Relationship between stereochemistry and the beta3-adrenoceptor agonistic activity of 4'-hydroxynorephedrine derivative as an agent for treatment of frequent urination and urinary incontinence.

Article abstract of DOI:10.1021/jm020177z

This report proposes a beta(3)-adrenoceptor (AR) selective agonist, 2-[2-chloro-4-(2-([(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino)ethyl)phenoxy]acetic acid (1a), as a novel agent for treating urinary bladder dysfunction. This compound and i

Full text of DOI:10.1021/jm020177z

J . Med. Chem. 2003, 46, 105-112
105
Rela tion sh ip betw een Ster eoch em istr y a n d th e â3-Ad r en ocep tor Agon istic
Activity of 4′-Hyd r oxyn or ep h ed r in e Der iva tive a s a n Agen t for Tr ea tm en t of
F r equ en t Ur in a tion a n d Ur in a r y In con tin en ce
Nobuyuki Tanaka,* Tetsuro Tamai, Harunobu Mukaiyama, Akihito Hirabayashi, Hideyuki Muranaka,
Takehiro Ishikawa, J unichi Kobayashi, Satoshi Akahane, and Masuo Akahane
Central Research Laboratory, Kissei Pharmaceutical Company Ltd., 4365-1, Hotaka, Nagano, 399-8304, J apan
Received April 25, 2002
This report proposes a â3-adrenoceptor (AR) selective agonist, 2-[2-chloro-4-(2-{[(1S,2R)-2-
hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]acetic acid (1a ), as a novel
agent for treating urinary bladder dysfunction. This compound and its relatives have a unique
feature among â3-AR agonists: two chiral carbons are adjacently structured on the left side of
the molecule. To study the relationship between the stereoconfiguration of the vicinal chiral
carbons in 1a and â-AR agonistic activity, the four stereoisomers were synthesized via
oxazolidinone prepared by intracyclization involving inversion of the â-hydroxy group. The in
vitro assays using rat atria for â1-AR, rat uteri for â2-AR, and ferret detrusor for â3-AR showed
that 1a possessed potent â3-AR agonistic activity (EC50 ) 3.85 nM) and 3700- and 1700-fold
selectivity for â3-AR relative to â1- and â2-AR, respectively. Comparison of the four isomers
revealed that the (RS,âR)-compound (1a ) was not only the most potent agonist but was also
the most selective for â3-AR. In the anesthetized rat, intravenous administration of 1a brought
about a sufficient decrement of the intrabladder pressure (ED50 ) 12 µg/kg), and intraduodenal
administration of 2a , which is the ethyl ester of 1a , led to same result (ED50 ) 0.65 mg/kg).
Moreover, no effects on the cardiovascular system were observed in either test.
Ta ble 1. Structure and Analytical Data of
4′-Hydroxynorephedrine Derivatives
In tr od u ction
Human urinary bladder is controlled by the sympa-
thetic and parasympathetic nervous systems.1 Urinary
bladder dysfunctions, such as frequent urination and
urinary incontinence, arise from disturbance of this dual
nervous mechanism, which can be caused by various
anal.
dataa
factors such as neurologic disease. Anti-muscarinic
drugs are the most commonly used to treat such
diseases.2,3 Many investigators have pursued alternative
therapeutic approaches not only to avoid the side effects
of anti-muscarinic agents but to eliminate the various
causes of urinary dysfunction, which has led to the
development of a new classes of drugs based on the
other mechanisms,4 including potassium channel open-
ers5 and 5-HT and NE reuptake inhibitors.6 â3-Adreno-
ceptor (AR) stimulation has attracted attention recently
as a new therapeutic approach. Some reports on the
distribution and function of â3-ARs in human bladder
have suggested that â3-ARs play a significant role in
urinary storage.7-9 Moreover, â3-AR agonists prolong
the micturition interval in a rat bladder hyperactivity
model.10 We have been exploring a new type of selective
â3-AR agonist to treat such diseases and assessed them
by novel assay using a ferret bladder strip, which is
relaxed via â3-AR subtype, as in the human bladder.11
Previously, we presented N-phenylglycine derivatives
as the new class of â3-AR agonists in our previous
report.12 Here, we report on a 2-(2-chlorophenoxy)acetic
acid derivative (1a ) and its ester (2a ) bearing 4′-
compd configuration
R
formula
MW
1a
1b
1c
1d
2a
RS,âR
RR,âS
RS,âS
RR,âR
RS,âR
H
H
H
H
Et
C
C
C
C
C
19H22ClNO5
19H22ClNO5
379.83 C, H, N
379.83 C, H, N
19H22ClNO5‚0.5H2O 388.84 C, H, N
19H22ClNO5‚0.5H2O 388.84 C, H, N
21H26ClNO5‚HCl
444.36 C, H, N
a
Analytical data were within (0.4% of the theoretical values.
hydroxynorephedrine as a potent and selective â3-AR
agonists (Table 1). Two vicinal asymmetric centers are
characteristic of these compounds, which occurred as
four stereoisomers. There are no reports on the â3-AR
agonist’s stereochemistry of the two vicinal chiral
carbons involved in the â3-AR agonistic activity except
for the study of ephedrine isomers, which are not
selective â3-AR agonists. Most of the â3-AR agonists do
not possess vicinal chiral carbons atoms but are con-
structed with separated asymmetrical carbon atoms
that are connected by the aminomethylene group.13-15
Some reports have examined the agonistic activity of
the four isomers on â-ARs.16,17 Our previous investiga-
tion of (RS,âR)-4′-hydroxynorephedrine-structured â3-
AR agonists was based on the evidence that â2-AR
agonistic activity was enhanced on the (RS,âR)-config-
uration.18 First, it was necessary to define whether the
(RS,âR)-configuration was important in determining â3-
AR agonistic activity.
* To whom correspondence should be addressed. Phone: +81-
263-82-8820. Fax: +81-263-82-8827. E-mail: nobuyuki_tanaka@
pharm.kissei.co.jp.
10.1021/jm020177z CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/02/2002

106 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Tanaka et al.
Sch em e 1. Synthesis of Ethyl
2-[4-(2-Bromoethyl)phenoxy]acetate (7)a
a
Reagents: (a) ethyl bromoacetate, K2CO3; (b) CBr4, Ph3P.
Sch em e 2. Synthesis of
(RS,âS)-4′-Hydroxynorephedrine (6c)a
a
Reagents: (a) t-Boc2O; (b) BnBr, Cs2CO3; (c) MsCl, Et3N; (d)
aqueous KOH; (e) H2, Pd-C.
which was converted to the 2-phenoxyacetic acid ester
(9) by alkylation using ethyl bromoacetate with K2CO3.
The hydroxy group of 9 was converted into bromine with
CBr4 and Ph3P to give the phenethyl bromide derivative
(7). Optical resolution of the racemic erythro-6 was
performed using optically active tartaric acid according
to a previous report by H. E. Smith et al.20 to give the
optically active 6a and 6b. The isolation of optically
active threo-6 by optical resolution with 10-camphor-
sulfonic acid was also described in that previous report.
However, the racemic threo-6 is not commercially avail-
able. We adopted inversion of configuration of the
hydroxy groups of 6a and 6b via oxazolidinones by
intramolecular cyclization of N-Boc-â-amino alcohols.21,22
The synthesis of 6c is outlined in Scheme 2. The erythro-
isomer (6a ) was protected with Boc and the benzyl
group. Treatment of 10 with methanesulfonyl chloride
led to ready intramolecular-cyclization to form oxa-
zolidinone (11). Following hydrolysis of the urethane
moiety of the oxazolidinone, the benzyl group was
removed by hydrogenation to obtain the optically active
threo-isomer (6c). The enantiomer 6d was derived from
6b with the exact same route as shown in Scheme 2.
F igu r e 1. Structures of the novel â3-adrenergic agonists (1a -
d , 2a ) and their synthetic intermediates (6a -d , 7), the
launched â2-adrenergic agonists (3 is ritodrine; 4 is procaterol),
and the classic â3-adrenergic agonist (5: BRL37344). The R
and S next to R or â indicate the absolute configuration of the
hydroxy and methyl groups. The R* and S* in parentheses
indicate the relative configuration of the hydroxy and methyl
groups.
In this study, we synthesized the four stereoisomers
of 2-(2-chlorophenoxy)acetic acid derivatives (1a -d ) via
oxazolidinone prepared by cyclization-inversion from
4′-hydroxynorephedrine (Scheme 2) and estimated their
agonistic activity for â1-, â2-, and â3-ARs. Stereochem-
istry vs activity relationship is discussed in the following
section. Additionally, we report on the pharmacological
tests that measured the antagonist activity of 3-(2-allyl-
phenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-
(2S)-2-propranol (SR-58894A)19 against compound 1a
and the in vivo measurements of the effects of intra-
duodenal administration of the ester of 1a .
Resu lts
â1-AR Agon istic Activity. The â1-AR agonistic ac-
tivity of the compounds was estimated from their
chronotropic effect on rat atria. The EC20 value is the
mean concentration required to increase the basal rate
of the rat atria by 20 beats per minute (Table 2). No
isomer of the acid (1a -d ) had remarkable effects at
concentrations up to 10 µM. Even the most active
(RS,âR)-isomer (1a ) was 90000-fold and 100-fold weaker
than isoproterenol and BRL37344, respectively. The
ethyl esterification of 1a resulted in an increase of â1-
AR agonistic activity by 1 order of magnitude. The rank
order of potency for these compounds was the follow-
ing: ester (2a ) > (RS,âR)-compound (1a ) > (RR,âR)-
compound (1d ) > (RS,âS)-compound (1c) . (RR,âS)-
compound (1b).
Ch em istr y
The isomers of 2-(2-chlorophenoxy)acetic acid deriva-
tives (1a -d ), as shown in Figure 1, were synthesized
by N-alkylation of the corresponding 4′-hydroxynor-
ephedrine isomers (6a -d ) with ethyl 2-[4-(2-bromo-
ethyl)-2-chlorophenoxy]acetate (7) under heating, fol-
lowed by saponification via the corresponding esters.
The phenethyl bromide derivative (7) was prepared as
illustrated in Scheme 1. Reduction of 4′-hydroxy-2′-
chlorophenylacetic acid with a borane-methyl sulfide
complex afforded 2-chloro-4-(2-hydroxyethyl)phenol (8),

4′-Hydroxynorephedrine Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 107
Ta ble 2. â-AR Agonistic Activity and Selectivity of 4′-Hydroxynorephedrine Derivatives
â1-AR
â2-AR
â3-AR
â3-AR selectivitye
â1/â3 â2/â3
3700 1700
pEC20 ( SE
pIC50 ( SE
pEC50 ( SE
compd
(EC20, nM)a
(IC50, nM)b
IAc (EC50, nM)d
1a
1b
1c
1d
2a
4.86 ( 0.04
(14000)
5.19 ( 0.20
(6400)
8.42 ( 0.23
0.88 (3.8)
4>
4>
6.31 ( 0.15
0.77 (490)
6.21 ( 0.19
0.74 (610)
5.24 ( 0.13
0.66 (5800)
8.13 ( 0.38
0.81 (7.4)
>200
90
>200
>160
10
(>100000)
4.26 ( 0.18
(55000)
(>100000)
>4
(>100000)
4.22 ( 0.25
(60500)
6.28 ( 0.03
(520)
4.68 ( 0.07
(21000)
5.80 ( 0.10
(1600)
6.92 ( 0.04
(120)
3.6
220
55
70
BRL37344
8.04 ( 0.10
(9.1)
8.66 ( 0.08
0.96 (2.2)
4.1
isoproterenol
9.82 ( 0.08
(0.15)
10.0 ( 0.03
(0.1)
7.06 ( 0.11
0.99 (87)
0.002
0.001
a
The value in parentheses is the EC20 (nM), which is the mean concentration required to increase the heart rate of rat atrium by 20
b
beats per minute (n g 3). The value in parentheses is the IC50 (nM), which is the mean concentration required to produce 50% inhibition
of uterine contractions in the rat uterus (n g 3). c The intrinsic activity (IA) is given as the ratio of the maximum stimulation with
forskolin (10-5 M). The value in parentheses is the EC50 (nM), which is the mean concentration required to produce 50% relaxation of
d
the detrusor (n g 3). e The selectivity is the concentration ratio of â3 (EC50) to â1 (EC20) or â2 (IC50) for each drug.
â2-AR Agon istic Activity. The â2-AR agonistic ac-
tivity of the compounds was assessed by their inhibitory
effect on spontaneous contractions in isolated rat uterus.
The IC50 value is expressed as the concentration re-
quired to effect a 50% inhibition of the spontaneous
contraction of the rat uterus (Table 2). The two com-
pounds with the S-configuration at the benzyl carbon
(1b,c) had no â2-AR agonistic activity up to 100 µM. The
(RS,âR)-compound (1a ) was a full agonist with modest
potency (IC50 ) 6.40 µM, IA ) 0.94) while the (RR,âR)-
compound (1d ) was a partial agonist (IA ) 0.56) relative
to the maximal response to isoproterenol. The rank
order of potency for these compounds was the follow-
ing: ester (2a ) > (RS,âR)-compound (1a ) > (RR,âR)-
compound (1d ) . (RR,âS)-compound (1b), (RS,âS)-
compound (1c).
â3-AR Agon istic Activity. The â3-AR agonistic ac-
tivity of the compounds was evaluated by the relaxing
ability of the isolated ferret detrusor basal tone. The
EC50 value is expressed as the concentration required
to effect 50% relaxation of the ferret detrusor strip
(Table 2). The (RS,âR)-compound (1a ) was more potent
for â3-AR agonistic activity (EC50 ) 3.80 nM; IA ) 0.88)
with almost full agonism relative to isoproterenol. Its
potency was comparable to that of BRL37344. By
contrast, the other isomers (1b-d ) showed modest
potency with partial agonism (EC50 ) 0.49-5.80 µM;
IA ) 0.66-0.77). The rank order of the potency of these
compounds was the following: BRL37344 ) (RS,âR)-
compound (1a ) > ester (2a ) > isoproterenol > (RR,âS)-
compound (1b) ) (RS,âS)-compound (1c) > (RR,âR)-
compound (1d ).
F igu r e 2. Effect of SR58894A on the 1a -induced inhibition
of spontaneous contraction in isolated rat proximal colon
preparations. All experiments were carried out in the presence
of CGP-20712A (10-7 M), ICI-118,551 (10-7 M), and phentol-
amine (10-6 M). (a) Concentration-response relationships for
1a , either alone or in the presence of SR58894A (3 × 10-7
,
10-7, 3 × 10-6, and 10-6 M). Each point represents the mean
( SEM (n ) 7-12). The data are expressed as a percentage of
the maximal relaxation induced by 10-5 M forskolin. (b) Schild
plot for the inhibition of the 1a -induced relaxation produced
by SR58894A.
E ffect of a â3-AR An t a gon ist , SR 58894A, on
Com p ou n d 1a In d u ced In h ibition of Sp on ta n eou s
Con tr a ction in th e Ra t P r oxim a l Colon . The inter-
action between (RS,âR)-compound (1a ) and 3-(2-allyl-
phenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-
(2S)-2-propranol (SR-58894A), a specific â3-AR antagonist
developed by Sanofi Co., Ltd.,19 was set out in isolated
rat proximal colon. The (RS,âR)-compound (1a ) sup-
pressed spontaneous contraction of the colon. SR58894A
caused a rightward shift of the concentration-response
curve for 1a in a concentration-dependent manner, as
shown in Figure 2. A Schild plot analysis yielded a pA2
value of 8.1 and a slope of 0.96.
In Vivo Stu d ies of iv or id Ad m in istr a tion in th e
An esth etized Ra t. The results of the in vivo examina-
tions of the compound are shown in Figures 3 and 4. In
the test of 1a , the intravenous administration of iso-
proterenol decreased the intrabladder pressure from 6.5
( 0.6 to 3.8 ( 0.5 cmH2O. This change was defined as
the maximum (100%) response to which the effect of 1a
was compared (Figure 3a). The blood pressure is ex-
pressed as the percentage change from the initial blood

108 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Tanaka et al.
F igu r e 4. Time course of the changes in intrabladder pressure
(a) and heart rate (b) in anesthetized rats after intraduodenal
administration of saline (1 mL/kg) and 2a (0.1, 1.0, 10 mg/kg)
(n ) 3). Intrabladder pressure (a) is expressed as a percentage
of the maximal relaxation with isoproterenol (iv) at 2 min.
Heart rate (b) is expressed as the difference from the value
before drug administration.
Isoproterenol was 20-fold more potent (ED50 ) 0.6 µg/
kg) than 1a but with a shorter duration of action and
producing a significant increase in heart rate.
Intraduodenal administration of the ester compound
(2a ) produced sufficient decrement of intrabladder pres-
sure in the anesthetized rats in a dose-dependent
manner. Moreover, it did not increase the heart rate.
The ED50 value of 2a was 0.65 mg/kg when the maximal
response of the iv isoproterenol-induced decrement of
intrabladder pressure was 100%.
F igu r e 3. Time course of intrabladder pressure (a), heart rate
(b), and blood pressure (c) in anesthetized rats after intrave-
nous injection of saline (1 mL/kg), isoproterenol (10 µg/kg), or
1a (1.0 µg/kg, 10 µg/kg, 0.1 mg/kg, 1.0 mg/kg) (n ) 3).
Intrabladder pressure (a) is expressed as a percentage of the
maximal relaxation with isoproterenol. Heart rate (b) is
expressed as the difference from the value before drug
administration. Blood pressure (c) is expressed as a percentage
of the value before drug administration.
Discu ssion
It is well-known that â-AR agonists as phenyletha-
nolamines essentially require R-configuration at the
benzyl position with the hydroxy group to enhance their
affinities and agonist activity for â-ARs.23 However,
there are few studies of phenylpropanolamines, such as
ephedrine, ritodrine 3, and procaterol 4 (Figure 1), with
respect to the correlation of â-AR agonist activity with
the relative stereochemistry between the benzyl carbon
with its hydroxy group (â-carbon) and the vicinal carbon
with an amino group (R-carbon). Meiji Seika Company
and Otsuka Pharmaceutical Company researchers re-
ported on two ritodrine enantiomers18 and four pro-
caterol stereoisomers,24 respectively. (-)-(RS,âR)-erythro-
Ritodrine and procaterol are more potent â2-AR agonists
than the corresponding (+)-(RR,âS)-erythro-enanti-
omers, and threo-procaterols (RR- and SS-compounds)
showed mid â2-AR agonistic activity between the (-)-
pressure (92.9 ( 2.9 mmHg), and the heart rate (∆bpm,
change in beats per minute) is expressed as the change
from the initial rate (371.3 ( 5.8 beats/min). In the test
of 2a , the intrabladder pressure was initially 4.6 ( 0.4
cmH2O and changed to 3.1 ( 0.3 cmH2O after admin-
istering isoproterenol iv. The vertical axis in Figure 4a
is the same as in Figure 3a. The basal heart rate was
363.3 ( 6.7 beats/min. Intravenous administration of
the (RS,âR)-compound (1a ) decreased the bladder pres-
sure in the anesthetized rat in a dose-dependent manner
without increasing the heart rate (ED50 ) 12 µg/kg).

4′-Hydroxynorephedrine Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 109
erythro-isomer and the (+)-erythro-isomer. When com-
paring the â2-AR agonistic activity of the stereoisomers
of our compounds (1) even with their low potency, the
(-)-(RS,âR)-erythro-isomer (1a ) was the most potent.
The rank order for â2-AR agonistic activity was the
following: (-)-(RS,âR)-erythro-isomer > (-)-(RR,âR)-
threo-isomer . (+)-(RS,âS)-threo-isomer ) (+)-(RR,âS)-
erythro-isomer. It was apparent that â2-AR was ex-
tremely sensitive to the â-hydroxy group in the R-config-
uration. These results are in agreement with the results
reported on ritodrine and procaterol. Besides, the rank
order for â1-AR agonistic activity was almost the same
as for â2-AR. Thus, the R-configuration at the â-carbon
bearing the hydroxy group was required for the best
enhancement of â1- or â2-AR agonistic activity, and the
S-configuration at the R-carbon bearing the methyl and
amino group complementarily enhanced the activity. In
1999, Feller et al. reported the stereochemistry studies
of ephedrine for â3-AR agonistic activity.25 (RS,âR)-
Erythro-ephedrine was shown to have the most potent
â3-AR agonistic activity of four isomers on human â3-
AR expressed cells, whereas ephedrine shows signifi-
cantly lower â3-AR agonistic activity than â1- and â2-
AR with low intrinsic activity (IA ) 0.07-0.31). We
postulated that a more potent â3-AR agonist with vicinal
chiral carbons was necessary to decide the activity order
of its isomers. Our selective â3-AR agonists (1a -d ) had
EC50 values below 5.80 µM and intrinsic activities (IA)
greater than 0.6. The (RS,âR)-isomer (1a ) was the most
potent of the four; however, the rank order for â3-AR
agonistic activity was inconsistent with the orders for
â1- and â2-AR activity. The two erythro-isomers (1a ,b)
had greater â3-AR agonistic activity than the threo-
isomers (1c,d ) regardless of the configuration at the
benzyl position (â-carbon), although there was a slight
difference in activities of 1b and 1c. 1a -d were selective
compounds for â3-AR agonistic activity significantly
because of their low potency for â1- and â2-AR. The rank
order for the â3-AR selectivity depended largely on the
â3-AR agonistic activity. It is noteworthy that the
erythro-compounds (1a and 1b) were over 200-fold
selective for â3-AR agonistic activity relative to their â1-
and â2-AR activity.
had more potent â3-AR agonistic activity than isopro-
terenol, as shown in Table 2. This reversal in their
potency order was largely due to the potent â2-AR
agonistic activity of isoproterenol. A previous report
confirmed that the rat detrusor is relaxed through both
â2- and â3-AR stimulations.26 In fact, the in vitro study
on the isolated rat detrusor showed that the relaxing
effect of 1a (EC50 ) 140 nM) was weaker than that of
isoproterenol (EC50 ) 10 nM). We predict that a selec-
tive â3-AR agonist (1a ) would produce a sufficient effect
on the human bladder, which is predominately mediated
via the â3-AR subtype over the other â-ARs.
Esterification of 1a brought about retainment of â3-
AR agonistic activity and a slight increase of â1- and
â2-AR agonistic activity by 8- and 12-fold, respectively.
The ester compound (2a ) is hydrolyzed into 1a during
intestinal absorption. Intraduodenal administration of
2a significantly decreased the intrabladder pressure in
a dose-dependent manner. Moreover, no cardiac effect
was observed, even with complete relaxation by admin-
istration of 10 mg of 2a (Figure 4). On the basis of these
findings, the ester compound (2a ) appears to be an oral
prodrug of 1a without the cardiac effects.
Su m m a r y
We synthesized a novel â3-AR selective agonist (1a )
with two vicinal chiral carbons and asymmetric isomers
by inversion of the hydroxy group via oxazolidinone. The
correlation between the stereochemistry of 1a -d and
the â-AR agonist activity of each was investigated by
in vitro assay using rat atria for â1-AR, rat uteri for â2-
AR, and ferret detrusor for â3-AR. All four isomers were
selective for â3-AR over â1- and â2-AR. The (RS,âR)-
configuration proved to be essential for enhancing â-AR
agonism, although no stereoisomer had the same effect
on each â-AR. The (RS,âR)-isomer (1a ) was the most
potent and selective â3-AR agonist of the four. Intrave-
nous administration of 1a in the anesthetized rat
showed a decrement of intrabladder pressure without
influence on heart rate and blood pressure. The â3-AR
agonist activity of the ester (2a ) was equipotent to that
of 1a , although it was slightly less selective. Intraduode-
nal administration of 2a in the rat produced a sufficient
decrement of intrabladder pressure without an increase
of heart rate. In preliminary pharmacokinetic tests, the
ester (2a ) had an oral bioavailability of 26% in dogs with
a half-life of 90 min. One of the problems of conventional
â3-AR agonists, which were selected using rat adipose
tissue, was their low potency, low efficacy, or lack of
adequate selectivity against human â3-AR. Therefore,
we investigated the potency of our compounds, which
were selected using ferret detrusor, on human â3-AR
expressed by CHO cells.27 Compound 1a produced a
concentration-dependent increase in cAMP accumula-
tion with full agonistic activity, as did isoproterenol. The
EC50 of 1a and isoproterenol was 1.50 µM (IA ) 1.03)
and 0.13 µM (IA ) 1.00), respectively. We are now
working on the preclinical profiles of 1a and 2a as drug
candidates. The results of further pharmacokinetic and
toxicologic studies and the pharmacodynamics of these
compounds in several species will be reported in due
course.
The erythro-compound (1a ) with the best â3-AR ago-
nistic activity and selectivity of its isomers was 22-fold
more potent for â3-AR agonistic activity than isopro-
terenol and almost the same as BRL37344. Next, we
studied the interaction between 1a and the â3-AR
selective antagonist, SR58894A, on â3-AR. The respec-
tive pA2 and slope of the Schild plot of SR58894A are
6.24 ( 0.20 and 0.68 ( 0.31 in human detrusor,7b 7.64
( 0.36 and 0.43 ( 0.19 in ferret detrusor,11 and 8.06 (
0.43 and 1.06 ( 0.40 in rat proximal colon.19b Therefore,
the rat proximal colon is better for estimating the
antagonism of SR58894A on â3-AR. SR58894A produced
a parallel rightward shift of the concentration-response
curve for 1a (Figure 2). The slope 0.96 from Schild plots
indicates a competitive form of antagonism.
In the in vivo study using the anesthetized rat, the
intravenous injection of 1a significantly reduced the
intrabladder pressure, as shown in Figure 3, implying
that 1a increases the urine storage in the bladder. The
effect of 1a (ED50 ) 12 µg/kg) was 20-fold weaker than
that of isoproterenol (ED50 ) 0.6 µg/kg), although 1a

110 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Tanaka et al.
vacuo, and the residue was triturated with a mixture of Et2O
(60 mL) and hexane (20 mL), followed by filtration through a
pad of Celite. The filtrate was concentrated in vacuo, and the
residue was purified by MPLC on silica gel (eluent, hexane/
Et2O ) 3/1) to give 6.5 g (95%) of 7 as a colorless oil: IR (KBr)
2977, 1756, 1605 cm-1; 1H NMR (CDCl3) δ 1.29 (3H, t, J ) 7.1
Hz), 3.08 (2H, t, J ) 7.5 Hz), 3.54 (2H, t, J ) 7.5 Hz), 4.27
(2H, q, J ) 7.1 Hz), 4.69 (2H, s), 6.80 (1H, d, J ) 8.4 Hz), 7.04
(1H, dd, J ) 8.4, 2.2 Hz), 7.25 (1H, d, J ) 2.2 Hz).
2-[2-Ch lor o-4-(2-{[(1S ,2R )-2-h yd r oxy-2-(4-h yd r oxy-
ph en yl)-1-m eth yleth yl]am in o}eth yl)ph en oxy]acetic Acid
(1a ). To a solution of 6a (502 mg, 3.00 mmol) and 7 (984 mg,
3.06 mmol) in DMF (3 mL) was added diisopropylamine (0.46
mL, 3.30 mmol), and the mixture was stirred for 2 h at 80 °C.
The reaction mixture was concentrated in vacuo, and the
residue was partitioned between EtOAc (70 mL) and water
(70 mL). The organic layer was washed with brine (50 mL),
dried over anhydrous MgSO4, and concentrated in vacuo. The
residual oil was purified by MPLC on APS (eluent, CH2Cl2/
EtOH ) 20/1) to give 690 mg (56%) of ethyl 2-[2-chloro-4-(2-
{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]-
amino}ethyl)phenoxy]acetate as a glassy oil: 1H NMR (CDCl3)
δ 0.98 (3H, d, J ) 6.4 Hz), 1.33 (3H, t, J ) 7.1 Hz), 2.60-2.85
(4H, m), 2.90-3.05 (1H, m), 4.31 (2H, q, J ) 7.1 Hz), 4.47 (1H,
d, J ) 5.6 Hz), 4.69 (2H, s), 6.64-6.75 (3H, m), 6.91 (1H, dd,
J ) 8.4, 2.1 Hz), 7.06 (2H, d, J ) 8.6 Hz), 7.13 (1H, d, J ) 2.1
Hz).
Exp er im en ta l Section
Gen er a l Meth od s. 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
spectrophotometer 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 (δ) downfield from tetra-
methylsilane as the internal standard. 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) were carried out with a Thermo Quest FINNI-
GAN AQA electrospray ionization mass spectrometer. Elemen-
tal analyses were performed by the Yanaco CHN MT-5
analyzer. The analytical results obtained were within (0.4%
of the theoretical values unless otherwise stated. Silica gel 60
F254 precoated plates on glass from Merck KGaA or amino-
propyl silica gel (APS) precoated NH plates from Fuji Silysia
Chemical Ltd. were used for thin-layer chromatography (TLC).
Medium-pressure liquid column chromatography (MPLC) was
performed on silica gel 60 N (particle size 40-50 µm) from
Kanto Chemical Co., Inc. or APS Daisogel IR-60 (particle size
25-40 µm) from Daiso Co., Ltd. Analytical HPLC was run on
a Shimadzu LC-VP instrument equipped with an CHIRALPAK
AD-H, 4.6 mm × 250 mm column (Daicel Chemical Industries,
Ltd.) under two elution conditions: isocratic conditions in
hexane/EtOH/diethylamine/trifluoroacetic acid ) 940/60/1/1
(method a) and in hexane/2-propanol ) 88/12 (method b); flow
rate ) 1.0 mL/min, λ ) 225 nm. The column temperature was
maintained at 25 °C. All reagents and solvents were com-
mercially available unless otherwise indicated. Yields were not
optimized.
Ethyl 2-[2-chloro-4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxy-
phenyl)-1-methylethyl]amino}ethyl)phenoxy]acetate (690 mg,
1.69 mmol) was dissolved in 1 M NaOH (8.5 mL), and the
solution was stirred for 1 h at room temperature. To the
reaction mixture was added 1 M HCl (8.5 mL) under ice-cooling
with stirring, and collection of the resulting precipitates by
filtration gave 464 mg (99%) of 1a as a solid: mp 229-230 °C
2-Ch lor o-4-(2-h yd r oxyeth yl)p h en ol (8). To a solution of
3-chloro-4-hydroxyphenylacetic acid (30.0 g, 160 mmol) in THF
(300 mL) was added BH3‚Me2S complex (40.0 mL, 400 mmol)
dropwise at 10 °C. The mixture was stirred for 1 h at room
temperature and then heated under reflux for 1 h. After the
mixture was cooled by an ice bath, MeOH (50 mL) was
carefully added and the mixture was concentrated in vacuo.
The residue was partitioned between 1 M HCl (100 mL) and
EtOAc (200 mL). The EtOAc layer was washed successively
with water (100 mL), saturated aqueous NaHCO3 (100 mL),
and brine (50 mL). The organic layer was dried over anhydrous
MgSO4 and concentrated in vacuo. The residue was triturated
with hexane, and the resulting precipitates were collected by
filtration to give 25.6 g (92%) of 8 as a colorless solid: mp 75-
dec; [R]30 -5.7° (c 1.01, HOAc); IR (KBr) 3366, 3297, 3034,
D
2817, 1608, 1571 cm-1; 1H NMR (DMSO-d6 + D2O) δ 0.93 (3H,
d, J ) 6.7 Hz), 2.68-2.82 (2H, m), 3.00-3.17 (2H, m), 3.26-
3.35 (1H, m), 4.47 (2H, s), 5.06 (1H, d, J ) 2.2 Hz), 6.75 (2H,
d, J ) 8.5 Hz), 6.83 (1H, d, J ) 8.6 Hz), 6.91 (1H, dd, J ) 8.6,
2.1 Hz), 7.17 (2H, d, J ) 8.5 Hz), 7.26 (1H, d, J ) 2.1 Hz); MS
m/z (relative intensity) 380 (M + H)+, 382 (0.35). Anal. (C19H22
ClNO5, 379.83) C, H, N.
-
The following compounds were prepared from the corre-
sponding 4′-hydroxynorephedrine isomers (6b-d ) by a method
similar to that described here.
2-[2-Ch lor o-4-(2-{[(1R ,2S )-2-h yd r oxy-2-(4-h yd r oxy-
p h en yl)-1-m eth yleth yl]a m in o}eth yl)p h en oxy]a cetic a cid
1
76 °C; H NMR (CDCl3) δ 1.55 (1H, br), 2.78 (2H, t, J ) 6.5
(1b): mp 230-234 °C dec; [R]26 +8.3° (c 1.10, 1 M HCl); MS
D
Hz), 3.80-3.85 (2H, m), 5.64 (1H, br s), 6.94 (1H, d, J ) 8.2
Hz), 7.03 (1H, dd, J ) 8.2, 2.0 Hz), 7.19 (1H, d, J ) 2.0 Hz).
Eth yl 2-[2-Ch lor o-4-(2-h yd r oxyeth yl)p h en oxy]a ceta te
(9). To a solution of 8 (5.00 g, 29.0 mmol) in DMF (80 mL)
were added K2CO3 (4.80 g, 34.8 mmol) and ethyl bromoacetate
(3.86 mL, 34.8 mmol) at 5 °C, and the resulting suspension
was stirred for 4 h at room temperature. Diethylamine (3.00
mL, 29.0 mmol) was added, and the mixture was stirred for 1
h at room temperature. The reaction mixture was diluted with
Et2O (100 mL) and poured into ice-water (200 g). The aqueous
layer was extracted with Et2O (100 mL). The combined Et2O
layer was washed with water (50 mL) twice, 1 M HCl (50 mL),
saturated aqueous NaHCO3 (50 mL), and brine (50 mL)
successively. After being dried over anhydrous MgSO4, the
organic layer was filtrated through a short column of APS
(eluent, Et2O). The filtrate was concentrated in vacuo to give
6.0 g (80%) of 9 as a colorless oil: 1H NMR (CDCl3) δ 1.29
(3H, t, J ) 7.2 Hz), 1.44 (1H, t, J ) 6.2 Hz), 2.79 (2H, t, J )
6.2 Hz), 3.82 (2H, q, J ) 6.2 Hz), 4.27 (2H, q, J ) 7.2 Hz), 4.67
(2H, s), 6.80 (1H, d, J ) 8.4 Hz), 7.05 (1H, dd, J ) 8.4, 2.2
Hz), 7.77 (1H, d, J ) 2.2 Hz).
m/z (relative intensity) 380 (M + H)+, 382 (0.34). Anal. (C19H22
ClNO5, 379.83) C, H, N.
-
2-[2-Ch lor o-4-(2-{[(1S ,2S )-2-h yd r oxy-2-(4-h yd r oxy-
p h en yl)-1-m eth yleth yl]a m in o}eth yl)p h en oxy]a cetic a cid
(1c): mp 232-236 °C dec; [R]26 +43.7° (c 1.00, 1 M HCl); IR
D
(KBr) 3326, 3246, 1608, 1565 cm-1; 1H NMR (DMSO-d6 + CF3-
CO2D) δ 0.97 (3H, d, J ) 6.7 Hz), 2.80-3.40 (5H, m), 4.42 (1H,
d, J ) 9.3 Hz), 4.81 (2H, s), 6.80 (2H, d, J ) 8.6 Hz), 7.01 (1H,
d, J ) 8.5 Hz), 7.15-7.23 (3H, m), 7.41 (1H, d, J ) 2.1 Hz),
14.98 (4H, br); MS m/z (relative intensity) 380 (M + H)+, 382
(0.35). Anal. (C19H22ClNO5‚0.5H2O, 388.84) C, H, N.
2-[2-Ch lor o-4-(2-{[(1R ,2R )-2-h yd r oxy-2-(4-h yd r oxy-
p h en yl)-1-m eth yleth yl]a m in o}eth yl)p h en oxy]a cetic a cid
(1d ): mp 242-246 °C dec; [R]26 -44.9° (c 1.10, 1 M HCl); MS
D
m/z (relative intensity) 380 (M + H)+, 382 (0.37). Anal. (C19H22
ClNO5‚0.5H2O, 388.84) C, H, N.
-
Eth yl 2-[2-Ch lor o-4-(2-{[(1S,2R)-2-h ydr oxy-2-(4-h ydr oxy-
ph en yl)-1-m eth yleth yl]am in o}eth yl)ph en oxy]acetate Hy-
d r och lor id e (2a ). To a stirred solution of ethyl 2-[2-chloro-
4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]-
amino}ethyl)phenoxy]acetate (1.50 g, 3.68 mmol) in EtOAc (40
mL) was added 4 M HCl in EtOAc (1.84 mL, 7.36 mmol)
dropwise at 5 °C. The mixture was concentrated in vacuo, and
the residue was triturated with Et2O, followed by filtration to
Eth yl 2-[4-(2-Br om oeth yl)-2-ch lor oph en oxy]acetate (7).
To a stirred solution of 9 (5.5 g, 21.3 mmol) and Ph3P (5.86 g,
22.3 mmol) in CH2Cl2 (55 mL) was added CBr4 (7.40 g, 22.3
mmol) at 5 °C. After the reaction mixture was stirred for 2 h
at room temperature, EtOH (12 mL) was added, and the
mixture was the stirred for 1 h. The solvent was removed in
give 1.6 g (98%) of 2a as a white solid: mp 196-198 °C; [R]30
D
-10.3° (c 1.00, EtOH); IR (KBr) 3297, 3160, 1737 cm-1 1H
;

4′-Hydroxynorephedrine Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 111
NMR (DMSO-d6) δ 0.96 (3H, d, J ) 6.7 Hz), 1.21 (3H, t, J )
7.1 Hz), 2.90-3.05 (2H, m), 3.15-3.40 (3H, m), 4.17 (2H, q, J
) 7.1 Hz), 4.90 (2H, s), 5.08 (1H, br s), 5.90-6.00 (1H, m),
6.76 (2H, d, J ) 8.6 Hz), 7.02 (1H, d, J ) 8.6 Hz), 7.10-7.20
(3H, m), 7.40 (1H, d, J ) 2.1 Hz), 8.85 (2H, br), 9.41 (1H, s);
MS m/z (relative intensity) 408 (M + H)+, 410 (0.35). Anal.
(C21H27Cl2NO5, 444.36) C, H, N.
min before the addition of 1a . Concentration-response curves
for 1a were obtained for each concentration of SR-58894A. All
experiments were conducted in the presence of 10-6
M
phentolamine. The pA2 value for SR-58894A, as defined by
Arunlakshana and Shild,28 was obtained from linear regression
analysis of the plot of the mean value of log(CR - 1) vs the
negative log of the antagonist concentration (Figure 2).
In Vivo Exp er im en ts.12,29 Male rats (300-350 g in body
weight) were anesthetized with urethane (1.5 g kg-1, sc.), and
the midline abdomen was incised. The ureter on each side was
ligated and cut proximal to the ligature to allow urine to drain
into cotton wads. After ligation of the urethra, a polyethylene
catheter (PE-50, Nihon Becton Dickinson, Tokyo, J apan) was
inserted into the urinary bladder via the top of the bladder
dome and connected through a three-way connector to a
pressure transducer (SPB-108, NEC San-ei) and a syringe
filled with warmed saline. The initial bladder pressure was
adjusted to ca. 6 cmH2O by instillation of warmed saline
(37 °C) in 0.05 mL increments. An arterial catheter was
inserted into the left carotid artery (PE-90, Nihon Becton
Dickinson) and connected to a pressure transducer (SPB-108,
NEC San-ei) to measure blood pressure. The heart rate was
measured via a tachometer (132l, NEC San-ei) connected to a
transducer amplifier (1829, NEC San-ei). Bladder pressure,
blood pressure, and heart rate were recorded continuously on
a rectigraph (Recti-Horiz-8K, NEC San-ei). The drug was
injected through a venous catheter (PE-50, Nihon Becton
Dickinson) inserted into the left femoral vein or through a
catheter (PE-50, Nihon Becton Dickinson) inserted into the
duodenum. The relaxing effect of each drug was expressed as
a percentage of the maximal relaxation with isoproterenol. No
animal was exposed to more than one of the test drugs.
ter t-Bu tyl N-[(1S,2R)-2-(4-Ben zyloxyph en yl)-2-h ydr oxy-
1-m eth yleth yl]ca r ba m a te (10). To a solution of t-Boc2O (6.20
g, 28.4 mmol) in THF (60 mL) was added 6a (5.00 g, 29.9
mmol) at room temperature. The mixture was stirred over-
night and concentrated in vacuo. The residue was dissolved
in EtOAc and washed with 10% aqueous citric acid, saturated
aqueous NaHCO3, and brine successively. The organic layer
was dried over anhydrous MgSO4 and evaporated in vacuo.
The residue (7.57 g) was dissolved in DMF (140 mL). Cesium
carbonate (10.0 g, 30.7 mmol) and benzyl bromide (3.57 mL,
30.0 mmol) were added, and the mixture was stirred overnight
at room temperature. The reaction mixture was diluted with
Et2O and washed with water, 1 M NaOH, and brine succes-
sively. The organic layer was dried over MgSO4 and concen-
trated in vacuo to give 9.50 g (93%) of 10 as a white solid: mp
1
135-138 °C dec; IR (KBr) 3360, 1682 cm-1; H NMR (CDCl3)
δ 0.99 (3H, d, J ) 7.0 Hz), 1.46 (9H, s), 3.17 (1H, br), 3.90-
4.05 (1H, m), 4.55-4.65 (1H, m), 4.75-4.85 (1H, m), 5.06 (2H,
s), 6.96 (2H, d, J ) 8.6 Hz), 7.25 (2H, d, J ) 8.6 Hz), 7.30-
7.50 (5H, m).
(4S,5S)-5-(4-Ben zyloxyp h en yl)-4-m et h yloxa zolid in -2-
on e (11). To a stirred solution of 10 (9.00 g, 25.2 mmol) and
triethylamine (6.97 mL, 50.0 mmol) in CH2Cl2 (100 mL) was
added MsCl (2.16 mL, 27.9 mmol) dropwise at 5 °C, and the
mixture was stirred overnight at room temperature. The
reaction mixture was washed with 1 M HCl, saturated aqueous
NaHCO3, and brine successively. The organic layer was dried
over MgSO4 and evaporated in vacuo. The residual solid was
recrystallized with EtOAc to give 4.50 g (63%) of 11 as a white
crystal: mp 180-181 °C; IR (KBr) 3263, 1751, 1708 cm-1; 1H
NMR (CDCl3) δ 1.35 (3H, d, J ) 6.1 Hz), 3.80-3.86 (1H, m),
4.98 (1H, d, J ) 7.2 Hz), 5.08 (2H, s), 5.63 (1H, br), 7.00 (2H,
d, J ) 8.7 Hz), 7.30 (2H, d, J ) 8.7 Hz), 7.31-7.45 (5H, m).
Ack n ow led gm en t. We are grateful to our col-
leagues for analytical and spectral determinations and
for performing the screening assays. We thank Dr.
Yoshinobu Yamazaki and Mr. Hiroo Takeda for their
pharmacological contributions to this work. We also
thank Dr. Hiromu Harada for helpful comments while
reviewing the manuscript.
(rS,âS)-4′-Hyd r oxyn or ep h ed r in e (6c). The mixture of 11
(4.50 g, 15.9 mmol) and KOH (1.06 g, 16.0 mmol) in water (16
mL) and dioxane (16 mL) was heated under reflux overnight.
Additionally, KOH (1.06 g), water (16 mL), and dioxane (16
mL) were added and the mixture was heated under reflux for
5 h. The reaction mixture was concentrated in vacuo, and the
residue was triturated with water (ca. 20 mL). The suspension
was allowed to stand at 5 °C overnight. The precipitates were
collected by filtration. After the mixture was dried under
reduced pressure, the resulting solid (3.65 g) was dissolved in
MeOH (70 mL), and 10% Pd on activated carbon (700 mg, wet)
was added. The mixture was stirred overnight at room
temperature under hydrogen and filtrated to remove the
catalyst. The filtrate was concentrated in vacuo, and the
residue was triturated with EtOAc. Filtration gave 2.36 g
(89%) of 6c as a white solid: mp 149-152 °C dec; [R]26D +34.2°
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1
(c 1.06, MeOH); IR (KBr) 3389, 2577 cm-1; H NMR (DMSO-
d6) δ 0.73 (3H, d, J ) 6.4 Hz), 2.72 (1H, quint, J ) 6.7 Hz),
3.96 (1H, d, J ) 6.7 Hz), 6.69 (2H, d, J ) 8.5 Hz), 7.06 (2H, d,
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giving tR ) 25.0 min (method a), while nontreated 6c gave tR
)16.4 min (method b): 100% ee, 99.4% de.
(rR,âR)-4′-Hyd r oxyn or ep h ed r in e (6d ). The title com-
pound was prepared from 6b by same method described in
6c: mp 153-155 °C; [R]26 -34.7° (c 1.08, MeOH); tR ) 29.1
D
min, 100% ee, 99.7% de (method a).
In Vitr o Exp er im en ts. The in vitro functional experi-
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previously described in detail.12 The rat colon preparation was
used to examine the interaction of a â3-AR antagonist (SR-
58894A) with 1a . SR-58894A was added to a Magnus bath 30

112 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
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