Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and evaluation of physical properties as potential overactive bladder therapies: Part 5

Article abstract of DOI:10.1021/jm9000709

As an extension of research conducted on β₃-adrenergic receptor agonists as potential drugs for treating overactive bladder (OAB), novel series containing an acylsulfonamide moiety instead of the carboxylic acid moiety were evaluated. These com

Full text of DOI:10.1021/jm9000709

J. Med. Chem. 2009, 52, 3063–3072
3063
Discovery of Highly Potent and Selective Biphenylacylsulfonamide-Based ꢀ3-Adrenergic
Receptor Agonists and Evaluation of Physical Properties as Potential Overactive Bladder
Therapies: Part 5
Kouji Hattori,*,† Susumu Toda,† Masashi Imanishi,† Shinji Itou,† Yutaka Nakajima,† Kenichi Washizuka,† Takanobu Araki,†
Hitoshi Hamashima,† Yasuyo Tomishima,† Minoru Sakurai,† Shigeo Matsui,‡ Emiko Imamura,‡ Koji Ueshima,§
Takao Yamamoto,‡ Nobuhiro Yamamoto,‡ Hirofumi Ishikawa,‡ Keiko Nakano,‡ Naoko Unami,‡ Kaori Hamada,‡
Yasuhiro Matsumura,| and Fujiko Takamura|
Chemistry Research Laboratories, Pharmacology Research Laboratories, Applied Pharmacology Research Laboratories, and Analysis &
Pharmacokinetic Research Laboratories, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
ReceiVed January 20, 2009
As an extension of research conducted on ꢀ3-adrenergic receptor agonists as potential drugs for treating
overactive bladder (OAB), novel series containing an acylsulfonamide moiety instead of the carboxylic
acid moiety were evaluated. These compounds have been identified as potent and selective human ꢀ3-AR
agonists with improved oral bioavailability compared to the previous series. Results of structure-activity
relationship (SAR) studies and cassette dosing evaluation in dogs showed several analogues (namely, 6h,
6j, 6o, 7e, and 9e) to have an excellent balance of in vitro potency and selectivity, pharmacokinetic (PK)
profile, and an in vivo OAB model. Here we examined the relaxation response in dog detrusor muscle strips
to a KCl induced tonic concentration. Results showed that the potency of in vitro relaxation response was
not mirrored in the potency of the cAMP accumulation in CHO cell lines. Surprisingly, the EC50 values of
6e and 7e found to induce relaxation of isolated bladder strips were over 50-fold higher than the cAMP
accumulation in cell line. In general, increased lipophilicity led to decreased potency for the bladder relaxation
compared with cAMP accumulation in CHO cell lines, indicating that lipophilicity is crucial for OAB drug
candidates to improve ꢀ3 activity.
Introduction
identified in the urinary bladder of many species, including
humans.7 In most species, the ꢀ2-AR subtype was proposed to
have an important role in relaxation via activation of adenyl
cyclase. In humans, however, the detrusor muscle has recently
been reported to be the predominant site of ꢀ3-AR mRNA
expression. The relaxation induced by adrenergic stimulation
of the human detrusor is mediated mainly through ꢀ3-AR
activation, suggesting that ꢀ3-ARs may represent a new
therapeutic target for treating OAB.8
Overactive bladder (OABa) is defined as urinary urgency with
or without urgency incontinence.1 An estimated 16% of the adult
population in the U.S. suffers from OAB, with worldwide
numbers steadily increasing.2 The urinary bladder is innervated
by both the sympathetic and parasympathetic nervous systems,
and antimuscarinic agents that induce relaxation of the detrusor
muscle have been widely used in the treatment of OAB.3
However, these agents have several disadvantages, including
adverse events associated with anticholinergic effects such as
dry mouth, constipation, and the potential for voiding difficulty
in patients with poorly contractile bladders. Accordingly, a drug
lacking these adverse effects due to the blockage of cholinergic
neuron would be a significant improvement over current therapy.
Activation of sympathetic nerves contributes to urine storage
by relaxing the detrusor muscle via activation of ꢀ-adrenoceptors
(ꢀ-ARs).4 ꢀ-ARs are classified into three types; ꢀ1-, ꢀ2-, and
ꢀ3-ARs.5 The ꢀ3-AR has been demonstrated to mediate several
pharmacological and physiological effects such as lipolysis in
white adipocytes and thermogenesis (energy expenditure) in
brown tissue adipocytes.6 Both ꢀ1- and ꢀ2-ARs have been
In our previous study, we described a series of first generation
biphenyl (FGB) analogues, shown in Figure 1, which exhibited
good oral bioavailability and a long plasma half-life.9 The key
to success was finding a benzoic acid moiety and R substitution
on the right-hand side (RHS) which maximized ꢀ3-AR activity,
selectivity, and bioavailability. Removal of the carboxylic acid
from the biphenyl analogue resulted in significantly reduced
potency and bioavailability.10 On the other hand, SAR studies
of the R position on the terminal phenyl ring in the FGB
analogues indicated that introduction of lipophilic substitute
group led to increased potency for ꢀ3-AR activity (O-cyclo-
hexyls 1e, 2e > O-isopropyls 1b, 2b > O-methyl 1a), but a
decreased oral bioavailability (1a > 1b, 2b > 1e, 2e) while
displaying high passive permeability and good stability to liver
microsomes. To address this problem, a series of second
generation biphenyl (SGB) analogues were developed, with
adjustment of physical properties by replacement of the phenyl
moiety with pyridine analogues on the left-hand side (LHS),
resulting in enhanced potency and maintenance of good oral
availability as shown in Table 1.11 In a previous metabolism
study of FGB/SGB analogues conducted in rat and human
hepatocytes, we identified two main metabolites: a dealkylated
metabolite (M-1) and an acylglucuronide conjugated metabolite
* To whom correspondence should be addressed. Phone: +81-29-863-
7179. Fax: +81-29-852-5387. E-mail: kouji.hattori@jp.astellas.com.
†
Chemistry Research Laboratories.
Pharmacology Research Laboratories.
Applied Pharmacology Research Laboratories.
Analysis & Pharmacokinetic Research Laboratories.
‡
§
|
a Abbreviations: ꢀ-AR, ꢀ-adrenergic receptors; OAB, overactive bladder;
FGB, first generation biphenyl; SGB, second generation biphenyl; LHS,
left-hand side; RHS, right-hand side; SAR, structure-activity relationship;
cAMP, cyclic adenosine monophosphate; ISP, isoproterenol; CHO, Chinese
hamster ovary; IVP, intravesical pressure; PAMPA, parallel artificial
membrane permeation assay; PK, pharmacokinetic; PB, protein binding.
10.1021/jm9000709 CCC: $40.75
2009 American Chemical Society
Published on Web 04/14/2009

3064 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Hattori et al.
Figure 1. Design and discovery strategy of bipenyl analogues.
Table 1. SAR Results for FGB and SGB Analoguesa
also been previously described. Target compounds 6a-p were
obtained as hydrochloride salts after deprotection the Boc group
with 4 N HCl, and pyridine analogues 7 were obtained from
11 by the similar method. The aniline analogue 8 was obtained
from 12 through an additional step involving reduction of the
nitro group with iron powder in the presence of NH4Cl. The
amino pyridine analogues 9 were prepared by coupling 13 with
boronic acids (14b,e,o,q) in the presence of a catalytic amount
of PdCl2(dppf)·CHCl3, followed by alkaline hydrolysis and
subsequent deprotection of the Boc amine silyl ether using 4 N
HCl.
human EC50 (nM)
dog cassett assay,b
Cmax ratioc
compd
Ar
R1
O-Me
ꢀ3
ꢀ2
ꢀ1
1a 3-CIPh
4.8 >10000 250
1.1 >10000 720
1.7
1.0
0.0
0.90
0.24
0.0
0.24
0.10
0.32
0.65
0.36
0.48
0.49
0.36
0.09
1b
1e
2b Ph
2j
2e
2f
2h
2o
3b 3-Py
3j
3e
4b 3-(6-NH2Py) O-iso-Pr
4j
4e
O-iso-Pr
O-c-Hex
O-iso-Pr
iso-Bu
O-c-Hex
O-Ph
0.46 NT
55
2.0 >10000 >1000
0.60 >10000 >1000
Result and Discussion
All compounds were evaluated for the ability to produce
cAMP in Chinese hamster ovary (CHO) cell lines expressing
cloned human ꢀ3- and ꢀ1-ARs. Select compounds were also
evaluated for human ꢀ2 activity by a similar method. The results
for the reference compound, isoproterenol (ISP), a nonselective
ꢀ-AR agonist, are shown for comparison in Tables 2 and 3.
Pharmacokinetic (PK) properties of selected compounds were
evaluated by cassette dosing assay in dogs.15
0.30 NT
0.34 NT
NH-iso-Pro 0.29 586
260
980
170
S-iso-Pr
O-iso-Pr
iso-Bu
0.56 >10000 >1000
1.5 >10000 >1000
0.26 NT
0.26 >10000 480
0.19 >10000 130
>1000
O-c-Hex
iso-Bu
O-c-Hex
0.066 3200
0.035 1100
150
39
a See refs 9 and 11. b Dose 0.10 or 0.20 mg/kg po (n ) 2, 3). c The ratio
is defined as the Cmax of each compound divided by the Cmax of 1b. The
ratio value of 1b was presented as 1.0.
We initially investigated the effect of newly designed
acylsulfonamide analogues with a phenyl moiety on the LHS.
The modification from a carboxylic acid to the corresponding
acylsulfonamide resulted in SAR trends similar to those seen
in FGB analogues with regard to ꢀ3 activity and the selectivity
for ꢀ1 and ꢀ2. Substitution to the R position of the terminal
phenyl ring on the RHS with a more lipophilic group such as
an O-linker resulted in increased ꢀ3-AR activity (O-cycloheptyl
6g > O-cyclohexyl 6e > O-phenyl 6f > O-cyclopentyl 6d >
O-isopropyl 6b > O-methyl 6a). Substitution with a bulky
cyclopentyl group (6d) showed a 4.5-fold greater potency than
the corresponding analogue substituted with an n-pentyl group
(6c). The N-linker analogues with an isopropyl group (6h) and
a cyclohexyl group (6i) showed greater improvement in ꢀ3-AR
activity relative to the O-linker analogues 6b and 6e, respec-
tively. The C-linker analogues with isobutyl (6j), cyclopentyl
(6k), and cyclohexyl (6l) also showed good activity; however,
the most lipophilic compound 6m was less active than the other
C-linker analogues and the corresponding cyclohexyl analogues
6e and 6i. Among analogues with an isopropyl group (6b, 6h,
6j and 6o), the S-linker 6o showed the most potent ꢀ3 activity
and selectivity for ꢀ1 and ꢀ2.
(M-2) as shown in Scheme 1.12 Although glucuronide metabo-
lites from phase II metabolism generally enhance elimination
in bile and urine, they can also transform into their covalent
adducts which can lead to toxicity.13 In light of these findings,
we set out to further explore this series by examining modifica-
tion utilizing an acylsulfonamide moiety on the RHS instead
of the carboxylic acid moiety, a potentially useful strategy to
protect against metabolism via acylglucuronide conjugation.
Here, we describe SAR studies conducted on this series as
well as report on the pharmacokinetics. We also evaluate the
correlation between ꢀ3 activity and the physical properties of
this series of biphenyl analogues, which allowed identification
of potential clinical drug candidates with no loss of ꢀ3 potency
in vitro and in vivo.
Chemistry
The general synthetic route to the target compounds is shown
in Scheme 2. Synthesis of the requisite intermediate Boc amine
derivatives 10-13 was conducted as previously reported,9,11 and
acylsulfonamide derivatives 14 were prepared from the corre-
sponding carboxylic acid by a 1,1′-carbonyldiimidazole-
promoted coupling with methansulfonamide followed by treat-
ment with pinacol diborane.14 Suzuki cross-coupling of Boc
amine intermediates 10 with boronic acid derivatives 14 has
Of particular note is the fact that all of these compounds
displayed remarkable pharmacokinetic properties on dogs.16 The
compounds with O-cyclohexyl (6e), NH-isopropyl (6h), isobutyl
(6j), and S-isopropyl (6o) showed dramatically improved
bioavailability over the corresponding carboxylic acids (2e, 2h,
2j, and 2o, respectively), a finding that can be explain by

Biphenylacylsulfonamide Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3065
Scheme 1. Main Metabolites Pathway of FGB and SGB Analogues
Scheme 2. Synthesis Route to Target Compoundsa
a (a) Pd(PPh3)4, aq NaHCO3, DME, 70 °C; (b) 4 N HCl/AcOEt; (c) 4 N HCl/dioxane; (d) Fe (powder), NH4Cl, EtOH, reflux, then 4 N HCl/dioxane; (e)
PdCl2(dppf)·CH2Cl2, dppf, aq Na2CO3, toluene, EtOH, 75 °C; (f) 1 N NaOH aq, EtOH, 100 °C, then 4 N HCl/dioxane, MeOH.
protection from conjugation. Further, compounds 6h and 6k also
showed good bioavailability despite having a low passive
permeability as shown by parallel artificial membrane perme-
ation assay (PAMPA),17 indicating that uptake following oral
administration of these compounds is likely dependent on active
transport using a carrier proteins.
potent activity among this series, and bioavailability was
improved over that of the corresponding SGB analogues.
After SAR examination, we selected 6h, 6j, 6o, 7e, and
9e for PK profile evaluation. Table 4 shows the metabolic
stability as measured by in vitro clearance using liver
microsomes. Overall, all compounds showed good stability
to dog, monkey, and human microsomes, and poor stability
to rat microsomes. Table 5 shows the pharmacokinetic
profiles in rats, dogs, and monkeys. The phenyl analogue with
an S-isopropyl group (6o) and the pyridyl analogue with an
O-cyclohexyl group (7e) displayed good oral bioavailability
in all species (rats, F ) 41%, 25%; dogs, F ) 62%, 81%;
monkeys, F ) 24%, 38%, respectively) and a moderate
plasma half-life in dogs (t1/2ꢀ ) 4.8, 5.5 h, respectively).
Isobutyl analogue 6j displayed similar good PK profiles in
rats and dogs (rats, F ) 40%; dogs, F ) 67%). However,
the phenyl analogue with an N-isopropyl (6h) and the
aminopyridyl analogue with an O-cyclohexyl (9e) displayed
good oral bioavailability in dogs (dogs, F ) 45% and 54%,
respectively) but poor bioavailability in monkeys.
We next focused our attention on replacing the phenyl
moiety on the LHS. Transformation of the phenyl group to
a pyridyl group resulted in slightly increased in ꢀ3-AR activity
(3- to 1.5-fold) over the corresponding phenyl group and
increased potency for the cyclohexyl substitution over that
for the isopropyl substitution. The N- and S-linker compounds
with isopropyl (7h and 7j) showed 5-fold more potent activity
than the O-linker 7b. However, the shift to pyridyl analogues
led to a decreased bioavailability compared to the phenyl
analogues. Only the compound with an O-cyclohexyl group
(7e) displayed an excellent balance between bioavailability
and potency, with improved activity and a PK profile relative
to the corresponding phenyl analogue 6e. On replacement
of the phenyl moiety with a 4-aminophenyl moiety on the
LHS, compound 8e showed the same activity and PK profile
as the corresponding phenyl compound 6e. Although the
aminopyridyl derivatives on the LHS display greatly in-
creased ꢀ3 activity, the Cmax level after oral administration
is fairly reduced in contrast to the pyridyl derivatives. The
compound with an O-cyclohexyl (10e) showed the most
We then examined the inhibitory effect on a carbachol-induced
increase of intravesical pressure (IVP) in anesthetized dog OAB
model.18 Table 6 shows in vitro potency of these compounds as
well as FGB and SGB series for human and dog ꢀ3-AR activity in
CHO cell lines, with in vivo inhibition results as well. Phenyl
analogues on the LHS with an O-isopropyl group (6b), an

3066 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Hattori et al.
Table 2. Right Hand-SAR Results and ꢀ3-AR Activity and Pharmacokinetic Profiles for Acylsulfonamide Analoguesa
pharmacokinetic parameters in dogsb,c
human EC50 (nM)b
po (n ) 3)
iv (n ) 3)
CLtot
Cmax
(ng/mL)
AUC0-24h
(ng·h/mL)
t1/2
(hr)
Fd
(%)
PAMPAe/
clogPf
compd
R
ꢀ3
ꢀ2
ꢀ1
((mL/min)/kg)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
1b
ISPg
O-Me
18.0 ( 0.39
3.10 ( 0.10
2.80 ( 0.99
0.62 ( 0.11
0.43 ( 0.06
0.60 ( 0.08
0.38 ( 0.04
0.60 ( 0.05
0.14 ( 0.01
0.46 ( 0.08
0.41 ( 0.03
0.33 ( 0.12
0.54 ( 0.08
1.40 ( 0.05
0.32 ( 0.03
0.50 ( 0.06
1.10 ( 0.10
0.97 ( 0.14
NTh
730 ( 159
>1000
NTh
NTh
NTh
NTh
NTh
75
7.6/3.38
15/4.22
O-iso-Pr
O-n-Pen
O-c-Pen
O-c-Hex
O-Ph
O-c-Hep
NH-iso-Pr
NH-c-Hex
iso-Bu
NTh
33.4 ( 1.4
561.5 ( 60.6
12.0 ( 2.0
2.3 ( 0.1
NTh
160 ( 24
750 ( 70
>1000
NTh
NTh
NTh
NTh
NTh
NTh
70
NTh/5.50
NTh/4.86
24/5.41
NTh
NTh
NTh
NTh
NTh
NTh
22.2 ( 3.6
70.3 ( 13.2
NTh
363.4 ( 41.3
756.5 ( 203.6
NTh
7.8 ( 1.2
4.4 ( 0.1
NTh
6.3 ( 1.3
3.9 ( 0.2
3.5 ( 0.2
4.7 ( 0.2
NTh
3.0 ( 0.1
2.2 ( 0.3
NTh
7.5 ( 1.6
9.9 ( 0.5
5.3 ( 0.8
4.0 ( 1.1
NTh
NTh
>1000
98
18/5.26
NTh
220 ( 10
260 ( 55
900 ( 10
>1000
NTh
45
NTh/5.97
0.8/4.52
4.4/5.71
6.8/4.85
0.32/4.65
1.2/5.21
NTh/6.04
2.7/4.25
2.2/4.03
12.5/5.23
>30/3.30
>10000
8.6 ( 0.7
3.2 ( 1.0
24.9 ( 1.9
17.1 ( 0.53
13.1 ( 1.1
27.9 ( 2.9
8.1 ( 1.0
39.8 ( 7.8
8.0 ( 2.2
13.0 ( 1.1
95.8 ( 1.9
33.5 ( 7.3
210.5 ( 22.6
211.1 ( 25.7
105.6 ( 1.9
230.0 ( 43.1
91.9 ( 12.5
440.6 ( 91.2
56.3 ( 9.0
149 ( 8.7
NTh
22
67
>10000
NTh
c-Pen
c-Hex
>1000
>1000
370 ( 30
>1000
>1000
840 ( 154
720 ( 160
0.84 ( 0.02
50
NTh
NTh
70
CH-c-Hex
S-n-Pro
S-iso-Pro
S-c-Hex
O-iso-Pr
NTh
3.9 ( 0.1
4.9 ( 0.4
NTh
NTh
NTh
NTh
75
>10000
4.3 ( 0.3
NTh
9.9 ( 0.4
2.9 ( 0.3
NTh
4.9 ( 0.6
NTh
NTh
45
>1000
2.0 ( 0.9
a Details of experimental methods are in refs 9 and 11. b The results are the mean ( SE (n ) 3) or are presented as the average of two experiments.
c Cassette assay data. Dose of 0.10 mg/kg po and iv. d F ) bioavailability. e See ref 17. f Biobyte ClogP, version 4.3. g ISP ) isoproterenol. h NT ) not
tested.
Table 3. Left Hand-SAR Results and ꢀ3-AR Activity and Pharmacokinetic Profiles for Acylsulfonamide Analoguesa
pharmacokinetic parameters in dogsb,c
human EC50 (nM)b
po (n ) 3)
iv (n ) 3)
CLtot
((mL/min)/kg) (%)
Cmax
(ng/mL)
AUC0-24h
(ng·h/mL)
t1/2
(h)
Fd PAMPAe/
clogPf
compd
Ar
R
ꢀ3
ꢀ2
ꢀ1
7b
7e
7h
7i
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
3-Py
O-iso-Pr
O-c-Hex
NH-iso-Pr
1.0 ( 0.08 NTh
130 ( 20
NTh
NTh
NTh
NTh
NTh NTh/2.72
0.13 ( 0.005 >10000 800 ( 40
34.5 ( 3.8 384.6 ( 57.7 5.5 ( 0.4
3.2 ( 0.3
16.4 ( 2.4
12.4 ( 4.9
35.9 ( 5.9
21.8 ( 4.3
5.4 ( 0.7
NTh
81
12
21
21
18
14
15/3.92
0.20 ( 0.02 NTh
320 ( 35
1.9 ( 1.1 10.4 ( 5.2
10.3 ( 2.2 28.4 ( 9.3
2.6 ( 1.1 12.0 ( 9.4
1.4 ( 0.3 13.4 ( 0.6
2.7 ( 0.3
1.3 ( 0.6
0.6 ( 0.1
1.4 ( 0.2
0.10/3.02
1.5/4.22
0.50/3.35
0.41/3.16
1.4/2.54
NH-c-Hex 0.062 ( 0.001 >10000 >1000
iso-Bu
c-Pen
S-iso-Pro
O-iso-Pr
O-c-Hex
7j
0.32 ( 0.03 NTh
0.29 ( 0.02 NTh
0.19 ( 0.01 NTh
5.5 ( 0.70 NTh
0.50 ( 0.04 NTh
0.68 ( 0.02 NTh
0.14 ( 0.02 NTh
0.029 ( 0.003 290
0.080 ( 0.012 1200
490 ( 20
>1000
240 ( 35
>1000
>1000
93 ( 2.0
200 ( 35
140 ( 10
390 ( 35
7k
7o
8b
8e
9q
9b
9e
5.3 ( 0.7 50.9 ( 22.4 4.3 ( 0.7
4-(NH2Ph)
4-(NH2Ph)
3-(6-NH2Py) O-Et
3-(6-NH2Py) O-iso-Pr
3-(6-NH2Py) O-c-Hex
3-(6-NH2Py) S-iso-Pr
NTh NTh NTh
NTh NTh/2.99
17.5 ( 1.0 212.5 ( 14.4 5.7 ( 0.3
5.0 ( 0.2
68
3.8/4.19
4.8 ( 1.0 36.5 ( 6.3
3.9 ( 0.6 44.4 ( 6.9
2.5 ( 0.3 26.7 ( 1.0
0.9 ( 0.1 3.8 ( 2.1
NTh
1.6 ( 0.02
6.7 ( 1.3
NTh
NTh
NTh NTh/2.09
22.9 ( 1.6
34.9 ( 2.5
NTh
36
54
NTh/2.40
0.60/3.59
9o
NTh NTh/2.21
ISPg
0.97 ( 0.14 2.0 ( 0.9 0.84 ( 0.02
a Details of experimental methods are in refs 9 and 11. b The results are the mean ( SE (n ) 3) or are presented as the average of two experiments.
c Cassette assay data. Dose of 0.10 mg/kg p.o and iv. d F ) bioavailability. e See ref 17. f Biobyte ClogP, version 4.3. g ISP ) isoproterenol. h NT ) Not
Test.
O-cyclohexyl group (6e), and an O-phenyl group (6f) all showed
less inhibitory activity after intravenous administration than the
FGB analogue with an O-isopropyl group (2b), although the dog
EC50 of 6e was 3.5-fold improved. The N-linker with an isopropyl
group (6h), C-linker with an isobutyl group (6j), and S-linker with
an isopropyl group (6o) all showed potent in vivo activity (6h,
EC50 ) 7.1 nM; 6j, EC50 ) 14.2 nM; 6o, EC50 ) 8.2 nM). The
pyridyl analogue with an O-cyclohexyl group (7e) also exhibited
improved activity compared with phenyl analogue 6e; however,
the in vivo EC50 value of 26.5 nM was lower than those of 6h and
6o despite similar in vitro dog ꢀ3-AR activity and lower dog protein
binding. The aminopyridyl analogue on the LHS (9e) had the most
potent in vivo activity among the selected compounds, with an
EC50 ) 1.2 nM.

Biphenylacylsulfonamide Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3067
Table 4. In Vitro Metabolism in Liver Microsomes CLint ((mL/min)/
8 indicates the effect on potency after exchanging the phenyl moiety
on the LHS with various aromatic moieties. Replacement of the
phenyl moiety with a more lipophilic (+0.7 units of clogP) Cl-
phenyl moiety showed slightly improved potency for human and
dog cAMP level but decreased potency for bladder relaxation with
an overall mean change of approximately 0.91 units (pEC50).
Exchanging the phenyl group for a pyridyl group (1.5 units
reduction in lipophilicity) led to a loss of potency for dog cAMP
with a mean change of 0.39 units but allowed for maintenance of
dog bladder relaxation with 0.08 units. Exchanging the phenyl
group for an aminopyridyl group resulted in an improvement of
0.63 units in potency for dog bladder relaxation, despite a loss in
potency for dog cAMP, with an overall mean change of 0.31 units.
Lipophilicity in this aminopyridyl analogue was reduced by the
presence of a polar amine group on the pyridine ring. Given the
value of human cAMP, these results highlight that the aminopyridyl
analogue has promising pharmacological properties for use in the
human bladder.
These findings raise the issue that increased lipophilicity leads
to opposing affect from other types of interaction in isolated
dog bladder strips, reducing the compound’s overall efficacy.
Lipophilicity underlying the structural properties seems to be
complementary properties, and increasing lipophilicity would
also affect protein binding or result in undesirable drug targets.24
With regard to drug design, increasing potency without increas-
ing lipophilicity is important in achieving an optimum design.
kg)a
compd
rat
dog
monkey
human
6h
6j
6o
7e
9e
36.0
NTb
84.3
46.9
10.3
3.3
4.7
4.6
NDc
NDc
5.4
NTb
NDc
3.4
NDc
NDc
2.2
NDc
3.5
NDc
a Each compound was incubated at 37 °C with live microsomes from
rats, dogs, monkeys, and humans in the presence of the NADPH-generating
system. Results are presented as the average of two to three experiments.
b NT ) not tested. c ND ) not determined (<0.1 (mL/min)/kg).
The ED50 values of acylsulfonamide derivatives were im-
proved over those of the corresponding carboxylic acid deriva-
tives, suggesting that this improvement was due to improvement
of pharmacokinetic properties (as in 6j vs 2j, 6o vs 2o, 7e vs
3e). However, the EC50 value of compound 7e showed 7-fold
less activity compared to compound 3e. To investigate this
discrepancy more carefully, we examined the relaxation response
in dog detrusor muscle strips in a KCl induced tonic concentra-
tion.19 Differences in EC50 values between cAMP accumulation
and bladder relaxation for each compound are shown in Table
6. Our findings suggested that the potency of in vitro relaxation
response was not mirrored by the corresponding potency of the
cAMP accumulation in CHO cell lines. In particular, the EC50
values for the relaxation of isolated dog bladder strips of 6e
and 7e were over 50-fold higher than values for cAMP
accumulation in the dog CHO cell line, which may explain the
lower activity observed in vivo.
Conclusions
In the present study, we described potent and selective ꢀ3
agonists in novel series that contain an acylsulfonamide moiety
instead of a carboxylic acid moiety and demonstrated their
improved PK profile over the FGB and SGB series. Results of
our SAR study and cassette dosing showed that several analogues
(namely, 6h, 6j, 6o, 7e, and 9e) displayed an excellent balance of
potency, selectivity, and PK profiles. In a carbachol-induced IVP
model in dogs, these compounds showed improved ED50 or both
improved in vivo ED50 and EC50 values. On the basis of their
attractive pharmacological profiles, these compounds may merit
consideration for use in OAB treatment. Further, to clarify the
relationship between in vitro and in vivo activity, we evaluated
the relaxation response of biphenyl analogues in dog detrusor
muscle strips. Managing lipophilicity was found to be a crucial
aspect of drug design for improving ꢀ3 activity. Increased lipo-
philicity of the biphenyl analogues was found to reduce the
compound’s efficacy in relaxation of isolated dog bladder strips,
regardless of the nature of the cAMP accumulation of the
compounds. These findings will be useful in identifying clinical
We also investigated the correlation of in vitro ꢀ3 activity using
84 biphenyl analogues.20 Figure 2 shows a plot of the disparity
index21 between cAMP accumulation in the dog CHO cell line
and the relaxation of isolated dog bladder strips versus lipophilicity
(clogP and clogD).22 This reveals that the lipophilic properties of
the compounds account for the general trend seen in the difference
in pEC50 values. Increasing lipophilicity of the biphenyl analogues
led to a decrease in potency for the bladder relaxation compared
to cAMP accumulation in CHO cell.
In addition, we investigated the direct correlation between ꢀ3
activity and each structural transformation on RHS or LHS using
matched molecular pairs of molecules (Figure 3).23 Table 7 shows
the corresponding change in potency when exchanging an isopropyl
moiety on the terminal phenyl ring of the RHS for a cyclohexyl
moiety. Although this exchange results in markedly improved in
pEC50 values of the ꢀ3 activity of cAMP accumulation in humans
and dogs, the improvement observed in pEC50 values for relaxation
in dog bladder strips failed to meet expectations. Similarly, Table
Table 5. Pharmacokinetic Profiles of Selected Members of Acylsulfonamide Analoguesa
po (n ) 3)
iv (n ) 3)
CLtot ((mL/min)/kg)
compd
species
dose (mg/kg)
Cmax (ng/mL)
AUC0-24h (ng·h/mL)
t1/2ꢀ (h)
F (%)b
6h
rat
dog
monkey
rat
dog
rat
dog
monkey
rat
dog
0.32
0.10
0.32
0.32
0.10
0.32
0.10
0.32
0.32
0.10
0.32
0.32
0.1
14.9 ( 1.1
8.6 ( 0.7
2.7 ( 1.3
6.4 ( 0.8
24.9 ( 1.9
9.9 ( 0.8
18.2 ( 3.1
15.7 ( 3.8
17.0 ( 2.7
34.5 ( 3.8
35.1 ( 10.2
3.3 ( 0.6
2.5 ( 0.3
0
60.9 ( 5.1
95.8 ( 1.9
19.3 ( 4.1
64.8 ( 2.9
210.5 ( 22.6
108.3 ( 12.5
214.9 ( 63.0
143.0 ( 9.2
72.2 ( 6.2
384.6 ( 57.7
272.4 ( 90.0
6.3 ( 2.9
4.1 ( 0.9
6.3 ( 1.3
3.1 ( 0.5
2.5 ( 0.5
3.5 ( 0.2
2.3 ( 0.2
4.8 ( 0.34
5.8 ( 0.3
3.0 ( 0.90
5.5 ( 0.4
4.7 ( 0.2
1.7 ( 0.7
6.7 ( 1.3
ND
16.9 ( 4.0
7.5 ( 1.6
25.9 ( 4.8
35.2 ( 9.7
5.3 ( 0.8
19.6 ( 4.2
5.1 ( 1.0
9.2 ( 1.1
17.1 ( 0.13
3.2 ( 0.3
7.5 ( 1.4
36.4 ( 0.6
34.9 ( 2.5
52.0 ( 14.0
20
45
10
40
67
41
62
24
25
81
38
5
6j
6o
7e
9e
monkey
rat
dog
26.7 ( 1.0
0
54
0
monkey
0.32
a The results are shown as the mean ( SE (n ) 3). Cassette assay data except the dog PK data of 6o. ND ) not determined. b F ) bioavailability.

3068 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Hattori et al.
Table 6. Inhibitory Effect of ꢀ3 Agonists on Increase in IVP, Induced by Carbachol in Anesthetized Dogsa
in vivo
in vitrob
idb
human ꢀ3
EC50 (nM)
dog ꢀ3
EC50 (nM)
iv (10 µg/kg),c
inhibition (%)
ED50
(µg/kg)
EC50
(nM)
bladder-relaxation,e
EC50 (nM)
PBd
6b
6e
6f
6h
6j
6o
7e
9e
2b
2j
2o
3e
4j
3.1 ( 0.1
0.43 ( 0.06
0.60 ( 0.08
0.60 ( 0.05
0.46 ( 0.08
0.32 ( 0.03
0.13 ( 0.005
0.029 ( 0.003
2.0 ( 0.06
0.60 ( 0.12
0.56 ( 0.10
0.26 ( 0.02
0.066 ( 0.004
4.9 ( 2.0
0.83 ( 0.08
2.9 ( 1.0
1.1 ( 0.05
0.88 ( 0.12
0.68 ( 0.05
1.4 ( 0.05
1.2 ( 0.2
2.9 ( 0.4
2.2 ( 0.1
1.2 ( 0.1
1.3 ( 0.2
3.2 ( 0.4
51
58
35
NT
NT
NT
NT
NT
NT
NT
NT
NT
90
92
90
83
76
86
86
NT
>50
NT
>70
>70
>70
>70
>70
>70
NT
NT
NT
NT
34.9 ( 17.5
24.7 ( 6.6
14.5 ( 6.6
16.2 ( 4.6
14.3 ( 5.2
25.9 ( 6.9
45.9 ( 26.9
47.0 ( 19.2
65.0 ( 31.6
16.1 ( 9.5
7.1 ( 0.9
14.2 ( 1.4
8.2 ( 0.2
26.5 ( 0.8
1.2 ( 0.06
20.0 ( 4.0
18.7 ( 5.6
11.2 ( 3.9
4.0 ( 0.5
1.8 ( 1.0
1.2 ( 0.2
2.6 ( 1.7
11.4 ( 4.2
62.0 ( 3.8
4.4 ( 1.2
6.8 ( 0.2
8.7 ( 0.1
11.0 ( 0.5
14.1 ( 0.8
3.6 ( 0.1
NT
89
80
a Details of experimental methods are in refs 9 and 11. NT ) not tested. b The results are shown as the mean ( SE (n g 3). c The results are shown as
the average of two experiments (n ) 2). d PB ) dog protein binding (n ) 2). e The relaxing effect on the KCl-induced dog bladder strips (n ) 3). See
Experimental Section.
Figure 2. Plot of effect of target compounds on disparity index, showing pEC50(cAMP accumulation on dogs) - pEC50(bladder relaxation on
dogs) vs lipophilicity: clogP and clogD, pH 6.8.
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-3-isopropoxy-
N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride (6b).
Compound 6b was synthesized from 10 according to procedure A.
Anal. for C27H35ClN2O5S. ESI-MS (m/z): 497 (M + H)+. NMR
(200 MHz, DMSO-d6) δ: 1.37 (6H, d, J ) 5.7 Hz), 3.06-3.25
(6H, m), 3.38 (3H, s), 4.97-5.00 (2H, m), 6.23 (1H, broad s),
7.28-7.48 (9H, m), 7.72-7.79 (3H, m).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-N-(methyl-
sulfonyl)-3-(pentyloxy)biphenyl-4-carboxamide Hydrochloride (6c).
Compound 6c was synthesized from 10 according to procedure A.
Anal. for C29H37ClN2O5S. ESI-MS (m/z): 562 (M + H)+. NMR
(200 MHz, DMSO-d6) δ: 0.91 (6H, d, J ) 5.4 Hz), 1.25-1.58
(4H, m), 1.75-1.85 (2H, m), 3.0-3.35 (6H, m), 3.36 (3H, s), 4.24
(2H, t, J ) 5.4 Hz), 4.95-5.03 (1H, m), 6.23 (1H, broad s), 7.3-7.5
(9H, m), 7.73-7.76 (3H, m).
3-(Cyclopentyloxy)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride
(6d). Compound 6d was synthesized from 10 according to procedure
A. Anal. for C29H35ClN2O5S·0.2H2O. ESI-MS (m/z): 560 (M +
H)+. NMR (200 MHz, DMSO-d6) δ: 1.6-2.1 (8H, m), 1.75-1.85
(2H, m), 3.0-3.35 (6H, m), 3.37 (3H, s), 4.97-5.01 (1H, m), 5.22
(1H, m), 6.21 (1H, broad s), 7.3-7.5 (9H, m), 7.72-7.74 (3H, m).
3-(Cyclohexyloxy)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide hydrochloride
(6e). Typical Procedure A. (1) To a solution of tert-butyl [2-(4-
bromophenyl)ethyl][(2R)-2-hydroxy-2-phenylethyl]carbamate (550
mg) in 1,2-dimethoxyethane (10 mL) was added 2-cyclohexyloxy-
N-(methylsulfonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
candidates that provide best-in-class properties in addition to an
acceptable safety profile.
Experimental Section
Chemistry. General Methods. Reactions involving air- or
moisture-sensitive reagents were carried out under a nitrogen atmo-
sphere. If not specified, reactions were carried out at ambient
temperature. Silica gel (Kanto Chemical, 63-210 µm) was used for
chromatographic purification unless otherwise indicated. Anhydrous
solvents were obtained from commercial sources. Proton NMR spectra
were recorded on a Bruker BioSpin Avance 400 or DPX 200. Values
in ppm relative to tetramethylsilane are given. The following abbrevia-
tions are used to describe peak patterns when appropriate: br ) broad,
s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet. High
resolution mass spectra were recorded with Micromass LCT. Chemical
purity was given by HPLC analysis with a Shiseido Capcell pack C18
column (detection at 254 nm), confirming >95% purity. Results of
elemental analysis were recorded with Perkin-Elmer 2400II and were
within 0.4% of the theoretical values calculated for C, H, and N unless
otherwise noted.
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-3-methoxy-
N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride (6a).
Compound 6a was synthesized from 10 according to procedure A.
Anal. for C25H29ClN2O5S. ESI-MS (m/z): 469 (M + H)+. 1H NMR
(200 MHz, DMSO-d6) δ: 2.99-3.27 (6H, m), 3.36 (3H, s), 3.98
(3H, s), 4.95-5.06 (1H, m), 6.23 (1H, d, J ) 4.0 Hz), 7.29-7.44
(9H, m), 7.65-7.8 (3H, m).

Biphenylacylsulfonamide Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3069
Table 7. Demonstrating the Effect on Potency of Changing i-Pro to c-Hex on RHSa
compd
i-Pro f
h-ꢀ3 (pEC50)
d-ꢀ3 (pEC50)
d-mag (pEC50)b
pEC50 (c-Hex) - pEC50(i-Pro)
c-Hex
i-Pro
c-Hex
i-Pro
c-Hex
i-Pro
c-Hex
h-ꢀ3
d-ꢀ3
d-mag
3b
6h
8h
9b
3e
6i
8i
8.8
9.2
9.7
9.9
9.6
9.9
10.2
10.5
8.0
9.0
8.6
8.0
8.9
9.2
9.0
8.9
8.1
8.9
8.9
8.1
8.3
7.9
8.9
8.4
+0.8
+0.7
+0.5
+0.6
+0.9
+0.2
+0.4
+0.9
+0.2
-1.0
0
9e
+0.3
a Set of compounds illustrated in Figure 3. b The relaxing effect on the KCl-induced dog bladder strips.
Table 8. Demonstrating the Effect on Potency of Changing Aryl Moiety
on LHSa
h-ꢀ3
d-ꢀ3
d-magd
no. of
mean change
Ar
pairs meanb SDc meanb SDc meanb SDc in clogPe
3-CIPh
3-Py
3-(6-NH2Py)
3
8
4
+0.13 0.18 +0.25 0.19 -0.91 0.51
+0.31 0.18 -0.39 0.18 +0.08 0.21
+1.13 0.17 -0.31 0.13 +0.63 0.36
0.7
-1.5
-1.8
Figure 3. Matched molecular pairs for substitution on biphenyl
analogues in Tables 7 and 8.
a Set of compounds illustrated in Figure 3. b The mean value of (pEC50
for Ar-B) - (pEC50 for Ph-B). c Standard deviation. d The relaxing effect
on the KCl-induced dog bladder strips. e The mean of the distribution of
values of (clogP for Ar-B) - (clogP for Ph-B).
J ) -192.6 Hz), 6.86 (1H, d, J ) 8.4 Hz), 6.97 (1H, s), 7.3-7.42
(7H, m), 7.7 (2H, d, J ) 8.1 Hz), 7.83 (1H, d, J ) 8.4 Hz), 8.94
(1H, b.s), 9.35 (1H, b s).
benzamide (275 mg), tetrakis(triphenylphosphine)palladium (55
mg), and aqueous solution of sodium carbonate (2 M, 2.0 mL),
and the mixture was stirred at 80 °C for 6 h under nitrogen. The
mixture was diluted with ethyl acetate, washed with 1 N aqueous
hydrochloride solution, water, and brine, dried over magnesium
sulfate, and evaporated under reduced pressure. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate ) 2/1) to give tert-butyl [(2R)-2-hydroxy-2-phenylethyl][2-
(3′-(3-cyclohexyloxy)-4′-{[(methylsulfonyl)amino]carbonyl}-4-bi-
phenylyl)ethyl]carbamate (380 mg).
(2) To a solution of the product (89 mg) in AcOEt (2 mL) was
added hydrochloric acid in AcOEt solution (4 N, 4 mL) at room
temperature, and the mixture was stirred at the same temperature
for 2 h. The resultant solid was collected by filtration and dried to
give 3-(cycloheyloxy)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)-4-biphenylcarboxamide hydrochloride (76
mg). Anal. for C30H37ClS. ESI-MS (m/z): 537(M + H)+. NMR (200
MHz, DMSO-d6) δ: 1.32-1.81 (8H, m), 1.89-2.02 (2H, m),
2.98-3.30 (6H, m), 3.38 (3H, s), 4.75-4.87 (1H, m), 4.98-5.03
(1H, m), 6.23 (1H, d, J ) 3.8 Hz), 7.31-7.42 (9H, m), 7.71-7.80
(3H, m).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-N-(methyl-
sulfonyl)-3-phenoxybiphenyl-4-carboxamide Hydrochloride (6f).
Compound 6f was synthesized from 10 according to procedure A.
Anal. for C30H31ClN2O5S·3H2O. ESI-MS (m/z): 531 (M + 1)+.
NMR (200 MHz, DMSO-d6) δ: 3.0-3.3 (6H, m), 3.33 (3H, s),
4.82 (1H, m), 5.02 (1H, m), 6.20 (1H, m), 7.1-7.5 (12H, m),
7.5-7.9 (5H, m).
3-(Cycloheptyloxy)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride
(6g). Compound 6g was synthesized from 10 according to procedure
A. Anal. for C31H39ClN2O5S·0.2H2O. ESI-MS (m/z): 588 (M +
1)+. NMR (200 MHz, DMSO-d6) δ: 1.4-1.84 (10H, m), 2.02-2.12
(2H, m), 2.98-3.30 (6H, m), 3.38 (3H, s), 4.75-4.87 (1H, m),
4.98-5.03 (1H, m), 6.21 (1H, d, J ) 3.8 Hz), 7.33-7.43 (9H, m),
7.71-7.80 (3H, m).
3-(Cyclohexylamino)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydrochloride
(6i). Compound 6i was synthesized from 10 according to procedure
A. Anal. for C30H39Cl2N3O4S·4H2O. ESI-MS (m/z): 536.2 (M +
H)+. 1H NMR (200 MHz,) δ: 1.14-1.66 (8H, m), 1.91-1.99 (2H,
m), 3.08-3.37 (6H, m), 3.37 (3H, s), 3.55-3.72 (1H, m), 5 (1H,
d, J ) 7.6 Hz), 6.83 (1H, d, J ) 8.2 Hz), 6.97 (1H, s), 7.32-7.42
(7H, m), 7.68 (2H, d, J ) 8 Hz), 7.82 (1H, d, J ) 8.4 Hz), 8.93
(1H, b s), 9.29 (1H, b s).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-3-isobutyl-
N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride (6j). Com-
pound 6j was synthesized from 10 according to procedure A. Anal.
for C28H35ClN2O4S. ESI-MS (m/z):493 (M -H)-. NMR (400 MHz,
DMSO-d6) δ: 0.87 (6H, d, J ) 6.5 Hz), 1.82-1.86 (1H, m), 2.73
(2H, d, J ) 7.0 Hz), 3.02-3.08 (3H, m), 3.19-3.23 (3H, m), 3.36
(3H,s), 4.95-5.00 (1H, m), 6.22 (1H, br), 7.32-7.41 (7H, m),
7.53-7.61 (3H, m), 7.70 (2H, d, J ) 8.0 Hz), 8.83 (1H, br), 9.12
(1H, br).
3-Cyclopentyl-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)-
N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride (6k).
Compound 6k was synthesized from 10 according to procedure A.
Anal. for C29H35ClN2O4S·0.2H2O. ESI-MS (m/z):505 (M - H)-.
NMR (200 MHz, DMSO-d6) δ: 1.65-1.81 (6H, m), 1.99-2.05
(2H, m), 3.04-3.33 (7H, m), 3.38 (3H, s), 4.95-5.00 (1H, m),
6.22 (1H, d, J ) 3.8 Hz), 7.31-7.42 (7H, m), 7.46-7.58 (2H, m),
7.67 (1H, d, J ) 7.3 Hz), 7.69 (2H, d, J ) 8.1 Hz), 8.86 (1H, br),
9.10 (1H, br), 12.21 (1H, br).
3-Cyclohexyl-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)-
N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride (6l). Com-
pound 6l was synthesized from 10 according to procedure A. Anal.
for C30H37ClN2O4S. ESI-MS (m/z): 519 (M - H)-. NMR (200
MHz, DMSO-d6) δ: 1.28-1.84 (10H, m), 2.84-2.95 (2H, m),
3.04-3.24 (6H, m), 3.38 (3H, S), 4.95-5.00 (1H, m), 6.23 (1H, d,
J ) 3.8 Hz), 7.31-7.40 (7H, m), 7.42-7.71 (5H, m), 8.86 (1H,
br), 9.10 (1H, br), 12.2 (1H, br).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-3-(isopropy-
lamino)-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydrochlo-
ride (6h). Compound 6h was synthesized from 10 according to
procedure A. Anal. for C27H35Cl2N3O4S·H2O. ESI-MS (m/z): 494.2
3-(Cyclohexylmethyl)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride
(6m). Compound 6mwas synthesized from 10 according to proce-
dure A. Anal. for C31H39ClN2O4S·0.5H2O. ESI-MS (m/z):533 (M
- H)-. NMR (200 MHz, DMSO-d6) δ: 0.91-1.17 (5H, m),
1
(M - H)-. H NMR (200 MHz,) δ: 1.24 (6H, d, J ) 6.2 Hz),
2.98-3.32 (6H, m), 3.37 (3H, s), 3.89-3.98 (1H, m), 5.51 (1H, d,

3070 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Hattori et al.
1.53-1.72 (6H, m), 2.74 (2H, d, J ) 6.3 Hz), 3.05-3.31 (6H, m),
3.37 (3H, s), 4.93-5.00 (1H, m), 6.22 (1H, d, J ) 3.8 Hz),
7.28-7.42 (7H, m), 7.51-7.61 (3H, m), 7.69 (2H, d, J ) 8.1 Hz),
8.90 (1H, br), 9.22 (1H, br).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-N-(methyl-
sulfonyl)-3-(propylsulfanyl)biphenyl-4-carboxamide Hydrochloride
(6n). Compound 6n was synthesized from 10 according to procedure
A. Anal. for C27H33ClN2O4S2. ESI-MS (m/z):511 (M - H)-. NMR
(200 MHz, DMSO-d6) δ: 1.25 (3H, t, J ) 7.3 Hz), 1.57-1.68 (2H,
m), 3.03 (2H, t, J ) 7.2 Hz), 3-3.34 (6H, m), 3.37 (3H, s), 4.96-5.01
(1H, m), 6.23 (1H, d, J ) 3.8 Hz), 7.3-7.42 (7H, m), 7.54 (1H, d, J
) 8.1 Hz), 7.63-7.67 (2H, m), 7.73 (2H, d, J ) 8.2 Hz).
4′-(2-{[(2R)-2-Hydroxy-2-phenylethyl]amino}ethyl)-3-(isopropyl-
sulfanyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochlo-
ride (6o). Compound 6o was synthesized from 10 according to
procedure A. Anal. for C27H33ClN2O4S2. ESI-MS (m/z): 511 (M -
H)-. NMR (200 MHz, DMSO-d6) δ: 1.26 (6H, d, J ) 6.6 Hz),
3.00-3.30 (6H, m), 3.65 (1H, m), 4.95-5.00 (1H, m), 6.22 (1H, d, J
) 3.7 Hz), 7.30-7.42 (7H, m), 7.61 (2H, s), 7.70-7.74 (3H, m).
3-(Cyclohexylsulfanyl)-4′-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Hydrochloride
(6p). Compound 6p was synthesized from 10 according to procedure
A. Anal. for C30H37ClN2O4S2. ESI-MS (m/z): 551 (M - H)-. NMR
(200 MHz, DMSO-d6) δ: 1.14-1.99 (10H, m), 3.04-3.42 (6H,
m), 3.36 (3H, s), 4.94-4.99 (1H, m), 6.22 (1H, d, J ) 3.8 Hz),
7.28-7.42 (7H, m), 7.6 (2H, s), 7.69 (1H, s), 7.73 (2H, s), 8.85
(1H, b s), 9.04 (1H, b s), 12.19 (1H, b s).
4′-(2-{[(2R)-2-Hydroxy-2-pyridin-3-ylethyl]amino}ethyl)-3-isobu-
tyl-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydrochloride (7j).
Compound 7j was synthesized from 11 according to procedure B.
Anal. for C27H35Cl2N3O4S·2H2O. ESI-MS (m/z): 494 (M - H)-.
NMR (200 MHz, DMSO-d6) δ: 0.87 (6H, d, J ) 6.5 Hz), 1.77-1.87
(1H, m), 2.74 (2H,d, J ) 7.0 Hz), 3.06-3.37 (6H, m), 3.57 (3H,
s), 5.22-5.25 (1H, m), 7.37-7.73 (8H, m), 7.82-7.89 (1H, m),
8.31-8.35 (1H, m), 8.77 (1H, d,J ) 4.3 Hz), 8.83 (1H, s), 9.14
(1H, br), 9.26 (1H, br), 12.2 (1H, br).
3-Cyclopentyl-4′-(2-{[(2R)-2-hydroxy-2-pyridin-3-ylethyl]amino}-
ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydrochloride
(7k). Compound 7k was synthesized from 11 according to procedure
B. Anal. for C28H35Cl2N3O4S2 ·2H2O. ESI-MS (m/z): 507 (M - H)-.
NMR (200 MHz, DMSO-d6) δ: 1.65-1.81 (6H, m), 1.99-2.05
(2H, m), 3.06-3.36 (7H, m), 3.40 (3H, s), 5.24-5.28 (1H,m),
7.37-7.71 (8H, m), 7.88-7.95 (1H, m), 8.38-8.42 (1H, m), 8.80
(1H, d, J ) 4.3 Hz), 8.86 (1H, s), 9.20 (1H, br), 9.33 (1H, br),
12.2 (1H, br).
4′-(2-{[(2R)-2-Hydroxy-2-pyridin-3-ylethyl]amino}ethyl)-3-(iso-
propylsulfanyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Dihy-
drochloride (7o). Compound 7o was synthesized from 11 according
to procedure B. Anal. for Anal. for C26H33Cl2N3O4S2 ·1.8H2O. ESI-
MS (m/z): 514 (M + H)+. NMR (200 MHz, DMSO-d6) δ: 1.26
(6H, d, J ) 6.6 Hz), 2.99-3.75 (10H, m), 4.93-5.08 (1H, m),
6.33 (1H, bs), 7.36-7.45 (4H, m), 7.58 (1H, s), 7.69 (3H, d, J )
7.5 Hz), 8.01 (1H, d, J ) 8.4 Hz), 8.55 (1H, dd, J ) 1.5, 4.8 Hz),
8.62 (1H, d, J ) 1.7 Hz).
4′-(2-{[(2R)-2-Hydroxy-2-pyridin-3-ylethyl]amino}ethyl)-3-iso-
propoxy-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydrochlo-
ride (7b). Typical Procedure B. (1) Suzuki coupling was performed
with 11 instead of 10 in the same procedure as procedure A.
(2) To a solution of the product (100 mg) in 1,4-dioxane (2 mL)
was added hydrochloric acid in 1,4-dioxane solution (4 N, 4 mL)
at room temperature, and the mixture was stirred at the same
temperature for 2 h. The solution was evaporated under reduced
pressure. The resultant solid was washed with ether and collected
to give 4′-(2-{[(2R)-2-hydroxy-2-pyridin-3-ylethyl]amino}ethyl)-
3-isopropoxy-N-(methylsulfonyl)biphenyl-4-carboxamide dihydro-
chloride (92 mg). Anal. for C26H33Cl2N3O5S·1.5H2O. ESI-MS (m/
z): 498 (M + H)+. NMR (200 MHz, DMSO-d6) δ: 1.30 (6H, d, J
) 6.0 Hz), 2.9-3.4 (6H, m), 3.48 (3H, s), 4.6-5.3 (2H, m), 7.2-8.0
(8H, m), 8.4-8.9 (3H, m).
3-(Cyclohexyloxy)-4′-(2-{[(2R)-2-hydroxy-2-pyridin-3-
ylethyl]amino}ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Di-
hydrochloride (7e). Compound 7e was synthesized from 11
according to procedure B. Anal. for C29H37Cl2N3O5S·1.3H2O. ESI-
MS (m/z): 538 (M + 1)+. NMR (200 MHz, DMSO-d6) δ: 1.2-2.1
(10H, m), 3.0-3.6 (5H, m), 4.81 (1H, m), 5.20 (1H, m), 7.2-7.4
(4H, m), 7.7-7.7.9 (3H, m), 7.9-8.0 (1H, m), 8.46 (1H, m),
8.82-8.89 (2H, m).
4′-(2-{[(2R)-2-Hydroxy-2-pyridin-3-ylethyl]amino}ethyl)-3-(iso-
propylamino)-N-(methylsulfonyl)biphenyl-4-carboxamide Trihy-
drochloride (7h). Compound 7 h was synthesized from 11 according
to procedure B. Anal. for C26H35Cl3N4O4S·2.1H2O. ESI-MS (m/
z): 495.2 (M - H)-. NMR (200 MHz, DMSO-d6) δ: 1.24 (6H, d,
J ) 3.1 Hz), 3.07-3.11 (2H, m), 3.18-3.31 (3H, m), 3.37 (3H, s),
3.37-3.43 (1H, m), 3.9-3.93 (1H, m), 5.35 (1H, dd, J ) 1.5, 4.4
Hz), 6.86 (1H, d, J ) 4.2 Hz), 6.97 (1H, s), 7.38 (2H, d, J ) 4.1
Hz), 7.7 (2H, d, J ) 4.1 Hz), 7.83 (1H, d, J ) 4.2 Hz), 8.08 (1H,
dd, J ) 2.8, 4 Hz), 8.6 (1H, d, J ) 4 Hz), 8.89 (1H, d, J ) 2.8
Hz), 8.95 (1H, s), 9.34 (1H, br), 9.44 (1H, br).
3-(Cyclohexylamino)-4′-(2-{[(2R)-2-hydroxy-2-pyridin-3-
ylethyl]amino}ethyl)-N-(methylsulfonyl)biphenyl-4-carboxamide Tri-
hydrochloride (7i). Compound 7i was synthesized from 11 accord-
ing to procedure B. Anal. for C29H39Cl3N4O4S·1.8H2O. ESI-MS
(m/z): 535.2 (M - H)-. 1H NMR (200 MHz, DMSO-d6) δ:
1.61-1.67 (8H, m), 1.91-1.99 (2H, m), 2.99-3.48 (6H, m),
3.55-3.72 (1H, m), 6.84 (1H, d, J ) 8.4 Hz), 6.97 (1H, s), 7.38
(2H, d, J ) 8.2 Hz), 7.69 (2H, d, J ) 8.1 Hz), 7.82 (1H, d, J ) 8.4
Hz), 8.08 (1H, dd, J ) 5.7, 8.1 Hz), 8.61 (1H, d, J ) 8.2 Hz), 8.89
(1H, d, J ) 5.5 Hz), 8.95 (1H, s), 9.34 (2H, b s).
4′-(2-{[(2R)-2-(4-Aminophenyl)-2-hydroxyethyl]amino}ethyl)-3-
isopropoxy-N-(methylsulfonyl)biphenyl-4-carboxamide Dihydro-
chloride (8b). Compound 8b was synthesized from 12 according
to procedure C. Anal. for C27H35Cl2N3O5S·1.2H2O. ESI-MS (m/
z): 512 (M + H)+. NMR (200 MHz, DMSO-d6) δ: 1.31 (6H, d, J
) 6.0 Hz), 3.0-3.3 (6H, m), 3.34 (3H, s), 5.02 (1H, m), 7.1-7.5
(8H, m), 7.6-7.9 (3H, m), 8.9 (1H, m), 9.2 (1H, m).
4′-(2-{[(2R)-2-(4-Aminophenyl)-2-hydroxyethyl]amino}ethyl)-3-
(cyclohexyloxy)-N-(methylsulfonyl)biphenyl-4-carboxamide Dihy-
drochloride (8e). Typical Procedure C. (1) tert-Butyl [2-[3′-
(cyclohexyloxy)-4′-[[(methylsulfonyl)amino]carbonyl]-4-biphenylyl]-
ethyl][(2R)-2-hydroxy-2-(4-nitrophenyl)ethyl]carbamate was ob-
tained from 12 in a method similar to procedure A.
(2) A mixture of the product (281 mg), iron powder (69.1 mg),
ammonium chloride (11 mg), ethanol (4.2 mL), and water (1.4 mL)
was refluxed for 1 h. After the mixture was cooled to room
temperature, the insoluble solid was filtered off through a Celite
pad and washed with ethyl acetate (20 mL). The filtrate was washed
with brine (20 mL) and dried over magnesium sulfate. Filtration
followed by evaporation gave a yellow foam (271 mg) which was
chromatographed on silica gel (eluent, hexane/ethyl acetate) to give
tert-butyl [(2R)-2-(4-aminophenyl)-2-hydroxyethyl][2-[3′-(cyclo-
hexyloxy)-4′-[[(methylsulfonyl)amino]carbonyl]-4-biphenylyl]eth-
yl]carbamate (104 mg) as a pale-yellow solid.
(3) To a solution of the product (81.8 mg) in dioxane (1 mL)
was added 4 N hydrogen chloride in ethyl acetate (1 mL), and the
mixture was stirred at room temperature for 3 h. The precipitates
were collected by filtration, washed with ethyl acetate, and dried
under reduced pressure to give 4′-(2-{[(2R)-2-(4-aminophenyl)-
2-hydroxyethyl]amino}ethyl)-3-(cyclohexyloxy)-N-(methylsulfonyl)-
biphenyl-4-carboxamide dihydrochloride (64.5 mg) as an off-white
solid. Anal. for C30H39Cl2N3O5S·1.4H2O. ESI-MS (m/z): 550 (M
- H)-. NMR (400 MHz, DMSO-d6,) δ: 1.31-1.64 (6H, m),
1.69-1.77 (2H, m), 1.92-1.98 (2H, m), 3.01-3.26 (6H, m), 3.39
(3H, s), 4.79-4.84 (1H, m), 4.99 (1H, dd, J ) 2.2, 10.3 Hz), 7.25
(2H, d, J ) 7.7 Hz), 7.35-7.45 (6H, m), 7.74 (2H, d, J ) 8.1 Hz),
7.78 (1H, d, J ) 8.1 Hz), 8.91 (1H, br), 9.25 (1H, br), 9.74 (1H,
br), 11.2 (1H, br).
4′-(2-{[(2R)-2-(6-Aminopyridin-3-yl)-2-hydroxyethyl]amino}ethyl)-
3-ethoxy-N-(methylsulfonyl)biphenyl-4-carboxamide (9q). Typi-
cal Procedure D. (1) A mixture of tert-butyl [(2R)-2-hydroxy-2-
phenylethyl][2-(4-iodophenoxy)ethyl]carbamate (300 mg), 2-ethoxy-
N-(methylsulfonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzamide (174 mg), [1,1′-bis(diphenylphosphino)ferrocene]-

Biphenylacylsulfonamide Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3071
dichloropalladium(II) complex with dichloromethane (1:1, 62 mg),
1,1′-bis(diphenylphosphino)ferrocene (42.1 mg), N,N-dimethylfor-
mamide (6 mL), and 2 N sodium carbonate solution (1.01 mL)
was stirred at 80 °C for 3 h. After cooling to room temperature,
the mixture was quenched by the addition of pH 6.86 buffer (30
mL) and extracted with ethyl acetate (20 mL × 1, 10 mL × 1).
The extracts were combined and washed with pH 6.86 buffer
(20 mL × 2) and brine (20 mL) and dried over magnesium
sulfate. Filtration followed by evaporation gave a light-brown
solid which was chromatographed on silica gel (eluent, hexane/
ethyl acetate) to give tert-butyl [(2R)-2-[6-(acetylamino)-3-
pyridinyl]-2-[[tert-butyl(dimethyl)silyl]oxy]ethyl][2-[3′-ethoxy-4′-
[[(methylsulfonyl)amino]carbonyl]-4-biphenylyl]ethyl]carbam-
ate (259 mg) as a white solid.
detrusor of about 2 mm width and 8 mm length were prepared.
Detrusor strips were suspended in orgon baths containing 25
mL of oxygenated Krebs-Henseleit solution (118 mM NaCl,
4.7 mM KCl, 1.2mM KH2PO4, 25 mM NaHCO3, 1.2 mM
MgSO4, 2.5 mM CaCl2, 11.1 mM glucose) at 37 °C. The tension
of the strips was measured isometrically with a force displace-
ment transducer coupled to a carrier amplifier. Resting tension
was adjusted to 0.5 g.
KCl (20 nM) was added at about 30 min intervals. After
confirmation of reproducibility of the response, test compound was
added 15 min prior to addition of KCl. The protocol was
noncumulative with rinse cycles between each concentration of the
test compounds. Test compound was added several times, increasing
from the lowest concentration. Contractile responses were expressed
as a percentage of the contraction before addition of the test
compound.
(2) To a solution of the product (237 mg) in ethanol (2.4 mL)
was added 1 N sodium hydroxide (3.14 mL), and the mixture was
refluxed for 18 h. After cooling to room temperature, the mixture
was quenched by the addition of 1 N hydrochloric acid (3.14 mL)
and the solvent was removed by evaporation.
The compound was dissolved in DMSO (10-2 M), then diluted
with distilled water and added at final concentrations of 10-10, 10-9
,
10-8, 10-7, 10-6, and 10-5 M. Vehicle was added at corresponding
concentrations. Volumes added were all 25 µL.
(3) To the residue were added 4 N hydrogen chloride in dioxane
(4 mL) and methanol (1 mL), and the mixture was stirred at room
temperature for 15 h. The solvent was concentrated in vacuo, and
the residue was dissolved in water (5 mL) and treated with activated
carbon. After stirring for 2 h, the mixture was filtered and the pH
of the filtrate was adjusted to 7 by the addition of 1 N NaOH. The
precipitates were collected by filtration, washed with water, and
dried under reduced pressure at 50 °C to give 4′-[2-[[(2R)-2-(6-
amino-3-pyridinyl)-2-hydroxyethyl]amino]ethyl]-3-ethoxy-N-(me-
thylsulfonyl)-4-biphenylcarboxamide (123 mg) as an off-white solid.
Anal. for C25H30N4O5S·2.8H2O. ESI-MS (m/z): 497 (M - H)-.
NMR (400 MHz, DMSO-d6) δ: 1.27 (3H, t, J ) 7.0 Hz), 2.88-3.14
(9H, m), 4.15 (2H, q, J ) 7.0 Hz), 4.67 (1H, t, J ) 6.6 Hz), 5.73
(1H, br), 5.92 (2H, br), 6.43 (1H, d, J ) 8.4 Hz), 7.18-7.20 (2H,
m), 7.32 (2H, d, J ) 8.4 Hz), 7.37 (1H, dd, J ) 2.2, 8.4 Hz), 7.46
(1H, d, J ) 8.4 Hz), 7.65 (2H, d, J ) 8.4 Hz), 7.88 (1H, d, J ) 2.2
Hz).
Percentage inhibition was expressed as the mean ( SE. EC50
values were calculated by linear regression analysis, if possible.
Statistical analysis was performed using analysis of variance, based
on randomized block design, followed by Dunnutt’s multiple
comparisons.
In Vitro Metabolism. Incubation mixtures (0.5 mL) contained
rat hepatocytes (2 × 106 cells/mL) and 100 mM phosphate buffer
(pH 7.4). After preincubation at 37 °C for 5 min, the reaction was
started by addition of 1 (substrate concentration of 10 µM).
Incubations were carried out at 37 °C for 120 min, and the reactions
were stopped by adding acetonitrile (0.5 mL). The mixtures were
vortexed and centrifuged at 22000g for 5 min. The supernatant was
removed and analyzed by LC/MS/MS.
Acknowledgment. We express our thanks to Dr. David
Barrett for critical reading of the manuscript.
4′-(2-{[(2R)-2-(6-Aminopyridin-3-yl)-2-hydroxyethyl]amino}ethyl)-
3-isopropoxy-N-(methylsulfonyl)biphenyl-4-carboxamide (9b). Com-
pound 9b was synthesized from 13 according to procedure D. Anal.
for C26H32N4O5S·2.7H2O. ESI-MS (m/z): 511 (M - H)-. NMR
(400 MHz, DMSO-d6) δ: 1.27 (6H, d, J ) 6.2 Hz), 2.88-2.99
(4H, m), 2.98 (3H, s), 3.06-3.13 (2H, m), 4.63-4.73 (1H, m),
5.71 (1H, br), 5.91 (2H, brs), 6.43 (1H, d, J ) 8.4 Hz), 7.18-7.22
(2H, m), 7.32 (2H, d, J ) 8.4 Hz), 7.37 (1H, dd, J ) 2.6, 8.4 Hz),
7.47 (1H, d, J ) 7.7 Hz), 7.63 (2H, d, J ) 8.4 Hz), 7.88 (1H, d,
J ) 2.6 Hz), 8.02 (2H, br).
4′-(2-{[(2R)-2-(6-Aminopyridin-3-yl)-2-hydroxyethyl]amino}ethyl)-
3-(cyclohexyloxy)-N-(methylsulfonyl)biphenyl-4-carboxamide Di-
hydrochloride (9e). Compound 9e was synthesized from 13
according to procedure D. Anal. for C29H38Cl2N4O5S·3H2O. ESI-
MS (m/z): 551 (M - H)-. NMR (400 MHz, DMSO-d6) δ:
1.27-1.37 (3H, m), 1.43-1.58 (3H, m), 1.70-1.79 (2H, m),
1.82-1.91 (2H, m), 2.86-2.97 (2H, m), 2.99 (3H, s), 3.00-3.17
(4H, m), 4.47-4.53 (1H, m), 4.65-4.68 (1H, m), 5.73 (1H, br),
5.92 (2H, brs), 6.44 (1H, d, J ) 8.4 Hz), 7.18-7.21 (2H, m), 7.32
(2H, d, J ) 8.4 Hz), 7.37 (1H, dd, J ) 2.2, 8.4 Hz), 7.46 (1H, d,
J ) 8.4 Hz), 7.63 (2H, d, J ) 8.4 Hz), 7.88 (1H, d, J ) 2.2 Hz),
8.21 (2H, br).
4′-[2-[[(2R)-2-(6-Amino-3-pyridinyl)-2-hydroxyethyl]amino]et-
hyl]-3-(isopropylthio)-N-(methylsulfonyl)-4-biphenylcarboxam-
ide (9o). Compound 9o was synthesized from 13 according to
procedure D. Anal. for C26H32N4O4S2. ESI-MS (m/z): 527 (M -
H)-. NMR (400 MHz, DMSO-d6) δ: 1.26 (6H, d, J ) 6.6 Hz),
2.96-3.24 (9H, m), 3.64 (1H, heptuplet, J ) 6.6 Hz), 4.77 (1H,
m), 5.97 (1H, d, J ) 3.7 Hz), 6.03 (2H, s), 6.47 (1H, d, J ) 8.4
Hz), 7.36-7.42 (3H, m), 7.48 (1H, d, J ) 8.4 Hz), 7.61 (1H, s),
7.66-7.70 (3H, m), 7.90 (1H, d, J ) 2.2 Hz), 8.74 (2H, br).
Relaxation Response in a KCl Induced Tonic Concentration
in Dog Detrusor Muscle Strips. Female beagle dogs were
anesthetized with pentobarbital. The urinary bladder was col-
lected, and top, trigon, and mucosa were removed. Strips of the
Supporting Information Available: Biological materials and
methods and combustion analysis data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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