A New Class of Nonpeptide Bradykinin B2 Receptor Ligand, Incorporating a 4-Aminoquinoline Framework. Identification of a Key Pharmacophore to Determine Species Difference and Agonist/Antagonist Profile

Article abstract of DOI:10.1021/jm030326t

Introduction of various aliphatic amino groups at the 4-position of the quinoline moiety of our nonpeptide bradykinin (BK) B₂ receptor antagonists afforded highly potent ligands for human B₂ receptor with various affinities for guine

Full text of DOI:10.1021/jm030326t

J . Med. Chem. 2004, 47, 2667-2677
2667
A New Cla ss of Non p ep tid e Br a d yk in in B2 Recep tor Liga n d , In cor p or a tin g a
4-Am in oqu in olin e F r a m ew or k . Id en tifica tion of a Key P h a r m a cop h or e To
Deter m in e Sp ecies Differ en ce a n d Agon ist/An ta gon ist P r ofile
Yuki Sawada,† Hiroshi Kayakiri,*,‡ Yoshito Abe,† Tsuyoshi Mizutani,† Noriaki Inamura,‡ Masayuki Asano,§
Ichiro Aramori,§ Chie Hatori, Teruo Oku, and Hirokazu Tanaka|
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, J apan
Received J uly 3, 2003
Introduction of various aliphatic amino groups at the 4-position of the quinoline moiety of our
nonpeptide bradykinin (BK) B2 receptor antagonists afforded highly potent ligands for human
B2 receptor with various affinities for guinea pig B2 receptor, indicating remarkable species
difference. A representative 4-dimethyamino derivative 40a exhibited subnanomolar and
nanomolar binding affinities for human and guinea pig B2 receptors, respectively, and
significantly inhibited BK-induced bronchoconstriction in guinea pigs at 10 µg/kg by intravenous
administration. Further chemical modification led us to discover unique partial agonists for
the human B2 receptor that increase inositol phosphates (IPs) production by themselves in
Chinese hamster ovary (CHO) cells expressing the cloned human B2 receptor. Although their
potency and efficacy were much lower than those of BK, we identified them as screening leads
for nonpeptide B2 agonists. In these studies it was revealed the 4-substituent of the quinoline
moiety is the key pharmacophore to determine species difference and agonist/antagonist profiles.
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). There are at least
two subtypes of specific cell surface receptors, desig-
nated as B1 and B2, both of which have been identified
by molecular cloning and pharmacological means.1-3
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.1,3 The B2 receptor is coupled with G-protein which
stimulates phosphatidylinositol (PI) hydrolysis.
BK exhibits highly potent and diverse proinflamma-
tory activities and is believed to play important roles
in a variety of inflammatory diseases.4 Recently, it was
also suggested that BK may be involved in the pathology
of small cell lung cancer (SCLC),5,6 bacterial and viral
infections,7,8 and Alzheimer’s disease.9 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.
second generation antagonists, 1 (HOE140; icatibant;
[D-Arg0,Hyp3,Thi,5D-Tic7,Oic8]BK) and CP0127 (brady-
cor) (Chart 1). On the other hand, in 1993 WIN64338
was disclosed as the first nonpeptide B2
antagonist.15
However, it was reported to be not so selective16 and to
be practically inactive against B2 receptors in isolated
human umbilical vein.17 In 1996 we presented 3a (FR
173657) as the first potent, selective, and orally active
nonpeptide B2 receptor antagonist.18 Since then, we
have reported several novel classes of nonpeptide B2
antagonists,19-25 represented by 2, 3b,c, and 4a (Chart
2). Recently, a pseudopeptide26-28 and several new
nonpeptide compounds, some of which also incorporate
similar skeleton to that of our FR compounds,29-31 were
described as potent B2 receptor antagonists.
Since 1985 a number of peptide B2 antagonists have
been synthesized,10-14 including the clinically evaluated
* 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., 2-1-6, Kashima, Yodogawa-ku,
Osaka 532-8514, J apan.
In the preceding paper32 we reported that the 4-sub-
stituent of the quinoline ring is important not only to
improve aqueous solubility but also to increase the
affinity for the human B2 receptor. In this article we
would like to report the SAR of the 4-alkylamino
quinoline derivatives, possessing characteristic binding
profiles and/or agonist/antagonist properties.
‡ Present address: Research Division, 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.
| Present address: Faculty of Pharmaceutical Sciences, Setsunan
University, 45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, J apan.
10.1021/jm030326t CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/01/2004

2668 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Sawada et al.
Ch a r t 2. Representative Fujisawa Nonpeptide B2 Antagonists (2, 3a -c and 4a ,b)
Sch em e 1a
in Scheme 3. Methyl 4-(acetyloxy)methyl-2-methoxy-
benzoate (15) was hydrolyzed with 1 N NaOH to afford
16. Oxidation of the alcohol group of 16 with sulfur
trioxide pyridine complex in a mixture of DMSO and
CH2Cl2 followed by a Wittig reaction with methyl
(triphenylphosphoranylidene)acetate afforded the ester
17. The ester 17 was condensed with methylamine
hydrochloride providing the amide 18, which was hy-
drolyzed to give the cinnamic acid 19.
On the other hand, acetylation of (4-amino-3-methyl-
phenyl)methanol (20)33 with acetic anhydride in MeOH
gave the acetamide 21. Oxidation of the alcohol group
of 21 with sulfur trioxide pyridine complex in a mixture
of DMSO and CH2Cl2 followed by a Perkin reaction of
the aldehyde 22 with malonic acid in the presence of
pyridine in EtOH afforded the cinnamic acid 23.
a
Reagents (a) corresponding amines, phenol, 125 °C; (b) hex-
amethyleneimine, n-Bu4NI
Ch em istr y
Modifications of the terminal cinnamamide moiety of
4-(substituted)-2-methylquinoline derivatives are shown
in Scheme 4. Compound 24 was treated with methane-
sulfonyl chloride followed by coupling with 6b,d and
4-(dimethylamino)-2-methyl-8-quinolinol, respectively.
Removal of the N-phthaloyl group of 25a -c with hy-
drazine monohydrate and coupling with the (E)-4-
(substituted)cinnamic acids afforded the corresponding
cinnamamides 27b,c, 28, 29a -c, 30a -c, 31a ,b, 32,
33a ,c, 37b, and 37c, respectively. The ethyl esters 33a
and 33c were hydrolyzed with 1 N NaOH to give the
corresponding carboxylic acids 34a and 34c, which were
condensed with the appropriate amine hydrochloride to
furnish the corresponding amides 35a ,c and 36, respec-
tively. The cinnamamide derivatives 27b,c, 28, 29a -c,
30a -c, 31a ,b, 32, 35a ,c, 36, 37b, and 37c were treated
with 10% HCl in MeOH to afford the corresponding
hydrochlorides 38b,c, 39, 40a -c, 41a -c, 42a ,b, 43,
44a ,c, 45, 46b, and 46c, respectively.
The compounds described in this study are shown in
Tables 1-4, and the methods for their synthesis are
outlined in Schemes 1-4.
Preparation of the 4-(substituted)-2-methylquinolinol
derivatives 6a -h is shown in Scheme 1. Reaction of the
4-chloroquinolinol 5 with the corresponding amines
yielded the 4-substituted-quinolinols 6a -h , respectively
(Scheme 1).
Preparation of the benzyl bromide 10b is shown in
Scheme 2. Condensation of 7 with (E)-3-(6-acetylamino-
pyridin-3-yl)acrylic acid20 in the presence of 1-ethyl-3-
[(dimethylamino)propyl]carbodiimide
hydrochloride
(WSCD‚HCl) and 1-hydroxybenzotriazole (HOBt) gave
the cinnamamide 8. Removal of the silyl protecting
group with tetrabutylammonium fluoride and subse-
quent treatment with triphenylphosphine and carbon
tetrabromide in CH2Cl2 provided the benzyl bromide
10b. The quinolinols 6a -h and 4-(dimethylamino)-2-
methyl-8-quinolinol32 were coupled with the benzyl
bromide 10a or 10b in the presence of K2CO3 to afford
11a -h , 12a , and 12b, respectively. The 4-substituted
quinoline derivatives 11a -h and 12a were treated with
10% HCl in MeOH to the corresponding hydrochlorides
13a -h and 14, respectively (Scheme 2).
Synthesis of compound 48 is shown in Scheme 4. The
amine 26a was coupled with phenyl 3-[(4-pyridinyl-
amino)carbonyl]phenylcarbamate32 in the presence of
Et3N to afford the urea 47. 47 was treated with 10%
HCl in MeOH to form the corresponding hydrochloride
48.
Preparation of the cinnamic acids 19 and 23 is shown

New Nonpeptide Bradykinin B2 Receptor Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2669
Sch em e 2a
a
Reagents (a) (E)-3-(6-acetylaminopyridin-3-yl)acrylic acid, WSCD‚HCl, HOBt, DMF; (b) n-Bu4NF, THF; (c) CBr4, Ph3P, CH2Cl2; (d)
6a -h , K2CO3, DMF; (e) 10% HCl in MeOH.
Sch em e 3a
of life-threatening inflammatory diseases, led to a new
class of nonpeptide B2 antagonists incorporating basic
heteroaromatic moieties at the 4-position of the quino-
line ring. One of the most potent 4-(1-imidazolyl)
derivatives, 4b, exhibited excellent subnanomolar bind-
ing affinity for the human B2 receptor superior to that
of 1, indicating that the 4-substituent of the quinoline
ring contributes not only to improvement of aqueous
solubility but also to increase in affinity for the human
B2 receptor as a novel pharmacophore.32
These results prompted us to carry out further
detailed investigation on the SAR of this critical sub-
stituent. At the outset we intended to introduce several
aliphatic amines at the 4-position of the quinoline ring
in order to probe steric and electrostatic effects to B2
binding affinities. As shown in Table 1, it was clearly
indicated that the 4-substituent affected binding affini-
ties to human and guinea pig B2 receptors in a highly
sensitive manner. 4-Dimethylamino derivative 3c re-
tained low nanomolar binding affinities for both B2
receptors. On the other hand, the ring size of cyclic
amines made drastic changes in the activities as follows,
suggesting quite strict steric requirements. Although
pyrrolidine 13a showed much lower affinities than 3b,
piperidine 13b recovered low nanomolar binding affini-
ties only for the human B2 receptor. However, homopi-
peridine 13c resulted in a great loss of the activities
again. Introduction of an oxygen atom (13d ) at the
4-position of the piperidine ring of 13b retained low
nanomolar binding affinity for the human B2 receptor
and brought 5-fold increase in affinity for the guinea
pig B2 receptor. In contrast, displacement of this oxygen
atom with methylamino moiety (13e) was not tolerated
by both B2 receptors. Morpholine and piperazine related
ring-opening derivatives 13f-h failed to show nano-
molar binding affinities. In general, a large aliphatic
amino substituent at the 4-position of the quinoline ring
decreased binding affinity for the guinea pig B2 receptor,
while some of them retained low nanomolar affinities
for the human B2 receptor. We also reported in the
previous paper that nitrogen containing heteroaromatic
moieties afforded subnanomolar human B2 affinity,
while they resulted in several times decrease in guinea
a
Reagents (a) 1 N NaOH, MeOH; (b) SO3‚Py, DMSO, CH2Cl2,
then methyl (triphenylphosphoranylidene)acetate, THF; (c) MeNH2‚
HCl, WSCD, HOBt, DMF; (d) 1 N NaOH, MeOH, 50 °C; (e) Ac2O,
MeOH; (f) SO3‚Py, DMSO; (g) malonic acid, pyridine, EtOH.
Biology
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,19,20,23-25 and they were also evaluated for inhibi-
tion of the specific binding of [3H]BK to the cloned
human B2 receptor expressed in CHO cells.24 Selected
compounds were then tested for in vivo functional
antagonistic activity in inhibiting BK-induced broncho-
constriction in guinea pigs by iv administration.19,20,23,25
Furthermore, some compounds were examined for their
B2 agonistic activity by measuring the agonist-induced
IPs formation in CHO cells expressing the human B2
receptor.
Resu lts a n d Discu ssion
We had reported the first selective and orally active
nonpeptide B2 receptor antagonists with subnanomolar
affinities for human and guinea pig B2 receptors.19-22,34
Successive investigation to improve aqueous solubility,
aiming at development of novel therapeutic drugs which
could be administered intravenously for the treatment

2670 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Sawada et al.
Sch em e 4a
a
Reagents (a) MsCl, Et3N, CH2Cl2; (b) 4-(dimethylamino)-8-hydroxy-2-methylquinoline, 6b or 6d , K2CO3, DMF; (c) N2H4‚H2O, EtOH;
(d) 4-substituted cinnamic acid, WSCD‚HCl, HOBt, DMF; (e) 10% HCl-MeOH, (f) 1N NaOH, EtOH, 60 °C; (g) corresponding amine‚HCl,
WSCD, HOBt, DMF; (h) phenyl 3-[(4-pyridinylamino)carbonyl]phenylcarbamate, Et3N, DMF, 80 °C.
Ta ble 1. SAR on the 4-Substituents of the Quinoline Ring
in the pocket, which accommodates the critical phar-
macophore at the 4-position of the quinoline ring.
Among the 4-aliphatic amino derivatives examined,
4-dimethylamino (3c), piperidino (13b), and morpholino
(13d ) derivatives, which exhibited low nanomolar bind-
ing affinities for the human B2 receptor, were selected
as lead compounds for further chemical modification.
Substitution of the cinnamoyl side chain of 3c with
representative acyl moieties, identified in our previous
studies,21,32 afforded potent B2 antagonists 14, 38a , 39,
40a , 41a , 42a , 43, 44a , 45, and 48 which could be
dissolved in 5% aqueous solution of citric acid up to the
in vitro IC50 (nM)
b
compd
X
n
GP ileuma cloned human B2
concentration of 10 mg/mL (Table 2). Compounds 14,
38a , 39, 40a , 41a , 44a , and 45 retained nanomolar
affinities for the guinea pig B2 receptor too, with 57 to
90-fold higher IC50 values compared to that of 1. Despite
such much lower affinities for the guinea pig B2 recep-
tor, they inhibited BK induced bronchoconstriction in
guinea pig at 10 µg/kg by intravenous administration
more efficaciously than 1 did at 1 µg/kg (iv). In the same
way compounds 42a , 43, and 48 with 133- to 311-fold
lower affinities for the guinea pig B2 receptor exhibited
almost complete in vivo inhibition at 100 µg/kg (iv).
Their in vivo effects were as strong as that of 1 at 10
µg/kg (iv). Since one of the representative compounds,
40a possesses comparable affinity for the human B2
receptor with that of 1, we are expecting that 40a could
exhibit equal or superior clinical effects to those of 1.
Table 3 shows the results of similar attempts in 4-(4-
morpholino) and 4-(1-piperidino) series. In comparison
with 4-dimethylamino derivatives 14, 40a , 42a , and 44a
(Table 2), similar solubility profile and larger species
difference between human and guinea pig B2 binding
affinities were observed. The most potent morpholino
3b
3c
H
NMe2
1
2
2
2
2
2
3
2
2
3
0.51c
1.1c
1.8d
3.2d
13a
13b
13c
13d
13e
13f
13g
13h
1-pyrrolidinyl
1-piperidinyl
2300
87
1.1
37
2.7
98
41
19
2500
68
840
12
1500
49
1-homopiperidinyl
4-morpholinyl
4-Me-1-piperazinyl
NH(CH2)2OMe
N[(CH2)2OMe]2
NH(CH2)2NMe2
560
2300
a
Concentration required to inhibit specific binding of [3H]BK
(0.06 nM) to B2 receptors in guinea pig ileum membrane prepara-
tions by 50%. Values are expressed as the average of at least three
determinations, with variation in individual values of <15%. See
b
the Experimental Section for further details. Concentration
required to inhibit specific binding of [3H]BK (1.0 nM) to human
B2 receptors that were expressed in CHO (Chinese hamster ovary)
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 Previously pub-
d
lished, see ref 21. Previously published, see ref 32.
pig affinity.32 Taken together, these results clearly
indicated that human and guinea pig B2 receptors differ

New Nonpeptide Bradykinin B2 Receptor Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2671
Ta ble 2. Binding and in Vivo Antagonistic Activities of 4-Dimethylaminoquinoline Derivatives
a,b
See corresponding footnotes in Table 1. 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 method36 as previous 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 Section
for further details. NT, not tested. e Previously published, see ref 32.
d

2672 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Sawada et al.
Ta ble 3. Binding and in Vivo Antagonistic Activities of Morpholinyl- and Piperidinylquinoline Derivatives
inhibition (%) against
IC50(nM)
cloned human B2
BK-induced bronchoconstrictionc
GP ileuma
10 µg/kg (iv)
100 µg/kg (iv)
b
compds
X
Y
R
n
40c
12b
44c
42b
1
O
O
O
CH
CH
N
N
CONH-4Py
NHAc
CONHMe
CONHCH2-2Py
3
0
3
3
37
65
85
40
0.64
1.8
1.1
2.5
0.49
46.2 ( 9.3*
NTd
NT
91.8 ( 2.1***
47.8 ( 9.3***
72.3 ( 9.9**
72.6 ( 8.0*
NT
CH
NT
0.09
96.7 ( 0.3***
a,b,c,d
See corresponding footnotes in Table 1 and Table 2.
Ta ble 4. Binding and B2 Agonistic Activities of Morpholinyl- and Piperidinylquinoline Derivatives
relative agonistic activity (%)
in IP formation compared to BK (10 nM)c
IC50 (nM)
cloned human B2
b
compds
R
X
n
GP ileuma
1 µM
10 µM
13b
13d
38b
38c
40b
40c
41b
41c
46b
46c
CONHMe
CONHMe
CONMe2
CONMe2
CONH-4Py
CONH-4Py
NHCO-4Py
NHCO-4Py
CH2
O
CH2
O
CH2
O
CH2
O
2
2
2
2
3
3
3
3
2
2
68
12
30
43
240
37
75
29
28
13
1.1
2.7
4.4 ( 0.9
0.9 ( 2.7
17.2 ( 0.9
5.9 ( 0.5
5.7 ( 2.2
0.1 ( 0.5
5.6 ( 0.6
-0.10 ( 0.4
14.8 ( 0.4
6.0 ( 0.2
4.2 ( 0.6
-1.9 ( 2.0
21.8 ( 1.4
7.4 ( 0.3
12.7 ( 1.2
-0.3 ( 0.2
5.3 ( 0.7
0.9 ( 0.2
18.7 ( 2.1
9.2 ( 0.6
0.76
0.88
0.52
0.64
1.6
1.0
1.2
1.8
2-oxopyrrolidin-1-yl
2-oxopyrrolidin-1-yl
CH2
O
a,b
See corresponding footnotes in Table 1. c Inositol phosphates (IPs) production was measured essentially as described previously.24
See the Experimental Section for further details.
derivative 40c significantly inhibited BK induced bron-
choconstriction at 10 µg/kg (iv) despite 411-fold lower
affinity for the guinea pig B2 receptor compared to that
of 1. A piperidino derivative 42b with similar affinity
for the guinea pig B2 receptor also showed significant
inhibition at 100 µg/kg (iv).
Our representative nonpeptide B2 antagonists previ-
ously reported20,21,32 including compounds 2, 3a -c, 4a ,
and 4b had been confirmed not to affect the formation
of the second messengers, IPs in CHO cells expressing
the cloned human B2 receptor. Namely, they had been
proven not to have any agonistic or inverse agonistic
properties. The B2 ligands described above in this paper
except for 13b also showed pure antagonistic profiles.
However, some of the 4-(4-morpholino) and 4-(1-piperi-
dino) derivatives 13b, 38b,c, 40b, 41b, and 46b,c (Table
4) were revealed to increase IPs formation significantly
at the concentrations of 1 and 10 µM compared with
the control IPs formation determined in the absence of
the compounds (by Student’s t-test, at 1 µM 40b: P <
0.05, 13b: P < 0.01, 38b,c, 41b, 46b,c: P < 0.001; at
10 µM 40b: P < 0.01, 13b, 38b,c, 41b, 46b,c: P <
0.001). The B2 agonistic activity was expressed as a
percentage relative to the maximum effect of BK exerted
at 10 nM concentration. Generally, piperidino deriva-
tives were more efficacious than morpholino congeners.
The 4-(4-morpholino) and 4-(1-piperidino) moieties seem
to be critical for B2 agonistic activity, while the acyl side
chains of each series also make contribution to modify
the efficacy. Among the compounds tested, the best
combination to produce agonistic activity was that of
4-(1-piperidino) moiety and 4-(dimethylcarbamoyl)cin-

New Nonpeptide Bradykinin B2 Receptor Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2673
rated in vacuo. The resulting residue was crystallized from
Et2O to afford 6a (109 mg, 30.8%) as pale-brown crystals: mp
135-137 °C; 1H NMR (300 MHz, CDCl3): δ 1.99-2.10 (m, 4H),
2.56 (s, 3H), 3.65-3.76 (m, 4H), 6.32 (s, 1H), 7.03 (d, J ) 8
Hz, 1H), 7.16 (dd, J ) 8, 8 Hz, 1H), 7.65 (d, J ) 8 Hz, 1H); MS
(ESI) m/z 229 (M + 1); Anal. (C14H16N2O) C, H, N.
namide side chain in compound 38b. Although the
potency and efficacy of these partial agonists were much
lower than those of BK, their B2 agonistic activity was
significant enough to afford a new insight into our study
on nonpeptide B2 receptor ligands. Thus we identified
screening leads for nonpeptide B2 agonists.
Compounds 6b, 6d -g, and 6h were prepared following the
procedure described above for 6a .
Con clu sion
4-(1-Azep a n yl)-2-m eth yl-8-qu in olin ol (6c). A mixture of
5 (200 mg, 1.03 mmol), tetrabutylammonium iodide (10 mg),
and hexamethyleneimine (1.02 g, 10.3 mmol) was refluxed for
2 h. After cooling, the resulting residue was dissolved in CHCl3
and the solution washed with aqueous NaHCO3, dried over
MgSO4, and evaporated in vacuo. The residue was purified by
flash silica gel chromatography (NH-DM 1020, CHCl3) to afford
6c (259 mg, 97.8%) as a brown oil: 1H NMR (300 MHz,
CDCl3): δ 1.70-1.80 (m, 4H), 1.87-1.99 (m, 4H), 2.59 (s, 3H),
3.49-3.58 (m, 4H), 6.63 (s, 1H), 7.03 (d, J ) 8 Hz, 1H), 7.21
(dd, J ) 8, 8 Hz, 1H), 7.46 (d, J ) 8 Hz, 1H); MS (ESI) m/z
257 (M + 1); Anal. (C16H20N2O) C, H, N.
(2E )-3-[6-(Ace t yla m in o)-3-p yr id in yl]-N -{2-[3-({[ter t -
b u t yl(d ip h en yl)silyl]oxy}m et h yl)-2,4-d ich lor om et h yl-
a n ilin o]-2-oxoeth yl}-2-p r op en a m id e (8). To a solution of
7 (1.20 g, 2.39 mmol) in dry DMF (12 mL) were added (E)-3-
(6-acetylaminopyridin-3-yl)acrylic acid (501 mg, 2.44 mmol),
Various aliphatic amines were introduced to the
4-position of the quinoline ring. Their SAR clearly
indicated that the 4-substituent affected binding affini-
ties to both human and guinea pig B2 receptors in a
highly sensitive manner. Among them 4-dimethylamino
(3c), piperidino (13b), and morpholino (13d ) derivatives
exhibited low nanomolar binding affinities for the
human B2 receptor. Substitution of their acyl side chains
with representative ones, identified in our previous
studies, afforded potent and pure antagonists for iv use
as well as unique partial agonists for the human B2
receptor. Representative 4-dimethylamino derivative
40a inhibited BK induced bronchoconstriction in vivo
at 10 µg/kg by intravenous administration more ef-
ficaciously than 1 did at 1 µg/kg (iv), despite 57-fold
lower binding affinity for the guinea pig B2 receptor
compared to that of 1. Since 40a possesses comparable
affinity for the human B2 receptor with that of 1, it is
expected that 40a could exhibit equal or superior clinical
effects to those of 1. On the other hand several 4-(4-
morpholino) and 4-(1-piperidino) derivatives were re-
vealed to increase IPs formation by themselves in CHO
cells expressing the cloned human B2 receptor. They
were the first examples with agonistic activities in our
nonpeptide B2 ligands. Although the potency and ef-
ficacy of these partial agonists were much lower than
those of BK, we identified them as screening leads for
nonpeptide B2 agonists. Extensive investigation for
nonpeptide B2 full agonists and their in vivo pharmacol-
ogy will be discussed in due course.
1-ethoxy-3-[3-(dimethylamino)propyl]carbodiimide
hydro-
chloride (WSCD•HCl; 550 mg, 2.87 mmol), and 1-hydroxyben-
zotriazole (HOBt; 420 mg, 3.11 mmol) at ambient temperature.
After 3 h, this mixture was partitioned between CH2Cl2 and
water. The organic layer was washed with saturated aqueous
NaHCO3, water, and brine, dried over MgSO4, and evaporated
in vacuo. The resulting residue was crystallized from EtOAc
to afford 8 (1.24 g, 75.4%) as pale-yellow crystals: mp 194-
1
196 °C; H NMR (300 MHz, CDCl3): δ 1.06 (s, 9H), 2.22 (s,
3H), 3.23 (s, 3H), 3.57 (dd, J ) 17, 4 Hz, 1H), 3.94 (dd, J ) 17,
5 Hz, 1H), 4.92 (d, J ) 10 Hz, 1H), 4.98 (d, J ) 10 Hz, 1H),
6.44 (d, J ) 15 Hz, 1H), 6.63 (br s, 1H), 7.22 (d, J ) 8 Hz, 1H),
7.35-7.48 (m, 6H), 7.52 (d, J ) 15 Hz, 1H), 7.70-7.77 (m, 4H),
7.83 (dd, J ) 8, 3 Hz, 1H), 8.05 (br s, 1H), 8.22 (d, J ) 8 Hz,
1H), 8.36 (d, J ) 3 Hz, 1H); Anal. (C36H38Cl2N4O4Si) C, H, N.
(2E)-3-[6-(Acetylam in o)-3-pyr idin yl]-N-{2-[2,4-dich lor o-
3-(h yd r oxym eth yl)m eth yla n ilin o]-2-oxoeth yl}-2-p r op en -
a m id e (9). To a suspension of 8 (940 mg, 1.36 mmol) in THF
(9.4 mL) was added 1 M tetrabutylammonium fluoride in THF
(2.04 mL) at ambient temperature. After 1 h, the mixture was
partitioned between CH2Cl2 and water. The organic layer was
washed with 1 N HCl, water, saturated aqueous NaHCO3 and
brine, dried over MgSO4, and evaporated in vacuo. The residue
was crystallized from CH3CN to afford 9 (569 mg, 92.4%) as
colorless crystals: mp 207-209 °C; 1H NMR (300 MHz, DMSO-
d6): δ 2.10 (s, 3H), 3.10 (s, 3H), 3.47 (dd, J ) 17, 4 Hz, 1H),
3.76 (dd, J ) 17, 5 Hz, 1H), 4.74 (d, J ) 5 Hz, 1H), 5.35 (br s,
1H), 6.79 (d, J ) 15 Hz, 1H), 7.37 (d, J ) 15 Hz, 1H), 7.61 (d,
J ) 8 Hz, 1H), 7.65 (d, J ) 8 Hz, 1H), 7.98 (dd, J ) 8, 3 Hz,
1H), 8.11 (d, J ) 8 Hz, 1H), 8.21 (t, J ) 5 Hz, 1H), 8.47 (s,
1H); Anal. (C20H20Cl2N4O4) C, H, N.
(2E)-3-[6-(Acet yla m in o)-3-p yr id in yl]-N-{2-[3-(b r om o-
m et h yl)-2,4-d ich lor om et h yla n ilin o]-2-oxoet h yl}-2-p r o-
p en a m id e (10b). To a solution of 9 (555 mg, 1.23 mmol) in
CH2Cl2 (10 mL) were added triphenylphosphine (387 mg, 1.48
mmol) and carbon tetrabromide (612 mg, 1.84 mmol) in an
ice-water bath. After 10 min, the reaction mixture was stirred
at ambient temperature for 3 h. To the mixture were added
triphenylphosphine (96.8 mg, 0.369 mmol) and carbon tetra-
bromide (163 mg, 0.49 mmol), and the reaction mixture was
stirred for another 2 h. The reaction mixture was washed with
saturated aqueous NaHCO3, water, and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by
flash silica gel chromatography (CH2Cl2/MeOH, 20:1) to afford
10b (400 mg, 63.3%) as pale-yellow crystals: mp 222-223 °C;
1H NMR (300 MHz, CDCl3-CD3OD): δ 2.22 (s, 3H), 3.27 (s,
3H), 3.60 (dd, J ) 17, 3 Hz, 1H), 3.94 (dd, J ) 17, 3 Hz, 1H),
4.78 (s, 2H), 6.49 (d, J ) 15 Hz, 1H), 7.31 (d, J ) 8 Hz, 1H),
7.49 (d, J ) 8 Hz, 1H), 7.51 (d, J ) 15 Hz, 1H), 7.88 (dd, J )
Exp er im en ta l Section
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
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 Micro-
mass Platform (ESI) system. Elemental analyses were per-
formed on a Perkin-Elmer 2400 CHN analyzer. Analytical
results were within (0.4% of the theoretical values unless
otherwise noted. Silica gel 60F254 precoated plates on glass
from Merck KGaA or amonopropyl silicagel (APS) precoated
NH plates from Fuji Silysia Chemical Ltd. were used for thin-
layer chromatography. Silica gel chromatographies were per-
formed with Kieselgel 60 (230-400 mesh) (Merck KGaA,
Darmstadt, Germany) or NH-DM 1020 (Fuji Silysia Chemical
Ltd., J apan). Yields were not optimized.
2-Meth yl-4-(1-p yr r olid in yl)-8-qu in olin ol (6a ). A mixture
of 4-chloro-2-methyl-8-quinolinol (5)32 (300 mg, 1.55 mmol),
pyrrolidine (165 mg, 2.32 mmol), and phenol (292 mg, 3.1
mmol) was stirred at 125 °C for 5 h. Pyrrolidine (165 mg, 2.32
mmol) was added to the reaction mixture, and the mixture
was stirred at 125 °C for additional 5 h. After cooling, the
resulting residue was crystallized from acetone. The solid was
dissolved in CHCl3 and the solution washed with a mixture of
aqueous NaHCO3 and brine, dried over MgSO4, and evapo-

2674 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Sawada et al.
8, 3 Hz, 1H), 8.23 (br d, J ) 8 Hz, 1H), 8.33 (d, J ) 3 Hz, 1H);
62.1%) to a solid: 1H NMR (300 MHz, CDCl3): δ 3.83 (s, 3H),
4.12 (s, 3H), 6.53 (d, J ) 15 Hz, 1H), 7.13 (s, 1H), 7.30 (d, J )
8 Hz, 1H), 7.68 (d, J ) 15 Hz, 1H), 8.20 (d, J ) 8 Hz, 1H); MS
(ESI) m/z 237 (M + 1); Anal. (C12H12O5) C, H.
Anal. (C20H19BrCl2N4O3) C, H, N.
4-[(1E)-3-({2-[2,4-Dich lor o(m et h yl)-3-({[2-m et h yl-4-(1-
p yr r olid in yl)-8-q u in olin yl]oxy}m et h yl)a n ilin o]-2-oxo-
eth yl}am in o)-3-oxo-1-pr open yl]-N-m eth ylben zam ide (11a).
To a mixture of compound 10a (80.9 mg, 0.158 mmol) and 6a
(36.0 mg, 0.158 mmol) in dry DMF (1.0 mL) was added K2CO3
(65.5 mg, 0.474 mmol) at ambient temperature, and the
mixture was stirred at same temperature for 5 h. The reaction
mixture was poured into water and extracted with CH2Cl2
twice. The extracts were washed with water and brine, dried
over MgSO4, and evaporated in vacuo. The residue was
purified by preparative thin-layer chromatography (CHCl3/
MeOH, 10:1) to afford 11a (85.0 mg, 81.6%) as a colorless
amorphous solid: mp 169-172 °C; 1H NMR (300 MHz,
CDCl3): δ 1.98-2.06 (m, 4H), 2.54 (s, 3H), 2.99 (d, J ) 5 Hz,
3H), 3.24 (s, 3H), 3.59-3.72 (m, 5H), 3.93 (dd, J ) 17, 5 Hz,
1H), 5.56 (d, J ) 10 Hz, 1H), 5.60 (d, J ) 10 Hz, 1H), 6.36-
6.41 (m, 2H), 6.52 (d, J ) 15 Hz, 1H), 6.85 (br s, 1H), 7.11-
7.30 (m, 3H), 7.41-7.50 (m, 3H), 7.55 (d, J ) 15 Hz, 1H), 7.71
Meth yl (2E)-3-{3-Meth oxy-4-[(m eth yla m in o)ca r bon yl]-
p h en yl}-2-p r op en oa te (18). To a solution of 17 (500 mg, 2.12
mmol) in dry DMF (20 mL) were added methylamine hydro-
chloride (153 mg, 2.33 mmol), WSCD (487 mg, 2.54 mmol),
and HOBt (372 mg, 2.75 mmol) at ambient temperature. After
5 h, this mixture was partitioned between EtOAc and satu-
rated aqueous sodium bicarbonate. The organic layer was
washed with water and brine, dried over MgSO4, and evapo-
rated in vacuo. The residue was crystallized from IPE to afford
18 (448 mg, 84.9%) as a colorless amorphous solid: mp 127-
128 °C; 1H NMR (300 MHz, CDCl3): δ 3.01 (d, J ) 5 Hz, 3H),
3.82 (s, 3H), 4.00 (s, 3H), 6.49 (d, J ) 15 Hz, 1H), 7.08 (s, 1H),
7.25 (d, J ) 8 Hz, 1H), 7.67 (d, J ) 15 Hz, 1H), 7.78 (br s, 1H),
8.23 (d, J ) 8 Hz, 1H); MS (ESI) m/z 250 (M + 1); Anal. (C13H15
NO4) C, H, N.
-
Compounds 35a ,c and 36 were prepared following the
procedure described above for 18.
(br d, J ) 8 Hz, 2H), 7.84 (br d, J ) 8 Hz, 2H); Anal. (C35H35
Cl2N5O4) C, H, N.
-
(2E)-3-[3-Meth oxy-4-[(m eth yla m in o)ca r bon yl]p h en yl]-
2-p r op en oic Acid (19). To a solution of 18 (300 mg, 1.20
mmol) in MeOH (6 mL) was added 1 N NaOH (1.5 mL) at
ambient temperature. The reaction mixture was stirred at 50
°C for 5 h. The solvent was evaporated in vacuo, and the
residue was dissolved in water. The water layer was washed
with Et2O and adjusted to pH 4 with 1 N HCl. The solid that
precipitated was collected by vacuum filtration and washed
with water to afford 19 (250 mg, 88.3%) as a pale-yellow
amorphous solid: 1H NMR (300 MHz, DMSO-d6): δ 2.79 (d, J
) 5 Hz, 3H), 3.91 (s, 3H), 6.66 (d, J ) 15 Hz, 1H), 7.31 (d, J
) 8 Hz, 1H), 7.43 (s, 1H), 7.60 (d, J ) 15 Hz, 1H), 7.73 (d, J
) 8 Hz, 1H), 8.16 (q, J ) 5 Hz, 1H); MS (ESI) m/z 236 (M +
1); Anal. (C12H13NO4) C, H, N.
Compounds 11b-h , 12a , and 12b were prepared following
the procedure described above for 11a .
4-[(1E)-3-({2-[2,4-Dich lor o(m et h yl)-3-({[2-m et h yl-4-(1-
p yr r olid in yl)-8-q u in olin yl]oxy}m et h yl)a n ilin o]-2-oxo-
eth yl}a m in o)-3-oxo-1-p r op en yl]-N-m eth ylben za m id e Di-
h yd r och lor id e (13a ). To a solution of 11a (60.0 mg, 0.091
mmol) in MeOH (0.9 mL) was added 10% HCl in MeOH (0.5
mL) at ambient temperature. After 5 min, the solution was
evaporated in vacuo. The resulting residue was washed with
Et2O to afford 13a (52.0 mg, 78.1%) as colorless crystals: mp
203-206 °C; 1H NMR (300 MHz, CDCl3-CD3OD): δ 2.14-
2.26 (m, 4H), 2.67 (s, 3H), 2.99 (s, 3H), 3.29 (s, 3H), 3.87 (d, J
) 17 Hz, 1H), 3.89-4.08 (m, 4H), 4.13 (d, J ) 17 Hz, 1H),
5.48 (d, J ) 10 Hz, 1H), 5.65 (d, J ) 10 Hz, 1H), 6.51 (s, 1H),
6.62 (d, J ) 15 Hz, 1H), 7.33-7.64 (m, 7H), 7.81 (d, J ) 8 Hz,
2H), 8.02 (d, J ) 8 Hz, 1H); Anal. (C35H35Cl2N5O4¨ı2HCl) C, H,
N.
N-[4-(Hydr oxym eth yl)-2-m eth ylph en yl]acetam ide (21).
To a solution of (4-amino-3-methylphenyl)methanol (20) (1.77
g, 12.9 mmol) in MeOH (18 mL) was added Ac2O (3.7 mL, 39.2
mmol) at ambient temperature, and the reaction mixture was
stirred at same temperature for 2 h. The reaction mixture was
evaporated in vacuo, and the residue was dissolved in EtOAc.
The solution was concentrated in vacuo, and the residue was
washed with Et2O to afford 21 (1.90 g, 82.2%) as a solid: mp
Compounds 13b-h , 14, 38b,c, 39, 40a -c, 41a -c, 42a ,b,
43, 44a ,c, 45, 46b,c, and 48 were prepared following the
procedure described above for 13a .
4-(Hyd r oxym eth yl)-2-m eth oxyben zoic Acid (16). To a
solution of methyl 4-[(acetyloxy)methyl]-2-methoxybenzoate
(15) (8.82 g, 37.0 mmol) in MeOH (200 mL) was added 1 N
NaOH (75 mL) at ambient temperature. The reaction mixture
was stirred at same temperature for 3 h. The solvent was
evaporated, and the residue was dissolved in water. The water
layer was washed with Et2O and adjusted to pH 4 with
concentrated HCl. The mixture was concentrated in vacuo and
EtOH was added to the residue. The precipitates were filtered
off. The filtrate was evaporated and the residue dried in a
vacuum to afford 16 (4.62 g, 78.5%) as a pale yellow solid: 1H
NMR (300 MHz, DMSO-d6): δ 3.81 (s, 3H), 4.52 (s, 2H), 5.30
(br s, 1H), 6.92 (d, J ) 8 Hz, 1H), 7.08 (s, 1H), 7.61 (d, J ) 8
Hz, 1H); MS (ESI) m/z 183 (M + 1); Anal. (C9H10O4) C, H.
2-Meth oxy-4-[(1E)-3-m eth oxy-3-oxo-1-p r op en yl]ben zo-
ic Acid (17). To a mixture of 16 (1.5 g, 8.23 mmol) and Et3N
(5.00 g, 49.4 mmol) in a mixture of CH2Cl2 (7.5 mL) and DMSO
(7.5 mL) was added sulfur trioxide pyridine complex (3.93 g,
24.7 mmol) portionwise in water bath. The mixture was stirred
at ambient temperature for 2 h. The reaction mixture was
poured into water and extracted with CH2Cl2. The organic
layer was washed with water and brine, dried over MgSO4,
and evaporated in vacuo. To a solution of the residue in THF
(10 mL) was added methyl (triphenylphosphoranylidene)-
acetate (3.30 g, 9.88 mmol) at ambient temperature under
nitrogen. The reaction mixture was stirred at same temper-
ature for 1 h. The reaction mixture was concentrated in vacuo
and partitioned between EtOAc and saturated aqueous NaH-
CO3. The water layer was adjusted to pH 4 with 1 N HCl and
extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, and evaporated in vacuo.
The residue was washed with hot IPE to afford 17 (1.21 g,
1
103-104 °C; H NMR (300 MHz, DMSO-d6): δ 2.03 (s, 3H),
2.18 (s, 3H), 4.42 (d, J ) 6 Hz, 2H), 5.09 (t, J ) 6 Hz, 1H),
7.07 (d, J ) 8 Hz, 1H), 7.13 (s, 1H), 7.31 (d, J ) 8 Hz, 1H),
9.24 (s, 1H); Anal. (C10H13NO2) C, H, N.
N-(4-F or m yl-2-m eth ylp h en yl)a ceta m id e (22). To a mix-
ture of 21 (1.90 g, 10.6 mmol) and triethylamine (7.4 mL, 53.1
mmol) in DMSO (9.5 mL) was added sulfur trioxide pyridine
complex (3.71 g, 23.3 mmol) portionwise in water bath. The
mixture was stirred at ambient temperature for 2 h. The
reaction mixture was poured into water and extracted with
EtOAc. The organic layer was washed with water and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
crystallized from IPE to afford 22 (1.25 g, 66.2%) as colorless
crystals: mp 110-112 °C; 1H NMR (300 MHz, CDCl3): δ 2.26
(s, 3H), 2.33 (s, 3H), 7.16 (br s, 1H), 7.70-7.78 (m, 2H), 8.30
(br s, 1H), 9.91 (s, 1H); Anal. (C10H11NO2) C, H, N.
(2E)-3-[4-(Acet yla m in o)-3-m et h ylp h en yl]-2-p r op en o-
ic Acid (23). A mixture of 22 (1.23 g, 6.94 mmol) and malonic
acid (795 mg, 7.64 mmol) in dry pyridine (549 mg, 6.94 mmol)
and EtOH (3.4 mL) was refluxed for 3 h. After the mixture
was cooled, the solid that precipitated was collected by vacuum
filtration to afford 23 (1.08 g, 71.0%) as a pale yellow
amorphous solid: mp 262-263 °C; 1H NMR (300 MHz, DMSO-
d6): δ 2.09 (s, 3H), 2.23 (s, 3H), 6.43 (d, J ) 16 Hz, 1H), 7.43-
7.61 (m, 4H), 9.33 (s, 1H); Anal. (C12H13NO3) C, H, N.
N -[2,4-Dich lor o-3-({[4-(d im e t h yla m in o)-2-m e t 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 (25a ). Step 1. To a
solution of 24 (2.00 g, 5.09 mmol) and Et3N (772 mg, 7.63
mmol) in dry CH2Cl2 (20 mL) was added dropwise methane-
sulfonyl chloride (641 g, 5.60 mmol) in an ice-water bath

New Nonpeptide Bradykinin B2 Receptor Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2675
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 methanesulfonate intermediate (2.40 g, ∼100%)
as a pale-yellow oil.
solid: 1H NMR (300 MHz, DMSO-d6): δ 2.60 (s, 3H), 3.13 (s,
3H), 3.20-3.42 (m, 6H), 3.58 (br dd, J ) 17, 4 Hz, 1H), 3.90
(br dd, J ) 17, 5 Hz, 1H), 5.51 (d, J ) 10 Hz, 1H), 5.58 (d, J
) 10 Hz, 1H), 6.90 (br s, 1H), 7.01 (d, J ) 15 Hz, 1H), 7.44-
7.93 (m, 6H), 8.07 (d, J ) 8 Hz, 1H), 8.14 (br d, J ) 8 Hz, 1H),
8.45 (br t, J ) 5 Hz, 1H), 8.88 (br s, 1H); MS (ESI) m/z 622 (M
+ 1); Anal. (C31H29Cl2N5O5) C, H, N.
Step 2. To a mixture of 4-(dimethylamino)-8-hydroxy-2-
methylquinoline33 (1.02 g, 5.04 mmol) and K2CO3 in dry DMF
(25 mL) was added dropwise a solution of methanesulfonate
intermediate (2.40 g, 5.09 mmol) in dry DMF (25 mL), and
the mixture was stirred at ambient temperature for 1 day. The
reaction mixture was poured into water and extracted with
CHCl3 twice. The extracts were washed with water and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
crystallized from a mixture of isopropyl alcohol and water to
afford 25a (2.36 g, 81.0%) as colorless crystals: mp 137-139
Compound 34c was prepared following the procedure de-
scribed above for 34a .
3-{[({2-[2,4-Dich lor o-3-({[4-(d im eth yla m in o)-2-m eth yl-
8-q u in olin yl]oxy}m e t h yl)m e t h yla n ilin o]-2-oxoe t h 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 (47).
A mixture of 26a (80 mg, 0.179 mmol), phenyl 3-[(4-pyridiny-
lamino)carbonyl]phenylcarbamate (62.6 mg, 0.188 mmol), and
Et3N (109 mg, 1.07 mmol) in dry DMF (1.5 mL) was stirred at
ambient temperture for 5 h. The reaction mixture was poured
into water and extracted with CHCl3. The organic layer was
washed with water three times, dried over MgSO4, and
evaporated in vacuo. The residue was purified by preparative
thin-layer chromatography (CHCl3/MeOH/NH4OH, 10:1:0.1) to
afford 47 (61.0 mg, 49.7%) as a pale-yellow amorphous solid:
1H NMR (300 MHz, CDCl3): δ 2.50 (s, 3H), 3.01 (s, 6H), 3.19
(s, 3H), 3.87-3.98 (m, 2H), 5.35-5.49 (m, 2H), 6.32 (br peak,
1H), 6.65 (s, 1H), 7.03 (dd, J ) 8, 8 Hz, 1H), 7.10-7.20 (m,
2H), 7.28-7.42 (m, 4H), 7.59-7.68 (m, 1H), 7.70 (d, J ) 8 Hz,
1H), 7.78 (d, J ) 6 Hz, 1H), 8.40-8.52 (m, 2H), 8.69 (br s,
1H), 9.47 (br peak, 1H); MS (ESI) m/z 686 (M + 1); Anal.
(C35H33Cl2N7O4) 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
described35 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
Polytron. The homogenate was centrifuged to remove cellular
debris (1000g, 20 min, 4 °C), and the supernatant was
centrifuged (100 000g, 60 min, 4 °C). The pellet was then
resuspended in ice-cooled binding buffer (50 mM TES, 1 mM
1,10-phenanthroline, 140 µg/mL bacitracin, 1 mM dithiothrei-
tol, 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. All experiments
were carried out three times.
Clon ed Hu m a n B2 Recep tor s Exp r essed in CHO Cells.
CHO (dhfr-) cells that were transfected with and stably
expressed with human B2 receptors have been described
previously.24 Cells were maintained in an 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,
1
°C; H NMR (300 MHz, CDCl3): δ 2.66 (s, 3H), 2.96 (s, 6H),
3.21 (s, 3H), 4.07 (s, 2H), 5.63 (d, J ) 10 Hz, 1H), 5.71 (d, J )
10 Hz, 1H), 6.69 (s, 1H), 7.20 (d, J ) 8 Hz, 1H), 7.30 (d, J )
8 Hz, 1H), 7.46 (d, J ) 8 Hz, 1H), 7.53 (d, J ) 8 Hz, 1H), 7.65-
7.75 (m, 3H), 7.82-7.90 (m, 2H); Anal. (C30H26Cl2N4O4) C, H,
N.
Compounds 25b and 25c were prepared following the
procedure described above for 25a .
2-Am in o-N -[2,4-d ich lor o-3-({[4-(d im e t h yla m in o)-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 (26a ). To a suspension of 25a (2.44 g, 4.23 mmol)
in EtOH (25 mL) was added hydrazine monohydrate (423 mg,
8.45 mmol) at ambient temperature, and the mixture was
refluxed for 1 h. After the reaction mixture was cooled, the
precipitates formed were filtered off. The filtrate was evapo-
rated in vacuo, to the residue was added CHCl3 (20 mL), and
precipitates were filtered off. The filtrate was evaporated in
vacuo, and the residue was purified by silica gel chromatog-
raphy (NH-DM1020, CHCl3) to afford 26a (1.40 g, 74.1%) as a
colorless amorphous solid: 1H NMR (300 MHz, CDCl3): δ 2.66
(s, 3H), 2.91-3.13 (m, 8H), 3.21 (s, 3H), 5.61 (s, 2H), 6.70 (s,
1H), 7.12-7.36 (m, 3H), 7.45 (d, J ) 8 Hz, 1H), 7.70 (d, J ) 8
Hz, 1H); MS (ESI) m/z 447 (M + 1); Anal. (C22H24Cl2N4O2) C,
H, N.
Compounds 26b and 26c were prepared following the
procedure described above for 26a .
4-[(1E)-3-({2-[2,4-Dich lor o(m et h yl)-3-({[2-m et h yl-4-(1-
p ip e r id in yl)-8-q u in olin yl]oxy}m e t h yl)a n ilin o]-2-oxo-
e t h y l}a m in o )-3-o x o -1-p r o p e n y l]-N ,N -d im e t h y lb e n z-
a m id e (27b). To a solution of 26b (30 mg, 0.062 mmol) in dry
DMF (1 mL) were added (E)-4-(N,N-dimethylcarbamoyl)-
cinnamic acid19 (14.8 mg, 0.068 mmol), WSCD•HCl (14.2 mg,
0.074 mmol), and HOBt (10.8 mg, 0.08 mmol) at ambient
temperature. After 3 h, this mixture was partitioned between
EtOAc and saturated aqueous NaHCO3. The organic layer was
washed with water and brine, dried over MgSO4, and evapo-
rated in vacuo. The residue was purified by preparative thin-
layer chromatography (CHCl3/MeOH, 10:1) to afford 27b (42
mg, 99.1%) as a pale-yellow amorphous solid: 1H NMR (300
MHz, CDCl3): δ 1.60-1.75 (m, 2H), 1.79-1.90 (m, 4H), 2.68
(br s, 3H), 2.98 (br s, 3H), 3.06-3.29 (m, 10H), 3.70 (br d, J )
17 Hz, 1H), 3.97 (br d, J ) 17 Hz, 1H), 5.60 (br s, 2H), 6.52 (br
d, J ) 15 Hz, 1H), 6.71 (s, 1H), 7.20 (br d, J ) 8 Hz, 1H),
7.27-7.60 (m, 9H), 7.62 (br d, J ) 8 Hz, 1H); MS (ESI) m/z
688 (M + 1); Anal. (C37H39Cl2N5O4) C, H, N.
Compounds 27c, 28, 29a -c, 30a -c, 31a ,b, 32, 33a ,c, 37b,
and 37c were prepared following the procedure described
above for 27b.
5-[(1E)-3-({2-[2,4-Dich lor o-3-({[4-(d im et h yla m in o)-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-
oxoeth yl}a m in o)-3-oxo-1-p r op en yl]-2-p yr id in eca r boxy-
lic a cid (34a ). A suspension of 33a (948 mg, 1.46 mmol) in
EtOH (8 mL) containing 1N NaOH (1.6 mL) was heated at 60
°C for 2 h. Upon cooling, the reaction mixture was adjusted to
pH 7 with 1 N HCl and concentrated in vacuo. The residue
was partitioned between a mixture of CHCl3 and MeOH (5:1)
and brine. The organic layer was dried over MgSO4 and
evaporated in vacuo. The residue was triturated with CH3CN
to afford 34a (771 mg, 85.0%) as a pale-yellow amorphous

2676 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Sawada et al.
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determined in the presence of 1 µM unlabeled BK, from the
total binding. Bound radioactivity was determined by solubi-
lizing with 1% sodium doedecyl sulfate containing 0.05 N
NaOH and quantified in a liquid scintillation counter.
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
intraperitoneal 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 admin-
istered subcutaneously. 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 method36 as
the peak increase of pulmonary insufflation pressure (PIP).37
Each dose of the compound dissolved in 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 then the bronchoc-
onstriction 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 calculated from the following formula:
%
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Allen, L. G.; Duggan, M., J .; Loy, S.; Bonham, K. A.; Blodgett,
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Topography Using Ethers of Hydroxyproline in Novel Peptides.
J . Med. Chem. 1991, 34, 2649-2653.
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.
Agon ist-In d u ced In ositol P h osp h a tes F or m a tion . Inos-
itol phosphate (IPs) formation was measured essentially as
described previously.24 CHO cells expressing the human B2
receptor were seeded in 12-well plates at density of 1 × 105
cells/well and cultured for 1 day. The cells were labeled with
[3H]inositol (1 µCi/mL) for 24 h. The cells were washed twice
with PBS containing 0.2% BSA and incubated with the same
solution for 30 min and then with PBS containing 0.2% BSA
and 10 mM LiCl for 30 min at 37°C. Agonist stimulation was
started by replacing the medium with fresh PBS containing
0.2% BSA, 10 mM LiCl, and test compounds. The reaction was
terminated by 5% (w/v) trichloroacetic acid after incubation
for 30 min at 37 °C. Separation of [3H]inositol phosphates was
carried out by Bio-Rad AG 1-X8 chromatography essentially
as described elsewhere.38 A mixture of 3H-labeled inositol
monophosphate (IP1), inositol bisphosphate (IP2), and inositol
trisphosphate (IP3) was eluted from the column with 0.1 M
formic acid/1.0 M ammonium formate. The radioactivity in the
eluates was determined by a liquid scintillation spectrometer.
The agonist-induced IPs formation was calculated by subtract-
ing the control radioactivity determined in the absence of the
compound. The efficacy of the compound was expressed as the
relative agonistic activity in IPs formation compared to that
of BK (10 nM).
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1996.
(19) Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.; Imai, K.;
Inamura, N.; Asano, M.; Hatori C.; Katayama, A.; Oku T.;
Tanaka H. A Novel Class of Orally Active Non-Peptide Brady-
kinin B2 Receptor Antagonist. 1. Construction of the Basic
Framework J . Med. Chem. 1998, 41, 564-578.
(20) Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.; Inamura,
N.; Asano, M.; Hatori C.; Sawai H.; Oku T.; Tanaka H. A Novel
Class of Orally Active Non-Peptide Bradykinin B2 Receptor
Antagonists. 2. Overcoming the Species Difference between
Guinea Pig and Man. J . Med. Chem. 1998, 41, 4053-4061.
(21) Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.; Inamura,
N.; Asano, M.; Aramori I.; Hatori C.; Sawai H.; Oku T.; Tanaka
H. A Novel Class of Orally Active Non-Peptide Bradykinin B2
Receptor Antagonists. 3. Discovering Bioisosteres of the Imidazo-
[1,2-a]pyridine Moiety. J . Med. Chem. 1998, 41, 4062-4079.
(22) Abe, Y.; Kayakiri, H.; Satoh, S.; Inoue, T.; Sawada, Y.; Inamura,
N.; Asano, M.; Aramori I.; Hatori C.; Sawai H.; Oku T.; Tanaka
H. A Novel Class of Orally Active Non-Peptide Bradykinin B2
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4098.
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 values were obtained by
using nonlinear curve-fitting methods with a computer pro-
gram developed in-house.
Su p p or tin g In for m a tion Ava ila ble: Physical data of 6b,
6d -h , 11b-h , 12a ,b, 13b-h , 14, 25b,c, 26b,c, 27c, 28, 29a -
c, 30a -c, 31a ,b, 32, 33a ,c, 34c, 35a ,c, 36, 37b,c, 38b,c, 39,
40a -c, 41a -c, 42a ,b, 43, 44a ,c, 45, 46b,c, and 48. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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