A New Series of Highly Potent Non-Peptide Bradykinin B2 Receptor Antagonists Incorporating the 4-Heteroarylquinoline Framework. Improvement of Aqueous Solubility and New Insights into Species Difference

Article abstract of DOI:10.1021/jm030159x

Introduction of nitrogen-containing heteroaromatic groups at the 4-position of the quinoline moiety of our non-peptide B₂ receptor antagonists resulted in enhancing binding affinities for the human B ₂ receptor and reducing binding affinities for the guinea pig one, providing new structural insights into species difference. A CoMFA study focused on the diversity of the quinoline moiety afforded correlative and predictive QSAR models of binding for the human B₂ receptor but not for the guinea pig one. A series of 4-(1-imidazolyl)quinoline derivatives could be dissolved in a 5% aqueous solution of citric acid up to a concentration of 10 mg/mL. A representative compound 48a inhibited the specific binding of [ ³H]BK to the cloned human B₂ receptor expressed in Chinese hamster ovary cells with an IC₅₀ value of 0.26 nM and significantly inhibited BK-induced bronchoconstriction in guinea pigs even at 1 μg/kg by intravenous administration.

Full text of DOI:10.1021/jm030159x

J . Med. Chem. 2004, 47, 1617-1630
1617
Articles
A New Ser ies of High ly P oten t Non -P ep tid e Br a d yk in in B2 Recep tor
An ta gon ists In cor p or a tin g th e 4-Heter oa r ylqu in olin e F r a m ew or k . Im p r ovem en t
of Aqu eou s Solu bility a n d New In sigh ts in to Sp ecies Differ en ce
Yuki Sawada,† Hiroshi Kayakiri,*,‡ Yoshito Abe,† Keisuke Imai,§ Tsuyoshi Mizutani,† Noriaki Inamura,‡
Masayuki Asano,# Ichiro Aramori,# Chie Hatori, Akira Katayama, Teruo Oku, and Hirokazu Tanaka
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, J apan
Received April 8, 2003
Introduction of nitrogen-containing heteroaromatic groups at the 4-position of the quinoline
moiety of our non-peptide B2 receptor antagonists resulted in enhancing binding affinities for
the human B2 receptor and reducing binding affinities for the guinea pig one, providing new
structural insights into species difference. A CoMFA study focused on the diversity of the
quinoline moiety afforded correlative and predictive QSAR models of binding for the human
B2 receptor but not for the guinea pig one. A series of 4-(1-imidazolyl)quinoline derivatives
could be dissolved in a 5% aqueous solution of citric acid up to a concentration of 10 mg/mL.
A representative compound 48a inhibited the specific binding of [3H]BK to the cloned human
B2 receptor expressed in Chinese hamster ovary cells with an IC50 value of 0.26 nM and
significantly inhibited BK-induced bronchoconstriction in guinea pigs even at 1 µg/kg by
intravenous administration.
Ch a r t 1. Representative Peptide B2 Receptor
Antagonists
In tr od u ction
Human kinins consist of two endogenous peptides,
bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-
Phe8-Arg9) and kallidin (KD; [Lys0]BK; Lys1-Arg2-Pro3-
Pro4-Gly5-Phe6-Ser7-Pro8-Phe9-Arg10). BK is generated
from high molecular weight kininogen by plasma kal-
likrein, while kallidin is released by tissue kallikrein
from low molecular weight kininogen and is successively
converted to BK through the action of aminopeptidases.
Kinins can be degraded by a variety of enzymes called
kininases, including kininase I, which cleaves the C-
terminal arginine from kinins to produce [des-Arg9]BK
and [des-Arg10]KD.1
There are at least two subtypes of specific G-protein-
coupled cell surface receptors, designated as B1 and B2,
both of which have been identified by molecular cloning
and pharmacological means.2-4 C-Terminal des-argin-
ated derivatives of kinins have strong affinities for the
B1 receptor, which is induced under stressful and
inflammatory conditions.5-7 On the other hand, kinins
are highly potent agonists of the B2 receptor, which is
expressed constitutively in many tissues and is thought
to mediate most of the biological actions of BK.2,4
BK exhibits highly potent and diverse proinflamma-
tory activities and is believed to play important roles
in a variety of inflammatory diseases including asthma,
rhinitis, pancreatitis, sepsis, rheumatoid arthritis, brain
edema, angioneurotic edema, hepatorenal syndrome,8
and ulcerative colitis.9 Recently, it was also suggested
that BK may be involved in the pathology of small-cell
lung cancer (SCLC),10,11 bacterial and viral infec-
tions,12,13 and Alzheimer’s disease.14 Therefore, the
development of specific BK antagonists has been of great
importance for investigating the pathophysiological
roles of BK and for developing a novel class of thera-
peutic drugs.
* To whom correspondence should be addressed. Phone: +81-6-
6390-1247. Fax: +81-6-6304-5385. E-mail: hiroshi_kayakiri@
po.fujisawa.co.jp.
† Present address: Medicinal Chemistry Research Laboratories,
Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki
300-2698, J apan.
‡ Present address: Research Division, Fujisawa Pharmaceutical Co.
Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, J apan.
§ Present address: Medicinal Chemistry Research Laboratories,
Fujisawa Pharmaceutical Co. Ltd., 2-1-6, Kashima, Yodogawa-ku,
Osaka 532-8514, J apan.
# Present address: Medicinal Biology Research Laboratories, Fujisa-
wa Pharmaceutical Co. Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-
8514, J apan.
Since the discovery of the first peptide BK B2 receptor
antagonist, NPC567 ([D-Arg0, Hyp3, D-Phe7]BK), by
Vavrek and Stewart in 1985,15 a number of peptide B2
Present address: Faculty of Pharmaceutical Sciences, Setsunan
University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, J apan.-
10.1021/jm030159x CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

1618 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
Ch a r t 2. Representative Fujisawa Non-Peptide B2 Antagonists (2, 3a -d , and 4)
Sch em e 1a
Sch em e 2a
a
Reagents: (a) 1-phenyl-2-buten-1-one, concentrated HCl.
antagonists have been synthesized,16-20 including the
clinically evaluated second-generation antagonists 1
(HOE140; icatibant; [D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]BK)
and CP0127 (bradycor) (Chart 1). On the other hand,
WIN64338 was disclosed as the first non-peptide B2
antagonist in 1993.21 However, this compound was not
so selective22 and was practically inactive against the
B2 receptor in isolated human umbilical vein.23 In 1996,
we presented 3a (FR 173657) as the first potent,
selective, and orally active non-peptide BK B2 receptor
antagonist. Since then, we have reported several novel
classes of non-peptide B2 antagonists,24-30 represented
by 2, 3b,c, and 4 (Chart 2). Recently, several new
pseudopeptide31-33 and non-peptide34-39 compounds
were described as B2 receptor antagonists.
We recently initiated a new research program to
discover non-peptide B2 antagonists that would be
suitable for intravenous infusion for the treatment of
life-threatening inflammatory disorders such as acute
pancreatitis, septic shock, and traumatic brain edema.
Herein, we report the SAR and new insights into species
difference revealed in the course of discovery of a new
class of highly potent non-peptide B2 antagonists with
improved aqueous solubility.
a
Reagents: (a) ethyl acetoacetate, AcOH, benzene; (b) biphenyl,
phenyl ether, 235 °C; (c) PhNMe2, POCl3, reflux; (d) BBr3, CH2Cl2;
(e) five-membered heterocycles, 1,4-dioxane, reflux; (f) DMF, reflux.
The crotonate 8, which was prepared by condensing
o-anisidine (7) with ethyl acetoacetate, was cyclized to
give 9. The quinolinol 9 was treated with phosphorus
oxychloride to give the 4-chloroquinoline 10, which was
deprotected to yield 11. The quinolinol 11 was heated
with imidazole, pyrazole, or triazole in 1,4-dioxane to
give 12a , 12b, or 12c, respectively. On the other hand,
4-(dimethylamino)-2-methyl-8-quinolinol (12d ) was ob-
tained from 11 by heating in DMF (Scheme 2).
The quinolinols 6 and 12a -d were coupled with the
benzyl bromide 13a 26 or the benzyl chloride 13b26 in the
presence of K2CO3 to give 14, 15a -c, and 49a , respec-
tively. The 4-substituted quinoline derivatives 14, 15a -
c, and 49a were treated with 10% HCl in MeOH to
afford the corresponding hydrochlorides 16, 17a -c, and
50a , respectively (Scheme 3).
Ch em istr y
Preparation of the 4-pyrazol-1-yl or the 4-triazol-1yl
derivatives 23b and 23c are shown in Scheme 4. The
amine 1826 was acylated and deprotected to provide
the benzyl alcohol 20. Treatment of 20 with methane-
sulfonyl chloride and successive condensation with the
quiunolinols 12b and 12c yielded the 4-heteroaromatic
quinoline derivatives 22b and 22c, respectively. The
compounds 22b and 22c were treated with 10% HCl in
The compounds described herein are presented in
Tables 1-3, and the methods for their syntheses are
outlined in Schemes 1-7.
Schemes 1 and 2 show the synthetic routes for the
4-substituted quinolinol derivatives. Cyclization of 2-
aminophenol (5) with 1-phenyl-2-buten-1-one provided
2-methyl-4-phenyl-8-quinolinol (6) (Scheme 1).

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1619
Sch em e 3a
a
Reagents: (a) 4-substituted quinolinols (6, 12a -d ), K2CO3, DMF; (b) 10% HCl/MeOH.
Sch em e 4a
25a was obtained from the acid 2425 via its acid chloride.
On the other hand, condensation of 24 with 2-(amino-
methyl)pyridine gave methyl (E)-4-{[(2-pyridinylmethyl)-
amino]carbonyl}cinnamate (25b). The methyl esters 25a
and 25b were hydrolyzed to the give (E)-4-(substituted)-
cinnamic acids 26a and 26b, respectively. Ethyl (E)-4-
aminocinnamate (27)25 was treated with isonicotinoyl
chloride and was saponified to give the acid 29. Coupling
of 3-nitrobenzoyl chloride (30) with 4-aminopyridine
followed by reduction of the nitro group and treatment
with phenyl chloroformate yielded the phenyl carbamate
33.
Modifications of the terminal cinnamamide moiety of
the 4-(imidazol-1-yl) or 4-dimethylaminoquinoline de-
rivatives are shown in Scheme 6. The benzyl alcohol 34
was mesylated and coupled with the quinolinols 12a and
12d to give 35a and 35b, respectively. Removal of the
N-phthaloyl groups of 35a and 35b and coupling with
(E)-4-(substituted)cinnamic acids afforded the corre-
sponding cinnamamides 22a , 37, and 49b. They were
treated with 10% HCl in MeOH to afford the corre-
sponding hydrochlorides 23a , 38, and 50b.
a
Reagents: (a) (E)-4-(N,N-dimethylcarbamoyl)cinnamic acid,
WSCD‚HCl, HOBt, DMF; (b) n-Bu4NF, THF; (c) MsCl, Et3N,
CH2Cl2; (d) 4-substituted quinolinols (12b or 12c), K2CO3, DMF;
(e) 10% HCl/MeOH.
On one hand, the amine 36a was coupled with
phenylcarbamate 33 and was treated with 10% HCl in
MeOH to give the hydrochloride 40 (Scheme 6).
Preparation of 4-(substituted)pyrrole derivatives 48a
and 48b is outlined in Scheme 7. The pyrrole 42 was
MeOH to afford the corresponding hydrochlorides 23b
and 23c, respectively.
The fragments of the terminal cinnamide and ureas
were prepared as shown in Scheme 5. The cinnamate
Sch em e 5a
a
Reagents: (a) SOCl2, DMF, RNH2; (b) RNH2, WSCD‚HCl, HOBt, DMF; (c) 1 N NaOH, MeOH; (d) isonicotinoyl chloride, Et3N, CH2Cl2;
(e) 4-aminopyridine, Et3N, CH2Cl2; (f) H2, Pd/C, MeOH, 1,4-dioxane; (g) phenyl chloroformate, 1 N NaOH, 1,4-dioxane.

1620 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
Sch em e 6a
a
Reagents: (a) MsCl, Et3N, CH2Cl2; (b) 12a or 12d , n-Bu4NI, K2CO3, DMF; (c) N2H4‚H2O, EtOH; (d) 4-substituted cinnamic acid,
WSCD‚HCl, HOBt, DMF; (e) 10% HCl/MeOH; (f) 33, Et3N, DMF, 80 °C.
Sch em e 7a
a
Reagents: (a) 2,5-dimethoxytetrahydrofuran, AcOH, 90 °C; (b) ClSO2NCO, DMF, CH2Cl2; (c) LAH, THF; (d) 4-substituted cinnamic
acid, WSCD‚HCl, HOBt, DMF; (e) n-Bu4NF, THF; (f) MsCl, Et3N, CH2Cl2; (g) 12a , K2CO3, DMF; (h) 10% HCl/MeOH.
obtained from the aniline 4126 by heating with 2,5-
dimethoxytetrahydrofuran in AcOH. Reaction of 42 with
chlorosulfonyl isocyanate provided the 2-cyano deriva-
tive 43. The cyano group of 43 was reduced to give the
amine 44, which was condensed with the (E)-4-(substi-
tuted)cinnamic acids followed by deprotection to give the
benzyl alcohols 46a and 46b, respectively. Reaction of
the benzyl alcohols 46a and 46b with methanesulfonyl
chloride and successive condensation with 12a yielded
the quinoline derivatives 47a and 47b, which were
converted to the corresponding hydrochlorides 48a and
48b, respectively.
Com p u ta tion a l Ch em istr y
Molecu la r Mod elin g. All computational studies
were performed using the molecular modeling program
SYBYL 6.7,40 running on a Silicon Graphics Octane
workstation. Structures were energy-minimized using
a conjugate gradient minimization algorithm with the
Tripos force field41 until a gradient convergence of 0.001
kcal/(mol‚Å) was reached, without solvent, using default
parameters. Partial atomic charges of the molecules
were calculated using the PM3 model Hamiltonian
within MOPAC 6.0.42

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1621
Ta ble 1. Introduction of 4-Substituents
Com p ou n d Selection a n d Align m en t. Twenty-four
compounds, incorporating various heteroaromatic groups
as the “quinoline part” and the common benzyl moiety
of 3b, were selected from our non-peptide B2 antagonist
library.25,26 Their structures and binding affinities for
human and guinea pig B2 receptors are summarized in
Table 2. The alignment was performed using the heavy
atoms of the identical substituent by least-squares
fitting.
Biology
in vitro IC50 (nM)
All compounds were tested for inhibition of the
specific binding of [3H]BK to the B2 receptor in guinea
pig ileum membrane preparations as previously re-
ported,24,25,28-30 and they were also evaluated for inhibi-
tion of the specific binding of [3H]BK to the cloned
human B2 receptor expressed in Chinese hamster ovary
(CHO) cells.29 Selected compounds were then tested for
in vivo functional antagonistic activity in inhibiting BK-
induced bronchoconstriction in guinea pigs by intra-
venous administration.24,25,28,30 Pharmacokinetics by
intravenous administration of the representative com-
pound was also studied in rats.
guinea pig
ileuma
cloned
human B2
b
compds
R
X
Y
n
3b
3d
16
H
H
Ph
Cl
Me
Me
H
H
H
H
H
H
Me
Me
Me
Me
H
0
1
1
2
2
2
1
2
2
2
2
2
0.51c
0.97c
3.1
3.7
2.7
1.1c
4.8c
1.0
0.28
0.17
0.23
3.9c
0.35
0.18
0.28
3.2
17a
17b
17c
3c
23a
23b
23c
50a
50b
1-imidazolyl Cl
1-pyrazolyl
1-triazolyl
H
Cl
Cl
Cl
4.4
1.3c
4.3
1-imidazolyl Cl
1-pyrazolyl
1-triazolyl
NMe2
Cl
Cl
Cl
Cl
3.3
9.1
1.8
2.9
NMe2
Me
2.1
Concentration required to inhibit specific binding of [3H]BK
(0.06 nM) to the B2 receptor in guinea pig ileum membrane
preparations by 50%. Values are expressed as the average of at
least three determinations, with variation in individual values of
a
Resu lts a n d Discu ssion
We reported earlier the first selective and orally active
non-peptide BK B2 receptor antagonists with subnano-
molar affinities for both human and guinea pig B2 re-
ceptors, incorporating 8-[[2,6-dichloro-3-[N-substituted]-
benzyl]oxy]-2-methylimidazo[1,2-a]pyridine and 8-[[2,6-
dichloro-3-[N-substituted]benzyl]oxy]-2-methylquino-
line frameworks.24-27,43 Representative compounds
significantly inhibited BK-induced bronchoconstriction
in guinea pigs by oral administration at 0.1 mg/kg or
less; however, it was difficult to administer them
intravenously because of poor aqueous solubility. The
saturated concentration of 3b was 0.55 mg/mL even in
a pH 2 buffer. Therefore, we started a new research
program to improve the aqueous solubility of our non-
peptide B2 antagonists, aiming at development of novel
therapeutic drugs that could be administered by intra-
venous infusion for the treatment of life-threatening
inflammatory disorders such as acute pancreatitis,
septic shock, and traumatic brain edema.
b
<15%. See the Experimental Section for further details. Con-
centration required to inhibit specific binding of [3H]BK (1.0 nM)
to the human B2 receptor that was expressed in CHO cells by 50%.
Values are expressed as the average of at least three determina-
tions, with variation in individual values of <15%. See the
Experimental Section for further details. c Previously published
(see ref 26).
pig B2 receptors was evaluated (Table 1). As a result,
16 displayed nanomolar affinity for both human and
guinea pig B2 receptors, indicating that both B2 recep-
tors have sufficient space to accommodate 4-substitu-
ents on the quinoline ring. Specifically, its IC50 values
for the human and guinea pig B2 receptors are 5 times
lower and 3 times higher, respectively, compared with
those of the 4-H counterpart 3d . The 4-phenyl group
seems to be favorable for binding to the human B2
receptor and unfavorable for binding to the guinea pig
B2 receptor.
The amino acid sequence of the B2 receptors is highly
conserved between species. For example, the guinea pig
B2 receptor is reported to share high sequence homology
with the human (81.5%), rat (82.6%), mouse (81.5%),
and rabbit (80.7%) B2 receptors.44 However, the phar-
macological profiles of the B2 receptors to various
ligands are markedly different.45 Therefore, it is es-
sential to overcome this “binding paradox”45 in order to
develop a B2 ligand as a therapeutic drug. As part of
our efforts to address this issue, we have carefully
investigated the SAR for both human and guinea pig
B2 receptors.
Since a halogen atom at the 3-position of the imidazo-
[1,2-a]pyridine ring in our former series enhanced
binding affinities for the human and guinea pig B2
receptors, we presumed that both B2 receptors could
accommodate a rather large substituent around the
4-position of the quinoline ring. As a probe, the 4-phenyl
derivative 16 was synthesized and inhibition of the
specific binding of [3H]BK to cloned human and guinea
As the next step, we investigated conversion of the
phenyl group of 16 to heteroaromatic rings containing
nitrogen atoms, aiming to improve aqueous solubility.
Introduction of 1-imidazolyl, 1-pyrazolyl, and 1-triazolyl
moieties to the 4-position of the quinoline ring of
compounds 3b and 3c afforded highly potent ligands
17a -c and 23a -c, respectively. These compounds
displayed excellent subnanomolar binding affinities for
the human B2 receptor compared with the low nano-
molar affinities of parent compounds 3b and 3c. In
contrast, affinities for the guinea pig B2 receptor were
several times lower than those of 3b and 3c. The quite
similar in vitro profiles of imidazole, pyrazole, and
triazole derivatives suggested that the 2-, 3-, and
4-nitrogen atoms on the heterocyclic rings do not make
important contributions to binding. To elucidate the
effect of the common 1-nitrogen atom, 4-dimethylamino
derivatives 50a and 50b were investigated. They showed
low nanomolar binding affinities for both human and
guinea pig B2 receptors, indicating that the nitrogen

1622 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
Ta ble 2. Test Set of Benzamide Derivatives Used To Test the Predictive Ability of the CoMFA Model
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to the B2 receptor in guinea pig ileum membrane preparations
by 50%. Values are expressed as the average of at least three determinations, with variation in individual values of <15%. See the
b
Experimental Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.0 nM) to the human B2 receptor
that was expressed in CHO cells by 50%. Values are expressed as the average of at least three determinations, with variation in individual
d
values of <15%. See the Experimental Section for further details. c Previously published (see ref 25). Previously published (see ref 26).
atom at the 4-position of the quinoline ring does not
contribute to the species difference. On the basis of these
results, we speculated that the imidazolyl, pyrazolyl,
and triazolyl moieties could increase binding affinity for
the human B2 receptor and decrease that for the guinea
pig B2 receptor mainly in a steric manner.
using SYBYL, version 6.7, using 24 compounds chosen
from our B2 ligand library on the basis of the structural
features in the “quinoline part”. Table 2 summarizes
their structures and binding affinities. In the analysis
of binding affinities for the human B2 receptor, the
CoMFA-derived QSAR models exhibited considerably
correlative and predictive properties. A cross-validated
r2 ) 0.643 with five components and a non-cross-
To obtain more detailed information on SAR around
the quinoline moiety, we carried out a CoMFA46 study

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1623
Å. The r2(cv) values for each orientation ranged from
0.52 to 0.62, suggesting a slight dependence of the model
on absolute orientation. The effect of the grid size was
tested by the calculation at 2 Å (default) and 1 Å grid
distances. No significant differences were found between
the models obtained at 2 or 1 Å grid distances (for 2 Å,
r2(cv) ) 0.643; for 1 Å, r2(cv) ) 0.608).
Figure 2 illustrates steric and electrostatic fields
generated by CoMFA. The steric and electrostatic fields
were analyzed for areas that might improve binding
affinity with changes in substituent sizes or charges.
The green (sterically favorable) and yellow (sterically
unfavorable) contours represent 80% and 20% level
contributions, respectively. Figure 2 also shows CoMFA
electrostatic fields, with blue and red regions indicative
of areas where affinity could be improved by more
positively and negatively charged substituents, respec-
tively. The blue and red contours represent 80% and
20% level contributions, respectively. Examination of
the CoMFA steric and electrostatic map revealed that
near the 4-substituent of the quinoline ring, a favorable
steric region was located and no electrostatic region was
found, supporting the speculation discussed above. On
the other hand, in the CoMFA study of binding affinities
for the guinea pig B2 receptor, no correlative or predic-
tive CoMFA-derived QSAR model was obtained. Trials
to modify the methods of charge calculations, grid
spacing, CoMFA regions, and filtering in the PLS
calculation were performed, but the best cross-validated
r2 was 0.19. This result shows that affinities for the
guinea pig B2 receptor could not be explained by only
steric and electrostatic interactions, clearly indicating
the species difference between the human and guinea
pig B2 receptors.
F igu r e 1. CoMFA predicted vs experimental pIC50 values.
validated r2 ) 0.979 with F ) 167.20 (n1 ) 5, N2 ) 18)
were observed with the model. The graph depicting the
calculated vs observed activities of the molecules used
in building the QSAR model is shown in Figure 1. In
this analysis, steric fields were observed to be more
contributive than electrostatic fields (steric:electrostatic
) 0.62:0.38). A bootstrap analysis (100 samples) was
performed to obtain the confidence limits for this
analysis. An r2 bootstrap of 0.986 with a standard
deviation of 0.007 suggested that a similar relationship
exists for all compounds.
The model with 2 Å grid spacing was further validated
to assess its predictive power. The effect of the align-
ment relative to the grid position was investigated by
consistently moving all compounds in increments of 0.5
A preliminary solubility study of these 4-substituted
derivatives was carried out to assess the possibility for
intravenous administration. Among the tested 4-aryl-
substituted derivatives, only the imidazole derivative
F igu r e 2. Contour map of steric and electrostatic fields from the B2 receptor CoMFA models. Color coding is as follows: blue )
positive charge favors high affinity; red ) negative charge favors high affinity; green ) steric bulk favors high affinity; yellow )
steric bulk does not favor high affinity.

1624 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
Ta ble 3. Representative Imidazolyl Derivatives with Improved Aqueous Solubility
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to the B2 receptor in guinea pig ileum membrane preparations
by 50%. Values are expressed as the average of at least three determinations, with variation in individual values of <15%. See the
b
Experimental Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.0 nM) to the human B2 receptor
that was expressed in CHO cells by 50%. Values are expressed as the average of at least three determinations, with variation in individual
values of <15%. See the Experimental Section for further details. c BK (5 µg/kg) was administered intravenously to anesthetized guinea
pigs, and bronchoconstriction induced by the BK administration was measured by the modified Konzett and Ro¨sseler method48 as previously
reported. After 5 min, compounds were intravenously administrated. After 25 min, BK was administered again and bronchoconstriction
was measured. Percent inhibition was calculated from the values of percent responses of drug-treated and controlled groups (n ) 3-4).
The results are expressed as the mean ( SEM: (/) P < 0.05, (//) P < 0.01, (///) P < 0.001 vs control (Student’s t-test). See the Experimental
d
Section for further details. NT, not tested.
23a could be dissolved up to 10 mg/mL in a 5% aqueous
solution of citric acid. Thus, we investigated further
optimization of the 4-imidazolyl series. As shown in
Table 3, introduction of representative side chains,
identified in our previous studies, to the 3-position of
the benzyl moiety afforded highly potent ligands 38, 40,
48a , and 48b for the human B2 receptor with sub-
nanomolar IC50 values. On the other hand, their binding
affinities for the guinea pig B2 receptor were 3-17 times
lower than that of the 4-H derivative 3b, indicating the
species difference more clearly. All of them could be
dissolved in a 5% aqueous solution of citric acid at up
to 10 mg/mL, indicating potential for iv administration.
Compounds 23a , 38, 40, 48a , and 48b did not affect
by themselves the formation of the second messenger
inositol phosphates in CHO cells expressing the cloned
human B2 receptor up to 10 µM, indicating that they
do not have agonistic or inverse agonistic properties
(data not shown). Furthermore, their antagonistic prop-
erties were proven in vivo. We used a BK-induced
bronchoconstriction model in guinea pigs for quick and
precise in vivo screening. Compounds 23a , 38, 40, 48a ,
and 48b significantly inhibited BK-induced broncho-
constriction in guinea pigs by intravenous administra-
tion at 10 µg/kg or less. In particular, the pyrrole
derivative 48a exhibited comparable inhibitory activity
to that of 1, a representative second-generation peptide
B2 antagonist, even at 1 µg/kg (iv), despite its more than
30 times lower binding affinity for the guinea pig B2
receptor compared with that of 1. Since its binding
affinity for the human B2 receptor is superior to that of
1, we expect that compound 48a should be more
effective than 1 in the clinic. In a preliminary pharma-
cokinetic study in rats, 48a showed a t1/2 value of 22
min at 3.2 mg/kg (iv) (Figure 3).
Further investigations of the 4-substituent of the
quinoline ring are revealing its critical role in determin-
ing agonist/antagonist profiles. These studies will be
reported in due course.
Con clu sion s
The 4-phenylquinoline derivative 16 revealed that
human and guinea pig B2 receptors have a pocket that
can accommodate rather large substituents at the

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1625
components, which were used to generate the final CoMFA
model without cross-validation. Equal weights were assigned
to steric and electrostatic descriptors using the CoMFA scaling
option. All cross-validated PLS analyses were performed with
a
minimum value of 2.0 kcal/mol, which minimized the
influence of column noise and reduced the computation time.
To obtain the statistical confidence limits on the analysis, PLS
analysis using 100 bootstrap groups was performed with
randomly interchanged biological activity.
2-Meth yl-4-p h en yl-8-qu in olin ol (6). To a suspension of
2-aminophenol (5) (2.00 g, 18.3 mmol) in concentrated HCl (8
mL) was added dropwise 1-phenyl-2-buten-1-one (8.03 g, 55.0
mmol) at 100 °C. This mixture was refluxed for 24 h, cooled
to 0 °C, adjusted to pH 7 with 28% ammonium hydroxide
solution, and extracted with CHCl3. The organic layer was
washed with brine, dried over MgSO4, and evaporated in
vacuo. The residue was purified by flash silica gel chromatog-
raphy (CHCl3/MeOH, 97:3) to afford 6 (2.40 g, 55.7%) as a
brown oil: 1H NMR (300 MHz, CDCl3) δ 2.75 (s, 3H), 7.14 (m,
1H), 7.27-7.36 (m, 2H), 7.40-7.61 (m, 6H), 7.95 (d, J ) 8 Hz,
1H); MS (ESI) m/z 236 (M + 1). Anal. (C16H13NO) C, H, N.
Eth yl (2E)-3-(2-Meth oxya n ilin o)-2-bu ten oa te or Eth yl
(2Z)-3-(2-Meth oxya n ilin o)-2-bu ten oa te (8). A solution of
o-anisidine (7) (30.0 g, 0.244 mol), ethyl acetoacetate (31.7 g,
0.244 mol), and AcOH (1 mL) in benzene (90 mL) was refluxed
for 8 h, removing water with a Dean-Stark apparatus. This
mixture was cooled to room temperature and concentrated in
vacuo. The residue was purified by flash silica gel chromatog-
raphy (hexane/EtOAc, 20:1) to afford 8 (57.2 g, 99.8%) as a
pale-yellow oil: 1H NMR (300 MHz, CDCl3) δ 1.29 (t, J ) 7.5
Hz, 3H), 2.00 (s, 3H), 3.86 (s, 3H), 4.15 (q, J ) 7.5 Hz, 2H),
4.71 (s, 1H), 6.82-6.93 (m, 2H), 7.02-7.16 (m, 2H). Anal.
(C13H17NO3) C, H, N.
8-Meth oxy-2-m eth yl-4-qu in olin ol (9). To a mixture of
biphenyl (90 g) and phenyl ether (90 mL) was added dropwise
8 (60.2 g, 0.256 mol) at 230-235 °C over 15 min. After 3 h of
being stirred at 235 °C, the reaction mixture was cooled at
room temperature. The mixture was diluted with hexane (200
mL) and the precipitate was collected by vacuum filtration
followed by crystallization from CH3CN to afford 9 (23.6 g,
48.7%) as pale-brown crystals: mp 230-232 °C; 1H NMR (300
MHz, DMSO-d6) δ 2.38 (s, 3H), 3.99 (s, 3H), 5.90 (s, 1H), 7.18-
7.23 (m, 2H), 7.54-7.63 (m, 1H). Anal. (C11H11NO2) C, H, N.
F igu r e 3. 48a plasma concentration-time curve.
4-position of the quinoline ring. Introduction of various
heteroaromatic substituents at this position enabled
discovery of a new class of highly potent non-peptide
B2 receptor antagonists. For the human B2 receptor, the
4-heteroaromatic substituents functioned as a new
pharmacophore and increased binding affinities, al-
though they reduced those for the guinea pig receptor,
indicating the species difference between human and
guinea pig B2 receptors. CoMFA studies suggested that
the 4-heteroaromatic substituents increased binding
affinities for the human B2 receptor mainly in a steric
manner and that there is a clear species difference
between the human and guinea pig B2 receptors. The
4-(1-imidazolyl) derivatives 23a , 38, 40, 48a , and 48b
could be dissolved in a 5% citric acid aqueous solution
up to 10 mg/mL and significantly inhibited BK-induced
bronchoconstriction in guinea pigs by intravenous ad-
ministration at 10 µg/kg. In particular, 48a afforded
comparable potency in vivo to that of 1, despite its more
than 30 times lower binding affinity for the guinea pig
B2 receptor compared to that of 1. Since 48a exhibited
superior affinity for the human B2 receptor compared
to 1, it is expected that this compound should be more
potent than 1 in the clinic.
Exp er im en ta l Section
4-Ch lor o-8-m eth oxy-2-m eth ylqu in olin e (10). To a sus-
pension of 8-methoxy-2-methyl-4-quinolinol (9) (23.5 g, 0.124
mol) in phosphorus oxychloride (92 mL, 0.987 mol) was added
dropwise N,N-dimethylaniline (31.5 mL, 0.248 mol) in an ice/
water bath over 20 min under nitrogen. After 15 min, the
reaction mixture was stirred at ambient temperature for 2 h
and refluxed for 2 h. The reaction mixture was concentrated
in vacuo, adjusted to pH 8 with saturated aqueous NaHCO3
and extracted with CH2Cl2 twice. The organic layer was
washed with saturated aqueous NaHCO3, water, and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
purified by flash silica gel column chromatography (CH2Cl2/
EtOAc, 5:1) followed by crystallization from hexane to afford
Ch em istr y. Melting points were determined on a Mel-Temp
instrument (Mitamura Riken Kogyo, J apan) and are uncor-
rected. Proton NMR spectra were recorded at 200 or 300 MHz
with a Bruker AM200 or a Varian Gemini 300 spectrometer,
and chemical shifts are expressed in δ (ppm) with TMS as the
internal standard. The peak patterns are shown as the
following abbreviations: s ) singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad. The mass spectra (MS)
were recorded with a VG (Fisons) ZAB-SE (FAB) or Micromass
Platform (ESI) system. Elemental analyses were performed
on a Perkin-Elmer 2400 CHN analyzer. Analytical results were
within (0.4% of the theoretical values unless otherwise noted.
Silica gel thin-layer chromatography was performed on pre-
coated plates Kieselgel 60F254 (E. Merck, AG, Darmstadt,
Germany). Silica gel flash chromatography was performed
with Kieselgel 60 (230-400 mesh) (E. Merck, AG, Darmstadt,
Germany). Yields were not optimized.
Com p u ta tion a l Ch em istr y. CoMF A In ter a ction En er -
gies. The CoMFA grid spacing was 2.0 Å in the x, y, and z
directions, and the grid region was automatically generated
by the CoMFA routine to encompass all molecules with an
extension of 4.0 Å in each direction. An sp3 carbon and a charge
of +1.0 were used as probes to generate the interaction
energies at each lattice point. The steric and electrostatic
contributions were truncated to (30 kcal/mol, and the elec-
trostatic contributions were ignored at lattice intersections
with maximal steric interactions.
1
10 (21.1 g, 81.9%) as pale-yellow crystals: mp 80-82 °C; H
NMR (300 MHz, CDCl3) δ 2.78 (s, 3H), 4.09 (s, 3H), 7.10 (d, J
) 8 Hz, 1H), 7.44 (s, 1H), 7.50 (dd, J ) 8, 8 Hz, 1H), 7.75 (d,
J ) 8 Hz, 1H). Anal. (C11H10ClNO) C, H, N.
4-Ch lor o-2-m eth yl-8-qu in olin ol (11). To a solution of
4-chloro-8-methoxy-2-methylquinoline (10) (16.0 g, 77 mmol)
in dry CH2Cl2 (160 mL) was added dropwise boron tribromide
(38.6 g, 154 mmol) below -50 °C under nitrogen. After 15 min,
the reaction mixture was stirred at ambient temperature for
2 h and refluxed for 2 h. The cooled mixture was adjusted to
pH 8 with saturated aqueous NaHCO3 and stirred in an ice/
water bath for 4 h. The organic layer was separated, and the
water layer was extracted with CH2Cl2. The organic layer was
combined, washed with water and brine, dried over MgSO4,
and evaporated in vacuo. The residue was dissolved in CH2Cl2
(100 mL), and silica gel (5 g) was added to the solution. The
mixture was stirred at ambient temperature for 1 h, and silica
gel was removed by filtration. The filtrate was concentrated
P a r tia l Lea st Squ a r es An a lysis. The partial least-squares
algorithm was used in conjugation with the cross-validation
(leave one out) option to obtain the optimum number of

1626 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
in vacuo followed by crystallization from hexane to afford 11
(10.5 g, 70.4%) as pale-yellow crystals: mp 57-59 °C; 1H NMR
(300 MHz, CDCl3) δ 2.70 (s, 3H), 7.20 (d, J ) 8 Hz, 1H), 7.40
(s, 1H,), 7.47 (dd, J ) 8, 8 Hz, 1H), 7.61 (d, J ) 8 Hz, 1H).
Anal. (C10H8ClNO) C, H, N.
4-(1H-Im id a zol-1-yl)-2-m eth yl-8-qu in olin ol (12a ). A so-
lution of 4-chloro-2-methyl-8-quinolinol (11) (2.00 g, 10.3 mmol)
and imidazole (3.52 g, 51.6 mmol) in 1,4-dioxane (20 mL) was
refluxed for 6 h. After cooling, this mixture was partitioned
between CHCl3 and aqueous NaHCO3. The organic layer was
washed with water and brine, dried over MgSO4, and evapo-
rated in vacuo. The resulting residue was crystallized from
Et2O to afford 12a (1.99 g, 85.5%) as colorless crystals: mp
192-196 °C; 1H NMR (300 MHz, CDCl3) δ 2.79 (s, 3H), 7.20-
7.37 (m, 5H), 7.45 (dd, J ) 8, 8 Hz, 1H), 7.86 (s, 1H), 7.61 (d,
J ) 8 Hz, 1H); MS (ESI) m/z 224 (M - 1). Anal. (C13H11N3O)
C, H, N.
Compounds 15b,c and 49a were prepared following the
procedure described above for 15a .
N -Me t h yl-4-((1E )-3-oxo-3-{[2-oxo-2-(2,4-t r im e t h yl-3-
{[(2-m eth yl-4-p h en yl-8-qu in olin yl)oxy]m eth yl}a n ilin o)-
eth yl]a m in o}-1-p r op en yl)ben za m id e Hyd r och lor id e (16).
To a solution of 14 (160 mg, 0.255 mmol) in MeOH (2 mL)
was added 10% HCl in MeOH (2 mL) at ambient temperature.
The reaction mixture was stirred at the same temperature for
10 min. The solution was evaporated in vacuo and the residue
was washed with EtOAc to afford 16 (139 mg, 82.1%) as a
yellow amorphous solid: 1H NMR (300 MHz, CDCl3/CD3OD)
δ 2.32 (s, 3H), 2.50 (s, 3H), 2.96 (s, 3H), 3.12 (s, 3H), 3.29 (s,
3H), 3.80 (d, J ) 17 Hz, 1H), 3.87 (d, J ) 17 Hz, 1H), 5.42 (d,
J ) 9 Hz, 1H), 5.55 (d, J ) 9 Hz, 1H), 6.64 (d, J ) 15 Hz, 1H),
7.33 (s, 1H), 7.40-7.88 (m, 15H). Anal. (C39H38N4O4¨ıHCl) C,
H, N.
Compounds 17a -c, 23a -c, 38, 40, 48a b, 50a , and 50b were
prepared following the procedure described above for 16.
Compounds 12b and 12c were prepared following the
procedure described above for 12a .
4-[(1E)-3-({2-[3-({[ter t-Bu tyl(diph en yl)silyl]oxy}m eth yl)-
2,4-d ich lor om et h yla n ilin o]-2-oxoet h yl}a m in o)-3-oxo-1-
p r op en yl]-N,N-d im eth ylben za m id e (19). To a solution of
1826 (2.10 g, 4.19 mmol) in dry DMF (20 mL) were added
(E)-4-(N,N-dimethylcarbamoyl)cinnamic acid (1.01 g, 4.61
mmol), 1-ethoxy-3-[3-(dimethylamino)propyl]carbodiimide hydro-
chloride (WSCD‚HCl; 963 mg, 5.02 mmol), and 1-hydroxy-
benzotriazole (HOBt; 792 mg, 5.86 mmol) at ambient temper-
ature. After 3 h, this mixture was partitioned between EtOAc
and water. The organic layer was washed with saturated
aqueous NaHCO3, water, and brine, dried over MgSO4, and
evaporated in vacuo. The residue was purified by flash silica
gel column chromatography (CHCl3/MeOH, 50:1) to afford 19
(2.94 g, 99.9%) as a colorless amorphous solid: 1H NMR (300
MHz, CDCl3) δ 1.05 (s, 9H), 2.98 (br s, 3H), 3.10 (br s, 3H),
3.22 (s, 3H), 3.56 (dd, J ) 17, 4 Hz, 1H), 3.94 (dd, J ) 17, 5
Hz, 1H), 4.91 (d, J ) 10 Hz, 1H), 4.97 (d, J ) 10 Hz, 1H), 6.49
(d, J ) 15 Hz, 1H), 6.60 (br s, 1H), 7.22 (d, J ) 8 Hz, 1H),
7.34-7.60 (m, 12H), 7.69-7.78 (m, 4H); MS (ESI) m/z 702 (M
+ 1). Anal. (C38H41Cl2N3O4Si) C, H, N.
4-(Dim eth yla m in o)-2-m eth yl-8-qu in olin ol (12d ). A solu-
tion of 4-chloro-2-methyl-8-quinolinol (11) (3.50 g, 18.1 mmol)
in DMF (5 mL) was refluxed for 16 h. After cooling, the
reaction mixture was evaporated in vacuo and the resulting
residue was washed with acetone and collected by vacuum
filtration. The residue was partitioned between CHCl3 and
aqueous NaHCO3. The organic layer was washed with water
and brine, dried over MgSO4, and evaporated in vacuo. The
resulting residue was crystallized from hexane to afford 12d
(2.53 g, 69.2%) as colorless crystals: mp 192-196 °C; 1H NMR
(300 MHz, CDCl3) δ 2.60 (s, 3H), 3.03 (s, 6H), 6.62 (s, 1H),
7.03 (d, J ) 8 Hz, 1H), 7.25 (dd, J ) 8, 8 Hz, 1H), 7.46 (d, J )
8 Hz, 1H); MS (ESI) m/z 203 (M + 1). Anal. (C12H14N2O) C, H,
N.
N -Me t h yl-4-((1E )-3-oxo-3-{[2-oxo-2-(2,4-t r im e t h yl-3-
{[(2-m eth yl-4-p h en yl-8-qu in olin yl)oxy]m eth yl}a n ilin o)-
eth yl]a m in o}-1-p r op en yl)ben za m id e (14). To a mixture of
13b26 (165 mg, 0.386 mmol) and 2-methyl-4-phenyl-8-quino-
linol (6) (99.8 mg, 0.424 mmol) in dry DMF (1.5 mL) were
added K2CO3 (160 mg, 1.16 mmol) and tetrabutylammonium
iodide (10 mg) at ambient temperature, and the mixture was
stirred at the same temperature for 18 h. The reaction mixture
was poured into saturated aqueous NaHCO3 and extracted
with EtOAc twice. The extracts were washed with brine. The
water layer was extracted with CHCl3. The organic layer was
combined, dried over MgSO4, and evaporated in vacuo. The
residue was purified by flash silica gel chromatography
(EtOAc/MeOH, 9:1) to afford 14 (181 mg, 74.9%) as a colorless
amorphous solid: 1H NMR (300 MHz, CDCl3) δ 2.39 (s, 3H),
2.55 (s, 3H), 2.74 (s, 3H), 2.99 (d, J ) 5 Hz, 3H), 3.26 (s, 3H),
3.64 (dd, J ) 17, 4 Hz, 1H), 3.88 (dd, J ) 17, 5 Hz, 1H), 5.37
(s, 2H), 6.25 (br q, J ) 5 Hz, 1H), 6.50 (d, J ) 15 Hz, 1H), 6.73
(br t, J ) 5 Hz, 1H), 7.07 (d, J ) 8 Hz, 1H), 7.16 (d, J ) 8 Hz,
1H), 7.20-7.30 (m, 3H), 7.38 (dd, J ) 8, 8 Hz, 1H), 7.44-7.60
(m, 8H), 7.74 (d, J ) 8 Hz, 1H); MS (ESI) m/z 627 (M + 1).
Anal. (C39H38N4O4) C, H, N.
Compounds 22a , 37, 45a ,b, and 49b were prepared follow-
ing the procedure described above for 19.
4-[(1E)-3-({2-[2,4-Dich lor o-3-(h yd r oxym et h yl)m et h yl-
a n ilin o]-2-oxoet h yl}a m in o)-3-oxo-1-p r op en yl]-N,N-d i-
m eth ylben za m id e (20). To a solution of 19 (3.0 g, 4.27 mmol)
in THF (30 mL) was added 1 M tetrabutylammonium fluoride
in THF (6.4 mL) at ambient temperature. After 1 h, the
mixture was partitioned between CHCl3 and water. The
organic layer was washed with water and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by
flash silica gel column chromatography (CHCl3/MeOH, 15:1)
to afford 20 (1.95 g, 98.4%) as a colorless amorphous solid:
1H NMR (300 MHz, CDCl3) δ 2.99 (br s, 3H), 3.12 (br s, 3H),
3.26 (s, 3H), 3.66 (dd, J ) 17, 4 Hz, 1H), 3.90 (dd, J ) 17, 5
Hz, 1H), 5.02 (br s, 2H), 6.49 (d, J ) 15 Hz, 1H), 6.64 (br s,
1H), 7.28 (d, J ) 8 Hz, 1H), 7.39-7.62 (m, 6H); MS (ESI) m/z
464 (M + 1). Anal. (C22H23Cl2N3O4) C, H, N.
4-[(1E)-3-({2-[2,4-Dich lor o-3-({[4-(1H -im id a zol-1-yl)-2-
m e t h y l-8-q u in o lin y l]o x y }m e t h y l)m e t h y la n ilin o ]-2-
o x o e t h y l}a m in o )-3-o x o -1-p r o p e n y l]-N -m e t h y lb e n z-
a m id e (15a ). To a mixture of 13a 26 (52.4 mg, 0.102 mmol)
and 4-(1H-imidazol-1-yl)-2-methyl-8-quinolinol (12a ) (23.0 mg,
0.102 mmol) in dry DMF (0.5 mL) was added K2CO3 (42.3 mg,
0.306 mmol) at ambient temperature, and the mixture was
stirred at the same temperature for 3 h. The reaction mixture
was poured into water and extracted with EtOAc. The extracts
were washed with brine, dried over MgSO4, and evaporated
in vacuo. The residue was purified by preparative thin-layer
chromatography (CHCl3/MeOH, 10:1) to afford 15a (60 mg,
89.4%) as a colorless amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 2.79 (s, 3H), 3.02 (d, J ) 5 Hz, 3H), 3.29 (s, 3H), 3.67
(dd, J ) 17, 4 Hz, 1H), 3.93 (dd, J ) 17, 5 Hz, 1H), 5.64 (d, J
) 10 Hz, 1H), 5.69 (d, J ) 10 Hz, 1H), 6.20 (br d, J ) 5 Hz,
1H), 6.52 (d, J ) 15 Hz, 1H), 6.68 (br t, J ) 5 Hz, 1H), 7.30-
7.61 (m, 10H), 7.75 (br d, J ) 7.5 Hz, 2H), 7.83 (s, 1H); MS
(ESI) m/z 657 (M + 1). Anal. (C34H30Cl2N6O4) C, H, N.
Compounds 46a and 46b were prepared following the
procedure described above for 20.
2,6-Dich lor o-3-({[((2E)-3-{4-[(d im eth yla m in o)ca r bon yl]-
p h e n yl}-2-p r op e n oyl)a m in o]a ce t yl}(m e t h yl)a m in o)-
ben zyl Meth a n esu lfon a te (21). To a solution of 20 (300 mg,
0.646 mmol) and Et3N (78.5 mg, 0.775 mmol) in dry CH2Cl2
(3 mL) was added dropwise methanesulfonyl chloride (81.4 mg,
0.711 mmol) in an ice/water bath under nitrogen. After 10 min,
the reaction mixture was stirred at ambient temperature for
30 min. This mixture was partitioned between CHCl3 and
water. The organic layer was washed with water, saturated
aqueous NaHCO3 and brine, dried over MgSO4, and evapo-
rated in vacuo to afford 21 (330 mg, 94.2%) as a pale-yellow
amorphous solid: 1H NMR (300 MHz, CDCl3) δ 2.98 (br s, 3H),
3.06-3.18 (m, 6H), 3.26 (s, 3H), 3.62 (dd, J ) 17, 4 Hz, 1H),
3.92 (dd, J ) 17, 5 Hz, 1H), 5.54 (br s, 2H), 6.49 (d, J ) 15 Hz,
1H), 6.62 (br s, 1H), 7.35-7.44 (m, 3H), 7.48-7.61 (m, 4H);
MS (ESI) m/z 542 (M + 1). Anal. (C23H25Cl2N3O6S) C, H, N.

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1627
4-[(1E )-3-({2-[2,4-Dich lor o(m e t h yl)-3-({[2-m e t h yl-4-
(1H -p yr a zol-1-yl)-8-q u in olin yl]oxy }m e t h yl)a n ilin o]-
2-oxoe t h yl}a m in o)-3-oxo-1-p r op e n yl]-N ,N -d im e t h yl-
ben za m id e (22b). To a solution of 21 (101 mg, 0.186 mmol)
and 2-methyl-4-(1H-pyrazol-1-yl)-8-quinolinol (12b ) (42 mg,
0.186 mmol) in dry DMF (1 mL) was added K2CO3 (77.3 mg,
0.559 mmol) at ambient temperature, and the reaction mixture
was stirred at the same temperature for 18 h. The mixture
was poured into water and extracted with EtOAc. The organic
layer was washed with water three times, dried over MgSO4,
and evaporated in vacuo. The residue was purified by prepara-
tive thin-layer chromatography (CHCl3/MeOH, 10:1) to afford
22b (110 mg, 87.8%) as a colorless amorphous solid: 1H NMR
(300 MHz, CDCl3) δ 2.79 (s, 3H), 2.98 (br s, 3H), 3.11 (br s,
3H), 3.28 (s, 3H), 3.58 (dd, J ) 17, 4 Hz, 1H), 3.92 (dd, J ) 17,
5 Hz, 1H), 5.68 (s, 2H), 6.50 (d, J ) 15 Hz, 1H), 6.58 (br s,
1H), 6.67 (br s, 1H), 7.32 (d, J ) 8 Hz, 2H), 7.38-7.61 (m,
8H), 7.78 (br d, J ) 8 Hz, 1H), 7.84 (br s, 1H); MS (ESI) m/z
671 (M + 1). Anal. (C35H32Cl2N6O4) C, H, N.
in MeOH (5.4 mL) containing 1 N NaOH (1.7 mL) was heated
at 60 °C for 2 h. Upon cooling, the reaction mixture was
adjusted to pH 5 with 1 N HCl and diluted with water. The
solid that precipitated was collected by vacuum filtration and
washed with water to afford 26a (401 mg, 78.2%) as crystals:
mp >250 °C; 1H NMR (200 MHz, DMSO-d6) δ 6.69 (d, J ) 16
Hz, 1H), 7.52-8.08 (m, 7H), 8.49 (d, J ) 6 Hz, 2H). Anal.
(C15H12N2O3) C, H, N.
Compounds 26b and 29 were prepared following the pro-
cedure described above for 26a .
3-Nitr o-N-(4-p yr id in yl)ben za m id e (31). To a mixture of
4-aminopyridine (1.83 g, 19.4 mmol) and Et3N (2.45 g, 24.2
mmol) in CH2Cl2 (25 mL) was added a solution of 3-nitro-
benzoyl chloride (30) (3.00 g, 16.2 mmol) in CH2Cl2 (5 mL) in
an ice/water bath under nitrogen. The reaction mixture was
stirred at the same temperature for 10 min and stirred at
ambient temperature for 2 h. The precipitate was collected by
vacuum filtration and washed with water and MeOH to afford
31 (3.42 g, 87.0%) as a solid: mp >250 °C; 1H NMR (200 MHz,
DMSO-d6) δ 7.80 (d, J ) 6 Hz, 2H), 7.89 (dd, J ) 7, 7 Hz, 1H),
8.38-8.58 (m, 4H), 8.80 (t, J ) 1 Hz, 1H). Anal. (C12H9N3O3)
C, H, N.
The compound 22c was prepared following the procedure
described above for 22b.
Meth yl (2E)-3-{4-[(4-P yr idin ylam in o)car bon yl]ph en yl}-
2-p r op en oa te (25a ). To a suspension of 4-[(1E)-3-methoxy-
3-oxo-1-propenyl]benzoic acid (24)25 (400 mg, 1.94 mmol) in
thionyl chloride (1.4 mL, 19.4 mmol) was added DMF (1drop)
at ambient temperature, and the mixture was refluxed for 20
min. The reaction mixture was evaporated in vacuo and
azeotropically removed with toluene. The residue was dissolved
in CH2Cl2 (10 mL). To this solution were added 4-amino-
pyridine (201 mg, 2.13 mmol) and Et3N (588 mg, 5.82 mmol)
in an ice/water bath, and the reaction mixture was stirred at
the same temperature for 3 h. The reaction mixture was
poured into water and extracted with a mixture of CHCl3 and
MeOH (5:1). The organic layer was washed with saturated
aqueous NaHCO3, water, and brine, dried over MgSO4, and
evaporated in vacuo. The residue was crystallized from EtOAc
to afford 25a (555 mg, 82.5%) as crystals: mp 209-211 °C;
1H NMR (200 MHz, DMSO-d6) δ 3.76 (s, 3H), 6.82 (d, J ) 15
Hz, 1H), 7.69-7.83 (m, 3H), 7.92 (d, J ) 9 Hz, 2H), 8.01 (d, J
) 9 Hz, 2H), 8.50 (d, J ) 7 Hz, 2H). Anal. (C16H14N2O3) C, H,
N.
3-Am in o-N-(4-p yr id in yl)ben za m id e (32). A mixture of 31
(1.09 g, 4.49 mmol) and 10% palladium on carbon (109 mg) in
a mixture of MeOH (10 mL) and 1,4-dioxane (20 mL) was
hydrogenated at ambient temperature. After completion of the
reaction, the catalyst in the reaction mixture was removed by
filtration. The solvent was evaporated in vacuo. The resulting
residue was crystallized from EtOAc to afford 32 (901 mg,
94.2%) as pale-yellow crystals: mp 232-234 °C; 1H NMR (200
MHz, DMSO-d6) δ 5.39 (br s, 2H), 6.79 (br d, J ) 8 Hz, 1H),
7.02-7.11 (m, 2H), 7.19 (dd, J ) 8, 8 Hz, 1H), 7.78 (d, J ) 7
Hz, 2H), 8.46 (d, J ) 7 Hz, 2H). Anal. (C12H11N3O) C, H, N.
P h en yl 3-[(4-P yr id in yla m in o)ca r bon yl]p h en ylca r ba m -
a te (33). To a solution of 32 (295 mg, 1.38 mmol) and 1 N
NaOH (2.76 mL) in 1,4-dioxane (3 mL) was added phenyl
chloroformate (260 mg, 1.66 mmol) in an ice/water bath. The
reaction mixture was stirred for 30 min at the same temper-
ature and poured into a mixture of water and CHCl3. The
precipitate was collected by vacuum filtration to afford 33 (460
1
mg, 99.7%) as a solid: mp 186-188 °C; H NMR (300 MHz,
DMSO-d6) δ 6.75 (d, J ) 6 Hz, 2H), 7.12 (dd, J ) 7, 7 Hz, 1H),
7.20-7.30 (m, 2H), 7.35-7.50 (m, 2H), 7.55 (dd, J ) 7, 7 Hz,
1H), 7.77 (d, J ) 8 Hz, 2H), 8.16 (s, 1H), 8.30 (d, J ) 6 Hz,
2H), 8.75 (d, J ) 6 Hz, 2H). Anal. (C19H15N3O3) C, H, N.
N-[2,4-Dich lor o-3-({[4-(1H -im id a zol-1-yl)-2-m et h yl-8-
qu in olin yl]oxy}m eth yl)p h en yl]-2-(1,3-d ioxo-1,3-d ih yd r o-
2H-isoin d ol-2-yl)-N-m eth yla ceta m id e (35a ). Step 1. To a
solution of 34 (1.95 g, 4.96 mmol) and Et3N (753 mg, 7.44
mmol) in dry CH2Cl2 (20 mL) was added dropwise methane-
sulfonyl chloride (625 mg, 5.46 mmol) in an ice/water bath
under nitrogen. After 30 min, the reaction mixture was washed
with water, saturated aqueous NaHCO3, and brine. The
organic layer was dried over MgSO4 and evaporated in vacuo
to afford the mesylate intermediate (2.35 g, ∼100%) as a pale-
yellow oil.
Meth yl (2E)-3-(4-{[(2-P yr idin ylm eth yl)am in o]car bon yl}-
p h en yl)-2-p r op en oa te (25b). To a solution of 24 (1.00 g, 4.85
mmol), 2-(aminomethyl)pyridine (577 mg, 5.33 mmol), and
HOBt (852 mg, 6.30 mmol) in dry DMF (20 mL) was added
WSCD‚HCl (1.12 g, 5.82 mmol) at ambient temperature under
nitrogen. After 4 h of stirring, this mixture was partitioned
between EtOAc and water. The organic layer was separated,
washed with saturated aqueous NaHCO3, water, and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
purified by flash silica gel column chromatography (CH2Cl2/
MeOH, 30:1) to afford 25b (1.15 g, 79.7%) as a colorless
1
amorphous solid: H NMR (200 MHz, CDCl3) δ 3.82 (s, 3H),
4.78 (d, J ) 5 Hz, 1H), 6.50 (d, J ) 15 Hz, 1H), 7.22 (dd, J )
9, 6 Hz, 1H), 7.32 (d, J ) 9 Hz, 1H), 7.56-7.76 (m, 5H), 7.90
(d, J ) 9 Hz, 2H), 8.58 (d, J ) 6 Hz, 1H). Anal. (C17H16N2O3)
C, H, N.
Step 2. To a solution of the crude mesylate intermediate
(2.35 g, 4.99 mmol), tetrabutylammonium iodide (123 mg,
0.333 mmol), and 4 Å molecular sieves (340 mg) in dry DMF
(38 mL) were added 4-(1H-imidazol-1-yl)-2-methyl-8-quinolinol
(12a ) (750 mg, 3.33 mmol) and K2CO3 (2.30 g, 16.6 mmol) in
an ice/water bath, and the mixture was stirred at ambient
temperature for 18 h. The reaction mixture was poured into
water and extracted with CHCl3. The organic layer was
washed with water and brine, dried over MgSO4, and evapo-
rated in vacuo. The resulting residue was crystallized from
EtOAc to afford 35a (1.92 g, 96.0%) as pale-yellow crystals:
mp 211-213 °C; 1H NMR (300 MHz, CDCl3) δ 2.81 (s, 3H),
3.25 (s, 3H), 4.09 (s, 2H), 5.70 (d, J ) 10 Hz, 1H), 5.77 (d, J )
10 Hz, 1H), 7.24-7.64 (m, 8H), 7.69-7.78 (m, 2H), 7.81-7.90
(m, 3H); MS (ESI) m/z 600 (M + 1). Anal. (C31H23Cl2N5O4) C,
H, N.
Eth yl (2E)-3-[4-(Ison icotin oyla m in o)p h en yl]-2-p r op en -
oa te (28). To a solution of ethyl (2E)-3-(4-aminophenyl)-2-
propenoate (27)25 (2.00 g, 10.46 mmol) in CH2Cl2 (30 mL) were
added Et3N (5.83 mL, 41.84 mmol) and isonicotinoyl chloride
hydrochloride (2.23 g, 12.55 mmol) in an ice/water bath under
nitrogen. The mixture was stirred at the same temperature
for 30 min and stirred at ambient temperature for 3 h. The
reaction mixture was washed with water, saturated aqueous
NaHCO3, and brine, dried over MgSO4, and evaporated in
vacuo. The residue was washed with EtOAc to afford 28 (2.00
g, 66.0%) as a colorless amorphous solid: mp 179-188 °C; 1H
NMR (200 MHz, CDCl3) δ 1.34 (t, J ) 7.5 Hz, 3H), 4.26 (q, J
) 7.5 Hz, 2H), 6.40 (d, J ) 16 Hz, 1H), 7.52 (d, J ) 9 Hz, 2H),
7.57 (d, J ) 16 Hz, 1H), 7.65-7.78 (m, 4H), 8.19 (br s, 1H),
8.31 (dd, J ) 6, 1 Hz, 2H). Anal. (C17H16N2O3) C, H, N.
(2E)-3-{4-[(4-P yr id in yla m in o)ca r bon yl]p h en yl}-2-p r o-
p en oic Acid (26a ). A suspension of 25a (535 mg, 1.54 mmol)
Compounds 35b, 47a , and 47b were prepared following the
procedure described above for 35a .

1628 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Sawada et al.
2-Am in o-N-[2,4-d ich lor o-3-({[4-(1H -im id a zol-1-yl)-2-
m e t h y l-8-q u in o lin y l]o x y }m e t h y l)p h e n y l]-N -m e t h y l-
a ceta m id e (36a ). To a suspension of 35a (1.91 g, 3.18 mmol)
in EtOH (19 mL) was added hydrazine monohydrate (318 mg,
6.38 mmol) at ambient temperature, and the mixture was
refluxed for 1 h. After the reaction mixture was cooled, the
precipitates were filtered off. The filtrate was evaporated in
vacuo, and to the residue was added CHCl3 (20 mL). The
precipitates were filtered off. The filtrate was evaporated in
vacuo and the solid was washed with IPE to afford 36a (1.50
g, 100%) as a pale-yellow amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 2.70 (br s, 3H), 2.92-3.12 (m, 2H), 3.24 (br s, 3H),
5.68 (br s, 2H), 7.18-7.55 (m, 8H), 7.85 (br s, 1H); MS (ESI)
m/z 470 (M + 1). Anal. (C23H21Cl2N5O2) C, H, N.
mixture was added water (2 mL) dropwise in an ice/water bath.
The precipitate was removed by vacuum filtration through
Celite, which was then washed with EtOAc. The filtrate and
washings were combined, and the organic layer was separated,
dried over MgSO4, and evaporated in vacuo. The residue was
purified by flash silica gel column chromatography (CHCl3/
MeOH, 9:1) to afford 44 (414 mg, 50.2%) as a pale-yellow oil:
1H NMR (300 MHz, CDCl3) δ 1.06 (s, 9H), 3.52 (d, J ) 15 Hz,
1H), 3.63 (d, J ) 15 Hz, 1H), 4.97 (s, 2H), 6.30-6.19 (m, 2H),
6.63 (d, J ) 4 Hz, 1H), 7.30 (d, J ) 8 Hz, 1H), 7.34-7.48 (m,
7H), 7.68-7.79 (m, 4H). Anal. (C28H30Cl2N2OSi) C, H, N.
Biologica l Met h od s. R ecep t or Bin d in g: Gu in ea P ig
Ileu m . The specific binding of [3H]BK (a high-affinity B2
ligand) was assayed according to the method previously
described47 with minor modifications. Male Hartley guinea pigs
(from Charles River J apan, Inc.) were killed by exsanguination
under anesthesia. The ilea were removed and homogenized
in ice-cooled buffer (50 mM sodium (trimethylamino)ethane-
sulfonate (TES) and 1 mM 1,10-phenanthroline, pH 6.8) with
a Polytron. The homogenate was centrifuged to remove cellular
debris (1000g, 20 min, 4 °C), and the supernatant was
centrifuged (100000g, 60 min, 4 °C). The pellet was then resus-
pended in ice-cooled binding buffer (50 mM TES, 1 mM 1,10-
phenanthroline, 140 µg/mL bacitracin, 1 mM dithiothreitol, 1
µM captopril, and 0.1% bovine serum albumin (BSA), pH 6.8)
and was stored at -80 °C until use.
In the binding assay, the membranes (0.2 mg of protein/
mL) were incubated with 0.06 nM of [3H]BK and varying
concentrations of test compounds or unlabeled BK at room
temperature for 60 min. Receptor-bound [3H]BK was harvested
by filtration through Whatman GF/B glass fiber filters under
reduced pressure, and the filter was washed five times with
300 µL of ice-cooled buffer (50 mM Tris-HCl). The radioactivity
retained on the washed filter was measured with a liquid
scintillation counter. Specific binding was calculated by sub-
tracting the nonspecific binding (determined in the presence
of 1 µM unlabeled BK) from total binding.
Clon ed Hu m a n B2 Recep tor s Exp r essed in CHO cells.
CHO (dhfr-) cells that were transferred with, and stably
expressed the human B2 receptor, have been described previ-
ously.29 Cells were maintained in a R-minimum essential
medium supplemented with penicillin (100 µg/mL), strepto-
mycin (100 µg/mL), and 10% fetal bovine serum. The cells were
seeded in 48-well tissue culture plates at a density of 3.0 ×
104 cells/well and cultured for 1 day. The cells were washed
three times with phosphate-buffered saline containing 0.1%
BSA and incubated with 1.0 nM of [3H]BK and test compounds
for 2 h at 4 °C in 0.25 mL of binding buffer III (20 mM HEPES,
125 mM N-methyl-D-glucamine, 5.0 mM KCl, 1.8 mM CaCl2,
0.8 mM MgSO4, 0.05 mM bacitracin, 5 µM enalaprilat, and
0.1% BSA, pH 7.2). All experiments were carried out three
times. Nonspecific binding was determined in the presence of
1 µM unlabeled BK. At the end of the incubation, the buffer
was aspirated, and the cells were washed twice with ice-cooled
phosphate-buffered saline containing 0.1% BSA. The specific
binding was calculated by subtracting the nonspecific binding,
determined in the presence of 1 µM unlabeled BK, from the
total binding. Bound radioactivity was determined by solubi-
lizing with 1% sodium dodecyl sulfate containing 0.05 N NaOH
and quantified in a liquid scintillation counter.
Compound 36b was prepared following the procedure
described above for 36a .
3-{[({2-[2,4-Dich lor o-3-({[4-(1H-im id a zol-1-yl)-2-m eth -
yl-8-qu in olin yl]oxy}m eth yl)m eth yla n ilin o]-2-oxoeth yl}-
a m in o)ca r bon yl]a m in o}-N-(4-p yr id in yl)ben za m id e (39).
A mixture of 36a (60.0 mg, 0.128 mmol), phenyl 3-[(4-
pyridinylamino)carbonyl]phenylcarbamate (33) (44.6 mg, 0.134
mmol), and Et3N (25.8 mg, 0.255 mmol) in dry DMF (0.6 mL)
was stirred at 80 °C for 2 h. After cooling to ambient
temperature, the reaction mixture was poured into water and
extracted with EtOAc. The organic layer was washed with
water three times, dried over MgSO4, and evaporated in vacuo.
The residue was purified by preparative thin-layer chroma-
tography (CHCl3/MeOH, 10:1) to afford 39 (57.0 mg, 63.0%)
as a pale-yellow amorphous solid: 1H NMR (300 MHz, CDCl3)
δ 2.76 (s, 3H), 3.27 (s, 3H), 3.91-4.01 (m, 2H), 5.39 (br d, J )
10 Hz, 1H), 5.54 (br d, J ) 10 Hz, 1H), 6.50 (br s, 1H), 6.90 (s,
1H), 7.04 (dd, J ) 8, 8 Hz, 1H), 7.23-7.58 (m, 10H), 7.84 (s,
1H), 7.90 (br d, J ) 7 Hz, 2H), 8.31 (br s, 1H), 8.52 (br d, J )
7 Hz, 2H), 9.64 (br s, 1H); MS (ESI) m/z 709 (M + 1). Anal.
(C36H30Cl2N8O4) C, H, N.
1-(3-{[t er t -Bu t yl(d ip h e n yl)silyl]oxy}m e t h yl)-2,4-d i-
ch lor op h en yl)-1H-p yr r ole (42). A solution of 3-({[tert-butyl-
(diphenyl)silyl]oxy}methyl)-2,4-dichloroaniline (41)26 (6.08 g,
14.1 mmol) and 2,5-dimethoxytetrahydrofuran (1.87 g, 14.1
mmol) in AcOH (15 mL) was heated at 90 °C for 1 h. The
mixture was evaporated in vacuo, and the residue was purified
by flash silica gel column chromatography (hexane/EtOAc, 19:
1) to afford 42 (5.82 g, 85.9%) as a pale-yellow oil: 1H NMR
(300 MHz, CDCl3) δ 1.06 (s, 9H), 4.98 (s, 2H), 6.32 (d, J ) 4
Hz, 2H), 6.83 (d, J ) 4 Hz, 2H), 7.22 (d, J ) 8 Hz, 1H), 7.32-
7.48 (m, 7H), 7.74 (d, J ) 8 Hz, 4H). Anal. (C27H27Cl2NOSi) C,
H, N.
1-[3-({[ter t-Bu t yl(d ip h en yl)silyl]oxy}m et h yl)-2,4-d i-
ch lor op h en yl]-1H-p yr r ole-2-ca r bon itr ile (43). To a solu-
tion of 42 (1.201 g, 2.50 mmol) in dry CH2Cl2 (12 mL) was
added dropwise a solution of chlorosulfonyl isocyanate (455
mg, 3.22 mmol) in dry CH2Cl2 (2.8 mL) in a dry ice/CCl4 bath
below -20 °C under nitrogen. The reaction mixture was stirred
in a dry ice/CCl4 bath for 30 min and then at ambient
temperature for 1 h. The mixture was cooled to -20 °C and
treated with dry DMF (0.4 mL, 5.17 mmol). The reaction
mixture was stirred at the same temperature for 30 min and
then at ambient temperature for 1 h. To the mixture was
added 4 N HCl (15 mL) in an ice/water bath, and the mixture
was stirred for 30 min at that temperature. The organic layer
was separated, washed with brine and saturated aqueous
NaHCO3, dried over MgSO4, and evaporated in vacuo. The
residue was purified by flash silica gel column chromatography
(hexane/EtOAc, 9:1) to afford 43 (930 mg, 73.6%) as a pale-
yellow oil: 1H NMR (300 MHz, CDCl3) δ 1.07 (s, 9H), 4.98 (s,
2H), 6.38 (t, J ) 4 Hz, 1H), 6.93 (d, J ) 4 Hz, 1H), 6.98 (d, J
) 4 Hz, 1H), 7.31 (d, J ) 8 Hz, 1H), 7.33-7.48 (m, 7H), 7.68-
7.74 (m, 4H). Anal. (C28H26Cl2N2OSi) C, H, N.
BK-In d u ced Br on ch ocon str iction in Gu in ea P igs. Male
Hartley guinea pigs weighing 470-750 g (from Charles River
J apan, Inc.) were fasted overnight and anesthetized by intra-
peritoneal injection of sodium pentobarbital (30 mg/kg). Then
the trachea and jugular vein were cannulated. The animals
were ventilated at a tidal volume of 10 mL/kg with a frequency
of 60 breaths/min through the tracheal cannula. To suppress
spontaneous respiration, alcuronium chloride (0.5 mg/kg) was
administered intravenously through the jugular vein cannula.
Then, propranolol (10 mg/kg) was also administered subcuta-
neously. After 10 min, BK (5 µg/kg, dissolved in saline with
0.1% BSA) was administered intravenously through the
jugular vein cannula. Bronchoconstriction was measured by
the modified Konzett and Rossler method48 as the peak
{1-[3-({[ter t-Bu t yl(d ip h en yl)silyl]oxy}m et h yl)-2,4-d i-
ch lor op h en yl]-1H -p yr r ol-2-yl}m et h yla m in e (44). To a
solution of 43 (820 mg, 1.62 mmol) in dry THF (16 mL) was
added portionwise lithium aluminum hydride (74 mg, 1.95
mmol) at ambient temperature under nitrogen. The reaction
mixture was stirred at the same temperature for 1 h. To the

Bradykinin B2 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1629
increase of pulmonary insufflation pressure (PIP).49 Each dose
of the compound dissolved in a 5% (w/v) citric acid solution or
vehicle was administered through the same cannula 25 min
after the first BK administration. BK was administered again
5 min after the drug injection, and bronchoconstriction was
measured in the same manner. A 0% response was determined
as PIP before the administration of BK, and the 100% response
was determined as the first BK-induced bronchoconstriction
before drug administration. The percent response was calcu-
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lated from the following formula: % response ) (∆PIPafter
/
drug
∆PIPbefore drug) × 100. The efficacy of the drug was expressed
as % inhibition, which was calculated from the values of %
responses of drug-treated and vehicle groups as follows: %
inhibition ) [1 - (% responsedrug)/(% responsevehicle] × 100.
Qu a n tita tive Deter m in a tion of 48a in Ra t P la sm a by
LC/MS/MS. Male Lewis rats (n ) 5) were used. Compound
48a was dissolved in 5% aqueous solution of citric acid and
was injected into the femoral vein (3.2 (mg/1 mL)/kg). Blood
samples were taken from the femoral artery at 5, 10, 15, 30,
60, and 120 min after dosing followed by centrifugation.
Plasma samples were prepared on the basis of an MeOH
extraction and were analyzed on an HPLC (Alliance 2690,
Waters, Milford, MA). Detection was performed on a tandem
MS (Quattro Ultima, Micromass, Manchester, U.K.) by mul-
tiple reaction monitoring (MRM) mode via positive electrospray
ionization (ESI). The precursor ion was m/z 679 and the
product ion was m/z 461 in the MRM mode. The limit of
quantitation was 10 ng/mL for plasma.
Sta tistica l An a lysis. The results are expressed as the
mean ( SEM, and statistical significance between groups was
analyzed by Student’s t test. IC50 value was obtained by using
nonlinear curve-fitting methods with a computer program
developed in house.
Ack n ow led gm en t. We are grateful to Dr. D. Barrett
(Medicinal Chemistry Research Laboratories, Fujisawa
Pharmaceutical Co. Ltd., Osaka) for his valuable sug-
gestions.
Su p p or tin g In for m a tion Ava ila ble: Physical data for
12b,c, 15b,c, 17a -c, 22a ,c, 23a -c, 26b, 29, 35b, 36b, 37,
38, 40, 45a ,b, 46a ,b, 47a ,b, 48a ,b, 49a ,b, 50a , and 50b. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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