Discovery of the first non-peptide full agonists for the human bradykinin B2 receptor incorporating 4-(2-picolyloxy)quinoline and 1-(2-picolyl)benzimidazole frameworks

Article abstract of DOI:10.1021/jm030468n

In the course of our studies on non-peptide bradykinin (BK) B₂ receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B₂ rec

Full text of DOI:10.1021/jm030468n

J . Med. Chem. 2004, 47, 2853-2863
2853
Discover y of th e F ir st Non -P ep tid e F u ll Agon ists for th e Hu m a n Br a d yk in in B2
Recep tor In cor p or a tin g 4-(2-P icolyloxy)qu in olin e a n d
1-(2-P icolyl)ben zim id a zole F r a m ew or k s
Yuki Sawada,† Hiroshi Kayakiri,*,‡ Yoshito Abe,† Tsuyoshi Mizutani,† Noriaki Inamura,‡ Masayuki Asano,§
Chie Hatori, Ichiro Aramori,§ Teruo Oku, and Hirokazu Tanaka|
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, J apan
Received September 18, 2003
In the course of our studies on non-peptide bradykinin (BK) B2 receptor ligands, it was suggested
that the 4-substituent of the quinoline ring may play a critical role in determining binding
affinities for human and guinea pig B2 receptors, as well as agonist/antagonist properties. We
carried out an extensive investigation to elucidate the structure-activity relationships (SAR)
for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the
quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist.
This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster
ovary cells expressing the cloned human B2 receptor at concentrations greater than 10 nM
and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective
for the B2 receptor and was inhibited by selective peptide and non-peptide B2 antagonists. On
the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human
B2 receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy
moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-
peptide B2 agonist, 19a . Successive optimization of the acyl side chain afforded 38, which
exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could
be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimid-
azole derivative 47c increased PGE2 production at a 1 µM concentration to the same level as
the maximum effect of BK. Thus, we have established the medicinal chemistry modifications
required to convert our highly potent non-peptide B2 antagonists to agonists with potent efficacy.
In tr od u ction
by B2 receptors. It is well accepted that the antihyper-
tensive effect of angiotensin converting enzyme (ACE)
inhibitors is partly attributed to increased BK levels.7
Additionally, blockade of degradation of BK was re-
ported to protect cardiac tissue against ischemic dam-
age.8 Furthermore, a representative peptide B2 agonist,
RMP-7 (Cereport, [Hyp3, Thi5-4-Me-Tyr8Ψ(CH2NH)]-
BK), was shown to enhance the delivery of antitumor
agent to the brain.9,10 Therefore, the development of
specific B2 receptor agonists and antagonists has been
of great importance for investigating the pathophysio-
logical roles of BK and for developing novel classes of
therapeutic drugs.
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). Kinins are highly
potent agonists of G-protein-coupled cell surface recep-
tors, designated as B2 receptors, which are expressed
constitutively in many tissues and are thought to
mediate most of the biological actions of BK.1,2
BK exhibits highly potent and diverse biological
activities through B2 receptors, such as bronchocon-
striction, vasodilation, plasma extravasation, stimula-
tion of nociceptive neurons, release of various mediators
and cytokines, and growth stimulation of small-cell lung
cancer.3-5 On the basis of these strong proinflammatory
properties, BK is believed to play important roles in a
variety of inflammatory diseases represented by asthma,
rhinitis, brain edema, and hyperalgesia.1,3,6 On the other
hand, certain beneficial effects of BK are also mediated
We earlier reported the first selective and orally active
non-peptide B2 receptor antagonists with low nanomolar
to subnanomolar binding affinities for both the guinea
pig and cloned human B2 receptors (1-4, Chart 1).11-15
Subsequent investigations aimed at development of
novel therapeutic drugs for iv use led us to identify
highly potent antagonists with improved aqueous solu-
bility that significantly inhibited BK-induced broncho-
constriction, even at 1-10 µg/kg by intravenous admin-
istration (5, 6, Chart 1, refs 16 and 17) as well as partial
agonists, represented by 4-(1-piperidino)quinoline de-
rivative 7. These studies suggested the critical role of
the 4-substituent on the quinoline ring to determine
binding affinities for human and guinea pig B2 receptors
and agonist/antagonist properties. In this article, we
disclose results from extensive investigations to eluci-
* To whom correspondence should be addressed. Phone: +81-
6-6390-1247. Fax: +81-6-6304-5385. E-mail: hiroshi_kayakiri@
po.fujisawa.co.jp.
† Present address: Medicinal Chemistry Research Laboratories,
Fujisawa Pharmaceutical Co. Ltd., 5-2-3 Tokodai, Tsukuba, Ibaraki
300-2698, J apan.
‡ Present address: Research Division, Fujisawa Pharmaceutical Co.
Ltd., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, J apan.
§ Present address: Medicinal Biology Research Laboratories, Fuji-
sawa 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/jm030468n CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/16/2004

2854 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Sawada et al.
Ch a r t 1. Representative Fujisawa Non-Peptide B2 Antagonists and Non-Peptide B2 Partial Agonist
Sch em e 1a
Sch em e 2a
a
Reagents: (a) 28% NaOMe, MeOH; (b) corresponding alcohols,
NaH, 1,3-dimethyl-2-imidazolidinone.
date the SAR of this critical pharmacophore, leading to
the discovery of a unique partial agonist as well as a
powerful agonist switch that enabled us to convert our
potent non-peptide B2 antagonists to efficacious, potent
agonists.
a
Reagents: (a) ethyl acetoacetate, AcOH, benzene; (b) biphenyl,
phenyl ether, 235 °C; (c) ethyl bromoacetate, K2CO3, DMF; (d) H2,
Pd/C, EtOH, 1,4-dioxane.
Ch em istr y
The compounds described in this study are shown in
Tables 1-4, and their synthetic methods are outlined
in Schemes 1-6.
ester 18 was hydrolyzed with 1 N NaOH to afford the
carboxylic acid 20, which was condensed with dimeth-
ylamine hydrochloride to furnish the amide 21. The
4-substituted quinoline derivatives 17a -e, 18, and 21
were treated with 10% HCl in MeOH to afford the
corresponding hydrochlorides 22a -e, 23, and 24, re-
spectively (Scheme 3).
Modifications of the terminal cinnamamide moiety of
the 4-(2-pyridinylmethoxy)quinoline derivatives are
shown in Scheme 4. The benzyl alcohol 2513 was treated
with methanesulfonyl chloride followed by coupling with
the quinolinol 9f. Removal of the N-phthaloyl group of
26 with hydrazine monohydrate and coupling with (E)-
4-(substituted)cinnamic acids or (E)-3-[6-(substituted)-
pyridin-3-yl]acrylic acids afforded the corresponding
cinnamamides 28-33, 35, and 38. The amine 33 was
treated with 4-pyridylacetyl chloride to afford the amide
34. The acid 36, which was obtained by saponification
of the ester 35, was condensed with methylamine
hydrochloride to furnish the amide 37.
Preparation of the 4-alkoxy-2-methylquinolinol de-
rivatives 9a -f and 14 are shown in Schemes 1 and 2.
Reaction of 816 with the corresponding alcohols yielded
the 4-alkoxyquinolinols 9a -f (Scheme 1). The crotonate
11, which was prepared by condensation of 2-benzyl-
oxyaniline (10) with ethyl acetoacetate in benzene in
the presence of a catalytic amount of AcOH, was cyclized
in Dowtherm at 230 °C to give 12. The quinoline 12 was
condensed with ethyl bromoacetate to give 13, followed
by catalytic deprotection of the benzyl group to yield the
8-hydroxyquinoline 14 (Scheme 2).
Preparation of the 4-alkoxy-2-methylquinoline deriva-
tives 22a -e, 23, and 24 is shown in Scheme 3. Benzyl
alcohol 1517 was treated with methanesulfonyl chloride
to afford the benzyl chloride 16b. The quinolinols 9a -f
and 14 were coupled with 16a 13 or 16b in the presence
of K2CO3 to give 17a -e, 18, 19a , and 19b. The ethyl

Non-Peptide Full Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2855
Sch em e 3a
a
Reagents: (a) MsCl, Et3N, CH2Cl2; (b) 4-substituted quinolinols (9a -f and 14), K2CO3, DMF; (c) 10% HCl-MeOH; (d) 1 N NaOH,
EtOH; (e) Me2NH‚HCl, WSCD, HOBt, DMF.
Sch em e 4a
Sch em e 5a
a
Reagents: (a) 2-chloroethyl methyl ether, K2CO3, Bu4NI, DMF;
(b) 4 N HCl in EtOAc; (c) H2, Pd/C, EtOH, 1,4-dioxane; (d) NaH,
16b, DMF; (e) C(OMe)4, AcOH.
Sch em e 6a
a
Reagents: (a) MsCl, Et3N, CH2Cl2; (b) 9f, K2CO3, DMF; (c)
N2H4‚H2O, EtOH; (d) (E)-4-(substituted)cinnamic acids or (E)-3-
[6-(substituted)pyridin-3-yl]acrylic acids, WSCD‚HCl, HOBt, DMF;
(e) 4-pyridylacetyl chloride, Et3N, CH2Cl2; (f) 1 N NaOH, EtOH;
(g) MeNH2‚HCl, WSCD, HOBt, DMF.
Alkylation of 39 with 2-chloroethyl methyl ether using
K2CO3 as a base gave 40. After removal of the N-Boc
group of 40, compound 41 was hydrogenated to give the
diamine 42. Alkylation of the phenol 42 with the benzyl
chloride 16b in the presence of NaH and subsequent
cyclization with tetramethyl orthocarbonate gave the
2-methoxybenzimidazole derivative 43b (Scheme 5).
Preparation of the 3-substituted-2-methoxybenzim-
idazole derivatives 47a ,b and 47c are shown in Scheme
6. Alkylation of 2,3-diaminophenol (44) with the benzyl
chloride 16b in the presence of NaH and subsequent
cyclization with tetramethyl orthocarbonate gave the
2-methoxybenzimidazole derivative 46. Alkylation of 46
a
Reagents: (a) NaH, 16b, DMF; (b) C(OMe)4, AcOH; (c) alkyl
halide, K2CO3, DMF.
with the appropriate alkyl bromide or alkyl chloride
gave the 3-substituted 2-methoxybenzimidazole deriva-
tives 47a ,b or 47c, respectively.
Biology
All compounds were tested for inhibition of the
specific binding of [3H]BK to B2 receptors in guinea
pig ileum membrane preparations, as previously re-
ported,11,12,18-20 and they were also evaluated for inhibi-

2856 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Sawada et al.
Ta ble 1. Binding of 4-Alkoxy Derivatives to Guinea Pig and
Cloned Human B2 Receptors
IC50 (nM)
b
compd
X
GP ileuma
cloned human B2
3
H
OMe
OEt
O-n-propyl
O-isopropyl
O-cyclopentyl
0.51c
0.23
0.19
1.2
1.2
25
1.1c
8.1
34
77
13
F igu r e 1. Agonist activity of the compound 22b.
22a
22b
22c
22d
22e
61
a
Concentration required to inhibit specific binding of [3H]BK
(0.06 nM) to the B2 receptor in guinea pig ileum membrane
preparations by 50%. Values are expressed as the average of at
least three determinations, with variation in individual values of
b
<15%. See Experimental Section for further details. Concentra-
tion required to inhibit specific binding of [3H]BK (1.0 nM) to the
human B2 receptor that was expressed in CHO cells by 50%.
Values are expressed as the average of at least three determina-
tions, with variation in individual values of <15%. See Experi-
mental Section for further details. c Previously published (see
ref 13).
tion of the specific binding of [3H]BK to cloned human
B2 receptors expressed in Chinese hamster ovary (CHO)
cells.19 Furthermore, some compounds were examined
for their B2 agonistic activity by measuring both agonist-
induced inositol phosphates (IPs) formation in CHO cells
expressing human B2 receptors and agonist-induced
PGE2 production in human fibroblasts, WI-38 cells.
F igu r e 2. Effect of bradykinin antagonists on compound 22b
induced inositol phosphate formation.
the range from 100 nM to 10 µM, enhancing IPs
formation twice over the basal level. The intrinsic
activity was about one-tenth of the maximum effect
elicited by BK. 22b showed no stimulatory effect on IPs
formation in CHO cells expressing the cloned human
vasopressin V2 receptor instead of the B2 receptor
(Figure 1). In addition, the enhancement of IPs forma-
tion by 100 nM 22b was significantly inhibited by the
same concentrations of the representative second-
generation peptide B2 antagonist 48 (HOE140; icati-
bant; [D-Arg0,Hyp3,Thi,5D-Tic7,Oic8]BK) or our non-
peptide B2 antagonist 3 (Figure 2). These results clearly
indicate that the effect of 22b on IPs formation is
mediated by the human B2 receptor. On the other hand,
22b inhibited the IPs formation elicited by 1-100 nM
of BK (Figure 3). It is noteworthy that the maximum
effect of BK was remarkably decreased by 22b. Prelimi-
nary studies revealed that the agonistic activity of 22b
partly remained even after removal of the ligand by
washing the cells, suggesting a tight binding profile of
22b to the cloned human B2 receptor (data not shown).
This property might contribute to the strong inhibitory
activity against a high concentration of BK, although
other possibilities, such as desensitization of the recep-
tor, cannot be excluded at this stage.
Resu lts a n d Discu ssion
Introduction of nitrogen-containing heteroaromatic
moieties and aliphatic amines at the 4-position of the
quinoline ring enabled us to identify highly potent B2
antagonists with improved aqueous solubility for iv use
as well as the first partial agonists. These results
prompted us to carry out a more extensive investigation
to elucidate the SAR for this key pharmacophore to
determine binding profiles and agonist/antagonist prop-
erties.
At first, we introduced several alkoxy groups to the
4-position of the quinoline ring of 3. As shown in Table
1, these 4-alkoxyquinoline derivatives showed remark-
ably higher affinities for the guinea pig B2 receptor than
for the cloned human B2 receptor. It is interesting that
this species difference was in marked contrast to that
observed with 4-alkylamino- or 4-nitrogen-containing
heteroaromatic derivatives.17 It seemed that small
4-alkoxy groups are favorable for interaction with
guinea pig B2 receptors, while they are unfavorable for
human B2 receptor.
As the next step, we introduced several functional
groups on the alkoxy moiety, aiming at enhancing the
efficacy of our non-peptide B2 partial agonists (Table 2).
In addition to a direct measurement of the formation of
IPs in CHO cells expressing the human B2 receptor, we
also used a functional assay system to determine the
effect on PGE2 production in human fibroblast WI-38
cells. BK showed the maximum effect on the formation
Detailed pharmacological investigation of the in vitro
activity of 4-ethoxy derivative 22b to cloned human B2
receptors revealed a highly interesting profile. In re-
sponse to this compound, a significant increase in IPs
formation was observed in CHO cells expressing the
cloned human B2 receptor at a concentration of 10 nM
(Figure 1). The maximum stimulation was achieved in

Non-Peptide Full Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2857
Ta ble 2. Binding and B2 Agonistic Activities of 4-Alkoxy Derivatives-1
relative agonistic activity (%)
relative agonistic activity (%)
in PGE2 production
in IPs formation
IC50 (nM)
compared to BK (10 nM)c
compared to BK (100 nM)d
b
compd
X
n
GP ileuma cloned human B2
0.1 µM
1 µM
10 µM
1 µM
10 µM
23
24
20
19a
OCH2COOEt
OCH2CONMe2
OCH2COOH
OCH2-2Py
1
1
0
0
20
30
50
2.3
2.6
3.7
>1000
0.41
NTe
NTe
NTe
53.7 ( 3.3 66.4 ( 5.0 NTe
NTe
NTe
NTe
30.8 ( 0.9 24.4 ( 2.1 NTe
NTe
NTe
NTe
51.3 ( 2.5 59.5 ( 3.5 56.9 ( 2.3 81.2 ( 3.4
140 ( 11.3
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to the B2 receptor in guinea pig ileum membrane preparations
by 50%. Values are expressed as the average of at least three determinations, with variation in individual values of <15%. See Experimental
b
Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.0 nM) to the human B2 receptor that was
expressed in CHO cells by 50%. Values are expressed as the average of at least three determinations, with variation in individual values
of <15%. See Experimental Section for further details. c IPs production was measured essentially as described previously.19 See
d
Experimental Section for further details. Human fibroblasts (WI-38 cell) were used. After incubation, the supernatant was collected
and the PGE2 level included therein was measured by a PGE2 EIA kit. See Experimental Section for further details. e NT, not tested.
was the first example to afford full agonist activity for
IPs formation. Thus, we identified the 4-(2-picolyloxy)
moiety on the quinoline ring as a powerful agonist
switch.
Furthermore, we examined the applicability of this
strategy to other frameworks. The 2-methoxybenzim-
idazole series was selected as a model of our non-peptide
B2 ligands, incorporating five to six condensed ring
systems. The representative agonist pharmacophores
and related substituents were introduced at the 1-posi-
tion of the benzimidazole ring. As shown in Table 4, an
ester derivative 47a exhibited about 90% efficacy at
1 µM concentration and full agonist activity at 10 µM
on PGE2 production, while a methoxyethyl derivative
43b also significantly increased PGE2 production from
1 µM with lower efficacy. The 1-benzyl derivative 47b
showed its maximum efficacy, 80% of BK, at 1 µM.
Finally, the 1-(2-picolyl) derivative 47c afforded full
agonist activity on PGE2 production at 1 µM concentra-
tion. These results indicated that this strategy has a
wide range of applicability. Thus, we have established
the medicinal chemistry to convert our highly potent
non-peptide B2 antagonists to agonists with strong
efficacies.
F igu r e 3. Effect of 22b on BK-induced inositol phosphate
formation in CHO cells expressing the human B2 receptor.
of IPs at 10 nM and on the production of PGE2 at 100
nM. Ester derivative 23 stimulated IPs formation more
efficaciously than 22b. It exhibited about a half and two-
thirds efficacy of the maximum effect of BK at concen-
trations of 1 and 10 µM. The dimethylamide derivative
24 also showed significant agonistic activities in the
same concentration range, albeit with lower efficacies,
and the carboxylic acid 20 completely lost binding
affinity for the human B2 receptor. In contrast, intro-
duction of a 2-pyridyl moiety led to the potent agonist
19a , which afforded half the efficacy of BK for IPs
formation at 0.1 µM, as well as full agonist activity for
PGE2 production at 10 µM. Since we disclosed 19a as
the first non-peptide B2 agonist in 1997,21 this compound
has been used worldwide as a probe and has been shown
to selectively mimic BK activity mediated by B2 recep-
tors both in vitro and in vivo. Extensive reports on the
pharmacology of 19a have been published by others and
us.21-31 According to these reports, the binding mode of
19a to the B2 receptor seems to depend on the species,
tissues, and experimental conditions.25
Con clu sion
We carried out extensive investigations to elucidate
the structural features of the key pharmacophore to
determine the species difference in terms of binding
affinities and agonist/antagonist properties. Introduc-
tion of lower alkoxy groups to the 4-position of the
quinoline ring of 3 resulted in increasing affinities for
the guinea pig B2 receptor and decreasing affinities for
human B2 receptors, leading to partial agonists, as
represented by the 4-ethoxy derivative 22b. This com-
pound significantly stimulated IPs formation in CHO
cells expressing cloned human B2 receptors, from a
concentration of 10 nM with about 10% of the intrinsic
activity of BK. Its agonistic activity was selective for
the human B2 receptor and was inhibited by the
Table 3 summarizes the results of optimization stud-
ies on the acyl side chain of 19a . These results indicated
that a variety of side chains could be accommodated in
the agonist binding pocket and that they could also
modify the efficacy. The most efficacious compound, 38,

2858 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Sawada et al.
Ta ble 3. Binding and B2 Agonistic Activities of 4-(2-Picolyloxy)quinolines
relative agonistic activity (%)
in IPs formation compared to BK (10 nM)c
IC50 (nM)
b
compd
X
R
GP ileuma
cloned human B2
0.1 µM
1 µM
19a
28
29
30
31
32
19b
34
37
38
CH
CH
C-OMe
CH
CH
CH
N
N
N
N
CONHMe
CONMe2
CONHMe
CONH-4Py
NHCO-4Py
2-oxopyrrolidin-1-yl
NHAc
NHCOCH2-4Py
CONHMe
2.3
0.41
1.4
51.3 ( 2.5
22.3 ( 1.2
54.9 ( 6.4
36.7 ( 4.1
42.3 ( 2.4
42.9 ( 9.0
43.4 ( 10.1
39.8 ( 2.7
50.5 ( 8.4
81.2 ( 3.1
59.5 ( 3.5
34.3 ( 1.7
73.9 ( 4.4
47.4 ( 7.1
26.9 ( 2.6
67.4 ( 5.4
57.0 ( 11.8
40.3 ( 5.9
57.6 ( 8.5
99.3 ( 1.9
NTd
NTd
NTd
6.1
0.69
0.40
0.56
0.83
1.2
2.4
1.4
0.36
NTd
1.2
NTd
NTd
NTd
HCdCH-4Py (E)
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to the B2 receptor in guinea pig ileum membrane preparations
by 50%. Values are expressed as the average of at least three determinations, with variation in individual values of <15%. See Experimental
b
Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.0 nM) to the human B2 receptor that was
expressed in CHO cells by 50%. Values are expressed as the average of at least three determinations, with variation in individual values
of <15%. See Experimental Section for further details. c IPs production was measured essentially as described previously.19 See
d
Experimental Section for further details. Human fibroblasts (WI-38 cell) were used. After incubation, the supernatant was collected
and the PGE2 level included therein was measured by a PGE2 EIA kit. See Experimental Section for further details.
Ta ble 4. Binding and B2 Agonistic Activities of 1-Substituted Benzimidazoles
relative agonistic activity (%)
IC50 (nM)
cloned human B2
in PGE2 production compared to BK (100 nM)c
b
compd
X
GP ileuma
1 µM
10 µM
43a
43b
47a
47b
47c
Me
0.65d
4.6
4.2
26
6.3
4.2d
3.5
0.91
2.5
4.2 ( 3.5
24.2 ( 4.3
54.8 ( 11.0
121 ( 18.5
73.9 ( 8.5
89.5 ( 15.8
CH2CH2OMe
CH2COOEt
CH2Ph
38.3 ( 13.0
90.0 ( 3.5
84.5 ( 2.3
114 ( 2.4
CH2-2Py
0.68
a
Concentration required to inhibit specific binding of [3H]BK (0.06 nM) to the B2 receptor in guinea pig ileum membrane preparations
by 50%. Values are expressed as the average of at least three determinations, with variation in individual values of <15%. See Experimental
b
Section for further details. Concentration required to inhibit specific binding of [3H]BK (1.0 nM) to the human B2 receptor that was
expressed in CHO cells by 50%. Values are expressed as the average of at least three determinations, with variation in individual values
of <15%. See Experimental Section for further details. c IPs production was measured essentially as described previously.19 See
d
Experimental Section for further details. Previously published (see ref 13).
selective B2 antagonists 48 and 3. On the other hand,
22b inhibited IPs formation elicited by BK through the
human B2 receptor, remarkably suppressing the efficacy
of a high concentration of BK. Further SAR studies on
the key pharmacophore allowed us to identify a 2-pico-
lyloxy moiety as a powerful agonist switch, leading to
the discovery of the potent and efficacious non-peptide
B2 agonist 19a . Successive optimization of the acyl side
chain afforded 38, which exhibited full agonist activity
on the second messenger, IPs formation. Furthermore,
this strategy could also be applied successfully to the
benzimidazole series. The representative 1-(2-picolyl)-
benzimidazole derivative 47c increased PGE2 produc-
tion at 1 µM to the same level as the maximum effect
of BK. Thus, we have established the medicinal chem-
istry to convert our highly potent non-peptide B2
antagonists to agonists with strong efficacies.
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 the

Non-Peptide Full Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2859
internal standard. The peak patterns are shown as the
following abbreviations: s ) singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad. The mass spectra (MS)
were recorded with a VG (Fisons) ZAB-SE (FAB) or Micromass
Platform (ESI) system. Elemental analyses were performed
on a Perkin-Elmer 2400 CHN analyzer. Analytical results were
within (0.4% of the theoretical values unless otherwise noted.
Silica gel thin-layer chromatography was performed on pre-
coated plates, Kieselgel 60F254 (E. Merck, AG, Darmstadt,
Germany). Silica gel flash chromatography was performed
with Kieselgel 60 (230-400 mesh) (E. Merck, AG, Darmstadt,
Germany). Yields were not optimized.
reaction mixture was stirred at ambient temperature for 4 h.
The mixture was then partitioned between CH2Cl2 and water.
The organic layer was washed with water, saturated aqueous
NaHCO3, and brine, dried over MgSO4, and evaporated in
vacuo. The residue was washed with Et2O to afford 13 (1.47
g, 73.9%) as a pale-yellow solid: mp 138-140 °C; 1H NMR (300
MHz, CDCl3) δ 1.31 (t, J ) 7.5 Hz, 3H), 2.74 (s, 3H), 4.31 (q,
J ) 7.5 Hz, 2H), 4.81 (s, 2H), 5.43 (s, 2H), 6.53 (s, 1H), 7.02
(d, J ) 8 Hz, 1H), 7.22-7.40 (m, 4H), 7.51 (d, J ) 8 Hz, 2H),
7.79 (d, J ) 8 Hz, 1H). Anal. (C21H21NO4) C, H, N.
Eth yl [(8-Hyd r oxy-2-m eth yl-4-qu in olin yl)oxy]a ceta te
(14). A mixture of 13 (1.30 g, 3.70 mmol) and 10% palladium
4-Meth oxy-2-m eth yl-8-qu in olin ol (9a ). To a solution of
4-chloro-2-methyl-8-quinolinol (8)16 (49.0 g, 253 mmol) in 1,3-
dimethyl-2-imidazolidinone (DMI, 490 mL) was added a solu-
tion of 28% NaOMe in MeOH (244 mL, 1.265 mol) at ambient
temperature, and the mixture was refluxed for 5 h. The cooled
mixture was adjusted to pH 7 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
resulting residue was crystallized from hexane to afford 9a
(39.0 g, 81.6%) as colorless crystals: mp 111-112 °C; 1H NMR
(200 MHz, CDCl3) δ 2.68 (s, 3H), 4.01 (s, 3H), 6.62 (s, 1H),
7.10 (d, J ) 8 Hz, 1H), 7.30 (dd, J ) 8, 8 Hz, 1H), 7.58 (d, J )
8 Hz, 1H). Anal. (C11H11NO2) C, H, N.
4-Eth oxy-2-m eth yl-8-qu in olin ol (9b). To EtOH (2 mL)
was added 60% NaH in oil (200 mg, 5.0 mmol) portionwise in
an ice/water bath under nitrogen, and the mixture was stirred
at the same temperature for 15 min. 4-Chloro-2-methyl-8-
quinolinol (8) (194 mg, 1.00 mmol) was added, and the solvent
was evaporated in vacuo. DMI (2 mL) was added to the
residue, and then the mixture was stirred at 100 °C for 23 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
resulting residue was crystallized from IPE to afford 9b (142
mg, 69.9%) as colorless crystals: mp 85-86 °C; 1H NMR (300
MHz, CDCl3) δ 1.55 (t, J ) 7.5 Hz, 3H), 2.66 (s, 3H), 4.23 (q,
J ) 7.5 Hz, 2H), 6.61 (s, 1H), 7.10 (d, J ) 8 Hz, 1H), 7.30 (dd,
J ) 8, 8 Hz, 1H), 7.59 (d, J ) 8 Hz, 1H). Anal. (C12H13NO2) C,
H, N.
on carbon (130 mg) in a mixture of EtOH (8 mL) and
1,4-dioxane (7 mL) was hydrogenated at ambient temperature.
After completion, the catalyst was removed by filtration, and
solvent was evaporated in vacuo. The resulting residue was
washed with Et2O to afford 14 (539 mg, 55.8%) as a pale-yellow
1
solid: mp 97-98 °C; H NMR (300 MHz, DMSO-d6) δ 1.23 (t,
J ) 7.5 Hz, 3H), 2.60 (s, 3H), 4.22 (q, J ) 7.5 Hz, 2H), 5.07 (s,
2H), 6.92 (s, 1H), 7.04 (d, J ) 8 Hz, 1H), 7.34 (dd, J ) 8, 8 Hz,
1H), 7.52 (d, J ) 8 Hz, 2H). Anal. (C14H15NO4) C, H, N.
Compound 42 was prepared following the procedure de-
scribed above for 14.
(2E)-3-[6-(Acet yla m in o)-3-p yr id in yl]-N-{2-[3-(ch lor o-
m eth yl)-2,4-dich lor om eth ylan ilin o]-2-oxoeth yl}-2-pr open -
a m id e (16b). To a solution of 15 (5.36 g, 11.9 mmol) and Et3N
(2.40 g, 23.8 mmol) in dry DMF (30 mL) was added methane-
sulfonyl chloride (1.90 g, 16.6 mmol) in an ice/water bath under
nitrogen. After 30 min, the reaction mixture was stirred at
ambient temperature for 18 h. The mixture was partitioned
between CHCl3 and water, and the organic layer was washed
with water and brine, dried over MgSO4, and evaporated in
vacuo. The residue was washed with EtOAc to afford 16b (5.08
g, 91.1%) as a pale-yellow amorphous solid: 1H NMR (300
MHz, DMSO-d6) δ 2.10 (s, 3H), 3.10 (d, J ) 5 Hz, 3H), 3.45
(dd, J ) 17, 3 Hz, 1H), 3.74 (dd, J ) 17, 3 Hz, 1H), 4.96 (s,
2H), 6.76 (d, J ) 15 Hz, 1H), 7.35 (d, J ) 15 Hz, 1H), 7.70-
7.40 (m, 2H), 7.98 (d, J ) 8 Hz, 1H), 8.11 (d, J ) 8 Hz, 1H),
8.23 (t, J ) 5 Hz, 1H), 8.45 (d, J ) 3 Hz, 1H); MS (ESI) m/z
469, 471 (M + 1). Anal. (C20H19Cl3N4O3) C, H, N.
4-((1E)-3-{[2-(2,4-Dich lor o-3-{[(4-m eth oxy-2-m eth yl-8-
qu in olin yl)oxy]m eth yl}m eth ylan ilin o)-2-oxoeth yl]am in o}-
3-oxo-1-p r op en yl)-N-m eth ylben za m id e (17a ). To a mixture
of 16a (80.0 mg, 0.156 mmol) and 9a (31.0 mg, 0.164 mmol)
in dry DMF (1.0 mL) was added K2CO3 (64.6 mg, 0.467 mmol)
at ambient temperature, and the mixture was stirred at the
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 evapo-
rated in vacuo. The residue was purified by preparative thin-
layer chromatography (CHCl3/MeOH, 10:1) to afford 17a (76.0
mg, 78.4%) as a colorless amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 2.67 (s, 3H), 3.00 (d, J ) 5 Hz, 3H), 3.26 (s, 3H), 3.15
(dd, J ) 17, 4 Hz, 1H), 3.92 (dd, J ) 17, 5 Hz, 1H), 4.02 (s,
3H), 5.59 (d, J ) 10 Hz, 1H), 5.63 (d, J ) 10 Hz, 1H), 6.38 (br
d, J ) 5 Hz, 1H), 6.52 (d, J ) 15 Hz, 1H), 6.65 (s, 1H), 6.76 (br
s, 1H), 7.21-7.31 (m, 2H), 7.38 (dd, J ) 8, 8 Hz, 1H), 7.43-
7.61 (m, 4H), 7.75 (d, J ) 8 Hz, 2H), 7.83 (d, J ) 8 Hz, 1H);
MS (FAB) m/z 621.3 (M + 1). Anal. (C32H30Cl2N4O5) C, H, N.
Compounds 9c-f were prepared following the procedure
described above for 9b.
Eth yl (2E)-3-[2-(Ben zyloxy)an ilin o]-2-bu ten oate or Eth -
yl (2Z)-3-[2-(Ben zyloxy)a n ilin o]-2-bu ten oa te (11). A solu-
tion of 2-benzyloxyaniline (10) (15.0 g, 75.3 mmol), ethyl
acetoacetate (10.3 g, 10.1 mL, 79.2 mmol), and AcOH (1.5 mL)
in benzene (45 mL) was refluxed for 6 h, removing water with
a Dean-Stark apparatus. The mixture was then cooled to
room temperature and concentrated in vacuo. The residue was
purified by flash silica gel chromatography (hexane/EtOAc, 5:1)
to afford 11 (20.9 g, 89.3%) as a yellow oil: 1H NMR (300 MHz,
CDCl3) δ 1.28 (t, J ) 7 Hz, 3H), 1.99 (s, 3H), 4.16 (q, J ) 7 Hz,
2H), 4.73 (s, 1H), 5.11 (s, 2H), 6.88-6.99 (m, 2H), 7.03-7.15
(m, 2H), 7.26-7.40 (m, 3H), 7.47 (d, J ) 8 Hz, 2H). Anal.
(C19H21NO3) C, H, N.
8-(Ben zyloxy)-2-m eth yl-4-qu in olin ol (12). To a mixture
of biphenyl (9.6 g) and phenyl ether (22.4 mL) was added
dropwise crotonate 11 (20 g, 64.2 mmol) at 230-235 °C over
15 min. After 1 h of being stirred at 235 °C, the reaction
mixture was cooled at room temperature. The mixture was
diluted with hexane (200 mL), and the precipitate was collected
by vacuum filtration. The residue was purified by flash silica
gel chromatography (CHCl3/MeOH, 10:1) followed by crystal-
lization from CH3CN to afford 12 (4.21 g, 24.8%) as pale-brown
crystals: mp 155-164 °C; 1H NMR (300 MHz, DMSO-d6) δ
2.40 (s, 3H), 5.38 (s, 2H), 5.90 (s, 1H), 7.13 (dd, J ) 8, 8 Hz,
1H), 7.22 (d, J ) 8 Hz, 1H), 7.28-7.43 (m, 3H), 7.53 (d, J ) 8
Hz, 2H), 7.57 (d, J ) 8 Hz, 1H). Anal. (C17H15NO2) C, H, N.
Compounds 17b-e, 18, and 19a ,b were prepared following
the procedure described above for 17a .
[(8-{[2,6-Dich lor o-3-(m eth yl{[((2E)-3-{4-[(m eth ylam in o)-
ca r bon yl]p h en yl}-2-p r op en oyl)a m in o]a cetyl}a m in o)ben -
zyl]oxy}-2-m eth yl-4-qu in olin yl)oxy]a cetic Acid (20). To
a solution of 18 (207 mg, 0.298 mmol) in EtOH (2 mL) was
added 1 N NaOH (0.36 mL) at ambient temperature, and the
mixture was stirred at the same temperature for 2 h. The
reaction mixture was adjusted to pH 4 with 1 N HCl and
diluted with water. The precipitate was collected by vacuum
filtration and washed with water to afford 20 (176 mg, 88.9%)
as a colorless solid: mp 233-257 °C; 1H NMR (300 MHz,
DMSO-d6) δ 2.54 (s, 3H), 2.78 (d, J ) 5 Hz, 3H), 3.17 (s, 3H),
3.51 (dd, J ) 17, 5 Hz, 1H), 3.82 (dd, J ) 17, 5 Hz, 1H), 4.96
(s, 2H), 5.47 (d, J ) 10 Hz, 1H), 5.53 (d, J ) 10 Hz, 1H), 6.89
Eth yl {[8-(Ben zyloxy)-2-m eth yl-4-qu in olin yl]oxy}a ce-
ta te (13). To a mixture of 8-(benzyloxy)-2-methyl-4-quinolinol
(12) (1.50 g, 5.65 mmol) and K2CO3 (860 mg, 6.22 mmol) in
DMF (15 mL) was added ethyl bromoacetate (0.69 mL, 6.22
mmol) in an ice/water bath under nitrogen. After 10 min, the

2860 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Sawada et al.
(d, J ) 15 Hz, 1H), 6.93 (s, 1H), 7.33-7.50 (m, 3H), 7.60-7.70
(m, 2H), 7.73-7.81 (m, 3H), 7.85 (d, J ) 8 Hz, 2H), 8.32 (t,
J ) 4.5 Hz, 1H), 8.49 (q, J ) 4.5 Hz, 1H). Anal. (C33H30Cl2N4-
O7) C, H, N.
Compound 36 was prepared following the procedure de-
scribed above for 20.
Hz, 1H), 6.70 (s, 1H), 7.21-7.32 (m, 2H), 7.37 (t, J ) 8 Hz,
1H), 7.45 (d, J ) 8 Hz, 1H), 7.52 (d, J ) 8 Hz, 1H), 7.57 (d,
J ) 8 Hz, 1H), 7.64-7.80 (m, 3H), 7.80-7.89 (m, 2H), 7.95 (d,
J ) 8 Hz, 1H), 8.64 (d, J ) 6 Hz, 1H); MS (ESI) m/z 641
(M + 1). Anal. (C34H26Cl2N4O5) C, H, N.
2-Am in o-N-[2,4-d ich lor o-3-({[2-m eth yl-4-(2-p yr id in yl-
m eth oxy)-8-qu in olin yl]oxy}m eth yl)p h en yl]-N-m eth yla c-
eta m id e (27). A mixture of 26 (750 mg, 1.20 mmol) and
hydrazine monohydrate (117 mg, 2.34 mmol) in EtOH (7.5 mL)
was heated under reflux for 4 h. The precipitate was removed
by vacuum filtration, and the filtrate was evaporated in vacuo.
The residue was dissolved in a mixture of CHCl3 and MeOH
(10:1), the precipitate was removed by vacuum filtration, and
the filtrate was concentrated in vacuo again. The residue was
purified by flash silica gel column chromatography (CHCl3/
MeOH, 10:1) to afford 27 (390 mg, 65.2%) as a pale-yellow
amorphous solid: 1H NMR (300 MHz, DMSO-d6) δ 2.55 (s, 3H),
2.76 (d, J ) 18 Hz, 1H), 3.01 (d, J ) 18 Hz, 1H), 3.11 (s, 3H),
3.40-3.60 (m, 1H), 4.10 (br peak, 1H), 5.37-5.63 (m, 4H), 7.07
(s, 1H), 7.33-7.53 (m, 3H), 7.62-7.94 (m, 5H), 8.62 (d, J )
6 Hz, 1H); MS (ESI) m/z 511 (M + 1). Anal. (C26H24Cl2N4O3)
C, H, N.
4-[(1E)-3-[[2-[2,4-Dich lor o(m et h yl)-3-({[2-m et h yl-4-(2-
p yr id in ylm eth oxy)-8-qu in olin yl]oxy}m eth yl)a n ilin o]-2-
oxoeth yl]a m in o]-3-oxo-1-p r op en yl]-N,N-d im eth ylben za -
m id e (28). To a solution of 27 (23.0 mg, 0.045 mmol) in DMF
(2 mL) were added (E)-4-(N,N-dimethylcarbamoyl)cinnamic
acid (10.8 mg, 0.049 mmol), WSCD‚HCl (10.3 mg, 0.054 mmol),
and HOBt (9.12 mg, 0.067 mmol) at ambient temperature.
After 3 h, this mixture was partitioned between EtOAc and
water. The organic layer was washed with saturated aqueous
NaHCO3 solution, water (3×), and brine, dried over MgSO4,
and evaporated in vacuo. The residue was purified by prepara-
tive thin-layer chromatography (CHCl3/MeOH, 10:1) to afford
28 (17.0 mg, 53.0%) as a colorless amorphous solid: 1H NMR
(300 MHz, CDCl3) δ 2.64 (s, 3H), 2.98 (br s, 3H), 3.10 (br s,
3H), 3.25 (s, 3H), 3.61 (dd, J ) 18, 4 Hz, 1H), 3.91 (dd, J ) 18,
4 Hz, 1H), 5.40 (s, 2H), 5.64 (s, 2H), 6.51 (d, J ) 16 Hz, 1H),
6.71 (s, 1H), 6.75 (t, J ) 5 Hz, 1H), 7.21-7.35 (m, 3H), 7.35-
7.45 (m, 3H), 7.45-7.64 (m, 5H), 7.76 (dd, J ) 8, 8 Hz, 1H),
7.96 (d, J ) 8 Hz, 1H), 8.65 (d, J ) 6 Hz, 1H); MS (ESI) m/z
712 (M + 1). Anal. (C38H35Cl2N5O5) C, H, N.
4-{(1E)-3-[(2-{2,4-Dich lor o-3-[({4-[2-(d im eth yla m in o)-2-
oxoet h oxy]-2-m et h yl-8-q u in olin yl}oxy)m et h yl]m et h yl-
a n ilin o}-2-oxoet h yl)a m in o]-3-oxo-1-p r op en yl}-N-m et h -
ylben za m id e (21). To a solution of 20 (82.3 mg, 0.124 mmol),
dimethylamine hydrochloride (12.1 mg, 0.148 mmol), and
1-hydroxybenzotriazole (HOBt, 26.7 mg, 0.198 mmol) in dry
DMF (1 mL) was added 1-ethoxy-3-[3-(dimethylamino)propyl]-
carbodiimide (WSCD, 26.9 mg, 0.173 mmol) in an ice/water
bath under nitrogen, and the mixture was stirred at ambient
temperature for 14 h. The reaction mixture was partitioned
between CHCl3 and water. The organic layer was separated,
washed with saturated aqueous NaHCO3, water, and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
purified by preparative thin-layer chromatography (CHCl3/
MeOH, 10:1) to afford 21 (80.5 mg, 94.0%) as a colorless
amorphous solid: 1H NMR (300 MHz, DMSO-d6) δ 2.53 (s, 3H),
2.76 (d, J ) 5 Hz, 3H), 2.86 (s, 3H), 3.04 (s, 3H), 3.15 (s, 3H),
3.50 (dd, J ) 17, 5 Hz, 1H), 3.80 (dd, J ) 17, 5 Hz, 1H), 5.10
(s, 2H), 5.45 (d, J ) 9 Hz, 1H), 5.51 (d, J ) 9 Hz, 1H), 6.87 (d,
J ) 15 Hz, 1H), 6.88 (s, 1H), 7.32-7.48 (m, 3H), 7.61-7.69
(m, 2H), 7.73-7.81 (m, 3H), 7.87 (d, J ) 8 Hz, 2H), 8.33 (t,
J ) 5.5 Hz, 1H), 8.48 (q, J ) 5.5 Hz, 1H). Anal. (C35H35Cl2N5-
O6) C, H, N.
Compound 37 was prepared following the procedure de-
scribed above for 21.
4-((1E)-3-{[2-(2,4-Dich lor o-3-{[(4-m eth oxy-2-m eth yl-8-
qu in olin yl)oxy]m eth yl}m eth ylan ilin o)-2-oxoeth yl]am in o}-
3-oxo-1-p r op en yl)-N-m et h ylb en za m id e H yd r och lor id e
(22a ). To a solution of 17a (70.0 mg, 0.113 mmol) in MeOH (2
mL) was added 10% HCl in MeOH (2 mL) at ambient
temperature. The reaction mixture was stirred at the same
temperature for 10 min. The solution was evaporated in vacuo,
and the residue was washed with EtOAc to afford 22a (73.0
mg, 98.5%) as a pale-yellow amorphous solid: 1H NMR (300
MHz, CDCl3/CD3OD) δ 2.99 (s, 3H), 3.00 (br s, 3H), 3.29 (s,
3H), 3.89 (d, J ) 17 Hz, 1H), 4.10 (d, J ) 17 Hz, 1H), 4.36 (s,
3H), 5.51 (d, J ) 10 Hz, 1H), 5.68 (d, J ) 10 Hz, 1H), 6.63 (d,
J ) 15 Hz, 1H), 7.35-7.43 (m, 2H), 7.48-7.59 (m, 6H), 7.70-
7.81 (m, 4H), 7.95 (d, J ) 8 Hz, 1H). Anal. (C32H30Cl2N4O5‚
HCl) C, H, N.
Compounds 29-33, 35, and 38 were prepared following the
procedure described above for 28.
(2E)-N-{2-[2,4-Dich lor o(m eth yl)-3-({[2-m eth yl-4-(2-p yr -
id in ylm eth oxy)-8-qu in olin yl]oxy}m eth yl)a n ilin o]-2-oxo-
eth yl}-3-{6-[(4-pyr idin ylacetyl)am in o]-3-pyr idin yl}-2-pr o-
p en a m id e (34). To an ice-cooled mixture of 33 (90 mg, 0.14
mmol) and Et3N (856 mg, 0.55 mmol) in CH2Cl2 (2 mL) were
added 4-pyridylacetyl chloride (53 mg, 0.27 mmol) and nitro-
gen, and the mixture was stirred at the same temperature for
1 h and allowed to stand at ambient temperature for 1 day.
The reaction mixture was poured into water and extracted
with CHCl3. The organic layer was 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 give 34 (10 mg, 9%) as an amorphous solid:
1H NMR (300 MHz, CDCl3) δ 2.65 (s, 3H), 3.25 (s, 3H), 3.63
(dd, J ) 18, 4 Hz, 1H), 3.74 (s, 2H), 3.93 (dd, J ) 18, 4 Hz,
1H), 5.41 (s, 2H), 5.63 (s, 2H), 6.46 (d, J ) 16 Hz, 1H), 6.71 (s,
1H), 6.75 (br peak, 1H), 7.24-7.33 (m, 5H), 7.34 (dd, J ) 8, 8
Hz, 1H), 7.40-7.55 (m, 2H), 7.59 (d, J ) 8 Hz, 1H), 7.75 (dd,
J ) 8, 8 Hz, 1H), 7.81 (d, J ) 8 Hz, 1H), 7.95 (d, J ) 8 Hz,
1H), 8.12-8.21 (m, 2H), 8.31 (d, J ) 2 Hz, 1H), 8.59-8.67
(m, 3H); MS (ESI) m/z 776 (M + 1). Anal. (C41H35Cl2N7O5)
C, H, N.
Compounds 22b-e, 23, and 24 were prepared following the
procedure described above for 22a .
N-[2,4-Dich lor o-3-({[2-m eth yl-4-(2-p yr id in ylm eth oxy)-
8-qu in olin yl]oxy}m eth yl)ph en yl]-2-(1,3-dioxo-1,3-dih ydr o-
2H-isoin d ol-2-yl)-N-m eth yla ceta m id e (26). Step 1. To a
solution of 25 (900 mg, 2.29 mmol) and Et3N (279 mg, 2.76
mmol) in dry CH2Cl2 (20 mL) was added dropwise methane-
sulfonyl chloride (288 mg, 2.51 mmol) in an ice/water bath
under nitrogen. After 30 min, the reaction mixture was washed
with water, saturated aqueous NaHCO3, and brine. The
organic layer was dried over MgSO4, and evaporated in vacuo
to afford the methanesulfonate intermediate (1.08 g, ∼100%)
as a pale-yellow oil.
Step 2. To a suspension of 60% NaH in oil (99.0 mg, 2.48
mmol) in DMF (0.5 mL) was added a solution of 9f (600 mg,
2.25 mmol) in DMF (5 mL) in an ice/water bath under nitrogen,
and the mixture was stirred under the same conditions for 30
min. A solution of methanesulfonate (1.08 g, 2.29 mmol) in
dry DMF (25 mL) was added dropwise to the mixture under
the same conditions, 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 evapo-
rated in vacuo. The residue was purified by flash silica gel
chromatography (CHCl3/MeOH, 20:1) followed by trituration
with EtOAc to afford 26 (772 mg, 53.4%) as a colorless solid:
1H NMR (300 MHz, CDCl3) δ 2.67 (s, 3H), 3.21 (s, 3H), 4.01
(s, 2H), 5.38 (s, 3H), 5.67 (d, J ) 10 Hz, 1H), 5.73 (d, J ) 10
ter t-Bu tyl 3-(Ben zyloxy)-2-n itr op h en yl(2-m eth oxyeth -
yl)ca r ba m a te (40). To a solution of tert-butyl 3-(benzyloxy)-
2-nitrophenylcarbamate (39) (600 mg, 1.74 mmol) and 2-chlo-
roethyl methyl ether (666 mg, 6.97 mmol) in dry DMF (9 mL)
were added K2CO3 (1.20 g, 8.68 mmol) and tetrabutylammo-
nium iodide (60 mg) at ambient temperature under nitrogen,
and the reaction mixture was stirred at 100 °C for 6 h. The
mixture was poured into water and extracted with EtOAc. The

Non-Peptide Full Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11 2861
organic layer was washed with water and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by
flash silica gel chromatography (hexane/EtOAc, 3:1) to afford
40 (545 mg, 77.7%) as a pale-yellow oil: 1H NMR (300 MHz,
CDCl3) δ 1.35 (br peak, 9H), 3.33 (s, 3H), 3.39-4.03 (m, 4H),
5.19 (br s, 2H), 6.91-7.05 (m, 2H), 7.27-7.45 (m, 6H); MS
(ESI) m/z 403.1. Anal. (C21H26N2O6) C, H, N.
3-Ben zyloxy-N-(2-m eth oxyeth yl)-2-n itr oa n ilin e (41). A
mixture of 40 (542 mg, 1.35 mmol) in 4 N HCl in EtOAc (3
mL, 12 mmol) was stirred at ambient temperature for 1 h. The
reaction mixture was evaporated in vacuo. To the residue was
added saturated aqueous NaHCO3, and then the mixture was
extracted with EtOAc. The organic layer was washed with
water and brine, dried over MgSO4, and evaporated in vacuo
to give 41 (396 mg, 97.3%) as an oil: 1H NMR (300 MHz,
CDCl3) δ 3.35 (q, J ) 6 Hz, 2H), 3.40 (s, 3H), 3.61 (t, J ) 6 Hz,
2H), 5.16 (s, 2H), 6.21 (t, J ) 5 Hz, 1H), 6.30-6.42 (m, 2H),
7.20 (dd, J ) 8, 8 Hz, 1H), 7.27-7.47 (m, 5H); MS (ESI) m/z
303.2 (M + 1). Anal. (C16H18N2O4) C, H, N.
(2E)-3-[6-(Acetylam in o)-3-pyr idin yl]-N-{2-[2,4-dich lor o-
3-({[2-m et h oxy-1-(2-m et h oxyet h yl)-1H -b en zim id a zol-4-
yl]oxy}m et h yl)m et h yla n ilin o]-2-oxoet h yl}-2-p r op en a -
m id e (43b). Step 1. To an ice-cooled solution of 42 (232 mg,
1.27 mmol) in DMF (3 mL) was added 60% sodium hydride in
oil (53 mg, 1.33 mmol) under nitrogen. After 30 min, 16b was
added, followed by stirring at ambient temperature for 1 h.
The reaction mixture was then poured into water and ex-
tracted with CHCl3 twice. The extracts were washed with
water and brine, dried over MgSO4, and evaporated in vacuo
to give intermediate diamine.
Step 2. To a solution of diamine in AcOH (2 mL) was added
tetramethyl orthocarbonate (304 mg, 2.23 mmol) at ambient
temperature, and the mixture was stirred at the same tem-
perature for 20 h. The reaction mixture was evaporated in
vacuo, and AcOH was azeotropically removed with toluene.
The residue was purified by flash silica gel column chroma-
tography (CHCl3/MeOH, 50:1) and by preparative thin-layer
chromatography (EtOAc/MeOH, 12:1) to afford 43b (96.0 mg,
11.5%) as an amorphous solid: 1H NMR (300 MHz, CDCl3) δ
2.23 (s, 3H), 3.27 (s, 3H), 3.32 (s, 3H), 3.62-3.73 (m, 2H), 3.95
(dd, J ) 18, 4 Hz, 1H), 4.13 (t, J ) 7 Hz, 2H), 4.20 (s, 3H),
5.65 (s, 2H), 6.46 (d, J ) 16 Hz, 1H), 6.67 (t, J ) 5 Hz, 1H),
6.83 (t, J ) 8 Hz, 1H), 6.92 (d, J ) 8 Hz, 1H), 7.10 (dd, J ) 8,
8 Hz, 1H), 7.30 (d, J ) 8 Hz, 1H), 7.45-7.58 (m, 2H), 7.85
(dd, J ) 8, 2 Hz, 1H), 8.21 (d, J ) 8 Hz, 1H), 8.35 (d, J )
2 Hz, 1H); MS (ESI) m/z 655 (M + 1). Anal. (C31H32Cl2N6O6)
C, H, N.
(2E)-3-[6-(Acetylam in o)-3-pyr idin yl]-N-(2-{2,4-dich lor o-
3-[(2,3-d ia m in op h en oxy)m et h yl]m et h yla n ilin o}-2-oxo-
eth yl)-2-p r op en a m id e (45). To a solution of 3-hydroxyphen-
ylenediamine (44) (250 mg, 2.01 mmol) in DMF (5 mL) was
added 60% NaH in oil (84.6 mg, 2.11 mmol) with cooling in
an ice/water bath under a nitrogen atmosphere. After 20 min,
16b (993 mg, 2.11 mmol) was added to the reaction mixture.
The reaction mixture was stirred at 0 °C for 30 min and at
ambient temperature for 2 h, then poured into water, and the
precipitate was collected by vacuum filtration. The precipitate
was purified by flash silica gel chromatography (CHCl3/MeOH,
20:1) to afford 45 (748 mg, 66.6%) as a brown solid: 1H NMR
(300 MHz, CDCl3) δ 2.22 (s, 3H), 3.28 (s, 3H), 3.50 (br s, 4H),
3.69 (dd, J ) 17, 4 Hz, 1H), 3.94 (dd, J ) 17, 5 Hz, 1H), 5.34
(s, 2H), 6.40-6.50 (m, 2H), 6.60-6.76 (m, 3H), 7.32 (d, J )
7.5 Hz, 1H), 7.50 (d, J ) 7.5 Hz, 1H), 7.53 (d, J ) 15 Hz, 1H),
7.85 (dd, J ) 7.5, 2 Hz, 1H), 7.27-7.36 (m, 4H), 7.49 (d, J )
7.5 Hz, 1H), 7.51 (d, J ) 15 Hz, 1H), 7.84 (br d, J ) 7.5 Hz,
1H), 8.09 (br s, 1H), 8.22 (br d, J ) 7.5 Hz, 1H), 8.36
(br s, 1H); MS (ESI) m/z 557 (M + 1). Anal. (C26H26Cl2N6O4)
C, H, N.
reaction mixture was concentrated in vacuo, and the residue
was dissolved in CHCl3. The organic layer was washed with
saturated aqueous NaHCO3, water, and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by
flash silica gel chromatography (CHCl3/MeOH, 30:1) to afford
46 (654 mg, 83.0%) as a pale-brown solid: 1H NMR (300 MHz,
CDCl3) δ 2.21 (s, 3H), 3.29 (s, 3H), 3.59 (br d, J ) 17 Hz, 1H),
4.10-4.22 (m, 4H), 5.30 (d, J ) 10 Hz, 1H), 5.59 (d, J ) 10
Hz, 1H), 6.48 (d, J ) 15 Hz, 1H), 6.78 (br s, 1H), 6.83 (d, J )
7.5 Hz, 1H), 7.12 (dd, J ) 7.5, 7.5 Hz, 1H), 7.20-7.29 (m, 1H),
7.32 (d, J ) 7.5 Hz, 1H), 7.49 (d, J ) 7.5 Hz, 1H), 7.65 (d, J )
15 Hz, 1H), 7.85 (br d, J ) 7.5 Hz, 1H), 8.09 (br s, 1H), 8.23
(br d, J ) 7.5 Hz, 1H), 8.37 (br s, 1H); MS (ESI) m/z 597
(M + 1). Anal. (C28H26Cl2N6O5) C, H, N.
Eth yl {4-[(3-{[({(2E)-3-[6-(Acetyla m in o)-3-p yr id in yl]-2-
pr open oyl}am in o)acetyl](m eth yl)am in o}-2,6-dich lor oben -
zyl)oxy]-2-m eth oxy-1H-ben zim id a zol-1-yl}a ceta te (47a ).
To a solution of 46 (100 mg, 0.168 mmol) in DMF (1.5 mL)
were added ethyl bromoacetate (28.7 mg, 0.172 mmol) and K2-
CO3 (69.5 mg, 0.503 mmol) at ambient temperature, and the
mixture was stirred at the same temperature for 15 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
purified by preparative thin-layer chromatography (CHCl3/
MeOH, 10:1) to afford 47a (51.1 mg, 44.5%) as a colorless
amorphous solid: 1H NMR (300 MHz, CDCl3) δ 1.27 (t, J )
7 Hz, 3H), 2.20 (s, 3H), 3.28 (s, 3H), 3.65 (dd, J ) 17, 4 Hz,
1H), 3.95 (dd, J ) 17, 4 Hz, 1H), 4.20 (s, 2H), 4.24 (q, J )
7 Hz, 2H), 4.68 (s, 2H), 5.66 (s, 2H), 6.47 (d, J ) 15 Hz, 1H),
6.70 (br s, 1H), 6.77 (d, J ) 8 Hz, 1H), 6.86 (d, J ) 8 Hz, 1H),
7.10 (dd, J ) 8, 8 Hz, 1H), 7.30 (d, J ) 8 Hz, 1H), 7.48-7.56
(m, 2H), 7.83 (dd, J ) 8, 2 Hz, 1H), 8.15-8.22 (m, 2H), 8.35
(d, J ) 2 Hz, 1H); MS (ESI) m/z 683 (M + 1). Anal. (C32H32
Cl2N6O7) C, H, N.
-
Compounds 47b and 47c were prepared following the
procedure described above for 47a .
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
described32 with minor modifications. Male Hartley guinea pigs
(from Charles River J apan, Inc.) were killed by exsanguination
under anesthesia. The ilea were removed and homogenized
in ice-cooled buffer (50 mM sodium (trimethylamino)ethane-
sulfonate (TES) and 1 mM 1,10-phenanthroline, pH 6.8) with
a Polytron. The homogenate was centrifuged to remove cellular
debris (1000g, 20 min, 4 °C), and the supernatant was
centrifuged (100000g, 60 min, 4 °C). The pellet was then
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 [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 are transfected with, and stably express
human B2 receptors, have been described previously.19 Cells
were maintained in an R-minimum essential medium supple-
mented 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 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
(2E)-3-[6-(Acetyla m in o)-3-p yr idin yl]-N-[2-(2,4-d ich lor o-
3-{[(2-m eth oxy-1H-ben zim id a zol-7-yl)oxy]m eth yl}m eth -
yla n ilin o)-2-oxoeth yl]-2-p r op en a m id e (46). To a solution
of 45 (735 mg, 1.32 mmol) in AcOH (7.4 mL) was added
tetramethyl orthocarbonate (270 mg, 1.98 mmol), and the
mixture was stirred at ambient temperature for 5 h. The

2862 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 11
Sawada et al.
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0.25 mL of binding buffer (20 mM HEPES, 125 mM N-methyl-
D-glucamine, 5.0 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.05
mM bacitracin, 5 µM enalaprilat, and 0.1% BSA, pH 7.2). All
experiments were carried out three times. Nonspecific binding
was determined in the presence of 1 µM unlabeled BK. At the
end of the incubation, the buffer was aspirated, and the cells
were washed twice with ice-cooled phosphate-buffered saline
containing 0.1% BSA. The specific binding was calculated by
subtracting the nonspecific binding, determined in the pres-
ence of 1 µM unlabeled BK, from the total binding. Bound
radioactivity was determined by solubilizing with 1% sodium
doedecyl sulfate containing 0.05 N NaOH and quantified in a
liquid scintillation counter.
Agon ist-In d u ced In ositol P h osp h a te P r od u ction . Inos-
itol phosphate formation was measured essentially as de-
scribed previously.19 CHO cells expressing the human B2
receptor were seeded in 12-well plates at a 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.33 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).
Agon ist-In d u ced P GE2 P r od u ction . Human fibroblasts
(WI-38, ATCC CCL75) were grown in Eagle’s essential medium
(E-MEM) with 10% fetal bovine serum (FBS). The fibroblasts
were seeded in 24-well plates at a density of 1 × 105 cells/well
with 0.5 mL of E-MEM including 1% FBS and were cultured
for 24 h at 37 °C. Then the medium in the culture was removed
by aspiration, and Hanks’ balanced salt solution containing
0.1% BSA (Hanks-BSA buffer) was added. After the Hanks-
BSA buffer was removed by aspiration, different concentra-
tions of a test compound in Hanks-BSA buffer was added ,
and the cells were incubated for 30 min at 37 °C. After the
incubation, the supernatant was collected, and the PGE2 level
included therein was measured by a PGE2 EIA kit (Cayman
Chemical Co.). The agonist-induced PGE2 production was
calculated by subtracting the control PGE2 production deter-
mined in the absence of the compound. The efficacy of the
compound was expressed as the relative agonistic activity in
PGE2 production compared to that of BK (100 nM).
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Sta tistica l An a lysis. The results are expressed as the
mean ( SEM, and the statistical significance between groups
was analyzed by the Student’s t test. The IC50 value was
obtained by using nonlinear curve-fitting methods with a
computer program developed in house.
Ack n ow led gm en t. We are grateful to Dr. D. Barrett
(Medicinal Chemistry Research Laboratories, Fujisawa
Pharmaceutical Co. Ltd., Osaka) for his valuable sug-
gestions.
Su p p or tin g In for m a tion Ava ila ble: Physical data for
9c-f, 17b-e, 18, 19a ,b, 22b-e, 23, 24, 29-33, 35-38, 42,
47b, and 47c. This material is available free of charge via the
Internet at http://pubs.acs.org.
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