Beta(3)-adrenoceptor agonists for the treatment of frequent urination and urinary incontinence: 2-[4-(2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl)phenoxy]-2-methylpropionic acid.

Article abstract of DOI:10.1016/S0968-0896(01)00240-1

In a search for novel analogues of beta(3)-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-[[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino]ethyl)phenoxy]-2-methylpropionic acids (1a-e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective beta(3)-AR agonist in functional assays using the ferret detrusor (beta(3)-AR), rat uterus (beta(2)-AR), and rat atrium (beta(1)-AR); beta(3): EC(50)=7.8 nM, beta(2): IC(50)=7,300 nM, beta(1): EC(20)=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved beta(3)-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED(50)=31 microg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b-e), possess beta(3)-AR agonistic activity in the absence of undesirable beta(1)- or beta(2)-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.

Full text of DOI:10.1016/S0968-0896(01)00240-1

Bioorganic & Medicinal Chemistry 9 (2001) 3265–3271
ꢀ3-Adrenoceptor Agonists for the Treatment of Frequent
Urination and Urinary Incontinence: 2-[4-(2-{[(1S,2R)-2-
Hydroxy-2-(4-hydroxyphenyl)-1-
methylethyl]amino}ethyl)phenoxy]-2-methylpropionic Acid
Nobuyuki Tanaka,* Tetsuro Tamai, Harunobu Mukaiyama, Akihito Hirabayashi,
Hideyuki Muranaka, Takehiro Ishikawa, Satoshi Akahane and Masuo Akahane
Central Research Laboratory, Kissei Pharmaceutical Company Ltd., 4365-1, Hotaka, Nagano, 399-8304, Japan
Received 10 May 2001;accepted 27 June 2001
Abstract—In a search for novel analogues of b3-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dys-
function, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e),
into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective b3-AR ago-
nist in functional assays using the ferret detrusor (b3-AR), rat uterus (b2-AR), and rat atrium (b1-AR); b3: EC50=7.8 nM, b2:
IC50=7,300 nM, b1: EC20=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the
phenyl ring of 1a further improved b3-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED50=31 mg/kg) in
rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e),
possess b3-AR agonistic activity in the absence of undesirable b1- or b2-AR mediated actions, and may be useful for clinical treat-
ment and pharmacological studies. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
years ago.10,11 Since that time, a number of b3-AR ago-
nists have been developed for the treatment of obesity
and type II diabetes.12,13 Unfortunately, some drugs
developed for such metabolic diseases failed to produce
similar effects in humans, due to differences between
humans and animals with regard to amino acid sequen-
ces of receptors, adverse reactions and/or pharmacoki-
netic actions of the drugs.14 New generations of b3-AR
agonists have been found using the human b3-AR
expressed in Chinese hamster ovary (CHO) cells.13
Recent reports suggest that the b3-AR mRNA is
expressed in human bladder and that b3-AR in human
bladder could play a significant role in urinary
storage.1ꢀ4 Moreover, exogenous b3-AR agonists pro-
long the micturition interval in a rat bladder hyper-
activity model.5,6 These findings offer the opportunity
for a new approach to the treatment of urinary
disorders using b3-AR agonists.
The b3-AR belongs to the Gs protein-coupled receptor
family that shares a common structure, consisting of
seven transmembrane-spanning helices connected by
interhelical loops. The endogenous catecholamines,
adrenaline and noradrenaline, stimulate this receptor,
which mediates metabolic functions such as lipolysis,
thermogenesis and motility process control in the gas-
trointestinal tract.7ꢀ9 The characterisation and expres-
sion of the gene encoding the b3-AR was established ten
It has not yet been determined whether the recombinant
human b3-AR assay is applicable for evaluation of
relaxation of smooth muscle in the human bladder. We
have reported that various b3-AR agonists mediate
relaxation of the ferret detrusor, as seen in humans.15
To discover a new class of potent and selective b3-AR
agonists as therapeutic drugs for urological dysfunction,
we have evaluated our synthetic compounds by a novel
functional assay using the ferret detrusor preparation
for analysis of b3-AR agonistic activity. We have also
employed conventional assays using rat atrium and rat
uterus for estimation of agonistic activity at b1-AR and
b2-AR respectively.16 A previous report showed that
*Corresponding author. Tel.: +81-263-82-8820;fax: +81-263-82-
8827;e-mail: nobuyuki_tanaka@pharm.kissei.co.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00240-1

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N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
2-(phenylamino)isobutyric acid derivatives had high b3-
AR selectivity against b1- and b2-AR.17 Optimising
these compounds, we found that the 2-phenoxy-
isobutyric acid derivative (1a) possessed potent b3-AR
agonistic activity and significant selectivity against b1-
and b2-AR. Replacement of the nitrogen with oxygen
preserved the b3-AR activity and selectivity, in spite of
the presence of a fibrate-like (anti-hyperlipidemic18)
component in the structure. The effect of substituents
on the phenyl ring of 1a on the b-ARs agonistic activity,
and the in vivo effects of 1a in rats are reported below.
reaction of 5a–e with bromoacetyl bromide provided
phenacyl bromides (6a–e). The phenethyl bromides (3a–e)
were obtained by reduction of 6a–e with triethylsilane
under acidic conditions.
Results and Discussion
All compounds in the in vitro study were evaluated for
ability to increase heart rate, using isolated rat atrium
(b1-AR agonistic activity), inhibition of spontaneous
motility on rat uterus (b2-AR agonistic activity), and
relaxation of ferret detrusor (b3-AR agonistic activity).
Selectivity was calculated by dividing b1- or b2-AR
activity by b3-AR activity. The results of the in vitro
study are shown in Table 1.
Chemistry
The 2-phenoxyisobutyric acid derivatives 1a–e, as
shown in Figure 1, were synthesized by N-alkylation of
(ꢀ)-40-hydroxynorephedrine (2)19 with phenethyl bro-
mide derivatives 3a–e under heating, followed by sapo-
nification via the corresponding esters. The phenethyl
bromides (3a–e) were prepared as shown in Scheme 1.
Phenols (4a–e) were converted to 2-phenoxyisobutyric
acid esters (5a–e) using 1,1,1-trichloro-2-methyl-2-pro-
panol hemihydrate (Chloretone).20 Friedel–Crafts
The key compound 1a consists of phenylethanolamine,
a structure known to be required for b-agonistic activ-
ity, and 2-phenoxyisobutyrate, a structure characteristic
of conventional fibrates. Interestingly, this simple struc-
ture led to 10-fold more potent b3-AR agonistic activity
and significantly higher selectivity for b3-AR against b1-
and b2-AR than isoproterenol (1a: EC50=7.8 nM, b1/
b3=2,940-fold, b2/b3=930-fold). It has previously been
convincingly shown that improvement of b3-AR ago-
nistic activity or selectivity was dependent on the posi-
tion of halogens on the phenyl ring bearing the
Scheme 1. General synthesis of intermediates 3. Reagents: (a) Chlor-
etone, NaOH;(b) SOCl 2, EtOH;(c) bromoacetylbromide, AlCl 3;(d)
Et3SiH, TFA.
Figure 1. General formula of 2-phenoxyisobutyric acids (1) as a novel
b3-adrenoceptor agonist and synthetic intermediates.
Table 1. Structure and b-AR Agonistic Activity of 2-Phenoxyisobutyric acids (1)
Compounds
R1
R2
Anal.
b1-AR
b2-AR
b3-AR
Selectivityf
(CHN)a
pEC20ꢁS.E.
pIC50ꢁS.E.
pEC50ꢁS.E.
b1/b3
b2/b3
(EC20:nM)b
(IC50:nM)c
IA.d (EC50:nM)e
.
C21H27NO5 0.8H2O
1a
H
Me
H
H
H
4.64ꢁ0.03
(23,000)
4.96ꢁ0.02
(11,000)
< 4
(100,000<)
5.19ꢁ0.08
(6,500)
5.14ꢁ0.58
(7,300)
8.11ꢁ0.08
0.87 (7.8)
8.32ꢁ0.10
0.86 (4.8)
7.85ꢁ0.09
0.97 (14)
2,949
2,292
936
438
.
C22H29NO5 1.2H2O
1b
5.68ꢁ0.77
(2,100)
< 4
1c
Me
H
C22H29NO5
7,143<
342
7,143<
163
(100,000<)
5.51ꢁ0.03
(3,100)
4.42ꢁ0.09
(38,000)
10.0ꢁ0.03
(0.1)
.
C21H26ClNO5 0.8H2O
1d
Cl
H
7.72ꢁ0.17
0.85 (19)
.
C21H26ClNO5 H2O
1e
Cl
< 4
8.62ꢁ0.10
0.96 (2.4)
7.06ꢁ0.11
0.99 (87)
41,667<
15,833
(100,000<)
9.82ꢁ0.08
(0.15)
Isoproterenol
0.002
0.001
aElemental analyses were withinꢁ0.4% of the theoretical values.
bIn parentheses is the EC20 value (nM), the mean concentration required to increase heart rate by 20 beats per min in rat atrium (nꢂ3).
cIn parentheses is the IC50 value (nM), the mean concentration required to produce 50% inhibition of uterine contraction in the rat uterus (nꢂ3).
dThe intrinsic activity (IA) given as a ratio of the maximum stimulation with forskolin (10ꢀ5 M).
eIn parentheses is the EC50 value (nM), the mean concentration required to produce 50% relaxation of the detrusor (nꢂ3).
fThe selectivity is the concentration-ratio of b3 (EC50) to b1 (EC20) or b2 (IC50) for each drug.

N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
3267
carboxyalkylamino group.17 Thus, further optimisation
of the b3-AR agonistic activity and selectivity of 1a was
carried out by chlorine (Cl) and methyl group (Me)
substitutions. Introduction of a Me into the phenyl ring
at the meta-position (1b: EC50=4.8 nM) resulted in a
1.6-fold increase of b3-AR agonistic activity, while
ortho-substitution led to a 1.8-fold decrease in activity
(1c: EC50=14 nM). Replacement of Me with Cl at the
meta-position (1d: EC50=19 nM) resulted in a 2.4-fold
decrease of b3-AR agonistic activity, while ortho-substitu-
tion led to a 3.2-fold increase in activity (1e: EC50=2.4
nM), when compared with 1a. We conclude that enhance-
ment of b3-AR agonistic activity requires an electron-
donating group (Me) at the meta-position or an electron-
withdrawing group (Cl) at the ortho-position. On the
other hand, substitution of either Cl or Me at the meta-
position led to a 2–3.5-fold increase of b1- and b2-AR
agonistic activity. In the case of ortho-substitution, a more
than 5-fold decrease of b1- and b2-AR agonistic activity
occurred. It was apparent that b3-AR selectivity was
improved by ortho-substitution but lowered by meta-
substitution, regardless of their electric characterisations.
alkoxy group on acids-3c, e was more severely limited
than that of the unsubstituted or the meta-substituted
analogues, since the population of conformers at the
local minimum was decreased. The consequent differ-
ence in the conformational disposition of each carboxyl
group at the global minimum would lead to the low
affinity of ortho-substituted compounds for b1- and b2-
AR, so that the b1- and b2-AR agonistic activities of 1c
and 1e would be significantly decreased. Despite the
conformational distinction, the b3-AR agonistic activity
of all compounds was retained, i.e., it resulted in an
alteration of between half to twice the potency of the
unsubstituted compound (1a). It can be assumed that
there is a relatively extensive domain to which the iso-
butyric acid moiety binds for enhancement of b3-AR
agonistic activity. When comparing the substituents at
the same position on the phenyl ring, Cl and Me had
almost the same conformational effect, due to their
similar size, though their inductive effects differed.
However, the electrostatic potential charge on the
phenyl ring or ether oxygen in acids-3a–e did not cor-
relate with b3-AR activity (data not shown). The
In an attempt to assess structure–activity relationships
(SAR) by molecular modelling, the geometry optimisa-
tions of carboxylic acid derivatives of compounds 3a–e,
as simple models for 1a–e, were calculated via semi-
empirical AM1 utilising WinMOPAC1 (Fujitsu Lim-
ited21). The most stable conformer of acid-3c, as a
representative, is illustrated in Figure 2. Both the 2-
bromoethyl and alkoxy groups (ether of the isobutyric
acid moiety) of acids-3a–e were not coplanar with the
phenyl ring, but almost located perpendicular to it, in
an antiperiplanar relationship. The dihedral angles
between the phenyl and 2-bromoethyl groups (CP5–CP4–
CE1–CE2) on acids-3a–e were same (98.2ꢁ0.4ꢃ). The
dihedral angles between the phenyl and alkoxy groups
(CA1–CA2–O1–CP1) on acids-3a,b,d were also same
(96.3ꢁ0.3ꢃ). However, the dihedral angles on acid-3c, e
were changed by ortho-substitution to constrain a little
movement of the alkoxy group (to 103.2ꢁ0.1ꢃ from
96.3ꢁ0.3ꢃ). In addition, the isobutyric acid moiety was
slightly rotated around the CA1–O1 bond (by 5.4ꢁ0.1ꢃ).
Simultaneously, the conformational flexibility of the
Figure 3. Time course of changes in intravesical pressure (a) and heart
rate (b) in anaesthetized rats after administration of iv saline (1 mL/
kg), isoproterenol (10 mg/kg), and 1a (10 mg, 0.1 mg, 1.0 mg/kg) (n=3).
Intravesical pressure (a) is expressed as a percentage of the maximal
relaxation with isoproterenol at 2 min. Heart rate (b) is expressed as
the difference from the value before drug administration.
Figure 2. Structure of 2-[4-(2-bromoethyl)-2-methylphenoxy]-2-
methylpropionic acid (right), the hydrolysed derivative of 3c, and its
conformer at the global minimum (left).

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N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
hypothesis as described above, which proposes con-
tributions of electron distribution to b3-AR agonistic
activity, based on the profiles and positions of Cl and
Me, was not supported by the calculated molecular
charge. To analyse the SAR for b3-AR agonistic activity
using small molecular modelling would have needed a
wider range of potencies of compounds examined. We
conclude that construction of receptor models based on
the rhodopsin structure as a template, together with
more information on various phenyl ring substituents
might be necessary. We are still investigating the
synthesis and SAR of these analogues bearing other
substituents.
activity in CHO cells expressing the cloned human b3-
AR (data not shown). Thus, 1a should be a potent and
selective b3-AR agonist, acting as a urine-storage
agent. This action would be mediated via relaxation of
detrusor, without undesirable effects via b1- and b2-
ARs, and PPAR-a. Therefore, we expect 1a and its
analogues will have roles in the treatment of bladder
dysfunction and as new tools for studies of human
bladder function.
Experimental
Melting points were taken on a Yanaco MP-3S Micro
melting point apparatus and are uncorrected. Infrared
spectra were measured on a Nicolet 510 FT-IR spectro-
photometer and are reported in reciprocal centimeters.
Proton NMR spectra were recorded at 400 or 500 MHz
with a Bruker AMX 400 or DRX 500 instrument, and
chemical shifts are reported in parts per million (d)
downfield from tetramethysilane as the internal stan-
dard. The peak patterns are shown as the following
abbreviations: br=broad, d=doublet, m=multiplet,
s=singlet, t=triplet, q=quartet. The mass spectra
(MS) and the high resolution mass spectra (HRMS)
were carried out with a JEOL JMS-SX102A mass spec-
trometer with capabilities for fast atom bombardment.
Elemental analyses were performed by the Yanaco CHN
corder MT-5 analyzer. The analytical results obtained
were within 0.4% of the theoretical values (Table 1).
Silica gel 60 F254 pre-coated plates on glass from Merck
KGaA or aminopropyl silica gel (APS) pre-coated NH
TLC from Fuji Silysia Chemical Ltd. was used for Thin-
layer chromatography (TLC). Flash or medium pressure
liquid column chromatography (MPLC) was performed
on silica gel BW350 (particle size 25–44 mm) from Fuji
Silysia Chemical Ltd. or APS Daisogel IR60 (particle size
25–40 mm) from Daiso Co., Ltd. All reagents and sol-
vents were commercially available unless otherwise indi-
cated. Yields were not optimised.
The unsubstituted compound 1a appeared to have the
advantages of easier synthetic process and lower cost
compared with the substituted compounds. It was
therefore the first of these fibrate analogues assessed in
vivo, using intravenously administered 1a in anaes-
thetized rats. Compound 1a lowered the intrabladder
pressure of anaesthetized rats (ED50=31 mg/kg) with a
50-fold lower potency than isoproterenol (ED50=0.6
mg/kg), as shown in Figure 3a. The greater potency of
isoproterenol may have been due to b2-AR mediated
relaxation, since the rat detrusor is relaxed via both b2-
AR and b3-AR.22 The b3-AR selective compound 1a
exhibited sufficient intrabladder pressure lowering, in
spite of being able to relax the detrusor via b3-AR. In
addition, 1a showed little effect on heart rate, whereas
isoproterenol increased heart rate (42 beats as a max-
imum value at 2 min) as shown in Figure 3b. It
appeared that the in vivo results were closely related to
those derived from in vitro assays, where 1a was a
potent and selective b3-AR agonist.
Conclusion
Compound 1a and its analogues, bearing a portion of
the fibrate-like structure, produced significant relaxation
of the ferret detrusor (b3) and had little effect on both
discharge rate of rat atrium (b1) and spontaneous moti-
lity of rat uterus (b2). In fact, b3-AR agonistic activity
exceeded b1- or b2-AR agonistic activity by three orders
of magnitude. This evidence indicated that compounds
1a–e were potent and selective b3-AR agonists. Investi-
gations of the substitution pattern on the phenyl ring
suggested that Cl at the ortho-position, or Me at the
meta-position, led to a slight increase of b3-AR agonis-
tic activity. Furthermore, substitution of either Cl or
Me at the ortho-position led to significant improvement
of selectivity for the b3-AR with respect to b1- or b2-AR.
Intravenous administration of 1a in anaesthetized rats
produced a lowering of intrabladder pressure. In a pre-
liminary study, a reporter gene assay using the peroxi-
some proliferator response element (PPRE) upstream of
the luciferase gene,23 involving formation of PPAR-a
and retinoid X receptor (RXR) heterodimers, showed
no effect up to 10ꢀ4 M of 1a, using bezafibrate and
WAY14,643 as positive controls. It was presumed that
1a would lack fibrate-like functions such as modulation
of lipid metabolism, even if it possessed a fibrate-like
structure. Additionally, 1a showed nearly full agonistic
Ethyl 2-phenoxy-2-methylpropionate (5a)
To a solution of phenol (10.0 g, 106 mmol) and 1,1,1-
trichloro-2-methyl-2-propanol hemihydrate (39.6 g, 212
mmol) in acetone (500 mL) was added NaOH (34.0 g,
850 mmol), and the mixture was stirred overnight at
room temperature. After the solvent was removed under
reduced pressure, the residue was dissolved in water and
washed with Et2O. The aqueous layer was acidified with
concentrated HCl and extracted with Et2O. The extract
was washed twice with brine, dried over anhydrous
MgSO4, and the solvent was removed under reduced
pressure. The residue was dissolved in EtOH (500 mL),
and SOCl2 (7.75 mL, 106 mmol) was carefully added
dropwise to the stirred solution at room temperature.
The mixture was heated under reflux for 6 h. After the
reaction mixture was concentrated under reduced pres-
sure, the residue was dissolved in Et2O and washed with
water, saturated aqueous NaHCO3, and brine succes-
sively. The organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The
residue was dissolved in hexane (200 mL) and filtrated

N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
3269
through a short column of APS (eluent:hexane). The
filtrate was concentrated under reduced pressure to give
17.0 g of 5a as a colourless oil (77%): 1H NMR(CDCl3)
d 1.25 (3H, t, J=7.1 Hz), 1.60 (6H, s), 4.24 (2H, q,
J=7.1 Hz), 6.82–6.87 (2H, m), 6.95–7.01 (1H, m), 7.20–
7.27 (2H, m).
Ethyl 2-[4-(Bromoacetyl)-2-methylphenoxy]-2-methylpro-
1
pionate (6c). H NMR (CDCl3) d 1.26 (3H, t, J=7.1
Hz), 1.67 (6H, s), 2.28 (3H, s), 4.23 (2H, q, J=7.1 Hz),
4.39 (2H, s), 6.62 (1H, d, J=8.6 Hz), 7.72 (1H, dd,
J=8.6, 2.3 Hz,), 7.81 (1H, d, J=2.3 Hz).
Ethyl 2-[4-(Bromoacetyl)-3-chlorophenoxy]-2-methylpro-
1
The following compounds (5b–e) were prepared as
described for the preparation of 5a, using the corre-
sponding phenol derivatives (4b–e).
pionate (6d). H NMR (CDCl3) d 1.24 (3H, t, J=7.1
Hz), 1.65 (6H, s), 4.24 (2H, q, J=7.1 Hz), 4.53 (2H, s),
6.74 (1H, dd, J=8.7, 2.5 Hz), 6.89 (1H, d, J=2.5 Hz),
7.62 (1H, d, J=8.7 Hz).
1
Ethyl 2-(3-Methylphenoxy)-2-methylpropionate (5b). H
NMR (CDCl3) d 1.25 (3H, t, J=7.1 Hz), 1.59 (6H, s),
2.29 (3H, s), 4.24 (2H, q, J=7.1 Hz), 6.60–6.65 (1H, m),
6.67–6.69 (1H, m), 6.78–6.82 (1H, m), 7.11 (1H, t,
J=7.8 Hz).
Ethyl 2-[4-(Bromoacetyl)-2-chlorophenoxy]-2-methylpro-
1
pionate (6e). H NMR (CDCl3) d 1.24 (3H, t, J=7.1
Hz), 1.70 (6H, s), 4.24 (2H, q, J=7.1 Hz), 4.36 (2H, s),
6.81 (1H, d, J=8.7 Hz), 7.78 (1H, dd, J=8.7, 2.3 Hz),
8.04 (1H, d, J=2.3 Hz).
1
Ethyl 2-(2-Methylphenoxy)-2-methylpropionate (5c). H
NMR (CDCl3) d 1.25 (3H, t, J=7.1 Hz), 1.59 (6H, s),
2.24 (3H, s), 4.25 (2H, q, J=7.1 Hz), 6.64–6.68 (1H, m),
6.88 (1H, dt, J=7.5, 1.0 Hz), 7.01–7.07 (1H, m), 7.12–
7.16 (1H, m).
Ethyl 2-[4-(2-Bromoethyl)phenoxy]-2-methylpropionate
(3a). A solution of 6a (700 mg, 2.13 mmol) and trie-
thylsilane (747 mL, 4.68 mmol) in TFA (10 mL) was
stirred for 1 h at 70 ꢃC. After the reaction mixture was
concentrated under reduced pressure, the residue was
purified by MPLC on silica gel (eluent: hexane/
CH2Cl2=1:1) to give 640 mg of 3a as a colourless oil
1
Ethyl 2-(3-Chlorophenoxy)-2-methylpropionate (5d). H
NMR (CDCl3) d 1.25 (3H, t, J=7.1 Hz), 1.60 (6H, s),
4.24 (2H, q, J=7.1 Hz), 6.72 (1H, ddd, J=8.1, 2.1, 0.9
Hz), 6.86 (1H, t, J=2.1 Hz), 6.97 (1H, ddd, J=8.1, 2.1,
0.9 Hz), 7.15 (1H, t, J=8.1 Hz).
1
(96%): H NMR (CDCl3) d 1.25 (3H, t, J=7.1 Hz),
1.59 (6H, s), 3.09 (2H, t, J=7.8 Hz), 3.52 (2H, t, J=7.8
Hz), 4.23 (2H, q, J=7.1 Hz), 6.79 (2H, d, J=8.7 Hz),
7.07 (2H, d, J=8.7 Hz).
Ethyl 2-(2-Chlorophenoxy)-2-methylpropionate (5e). 1H
NMR (CDCl3) d 1.27 (3H, t, J=7.1 Hz), 1.62 (6H, s),
4.26 (2H, q, J=7.1 Hz), 6.89 (1H, dd, J=8.2, 1.5
Hz), 6.95 (1H, ddd, J=7.9, 7.5, 1.5 Hz), 7.12 (1H,
ddd, J=8.2, 7.5, 1.7 Hz), 7.37 (1H, dd, J=7.9, 1.7
Hz).
The following compounds (3b–e) were prepared
according as described for the preparation of 3a, using
the corresponding phenacyl bromides (6b–e).
Ethyl 2-[4-(2-Bromoethyl)-3-methylphenoxy]-2-methyl-
propionate (3b). 1H NMR (CDCl3) d 1.25 (3H, t,
J=7.1 Hz), 1.58 (6H, s), 2.26 (3H, s), 3.09 (2H, t, J=8.1
Hz), 3.46 (2H, t, J=8.1 Hz), 4.23 (2H, q, J=7.1 Hz),
6.61 (1H, dd, J=8.4, 2.7 Hz), 6.68 (1H, d, J=2.7 Hz),
6.99 (1H, d, J=8.4 Hz).
Ethyl
2-[4-(Bromoacetyl)phenoxy]-2-methylpropionate
(6a). To a stirred suspension of AlCl3 (3.84 g, 28.8
mmol) in CH2Cl2 (30 mL) was added bromoacetyl bro-
mide (2.51 mL, 28.8 mmol) at room temperature, and
the mixture was stirred for 1 h. Compound 5a (2.0 g,
9.60 mmol) was added dropwise to the stirred mixture
in an ice bath. The mixture was stirred overnight at
room temperature and heated under reflux for 6 h. The
reaction mixture was poured into ice–water and extrac-
ted with EtOAc. The extract was washed with water and
dried over anhydrous MgSO4 followed by concentration
under reduced pressure. The residue was purified by
MPLC on silica gel (eluent: hexane/EtOAc=10/1) to
give 0.74 g of 6a as a light yellow solid (23%): 1H NMR
(CDCl3) d 1.22 (3H, t, J=7.2 Hz), 1.67 (6H, s), 4.23
(2H, q, J=7.2 Hz), 4.39 (2H, s), 6.85 (2H, d, J=9.2
Hz,), 7.91 (2H, d, J=9.2 Hz).
Ethyl 2-[4-(2-Bromoethyl)-2-methylphenoxy]-2-methyl-
1
propionate (3c). H NMR (CDCl3) d 1.25 (3H, t, J=7.1
Hz), 1.58 (6H, s), 2.22 (3H, s), 3.05 (2H, t, J=7.8 Hz),
3.50 (2H, t, J=7.8 Hz), 4.24 (2H, q, J=7.1 Hz), 6.60
(1H, d, J=8.2 Hz), 6.87 (1H, dd, J=8.2, 1.9 Hz), 6.98
(1H, d, J=1.9 Hz).
Ethyl
2-[4-(2-Bromoethyl)-3-chlorophenoxy]-2-methyl-
1
propionate (3d). H NMR (CDCl3) d 1.26 (3H, t, J=7.1
Hz), 1.59 (6H, s), 3.20 (2H, t, J=7.7 Hz), 3.54 (2H, t,
J=7.7 Hz), 4.24 (2H, q, J=7.1 Hz), 6.69 (1H, dd, J=8.4,
2.6 Hz), 6.89 (1H, d, J=2.6 Hz), 7.11 (1H, d, J=8.4 Hz).
The following compounds (6b–e) were prepared as
described for the preparation of 6a, using the corre-
sponding esters (5b–e).
Ethyl
2-[4-(2-Bromoethyl)-2-chlorophenoxy]-2-methyl-
1
propionate (3e). H NMR (CDCl3) d 1.27 (3H, t, J=7.1
Hz), 1.61 (6H, s), 3.07 (2H, t, J=7.6 Hz), 3.51 (2H, t,
J=7.6 Hz), 4.25 (2H, q, J=7.1 Hz), 6.84 (1H, d, J=8.4
Hz), 6.96 (1H, dd, J=8.4, 2.2 Hz), 7.22 (1H, d, J=2.2 Hz).
Ethyl 2-[4-(Bromoacetyl)-3-methylphenoxy]-2-methylpro-
1
pionate (6b). H NMR (CDCl3) d 1.23 (3H, t, J=7.2
Hz), 1.65 (6H, s), 2.52 (3H, s), 4.23 (2H, q, J=7.2 Hz),
4.38 (2H, s), 6.65 (1H, dd, J=8.6, 2.4 Hz), 6.71 (1H, d,
J=2.4 Hz,), 7.67 (1H, d, J=8.6 Hz).
2-[4-(2-{[(1S,2R)-2-Hydroxy-2-(4-hydroxyphenyl)-1-methyl-
ethyl]amino}ethyl)phenoxy]-2-methylpropionic acid (1a).
A stirred mixture of 2 (630 mg, 2.0 mmol) and 3a (670

3270
N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
mg, 4.0 mmol) in DMF (2 mL) was heated at 70 ꢃC for 4
h. After cooling, the reaction mixture was diluted with
EtOAc and washed with saturated aqueous NaHCO3
and brine. The organic layer was dried over anhydrous
Na2SO4, and the solvent was removed under reduced
pressure. The residue was purified by MPLC on APS
(eluent: CH2Cl2/EtOH=40:1) to give 540 mg of ethyl 2-[4-
(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methyl-
ethyl]amino}ethyl)phenoxy]-2-methylpropionate (67%).
A solution of ethyl 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-
hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-
methylpropionate (530 mg) in 1 N NaOH (2.0 mL) was
stirred for 1 h at room temperature. To the reaction
mixture was added dropwise 2 N HCl (2.0 mL), and pre-
cipitates were collected by filtration and dried under
reduced pressure to give 380 mg of 1a (77%): mp 216–
219 ꢃC dec; [a]3D1=ꢀ13.1ꢃ (c=1.00, 1 N HCl);IR (KBr)
3429, 1611, 1568, 1511; 1H NMR (DMSO-d6) d 0.91 (3H,
d, J=6.6 Hz), 1.46 (6H, s), 2.60–2.80 (2H, m), 2.90–3.05
(2H, m), 3.15–3.35 (1H, m), 5.05 (1H, br s), 6.70–6.75 (4H,
m), 6.86 (2H, d, J=8.6 Hz), 7.14 (2H, d, J=8.6 Hz), 9.40
(1H, br);MS m/z 374 (M+H)+. HRMS: (M+H)+ calcd
for C21H28NO5, 374.1967;found, 374.1936.
2-[2-Chloro-4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphe-
nyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropio-
nic Acid (1e). Mp 182–184 ꢃC dec;[ a]D31=ꢀ6.9ꢃ
(c=0.75, AcOH);IR (KBr) 3491, 1631, 1611, 1568,
1
1511; H NMR (DMSO-d6) d 0.91 (3H, d, J=6.6 Hz),
1.50 (6H, s), 2.60–2.85 (2H, m), 2.90–3.50 (3H, m), 5.09
(1H, br s), 6.67 (1H, d, J=8.6 Hz), 6.72 (2H, d, J=8.5
Hz), 6.88 (1H, d, J=8.6 Hz), 7.15 (2H, d, J=8.5 Hz),
7.23 (1H, s), 9.35 (2H, br);MS m/z (relative intensity)
408 (M+H)+, 410 (0.36). HRMS: (M+H)+ calcd for
C21H27ClNO5, 408.1578, found: 408.1539.
Pharmacological experiments. In vitro and in vivo
experiments were performed as described pre-
viously.15ꢀ17 Using isolated preparations (rat atrium, rat
uterus, and ferret detrusor) in the in vitro experiments,
the effect of cumulative addition of compound 1 and
isoproterenol to a Magnus bath were measured and
estimated values calculated as follows: the EC20 value
for b1-AR was the concentration of each compound
required increase the heart rate by 20 beats per min. The
IC50 value for b2-AR was the concentration of each
compound required to produce a 50% inhibition of
spontaneous uterine contraction, calculated as the total
uterine contraction during 5 min following addition of
each compound, when the total before addition of the
compound was expressed as 100%. The EC50 value for
b3-AR was the concentration of each compound
required to produce a 50% relaxation of urinary blad-
der smooth muscle, taking the relaxation induced by
10ꢀ5 M forskolin to be 0%, while tension before addi-
tion of the compound was 100%. In the in vivo experi-
ment with 1a, a polyethylene catheter was inserted into
the urinary bladder of urethane-anaesthetized male rats.
The bladder pressure was measured via a pressure
transducer connected to the catheter and heart rate was
measured simultaneously. Drug effects on bladder pres-
sure (ED50 value) were quantified by expressing post-
administration value as a percentage of the value before
administration of 1a or isoproterenol.
The following compounds (1b–e) were prepared
according as described for the preparation of 1a, using
the corresponding phenethyl bromides (3b–e).
2-[4-(2-{[(1S,2R)-2-Hydroxy-2-(4-hydroxyphenyl)-1-methyl-
ethyl]amino}ethyl)-3-methylphenoxy]-3-methylpropionic
Acid (1b). Mp 209–212 ꢃC dec;[ a]3D0=ꢀ10.2ꢃ (c=0.90,
1N HCl);IR (KBr) 3405, 1613, 1570, 1513, 1502; 1H
NMR (DMSO-d6) d 0.91 (3H, d, J=6.6 Hz), 1.45 (3H,
s), 1.47 (3H, s), 1.99 (3H, s), 2.45–2.75 (2H, m), 2.80–
2.95 (2H, m), 3.20–3.35 (1H, m), 5.10 (1H, br s), 6.55
(1H, dd, J=8.3, 2.6 Hz), 6.59 (1H, d, J=2.6 Hz), 6.68
(1H, d, J=8.3 Hz), 6.73 (2H, d, J=8.7 Hz), 7.16 (2H, d,
J=8.7 Hz), 9.35 (1H, br);MS m/z 388 (M+H)+.
HRMS: (M+H)+ calcd for C22H30NO5, 388.2124,
found: 388.2169.
2-[4-(2-{[(1S,2R)-2-Hydroxy-2-(4-hydroxyphenyl)-1-me-
thylethyl]amino}ethyl)-2-methylphenoxy]-2-methylpropio-
nic acid (1c). Mp 209–211 ꢃC dec;[ a]D31=ꢀ10.0ꢃ
(c=0.36, AcOH);IR (KBr) 3280, 1616, 1594, 1565,
Acknowledgements
We are grateful to our colleagues for analytical and
spectral determinations and for performing the screen-
ing assays. We thank Dr. Yoshinobu Yamazaki and
Mr. Hiroo Takeda for their pharmacological contribu-
tions to this work, We also thank Dr. Hiromu Harada
for helpful comments while reviewing the manuscript.
1
1516; H NMR (DMSO-d6) d 0.92 (3H, d, J=6.6 Hz),
1.47 (6H, s), 2.11 (3H, s), 2.60–2.80 (2H, m), 2.85–3.05
(2H, m), 3.10–3.35(1H, m), 5.02 (1H, br s), 6.50–6.60
(1H, m), 6.65–6.75 (3H, m), 6.90 (1H, s), 7.13 (2H, d,
J=8.5 Hz);MS m/z 388 (M+H)+. HRMS: (M+H)+
calcd for C22H30NO5, 388.2124, found: 388.2117.
2-[3-Chloro-4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphe-
nyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropio-
nic acid (1d). Mp 208–210 ꢃC dec;[ a]3D0=ꢀ5.3ꢃ (c=0.15,
MeOH);IR (KBr) 3406, 1605, 1562, 1511; 1H NMR
(DMSO-d6) d 0.92 (3H, d, J=6.5 Hz), 1.48 (3H, s), 1.49
(3H, s), 2.70–3.10 (4H, m), 3.20–3.40 (1H, m), 5.05 (1H,
br s), 6.65–6.80 (3H, m), 6.85 (1H, d, J=2.4 Hz), 6.95
(1H, d, J=8.5 Hz), 7.16 (2H, d, J=8.5 Hz);MS m/z
(relative intensity) 408 (M+H)+, 410 (0.31). HRMS:
(M+H)+ calcd for C21H27ClNO5, 408.1578, found:
408.1539.
References and Notes
1. Igawa, Y.;Yamazaki, Y.;Takeda, H.;Akahane, M.;Aji-
sawa, Y.;Yoneyama, T.;Nishizawa, O. Acta Physiol. Scand.
1998, 164, 117.
2. Igawa, Y.;Yamazaki, Y.;Takeda, H.;Hayakawa, K.;
Akahane, M.;Ajisawa, Y.;Yoneyama, T.;Nishizawa, O.;
Anderrson, K.-E. Br. J. Pharmacol. 1999, 126, 819.
3. Takeda, M.;Obara, K.;Mizusawa, T.;Tomita, Y.;Arai,
K.;Tsutsui, T.;Hatano, A.;Takahashi, K.;Nomura, S.
Pharmacol. Exp. Ther. 1999, 288, 1367.
J.

N. Tanaka et al. / Bioorg. Med. Chem. 9 (2001) 3265–3271
3271
4. Fujimura, T.;Tamura, K.;Tsutsumi, T.;Yamamoto, T.; W. P.;Forrest, M. J.;Hom, G. J.;MacIntyre, D. E.;Miller,
Nakamura, K.;Koibuchi, Y.;Kobayashi, M.;Yamaguchi, O.
J. Urol. 1999, 161, 680.
R. R.;Stearns, R. A.;Tota, L.;Wyvratt, M. J.;Fisher, M. H.;
Weber, A. E. J. Med. Chem. 2000, 43, 3832. (b) Sher, P. M.;
Mathur, A.;Fisher, L. G.;Wu, G.;Skwish, S.;Michel, I. M.;
Seiler, S. M.;Dickinson, K. E. J. Bioorg. Med. Chem. Lett.
1997, 7, 1583. (c) Zheng, W.;Nikulin, V. I.;Konkar, A. A.;
Vansal, S. S.;Shams, G.;Feller, D. R.;Miller, D. D. J. Med.
Chem. 1999, 42, 2287.
5. Takeda, H.;Kaidoh, K.;Yamazaki, Y.;Akahane, S.;
Miyata, H.;Akahane, M.;Ajisawa, Y.;Igawa, Y.;Nishizawa,
O.;Anderrson, K.-E. In Neurourology and Urodynamics, 30th
Annual Meeting of the International Continence Society,
Tampere, Finland, August 28–31, 2000.
6. Kaidoh, K.;Takeda, H.;Yamazaki, Y.;Akahane, S.;
Miyata, H.;Akahane, M.;Ajisawa, Y.;Igawa, Y.;Nishizawa,
O.;Anderrson, K.-E. In Neurourology and Urodynamics, 30th
Annual Meeting of the International Continence Society,
Tampere, Finland, August 28–31, 2000
14. Strosberg, A. D. Annu. Rev. Pharmacol. Toxicol. 1997, 37,
421.
15. Takeda, H.;Igawa, Y.;Komatsu, Y.;Yamazaki, Y.;
Akahane, M.;Nishizawa, O.;Ajisawa, Y. Eur. J. Pharmacol.
2000, 403, 147.
7. Weyer, C.;Gautier, J. F.;Danforth, E., Jr.
Metabolism 1999, 25, 11.
8. Howe, R. Drugs of the Future 1993, 18, 529.
Diabetes &
16. Kobayashi, M.;Takeda, K.;Murata, S.;Kojima, M.;
Akahane, M.;Inoue, Y.;Kitamura, K.;Kawarabayashi, T. J.
Pharmacol. Exp. Ther. 2001, 297, 666.
9. (a) Arch, J. R.;Ainsworth, A. T.;Cawthorne, M. A.;
Piercy, V.;Sennitt, M. V.;Thod, V. E.;Wilson, C.;Wilson, S.
Nature 1984, 309, 163. (b) Wilson, C.;Wilson, S.;Piercy, V.;
Sennitt, M. V.;Arch, J. R. Eur. J. Pharmacol. 1984, 100, 309.
10. (a) Emorine, L. J.;Marullo, S.;Briend-Sutren, M.-M.;
Patey, G.;Tate, K.;Delavier-Klutchko, C.;Strosberg, A. D.
Science 1989, 245, 1118. (b) Emorine, L. J.;Feve, B.;Pairault,
J.;Briend-Sutren, M.-M.;Nahmias, C.;Marullo, S.;Delavier-
Klutchko, C.;Strosberg, D. A. Amer. J. Clin. Nutr. 1992, 55,
215S.
17. Tanaka, N.;Tamai, T.;Mukaiyama, H.;Hirabayashi, A.;
Muranaka, H.;Akahane, S.;Miyata, H.;Akahane, M.
Med. Chem. 2001, 44, 1436.
18. Staels, B.;Dallongeville, J.;Auwerx, J.;Schoonjans, K.;
Leitersdorf, E.;Fruchart, J. C. Circulation 1998, 98, 2088.
19. Sith, H. E.;Burrows, E. P. J. Med. Chem. 1977, 20, 978.
20. Corey, E. J.;Barcza, S.;Klotmann, G. J. Am. Chem. Soc.
1969, 91, 4782.
21. WinMOPAC ver. 3.0 is a Windows implementation of
MOPAC2000, semiempirical molecular orbital program.
Fujitsu Limited Home Page. http://venus.netlaboratory.com/
material/messe/winmopac/index_e.html (accessed August
2001).
J.
11. Tate, K. M.;Briend-Sutren, M.-M.;Emorine, L. J.;Dela-
vier-Klutchko, C.;Marullo, S.;Strosberg, A. D. Eur. J. Bio-
chem. 1991, 196, 357.
12. (a) Howe, R.;Rao, B. S.;Holloway, B. R.;Stribling, D. J.
Med. Chem. 1992, 35, 1751. (b) Bloom, J. D.;Dutia, M. D.;
Johnson, B. D. J. Med. Chem. 1992, 35, 3081. (c) Oriowo,
M. A.;Chapman, H.;Kirkham, D. M.;Sennitt, M. V.;
Ruffolo, R. R., Jr.;Cawthorne, M. A. J. Pharmacol. Exp.
Ther. 1996, 277, 22.
22. Yamazaki, Y.;Takeda, H.;Akahane, M.;Igawa, Y.;
Nishizawa, O.;Ajisawa, Y. Br. J. Pharmacol. 1998, 124, 593.
23. (a) Kliewer, S. A.;Umesono, K.;Noonan, D. J.;Heyman,
R. A.;Evans, R. M. Nature 1992, 358, 771. (b) Sher, T.;
Yi, H.-F.;McBride, W.;Gonzalez, F. J.
Biochemistry
1993, 32, 5598. (c) Lehmann, J. M.;Lenhard, J. M.;Oliver,
B. B.;Ringold, G. M.;Kliewer, S. A. J. Biol. Chem. 1997, 272,
3406.
13. (a) Mathvink, R. J.;Tolman, J. S.;Chitty, D.;Candelore,
M. R.;Cascieri, M. A.;Colwell, L. F.;Deng, L.;Feeney,