Novel chiral isoxazole derivatives: Synthesis and pharmacological characterization at human β-adrenergic receptor subtypes

Article abstract of DOI:10.1016/j.bmc.2007.01.056

Isoxazole derivative (±)-4 and the three pairs of stereoisomeric 3-bromo-isoxazolyl amino alcohols (S, R)-(-)-7a/(R, R)-(+)-7b, (S, R)-(-)-8a/(R, R)-(+)-8b, and (S, R)-(-)-9a/(R, R)-(+)-9b were synthesized and assayed for their affinity and efficacy at hu

Full text of DOI:10.1016/j.bmc.2007.01.056

Bioorganic & Medicinal Chemistry 15 (2007) 2533–2543
Novel chiral isoxazole derivatives: Synthesis and pharmacological
characterization at human b-adrenergic receptor subtypes
Clelia Dallanoce,a Fabio Frigerio,a Marco De Amici,a,* Sandra Dorsch,b
Karl-Norbert Klotzb,* and Carlo De Michelia
a
`
Istituto di Chimica Farmaceutica e Tossicologica ‘‘Pietro Pratesi’’, Universita degli Studi di Milano,
Viale Abruzzi 42, 20131 Milano, Italy
bInstitut fu¨r Pharmakologie und Toxikologie, Universita¨t Wu¨rzburg, Versbacher Strasse 9, 97078 Wu¨rzburg, Germany
Received 27 November 2006; revised 19 January 2007; accepted 31 January 2007
Available online 2 February 2007
Abstract—Isoxazole derivative ( )-4 and the three pairs of stereoisomeric 3-bromo-isoxazolyl amino alcohols (S,R)-(ꢀ)-7a/(R,R)-
(+)-7b, (S,R)-(ꢀ)-8a/(R,R)-(+)-8b, and (S,R)-(ꢀ)-9a/(R,R)-(+)-9b were synthesized and assayed for their affinity and efficacy at
human b1-, b2-, and b3-adrenergic receptors (b-ARs) in membranes from Chinese hamster ovary (CHO) cells stably transfected with
the respective receptor subtype. Whereas derivative ( )-4 did not bind at all three b-ARs, stereoisomers (S,R)-7a-(S,R)-9a behaved
as high-affinity ligands at b1- and, particularly, at b2-ARs (Ki 2.82–66.7 nM). The Ki values of isomers (R,R)-7b-(R,R)-9b at b1- and
b2-subtypes were about 30–100 times higher than those of their (S,R)-7a–9a counterparts, indicating a sizable stereochemical effect.
The affinity at b3-ARs was negligible for all the investigated compounds. When submitted to a functional assay, the three stereo-
isomeric pairs showed a comparable pattern of efficacy at all three b-AR subtypes. The highest value of efficacy (75–90%) was
observed at b2-ARs, whereas all compounds behaved as partial agonists (30–60%) at the b3-subtype. The lowest degree of efficacy
(15–35%) was found at b1-ARs. The affinity/efficacy profile of the derivatives under study has been compared with that of the two
model compounds, Broxaterol [( )-1] and BRL 37344 [( )-6].
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Among the numerous b-ARs ligands that have found
therapeutic application, antagonists (‘b-blockers’) repre-
sent a well-defined class of drugs in clinical use for virtu-
ally all major cardiovascular diseases, even though some
uncertainties remain concerning their exact mechanisms
of action.3,10 On the other hand, b2-AR agonists are
routinely used in the treatment of asthma11 owing to
their regulatory effect on bronchial tone, and agonists
or partial agonists selective for the human b3-ARs are
investigated as potential anti-obesity and anti-diabetes
agents as well as novel drugs for urinary bladder
dysfunctions.12,13
b-Adrenergic receptors (b-ARs) are among the most
thoroughly studied members of G protein-coupled
receptors (GPCRs), the largest signaling family of the
human genome.1–4 At present b-ARs are classified as
b1-, b2-, and b3-subtypes based on their physiological
actions, their pharmacological response to selective
ligands as well as cloning of the receptors.5 Functional-
ly, significant levels of b1-ARs are found in the heart,
kidney, and brain,6 b2-ARs are predominant on vascu-
lar, uterine, and airway smooth muscles,7 and b3-ARs
are mainly expressed in adipose tissue.8 All of them
couple primarily to Gas to stimulate adenylyl cyclase,
even though they can also couple to Gai in some cells
under certain conditions.9
Although a number of amino acids have been identified
through different experimental techniques as key bind-
ing site residues for a variety of b-AR agonists and
antagonists,14 at present a detailed understanding for
b-AR subtype ligand-binding selectivity is far from
being clearly established. Moreover, activation of
GPCRs seems to be a multistep process further compli-
cated by the existence of these receptors in different
putative inactive and active conformations.15,16 As a
consequence, the molecular interaction with agonists
Keywords: Synthesis; Isoxazole derivatives; Hybrid compounds;
Human b-adrenergic receptor subtypes; Binding affinity; Efficacy.
Corresponding authors. Tel.: +39 02503 17555; fax: +39 02503 17565
(M.D.A.); tel.: +49 931 20148405; fax: +49 931 20148539 (K.-N.K.);
*
e-mail addresses:
wuerzburg.de
marco.deamici@unimi.it;
klotz@toxi.uni-
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.056

2534
C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
would lead to a stabilization of one or more of these
activated states. Such mechanisms complicate the dis-
cussion of the relationship between binding affinity
and functional efficacy at b-ARs and other GPCRs,
however they also open interesting perspectives for the
development of novel agonists with the potential of rec-
ognizing functionally distinct states of receptor
subtypes.17
the 3-methanesulfonylamino-isoxazole derivative ( )-4
and the evaluation of its affinity at the three b-AR sub-
types. Worth mentioning, the skeleton of Soterenol ( )-
3 has recently been incorporated in a number of deriva-
tives, such as 5 (Fig. 1),26 endowed with the profile of
selective b3-AR agonists.
Next, we considered the structures of ( )-1 and BRL
37344 ( )-6 (Fig. 1), one of the first reported b3-AR ago-
nists27 and still used as a lead compound,28 to design
hybrid derivatives (S,R)-(ꢀ)-7a and (R,R)-(+)-7b. The
goal was the evaluation of the impact on the pharmaco-
logical profile brought about by the replacement of the
3-chlorobenzene moiety of ( )-6 with the 3-bromoisox-
azole nucleus of Broxaterol ( )-1. The related ethyl
esters (S,R)-(ꢀ)-8a and (R,R)-(+)-8b, and their
O-n-butoxy analogs (S,R)-(ꢀ)-9a and (R,R)-(+)-9b
(Fig. 1) were also prepared. This paper reports the
synthesis of ( )-4 and of enantiopure 3-bromoisoxazoles
(ꢀ)-7a/(+)-7b, (ꢀ)-8a/(+)-8b, and (ꢀ)-9a/(+)-9b, and
the evaluation of their binding affinity and efficacy at
human b1-, b2-, and b3-receptor subtypes.
Our interest in the investigation of subtype-selective li-
gands for the human b-ARs has been focused on a series
of D2-isoxazoline and isoxazole derivatives structurally
related to Broxaterol ( )-1 (Fig. 1),18–20 a b2-selective
agonist initially developed as a potential bronchodilato-
ry agent for the therapy of asthma,21,22 which was later
on discontinued. Interestingly, we found that this ligand,
which binds with comparable affinity to all three b-ARs,
is an agonist at b2- and b3-receptors and an antagonist
at the b1-receptor, thus behaving as a functionally selec-
tive b2-/b3-AR agonist.18,23 Despite the qualitatively dif-
ferent pharmacological profile, Broxaterol and
Isoproterenol ( )-2 (Fig. 1), the reference non-selective
b-AR agonist, share the same stereochemical preference,
since the eutomers of both compounds, that is (S)-(ꢀ)-1
and (R)-(ꢀ)-2, respectively, have an identical spatial
arrangement of the substituents around the common
stereogenic center.24
2. Chemistry
As depicted in Scheme 1, the synthetic sequence to target
compound ( )-4 started with 3-amino-5-ethoxycarbon-
yl-isoxazole ( )-10, in turn prepared according to a
published method.29 The corresponding 3-metha-
nesulfonylamino-isoxazole derivative ( )-11, prepared
with a standard procedure, was submitted to the Wein-
reb reaction protocol30,31 using commercially available
N,O-dimethylhydroxylamine hydrochloride and a THF
solution of methylmagnesium chloride to afford meth-
ylketone ( )-12.32 Intermediate ( )-12 was sequentially
The goal of the present paper was to further investigate
the role of the isoxazole moiety in the interaction with
the b-AR subtypes. To this end, we initially considered
the structure of Soterenol ( )-3, a b2-selective agonist
studied about thirty years ago,25 which is characterized
by the replacement of the 3-hydroxy group of the cate-
chol moiety of Isoproterenol with the methanesulfonyl-
amino moiety. Therefore, we planned the synthesis of
NHSO2CH3
R
R
HO
HO
SO2
OnBu
N
N
NHiPr
N
H
NHtBu
O
OH
OH
OH
( )-1: R = Br
( )-2: R = OH
5
( )-4: R = NHSO2CH3
( )-3: R = NHSO2CH3
Cl
Br
OR
OCH2COOH
CH3
CH3
N
N
H
O
N
H
OH
OH
( )-6: (
R
*,
R
*)-BRL 37344
(
(
(
(
(
(
S
,
R
)-(−)-7a: R = CH2CO2H
)-(+)-7b: R = CH2CO2H
)-(−)-8a: R = CH2CO2Et
)-(+)-8b: R = CH2CO2Et
)-(−)-9a: R = (CH2)3CH3
R,R)-(+)-9b: R = (CH2)3CH3
R,R
S,R
R,R
S,R
Figure 1. Model and target compounds in this study.

C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
2535
H2N
H3CO2SHN
H3CO2SHN
a
b
N
OEt
N
OEt
N
CH3
O
O
O
O
O
O
( )-10
( )-11
( )-12
H3CO2SHN
H3CO2SHN
c
d
e
( )-12
( )-4
N
CH2Br
N
CH2Br
O
O
O
OH
( )-13
( )-14
Scheme 1. Reagents: (a) CH3SO2Cl, Et3N/Cl(CH2)2Cl; (b) CH3ONHCH3· HCl, CH3MgCl/THF; (c) ðCH3Þ C6H5NþBrꢀ=THF; (d) NaBH4/MeOH;
3
3
(e) t-BuNH2/MeOH.
converted into a-bromoketone ( )-13 and bromohydrin
( )-14 which, by treatment with an excess of tert-butyl-
amine in refluxing methanol, provided the desired isox-
azolyl ethanolamine ( )-4.
compounds (S,R)-(ꢀ)-8a and (R,R)-(+)-8b, (S,R)-(ꢀ)-
9a and (R,R)-(+)-9b by removal of the N-Boc protecting
group with a dichloromethane solution of trifluoroacetic
acid. Alternatively, intermediates (S,R)-(ꢀ)-23a and
(R,R)-(ꢀ)-23b were first hydrolyzed to the correspond-
ing carboxylic acids and then submitted to N-Boc cleav-
age to produce target compounds (S,R)-(ꢀ)-7a and
(R,R)-(+)-7b.
The synthesis of the stereoisomeric couples 7a–b, 8a–b,
and 9a–b was accomplished by using the known 3-bro-
mo-5-acetyl-isoxazole ( )-1524 as starting material,
which was transformed into the corresponding
3-bromo-5-oxiran-2-yl-isoxazole ( )-17 through the
intermediacy of bromohydrin ( )-16 (Scheme 2). Epox-
ide ( )-17 was subsequently reacted with known (R)-
(ꢀ)-1-methyl-2-(4-methoxyphenyl)ethylamine 1833,34 in
refluxing acetonitrile under the catalysis of calcium
trifluoromethanesulfonate,35 to produce an almost equi-
molar mixture of stereomeric amino alcohols (S,R)-19a
and (R,R)-19b in 60% overall yield. These experimental
conditions proved to be the most convenient among a
series of variants. The two stereoisomers could be sepa-
rated by preparative silica gel chromatography only
after their conversion into the corresponding N-Boc
derivatives (S,R)-(ꢀ)-20a and (R,R)-(ꢀ)-20b. The
absolute configurations were unambiguously assigned
to (ꢀ)-20a and (ꢀ)-20b by performing, on an analytical
scale, the nucleophilic substitution of (R)-(ꢀ)-18 onto
the enantiopure bromohydrin (R)-(ꢀ)-1624 in refluxing
methanol, which, after insertion of the N-Boc group,
afforded (S,R)-(ꢀ)-20a as the sole isomer.
3. Results and discussion
Isoxazole derivative ( )-4 and the three pairs of stereo-
isomeric 3-bromo-isoxazolyl derivatives (S,R)-(ꢀ)-7a/
(R,R)-(+)-7b, (S,R)-(ꢀ)-8a/(R,R)-(+)-8b, and (S,R)-
(ꢀ)-9a/(R,R)-(+)-9b were tested for their binding affinity
at human b1-, b2-, and b3-ARs in membranes from CHO
cells stably transfected with the respective receptor sub-
type.23 Table 1 reports the dissociation constants (Ki
values) of the investigated compounds from competition
experiments with 125I-cyanopindolol (125I-CYP) as the
radioligand. The corresponding Ki values for Broxaterol
( )-120,23 and the BRL 37344 ( )-6 have been included
for comparison. Since binding experiments were carried
out in the presence of 100 lM GTP all Ki values for
agonists reflect low-affinity binding.
Inspection of Table 1 reveals the lack of any detectable
affinity of derivative ( )-4 for b-ARs (Ki > 100,000 nM
at all three subtypes). Hence, the replacement of the
3-bromo moiety of ( )-1 by the methanesulfonylamino
group prevents any interaction with the binding sites
of the b-ARs. Conversely, within the group of structur-
ally related 3-bromoisoxazole derivatives, the (S,R)-7a,
(S,R)-8a, and (S,R)-9a analogs behaved as high-affinity
ligands at b1- and, particularly, at b2-ARs (Table 1). In-
deed, in the set of compounds under study, amino alco-
hol (S,R)-(ꢀ)-8a, bearing the ester function on the distal
chain, had the highest affinity at b1 and b2 subtypes
(Ki = 23.2 and 2.82 nM, respectively). The same set of
isomers showed a marginal affinity for the b3-ARs, with
the sole exception of the O-n-butoxy ether (S,R)-(ꢀ)-9a
which gave a value of Ki equal to 2500 nM. As a general
The synthetic pathway to the final compounds
proceeded along the reaction sequence is shown in
Scheme 3. Isomers (S,R)-(ꢀ)-20a and (R,R)-(ꢀ)-20b
were separately treated with a 1.0 M solution of boron
tribromide in dichloromethane to give intermediates
(S,R)-(ꢀ)-21a and (R,R)-(ꢀ)-21b, respectively. The cor-
responding N-Boc-reprotected derivatives (S,R)-(ꢀ)-22a
and (R,R)-(ꢀ)-22b were then suitably functionalized at
the 4-hydroxyphenyl group by treatment with ethyl
bromoacetate or 1-bromobutane in a refluxing acetone
suspension of potassium carbonate, to produce the pairs
of diastereomers (S,R)-(ꢀ)-23a/(R,R)-(ꢀ)-23b and
(S,R)-(ꢀ)-24a/(R,R)-(ꢀ)-24b, respectively. These diaste-
reomers were then converted into the desired final

2536
C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
Br
Br
Br
a,b
c
N
CH3
N
CH2Br
OH
N
O
O
O
O
O
( )-15
( )-16
( )-17
Br
N
OCH3
CH3
N
R
O
OH
OCH3
(S,R)-19a: R = H
CH3
d
e
( )-17
+
(S,R)-(—)-20a: R = Boc
H2N
(R)-(—)-18
Br
N
OCH3
CH3
N
O
OH
R
(R,R)-19b: R = H
e
(R,R)-(—)-20b: R = Boc
Br
f,e
(S,R)-(—)-20a
N
CH2Br
O
OH
(R)-(—)-16
Scheme 2. Reagents: (a) ðCH3Þ C6H5NþBrꢀ3 =THF; (b) NaBH4/MeOH; (c) NaH/toluene; (d) Ca(OTf)2/ CH3CN; (e) (Boc)2O, Et3N/CH2Cl2;
3
(f) (R)-(ꢀ)-18/MeOH.
trend, the (R,R)-stereoisomers showed an affinity lower
than that of the corresponding (S,R)-diastereomers. In
particular, the Ki values at b1- and b2-subtypes were usu-
ally 30–100 times higher than those of the corresponding
(S,R)-counterparts, indicating a substantial stereochem-
ical effect.
dictated at b1-ARs and to a lesser extent at b2-ARs
by the (S)-configuration at the stereogenic center local-
ized at the secondary alcohol function. The affinity at
b3-ARs is almost negligible for all the investigated
compounds and, consequently, is marginally affected
by the absolute configuration at the same stereogenic
center.
On the whole, the replacement of the 3-chlorobenzene
group of BRL 37344 ( )-6 with the 3-bromoisoxazole
moiety of Broxaterol ( )-1 brings about a considerable
change in the affinity profile, as evidenced by derivative
(S,R)-(ꢀ)-7a, the hybrid incorporating the structure of
the eutomer of Broxaterol, that is (S)-(ꢀ)-1, and retain-
ing the acidic group of BRL 37344 ( )-6. As a matter
of fact, analogs 7a–9a, characterized by the (S,R)-con-
figuration at the stereogenic centers, behave as high-af-
finity ligands at both b1- and b2-subtypes (Ki values in
the range 2.82–66.7 nM), at variance with ( )-1 (Ki
equal to 1310 nM at b1-ARs and 1290 nM at b2-ARs)
and, even more, with ( )-6 (Ki equal to 37,000 nM at
b1-ARs and 2850 nM at b2-ARs). Within this set of
new derivatives, which belong to the class of aryletha-
nolamines, the ligand-receptor recognition process is
In order to fully define their pharmacological profile, the
six isoxazolyl amino alcohols (S,R)-7a-(S,R)-9a and
(R,R)-7b-(R,R)-9b were submitted to a functional assay
in which stimulation of adenylyl cyclase (AC) was tested
in membranes from stably transfected CHO cells with
similar receptor expression for each b-AR subtype.23
The agonistic activity of these compounds was com-
pared to the maximal stimulation obtained with the full
agonist Isoproterenol (100%; basal activity 0%) as the
reference. From our previous characterization of the
transfected CHO cell clones used in this study, we know
that EC50 values for agonists tend to be lower than the
corresponding Ki values derived from binding experi-
ments.23 At a Ki-concentration, a signal of 80–90%
amplitude is expected from a compound with full

C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
2537
(
S,
R)-(—)-20a
(
R,
R)-(—)-20b
a
a
Br
Br
OH
OH
CH3
CH3
N
N
N
R
N
R
O
b
O
b
OH
OH
(
(
S
,
,
R
)-(—)-21a: R = H
(
(
R,R
)-(—)-21b: R = H
S
R
)-(—)-22a: R = Boc
R,R
)-(—)-22b: R = Boc
Br
OCH2CO2Et
d
c
CH3
(
(
S
,
R
)-(—)-22a
(
(
S
,
R
)-(—)-8a
N
O
R
,
R
)-(—)-22b
R
,
R
)-(+)-8b
N
OH BOC
e,d
(
(
S,R)-(—)-7a
(
(
S,R)-(—)-23a:
OH
OH
R,
R)-(+)-7b
R,
R)-(—)-23b:
Br
O(CH2)3CH3
d
f
CH3
N
(
(
S
R
,
R
)-(—)-22a
)-(—)-22b
(
(
S
R
,
R
)-(—)-9a
N
O
,
R
,
R
)-(+)-9b
OH BOC
(
(
S
,
R
)-(—)-24a:
OH
OH
R,R)-(—)-24b:
Scheme 3. Reagents: (a) BBr3/CH2Cl2; (b) (Boc)2O, Et3N/CH3CN; (c) BrCH2CO2Et, K2CO3/acetone; (d) CF3COOH/CH2Cl2; (e) K2CO3/EtOH–
H2O; (f) Br(CH2)3CH3, K2CO3/acetone.
Table 1. Binding affinities of the novel isoxazole derivatives from competition experiments at human b-adrenergic receptor subtypes
Compound
b1-receptors
b2-receptors
b3-receptors
Ki (nM)
95% confidence limits
Ki (nM)
95% confidence limits
Ki (nM)
95% confidence limits
( )-4
>105
61.1
—
>105
13.9
501
—
>105
—
8910–54,100
—
(S,R)-(ꢀ)-7a
(R,R)-(+)-7 b
(S,R)-(ꢀ)-8a
(R,R)-(+)-8 b
(S,R)-(ꢀ)-9a
(R,R)-(+)-9 b
( )-1
28.1–133
4540–9170
11.2–47.9
708–1750
37.6–118
1940–2400
930–1860
34,100–41,800
5.88–33.1
367–683
1.38–5.75
68.2–122
9.52–25.4
333–630
916–1810
2120–3830
22,000
>50,000
10,100
21,800
2485
6460
23.2
2.82
81.4
15.5
458
1290a
2850
3390–30,100
10,700–44,300
1820–3390
9680–11,800
3470–4590
389–475
1110
66.7
2160
1310a
37,900a
10,700
3990a
430a
( )-6
125I-CYP was used as radioligand at a concentration of 50–80 pM. Experiments were done in the presence of 100 lM GTP. Ki values were calculated
with the program SCTFIT and represent geometric mean values of at least three different experiments done in triplicate.
a Values are taken from Ref. 23.
agonistic activity. Therefore, compounds were tested at
a concentration of 100 lM since a maximal signal may
be expected even with the low-affinity interaction at the
b3-AR. The percent values reported in Table 2 for the
new isoxazole derivatives show a similar pattern of
efficacy at all b-AR subtypes. Higher efficacy
values (75–90%) were found at b2-ARs, whereas at the
b3-subtype all compounds behaved as partial agonists
(30–60%). The lowest range of efficacy (15–35%) was
found at b1-ARs. This profile resembles that of
Broxaterol ( )-1, although this parent compound was
previously characterized as an antagonist at b1-ARs.
Depending on the experimental conditions, its efficacy
ranged from some inverse agonism (–10%)23 to negligi-
ble partial agonism (+8%).20 In contrast, BRL 37344
( )-6 showed partial agonistic activity at b3-ARs
(28%) and an antagonist profile at the other two sub-
types.23 It is clearly shown that the introduction of the
3-bromoisoxazole for the 3-chlorophenyl moiety
increases the efficacy at all b-AR subtypes and thereby
converts the b1- and b2-antagonist BRL 37344 ( )-6 into
(partial) b1- and b2-agonists.

2538
C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
carboxylic acid ethyl ester ( )-10,29 2-bromo-1-(3-bro-
moisoxazol-5-yl)-ethanols ( )-1624 and (R)-(ꢀ)-16,24
(R)-(ꢀ)-1-methyl-2-(4-methoxyphenyl)ethylamine 18,33
and calcium trifluoromethanesulfonate35 were all pre-
pared according to published procedures. 1H NMR
and 13C NMR spectra were recorded with a Varian Mer-
cury 300 (1H, 300.063; 13C, 75.451 MHz) in CDCl3 (un-
less otherwise specified) solutions; chemical shifts (d) are
expressed in ppm and coupling constants (J) in Hz.
Table 2. Adenylyl cyclase responses of human b-adrenergic receptor
subtypes to the listed compounds
Compound
Efficacy % (isoproterenol = 100%)
b1-rec. b2-rec. b3-rec.
(S,R)-(ꢀ)-7a
(R,R)-(+)-7b
(S,R)-(ꢀ)-8a
(R,R)-(+)-8b
(S,R)-(ꢀ)-9a
(R,R)-(+)-9b
( )-1a
34
27
31
24
24
13
8
4
5
4
5
7
3
3
4
74
69
81
77
87
75
88
ꢀ7
6
50
34
56
39
4
4
4
9
0
3
3
7
4
7
2
51 12
Melting points were determined on a Mod. B 540 Buchi
¨
31
42
28
5
3
3
apparatus and are uncorrected. Liquid compounds were
characterized by the oven temperature for bulb to bulb
distillations. Rotary power determinations were carried
out with a Jasco J-810 spectropolarimeter coupled with
a Haake N3-B thermostat. TLC analyses were per-
formed on commercial silica gel 60 F254 aluminum
sheets: spots were further evidenced by spraying with a
dilute alkaline potassium permanganate solution.
Microanalyses (C,H,N) of new compounds agreed with
the theoretical value 0.4%.
( )-6b
ꢀ5
Membranes were prepared from cells with comparable receptor
expression level. Adenylyl cyclase stimulation represents the percent-
age of maximal stimulation achieved by 100 lM isoproterenol (positive
values) or percent inhibition of basal activity (negative values) SEM.
a Values are taken from Ref. 20.
b Values are taken from Ref. 23.
Therefore, based on these functional results, the novel hy-
brid compounds may be classified as full agonists at b2-
ARs and partial agonists at both b1- and b3-ARs. The
presence of the 3-bromoisoxazole moiety greatly affects
their functional profile which, at least at the b2 and b3 sub-
types, is similar to that of Broxaterol ( )-1. At variance
with ( )-1, which is an antagonist at b1-ARs, the new
derivatives are partial agonists at this receptor subtype.
The radioligand (ꢀ)3-125I-Iodocyanopindolol
(
125I-
CYP) was purchased from Amersham Biosciences
(specific activity, 2200 Ci/mmol). [a-32P]ATP was from
Perkin-Elmer LifeScience. Cell culture media and fetal
calf serum were from PanSystems, penicillin (100 U/mL),
streptomycin (100 lg/mL), L-glutamine, and G-418 were
purchased from Gibco-Life Technologies. All other
materials were from sources as described earlier.23
4. Conclusions
5.2. Chemical experimental section
The results of our study pinpoint that the replacement of
the 3-chlorobenzene moiety of BRL 37344 ( )-6 with the
3-bromoisoxazole nucleus of Broxaterol ( )-1 gives rise
to a series of isomeric amino alcohols which are b-AR
ligands. These hybrid compounds show a marked
change in both the binding and functional patterns at
the three b-adrenergic receptor subtypes if compared
to the profiles of reference compounds ( )-1 and ( )-6.
Most striking is the dramatic increase in b1- and b2-affin-
ity of some of the compounds. The stereoisomers with
the (S)-configuration at the stereocenter bearing the
secondary alcohol group turned out to be high-affinity
b1-/b2-ligands devoid of an appreciable affinity at the
b3-subtype. In addition, all members of the series
activated the three b-adrenergic receptor subtypes to
various degrees. Since structural modifications in this
set of related ligands caused interesting changes in effica-
cy, these compounds may be valuable tools for studies
of receptor activation. They might be particularly useful
in studies employing receptor mutants which were
recently shown to exhibit different functional responses
to compounds like Broxaterol ( )-1.36
5.2.1. N-{5-[2-(tert-Butylamino)-1-hydroxyethyl]isoxazol-
3-yl}methanesulfonamide ( )-4. (A) To a solution of
1.92 g (12.3 mmol) of ( )-1029 in 1,2-dichloroethane
(50 mL) were added triethylamine (5.15 mL, 36.9 mmol)
and methanesulfonyl cloride (1.43 mL, 18.5 mmol). The
reaction mixture was heated at reflux for 1 h, then
cooled at rt and washed with water. The organic layer
was dried over anhydrous sodium sulfate and the sol-
vent was removed under reduced pressure. A silica gel
column chromatography of the residue (eluant: 30% eth-
yl acetate/petroleum ether) gave 2.33 g (81% yield) of the
desired compound.
Ethyl 3-[(methylsulfonyl)amino]isoxazole-5-carboxylate
( )-11: colorless prisms (from ethyl acetate/n-hexane),
mp 99–101 ꢁC. Rf 0.33 (eluant: 30% ethyl acetate/petro-
leum ether). 1H NMR: 1.42 (t, 3H, J = 6.9), 3.19 (s, 3H),
4.44 (q, 2H, J = 6.9), 7.08 (s, 1H), 7.90 (br s, 1H). Anal.
Calcd for C7H10N2O5S: C, 35.89; H, 4.30; N, 11.96.
Found: C, 35.98; H, 4.51; N, 12.16.
(B) To a stirred suspension of N,O-dimethylhydroxyl-
amine hydrochloride (707 mg, 7.25 mmol) and ethyl
ester ( )-11 (1.40 g, 6.0 mmol) in anhydrous THF
(80 mL), maintained at ꢀ5 ꢁC under nitrogen, was add-
ed dropwise during 1 h 16.2 mL (48.6 mmol) of a 3 M
solution of methylmagnesium chloride in anhydrous
THF. The reaction mixture was further stirred at a
ꢀ5 ꢁC for 1 h and at rt for 4 h, then 50 mL of 1 N HCl
was added and the organic solvent was evaporated at
reduced pressure. After treatment with dichloromethane
5. Experimental
5.1. Materials and methods
[(R*,R*)-( )-4-[2-[2-(3-chlorophenyl)-2-hydroxyethylam-
ino]propyl] phenoxyacetic acid [BRL 37344, ( )-6]
was purchased from Tocris/Biotrend (Cologne, Germa-
ny). ( )-Broxaterol 1 (free base),24 3-aminoisoxazole-5-

C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
2539
(3 · 25 mL), the pooled organic extracts were dried over
anhydrous sodium sulfate and concentrated, and the
residue was purified by silica gel column chromatogra-
phy (eluant: 50% ethyl acetate/petroleum ether) to give
625 mg (51% yield) of the wanted methyl ketone.
5.2.2. 1-(3-Bromoisoxazol-5-yl)-2-{[2-(4-methoxyphenyl)-
1-methylethyl]tert-butoxycarbonylamino}ethanols (S,R)-
(ꢀ)-20a and (R,R)-(ꢀ)-20b. (A) To a suspension of
222 mg (9.23 mmol) of sodium hydride in dry toluene
(80 mL) was dropped a solution of bromohydrin ( )-
1624 (2.50 g, 9.23 mmol) in dry toluene (15 mL) under
nitrogen at rt After stirring for 1 h at rt, the suspension
was filtered and the concentrated residue was purified by
silica gel column chromatography (eluant: 5% ethyl ace-
tate/petroleum ether) giving 1.54 g (88% yield) of the
epoxide.
N-(5-Acetylisoxazol-3-yl)methanesulfonamide ( )-12:
colorless prisms (from ethyl acetate/n-hexane), mp 160–
162 ꢁC. Rf 0.36 (eluant: 80% ethyl acetate/petroleum
ether). 1H NMR: 2.60 (s, 3H), 3.18 (s, 3H), 7.02 (s, 1H).
7.80 (br s, 1H). Anal. Calcd for C6H8N2O4 S: C, 35.29;
H, 3.95; N, 13.72. Found: C, 35.08; H, 4.18; N, 13.90.
3-Bromo-5-oxiran-2-ylisoxazole ( )-17: colorless oil, bp
120–125 ꢁC/0.5 mmHg. Rf 0.49 (eluant: 20% ethyl ace-
tate/petroleum ether). H NMR: 3.12 (dd, 1H, J = 2.6
and 5.5), 3.20 (dd, 1H, J = 4.3 and 5.5), 3.96 (dd, 1H,
J = 2.6 and 4.3), 6.34 (s, 1H). Anal. Calcd for
C5H4BrNO2: C, 31.61; H, 2.12; N, 7.37. Found: C,
31.24; H, 2.42; N, 7.59.
(C) To a stirred solution of ( )-12 (429 mg, 2.10 mmol)
in anhydrous THF (20 mL), trimethylphenylammonium
tribromide (790 mg, 2.10 mmol) was added portionwise
at rt in 30 min. The mixture was further stirred for about
30 min until disappearance of the orange color, then
water (25 mL) was added, the solvent was evaporated
at reduced pressure, and the residue was extracted with
ethyl acetate (3 · 20 mL). After the usual work-up, the
crude bromoketone ( )-13 was used in the next step
without further purification.
1
(B) To a solution of epoxide ( )-17 (1.51 g, 7.95 mmol)
in acetonitrile (50 mL) were added calcium trifluoro-
methanesulfonate (1.35 g, 3.99 mmol) and (R)-(ꢀ)-
1-methyl-2-(4-methoxyphenyl)ethylamine 1833(1.31 g,
7.95 mmol). The reaction mixture was stirred and heated
at reflux under nitrogen for 24 h. After evaporation of
the solvent, the residue was purified by silica gel column
chromatography (eluant: 2% methanol/ dichlorometh-
ane) to afford 1.72 g (61% overall yield,) of a mixture
of stereoisomeric amino alcohols (S,R)-19a and (R,R)-
19b, Rf 0.54 (eluant: 10% methanol/dichloromethane).
The 1H NMR analysis allowed evaluation of the relative
amounts of the two isomers, by considering the two sin-
glets resonating at 6.21 d [(R,R)-19b, 53%] and at 6.30 d
[(S,R)-19a, 47%], which corresponded to the H-4 proton
of the isoxazole nucleus for each isomer.
(D) To a stirred solution of crude ( )-13 (505 mg,
1.78 mmol) in methanol (20 mL) sodium borohydride
(202 mg, 5.34 mmol) was added portionwise at 0 ꢁC.
After completion of the reduction (30 min, rt), the sol-
vent was removed under vacuum, the residue was treat-
ed with dilute HCl (20 mL) and extracted with ethyl
acetate (3 · 20 mL). After the usual work-up, the desired
bromohydrin (287 mg, 48% overall yield) was obtained
through purification on a silica gel column (eluant:
45% ethyl acetate/petroleum ether).
N-[5-(2-Bromo-1-hydroxyethyl)isoxazol-3-yl]methanesulf-
onamide ( )-14: colorless viscous oil, bp 195–200 ꢁC/
0.2 mmHg. Rf 0.30 (eluant: 45% ethyl acetate/petroleum
(C) To a stirred and ice cooled mixture of (S,R)-19a and
(R,R)-19b (3.27 g, 9.21 mmol) dissolved in dichloro-
methane (75 mL) were sequentially added di-tert-butyl
dicarbonate (2.21 g, 10.1 mmol) and triethylamine
(2.82 mL, 20.2 mmol). The reaction mixture was left un-
der stirring for 24 h at rt, then washed with 3 · 25 mL of
diluted HCl (pH 5). After the usual work-up, the residue
of the organic phase was submitted to a silica gel column
chromatography (eluant: 5% ethyl acetate/petroleum
ether), which allowed the isolation of the two stereoiso-
mers (S,R)-(ꢀ)-20a (1.36 g) and (R,R)-(ꢀ)-20b (1.74 g)
in 72% overall yield.
1
ether). H NMR: 1.92 (br s, 1H), 3.17 (s, 3H), 3.72 (dd,
1H, J = 6.2 and 10.6), 3.79 (dd, 1H, J = 4.0 and 10.6),
5.05 (dd, 1H, J = 4.0 and 6.2), 6.52 (s, 1H), 7.86 (br s,
1H). Anal. Calcd for C6H9BrN2O4S: C, 25.28; H, 3.18;
N, 9.83. Found: C, 24.90; H, 3.41; N, 9.53.
(E) A stirred solution of bromohydrin ( )-14 (250 mg,
0.88 mmol) and tert-butylamine (925 lL, 8.80 mmol) in
methanol (10 mL) was refluxed until TLC evidenced
the disappearance of the starting material (about 2 h).
The solvent and excess reagent were removed under vac-
uum, and the residue was directly purified on a silica gel
column (eluant: 30% methanol/ dichloromethane/0.1%
concentrated ammonia), which afforded the zwitterionic
final derivative (120 mg, 49% yield).
Isomer (S,R)-(ꢀ)-20a: thick pale yellow oil, Rf 0.48 (elu-
1
ant: 20% ethyl acetate/petroleum ether). H NMR: 1.22
(d, 3H, J = 6.6), 1.37 (s, 9H), 2.58 (dd, 1H, J = 5.9 and
13.6), 2.68 (dd, 1H, J = 8.1 and 13.6), 3.41 (dd, 1H,
J = 2.4 and 14.7), 3.51 (dd, 1H, J = 8.4 and 14.7), 3.77
(s, 3H), 4.06 (m, 1H), 4.81 (m, 1H), 5.77 (br s, 1H),
Compound ( )-4: pale pink prisms (from methanol), mp
232–234 ꢁC. Rf 0.22 (eluant: 20% methanol/dichloro-
methane/0.1% concentrated ammonia). 1H NMR
(D2O): 1.21 (s, 9H), 2.83 (s, 3H), 3.17 (dd, 1H, J = 9.2
and 13.2), 3.27 (dd, 1H, J = 3.3 and 13.2), 4.87 (dd,
1H, J = 3.3 and 9.2), 5.94 (s, 1H). 13C NMR (D2O):
24.9, 39.4, 44.7, 57.9, 62.7, 98.3, 164.8, 169.2. Anal.
Calcd for C10H19N3O4S: C, 43.31; H, 6.91; N, 15.15.
Found: C, 43.55; H, 7.12; N, 15.37.
6.39 (s, 1H), 6.80 (d, 2H, J = 8.1), 7.00 (d, 2H,
20
D
J = 8.1). ½aꢁ ꢀ61.6 (c 0.950, CHCl3). Anal. Calcd for
C20H27BrN2O5: C, 52.75; H, 5.98; N, 6.15. Found: C,
53.12; H, 5.65; N, 6.37.
Isomer (R,R)-(ꢀ)-20b: thick pale yellow oil, Rf 0.40 (elu-
1
ant: 20% ethyl acetate/petroleum ether). H NMR: 1.24

2540
C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
(d, 3H, J = 6.6), 1.37 (s, 9H), 2.65 (dd, 1H, J = 6.6 and
13.6), 2.77 (dd, 1H, J = 8.4 and 13.6), 3.44 (dd, 1H,
J = 2.7 and 14.7), 3.50 (dd, 1H, J = 7.4 and 14.7), 3.78
(s, 3H), 4.09 (m, 1H), 4.88 (m, 1H), 5.74 (br s, 1H),
(CD3OD): 1.06 (d, 3H, J = 6.2), 2.58 (dd, 1H, J = 5.8
and 12.5), 2.62 (dd, 1H, J = 7.1 and 12.5), 2.80-2.95
(m, 2H), 3.02 (dd, 1H, J = 4.0 and 12.1), 4.85 (dd, 1H,
J = 4.2 and 9.2), 6.41 (s, 1H), 6.70 (d, 2H, J = 8.4),
20
D
6.35 (s, 1H), 6.83 (d, 2H, J = 8.4), 7.06 (d, 2H,
7.01 (d, 2H, J = 8.4). ½aꢁ ꢀ4.25 (c 1.040, CH3OH).
20
D
J = 8.4). ½aꢁ ꢀ39.6 (c 0.900, CHCl3). Anal. Calcd for
Anal. Calcd for C14H17BrN2O3: C, 49.28; H, 5.02; N,
8.21. Found: C, 49.51; H, 4.82; N, 8.42.
C20H27BrN2O5: C, 52.75; H, 5.98; N, 6.15. Found: C,
52.87; H, 5.61; N, 6.50.
(B) To a stirred and ice cooled mixture of (S,R)-(ꢀ)-21a
(1.31 g, 3.84 mmol) dissolved in acetonitrile (15 mL) was
sequentially added a solution of di-tert-butyl dicarbon-
ate (1.09 g, 4.99 mmol) in acetonitrile (10 mL) and
triethylamine (1.18 mL, 8.45 mmol). The reaction mix-
ture was left under stirring for 6 h at rt, then concentrat-
ed at reduced pressure, treated with 20 mL of diluted
HCl (pH 5), and extracted with dichloromethane
(3 · 20 mL). After the usual work-up, the residue of
the organic phases was submitted to a silica gel column
chromatography (eluant: 5% ethyl acetate/petroleum
ether), which afforded 1.47 g of carbamate (S,R)-(ꢀ)-
22a (87% yield).
(D) A solution of bromohydrin (R)-(ꢀ)-1624 (271 mg,
1 mmol) and amine (R)-(ꢀ)-18(165 mg, 1.00 mmol) in
methanol (5 mL) was stirred and heated at reflux under
nitrogen for 24 h. The solvent was removed under vacu-
um, the brown oily residue was dissolved in 3 N HCl
(10 mL) and washed with ethyl ether (2 · 10 mL). The
aqueous layer was alkalinized with solid sodium carbon-
ate and extracted with dichloromethane (4 · 10 mL).
After the usual work-up, the residue was dissolved in
dichloromethane (5 mL) and treated with di-tert-butyl
dicarbonate (218 mg, 1.00 mmol) and triethylamine
(280 lL, 2.00 mmol). After stirring for 24 h at rt, the
reaction mixture was washed with a solution of diluted
HCl (pH 5) and, after the usual work-up, the residue
of the organic phase was purified on a silica gel column
chromatography (eluant: 10% ethyl acetate/petroleum
ether), which allowed isolation of compound (S,R)-
4-[2-{[2-(3-Bromoisoxazol-5-yl)-2-hydroxyethyl]tert-but-
oxycarbonylamino}propyl]phenol (S,R)-(ꢀ)-22a: thick
pale yellow oil, Rf 0.26 (eluant: 20% ethyl acetate/petro-
leum ether). 1H NMR: 1.23 (d, 3H, J = 6.9), 1.38 (s, 9H),
2.57 (dd, 1H, J = 6.2 and 13.2), 2.64 (dd, 1H, J = 8.4 and
13.2), 3.39 (dd, 1H, J = 2.2 and 15.0), 3.53 (dd, 1H,
J = 7.7 and 15.0), 4.04 (m, 1H), 4.80 (m, 1H), 5.0 (br s,
(ꢀ)-20a (118 mg, 26% overall yield) as a thick pale
20
yellow oil, ½aꢁ ꢀ62.15 (c 0.900, CHCl3). The 1H
D
NMR data matched those above reported for the same
isomer.
1H), 5.78 (br s, 1H), 6.40 (s, 1H), 6.74 (d, 2H, J = 8.4),
20
D
6.95 (d, 2H, J = 8.4). ½aꢁ ꢀ69.2 (c 0.910, CHCl3). Anal.
5.2.3. Ethyl {4-[2-{[2-(3-bromoisoxazol-5-yl)-2- hydroxy-
ethyl]amino}propyl]phenoxy} acetates (S,R)-(ꢀ)-8a and
(R,R)-(+)-8b. (A) To a stirred solution of (S,R)-(ꢀ)-20a
(2.16 g, 4.74 mmol) in dichloromethane (50 mL) was
added dropwise 25 mL of a 1 M solution of boron tribr-
omide. After stirring for 2 h at rt, water (30 mL) was
added to the reaction mixture cooled at ꢀ5 ꢁC. The
aqueous phase was separated, alkalinized (pH 8), and
extracted with ethyl acetate (4 · 25 mL). After the usual
work-up, the residue was purified by silica gel column
chromatography (eluant: 2% methanol/ dichlorometh-
ane) to give the desired intermediate (S,R)-(ꢀ)-21a
(1.34 g, 83% yield).
Calcd for C19H25BrN2O5: C, 51.71; H, 5.71; N, 6.35.
Found: C, 52.07; H, 5.95; N, 6.08.
Similarly, isomer (R,R)-(ꢀ)-21b was converted into the
corresponding carbamate (R,R)-(ꢀ)-22b in 77% yield.
4-[2-{[2-(3-Bromoisoxazol-5-yl)-2-hydroxyethyl]tert-but-
oxycarbonylamino}propyl]phenol (R,R)-(ꢀ)-22b: thick
colorless oil, Rf 0.39 (eluant: 30% ethyl acetate/petro-
1
leum ether). H NMR: 1.26 (d, 3H, J = 6.9), 1.45 (s,
9H), 2.65 (dd, 1H, J = 7.2 and 13.8), 2.78 (dd, 1H,
J = 8.1 and 13.8), 3.46 (m, 2H), 4.08 (m, 1H), 4.85 (m,
2H), 5.80 (br s, 1H), 6.35 (s, 1H), 6.75 (d, 2H, J = 8.4),
20
7.02 (d, 2H, J = 8.4). ½aꢁ ꢀ36.25 (c 0.960, CHCl3).
D
4-[2-{[2-(3-Bromoisoxazol-5-yl)-2-hydroxyethyl]amino}-
propyl]phenol (S,R)-(ꢀ)-21a: thick colorless oil, Rf 0.49
(eluant: 10% methanol/dichloromethane). 1H NMR
(CD3OD): 1.07 (d, 3H, J = 6.2), 2.52 (dd, 1H, J =7 .7
and 13.2), 2.72 (dd, 1H, J = 6.4 and 13.2), 3.00 (m,
Anal. Calcd for C19H25BrN2O5: C, 51.71; H, 5.71; N,
6.35. Found: C, 51.47; H, 6.05; N, 6.52.
(C) To a stirred solution of (S,R)-(ꢀ)-22a (487 mg,
1.10 mmol) in acetone (15 mL) were added potassium
carbonate (381 mg, 2.76 mmol) and ethyl bromoacetate
(122 lL, 1.10 mmol). The suspension was stirred and
heated at reflux for 6 h, then cooled at rt and concentrat-
ed under vacuum. Water (10 mL) was added and the
aqueous phase was extracted with ethlyl acetate
(4 · 20 mL). After the usual work-up, the crude reaction
mixture was purified by silica gel column chromatogra-
phy (eluant: 15% ethyl acetate/petroleum ether), afford-
ing 489 mg (84% yield) of ester (S,R)-(ꢀ)-23a.
3H), 4.88 (dd, 1H, J = 5.7 and 8.4), 6.47 (s, 1H), 6.71
20
D
(d, 2H, J = 8.4), 7.00 (d, 2H, J = 8.4). ½aꢁ ꢀ29.0
(c 1.080, CH3OH). Anal. Calcd for C14H17BrN2O3: C,
49.28; H, 5.02; N, 8.21. Found: C, 49.02; H, 5.21; N,
8.48.
The same procedure was applied to the transformation
of a comparable amount of isomer (R,R)-(ꢀ)-20b which
produced the free base (R,R)-(ꢀ)-21b in 94% yield.
4-[2-{[2-(3-Bromoisoxazol-5-yl)-2-hydroxyethyl]amino}-
propyl]phenol (R,R)-(ꢀ)-21b: thick colorless oil, Rf 0.60
(eluant: 10% methanol/dichloromethane). 1H NMR
Ethyl {4-[2-{[2-(3-bromoisoxazol-5-yl)-2-hydroxyeth-
yl]tert-butoxycarbonylamino}propyl] phenoxy}acetate
(S,R)-(ꢀ)-23a: thick pale yellow oil, Rf 0.66 (eluant:

C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
2541
1
15% ethyl acetate/petroleum ether). H NMR: 1.22 (d,
3H, J = 6.9), 1.29 (t, 3H, J = 7.3), 1.37 (s, 9H), 2.52-
275 (m, 2H), 3.40 (dd, 1H, J = 2.6 and 14.8), 3.48 (dd,
1H, J = 7.2 and 14.8), 4.09 (m, 1H), 4.26 (q, 2H,
J = 7.3), 4.58 (s, 2H), 4.79 (m, 1H), 5.77 (br s, 1H),
for C20H24BrF3N2O7: C, 44.38; H, 4.47; N, 5.18. Found:
C, 44.09; H, 4.71; N, 5.39.
5.2.4. {4-[2-{[2-(3-Bromoisoxazol-5-yl)-2-hydroxyethyl]-
amino}propyl]phenoxy}acetic acids (S,R)-(ꢀ)-7a and
(R,R)-(+)-7b. (A) A solution of ethyl ester (S,R)-(ꢀ)-
23a (260 mg, 0.49 mmol) in methanol (5 mL) was treat-
ed with 5 mL of a 10% aqueous solution of potassium
carbonate. The reaction mixture was stirred at rt for
1 h until disappearance of the starting material (TLC:
15% ethyl acetate/petroleum ether), then the solvent
was evaporated under vacuum. After addition of water
(10 mL) and treatment with diethyl ether (3 · 5 mL),
the residual aqueous phase was acidified with diluted
HCl (pH 4) and extracted with ethyl acetate
(3 · 5 mL). After the usual work-up, the thick oily resi-
due of the pooled organic extracts (215 mg, 87% yield)
was used in the following step without further
purification.
6.40 (s, 1H), 6.83 (d, 2H, J = 8.0), 7.02 (d, 2H,
20
D
J = 8.0). ½aꢁ ꢀ59.9 (c 0.950, CHCl3). Anal. Calcd for
C23H31BrN2O7: C, 52.38; H, 5.92; N, 5.31. Found: C,
52.47; H, 6.15; N, 5.50.
The same procedure was applied to the transformation
of a comparable amount of isomer (R,R)-(ꢀ)-22b,
which produced ester (R,R)-(ꢀ)-23b in 75% yield.
Ethyl
{4-[2-{[2-(3-bromoisoxazol-5-yl)-2-hydroxyeth-
yl]tert-butoxycarbonylamino}propyl] phenoxy}acetate
(R,R)-(ꢀ)-23b: thick pale yellow oil, Rf 0.48 (eluant:
1
30% ethyl acetate/petroleum ether). H NMR: 1.27 (d,
3H, J = 6.9), 1.30 (t, 3H, J = 7.3), 1.44 (s, 9H), 2.66
(dd, 1H, J = 7.0 and 13.9), 2.77 (m, 1H), 3.45 (dd, 1H,
J = 2.6 and 14.7), 3.52 (m, 1H), 4.09 (m, 1H), 4.25 (q,
2H, J = 7.3), 4.59 (s, 2H), 4.86 (m, 1H), 5.73 (br s,
(B) The crude above prepared N-Boc protected amino
acid (215 mg, 0.43 mmol) was treated with a 30% dichlo-
romethane solution of trifluoroacetic acid at 0 ꢁC, fol-
lowing the procedure described for (S,R)-(ꢀ)-8a. The
volatiles were removed under vacuum and the oily resi-
due was taken up with anhydrous diethyl ether and
dried under vacuum giving 185 mg (84% yield) of the
wanted trifluoroacetic acid salt.
1H), 6.35 (s, 1H), 6.83 (d, 2H, J = 8.4), 7.07 (d, 2H,
20
D
J = 8.4). ½aꢁ ꢀ26.4 (c 0.940, CHCl3). Anal. Calcd for
C23H31BrN2O7: C, 52.38; H, 5.92; N, 5.31. Found: C,
52.59; H, 5.65; N, 5.60.
(D) The N-Boc protected amino ester (S,R)-(ꢀ)-23a
(211 mg, 0.40 mmol) was treated with a 30% dichloro-
methane solution (1 mL) of trifluoroacetic acid
(308 lL, 4.15 mmol) at 0 ꢁC. The solution was stirred
at rt for 12 h until disappearance of the starting material
(TLC: 15% ethyl acetate/petroleum ether). The volatiles
were removed under vacuum and the thick oily residue
was taken up with anhydrous diethyl ether and dried un-
der vacuum to afford 175 mg (81% yield) of the desired
trifluoroacetic acid salt.
(S,R)-(ꢀ)-7a trifluoroacetate: colorless prisms (from
50% methanol/diethyl ether), mp 159–160 ꢁC, dec 1H
NMR (CD3OD): 1.22 (d, 3H, J = 6.9), 2.71 (dd, 1H,
J = 10.3 and 13.2), 3.10 (dd, 1H, J = 4.3 and 13.2),
3.40–3.56 (m, 3H), 4.59 (s, 2H), 5.18 (m, 1H), 6.64 (s,
1H), 6.92 (d, 2H, J = 8.4), 7.18 (d, 2H, J = 8.4). 13C
NMR (CD3OD): 14.2, 38.2, 56.3, 62.4, 65.4, 65.7,
105.8, 114.8, 128.6, 128.9, 130.3, 140.6, 157.8, 173.6.
½aꢁ
20
D
ꢀ31.7 (c 0.900, CH3OH). Anal. Calcd for
(S,R)-(ꢀ)-8a trifluoroacetate: gummy colorless solid. 1H
NMR: 1.22 (d, 3H, J = 6.9), 1.28 (t, 3H, J = 7.2), 2.58
(dd, 1H, J = 6.9 and 13.8), 2.66 (dd, 1H, J = 6.6 and
13.8), 2.89 (m, 1H), 3.0 (m, 2H), 3.63 (br s, 2H), 4.24
(q, 2H, J = 7.2), 4.57 (s, 2H), 4.76 (m, 1H), 6.30 (s,
1H), 6.83 (d, 2H, J = 8.4), 7.06 (d, 2H, J = 8.4). 13C
NMR: 14.4, 20.1, 42.4, 50.1, 55.1, 61.3, 65.1, 65.7,
C18H20BrF3N2O7: C, 42.12; H, 3.93; N, 5.46. Found:
C, 42.31; H, 4.12; N, 5.19.
In a parallel way, isomer (R,R)-(ꢀ)-23b (180 mg,
0.34 mmol) was transformed into the corresponding
trifluoroacetate of (R,R)-(+)-7b (116 mg, 66% overall
yield).
105.5, 115.0, 130.5, 131.9, 140.7, 156.7, 169.3, 175.5.
ꢀ21.5 (c 1.150, CH3OH). Anal. Calcd for
20
D
½aꢁ
(R,R)-(+)-7b trifluoroacetate: colorless prisms (from
20% methanol/diethyl ether), mp 152–153 ꢁC. 1H
NMR (CD3OD): 1.24 (d, 3H, J = 6.9), 2.66 (dd, 1H,
J = 9.9 and 13.2), 3.11 (dd, 1H, J = 4.5 and 13.2),
3.37–3.49 (m, 3H), 4.60 (s, 2H), 5.15 (m, 1H), 6.67 (s,
1H), 6.92 (d, 2H, J = 8.4), 7.18 (d, 2H, J = 8.4). 13C
NMR (CD3OD): 14.9, 37.6, 56.4, 62.5, 64.8, 65.0,
C20H24BrF3N2O7: C, 44.38; H, 4.47; N, 5.18. Found:
C, 44.68; H, 4.21; N, 5.35.
Similarly, isomer (R,R)-(ꢀ)-23b was converted into the
corresponding trifluoroacetic acid salt of (R,R)-(+)-8b
in 72% yield.
105.9, 114.9, 128.7, 129.0, 130.3, 140.7, 157.7, 173.4.
½aꢁ
20
D
(R,R)-(+)-8b trifluoroacetate: gummy pale yellow solid.
1H NMR: 1.23 (d, 3H, J = 6.9), 1.29 (t, 3H, J = 7.2),
2.72 (dd, 1H, J = 8.1 and 13.9), 3.18 (dd, 1H, J = 5.5
and 13.9), 3.23 (m, 1H), 3.44 (m, 2H), 4.23 (q, 2H,
J = 7.2), 4.58 (s, 2H), 4.83 (br s, 2H), 5.29 (m, 1H),
6.38 (s, 1H), 6.81 (d, 2H, J = 8.4), 7.02 (d, 2H,
J = 8.4). 13C NMR: 14.4, 15.9, 38.6, 48.8, 57.3, 61.8,
+1.5 (c 0.830, CH3OH). Anal. Calcd for
C18H20BrF3N2O7: C, 42.12; H, 3.93; N, 5.46. Found:
C, 41.95; H, 4.18; N, 5.58.
5.2.5. 1-(3-Bromoisoxazol-5-yl)-2-{[2-(4-butoxyphenyl)-
1-methylethyl]amino}ethanols (S,R)-(ꢀ)-9a and (R,R)-
(+)-9b. (A) To a stirred solution of (S,R)-(ꢀ)-22a
(300 mg, 0.68 mmol) in acetone (10 mL) were
added potassium carbonate (235 mg, 1.70 mmol) and
62.9, 65.5, 106.4, 115.4, 128.5, 130.6, 141.0, 157.4,
20
D
169.3, 172.5. ½aꢁ +2.1 (c 1.180, CH3OH). Anal. Calcd

2542
C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
1-bromobutane (330 lL, 3.07 mmol). The suspension
was stirred and heated at reflux for 2 d, then cooled at
rt and filtered under vacuum. The concentrated filtrate
was purified by silica gel column chromatography (elu-
ant: 10% ethyl acetate/petroleum ether) to produce
287 mg (85% yield) of ether (S,R)-(ꢀ)-24a.
(R,R)-(+)-9b trifluoroacetate: gummy colorless solid, Rf
0.47 (eluant: 5% methanol/ dichloromethane). 1H NMR:
0.97 (t, 3H, J = 7.3), 1.39 (d, 3H, J = 6.6), 1.49 (m, 2H),
1.76 (m, 2H), 2.82 (dd, 1H, J = 8.4 and 13.9), 3.04-3.19
(m, 2H), 3.36 (m, 1H), 3.52 (m, 1H), 3.93 (t, 2H,
J = 6.6), 5.32 (m, 1H), 5.50 (br s, 1H), 6.38 (s, 1H),
6.86 (d, 2H, J = 8.4), 7.06 (d, 2H, J = 8.4). 13C NMR:
1-(3-Bromoisoxazol-5-yl)-2-{[2-(4-butoxyphenyl)-1-meth-
ylethyl]tert-butoxycarbonylamino} ethanol (S,R)-(ꢀ)-
24a: thick pale yellow oil, Rf 0.24 (eluant: 10% ethyl
acetate/petroleum ether). 1H NMR: 0.96 (t, 3H, J = 7.4),
1.22 (d, 3H, J = 6.6), 1.36 (s, 9H), 1.47 (m, 2H), 1.74
(m, 2H), 2.58 (dd, 1H, J = 5.9 and 13.5), 2.67 (dd, 1H,
J = 8.0 and 13.5), 3.42 (dd, 1H, J = 1.8 and 15.0), 3.52
(dd, 1H, J = 7.3 and 15.0), 3.92 (t, 2H, J = 6.6), 4.04
14.1, 16.0, 19.5, 31.5, 38.7, 48.8, 57.5, 62.9, 67.9, 106.4,
20
D
115.3, 126.6, 130.4, 141.0, 158.8, 172.5. ½aꢁ +3.1 (c
1.0, CHCl3). Anal. Calcd for C20H26BrF3N2O5: C,
46.98; H, 5.13; N, 5.48. Found: C, 46.72; H, 5.41; N,
5.60.
5.3. Pharmacological experimental section
(m, 1H), 4.80 (m, 1H), 5.80 (br s, 1H), 6.40 (s, 1H),
5.3.1. Cell culture and membrane preparation. CHO cells
expressing the stably transfected human b-adrenergic
receptor subtypes were grown adherently and main-
tained in Dulbecco’s modified Eagle’s medium with
nutrient mixture F12 (DMEM/F12), containing 10% fe-
tal calf serum, penicillin (100 U/mL), streptomycin
(100 lg/mL), L-glutamine (2 mM), and Geneticin (G-
418, 0.2 mg/mL) at 37 ꢁC in 5% CO2/95% air. Details
have been described by Hoffmann et al.23 Membranes
for radioligand binding were prepared from frozen cells
in a two-step procedure as described previously.37 For
the measurement of adenylyl cyclase a one-step proce-
dure from fresh cells was used.23,37
20
D
6.80 (d, 2H, J = 8.4), 6.98 (d, 2H, J = 8.4). ½aꢁ ꢀ58.0
(c 1.050, CHCl3). Anal. Calcd for C23H33BrN2O5: C,
55.54; H, 6.69; N, 5.63. Found: C, 55.80; H, 6.41; N,
5.88.
The same procedure was applied to the transformation
of a comparable amount of isomer (R,R)-(ꢀ)-22b,
which produced ether (R,R)-(ꢀ)-24b in 89% yield.
1-(3-Bromoisoxazol-5-yl)-2-{[2-(4-butoxyphenyl)-1-meth-
ylethyl]tert-butoxycarbonylamino} ethanol (R,R)-(ꢀ)-
24b: thick pale yellow oil, Rf 0.48 (eluant: 20% ethyl
acetate/petroleum ether). 1H NMR: 0.97 (t, 3H,
J = 7.4), 1.26 (d, 3H, J = 6.6), 1.44 (s, 9H), 1.55 (m,
2H), 1.75 (m, 2H), 2.66 (dd, 1H, J = 7.0 and 13.9),
2.76 (dd, 1H, J = 7.9 and 13.9), 3.45 (dd, 1H, J = 2.2
and 14.6), 3.56 (m, 1H), 3.93 (t, 2H, J = 6.6), 4.09
5.3.2. Radioligand binding studies and adenylyl cyclase
activity. Radioligand binding experiments were carried
out as described recently.23 In brief, membranes from
CHO cells stably transfected with human b-AR sub-
types (b1, b2 about 5 lg, b3 about 25 lg of protein) were
incubated with the antagonist 125I-CYP in a concentra-
tion of about 50 pM in the case of b1- and b2-receptors,
or about 80 pM 125I-CYP for b3-receptors. Assays were
carried out in 50 mM Tris/HCl, pH 7.4 (assay buffer),
containing 100 lM GTP, in a total volume of 200 lL.
GTP was added to achieve monophasic binding curves
for agonists. Membranes were incubated for 90 min at
30 ꢁC. Bound ligand was separated from free ligand by
filtration through Whatman GF/C filters, which were
then washed three times with ice-cold assay buffer.
Non-specific binding was determined in the presence of
10 lM alprenolol. Ki values were calculated by non-
linear curve fitting with the program SCTFIT.38
(m, 1H), 4.87 (m, 1H), 5.75 (br s, 1H), 6.35 (s, 1H),
20
D
6.80 (d, 2H, J = 8.4), 7.04 (d, 2H, J = 8.4). ½aꢁ
ꢀ34.5 (c 1.020, CHCl3). Anal. Calcd for
C23H33BrN2O5: C, 55.54; H, 6.69; N, 5.63. Found: C,
55.29; H, 6.88; N, 5.90.
(B) The N-Boc protected amino ether (S,R)-(ꢀ)-24a
(220 mg, 0.44 mmol) was treated with a 30% dichloro-
methane solution of trifluoroacetic acid at 0 ꢁC follow-
ing the procedure described for (S,R)-(ꢀ)-8a. The
volatiles were removed under vacuum and the thick oily
residue was taken up with n-hexane and dried under vac-
uum to afford 142 mg (63% yield) of the corresponding
trifluoroacetic acid salt.
(S,R)-(ꢀ)-9a trifluoroacetate: colorless powder (from
n-hexane), mp 87–88 ꢁC. Rf 0.38 (eluant: 5% methanol/
dichloromethane). H NMR: 0.97 (t, 3H, J = 7.3), 1.35
(d, 3H, J = 6.6), 1.51 (m, 2H), 1.75 (m, 2H), 2.80 (dd,
1H, J = 8.8 and 13.6), 3.09 (dd, 1H, J = 5.3 and 13.6),
3.30–3.53 (m, 3H), 3.93 (t, 2H, J = 6.6), 5.32 (m, 1H),
5.78 (br s, 1H), 6.42 (s, 1H), 6.85 (d, 2H, J = 8.4), 7.06
(d, 2H, J = 8.4). 13C NMR: 14.1, 15.5, 19.4, 31.5, 38.8,
The activity of adenylyl cyclase in cell membranes was
determined as described recently.23 Briefly, the conver-
sion of [a-32P]ATP to [32P]cAMP was measured in an as-
say mixture containing membranes from CHO cells
expressing human b-AR subtypes (about 50 lg of pro-
tein), 100 lM cAMP, 0.2% BSA, 10 lM GTP, 100 lM
ATP, 1 mM mgCl2, 100 lM isobutylmethylxanthine,
and an ATP-regenerating system consisting of 15 mM
phosphocreatine and 300 U/ml of creatine kinase in
50 mM Tris/HCl, pH 7.4. The reaction was allowed to
proceed for 20 min at 37 ꢁC. The reaction was stopped
by precipitation with zinc acetate and sodium carbonate.
After centrifugation [32P]cAMP and remaining
[a-32P]ATP in the supernatant were separated by chro-
matography over (neutral) alumina columns and the
1
48.6, 57.4, 62.8, 67.9, 106.5, 115.2, 126.9, 130.4, 141.1,
20
D
158.8, 172.4. ½aꢁ ꢀ25.8 (c 0.960, CHCl3). Anal. Calcd
for C20H26BrF3N2O5: C, 46.98; H, 5.13; N, 5.48. Found:
C, 47.18; H, 5.31; N, 5.20.
Isomer (R,R)-(+)-9b was similarly obtained as trifluoro-
acetic acid salt in 73% yield.

C. Dallanoce et al. / Bioorg. Med. Chem. 15 (2007) 2533–2543
2543
amount of [32P]cAMP was determined in a b-counter.
The efficacy of compounds under investigation was test-
ed at a concentration of 100 lM and compared to the
stimulation by 100 lM isoproterenol (100%) over basal
adenylyl cyclase activity (0%). Inverse agonistic activity
is expressed as percent inhibition of basal activity.
15. Baker, J. G.; Hall, I. P.; Hill, S. J. Mol. Pharmacol. 2003,
63, 1312.
16. Alves, I. D.; Cowell, S. M.; Salamon, Z.; Devanathan, S.;
Tollin, G.; Hruby, V. J. Mol. Pharmacol. 2004, 65, 1248.
17. Kobilka, B. Mol. Pharmacol. 2004, 65, 1060.
18. Conti, P.; Dallanoce, C.; De Amici, M.; De Micheli, C.;
Klotz, K.-N. Bioorg. Med. Chem. 1998, 6, 401.
19. De Amici, M.; Conti, P.; Dallanoce, C.; Kassi, L.;
Castellano, S.; Stefancich, G.; De Micheli, C. Med. Chem.
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Acknowledgments
20. Dallanoce, C.; Meroni, G.; De Amici, M.; Hoffmann, C.;
Klotz, K.-N.; De Micheli, C. Bioorg. Med. Chem. 2006,
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21. Sala, R.; Moriggi, E.; Della Bella, D.; Carenzi, A. Eur. J.
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`
This work was financially supported by the Universita
degli Studi di Milano (FIRST 2004 and 2005). The expert
technical assistance of Sonja Kachler and Nico Falgner is
gratefully acknowledged. The financial support from
the Vigoni program (to C.D.M. and K.-N.K.), which
encourages German–Italian scientific collaborations, is
gratefully acknowledged.
24. De Amici, M.; De Micheli, C.; Carrea, G.; Spezia, S.
J. Org. Chem. 1989, 54, 2646.
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