A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 2. Overcoming the species difference between guinea pig and man

Article abstract of DOI:10.1021/jm980214f

Recently we reported the identification of a series of 8-[[3-(N- acylglycyl-N-methylamino)-2,6-dichlorobenzyl]oxy]-3-halo-2-methytimidazo[1,2- α]pyridines as the first orally active non-peptide bradykinin (BK)B₂ receptor antagonists (1-3). Thes

Full text of DOI:10.1021/jm980214f

J . Med. Chem. 1998, 41, 4053-4061
4053
A Novel Cla ss of Or a lly Active Non -P ep tid e Br a d yk in in B2 Recep tor
An ta gon ists. 2. Over com in g th e Sp ecies Differ en ce betw een Gu in ea P ig a n d
Ma n
Yoshito Abe,† Hiroshi Kayakiri,*,† Shigeki Satoh,† Takayuki Inoue,† Yuki Sawada,† Noriaki Inamura,‡
Masayuki Asano,§ Chie Hatori,§ Hiroe Sawai,§ Teruo Oku,† and Hirokazu Tanaka|
Exploratory Research Laboratories, Fujisawa Pharmaceutical Company, Ltd.,
5-2-3, Tokodai, Tsukuba, Ibaraki 300-2698, J apan
Received April 7, 1998
Recently we reported the identification of a series of 8-[[3-(N-acylglycyl-N-methylamino)-2,6-
dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide
bradykinin (BK) B2 receptor antagonists (1-3). These compounds inhibited the specific binding
of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors with nanomolar
IC50’s and also displayed in vivo functional antagonistic activities against BK-induced
bronchoconstriction in guinea pigs at 1 mg/kg by oral administration. However, it was found
that their affinities for the B2 receptors in human A-431 cells (human epidermoid carcinoma)
were much lower. Intensive modifications of the terminal substituents at the glycine moiety
elucidated the structure-activity relationships (SAR) for human B2 receptors, leading to an
extended basic framework which incorporated a novel key pharmacophore. Thus, we overcame
the species difference and identified the first clinical candidate 18c (FR167344) with IC50’s of
0.66 and 1.4 nM for guinea pig ileum and human A-431 cells, respectively. This compound
displayed in vivo functional antagonistic activity against BK-induced bronchoconstriction in
guinea pigs with an ED50 value of 0.17 mg/kg by oral administration. This novel non-peptide
B2 antagonist is extremely potent both in vitro and in vivo by oral administration and is expected
to be the first member of a new class of drug for the treatment of various inflammatory diseases.
Ch a r t 1
In tr od u ction
Bradykinin (BK) is an endogenous nonapeptide gen-
erated by tissue and plasma kallikreins. Because of its
highly potent proinflammatory activities, BK has been
implicated in a variety of pathophysiological responses,
including pain, inflammation, asthma, rhinitis, and
hypotension.1-8 Two types of BK receptors, designated
as B1 and B2, have been identified by molecular cloning
and pharmacological means.1,4,9-11 B2 receptors are
expressed constitutively in many tissues and are thought
to mediate most of the biological actions of BK.1,9
The search for antagonists of the B2 receptor has been
pursued for many years. A number of peptide BK B2
antagonists have been developed,12-16 including the
clinically evaluated second-generation antagonists Icati-
bant (Hoe140)12,13 and Bradycor (CP0127).14 Despite
their highly potent B2 antagonistic activity, their thera-
peutic use is still limited because of their peptidic
nature. On the other hand, only very few non-peptide
antagonists have been disclosed,17-19 and until our
earlier report20 there had been no description of potent
orally active non-peptide B2 antagonists.
8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichlorobenzyl]-
oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton as
the basic framework.20 The representative lead com-
pounds 1-3 (Chart 1) inhibited the specific binding of
[3H]BK to guinea pig ileum membrane preparations
expressing B2 receptors with nanomolar IC50’s and also
displayed in vivo functional antagonistic activities
against BK-induced bronchoconstriction in guinea pigs
by oral administration with ED50 values of <1 mg/kg.
However, it was found that these small non-peptide
antagonists bound to the B2 receptors in human A-431
cells rather weakly. The structure-activity relation-
ships (SAR) of the N-substituents of the glycine moiety
suggested that this group plays a key role in the
interaction with human A-431 B2 receptors and that an
additional pharmacophore might be necessary for stron-
ger binding. Intensive chemical modifications of this
terminal group enabled us to overcome the species
difference without loss of the potent oral in vivo
antagonistic activity leading to our first clinical candi-
Recently we reported the discovery of the first orally
active non-peptide BK B2 antagonists incorporating an
† Department of Medicinal Chemistry.
§ Department of Pharmacology.
‡ Present address: Exploratory Clinical Research, Development
Division, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yo-
dogawa-ku, Osaka 532-8514, J apan.
| Present address: New Drug Research Laboratories, Fujisawa
Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-
8514, J apan.
S0022-2623(98)00214-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/19/1998

4054 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Abe et al.
Sch em e 1a
a
(a) RH‚HCl, WSCD, HOBt, DMF; (b) 1 N NaOH, EtOH or MeOH; (c) CH2(CO2H)2, pyridine, EtOH; (d) Ac2O, CH2Cl2; (e) Br(CH2)3CO2H,
WSCD‚HCl, HOBt, DMF; (f) MeNCO, CH2Cl2; (g) MeSO2Cl, Et3N, CH2Cl2; (h) MeI, NaH, DMF; (i) K2CO3, DMF; (j) acrylic acid, Pd(OAc)2,
PPh3, n-Bu3N, xylene; (k) SOCl2, MeOH; (l) Br(CH2)3COCl, Et3N, CH2Cl2; (m) NaH, DMF.
Sch em e 2a
a
(a) RCOCl, Et3N, CH2Cl2; (b) Ph(CH2)3CO2H, WSCD‚HCl, HOBt, DMF; (c) HCl-MeOH; (d) RNCO, CH2Cl2; (e) SnCl2, EtOH; (f)
MeOCH2CO2H, WSCD‚HCl, HOBt, DMF; (g) 1 N NaOH, EtOH; (h) Me2NH‚HCl or MeNH2‚HCl, WSCD, HOBt, DMF; (i) MeO(CH2)2NH2,
WSCD‚HCl, HOBt, DMF; (j) cinnamoyl chloride, Et3N, CH2Cl2; (k) 6a -c or 10a -f,h ,k , WSCD‚HCl, HOBt, DMF.
date 18c (FR167344).21 Herein we wish to describe the
SAR culminating in the discovery of 18c.
in Scheme 1. Condensation of methyl (E)-4-carboxy-
cinnamate (4), which was synthesized by Wittig reaction
of terephthalaldehydic acid and methyl (triphenylphos-
phoranylidene)acetate, with the corresponding amines
in the presence of 1-ethyl-3-[(dimethylamino)propyl]-
carbodiimide (WSCD) and 1-hydroxybenzotriazole (HOBt)
followed by alkaline hydrolysis gave the acids 6a ,b.
Acetylation of ethyl (E)-4-aminocinnamate (8) and
subsequent alkylation of the acetamide 9a with methyl
Ch em istr y
The compounds described in this study are shown in
Tables 1-3, and their synthetic methods are outlined
in Schemes 1 and 2. Preparation of (E)-4-(substituted)-
cinnamic acids 6a ,b and 10a -f and (E)-3-[6-(substitut-
ed)pyridin-3-yl]acrylic acids 6c and 10h ,i,k are shown

Novel Non-Peptide BK B2 Receptor Antagonists. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4055
iodide gave the N-methylacetamide 9b. The acids 10a ,b
were obtained by saponification of the esters 9a ,b.
Coupling of 8 with 4-bromobutyric acid followed by
intramolecular cyclization of 9c in the presence of
potassium carbonate in DMF provided pyrrolidone 9d
which was hydrolyzed to 10d . Reaction of the amine 8
with methyl isocyanate followed by hydrolysis gave the
acid 10e. Sulfonylation of 8 with methanesulfonyl
chloride gave a mixture of mono- and disulfonamides
9f,g, which were hydrolyzed to yield the acid 10f. Two
types of alternative reactions were performed to prepare
the acids. The first was Perkin reaction of the alde-
hydes 7a -c with malonic acid in the presence of
pyridine in EtOH to give the acids 6a -c. The second
was Heck reaction of the bromopyridine 1122 with
acrylic acid to yield the acid 10h . Removal of the acetyl
group of 10h and successive esterification with SOCl2
in MeOH gave the amino ester 9i. Coupling of 9i with
4-bromobutyryl chloride and intramolecular cyclization
by sodium hydride followed by hydrolysis provided the
pyrrolidone derivative 10k .
Modifications of the substituent at the amino group
of 1220 are shown in Scheme 2. Acylation of 12 gave
the amides 13b-f. Compound 13c was treated with
10% hydrogen chloride in MeOH to afford the hydro-
chloride 14c. Reaction of 12 with various isocyanates
yielded the corresponding ureas 15a -j,m ,o. The nitro
group of 15j was reduced with SnCl2 in EtOH followed
by coupling with methoxyacetyl chloride to give the
amide 15l. Hydrolysis of the ester 15o and subsequent
coupling with appropriate amines afforded the carbam-
oyls 15p ,q. The ureas 15a ,c,e-j,m ,p ,q were converted
to the corresponding hydrochlorides 16a ,c,e-j,m ,p ,q,
respectively. Acylation of 12 with cinnamoyl chloride
gave the cinnamamide 17a . The acrylamide derivatives
17b-i,m were synthesized by coupling of 12 with the
acids 6a -c and 10a -f,g,k in the presence of WSCD‚
HCl and HOBt in DMF. Saponification of the ester 17i
followed by coupling with amines yielded the carbam-
oyls 17k ,l. The hydrochlorides 18a -h ,k -m were also
obtained by treatment of 17a -h ,k -m with 10% hydro-
gen chloride in MeOH.
induced bronchoconstriction in guinea pigs at 1 mg/kg
and above by oral administration (Table 1). Considering
the possibility of clinical studies, these compounds were
then advanced for evaluation of binding activity to
human B2 receptors in A-431 cells.23 Despite using a
20-fold higher concentration of [3H]BK in the human
A-431 cell system than in guinea pig ileum membrane
preparations, a representative second-generation pep-
tide B2 antagonist, Icatibant, potently inhibited the
specific binding with an IC50 value of 3.3 nM, ca. 37-
fold higher than that in guinea pigs. However, it was
revealed that our non-peptide B2 antagonists showed
only weak binding affinities in the human A-431 cell
system. The IC50 values of 1, 2, and 3 for the B2
receptors of human A-431 cells were ca. 70-, 350-, and
330-fold higher than those in guinea pigs, respectively.
To overcome this species difference, we initially inves-
tigated the SAR of the terminal N-acyl substituents of
the glycine moiety (Table 1). Replacement of the
terminal methyl group of 13a with a n-propyl (1) or
n-pentyl (13b) group increased their B2 binding affini-
ties in both the human A-431 cell and the guinea pig
ileum systems. Introduction of the phenyl group (14c)
to 13a also increased both affinities to the same level
of that of 1, indicating that a bulky substituent at the
glycine nitrogen is tolerated in the binding site of both
B2 receptors. Extending the methylene of 14c to an
ethylene (13d ) afforded a 2-fold increase in binding
affinity in the human A-431 cell system, while further
extension to the propylene (13e) resulted in a slight loss
of activity. Furthermore, the cinnamamide 18a resulted
in a ca. 2- and 3-fold improvement of the B2 binding
affinities in guinea pig ileum and human A-431 cell
systems with IC50’s of 0.78 and 120 nM, respectively.
On the other hand, as shown by 2 and 13f, introduction
of heteroatoms to the terminal chain failed to improve
affinities for the human receptor. It was interesting
that in the benzoylurea (15b), binding affinity in the
guinea pig system decreased much more severely than
in the human system compared with the phenylurea
(16a ). In the urea series, 16a exhibited more potent
B2 binding affinities than ethylurea (3) and benzylurea
(16c) in both systems. These results suggested that a
large planar terminal substituent, incorporating an
aromatic ring such as a cinnamamide or phenylurea
moiety, is favorable for interaction with both B2 recep-
tors. However, neither 18a nor 16a showed significant
antagonistic activities in vivo by oral administration.
Therefore we investigated the effects of substituents on
the terminal phenyl ring of 18a and 16a to improve both
binding affinity for human A-431 B2 receptors and in
vivo antagonistic activity by oral administration.
P h a r m a cology
All compounds were tested for inhibition of the
specific binding of [3H]BK to B2 receptors in guinea pig
ileum membrane preparations as previously reported,20,21
and they were also evaluated for inhibition of the
specific binding of [3H]BK to B2 receptors in human
A-431 cells. Compounds having potent binding affini-
ties were then tested for in vivo functional antagonistic
activity in inhibiting BK-induced bronchoconstriction in
guinea pigs by oral administration.20,21a
Introduction of substituents to the terminal phenyl
ring of the phenylurea (16a ) is summarized in Table 2.
Comparison of the B2 binding affinities of 15d and 16e,f
indicated that ortho and para substitutions at the
phenyl ring were not sterically tolerated in guinea pigs,
while ortho substitution retained and para substitution
only slightly decreased the activities in human A-431
cells. According to these results, we then introduced
various substituents at the 3-position of the phenyl ring,
where methyl substitution was tolerated in both guinea
pig ileum and human A-431 cell systems. Replacement
Resu lts a n d Discu ssion
Recently we reported on the first series of orally active
non-peptide BK B2 receptor antagonists incorporating
an 8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichloroben-
zyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton
as the basic framework.20 The representative lead
compounds 1-3 inhibited the specific binding of [3H]-
BK to guinea pig ileum membrane preparations with
nanomolar IC50’s and also displayed dose-dependent in
vivo functional antagonistic activities against BK-

4056 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Ta ble 1. Introduction of Acyl Substituents at the Glycine Moiety
Abe et al.
in vitro IC50 (nM)
in vivo % inhibn (po)c
1 mg/kg 3.2 mg/kg
guinea pig
ileuma
human
synth
compd
13a
1
13b
2
14c
13d
13e
13f
18a
3
16a
15b
16c
Icatibant
R
n
A-431 cellb
methodd
mp (°C)
formulae
Me
n-Pr
n-Pen
1
0
0
0
1
0
0
0
1
0
1
0
1
16
8.9
5.6
2.4
5.3
1.4
8.9
4.2
0.78
9.0
1.4
340
11
4100
610
240
830
670
350
480
510
120
3000
150
510
570
3.3
62.3 ( 15.6** 94.2 ( 2.2*** ref 20
179-180
158-159
131-132
175-177
C20H19BrCl2N4O3‚HCl
C22H23BrCl2N4O3
C24H27BrCl2N4O3
C22H24BrCl2N5O3
52.7 ( 14.6
NT
NTf
NT
ref 20
B
CH2NMe2
CH2Ph
72.4 ( 7.8**
92.8 ( 2.4*** ref 20
NT
19.8 ( 10.3
D
B
C
B
D
amorphous C26H23BrCl2N4O3‚HCl
amorphous C27H25BrCl2N4O3
amorphous C28H27BrCl2N4O3
amorphous C26H23BrCl2N4O4
amorphous C27H23BrCl2N4O3‚HCl
171-173
amorphous C25H22BrCl2N5O3‚HCl
187-190 26H22BrCl2N5O4
amorphous C26H24BrCl2N5O3‚HCl
(CH2)2Ph
(CH2)3Ph
CH2OPh
(E)-CHdCHPh
NHEt
NT
NT
NT
0 ( 19.2
NT
NT
NT
NT
57.2 ( 4.9*** 78.1 ( 6.2*** ref 20
C21H22BrCl2N5O3
NHPh
NT
NT
NT
NT
30.7 ( 11.1
NT
D
A
D
NHCOPh
NHCH2Ph
C
24.1 ( 9.8
NT
0.09
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to B2 receptors 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 Experimental
b
Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.2 nM) to A-431 cells (human epidermoid
carcinoma) which express B2 receptors 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 method27 as
previously reported. After 5 min, compounds were orally administered. After 30 min, BK was administered again and bronchoconstriction
was measured. Percent inhibition was calculated from the values of percent responses of drug-treated and control 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
d
for further details. For details, see the Experimental Section. e Analyses for C, H, and N were within (0.4% of the expected value for
the formula. f NT, not tested.
of the 3-methyl group of 16e with a chloro group (16h )
decreased the affinity in human A-431 cells selectively,
while amino (15k ) and carboxylic acid (15n ) resulted
in loss of affinity in both systems. A 3-methoxy (16g),
cyano (16i), nitro (16j), or ethoxycarbonyl (15o) group
retained activities. On the other hand, 3-methoxyac-
etamide (15l) and 3-acetyl (16m ) derivatives were twice
as potent as 16a in human A-431 cells. Furthermore,
3-carbamoyl derivatives (16p ,q) afforded a 2-fold and 1
order of magnitude increase in B2 binding affinities in
guinea pigs and human A-431 cells, respectively. These
results suggest that the carbonyl oxygen of the carbam-
oyl, amide, and ketone groups may have electrostatic
interaction with the B2 receptor. However, these com-
pounds failed to display significant in vivo antagonistic
activities at 1 mg/kg by oral administration.
orders of magnitude, while retaining the affinities in
guinea pig ileum. Furthermore, these compounds also
significantly inhibited BK-induced bronchoconstriction
at 1 mg/kg by oral administration. In particular,
compound 18c was quite potent even at the lower dose,
0.32 mg/kg, with an ED50 value of 0.17 mg/kg po. In
this series, an acetamide group afforded the same
degree of increase in human A-431 cell B2 affinity,
resulting in 18d . N-Methylation of the acetamide group
of 18d gave 18e which was 10- and 2-fold less potent in
human A-431 cells and guinea pigs, respectively. Al-
though 18d failed to show significant in vivo antago-
nistic activity at 1 mg/kg po, its N-methyl derivative,
18e, exhibited 84.3% inhibition at the same oral dose.
Cyclization of the N-methylamide group to the pyrroli-
done moiety (18f) regained the binding activity in both
systems and preserved the potent in vivo activity. In
addition, not only the carbamoyl (18b,c) and the amide
(18d ,f) but also the urea (18g) and the sulfonamide
(18h ) derivatives displayed potent binding activities in
human A-431 cells, whereas they exhibited only weak
in vivo activity. These results suggest that the oxygen
atom at the terminal carbonyl or the sulfonyl group of
the cinnamamide series may interact with the binding
site of the B2 receptor in human A-431 cells more
efficiently, maybe as a hydrogen bond acceptor, than
that of the phenylurea derivatives. Further investiga-
With these results in mind, we investigated substitu-
tions with carbamoyl, amide, and related functional
groups at the 4-position of the phenyl ring of the
cinnamamide 18a , which might correspond to the
3-position of the phenylurea with respect to the topology
(Table 3). Later, direct comparison of 3- and 4-(meth-
ylcarbamoyl)cinnamamide derivatives was carried out
in another series of B2 antagonists. These results which
support our hypothesis will be reported in due course.24
Introduction of the N-methylcarbamoyl (18b) and the
N,N-dimethylcarbamoyl (18c) groups increased B2 bind-
ing affinities in human A-431 cells by approximately 2

Novel Non-Peptide BK B2 Receptor Antagonists. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4057
Ta ble 2. Introduction of Substituents to the Phenylurea Moiety
in vitro IC50 (nM)
in vivo % inhibnc
synth
guinea pig
ileuma
human
compd
R
n
A-431 cellb
1 mg/kg, po
methodd
mp (°C)
formulae
16a
15d
16e
16f
16g
16h
16i
16j
15k
15l
16m
15n
15o
16p
16q
H
o-Me
1
0
1
1
1
1
1
1
0
0
1
0
0
1
1
1.4
83
2.7
12
2.3
3.8
1.4
4.2
15
2.7
0.79
63
150
150
140
260
200
1900
190
180
>1000
75
30.7 ( 11.1
NTf
D
A
D
D
D
D
D
D
Exh
C
D
E
A
D
D
amorphous
amorphous
amorphous
amorphous
amorphous
amorphous
amorphous
amorphous
amorphous
238-239
C25H22BrCl2N5O3‚HCl
C26H24BrCl2N5O3
C26H24BrCl2N5O3‚HCl
C26H24BrCl2N5O3‚HCl
m-Me
p-Me
m-OMe
m-Cl
m-CN
m-NO2
m-NH2
m-NHCOCH2OMe
m-Ac
m-COOH
27.4 ( 14.5g
35.0 ( 18.6g
0 ( 9.0
C
C
C
26H24BrCl2N5O4‚HCl
25H21BrCl3N5O3‚HCl
26H21BrCl2N6O3‚HCl
14.5 ( 8.4
4.7 ( 13.5
13.6 ( 15.7
NT
NT
24.9 ( 6.3*
NT
NT
23.2 ( 13.0
16.1 ( 6.9
C25H21BrCl2N6O5‚HCl
C25H23BrCl2N6O3
C28H27BrCl2N6O5
C27H24BrCl2N5O4‚HCl
C26H22BrCl2N5O5
C28H26BrCl2N5O5
C28H27BrCl2N6O4‚HCl
C29H29BrCl2N6O5‚HCl
61
amorphous
248-250
5200
150
20
m-COOEt
m-CONMe2
m-CONH(CH2)2OMe
2.2
0.76
0.79
amorphous
amorphous
amorphous
20
g
h
a-f See corresponding footnotes in Table 1. Percent inhibition at a doses of 3.2 mg/kg. Ex, experimental procedure described.
Ta ble 3. Introduction of Substituents to the Cinnamamide Moiety
in vitro IC50 (nM)
in vivo % inhibn (po)c
guinea pig
ileuma
human
synth
compd
R
X
n
A-431 cellb 0.32 mg/kg
1 mg/kg
methodd
mp (°C)
formulae
18a
18b
18c
18d
18e
18f
H
CH
CH
CH
CH
CH
CH
1
1
1
1
1
1
0.78
0.50
0.66
0.55
1.1
120
1.5
1.4
1.6
16
4.1
NTf
NT
0 ( 19.2
D
D
D
D
D
D
amorphous C27H23BrCl2N4O3‚HCl
CONHMe
CONMe2
NHAc
53.2 ( 3.6***
amorphous
143-146
C29H26BrCl2N5O4‚HCl
74.8 ( 2.9* 86.7 ( 4.9**
NT
NT
20.7 ( 2.0
C30H28BrCl2N5O4‚HCl‚2H2O
23.1 ( 10.1
84.3 ( 6.1**
82.0 ( 6.1***
amorphous
amorphous
C
C
29H26BrCl2N5O4‚HCl
30H28BrCl2N5O4‚HCl
NMeAc
0.59
amorphous C31H28BrCl2N5O4‚HCl
18g
18h
18k
18l
NHCONHMe CH
1
1
2
2
2
0.89
3.9
0.36
0.44
0.38
2.6
4.8
9.1
3.7
2.4
NT
NT
NT
NT
18.8 ( 5.7*
0 ( 16.1
D
D
D
D
D
amorphous
amorphous C28H26BrCl2N5O5S‚HCl
amorphous 28H25BrCl2N6O4‚2HCl
amorphous C29H27BrCl2N6O4‚2HCl
amorphous 28H25BrCl2N6O4‚2HCl
C29H27BrCl2N6O4‚HCl
NHSO2Me
CONHMe
CONMe2
CH
N
N
89.4 ( 4.1**
92.4 ( 5.3**
C
18m NHAc
N
74.6 ( 8.4** 76.4 ( 9.9*
C
a-f See corresponding footnotes in Table 1.
tions on this key pharmacophore will be discussed in
due course.24
able improvement in in vivo activity compared to the
corresponding cinnamamides 18b,d . Thus, we have
successfully overcome the species difference in affinity
between BK B2 receptors in guinea pigs and humans
by extending the basic framework leading to cinnama-
mide derivatives incorporating a novel pharmacophore.
On the basis of the most potent binding affinity for
human B2 receptors and the excellent in vivo antago-
Next we investigated replacement of the terminal
phenyl ring with a 3-pyridyl group, whose nitrogen atom
might correspond to the 3-substituent of the phenylurea
in terms of topology. This modification caused a slight
loss of binding affinities in human A-431 cells. How-
ever, it is noteworthy that 18k ,m displayed a remark-

4058 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Abe et al.
Hz), 8.02 (2H, d, J ) 9 Hz), 8.67 (1H, m), 10.06 (1H, s). Anal.
(C9H9NO2) C, H, N.
Compound 7b was prepared following a procedure similar
to the preparation of 7a .
nistic activities, compound 18c has been identified as a
candidate for clinical development. Further pharma-
cological characterization of 18c revealed its potent,
selective,21a,b and insurmountable antagonistic activity
for cloned human B2 receptors.21b
(E)-3-[6-(Eth oxycar bon yl)pyr idin -3-yl]acr ylic Acid (6c).
A mixture of 7c25 (2.14 g, 13.5 mmol) and malonic acid (1.54
g, 14.8 mmol) in dry pyridine (1.1 mL) and EtOH (3.2 mL)
was refluxed for 2 h. After the mixture was cooled, the solid
that precipitated was collected by vacuum filtration, washed
with AcOEt, and dried. The solid was crystallized from MeCN
to give 6c (2.04 g, 68.7%) as colorless crystals: mp 202-204
Con clu sion
We found that our first orally active non-peptide BK
B2 antagonists 1-3 were much less potent in the human
A-431 cell system than in guinea pigs. To overcome this
species difference, we investigated the SAR of the
terminal substituents on the glycine moiety. Intensive
chemical modifications revealed that the (E)-cinnamoyl
moiety with the carbamoyl or the amide groups at the
4-position or the (E)-3-(pyridin-3-yl)acryloyl moiety with
the carbamoyl or the amide groups at the 6-position of
the pyridine ring significantly enhanced the binding
affinity in human assay systems, leading to the discov-
ery of the first clinical candidate 18c. Since 18c is
selective, highly potent, and orally active in various
inflammatory models,21c it is expected to be the first of
a new class of drugs for the treatment of various
inflammatory diseases.
1
°C; H NMR (200 MHz, DMSO-d6) δ 1.33 (3H, t, J ) 7 Hz),
4.36 (2H, q, J ) 7 Hz), 6.80 (1H, d, J ) 16 Hz), 7.69 (1H, d, J
) 16 Hz), 8.07 (1H, d, J ) 9 Hz), 8.33 (1H, dd, J ) 9, 2 Hz),
9.00 (1H, d, J ) 2 Hz). Anal. (C11H11NO4) C, H, N.
Compounds 6a ,b were prepared following a procedure
similar to the preparation of 6c.
Eth yl (E)-4-Aceta m id ocin n a m a te (9a ). A mixture of 8
(2.00 g, 10.5 mmol) and acetic anhydride (1.28 g, 12.6 mmol)
in dry CH2Cl2 (20 mL) was stirred for 1 h. The reaction
mixture was evaporated in vacuo. The residue was partitioned
with CH2Cl2 and saturated aqueous sodium bicarbonate solu-
tion. The organic layer was washed with water and brine,
dried, and evaporated in vacuo. The residue was then crystal-
lized from isopropyl ether to give 9a (2.35 g, 96.3%) as colorless
1
crystals: mp 134-136 °C; H NMR (200 MHz, CDCl3) δ 1.32
(3H, t, J ) 7 Hz), 2.20 (3H, s), 4.26 (2H, q, J ) 7 Hz), 6.37
(1H, d, J ) 16 Hz), 7.36 (1H, br s), 7.49 (2H, d, J ) 8 Hz), 7.55
(2H, d, J ) 8 Hz), 7.63 (1H, d, J ) 16 Hz). Anal. (C13H15NO3)
C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Mel-
Temp (Mitamura Riken Kogyo, J apan) and are uncorrected.
The 200-MHz proton NMR spectra were recorded on a Brucker
AM200 or Varian Gemini 300 spectrometer, and shifts are
expressed in δ (ppm) with TMS as internal standard. Mass
spectra were recorded on a VG (Fisons) ZAB-SE (FAB) or
Micromass Platform (ESI). IR spectra were taken with a
Perkin-Elmer FTIR 1600 spectrometer in Nujol or KBr and
Eth yl (E)-4-(N-Meth yla ceta m id o)cin n a m a te (9b). To a
solution of 9a (1.2 g, 5.15 mmol) in dry DMF (12 mL) was
added portionwise 60% sodium hydride in oil (226 mg, 5.66
mmol) in an ice-water bath under nitrogen. After 30 min,
methyl iodide (803 mg, 5.66 mmol) was added therein, and
the reaction mixture was stirred at ambient temperature for
30 min. The mixture was poured into water and extracted
with AcOEt twice. The organic layer was washed with water
and brine, dried, and evaporated in vacuo. The residue was
recrystallized from isopropyl ether to give 9b (979 mg, 76.9%)
as colorless crystals: mp 74-75 °C; 1H NMR (200 MHz, CDCl3)
δ 1.35 (3H, t, J ) 7 Hz), 1.93 (3H, br s), 3.29 (3H, s), 4.29 (2H,
q, J ) 7 Hz), 6.44 (1H, d, J ) 16 Hz), 7.21 (2H, d, J ) 8 Hz),
are expressed in cm-1
. 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 precoated 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). Extraction solvents were dried over magnesium
sulfate.
Meth yl (E)-4-Ca r boxycin n a m a te (4). To a suspension
of phthalaldehydic acid (1.00 g, 6.66 mmol) in dry THF (15
mL) was added methyl (triphenylphosphoranylidene)acetate
(2.50 g, 7.33 mmol) in an ice-water bath under nitrogen. The
reaction mixture was stirred at the same temperature for 30
min and then stirred at ambient temperature. After 1 h, the
reaction mixture was poured into saturated aqueous sodium
bicarbonate solution and washed with AcOEt twice. The
aqueous layer was adjusted to pH 3 with 1 N HCl. The
precipitated solid was collected by vacuum filtration, washed
with water, and dried in vacuo. The solid was crystallized from
MeCN to give 4 (1.21 g, 88.4%) as colorless crystals: mp 242-
243 °C; 1H NMR (200 MHz, DMSO-d6) δ 3.74 (3H, s), 6.76 (1H,
d, J ) 16 Hz), 7.73 (1H, d, J ) 16 Hz), 7.85 (2H, d, J ) 8 Hz),
7.96 (2H, d, J ) 8 Hz). Anal. (C11H10O4) C, H.
7.58 (2H, d, J ) 8 Hz), 7.69 (1H, d, J ) 16 Hz). Anal. (C14H17
NO3) C, H, N.
-
Eth yl (E)-4-(2-Oxop yr r olid in -1-yl)cin n a m a te (9d ). To
a stirred solution of 9c (420 mg, 1.23 mmol) in DMF (5 mL)
was added potassium carbonate (552 mg, 3.99 mmol) at
ambient temperature, and the resulting mixture was warmed
at 50 °C for 3 h. The mixture was poured into water and
extracted with AcOEt twice. The organic layer was washed
with water and brine, dried, and evaporated in vacuo. The
residue was purified by flash column chromatography (CHCl3)
followed by crystallization from isopropyl ether to give 9d (281
1
mg, 87.8%) as a pale-yellow solid: mp 132-134 °C; H NMR
(200 MHz, CDCl3) δ 1.34 (3H, t, J ) 7.5 Hz), 2.19 (2H, quint,
J ) 7.5 Hz), 2.63 (2H, t, J ) 7.5 Hz), 3.88 (2H, t, J ) 7.5 Hz),
4.26 (2H, q, J ) 7.5 Hz), 6.38 (1H, d, J ) 15 Hz), 7.53 (2H, d,
J ) 8 Hz), 7.64 (1H, d, J ) 15 Hz), 7.68 (2H, d, J ) 8 Hz).
Anal. (C15H17NO3) C, H, N.
(E)-4-(Meth a n esu lfon a m id o)cin n a m ic Acid (10f). To a
solution of 8 (500 mg, 2.62 mmol) and triethylamine (530 mg,
5.24 mmol) in dry CH2Cl2 (5 mL) was added methanesulfonyl
chloride (450 mg, 3.93 mmol) 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 1 h. The reaction mixture was washed with saturated
aqueous sodium bicarbonate, water, and brine, dried, and
evaporated in vacuo. The residue was triturated with isopro-
pyl ether to give a mixture of 9f,g. This mixture was
hydrolyzed following a procedure similar to method E to afford
10f (416 mg, 65.9%) as colorless crystals after crystallization
from AcOEt: mp 247-249 °C; 1H NMR (200 MHz, DMSO-d6)
δ 3.05 (3H, s), 6.44 (1H, d, J ) 15 Hz), 7.21 (2H, d, J ) 8 Hz),
4-(N-Meth ylca r ba m oyl)ben za ld eh yd e (7a ). To a sus-
pension of terephthalaldehydic acid (3.00 g, 20.0 mmol) in dry
CH2Cl2 (6 mL) were added thionyl chloride (3.63 g, 24 mmol)
and DMF (0.5 mL) dropwise at ambient temperature under
nitrogen. The reaction mixture was refluxed for 2 h. After
cooling, this mixture was added to 40% methylamine-H2O (6.2
mL, 72 mmol) dropwise over 15 min in an ice-water bath,
and the reaction mixture stirred for 1 h at the same temper-
ature. The solid that precipitated was collected by vacuum
filtration, washed with water and hexane, and dried. The solid
was recrystallized from AcOEt-hexane to give 7a (2.08 g,
1
63.7%) as colorless crystals: mp 160-161 °C; H NMR (200
MHz, DMSO-d6) δ 2.97 (3H, d, J ) 6 Hz), 7.97 (2H, d, J ) 9

Novel Non-Peptide BK B2 Receptor Antagonists. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4059
7.53 (1H, d, J ) 15 Hz), 7.66 (2H, d, J ) 8 Hz). Anal. (C10H11
-
The filtrate was extracted with CH2Cl2 twice, and the organic
layer was washed with saturated sodium bicarbonate, water,
and brine, dried, and evaporated in vacuo. The residue was
purified by flash silica gel chromatography (CH2Cl2-MeOH,
50:1) to give 15k (539 mg, 70.6%) as a colorless amorphous
solid: 1H NMR (200 MHz, CDCl3) δ 2.42 (3H, s), 3.22 (3H, s),
3.56-3.75 (3H, m), 3.82 (1H, dd, J ) 18, 5 Hz), 5.48 (2H, s),
5.92 (1H, br t, J ) 4 Hz), 6.88 (1H, dd, J ) 8, 1 Hz), 6.53 (1H,
br d, J ) 7 Hz), 6.72 (1H, d, J ) 7 Hz), 6.79-6.91 (2H, m),
7.01 (1H, t, J ) 8 Hz), 7.09 (1H, br s), 7.33 (1H, d, J ) 9 Hz),
7.45 (1H, d, J ) 9 Hz), 7.78 (1H, d, J ) 7 Hz); MS (FAB) m/z
607 (M + 1). Anal. (C25H23BrCl2N6O3) C, H, N.
Meth od B. 3-Br om o-8-[[3-[N-(E)-cin n a m a m id oa cetyl-
N-m et h yla m in o]-2,6-d ich lor ob en zyl]oxy]-2-m et h ylim i-
d a zo[1,2-a ]p yr id in e (17a ). To a solution of 12 (160 mg,
0.339 mmol) and triethylamine (51 mg, 0.509 mmol) in dry
CH2Cl2 (1.6 mL) was added (E)-cinnamoyl chloride (62 mg,
0.373 mmol) in an ice-water bath under nitrogen. The
reaction mixture was stirred at the same temperature for 30
min and stirred at ambient temperature for 1 h. The reaction
mixture was washed with water, saturated aqueous sodium
bicarbonate, and brine, dried, and evaporated in vacuo. The
residue was purified by flash silica gel column chromatography
(CH2Cl2-MeOH, 50:1) to give 17a (127 mg, 60.7%) as a
colorless amorphous solid: 1H NMR (200 MHz, CDCl3) δ 2.43
(3H, s), 3.29 (3H, s), 3.69 (1H, dd, J ) 17, 5 Hz), 3.92 (1H, dd,
J ) 17, 5 Hz), 5.50 (2H, s), 6.49 (1H, d, J ) 15 Hz), 6.62 (1H,
br s), 6.72 (1H, d, J ) 7 Hz), 6.86 (1H, t, J ) 7 Hz), 7.30-7.64
(8H, m), 7.78 (1H, d, J ) 7 Hz). Anal. (C27H23BrCl2N4O3) C,
H, N.
NO4S) C, H, N.
(E)-3-[6-(Acetyla m in o)p yr id in -3-yl]a cr ylic Acid (10h ).
To a mixture of 1122 (2.15 g, 10.0 mmol) and tri-n-butylamine
(4.08 g, 22.0 mmol) in dry xylene (10.8 mL) were added
palladium(II) acetate (2.2 mg, 0.01 mmol) and triphenylphos-
phine (26 mg, 0.1 mmol) at ambient temperature under
nitrogen. Acrylic acid (865 mg, 12.0 mmol) was added drop-
wise over 15 min to the refluxed mixture, and the reaction
mixture was heated under reflux for 5 h. Upon cooling, H2O
(20 mL) was added therein, and the mixture was adjusted to
pH 4 with 6 N HCl. After 2 h of stirring, the precipitate was
collected by vacuum filtration, washed with water and isopro-
pyl ether, and dried. The solid was crystallized from AcOEt
to give 10h (1.55 g, 75.2%) as colorless crystals: mp 291-292
°C; 1H NMR (200 MHz, DMSO-d6) δ 2.10 (3H, s), 6.55 (1H, d,
J ) 16 Hz), 7.58 (1H, d, J ) 16 Hz), 8.07-8.21 (2H, m), 8.59
(1H, br s). Anal. (C10H10N2O3) C, H, N.
Meth yl (E)-3-(6-Am in op yr id in -3-yl)a cr yla te (9i). To dry
MeOH (5 mL) was added thionyl chloride (0.41 mL, 5.54 mmol)
dropwise over 5 min in a dry ice-acetone bath under nitrogen.
10i (700 mg, 4.26 mmol) was added therein, and the mixture
was refluxed for 2 h. After cooling, the reaction mixture was
concentrated in vacuo. To the residue was added water; the
mixture was adjusted to pH 8 with saturated aqueous sodium
bicarbonate and extracted with CH2Cl2. The organic layer was
dried and evaporated in vacuo. The residue was crystallized
from isopropyl ether to give 9i (725 mg, 95.4%) as colorless
crystals: mp 173-175 °C; 1H NMR (300 MHz, DMSO-d6) δ
3.67 (3H, s), 6.32 (1H, d, J ) 16 Hz), 6.45 (1H, d, J ) 8 Hz),
6.57 (2H, s), 7.51 (1H, d, J ) 16 Hz), 7.79 (1H, dd, J ) 8, 2
Hz), 8.15 (1H, d, J ) 2 Hz); MS (FAB) m/z 179 (M + 1). Anal.
(C9H10N2O2) C, H, N.
Compounds 9j and 13b-d ,f were prepared following a
procedure similar to method B.
Meth od C. 3-Br om o-8-[[2,6-d ich lor o-3-[N-[(E)-4-(N,N-
d im eth ylca r ba m oyl)cin n a m a m id oa cetyl]-N-m eth yla m i-
n o]ben zyl]oxy]-2-m eth ylim id a zo[1,2-a ]p yr id in e (17c). To
a solution of 12 (350 mg, 0.741 mmol), 6b (179 mg, 0.815
mmol), and 1-hydroxybenzotriazole (HOBt; 150 mg, 1.11 mmol)
in dry DMF (3.5 mL) was added 1-ethoxy-3-[3-(dimethylami-
no)propyl]carbodiimide hydrochloride (WSCD‚HCl; 164 mg,
0.852 mmol) portionwise at ambient temperature under
nitrogen. After 2 h of stirring, this mixture was partitioned
with AcOEt and saturated aqueous sodium bicarbonate solu-
tion. The organic layer was washed with water three times
and brine, dried, and evaporated in vacuo. The residue was
purified by flash silica gel column chromatography (AcOEt-
MeOH, 20:1) followed by trituration with isopropyl ether to
give 17c (480 mg, 96.2%) as a colorless amorphous solid, which
was crystallized from MeOH to afford colorless crystals (465
mg, 93.2%): mp 142-144 °C; 1H NMR (300 MHz, CDCl3) δ
2.45 (3H, s), 2.99 (3H, br s), 3.10 (3H, br s), 3.29 (3H, s), 3.69
(1H, dd, J ) 17, 4 Hz), 3.91 (1H, dd, J ) 17, 5 Hz), 5.47 (1H,
d, J ) 10 Hz), 5.52 (1H, d, J ) 10 Hz), 6.50 (1H, d, J ) 15 Hz),
6.65 (1H, br t, J ) 4 Hz), 6.71 (1H, d, J ) 7 Hz), 6.86 (1H, t,
J ) 7 Hz), 7.29-7.62 (7H, m), 7.78 (1H, d, J ) 7 Hz); MS (FAB)
m/z 674 (M + 1); IR (KBr) 3491, 3275, 3054, 2931, 1675, 1625,
1549, 1513. Anal. (C30H28BrCl2N5O4‚2H2O) C, H, N.
Meth yl (E)-3-[6-(2-Oxopyr r olidin -1-yl)pyr idin -3-yl]acr yl-
a te (9k ). To a solution of 9j (270 mg, 0.825 mmol) in dry DMF
(3 mL) was added 60% sodium hydride in oil (35 mg, 0.866
mmol) portionwise in an ice-water bath under nitrogen. The
mixture was stirred at the same temperature for 30 min and
then stirred at ambient temperature for 1 h. The mixture was
poured into water and extracted with AcOEt twice. The
extracts were washed with water twice and brine, dried, and
evaporated in vacuo. The residue was crystallized from
isopropyl ether to give 9k (190 mg, 93.6%) as colorless
1
crystals: mp 153-156 °C; H NMR (300 MHz, CDCl3) δ 2.16
(2H, quint, J ) 7.5 Hz), 2.69 (2H, t, J ) 7.5 Hz), 3.81 (3H, s),
4.11 (2H, t, J ) 7.5 Hz), 6.43 (1H, d, J ) 16 Hz), 7.65 (1H, d,
J ) 16 Hz), 7.87 (1H, dd, J ) 8, 2 Hz), 8.44-8.50 (2H, m); MS
(FAB) m/z 247 (M + 1). Anal. (C13H14N2O3) C, H, N.
Meth od A. 3-Br om o-8-[[2,6-d ich lor o-3-[N-m eth yl-N-
[(N′-ph en ylu r eido)acetyl]am in o]ben zyl]oxy]-2-m eth ylim -
id a zo[1,2-a ]p yr id in e (15a ). To a solution of 1220 (100 mg,
0.212 mmol) in dry CH2Cl2 (2 mL) was added phenyl isocyan-
ate (30 mg, 0.254 mmol) at ambient temperature under
nitrogen. The reaction mixture was stirred for 1 h and
evaporated in vacuo. The residue was purified by flash silica
gel column chromatography (CH2Cl2-MeOH, 50:1) followed
by crystallization from AcOEt to give 15a (74 mg, 60.3%) as
colorless crystals: mp 144-147 °C; 1H NMR (200 MHz, CDCl3)
δ 2.42 (3H, s), 3.22 (3H, s), 3.70 (1H, dd, J ) 17, 4 Hz), 3.86
(1H, dd, J ) 17, 4 Hz), 5.49 (2H, s), 5.79 (1H, br t, J ) 5 Hz),
6.71 (1H, d, J ) 7 Hz), 6.86 (1H, t, J ) 7 Hz), 7.02 (1H, m),
7.19-7.30 (5H, m), 7.32 (1H, d, J ) 9 Hz), 7.42 (1H, d, J ) 9
Hz), 7.78 (1H, d, J ) 7 Hz). Anal. (C25H22BrCl2N5O3) C, H,
N.
Compounds 9c, 13e, 15l,q, and 17b,d -i,m were prepared
following a procedure similar to method C.
Meth od D. 3-Br om o-8-[[2,6-d ich lor o-3-[N-[(E)-4-(N,N-
d im eth ylca r ba m oyl)cin n a m a m id oa cetyl]-N-m eth yla m i-
n o]ben zyl]oxy]-2-m eth ylim id a zo[1,2-a ]p yr id in e Hyd r o-
ch lor id e (18c). To a suspension of 17c (400 mg, 0.594 mmol)
in MeOH (4 mL) was added 10% hydrogen chloride in MeOH
(0.87 mL) at ambient temperature. After 10 min of stirring,
the solution was evaporated in vacuo. The residue was
crystallized from AcOEt, and the product was recrystallized
from MeCN to give 18c (422 mg, 79.5%) as colorless crystals:
Compounds 9e and 15b-j,m ,o were prepared following a
procedure similar to method A.
8-[[3-[N-[N′-(3-Am in op h en yl)u r eid oa cet yl]-N-m et h yl-
amino]-2,6-dichlorobenzyl]oxy]-3-bromo-2-methylimidazo[1,2-a]-
p yr id in e (15k ). To a suspension of 16j (800 mg, 1.26 mmol)
in EtOH (8 mL) was added tin(II) chloride (954 mg, 5.03 mmol)
at ambient temperature. The reaction mixture was refluxed
for 2 h. After cooling, the mixture was adjusted to pH 10 with
1 N NaOH solution. To this mixture was added CH2Cl2 (10
mL), and the precipitate was removed by filtration with Celite.
1
mp 143-146 °C; H NMR (300 MHz, CDCl3-CD3OD) δ 2.60
(3H, s), 3.02 (3H, br s), 3.12 (3H, br s), 3.30 (3H, s), 3.81 (2H,
s), 5.66 (1H, d, J ) 10 Hz), 5.77 (1H, d, J ) 10 Hz), 6.68 (1H,
d, J ) 15 Hz), 7.37-7.70 (9H, m), 8.21 (1H, dd, J ) 5, 2 Hz);
MS (FAB) m/z 674 (M + 1); IR (KBr) 3411, 3246, 3051, 2930,
1673, 1657, 1624, 1566, 1523. Anal. (C30H28BrCl2N5O4‚HCl‚
2H2O) C, H, N.

4060 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21
Abe et al.
Compounds 14c, 16a ,c,e-j,m ,p ,q, and 18a -h ,k -m were
2. Hu m a n A-431 Cells. Human A-431 cells, human
epidermoid carcinoma (obtained from the American Type
Culture Collection), were maintained in Dulbecco’s modified
Eagle’s minimum essential medium (DMEM) supplemented
with penicillin (100 µg/mL), streptomycin (100 µg/mL), and
10% fetal bovine serum. The cells were seeded in 48-well
tissue culture plates at a density of 1.5 × 105 cells/well and
cultured for 1 day. The cells were washed twice with phosphate-
buffered saline containing 0.1% BSA and incubated with 1.2
nM [3H]BK and test compounds for 3 h at 4 °C in 0.3 mL of
binding buffer II (120 mM N-methyl-D-glucamine, 2.68 mM
KCl, 9.79 mM Na2HPO4, 1.47 mM KH2PO4, 0.544 mM CaCl2,
0.295 mM MgCl2, 3.08 mM NaN3, 2 mM bacitracin, 1 mM
dithiothreitol, 1 µM captopril, and 0.1% BSA, pH 7.4). All
tested compounds (2-3 mg) were dissolved in DMSO (about
0.3 mL) and then diluted with DMSO and the assay buffer
(final concentration of DMSO was below 3%). All experiments
were carried out three times in duplicate. At the end of the
incubation, the buffer was aspirated, and the cells were
washed three times with ice-cooled phosphate-buffered saline
containing 0.1% BSA. The cells were solubilized with 1%
sodium dodecyl sulfate (SDS) and 0.1 N NaOH, and radioac-
tivity was determined by a liquid scintillation counter. The
specific binding was calculated by subtracting the nonspecific
binding, determined in the presence of 1 µM unlabeled BK,
from the total binding.
BK-In du 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), and
the trachea, jugular vein, and esophagus 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 Ro¨ssler method27 as
the peak increase of pulmonary insufflation pressure (PIP).28
Each dose of the compound suspended in 0.5% methylcellulose
solution (5 mL/kg) or vehicle was administered through the
esophageal cannula after the first BK-induced bronchocon-
striction. After 30 min, BK was administered again and the
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 calculated from the following formula:
% response ) (∆PIPafter drug/∆PIPbefore drug) × 100. Percent
response obtained from the vehicle-administered animals was
regarded as the control. Three or four animals were used in
each dose. The potency of the drug was expressed as percent
inhibition which was calculated from the values of percent
responses of drug-treated and control groups as follows: %
prepared following a procedure similar to method D.
Meth od E. 3-Br om o-8-[[3-[N-[(E)-3-(6-ca r boxyp yr id in -
3-yl)a cr yloylglycyl]-N-m eth yla m in o]-2,6-d ich lor oben zyl]-
oxy]-2-m eth ylim id a zo[1,2-a ]p yr id in e (17j). A solution of
17i (522 mg, 0.773 mmol) in EtOH (4.2 mL) containing 1 N
NaOH (0.85 mL) was heated at 60 °C for 1 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, washed with water, and dried. The solid
was crystallized from MeCN to give 17i (403 mg, 80.6%) as
colorless crystals: mp 226-228 °C; 1H NMR (200 MHz, DMSO-
d6) δ 2.30 (3H, s), 3.16 (3H, s), 3.45-3.62 (1H, overlapped with
H2O), 3.82 (1H, dd, J ) 18, 5 Hz), 5.49 (2H, s), 6.93-7.12 (3H,
m), 7.51 (1H, d, J ) 16 Hz), 7.79 (1H, d, J ) 9 Hz), 7.85 (1H,
d, J ) 9 Hz), 7.93 (1H, dd, J ) 5, 3 Hz), 8.01-8.20 (2H, m),
8.44 (1H, br t, J ) 5 Hz), 8.89 (1H, br s); MS (FAB) m/z 648
(M + 1). Anal. (C27H22BrCl2N5O5) C, H, N.
Compounds 10b,d ,e,i,k and 15n were prepared following a
procedure similar to method E.
Meth od F . 3-Br om o-8-[[2,6-d ich lor o-3-[N-m eth yl-N-
[(E)-3-[6-(N-m et h ylca r b a m oyl)p yr id in -3-yl]a cr yloylgly-
cyl]a m in o]ben zyl]oxy]-2-m eth ylim id a zo[1,2-a ]p yr id in e
(17k ). To a solution of 17j (100 mg, 0.154 mmol) in dry DMF
(1 mL) were added methylamine hydrochloride (13 mg, 0.185
mmol), WSCD (34 mg, 0.216 mmol), and HOBt (33 mg, 0.247
mmol) at ambient temperature. After 2 h of stirring, this
mixture was partitioned with AcOEt and saturated aqueous
sodium bicarbonate solution. The organic layer was washed
with water three times and brine, dried, and evaporated in
vacuo. The residue was purified by flash silica gel column
chromatography (CH2Cl2-MeOH, 20:1) to give 17k (77 mg,
75.7%) as a colorless amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 2.45 (3H, s), 3.05 (3H, d, J ) 5 Hz), 3.29 (3H, s), 3.70
(1H, dd, J ) 18, 4 Hz), 3.92 (1H, dd, J ) 18, 5 Hz), 5.49 (1H,
d, J ) 10 Hz), 5.52 (1H, d, J ) 10 Hz), 6.61 (1H, d, J ) 16 Hz),
6.70-6.79 (2H, m), 6.88 (1H, t, J ) 7.5 Hz), 7.34 (1H, d, J )
7.5 Hz), 7.50 (1H, d, J ) 7.5 Hz), 7.61 (1H, d, J ) 16 Hz), 7.78
(1H, d, J ) 7.5 Hz), 7.92-8.02 (2H, m), 8.02 (1H, d, J ) 7.5
Hz), 8.62 (1H, br s); MS (FAB) m/z 661 (M + 1). Anal. (C28H25
BrCl2N6O4) C, H, N.
-
Compounds 5a ,b, 15p , and 17l were prepared following a
procedure similar to method F.
Biologica l Meth od s. Recep tor Bin d in g: 1. 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
described26 with minor modifications. Male Hartley guinea
pigs (from Charles River J apan, Inc.) were killed by exsan-
guination under anesthesia. The ilea were removed and
homogenized in ice-cold buffer (50 mM sodium (trimethylami-
no)ethanesulfonate (TES) and 1 mM 1,10-phenanthroline, pH
6.8) with a Polytron homogenizer. 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 resuspended in ice-cold binding buffer I
(50 mM TES, 1 mM 1,10-phenanthroline, 140 µg/mL bacitra-
cin, 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 [3H]BK and varying
concentrations of test compounds or unlabeled BK at room
temperature for 60 min. All tested compounds (2-3 mg) were
dissolved in DMSO (about 0.3 mL) and then diluted with
DMSO and the assay buffer (final concentration of DMSO was
below 3%). 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-cold buffer (50 mM Tris-HCl). The radioactivity retained
on the washed filter was measured with a liquid scintillation
counter. Specific binding was calculated by subtracting the
nonspecific binding (determined in the presence of 1 µM
unlabeled BK) from total binding. All experiments were
carried out three times in duplicate.
inhibition ) (1 - % responsedrug/% responsemean
100.
of vehicle) ×
value
Sta tistica l An a lysis. Statistical significance was analyzed
with the results of percent inhibition between groups by
Student’s t-test. IC50 or ED50 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) and Dr. N. Maeda
(Department of Pharmacology, Fujisawa Pharmaceuti-
cal Co., Ltd., Tsukuba) for their valuable suggestions.
Su p p or tin g In for m a tion Ava ila ble: Physical Data of
5a ,b, 6a ,b, 7b, 9c,e,j, 10b,d ,e,i,k , 13b-f, 14c, 15b-j,l-q,
16a ,c,e-j,m ,p ,q, 17b,d -i,m , and 18a -h ,k -m (19 pages).
Ordering information is given on any current masthead page.

Novel Non-Peptide BK B2 Receptor Antagonists. 2
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 21 4061
(18) Salvino, J . M.; Seoane, P. R.; Douty, B. D.; Awad, M. M. A.; Dolle,
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Tanaka, H. A Novel Class of Orally Active Non-Peptide Brady-
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