d 7.0–7.6 (13H, m), 4.4 (1H, s), 4.3 (2H, d,
J ¼ 6 Hz), 4.2 (2H, d, J ¼ 4:8 Hz), 3.8 (2H, d,
J ¼ 18:4 Hz), 3.5–3.6 (4H, m), 2.3–2.4 (4H, m), 2.2 (3H,
s), 1.3 (9H, two s); FABMS m=z 176.0 (100), 605.20 (15).
1
m=z 57.5 (100), 425.93 (26).
4.2.4. N-((tert-Butyloxy)carbonyl)-N0-(4-methoxybenzyl)-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
diamide (A4B1). 0.67 g (89%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.6 (11H, m), 6.8 (2H, m), 4.4 (1H, s),
4.3 (2H, d, J ¼ 5:6 Hz), 4.2 (2H, d, J ¼ 3:2 Hz), 3.8 (2H,
d, J ¼ 20:4 Hz), 3.7 (3H, s), 3.5–3.6 (4H, m), 2.3–2.4
(4H, m), 1.4 (9H, two s); FABMS m=z 201.0 (100),
621.34 (14).
4.1.8. N-((tert-Butyloxy)carbonyl)-N0-(3-(5-tert-butylis-
oxazole))iminodiacetic acid monoamide (A8). 0.2 g
(26%); H NMR (acetone-d6, 400 MHz) d 6.0(1H, s),
4.4(4H, s), 1.4(9H, s), 1.2 (9H, s); FABMS m=z 57.5
(100), 356.03 (18).
1
4.1.9. N-((tert-Butyloxy)carbonyl)-N0-(2-(4-(4-bromophen-
yl)thiazole))iminodiacetic acid monoamide (A9). 0.27 g
1
(27%); H NMR (acetone-d6, 400 MHz) d 7.2–7.8 (5H,
m), 3.7–3.9 (4H, s), 1.2 and 1.3 (9H, two s); FABMS m=z
73.3 (100), 469.84 (7).
4.2.5. N-((tert-Butyloxy)carbonyl)-N0-(1-(methyl)-3-(phen-
yl)propyl)-N00-1-(4-chlorobenzhydryl)piperazine iminodi-
acetic acid diamide (A5B1). 0.78 g (94%); 1H NMR
(acetone-d6, 400 MHz) d 7.1–7.5 (14H, m), 4.4 (1H, s),
4.2–4.3 (2H, m), 3.9 (1H, m), 3.7–3.8 (2H, m), 3.5–3.7
(4H, m), 2.5–2.7 (2H, m), 2.3–2.5 (4H, m), 1.7 (2H, m),
1.4 (9H, two s), 1.0–1.2 (3H, dd, J ¼ 6:8 and 16.4 Hz);
FABMS m=z 201.0 (100), 633.24 (32).
4.1.10. N-((tert-Butyloxy)carbonyl)-N0-cyclohexylimino-
diacetic acid monoamide (A10). 0.3 g (12%); H NMR
1
(acetone-d6, 400 MHz) d 4.0 (4H, m), 3.7 (1H, m), 1.6–
1.9 (5H, m), 1.4 (9H, s), 1.2–1.4 (5H, m); FABMS m=z
153.9 (100), 315.02 (28).
4.2. General method for second diversification
4.2.6. N-((tert-Butyloxy)carbonyl)-N0-(3,4,5-trimethoxy-
benzyl)-N00-1-(4-chlorobenzhydryl)piperazine iminodiace-
tic acid diamide (A6B1). 0.31 g (81%); 1H NMR
(acetone-d6, 400 MHz) d 7.2–7.5 (9H, m), 6.6 (2H, s), 4.4
(1H, s), 4.3 (2H, d, J ¼ 6:0 Hz), 4.2 (2H, d, J ¼ 11:6 Hz),
4.0 (2H, d, J ¼ 7:2 Hz), 3.8 (9H, s), 3.5–3.6 (4H, m), 2.3–
2.5 (4H, m), 1.3 (9H, two s); FABMS (Na) m=z 132.9
(100), 703.28 (15).
The N-Boc-iminodiacetic monoamide (A1–A10 each,
1.31 mmol, 1.0 equiv), 1-(4-chloro-benzhydryl)pipera-
zine (P, 1.441 mmol, 1.1 equiv), and PyBOP
(1.441 mmol, 1.1 equiv) were combined in a 30 mL vial.
DMF (15 mL) was added followed by i-Pr2NEt
(2.62 mmol, 2.0 equiv). The reaction mixture was stirred
16 h at room temperature before it was poured into a
250 mL separatory funnel. The product was diluted with
EtOAc (100 mL), washed sequentially with 10% aqueous
HCl (3 ꢀ 50 mL), saturated aqueous NaHCO3
(2 ꢀ 50 mL), and saturated aqueous NaCl (50 mL), dried
(Na2SO4) and the solvent removed in vacuo to afford the
products A1B1–A10B1.
4.2.7. N-((tert-Butyloxy)carbonyl)-N0-(2-(4-(4-chlorophen-
yl)thiazole))-N00-1-(4-chlorobenzhydryl)piperazine imino-
diacetic acid diamide (A7B1). 0.34 g (69%); 1H NMR
(acetone-d6, 400 MHz) d 7.2–8.0 (14H, m), 4.45 (1H, s),
4.4 (2H, m), 4.1–4.15 (2H, m), 3.6–3.8 (4H, m), 2.4–2.5
(4H, m), 1.3 and 1.4 (9H, two s); FABMS m=z 132.9
(100), 694.11 (10.5).
4.2.1. N-((tert-Butyloxy)carbonyl)-N0-isopropyl-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid diamide
(A1B1). 0.47 g (77%); 1H NMR (acetone-d6, 400 MHz) d
7.2–7.5 (9H, m), 4.3 (1H, s), 3.8–4.0 (4H, m), 3.6 (1H, s),
3.4–3.5 (4H, m), 2.2–2.3 (4H, m), 1.4 (9H, s), 1.3 (6H,
dd, J ¼ 6:4 and 13.2 Hz); FABMS m=z 200.9 (100),
543.02 (18).
4.2.8. N-((tert-Butyloxy)carbonyl)-N0-(3-(5-tert-butylis-
oxazole))-N00-1-(4-chlorobenzhydryl)piperazine iminodi-
acetic acid diamide (A8B1). 0.29 g (83%); 1H NMR
(acetone-d6, 400 MHz) d 7.2–7.6 (9H, m), 6.6 (1H, s), 4.4
(1H, s), 4.3 (2H, d, J ¼ 5:2 Hz), 4.0 (2H, d, J ¼ 14 Hz),
3.5–3.7 (4H, m), 2.4–2.5 (4H, m), 1.4 (9H, two s), 1.3
(9H, two s); FABMS m=z 200.9 (100), 624.18 (46).
4.2.2.
N-((tert-Butyloxy)carbonyl)-N0-benzyl-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid diamide
(A2B1). 0.61 g (73%); 1H NMR (acetone-d6, 400 MHz) d
7.2–7.5 (14H, m), 4.4 (3H, m), 4.2 (2H, d, J ¼ 5:6 Hz),
3.8 (2H, d, J ¼ 18 Hz), 3.5–3.6 (4H, m), 2.3–2.5 (4H, m),
1.4 (9H, two s); FABMS m=z 201.0 (100), 591.3 (12.5).
4.2.9. N-((tert-Butyloxy)carbonyl)-N0-(2-(4-(4-bromophen-
yl)thiazole))-N00-1-(4-chlorobenzhydryl)piperazine imino-
diacetic acid diamide (A9B1). 0.23 g (92%); 1H NMR
(acetone-d6, 400 MHz) d 7.2–7.9 (14H, m), 4.45 (1H, s),
4.4 (2H, d, J ¼ 4:0 Hz), 4.1 (2H, d, J ¼ 20 Hz), 3.6–3.8
(4H, m), 2.4–2.5 (4H, m), 1.3 and 1.4 (9H, two s);
FABMS m=z 73.4 (100), 738.14 (3.3).
4.2.3. N-((tert-Butyloxy)carbonyl)-N0-(4-methylbenzyl)-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
diamide (A3B1). 0.73 g (86%); 1H NMR (acetone-d6,

3548
Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
4.2.10. N-((tert-Butyloxy)carbonyl)-N0-cyclohexyl-N00-1-
(4-chlorobenzhydryl)piperazine iminodiacetic acid di-
amide (A10B1). 0.47 g (65%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.9 (9H, m), 4.4 (1H, s), 4.2 (2H, d,
J ¼ 2:8 Hz), 3.7 (2H, d, J ¼ 15:2 Hz), 3.65 (1H, m), 3.5–
3.6 (4H, m), 2.3–2.5 (4H, m), 1.5–1.8 (5H, m), 1.4 (9H,
two s), 1.2–1.3 (5H, m); FABMS m=z 200.9 (100), 583.16
(60).
4.3.5.
N-Acetyl-N0-(3,4,5-trimethoxybenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A6B1C1). 7.3 mg (33%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.5 (9H, m), 6.6 (2H, m), 4.4 (1H, s),
4.2–4.4 (4H, m), 3.9–4.1 (2H, m), 3.8 (9H, s), 3.5–3.6
(4H, m), 2.3–2.4 (4H, m), 2.0 (3H, s); FABMS m=z 73.3
(100), 623.14 (1.0).
4.3.6. N-Acetyl-N0-(2-(4-(4-chlorophenyl)thiazole))-N00-1-
(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
4.3. General procedure for the third diversification
1
amide (A7B1C1). 10.0 mg (13%); H NMR (acetone-d6,
CHCl3 (1 mL) and 4 M HCl–dioxane (1 mL) were added
to A1B1–A10B1 (0.017 mmol) in a 4 mL vial and this
mixture was allowed to stand for 3 h. The solvent and
excess acid were removed under stream of N2. The
resulting residue was dissolved in 1 mL of DMF and
added i-Pr2NEt (0.055 mmol). After dissolution car-
boxylic acid (S1–S5, 0.019 mmol, 1.1 equiv) was added,
followed by PyBrOP (0.017 mmol, 1 equiv). After stir-
ring for 16 h, the reaction mixture was diluted with
EtOAc (40 mL) and washed with 10% aqueous HCl
(3 ꢀ 30 mL), saturated aqueous NaHCO3 (2 ꢀ 30 mL),
and saturated aqueous NaCl (30 mL), dried (NaSO4),
and concentrated to afford the products A2B1C1–
A10B1C5.
400 MHz) d 7.2–7.8 (14H, m), 4.3 (1H, s), 4.1–4.2 (4H,
m), 3.4–3.5 (4H, m), 2.4 (4H, m), 2.0 (3H, s); FABMS
m=z 148.9 (100), 635.96 (12).
4.3.7.
N-Acetyl-N0-(3-(5-tert-butylisoxazole))-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A8B1C1). 20.0 mg (17%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.6 (9H, m), 6.6 (1H, s), 4.6 (1H, s), 4.0–
4.4 (4H, m), 3.6–3.7 (4H, m), 2.4–2.5 (4H, m), 2.0 (3H,
s), 1.3–1.4 (9H, s); FABMS m=z 55.5 (100), 566.02 (15).
4.3.8. N-Acetyl-N0-(2-(4-(4-bromophenyl)thiazole))-N00-1-
(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
1
amide (A9B1C1). 40.0 mg (20%); H NMR (acetone-d6,
400 MHz) d 7.0–7.7 (14H, m), 4.2 (1H, s), 4.0–4.2 (4H,
m), 3.4–3.6 (4H, m), 2.4 (4H, m), 2.0 (3H, s); FABMS
m=z 55.5 (100), 679.92 (2.5).
4.3.1.
N-Acetyl-N0-benzyl-N00-1-(4-chlorobenzhydryl)-
piperazine iminodiacetic acid triamide (A2B1C1). 1 mg
1
(2%); H NMR (acetone-d6, 400 MHz) d 7.2–7.5 (14H,
m), 4.4 (3H, m), 4.2 (2H, d, J ¼ 5:6 Hz), 3.8 (2H, d,
J ¼ 18 Hz), 3.5 (4H, m), 2.4–2.5 (4H, m), 2.0 (3H, s);
FABMS m=z 148.9 (100), 533.02 (3.8).
4.3.9. N-Acetyl-N0-cyclohexyl-N00-1-(4-chlorobenzhydryl)-
piperazine iminodiacetic acid triamide (A10B1C1).
10.0 mg (30%); H NMR (acetone-d6, 400 MHz) d 7.2–
1
7.9 (9H, m), 4.4 (1H, s), 4.2–4.4 (4H, m), 3.8 (1H, s),
3.5–3.6 (4H, m), 2.5 (4H, m), 2.0 (3H, s), 1.8 (5H, m), 1.2
(5H, m); FABMS (Na) m=z 441.2 (100), 547.08 (5.4).
4.3.2. N-Acetyl-N0-(4-methylbenzyl)-N00-1-(4-chlorobenz-
hydryl)piperazine iminodiacetic acid triamide (A3B1C1).
58 mg (64%); H NMR (acetone-d6, 400 MHz) d 7.0–7.5
1
(13H, m), 4.6 (1H, s), 4.2–4.4 (4H, m), 3.9–4.0 (2H, m),
3.5–3.6 (4H, m), 2.3–2.4 (4H, m), 2.3 (3H, s), 2.0 (3H, s);
FABHRMS (NBA) m=z 547.2475 (MþH, C31H36O3N4Cl
requires 547.2476).
4.3.10. N-Benzoyl-N0-isopropyl-N00-1-(4-chlorobenzhydryl)-
piperazine iminodiacetic acid triamide (A1B1C2). 11 mg
(40%); H NMR (acetone-d6, 400 MHz) d 7.2–7.5 (14H,
1
m), 4.4 (1H, s), 4.2–4.4 (4H, m), 4.1 (1H, s), 3.5–3.6 (4H,
m), 2.3–2.5 (4H, m), 0.9–1.1 (6H, m); FABMS m=z 148.9
(100), 547.05 (1.9).
4.3.3.
benhydryl)piperazine
N-Acetyl-N0-(4-methoxybenzyl)-N00-1-(4-chloro-
iminodiacetic acid triamide
(A4B1C1). 11.9 mg (71%); 1H NMR (acetone-d6,
400 MHz) d 6.8–7.5 (13H, m), 4.4 (1H, s), 4.1–4.3 (4H,
m), 3.8 (2H, s), 3.7 (3H, s), 3.4–3.5 (4H, m), 2.2–2.3 (4H,
m), 1.9 (3H, s); FABMS m=z 154.0 (100), 563.05 (3.4).
4.3.11. N-Benzoyl-N0-benzyl-N00-1-(4-chlorobenzhydryl)-
piperazine iminodiacetic acid triamide (A2B1C2). 7 mg
(14%); H NMR (acetone-d6, 400 MHz) d 7.2–7.8 (19H,
m), 4.4 (3H, m), 4.0–4.3 (4H, m), 3.5–3.7 (4H, m), 2.3–
1
2.5 (4H, m); FABMS m=z 201.0 (100), 595.20 (15).
4.3.4. N-Acetyl-N0-(1-(methyl)-3-(phenyl)propyl)-N00-1-
(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
4.3.12. N-Benzoyl-N0-(4-methylbenzyl)-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A3B1C2). 67 mg (66%); 1H NMR (acetone-d6,
400 MHz) d 7.0–7.5 (18H, m), 4.4 (1H, s), 4.3–4.4 (4H,
m), 4.0–4.2 (2H, m), 3.5–3.6 (4H, m), 2.4–2.5 (4H, m),
2.2 (3H, s); FABHRMS (NBA) m=z 609.2630 (MþH,
C36H38O3N4Cl requires 609.2632).
1
amide (A5B1C1). 23 mg (76%); H NMR (acetone-d6,
400 MHz) d 7.1–7.5 (14H, m), 4.5 (1H, s), 4.3–4.4 (4H,
m), 4.2 (1H, s), 3.5–3.7 (4H, m), 2.5–2.7 (2H, m), 2.3–2.5
(4H, m), 2.0 (3H, s) 1.7–1.8 (2H, m), 1.0–1.2 (3H, dd,
J ¼ 6:4 and 25.2 Hz); FABMS m=z 200.9 (100), 575.05
(30).

Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
3549
4.3.13. N-Benzoyl-N0-(4-methoxybenzyl)-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A4B1C2). 9 mg (53%); 1H NMR (acetone-d6,
400 MHz) d 6.8–7.5 (18H, m), 4.4 (1H, s), 4.1–4.4 (4H,
m), 3.8–4.0 (2H, m), 3.7 (3H, s), 3.4–3.5 (4H, m), 2.2–2.4
(4H, m); FABHRMS (NBA) m=z 647.2401 (MþNa,
C36H37O4N4ClNa requires 647.2401).
(14H, m), 4.4 (1H, s), 4.2–4.4 (4H, m), 3.65 (1H, m), 3.6
(2H, s), 3.5–3.6 (4H, m), 2.3–2.4 (4H, m), 0.9–1.1 (6H,
m); FABMS m=z 149.0 (100), 561.14 (2.3).
4.3.21.
N-Phenylacetyl-N0-benzyl-N00-1-(4-chlorobenz-
hydryl)piperazine iminodiacetic acid triamide (A2B1C3).
1
6 mg (17%); H NMR (acetone-d6, 400 MHz) d 7.2–7.6
(19H, m), 4.6 (1H, s), 4.0–4.4 (6H, m), 3.6 (2H, m), 3.5–
3.6 (4H, m), 2.3–2.4 (4H, m); FABMS m=z 201.0 (100),
609.21 (18).
4.3.14. N-Benzoyl-N0-(1-(methyl)-3-(phenyl)propyl)-N00-
1-(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
1
amide (A5B1C2). 37 mg (77%); H NMR (acetone-d6,
400 MHz) d 7.2–7.6 (19H, m), 4.5 (1H, s), 4.3–4.4 (2H,
m), 4.0 (1H, s), 3.9–4.0 (2H, m), 3.5–3.7 (4H, m), 2.5–2.7
(2H, m), 2.2–2.5 (4H, m), 1.7–1.8 (2H, m), 1.0–1.2 (3H,
dd, J ¼ 6:4 and 16.4 Hz); FABMS m=z 200.9 (100),
637.03 (30).
4.3.22.
N-Phenylacetyl-N0-(4-methylbenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A3B1C3). 77 mg (74%); 1H NMR (acetone-d6,
400 MHz) d 7.0–7.5 (18H, m), 4.6 (1H, s), 4.3–4.4 (4H,
m), 4.0–4.1 (2H, m), 3.6 (2H, s), 3.5–3.6 (4H, m), 2.3–2.4
(4H, m), 2.3 (3H, s); FABHRMS (NBA) m=z 623.2786
(MþH, C37H40O3N4Cl requires 623.2789).
4.3.15. N-Benzoyl-N0-(3,4,5-trimethoxybenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A6B1C2). 5 mg (21%); 1H NMR (acetone-d6, 400 MHz)
d 7.2–7.6 (14H, m), 6.6 (2H, s), 4.4 (1H, s), 4.2–4.4 (4H,
m), 3.9–4.1 (2H, m), 3.8 (9H, s), 3.5–3.6 (4H, m), 2.2–2.4
(4H, m); FABMS (Na) m=z 132.8 (100), 707.06 (0.55).
4.3.23. N-Phenylacetyl-N0-(4-methoxybenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A4B1C3). 25 mg (77%); 1H NMR (acetone-d6,
400 MHz) d 6.8–7.5 (18H, m), 4.4 (1H, s), 4.2 (4H, m),
3.8–4.0 (2H, m), 3.7 (3H, s), 3.6 (2H, s), 3.4–3.5 (4H, m),
2.2–2.3 (4H, m); FABHRMS (NBA) m=z 661.2560
(MþNa, C37H39O4N4ClNa requires 661.2558).
4.3.16.
N-Benzoyl-N0-(2-(4-(4-chlorophenyl)thiazole))-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
triamide (A7B1C2). 35.0 mg (53%)1H NMR (acetone-
d6, 400 MHz) d 7.2–8.0 (19H, m), 4.4 (1H, s), 4.2–4.3
(4H, m), 3.5–3.7 (4H, m), 2.3–2.4 (4H, m); FABMS m=z
105.1 (100), 698.03 (24.2).
4.3.24. N-Phenylacetyl-N0-(1-(methyl)-3-(phenyl)propyl)-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
triamide (A5B1C3). 14 mg (34%); 1H NMR (acetone-
d6, 400 MHz) d 7.0–7.4 (19H, m), 4.5 (1H, s), 4.2–4.3
(2H, m), 3.9 (1H, m), 3.7–3.8 (2H, m), 3.4–3.5 (4H, m),
3.6 (2H, s), 2.4–2.6 (2H, m), 2.1–2.3 (4H, m), 1.5–1.7
(2H, m), 0.9–1.0 (3H, dd, J ¼ 6:8 and 22.4 Hz); FABMS
m=z 200.9 (100), 651.15 (20).
4.3.17. N-Benzoyl-N0-(3-(5-tert-butylisoxazole))-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A8B1C2). 40.0 mg (27%); 1H NMR (acetone-d6,
400 MHz) d 7.2–8.0 (14H, m), 6.6 (1H, s), 4.5 (1H, s),
4.2–4.4 (4H, m), 3.4–3.6 (4H, m), 2.4–2.5 (4H, m), 1.4
(9H, s); FABMS m=z 200.9 (100), 628.16 (48).
4.3.25. N-Phenylacetyl-N0-(3,4,5-trimethoxybenzyl)-N00-
1-(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
amide (A6B1C3). 2 mg (9%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.5 (14H, m), 6.6 (2H, s), 4.6 (1H, s),
4.2–4.4 (4H, m), 3.9–4.1 (2H, m), 3.8 (9H, s), 3.7 (2H, s),
3.5–3.6 (4H, m), 2.3–2.5 (4H, m); FABMS m=z 148.9
(100), 699.1 (10).
4.3.18.
N-Benzoyl-N0-(2-(4-(4-bromophenyl)thiazole))-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
triamide (A9B1C2). 40.0 mg (21%); H NMR (acetone-
1
d6, 400 MHz) d 7.2–7.9 (19H, m), 4.3 (1H, s), 4.1–4.3
(4H, m), 3.5–3.7 (4H, m), 2.3–2.5 (4H, m); FABMS m=z
154.0 (100), 741.95 (4).
4.3.26.
N-Phenylacetyl-N0-(2-(4-(4-chlorophenyl)thia-
zole))-N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic
acid triamide (A7B1C3). 80 mg (99%); H NMR (ace-
1
4.3.19. N-Benzoyl-N0-cyclohexyl-N00-1-(4-chlorobenzhydr-
yl)piperazine iminodiacetic acid triamide (A10B1C2).
tone-d6, 400 MHz) d 7.2–7.8 (14H, m), 7.0–7.2 (5H, m),
4.2 (1H, s), 4.0–4.2 (4H, m), 3.6 (2H, s), 3.4–3.5 (4H, m),
2.3–2.4 (4H, m); FABMS m=z 419.1 (100), 711.95 (16.2).
1
35 mg (95%); H NMR (acetone-d6, 400 MHz) d 7.2–
7.9 (14H, m), 4.4 (1H, s), 4.0–4.4 (4H, m), 3.8 (1H, s),
3.5–3.6 (4H, m), 2.4–2.6 (4H, m), 1.8–1.9 (2H, s), 1.6–1.8
(5H, m), 1.5 (5H, m); FABMS m=z 132.8 (100), 587.22
(15).
4.3.27. N-Phenylacetyl-N0-(3-(5-tert-butylisoxazole))-N00-
1-(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
1
amide (A8B1C3). 44 mg (32%); H NMR (acetone-d6,
400 MHz) d 7.2–7.6 (14H, m), 6.6 (1H, s), 4.6 (1H, s),
4.0–4.4 (4H, m), 3.8 (2H, s), 3.5–3.7 (4H, m), 2.4–2.5
(4H, m), 1.4 (9H, s); FABMS m=z 73.3 (100), 642.14
(14).
4.3.20. N-Phenylacetyl-N0-isopropyl-N00-1-(4-chlorobenz-
hydryl)piperazine iminodiacetic acid triamide (A1B1C3).
6 mg (64%); H NMR (acetone-d6, 400 MHz) d 7.2–7.5
1

3550
Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
4.3.28.
ole))-N00-1-(4-chlorobenzhydryl)piperazine
acid triamide (A9B1C3). 50.0 mg (23%); H NMR (ace-
tone-d6, 400 MHz) d 7.1–7.7 (19H, m), 4.2 (1H, s), 4.0–4.2
(4H, m), 3.6 (2H, s), 3.4–3.6 (4H, m), 2.3–2.4 (4H, m);
FABMS m=z 200.9 (100), 755.98 (11).
N-Phenylacetyl-N0-(2-(4-(4-bromophenyl)thiaz-
iminodiacetic
4.2–4.4 (4H, m), 4.0–4.2 (2H, m), 3.8 (9H, s), 3.5–3.6
(4H, m), 2.4–2.5 (4H, m); FABMS m=z 132.8 (100),
735.22 (2.0).
1
4.3.36. N-(2-Naphthoyl)-N0-(2-(4-(4-chlorophenyl)thia-
zole))-N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic
acid triamide (A7B1C4). 60 mg (61%); H NMR (ace-
1
4.3.29.
N-Phenylacetyl-N0-cyclohexyl-N00-1-(4-chloro-
tone-d6, 400 MHz) d 7.2–8.0 (21H, m), 4.4 (1H, s), 4.2–
4.3 (4H, m), 3.6–3.8 (4H, m), 2.4–2.5 (4H, m); FABMS
m=z 419.2 (100), 748.03 (11.5).
benzhydryl)piperazine iminodiacetic acid triamide
(A10B1C3). 10 mg (26%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.8 (14H, m), 4.3 (1H, s), 4.0–4.2 (4H,
m), 3.9 (1H, s), 3.7 (2H, s), 3.6 (4H, m), 2.4–2.5 (4H, m),
1.6–1.8 (5H, m), 1.2 (5H, m); FABMS m=z 148.9 (100),
601.00 (3.2).
4.3.37. N-(2-Naphthoyl)-N0-(3-(5-tert-butylisoxazole))-
N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid
triamide (A8B1C4). 40.0 mg (25%); H NMR (acetone-
1
d6, 400 MHz) d 7.3–8.2 (16H, m), 6.6 (1H, s), 4.5 (1H, s),
3.9–4.3 (4H, m), 3.5–3.7 (4H, m), 2.4–2.5 (4H, m), 1.3
(9H, s); FABMS m=z 154.9 (100), 678.00 (11.2).
4.3.30.
N-(2-Naphthoyl)-N0-isopropyl-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A1B1C4). 6 mg (45%); 1H NMR (acetone-d6,
400 MHz) d 7.2–8.0 (16H, m), 4.4 (1H, s), 4.2–4.3 (4H,
m), 4.0 (1H, s), 3.5–3.7 (4H, m), 2.2–2.5 (4H, m), 0.9–1.1
(6H, m); FABMS m=z 149.0 (100), 597.15 (2.0).
4.3.38. N-(2-Naphthoyl)-N0-(2-(4-(4-bromophenyl)thiaz-
ole))-N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic
acid triamide (A9B1C4). 40.0 mg (21%); H NMR (ace-
1
tone-d6, 400 MHz) d 7.2–8.0 (21H, m), 4.4 (1H, s), 4.1–
4.3 (4H, m), 3.4–3.6 (4H, m), 2.4 (4H, m); FABMS m=z
155.0 (100), 791.92 (3.18).
4.3.31. N-(2-Naphthoyl)-N0-benzyl-N00-1-(4-chlorobenz-
hydryl)piperazine iminodiacetic acid triamide (A2B1C4).
8 mg (19%); H NMR (acetone-d6, 400 MHz) d 7.3–8.0
(21H, m), 4.4 (3H, m), 3.8–4.2 (4H, m), 3.4–3.6 (4H, m),
1
2.4–2.5 (4H, m); FABMS m=z 201.0 (100), 645.2 (15).
4.3.39. N-(2-Naphthoyl)-N0-cyclohexyl-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A10B1C4). 80 mg (34%); 1H NMR (acetone-d6,
400 MHz) d 7.3–8.2 (16H, m), 4.4 (1H, s), 4.2–4.3 (4H,
m), 3.9 (1H, s), 3.4–3.6 (4H, m), 2.4–2.5 (4H, m), 1.8–1.9
(5H, m), 1.6 (5H, m); FABMS m=z 149.0 (100), 637.28
(6.8).
4.3.32.
N-(2-Naphthoyl)-N0-(4-methylbenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A3B1C4). 93 mg (85%); 1H NMR (acetone-d6,
400 MHz) d 7.0–8.0 (20H, m), 4.5 (1H, s), 4.3–4.4 (4H,
m), 4.0 (2H, s), 3.5–3.7 (4H, m), 2.3–2.5 (4H, m), 1.8
(3H, s); FABHRMS (NBA) m=z 659.2787 (MþH,
C40H40O3N4Cl requires 659.2789).
4.3.40. N-(2-Naphthylacetyl)-N0-isopropyl-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A1B1C5). 7 mg (26%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.8 (16H, m), 4.4 (1H, s), 4.2–4.3 (4H,
m), 3.8 (1H, m), 3.55 (2H, s), 3.4–3.5 (4H, m), 2.2–2.4
(4H, m), 0.9–1.1 (6H, m); FABMS m=z 154.0 (100),
611.18 (1.35).
4.3.33. N-(2-Naphthoyl)-N0-(4-methoxybenzyl)-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A4B1C4). 29 mg (77%); 1H NMR (acetone-d6,
400 MHz) d 6.8–8.0 (20H, m), 4.4 (1H, s), 4.2–4.3 (4H,
m), 3.9 (2H, s), 3.7 (3H, s), 3.5–3.6 (4H, m), 2.3–2.4 (4H,
m); FABMS m=z 57.5 (100), 675.24 (23).
4.3.41. N-(2-Naphthylacetyl)-N0-benzyl-N00-1-(4-chloro-
benzhydryl)piperazine iminodiacetic acid triamide
(A2B1C5). 8 mg (25%); 1H NMR (acetone-d6,
400 MHz) d 7.2–7.5 (21H, m), 4.6 (1H, s), 4.4 (4H, m),
4.0–4.2 (2H, m), 3.8 (2H, s), 3.5–3.6 (4H, m), 2.3–2.5
(4H, m); FABMS m=z 201.0 (100), 659.2 (10.5).
4.3.34. N-(2-Naphthoyl)-N0-(1-(methyl)-3-(phenyl)prop-
yl)-N00-1-(4-chlorobenzhydryl)piperazine
iminodiacetic
1
acid triamide (A5B1C4). 43 mg (97%); H NMR (ace-
tone-d6, 400 MHz) d 7.0–8.0 (21H, m), 4.4 (1H, s), 4.2–
4.4 (2H, m), 4.0 (1H, s), 3.8–3.9 (2H, m), 3.3–3.6 (4H,
m), 2.5–2.6 (2H, m), 2.2–2.4 (4H, m), 1.6–1.7 (2H, m),
1.0–1.2 (3H, dd, J ¼ 6:8 and 23.6 Hz); FABMS m=z
200.9 (100), 687.15 (18.2).
4.3.42. N-(2-Naphthylacetyl)-N0-(4-methylbenzyl)-N00-1-
(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
1
amide (A3B1C5). 91 mg (82%); H NMR (acetone-d6,
4.3.35. N-(2-Naphthoyl)-N0-(3,4,5-trimethoxybenzyl)-N00-
1-(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
amide (A6B1C4). 4 mg (14%); 1H NMR (acetone-d6,
400 MHz) d 7.2–8.0 (16H, m), 6.6 (2H, s), 4.5 (1H, s),
400 MHz) d 7.0–7.9 (20H, m), 4.6 (1H, s), 4.3–4.4 (4H,
m), 4.0–4.2 (2H, m), 3.8 (2H, s), 3.5–3.6 (4H, m), 2.3–2.4
(4H, m), 2.3 (3H, s); FABHRMS (NBA) m=z 673.2943
(MþH, C41H42O3N4Cl requires 673.2945).

Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
3551
4.3.43. N-(2-Naphthylacetyl)-N0-(4-methoxybenzyl)-N00-
1-(4-chlorobenzhydryl)piperazine iminodiacetic acid tri-
4.4. Bradykinin-induced contractions of guinea-pig ileum
1
amide (A4B1C5). 34 mg (84%); H NMR (acetone-d6,
Bradykinin, captopril, indomethacin, dithiothreitol,
atropine, and HOE 140 were obtained from Sigma (St.
Louis, MO, USA). All peptides were dissolved and di-
luted in physiological saline (0.9%). Captopril, dithio-
threitol and atropine were dissolved in DMSO and
diluted in physiological saline. Indomethacin and com-
pounds A2B1C1–A10B1C5 were dissolved and diluted
in DMSO. 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.
400 MHz) d 6.8–7.9 (20H, m), 4.4 (1H, s), 4.2–4.3 (4H,
m), 3.9–4.1 (2H, m), 3.8 (2H, s), 3.7 (3H, s), 3.5 (2H, s),
3.4–3.5 (4H, m), 2.1–2.3 (4H, m); FABMS m=z 200.9
(100), 689.01 (19.2).
4.3.44. N-(2-Naphthylacetyl)-N0-(1-(methyl)-3-(phenyl)-
propyl)-N00-1-(4-chlorobenzhydryl)piperazine iminodiace-
tic acid triamide (A5B1C5). 40 mg (90%); 1H NMR
(acetone-d6, 400 MHz) d 7.0–7.8 (21H, m), 4.4 (1H, s),
4.1–4.3 (2H, m), 4.0 (1H, m), 3.7–3.8 (2H, m), 3.4–3.6
(4H, m), 2.7 (2H, s), 2.4–2.6 (2H, m), 2.1–2.3 (4H, m),
1.5–1.7 (2H, m), 1.0–1.2 (3H, t, J ¼ 6:4 Hz); FABMS
m=z 200.9 (100), 701.13 (17).
Male Hartley guinea-pigs weighing 250–600 g (Jaeil,
Korea) were fasted overnight and decapitated. At a level
2 cm above the ileocecal 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 aer-
ated with 95% O2/5% CO2. Tissue contractions
were recorded isometrically on a Grass model 76E
polygraph.
4.3.45. N-(2-Naphthylacetyl)-N0-(3,4,5-trimethoxybenz-
yl)-N00-1-(4-chloro-benzhydryl)piperazine iminodiacetic
acid triamide (A6B1C5). 10 mg (39%); H NMR (ace-
1
tone-d6, 400 MHz) d 7.2–7.9 (16H, m), 6.6 (2H, s), 4.6
(1H, s), 4.3–4.4 (4H, m), 4.0–4.2 (2H, m), 3.8 (9H, s), 3.7
(2H, s), 3.5–3.6 (4H, m), 2.3–2.4 (4H, m); FABMS (Na)
m=z 132.8 (100), 771.18 (24.2).
After an equilibration period of about 60 min a stable
baseline tone was reached and two or three contractions
were obtained in response to BK (1 lM), every 20-min
interval, to assay sensibility and reproducibility of
the contractile response.18 Only segments producing
reproducible responses were used. The last control
response was taken as 100% and subsequent results
obtained with bradykinin antagonists expressed as a
percentage of this. The segments were incubated with
the bradykinin antagonists (0.1 lM) for 5 min before BK
was added.
4.3.46. N-(2-Naphthylacetyl)-N0-(2-(4-(4-chlorophenyl)-
thiazole))-N00-1-(4-chlorobenzhydryl)piperazine iminodi-
acetic acid triamide (A7B1C5) . 60 mg (47%); H NMR
(acetone-d6, 400 MHz) d 7.2–7.8 (21H, m), 4.3 (1H, s),
4.1–4.2 (4H, m), 3.8 (2H, s), 3.4–3.5 (4H, m), 2.3–2.4
(4H, m); FABMS m=z 201.0 (100), 762.11 (18.2).
1
4.3.47. N-(2-Naphthylacetyl)-N0-(3-(5-tert-butylisoxaz-
ole))-N00-1-(4-chlorobenzhydryl)piperazine iminodiacetic
acid triamide (A8B1C5). 44 mg (32%); H NMR (ace-
1
To minimize degradation of bradykinin and to
prevent responses due to neuronal activation or pros-
taglandin production, Tyrode solutions contained 1 lM
each of captopril, atropine, dithiothreitol, and indo-
methacin. In control tissues a corresponding volume of
physiological saline was applied in place of the antago-
nist.
tone-d6, 400 MHz) d 7.2–7.9 (16H, m), 6.6 (1H, s), 4.4
(1H, s), 3.9–4.1 (4H, m), 3.8 (2H, s), 3.5–3.7 (4H, m),
2.4–2.5 (4H, m), 1.3 (9H, s); FABMS m=z 57.5 (100),
692.10 (36).
4.3.48. N-(2-Naphthylacetyl)-N0-(2-(4-(4-bromophenyl)-
thiazole))-N00-1-(4-chlorobenzhydryl)piperazine iminodi-
acetic acid triamide (A9B1C5). 60 mg (22%); 1H
NMR (acetone-d6, 400 MHz) d 7.2–7.9 (21H, m), 4.7
(1H, s), 4.0–4.5 (4H, m), 3.8 (2H, s), 3.5–3.6 (4H,
m), 2.4–2.5 (4H, m); FABMS m=z 73.3 (100), 805.82
(2.05).
4.5. Statistical analysis
Pair-wise comparisons were done by the one-tailed
Student’s t test. Probability values p < 0:05 were con-
sidered to be statistically significant. All results are
expressed as the mean ꢁ SEM (n ¼ 6), and the sample
size, n, represents the number of individual strips of
ileum assayed. The experiments were designed such
that the sample size would also represent the same
number of guinea pigs. For example, two to four strips
from one guinea pig were used for two to four different
tests for an n ¼ 1 sample size, and those from another
guinea pig were also used for the same two to four tests
to increase the sample size for a given test to n ¼ 2, and
so on.
4.3.49.
N-(2-Naphthylacetyl)-N0-cyclohexyl-N00-1-(4-
chlorobenzhydryl)piperazine iminodiacetic acid triamide
(A10B1C5). 6 mg (13%); 1H NMR (acetone-d6,
400 MHz) d 8.0 (16H, m), 4.4 (1H, s), 4.0–4.3 (4H, m),
3.9 (1H, s), 3.8 (2H, s), 3.5–3.6 (4H, m), 2.4 (4H, m),
1.8–1.9 (5H, m), 1.6 (5H, m); FABMS m=z 73.3 (100),
651.02 (3.8).

3552
Y. L. Kam et al. / Bioorg. Med. Chem. 12 (2004) 3543–3552
10. Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.;
Acknowledgements
Inamura, N.; Asanno, M.; Hatori, C.; Oku, T.; Tanaka,
H. J. Med. Chem. 1998, 41, 4062.
11. Pruneau, D.; Luccarini, J.-M.; Fouchet, C.; Defrene, E.;
Franck, R. M.; Loilier, B.; Duclos, H.; Robert, C.;
Cremers, B.; Belichard, P.; Paquet, J. L. Br. J. Pharmacol.
1998, 125, 365.
This work was supported by the Korea Research
Foundation Grant (KRF-2002-041-E00291).
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