Stereospecific synthesis and bio-activity of novel β3-adrenoceptor agonists and inverse agonists

Article abstract of DOI:10.1016/j.bmc.2007.11.060

Since it is widely distributed into the body, β₃-adrenoceptor is becoming an attractive target for the treatment of several pathologies such as obesity, type 2 diabetes, metabolic syndrome, cachexia, overactive bladder, ulcero-inflammatory disorder of the gut, preterm labour, anxiety and depressive disorders, and heart failure. New compounds belonging to the class of arylethanolamines bearing one or two stereogenic centres were prepared in good yields as racemates and optically active forms. They were, then, evaluated for their intrinsic activity towards β₃-adrenoceptor and their affinity for β₁- and β₂-adrenergic receptors. Stereochemical features were found to play a crucial role in determining the behaviour of such compounds. In particular, α-racemic, (αR)- and (αS)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}-2- methylpropanoic acid, (α-rac, β-rac)-, (αR, βS)- and (αR, βR)- 2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid were found to be endowed with β₃-adrenoceptor agonistic activity. Whereas, (αS, βS)- and (αS, βR)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid behaved as β₃-adrenoceptor inverse agonists. Such compounds showed no affinity for β₁- and β₂-adrenergic receptor, respectively. Thus, resulting highly selective β₃-adrenoceptor ligands.

Full text of DOI:10.1016/j.bmc.2007.11.060

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2473–2488
Stereospecific synthesis and bio-activity of novel b3-adrenoceptor
agonists and inverse agonists
Maria Grazia Perrone,a Ernesto Santandrea,a Laura Bleve,a Paola Vitale,a
Nicola Antonio Colabufo,a Ralf Jockers,b Ferdinando Maria Milazzo,c
Anna Floriana Sciarronic and Antonio Scilimatia,*
a
`
Dipartimento Farmaco-Chimico, Universita di Bari, Via E.Orabona 4, 70125 Bari, Italy
bInstitut Cochin Inserm U567, CNRS 8104, Universite´ Paris, Descartes 22 rue Me´chain, 75014 Paris, France
cDipartimento di Endocrinologia e Metabolismo, Sigma-Tau S.p.A, Via Pontina km 30.400, 00040 Pomezia, Italy
Received 22 June 2007; revised 13 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—Since it is widely distributed into the body, b3-adrenoceptor is becoming an attractive target for the treatment of several
pathologies such as obesity, type 2 diabetes, metabolic syndrome, cachexia, overactive bladder, ulcero-inflammatory disorder of the
gut, preterm labour, anxiety and depressive disorders, and heart failure. New compounds belonging to the class of arylethanolam-
ines bearing one or two stereogenic centres were prepared in good yields as racemates and optically active forms. They were, then,
evaluated for their intrinsic activity towards b3-adrenoceptor and their affinity for b1- and b2-adrenergic receptors. Stereochemical
features were found to play a crucial role in determining the behaviour of such compounds. In particular, a-racemic, (aR)- and (aS)-
2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}-2- methylpropanoic acid, (a-rac, b-rac)-, (aR, bS)- and (aR, bR)- 2-{4-[2-(2-
hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid were found to be endowed with b3-adrenoceptor agonistic activity.
Whereas, (aS, bS)- and (aS, bR)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid behaved as b3-adrenocep-
tor inverse agonists. Such compounds showed no affinity for b1- and b2-adrenergic receptor, respectively. Thus, resulting highly
selective b3-adrenoceptor ligands.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
stimulation increases the production of cAMP.4a Recent
evidences show that in human heart b3-AR activity in-
duces a reduction in the contractility through a Gi pro-
tein activation that leads to impaired production of
cAMP and increase of nitric oxide levels.5
b-Adrenergic receptors (b-ARs) have been classified as
b1- and b2-AR since 1967.1 This subdivision led to the
discovery of selective drugs for the treatment of hyper-
tension and asthma. At the beginning of 1980s, a new
b-adrenergic receptor subtype,2 later called b3-AR, was
found in several species including man, bovine, rat and
mouse.3
b3-AR is expressed in several tissues including adipose
tissue, heart, uterus, bladder and gut, modulating differ-
ent physiological functions. Due to its wide distribution,
it can be considered a reasonable drug target for the
treatment of several pathologies (Table 1).
The b3-AR, like the other b-adrenergic receptors, is a se-
ven-transmembrane domain G-protein coupled recep-
tor. It is usually coupled to a Gs protein and its
Early developments of b3-AR agonists, for the treatment
of obesity and type 2 diabetes, based on rodent b3-AR
activity, are represented by the first agonist generation,
i.e. BRL 37344, CL 316243 and CGP 12177A (Fig. 1).7
Keywords: Agonist; Inverse agonist; b3-adrenoceptor; (aS; bS)- and
(aS; bR)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}prop-
anoic acid; Obesity; Type 2 diabetes; Heart failure; Cachexia; Meta-
bolic syndrome; Overactive bladder; Preterm labour; Anxiety and
depressive disorders; DELFIA.
Later, many other full and partial agonists like L-
770,6448,9 and BMS-19608510 AJ-9677,11 BMS
20162012 were reported and very recently, a new agonist
(GW427353, Fig. 2) endowed with a positive pharmaco-
*
Corresponding author. Tel.: +39 080 544 2762; fax: +39 080 544
2231; e-mail: ascilimati@farmchim.uniba.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.060

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M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
Table 1. Possible clinical use of b3-AR agents
Indication
Type of drug
Obesity
Type 2 diabetes
Agonist
Agonist
Metabolic (insulin resistance) syndrome
Cachexia
Overactive bladder
Antagonist, Inverse agonist
Antagonist, Inverse agonist
Agonist
Agonist
Ulcero-inflammatory disorder of the gut
Preterm labour
Agonist
Agonist
Anxiety and depressive disorders
Heart failure
Agonist, Antagonist/Inverse Agonista
a Agonists should serve in the first stage of the heart failure, whereas antagonists and inverse agonists in the late phase of the disease.6
OH
OH
H
N
H
N
O
O
COO Na
COO Na
O
COOH
Cl
Cl
CL 316, 243
BRL 37344
O
N
H
H
N
OH
O
N
H
CGP 12177A
Figure 1. First generation of b3-AR agonists.
OH
H
OH
H
N
N
O
O
S
CHF2O
NHSO2Me
O
N
N
H
HO
N
N
N
N
L-770,644
BMS-196085
OH
H
OH
H
O
O
P
N
OEt
OEt
CH3SO2HN
HO
N
N
H
N
H
O
OH
Cl
O
CH3O
AJ- 9677
BMS-201620
OH
O
H
N
N
H
OH
Cl
Figure 2. Second generation of b3-AR agonists.14
GW427353

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2475
logical profile has been chosen for clinical trials as drug
candidate for the treatment of type 2 diabetes and over-
active bladder.13
Another possible therapeutic relevance of b3-AR antag-
onists and inverse agonists appears clear by considering
first, the increased lipolytic action of b3-AR in visceral
adipose tissue of obese subjects, and second, the associ-
ation between increased b3-AR function and metabolic
syndrome.
Few b3-AR antagonists have been reported (Fig. 3),15–17
but none of them has been chosen for clinical trials.
Very little is known about b3-AR inverse agonists.7
In fact, in upper-body obese subjects with signs of
the metabolic syndrome, b3-AR blockade might pref-
erentially inhibit fatty acid release from visceral adi-
pose tissue and improve some of the metabolic
abnormalities associated with the high ‘portal’ fatty
acid flux.4b
Inverse agonism is not a new concept and it describes li-
gand behaviour displaying negative efficacy.18 In partic-
ular, for G-protein-coupled receptors (GPCRs), it has
been widely assumed that inverse agonists suppress the
agonist-independent activity of the receptor by stabiliz-
ing it in its inactive state. Whereas receptor activation
(receptor-G protein coupling) occurs in the absence of
the agonist, the inverse agonist suppresses this activity
in some way.19
As reported in Table 1, b3-AR antagonists and inverse
agonists would also be useful to prevent or control
cachexia.
In cachectic cancer patients, the remarkable loss in adi-
pose tissue (ꢀ30% of pre-illness stable weight at diagno-
sis) seems to be mediated by a lipid mobilizing factor
(LMF). Reported evidences demonstrate that, at least
in part, LMF produces this effect by interaction with
b3-AR.20
Novel data suggest that some classical b-AR antagonists
behave either as partial agonists, neutral antagonists or
inverse agonists in cell systems expressing the wild type
or a constitutively activated mutant of the human b-AR.
For example, b1-selective AR antagonists with signifi-
cant inverse agonistic activity, such as metoprolol, have
been proven to be safe in the treatment of heart failure
patients. Clinical studies with inverse agonists of the
b2- and b3-AR are not yet available.
Besides, very recently, the use of b3-AR agonists in the
treatment of preterm labour21 and, anxiety and depres-
sive disorders has been proven.22
Stimulation of b3-AR subtype inhibits cardiac contrac-
tility, thus opposing to the response of b1- and b2-AR.
In failing heart, b3-ARs are up-regulated. They probably
serve as a buffer, exerting a ‘rescue’ function from the ef-
fects of high plasma levels of catecholamines, as those
observed in hyperadrenergic states including heart fail-
ure. Upon disease progression, b3-adrenoceptor up-reg-
ulation may produce a depression in contractility, which
exacerbates heart failure.5,6
Thus, based on these considerations the development of
b3-AR agonists, antagonists and inverse agonists is of
great interest.
Most of b3-AR agents reported so far, share a similar
overall structure, in which three molecular portions
can be identified (Fig. 4): a left-hand side (LHS), a linker
(LK) and a right-hand side (RHS). LHS is typically an
arylethanolamine or aryloxypropanolamine, LK has
various structures including both aromatic and aliphatic
moieties, RHS typically contains polar and/or ionizable
functionalities including ureas, acylamides, sulfona-
mides and sulfonic, phosphonic and carboxylic
groups.12,23–29
Hence, selective b3-AR agonists should serve in the early
stage of heart failure, whereas highly selective antago-
nists/inverse agonists might be useful in the advanced
stage of the disease.
OH
H
OH
H
O
N
O
O
N
.
N
(COOH)2
.
HO
HCl
SR 59230 A
DPJ904
OH
H
O
N
O
O
S
N
H
R
L-748,328 R = SO2NH2
L-748,337 R = CH2NHCOCH3
Figure 3. b3-AR antagonists.

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M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
Catecholamine-like (Phenylethanolamine)
Fibric acid-like
OH
R
NH2
O
COOH
LK
RHS
LHS
OH
H
N
R
α
O
COOH
β
Compound
R
Absolute
Configuration
1a
1b
1c
1d
1e
1f
CH3
CH3
CH3
H
H
H
α
-rac
α
R
S
α
-rac,
α
β
-rac
S
S
α
α
α
α
S,
R,
S,
β
β
β
β
1g
1h
H
H
R
R
R,
Figure 4. Structures and absolute configurations of compounds 1a–h.
Up to now, several studies also dealing with the relation-
ship between stereochemical demand and b3-AR agonis-
tic activity have been carried out using optically active
compounds, proving that the activity towards the recep-
tor is higher when the stereogenic centre bearing the hy-
droxy group in the LHS has (R)- absolute configuration
in the series of arylethanolamines, (S) in that of aryloxy-
propanolamines. Besides, several research groups syn-
thesized and evaluated new optically active b3-AR
agonists, bearing additional stereogenic centres. They
were almost exclusively located on the carbon atoms
adjacent to the aminic nitrogen, in both LHS and
LK.10,11,26,30–35 Such compounds resulted full or partial
agonists and antagonists of the b3-AR.
man cloned b1- and b2-ARs, expressed from the same
CHO cells, was also determined.
2. Chemistry
Compounds 1a–h were prepared by N-alkylation of the
corresponding racemic-, (R)- or (S)-2-amino-1-phenyl-
ethanol with phenethyl bromide derivatives 3a–d, fol-
lowed by hydrolysis of the ester intermediates 2a–h
(not shown), as depicted in Scheme 1.40
Compounds 3a–d were obtained by Et3SiH reduction of
their precursors 4a–d, that in turn were prepared by Fri-
edel–Crafts acylation of 2-methyl-2-phenoxypropano-
ates (5a, R ¼ CH3) and racemic-, (R)- or (S)-2-
phenoxypropanoates (5b–d, R = H) with bromoacetyl
bromide (Scheme 2).
Herein, we present new b3-AR agonists bearing stereo-
genic centres in both the left- and the right-hand sides.
These compounds were found to be agonists or inverse
agonists at the receptor. Unlike b1-36,37 and b2-AR,38
few data on b3-AR inverse agonism7,39 have ever been
reported. The influence of the absolute configurations
of two stereogenic centres a=b in LHS/RHS of a series
of arylethanolamines (1a–h, Fig. 4) on b3-AR inverse
agonism is also described.
Different routes were followed for 5a–d preparation. 5a
was obtained in a two-step process, in which phenol was
first reacted with 1,1,1-trichloro-2-methyl-2-propanol
(Chloretoneꢁ)41 in alkaline conditions and then treated
with thionyl chloride in ethanol under reflux (Scheme 3).
The new compounds were prepared in both racemic and
optically active forms in order to evaluate the effect of
the stereochemistry on b3-AR activity, that in turn was
measured using Chinese Hamster Ovary (CHO)-K1 cells
stably expressing human cloned b3-AR. Affinity for hu-
Compound 5b was obtained from the reaction of phenol
with racemic ethyl 2-bromopropanoate.42 Its optically
active analogues (R)-5c and (S)-5d were prepared by
reacting the phenol with the p-toluenesulfonyl deriva-
tives 6c–d of commercially available ethyl (S)-lactate

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2477
OH
H
N
Br
α
i, ii
R
R
O
COOH
O
COOR'
β
β
3a: R = CH3, R' = C2H5
1a: α-rac, R = CH3 1d: α−rac
,
β−rac, R = H
3b: β-rac, R = H, R' = C2H5
1e: (αS,
1f: (αR,
1g: (αS,
1h: (αR,
βS), R = H
1b: (α
R
), R = CH3
), R = CH3
3c: (R
), R = H, R' = C2H5
), R = H, R' = CH3
1c: (αS
β
S
), R = H
), R = H
R), R = H
3d: (S
β
R
β
Scheme 1. General procedure for the preparation of 1a–h. Reagents and conditions: (i) racemic-, (R)- or (S)-2-amino-1-phenylethanol, DMF, 70 ꢂC;
(ii) NaOH, THF/H2O, rt.
rank order of potency and magnitude (BRL
37344 > noradrenaline ꢀ isoproterenol > adrenaline)
obtained by different assays, such as [a-32P]- and [3H]-
cAMP.7,45,46
O
Br
i
ii
R
R
3a - d
O
COOR'
O
COOR'
5a - d
4a - d
Compounds bearing two methyl groups at the b-posi-
tion (1a–c) showed a similar b3-AR agonistic activity.
Racemic 1a had an EC50 ¼ 4:9 nM, with 68% intrinsic
activity with respect to maximal effect (100%) by isopro-
terenol. Surprisingly, its enantiomers 1b and 1c proved
equally active as the racemic form, irrespective of the
absolute configuration at Ca, which is generally (R) in
b3-AR agonists (EC50 ¼ 3:9 and 3.4 nM; IA = 72 and
76%, respectively).
Scheme 2. Preparation of 3a–d. Reagents and conditions: (i) bromo-
acetyl bromide, AlCl3, CH2Cl2, reflux; (ii) Et3SiH, TFA, 70 ꢂC.
and methyl (R)-lactate, respectively, in the presence of
CsF (Scheme 4).43
3. Biology, results and discussion
A methyl group was then removed from Cb leading to
the racemic 1d. In order to evaluate the effect of this
structural modification and the corresponding stereo-
chemical implication on b3-AR agonistic activity, all
the four possible stereoisomers of 1d were separately
tested.
Novel compounds were assayed by measuring cAMP
levels in CHO cells expressing human cloned b3-AR to
determine their b3-AR activity (Table 2, Fig. 5). cAMP
was quantified by DELFIA, that has the advantage of
being a non radiolabelled ligand-based assay.44
DELFIA was found to be a suitable method to evaluate
b3-AR activity: isoproterenol, noradrenaline, adrenaline
and BRL 37344 were used as reference compounds, and
the found b3-AR EC50 values (Table 2) confirmed the
The racemic form 1d behaved as a strong agonist at the
b3-AR, with a potency (EC50 ¼ 3:8 nM, IA = 65%) com-
parable to that observed for compounds 1a–c.
i, ii
iii
O
COOC2H5
OH
O
COOC2H5
5a
5b
Scheme 3. Preparation of 5a–b. Reagents and conditions: (i) Chloretoneꢁ, NaOH, acetone, reflux; (ii) SOCl2, EtOH, reflux; (iii) ethyl 2-
bromopropanoate, K2CO3, acetone, reflux.
O
O
O
COOR'
ii
S
*
i
*
HO
COOR'
*
O
COOR'
ethyl (
methyl (
S
)-lactate, R' = C2H5
)-lactate, R' = CH3
6c: (
S
), R' = C2H5
), R' = CH3
5c: (
R
), R' = C2H5
R
6d: (R
5d: (S), R' = CH3
Scheme 4. Preparation of 5c–d. Reagents and conditions: (i) p-toluenesulfonyl chloride, CH2Cl2, pyridine, rt; (ii) phenol, CsF, DMF, rt.

2478
M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
Table 2. Evaluation of cAMP accumulation in CHO cells expressing human b3-AR by 1a–h
OH
H
N
α
R
O
COOH
β
1a-h
Compound
R
Absolute configuration at Ca- and Cb-stereocentre
EC50a (nM SEMb) (IA%)c
1a
1b
CH3
CH3
CH3
H
a-rac
aR
aS
a-rac, b-rac
aS, bS
aR, bS
aS, bR
aR, bR
4.9 0.25 (68)
3.9 2.1 (72)
3.4 0.8 (76)
3.8 0.7 (65)
181 19 (ꢁ64)d
2.7 0.7 (50)
136 20 (ꢁ73)d
235 37 (34)
5.8 1.2 (100)
5.5 1.2 (96)
31 10 (99)
1c
1d
1e
H
1f
1g
H
H
1h
H
Isoproterenole
Noradrenalinee
Adrenalinee
BRL 37344e
1.1 0.3 (76)
a EC50 = substance concentration which produces a cAMP response equal to 50% of its maximal response.
b SEM, standard error mean from at least three experiments ðn P 3Þ.
c IA is the fitted maximal value of the concentration-response curve, expressed as a percent of the maximal response to R-(ꢁ)-isoproterenol (10ꢁ4 M).
d Some further experiments were conducted in the presence of different concentrations (ranging from 10ꢁ9 to 10ꢁ7 M) of SR 59230A (Fig. 3), a well-
known neutral antagonist of b3-AR expressed in rat and human colon.15 Unfortunately, the curve did not shift to the right, as expected, because SR
59230A behaves as partial agonist in CHO expressing human cloned b3-AR.4c,47 On the other hand, very preliminary experiments conducted on rat
proximal colon showed that 1e and 1g are endowed with antagonist activity (in functional test in which colon motility is evaluated, it is not possible
to discriminate antagonism from inverse agonism).
e EC50 = 3.9, 6.3, 49 and 5.9 nM for isoproterenol, noradrenaline, adrenaline and BRL 37344, respectively, obtained by using [3H]-cyclic AMP assay
system46 instead of DELFIA see above. In both assay systems, the same intact cells expressing human b3-AR were used.
100
75
50
25
1d
0
1f
1h
1e
-25
1g
-50
-75
-100
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
Log Agonist [M]
Figure 5. Representative curves of adenylyl cyclase activity evaluation in CHO cells stably expressing human b3-AR subtype. Adenylyl cyclase
stimulation was calculated as percentage (%) of the maximum effect by isoproterenol.
Among 1d stereoisomers, the most potent one has
absolute configuration (R) at Ca (1f, EC50 ¼ 2.7 nM,
IA = 50%). This result is in agreement with data
published for many other b3-AR agonists. Opposite
configuration at Cb, as for 1h, dramatically decreased
b3-AR agonistic potency (EC50 ¼ 235 nM, IA = 34%).
In fact, 1h was found 87-fold less active than 1f.
Interesting results were found for 1e and 1g. These com-
pounds differ from the previous two (1f and 1h) in the
stereochemistry at Ca. In this case, the compounds be-
haved as inverse agonists on b3-AR. Such a finding
seems crucial, since few b3-AR inverse agonists have
been identified to date.7,48 The most potent inverse ago-
nist was (aS, bR)-1g with an EC50 ¼ 136 nM and

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2479
IA = ꢁ73%. Its epimer (aS, bS)-1e had a comparable
potency and intrinsic activity (EC50 ¼181 nM,
IA = ꢁ64%).
(%) of inhibition exerted by the compounds at the highest
concentration used (10ꢁ5 M) in competition binding
experiments was never greater than 35%. This indicates
that for all the compounds the affinity for both the b1-
and b2-AR was very weak and therefore their activity at
these receptors was not assessed.
To exclude that the compounds 1a–h were active at the b1-
and b2-ARs, we tested their ability to bind to these recep-
tors using [3H]-dihydroalprenolol on membranes of CHO
cells expressing the receptors. At the concentrations of
[3H]-dihydroalprenolol used in the competition-binding
experiments, specific binding was about 76% and 85% of
the total binding, for the b1- and b2-ARs, respectively
(Fig. 6A and B). The non specific binding was determined
in the presence of 10 lM Alprenolol for both saturation
binding experiments. As shown in Table 3, the percentage
Accurate determination of b3-AR binding affinities for
all the compounds using a [3H]-dihydroalprenolol was
not possible because of the low b3-receptor expression
level (about 100 fmol/mg of protein). On the other hand,
the functional and activity data above-reported provide
important informative about the behaviour of the novel
compounds (1a–h).
4000
A
Linear regression of non specific
binding values
7500
5000
3000
2500
0
0
100000
200000
300000
3
[
H]-DHA (cpm)
2000
1000
0
0
5
10
[3H]-DHA (nM)
Total binding; Specific binding; Non specific binding.
15
20
25
B
Linear regression of non-specific
binding values
3000
2000
1000
0
1500
1000
500
0
0
100000
200000
300000
3
[
H]-DHA (cpm)
0
1
2
3
4
5
[3H]-DHA (nM)
Figure 6. Saturation binding analysis with [3H]-DHA towards b1- (A) and b2-AR (B) expressed in CHO cell line. In CHO-b1 cells, Kd value for
Alprenolol was 12.49 nM and Bmax 2,970 fmol/mg of protein (A) while in CHO-b2-AR cells Kd value for Alprenolol was 0.50 nM and Bmax 540 fmol/
mg of protein (B). The inserts represent the linear regression of non-specific binding values.

2480
M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
Table 3. Percentage of inhibition of 1a–h for human b1- and b2-
adrenoceptor subtypes
4. Conclusions
Inhibition (%)a
A set of new compounds (1a–h) selective for the b3-AR
were prepared in good yields and evaluated for their
functional effect on the receptor. 1a–c resulted almost
equally active as noradrenaline and isoproterenol on this
receptor subtype, and their intrinsic activity was compa-
rable with that of BRL 37344. Compounds 1a–c showed
no significant correlation between stereochemistry and
potency as b3-AR agonists. Among 1d–h, isomer (aR,
bS)-1f was the most potent b3-AR agonist in 1a–h series,
though endowed with modest intrinsic activity (50%).
Some inverse agonists for b1- and b2-AR have already
been identified:48 Interestingly, the structural modifica-
tions introduced in this series of molecules, allowed
the identification of the first potent and selective b3-
AR inverse agonists.7,50 (S)-configuration at Ca seems
to be required for inverse agonistic activity, at least for
1e and 1g.
Compound
b1-AR
b2-AR
1a
1b
1c
1d
1e
1f
18
8
24
1
13
37
14
28
10
6
5
24
9
1
1g
1h
1
2
a Percentage of inhibition of 1a–h (10ꢁ5 M) for human b1- and b2-AR.
Since the compounds prepared share, in their RHS, a
structural similarity with fibrates,49 PPARa agonists,
the evaluation of the PPARa functional activities of
1a–h by a transactivation assay in eukaryotic COS-7
cells was performed. The results, summarized in Table
4, were expressed as a percent activation of the CAT re-
porter gene compared to that measured in the presence
of positive control Wy,14-643 (2 lM, Table 4), conven-
tionally taken as equal to 100%.
The recently developed DELFIA44 was efficiently used
to evaluate the activity of 1a–h; the observed order of
potency for reference compounds is in agreement with
data obtained by using other assays, such as [a-32P]-
and [3H]-cAMP.7,45,46
Though they present the molecular determinants for
PPARa agonistic activity, the compounds tested re-
sulted less active as PPARa activators (<4% activation)
than the reference compounds (clofibrate, clofibric acid
and Wy,14-643), thus proving to be highly selective on
b3-AR.
In binding experiments, 1a–h did not bind either b1- or
b2-ARs with high affinity, thus confirming that they
are selective b3-AR agonists or inverse agonists, being
resulted also less active than clofibric acid as PPARa
activators, although they present structural similarities
with fibrates.49
Table 4. Percentage of activation of the CAT reporter gene by 1a–h at the concentration of 50, 150 and 300 lM, compared to that measured in the
presence of reference compound Wy,14-643 (2 lM), conventionally taken as equal to 100%
Compound
50 lM (%)
150 lM (%)
300 lM (%)
1a
1b
1c
1d
1e
1f
2
1.7
1.5
1.5
1.9
1
1.8
1.5
2.1
1.8
1.8
1.5
3.6
2.6
1.3
1.7
1.5
1
1.6
1.3
1.1
1.8
2
1g
1h
1.5
Cl
12.6
11
89.9
86.5
—
O
COOEt
COOH
Clofibrate
Cl
Not tested
100 (2 lM)
Not tested
O
Clofibric acid
H
N
N
S
COOH
—
N
Wy, 14-643
Cl

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2481
Further studies are in progress aimed both at identifying
mechanism of action of 1e and 1g, and correlating
molecular structure modifications with their b3-AR in-
verse agonistic activity and pharmacological features
by model study of metabolic syndrome, heart failure
and cachexia.
solvent was removed under reduced pressure. SOCl2
(3.9 ml, 2.39 g, 20 mmol) was dropwise added to the ob-
tained residuedissolved in ethanol (250 mL). The reaction
mixture was refluxed for 6 h. The solvent was then re-
moved under reduced pressure. The residue was dissolved
in water and extracted four times with ethyl ether. The
combined extracts were washed three times with a satu-
rated solution of NaHCO3, three times with a saturated
solution of NaCl and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure. The
product was isolated as colourless oil (65% yield) by chro-
matography (petroleum ether/ethyl acetate = 9.5:0.5).
FT-IR (neat): 3063, 2996, 2945, 1760, 1594, 1499, 1458,
5. Experimental
5.1. General methods
Melting points taken on an Electrothermal apparatus
are uncorrected. H NMR spectra were recorded on a
1
1
1381, 1303, 1255, 1200, 1112, 1019, 964, 695 cmꢁ1. H
Varian Inova 400 MHz Mercury 300 MHz spectrometer
or on a Bruker Aspect 500 MHz, and chemical shifts are
reported in parts per million ðdÞ. Absolute values of the
coupling constant (J) are reported. IR spectra were re-
corded on a Perkin-Elmer 681 spectrometer. GC analy-
ses were performed by using an HP-5 MS column (5%
phenyl methyl siloxane; 30 m · 0.25 mm · 250 lm film
thickness) on a HP 6890 model, series II. Thin-layer
chromatography (TLC) was performed on silica gel
sheets with fluorescent indicator (Stratochrom SIF, 60
F254 Merk), the spots on the TLC were observed under
ultraviolet light or were visualized with I2 vapour. Chro-
matography was conducted by using silica gel 60 with a
particle size distribution of 40–63 lm and 230–400
ASTM. GC–MS analyses were performed on an HP
5995C model, and microanalyses were performed on
an elemental analyzer 1106 (Carlo Erba Instrument).
ESI-MS analyses were performed on an Agilent 1100
LC/MSD trap system VL. Optical rotations were mea-
sured at 20 ꢂC on a Perkin-Elmer model 341 polarime-
ter. Stereoisomeric purity of the final targets 1b–c and
1e–h was determined by using OD-R HPLC column
(Daicel Chemical Industries Ltd.); acetonitrile—(0.5N
percloric acid–sodium perclorate, pH 2.0) = 60:40 and
65:35 for 1b–c and 1e–h, respectively, as mobile phase;
detection at k = 254 nm; flow rate = 0.4 mL/min. Capac-
ity factors (k0) were calculated by using as tꢂR the reten-
tion time of D2O.
NMR (300 MHz, CDCl3, d): 1.24 (t, J = 7.2 Hz, 3H,
OCH2CH3), 1.59 (s, 6H, C(CH3)2), 4.23 (q, J = 7.2 Hz,
2H, OCH2CH3), 6.83–6.86 (m, 2H, aromatic protons),
6.95–7.00 (m, 1H, aromatic proton), 7.20–7.26 (m, 2H,
aromatic protons). 13C NMR (75 MHz, CDCl3, d):
14.27, 25.60, 61.60, 79.26, 119.36, 122.31, 129.35,
155.66, 174.56. GC–MS (70 eV) m/z (rel. int.): 208 (M+,
16), 136 (5), 135 (52), 115 (5), 107 (7), 95 (15), 94 (100),
87 (11), 77 (17), 65 (6). Anal. Calcd for C12H16O3: C,
69.19; H, 7.75. Found: C, 69.15; H, 7.72.
5.3. ( )-Ethyl 2-phenoxypropanoate (5b)42
( )-Ethyl 2-bromopropanoate (0.71 mL, 5.5 mmol) and
anhydrous K2CO3 (1.520 g, 11 mmol) were added to a
solution of phenol (471 mg, 5 mmol) in anhydrous ace-
tone (125 mL). The reaction mixture was refluxed for
2.5 days. Solvent was removed under reduced pressure,
and the residue was dissolved in ethyl acetate and
washed three times with water and once with 2N NaOH.
The organic layer was dried over anhydrous Na2SO4
and the solvent was removed under reduced pressure
to afford a colourless oil (95% yield). FT-IR (neat):
3065, 3042, 2987, 2939, 2902, 2876, 1763, 1588, 1496,
1457, 1375, 1343, 1272 1050, 1020, 946, 885, 860, 802,
758, 691 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.24
.
(t, J = 7.1 Hz, 3H, OCH2CH3), 1.62 (d, J = 6.7 Hz,
3H, CHCH3), 4.21 (q, J = 7.1 Hz, 2H, OCH2CH3),
4.75 (q, J = 6.7 Hz, 1H, CHCH3), 6.87–6.90 (m, 2H,
aromatic protons), 6.94–6.99 (m, 1H, aromatic proton),
7.23–7.30 (m, 2H, aromatic protons). 13C NMR
(75 MHz, CDCl3, d): 14.33, 18.78, 61.44, 72.82, 115.33,
121.76, 129.74, 157.85, 172.44. GC–MS (70 eV) m/z
(rel. int.): 194 (M+, 49), 122 (13), 121 (100), 103 (4), 94
(27), 93 (18), 77 (39), 65 (7), 51 (11). Anal. Calcd for
C11H14O3: C, 68.00; H, 7.27. Found: C, 67.98; H, 7.24.
DMF and CH2Cl2 from a commercial source were puri-
fied by distillation from CaH2. Commercially available
acetone was distilled from anhydrous K2CO3 prior to
use. All other chemicals and solvents purchased from
Aldrich & Co. were used as supplied without further
purification.
5.2. Ethyl 2-methyl-2-phenoxypropanoate (5a)41
5.4. 1-(Alkyloxycarbonyl)ethyl 4-methylbenzenesulfo-
nates (6c–d): General procedure43
Phenol (5 g, 53 mmol) and 1,1,1-trichloro-2-methyl-2-
propanol (19.8 g, 106 mmol) were dissolved in acetone
(250 mL). NaOH (17 g, 425 mmol) was carefully added.
The reaction mixture was stirred overnight at room tem-
perature. Then, the solvent was removed under reduced
pressure. Water was added to the crude residue and
washed three times with ethyl ether. The organic phase
was discarded and the aqueous layer was acidified with
37% HCl and extracted several times with ethyl ether.
The extracts were washed three times with a saturated
solution of NaCl, dried over anhydrous Na2SO4 and the
Alkyl lactate [(S)-(ꢁ)-ethyl lactate or (R)-(+)-methyl
lactate] (25 mmol) was dissolved in CH2Cl2 (10 mL)
and, p-toluenesulfonyl chloride (50 mmol) and pyridine
(5 mL) were added. The reaction mixture was stirred at
rt for 6 h. The solvent was removed under reduced pres-
sure, the residue was dissolved in ethyl acetate and washed
three times with 1N HCl and three times with water. The
product was isolated by chromatography (silica gel;
mobile phase: petroleum ether/ethyl acetate = 8:2).

2482
M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
5.4.1. (S)-(ꢁ)-1-(Ethoxycarbonyl)ethyl 4-methylbenzene-
sulfonate (6c)43. Mp: 33.5–34.6 ꢂC, white solid (85%
692 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.62 (d,
.
J = 6.9 Hz, 3H, CHCH3), 3.74 (s, 3H, OCH3), 4.77 (q,
J = 6.9 Hz, 1H, CHCH3), 6.87–6.90 (m, 2H, aromatic
protons), 6.95–7.00 (m, 1H, aromatic proton), 7.25–
7.30 (m, 2H, aromatic protons). 13C NMR (75 MHz,
CDCl3, d): 18.81, 52.49, 77.73, 115.29, 121.84, 129.80,
157.79, 172.93. GC–MS (70 eV) m/z (rel. int.): 180
(M+, 59), 122 (13), 121 (100), 94 (37), 93 (17), 91 (9),
77 (41), 65 (11), 59 (7), 51 (11). Anal. Calcd
for C10H12O3: C, 66.63; H, 6.72. Found: C, 66.61; H,
6.69.
yield). ½aꢃ ꢁ36.6 (c 1.45, CHCl3). FT-IR (KBr): 3075,
D
2988, 2935, 2877, 1756, 1598, 1495, 1449, 1370, 1307,
1191, 1179, 1122, 1083, 1029, 944, 888, 818, 785,
665 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.18 (t,
.
J = 7.1 Hz, 3H, OCH2CH3), 1.48 (d, J = 6.9 Hz, 3H,
CHCH3), 2.42 (s, 3H, CH3C6H4SO3), 4.09 (q,
J = 7.1 Hz, 2H, OCH2CH3), 4.90 (q, J = 6.9 Hz, 1H,
CHCH3), 7.31–7.34 (m, 2H, aromatic protons), 7.78–
7.82 (m, 2H, aromatic protons). 13C NMR (75 MHz,
CDCl3, d): 14.15, 18.62, 21.88, 62.04, 74.39, 128.23,
130.02, 133.56, 145.31, 169.29. GC–MS (70 eV) m/z
(rel. int.): 274 [(34S)M+, 1], 272 [(32S)M+, 11], 201 (3),
199 (48), 157 (9), 156 (18), 155 (100), 139 (5), 92 (22),
91 (71), 65 (21). Anal. Calcd for C12H16O5S: C, 52.91;
H, 5.93. Found: C, 52.88; H, 5.89.
5.6. Ethyl 2-[4-(2-bromoacetyl)phenoxy]-2-methylpro-
panoate (4a) and alkyl 2-[4-(bromoacetyl)phenoxy]pro-
panoates (4b–d): General procedure41
Anhydrous AlCl3 (11.54 g, 86.5 mmol) was suspended in
anhydrous CH2Cl2 (65 mL) under nitrogen atmosphere.
Bromoacetylbromide (7.5 mL, 86.5 mmol) was carefully
added and the mixture was stirred at 0 ꢂC for 1 h. Then,
5.4.2. (R)-(+)-1-(Methoxycarbonyl)ethyl 4-methylben-
zenesulfonate (6d)43. Colourless oil (quantitative yield).
½aꢃ +35.1 (c 1.05, CHCl3). FT-IR (neat): 3101, 2993,
ethyl
2-methyl-2-phenoxypropanoate
(5a,
6 g,
D
2957, 2928, 1762, 1598, 1458, 1433, 1369, 1311, 1221,
28.8 mmol) or alkyl 2-phenoxypropanoate (5b–d,
28.8 mmol) in anhydrous CH2Cl2 (25 mL) was added.
The resulting solution was stirred overnight at room
temperature under nitrogen atmosphere, and then re-
fluxed for 6 h. The mixture was then poured into
crushed ice and extracted three times with ethyl acetate.
The combined extracts were washed three times with a
saturated solution of NaHCO3, three times with water
and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure. The product was iso-
lated as a yellow oil by chromatography (silica gel;
mobile phase: petroleum ether/ethyl acetate = 8:2 for
4a and 1:1 for 4b–d).
1
1193, 1179, 1083, 1027, 975, 945, 824 cmꢁ1. H NMR
(300 MHz, CDCl3, d): 1.46 (d, J = 6.9 Hz, 3H, CHCH3),
2.41 (s, 3H, CH3C6H4), 3.62 (s, 3H, OCH3), 4.90 (q,
J = 6.9 Hz, 1H, CHCH3), 7.30–7.33 (m, 2H, aromatic
protons), 7.75–7.78 (m, 2H, aromatic protons). 13C
NMR (75 MHz, CDCl3, d): 18.56, 21.84, 52.77, 74.26,
128.18, 130.04, 133.51, 145.36, 169.70. GC–MS (70 eV)
m/z (rel. int.): 260 [(34S)M+, 1], 258 [(32S)M+, 13], 199
(45), 157 (7), 156 (12), 155 (100), 139 (10), 91 (79), 65
(23). Anal. Calcd for C11H14O5S: C, 51.13; H, 5.47.
Found: C, 51.11; H, 5.46.
5.5. Alkyl 2-phenoxypropanoates (5c–d): General
procedure43
5.6.1. Ethyl 2-[4-(2-bromoacetyl)phenoxy]-2-methylpro-
panoate (4a)41. Yield 21%. 1H NMR (300 MHz, CDCl3,
d): 1.19 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.64 (s, 6H,
C(CH3)2), 4.20 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.37 (s,
2H, CH2Br), 6.80–6.84 (m, 2H, aromatic protons),
7.87–7.90 (m, 2H, aromatic protons). 13C NMR
(75 MHz, CDCl3, d): 14.24, 25.59, 31.00, 61.97, 79.69,
117.65, 127.54, 131.14, 160.70, 173.70, 190.13. GC–MS
(70 eV) m/z (rel. int.): 330 [(81Br)M+, 8], 328
[(79Br)M+, 8], 257 (26), 255 (26), 216 (7), 214 (7), 177
(5), 122 (9), 121 (100), 115 (25), 107 (19), 87 (21), 59
(10). Anal. Calcd for C14H17BrO4: C, 51.06; H, 5.21.
Found: C, 51.08; H, 5.22.
CsF (11 mmol) was dried at 200 ꢂC in vacuum for
20 min, and it was then cooled at room temperature un-
der a nitrogen stream. Anhydrous DMF (34 mL) was
then added to CsF and the resulting suspension was stir-
red at room temperature under nitrogen atmosphere.
Phenol (11 mmol) was added and the resulting reaction
mixture was stirred at room temperature for 20 min. 1-
(Alkoxycarbonyl)ethyl 4-methylbenzenesulfonate (6c–d,
3.67 mmol) in anhydrous DMF (8.5 mL) was added
dropwise. The reaction mixture was stirred at room tem-
perature for 32 h. The mixture was then diluted with
ethyl acetate, washed three times with a saturated solu-
tion of NaHCO3 and with a saturated solution of NaCl.
The organic layer was dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure. The
product was isolated as a colourless oil by chromatogra-
phy (petroleum ether/ethyl acetate = 10:1).
5.6.2. ( )-Ethyl 2-[4-(bromoacetyl)phenoxy]propanoate
(4b). Yield 71%. FT-IR (neat): 3073, 3048, 2987, 2941,
2875, 1755, 1682, 1594, 1575, 1509, 1446, 1431, 1393,
1378, 1285, 1190, 1136, 1097, 1050, 1017, 949, 843,
763, 606 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.22
.
(t, J = 7.1 Hz, 3H, OCH2CH3), 1.62 (d, J = 6.7 Hz,
3H, CH3CH), 4.19 (q, J = 7.1 Hz, 2H, OCH2CH3),
4.36 (s, 2H, CH2Br), 4.80 (q, J = 6.7 Hz, 1H, CH3CH),
6.86–6.91 (m, 2H, aromatic protons), 7.89–7.93 (m,
2H, aromatic protons). 13C NMR (75 MHz, CDCl3,
d): 14.32, 18.59, 31.01, 61.78, 72.77, 115.13, 127.77,
131.54, 162.26, 171.48, 190.04. GC–MS (70 eV) m/z
(rel. int.): 316 [(81Br)M+, 10], 314 [(79Br)M+, 10], 243
(15), 241 (15), 222 (19), 221 (100), 163 (8), 121 (46),
5.5.1. (R)-(+)-Ethyl 2-phenoxypropanoate (5c)43. Yield
77%. ½aꢃ +27.9 (c 1.4, CHCl3). Spectroscopic data are
D
identical to those ones reported for 5b.
5.5.2. (S)-(ꢁ)-Methyl 2-phenoxypropanoate (5d). Yield
77%. ½aꢃ ꢁ22.5 (c 1.15, CHCl3). FT-IR (neat): 3065,
D
3042, 2993, 2955, 2848, 1760, 1601, 1589, 1495, 1456,
1376, 1287, 1244, 1205, 1136, 1100, 1053, 979, 754,

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2483
107 (17), 104 (8), 90 (7), 77 (4), 76 (7). Anal. Calcd for
C13H15BrO4: C, 49.51; H, 4.80. Found: C, 49.53; H,
4.84.
5.7.2. ( )-Ethyl 2-[4-(2-bromoethyl)phenoxy]propanoate
(3b). Colourless oil (83% yield). FT-IR (neat): 3033,
2986, 2938, 2874, 1752, 1612, 1585, 1512, 1448, 1376,
1297, 1265, 1243, 1199, 1135, 1098, 1052, 1016, 971,
5.6.3. (R)-(+)-Ethyl 2-[4-(bromoacetyl)phenoxy]propano-
ate (4c). Yield 79%. ½aꢃ +29.8 (c 1.1, CHCl3). Spectro-
823, 734 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.24
.
(t, J = 7.1 Hz, 3H, OCH2CH3), 1.60 (d, J = 6.9 Hz,
3H, CHCH3), 3.08 (t, J = 7.7 Hz, 2H, CH2CH2Br),
3.50 (t, J = 7.7 Hz, 2H, CH2Br), 4.21 (q, J = 7.1 Hz,
2H, OCH2CH3), 4.71 (d, J = 6.9 Hz, 1H, CHCH3),
6.80–6.84 (m, 2H, aromatic protons), 7.08–7.11 (m,
2H, aromatic protons). 13C NMR (75 MHz, CDCl3,
d): 14.36, 18.78, 33.41, 38.80, 61.48, 72.91, 115.45,
129.95, 132.18, 156.82, 172.40. GC–MS (70 eV) m/z
(rel. int.): 302 [(81Br)M+, 61], 300 [(79Br)M+, 60], 230
(12), 229 (100), 228 (13), 227 (99), 221 (24), 207 (38),
185 (4), 183 (5), 147 (20), 121 (23), 120 (14), 119 (26),
107 (70), 104 (21), 103 (21), 91 (22), 78 (11), 77 (22),
65 (7). Anal. Calcd for C13H17BrO3: C, 51.82; H, 5.69.
Found: C, 51.84; H, 5.70.
D
scopic data are identical to those ones reported for 4b.
5.6.4. (S)-(ꢁ)-Methyl 2-[4-(bromoacetyl)phenoxy]propan-
oate (4d). Yield 84%. ½aꢃ ꢁ32.7 (c 1.65, CHCl3). FT-IR
D
(neat): 3101, 3043, 2995, 2953, 2848, 1754, 1676, 1600,
1509, 1434, 1375, 1285, 1256, 1202, 1176, 1135, 1100,
1051, 978, 843, 606 cmꢁ1
.
1H NMR (300 MHz,
CDCl3, d): 1.63 (d, J = 6.9 Hz, 3H, CH3CH), 3.74 (s,
3H, OCH3), 4.37 (s, 2H, CH2Br), 4.84 (q,
J = 6.9 Hz, 1H, CH3CH), 6.87–6.90 (m, 2H, aromatic
protons), 7.91–7.94 (m, 2H, aromatic protons). 13C
NMR (75 MHz, CDCl3, d): 18.63, 30.96, 52.75,
72.69, 115.10, 127.84, 131.59, 162.19, 171.97, 190.04.
GC–MS (70 eV) m/z (rel. int.): 302 [(81Br)M+, 6],
300 [(79Br)M+, 6], 243 (7), 241 (8), 222 (6), 208 (14),
207 (100), 163 (8), 121 (46), 107 (15), 93 (6), 76 (6).
Anal. Calcd for C12H13BrO4: C, 47.84; H, 4.35.
Found: C, 47.86; H, 4.38.
5.7.3. (R)-(+)-Ethyl 2-[4-(2-bromoethyl)phenoxy]propan-
oate (3c). Yellow oil (81% yield). ½aꢃ +22.0 (c 1.1,
D
CHCl3). Spectroscopic data are identical to those ones
reported for 3b.
5.7. Ethyl 2-[4-(2-bromoethyl)phenoxy]-2-methylpropan-
oate (3a) and alkyl 2-[4-(2-bromoethyl)phenoxy]propan-
oates (3b–d): General procedure.41
5.7.4. (S)-(ꢁ)-Methyl 2-[4-(2-bromoethyl)phenoxy]pro-
panoate (3d). Yellow oil (75% yield). ½aꢃ ꢁ22.0 (c 1.1,
D
CHCl3). FT-IR (neat): 3033, 2993, 2953, 1761, 1613,
1586, 1507, 1448, 1376, 1231, 1134, 1100, 1053, 979,
1
Ethyl 2-[4-(2-bromoacetyl)phenoxy]-2-methylpropano-
ate (4a, 66 mg, 0.2 mmol) {or ( )- or (R)-(+)- or (S)-
(ꢁ)-alkyl 2-[4-(bromoacetyl)phenoxy]propanoate (4b–d,
1 g, 3.18 mmol)} was dissolved in trifluoroacetic acid
(1 mL or 15 mL). Triethylsilane (0.07 mL, 0.44 mmol
for 4a and 1.12 mL, 7 mmol for 4b–d) was added and
the reaction mixture was stirred at 70 ꢂC for 4 h. It
was, then, cooled at room temperature and ethyl acetate
was added. The organic phase was washed several times
with a saturated solution of NaHCO3, three times with
water and then dried over anhydrous Na2SO4. The sol-
vent was removed under reduced pressure and the prod-
uct was isolated by chromatography (petroleum ether/
ethyl acetate = 9:1).
823, 638 cmꢁ1. H NMR (300 MHz, CDCl3, d): 1.60
(d, J = 6.9 Hz, 3H, CHCH3), 3.08 (t, J = 7.7 Hz, 2H,
CH2CH2Br), 3.51 (t, J = 7.7 Hz, 2H, CH2Br), 3.75 (s,
3H, OCH3), 4.73 (q, J = 6.9 Hz, 1H, CHCH3), 6.80–
6.84 (m, 2H, aromatic protons), 7.09–7.13 (m, 2H, aro-
matic protons). 13C NMR (75 MHz, CDCl3, d): 18.81,
33.36, 38.80, 52.54, 72.84, 115.41, 130.01, 132.26,
156.75, 172.92. GC–MS (70 eV) m/z (rel. int.): 288
[(81Br)M+, 70], 286 [(79Br)M+, 70], 229 (94), 227 (93),
207 (33), 193 (74), 147 (21), 121 (31), 120 (18), 119
(28), 107 (100), 104 (21), 103 (23), 91 (32), 90 (9), 89
(8), 78 (14), 77 (26), 65 (11), 59 (13). Anal. Calcd for
C12H15BrO3: C, 50.15; H, 5.27. Found: C, 50.14; H,
5.31.
5.7.1. Ethyl 2-[4-(2-bromoethyl)phenoxy]-2-methylpro-
panoate (3a).41 Colourless oil (93% yield). FT-IR (neat):
2988, 2935, 2855, 1734, 1611, 1583, 1509, 1467, 1383,
1365, 1283, 1237, 1178, 1141, 1023, 971, 839, 738,
5.8. Ethyl 2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]-
phenoxy}-2-methylpropanoate (2a–c) and alkyl 2-{4-[2-
(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoates
(2d–h): General procedure
642 cmꢁ1 1H NMR (300 MHz, CDCl3, d): 1.24 (t,
.
J = 7.1 Hz, 3H, OCH2CH3), 1.58 (s, 6H, C(CH3)2),
3.08 (t, J = 7.8 Hz, 2H, CH2CH2Br), 3.51 (t,
J = 7.8 Hz, 2H, CH2Br), 4.23 (q, J = 7.1 Hz, 2H,
OCH2CH3), 6.77–6.80 (m, 2H, aromatic protons),
7.05–7.08 (m, 2H, aromatic protons). 13C NMR
(75 MHz, CDCl3, d): 14.30, 25.59, 33.31, 38.89, 61.64,
79.32, 119.45, 129.57, 132.75, 154.57, 174.50. GC–MS
(70 eV) m/z (rel. int.): 316 [(81Br)M+, 14], 314
[(79Br)M+, 14], 243 (29), 241 (30), 235 (9), 202 (46),
200 (47), 121 (32), 120 (10), 108 (9), 107 (100), 103
(10), 91 (15), 77 (13), 59 (7). Anal. Calcd for
C14H19BrO3: C, 53.32; H, 6.08. Found: C, 53.36; H,
6.11.
A mixture of ethyl 2-[4-(2-bromoethyl)phenoxy]-2-meth-
ylpropanoate (3a) or alkyl 2-[4-(2-bromoethyl)phen-
oxy]propanoate (3b–d) and racemic, (R)- or (S)-2-
amino-1-phenylethanol (ratio indicated in table below)
in anhydrous DMF was stirred under nitrogen atmo-
sphere at 70 ꢂC for 70 h. The reaction mixture was then
diluted with ethyl acetate, washed with a saturated solu-
tion of NaCl and dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure and the
product was isolated as yellow oil by chromatography
(silica gel; mobile phase: petroleum ether/ethyl ace-
tate = 8:2 for 2a–c and dichloromethane/ethanol = 40:1
for 2d–h).

2484
M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
5.8.5. (2S)-Methyl 2-{4-[2-((2S)-2-hydroxy-2-phenyleth-
Alkyl
propanoate 2-amino-
1-phenylethanol
1:1.2
Alkyl propanoate/ Alkyl propanoate
ylamino)ethyl]phenoxy}propanoate (2e). Yield 31%. ½aꢃ
D
concentration
(mol/L)
+11.8 (c 0.95, CHCl3). FT-IR (neat): 3600–3200, 3029,
2987, 2937, 2885, 1752, 1612, 1585, 1512, 1451, 1426,
1377, 1348, 1299, 1250, 1207, 1181,1137, 1100, 1071,
3a
0.85
1
0.85
1
1047, 825, 758, 700 cmꢁ1. H NMR (400 MHz, CDCl3,
3b, 3c
3d
1:1.3
1:1.1
d): 1.57 (d, J = 6.7 Hz, 3H, CHCH3), 2.69–2.93 (m, 6H,
CH2CHOH, CH2NH, CH2CH2NH), 3.45–3.64 (b s, 2H,
OH and NH: exchange with D2O), 3.71 (s, 3H, OCH3),
4.70 (q, J = 6.7 Hz, 2H, CHCH3), 4.74 (dd, J = 9.3 and
3.2 Hz, 1H, CHOH), 6.75–6.77 (m, 2H, aromatic pro-
tons), 7.03–7.05 (m, 2H, aromatic protons), 7.21–7.24
(m, 1H, aromatic proton), 7.27–7.32 (m, 4H, aromatic
protons). 13C NMR (100 MHz, CDCl3, d) 18.83,
35.05, 50.79, 52.56, 56.91, 71.53, 72.81, 115.36, 126.03,
127.80, 128.63, 129.99, 132.50, 142.51, 154.31, 173.01.
GC–MS (70 eV) m/z (rel. int.): 325 [(M-18)+, 9], 325
(41), 324 (25), 238 (31), 206 (24), 193 (40), 147 (30),
134 (30), 132 (100), 130 (21), 120 (20), 107 (44), 106
(21), 105 (50), 104 (45), 103 (30), 91 (69), 90 (20), 78
(20), 77 (38), 70 (25), 51 (19). MS-ESI m/z (%): 344
[M+H]+ (100%). Anal. Calcd for C20H25NO4: C, 69.93;
H, 7.34; N, 4.08. Found: C, 69.95; H, 7.31; N, 4.06.
5.8.1.
( )-Ethyl
2-{4-[2-(2-hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}-2-methylpropanoate (2a). Yield
80%. FT-IR (neat): 3600–3100, 3060, 3032, 2989, 2935,
2854, 1733, 1611, 1509, 1454, 1383, 1266, 1235, 1179,
1144, 1026, 914, 850, 736, 702 cmꢁ1
.
1H NMR
(300 MHz, CDCl3, d): 1.24 (t, J = 7.1 Hz, 3H,
OCH2CH3), 1.56 (s, 6H, C(CH3)2), 2.70–2.96 (m, 6H,
CH2CHOH, CH2NH, CH2CH2NH), 3.90–4.10 (b s,
2H, OH and NH: exchange with D2O), 4.22 (q,
J = 7.1 Hz, 2H, OCH2CH3), 4.75 (dd, J = 9.2 and
3.4 Hz, 1H, CHOH), 6.75–6.78 (m, 2H, aromatic pro-
tons), 7.00–7.03 (m, 2H, aromatic protons), 7.23–7.33
(m, 5H, aromatic protons). 13C NMR (75 MHz, CDCl3,
d): 14.31, 25.58, 35.54, 50.88, 57.12, 61.62, 71.82, 79.31,
119.67, 126.03, 127.73, 128.60, 129.54, 133.46, 142.84,
154.03, 174.59. GC–MS (70 eV) m/z (rel. int.): 353
[(M-18)+, 4], 298 (11), 264 (100), 238 (9), 235 (7), 150
(25), 132 (68), 121 (44), 107 (24), 91 (11), 77 (12), 43
(8). Anal. Calcd for C22H29NO4: C, 71.11; H, 7.88; N,
3.77. Found: C, 71.01; H, 7.85; N, 3.74.
5.8.6. (2S)-Methyl 2-{4-[2-((2R)-2-hydroxy-2-phenyleth-
ylamino)ethyl]phenoxy}propanoate (2f). Yield 47%. ½aꢃ
D
ꢁ58.5 (c 1.05, CHCl3). Spectroscopic data are identical
to those ones reported for 2e.
5.8.7. (2R)-(+)-Ethyl 2-{4-[2-((2S)-2-hydroxy-2-phenyl-
ethylamino)ethyl]phenoxy}propanoate (2g). Yield 66%.
5.8.2. Ethyl (ꢁ)-2-{4-[2-((R)-2-hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}-2-methylpropanoate (2b). Yield
½aꢃ +55.6 (c 1.01, CHCl3). Spectroscopic data are iden-
D
tical to those ones reported for 2d.
64%. ½aꢃ ꢁ32.1 (c 1.0, CHCl3). Spectroscopic data are
D
identical to those ones reported for 2a.
5.8.8. (2R)-(ꢁ)-Ethyl 2-{4-[2-((2R)-2-hydroxy-2-phenyl-
ethylamino)ethyl]phenoxy}propanoate (2h). Yield 54%.
5.8.3. Ethyl (+)-2-{4-[2-((S)-2-hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}-2-methylpropanoate (2c). Yield
½aꢃ ꢁ6.9 (c 0.96, CHCl3). Spectroscopic data are identi-
D
cal to those ones reported for 2d.
61%. ½aꢃ +32.0 (c 1.0, CHCl3). Spectroscopic data are
D
identical to those ones reported for 2a.
5.9. 2-{4-[2-(2-Hydroxy-2-phenylethylamino)ethyl]phenoxy}-
2-methylpropanoic acid (1a–c) and 2-{4-[2-(2-hydroxy-2-
phenylethylamino)ethyl]phenoxy}propanoic acid (1d–h):
General procedure.
5.8.4.
( )-Ethyl
2-{4-[2-(2-hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}propanoate (2d). Yield 49%. FT-
IR (neat): 3600–3100, 3058, 3022, 2986, 2935, 2855,
1750, 1669, 1612, 1581, 1511, 1449, 1375, 1292, 1239,
1N NaOH (8.4 mL, 8.4 mmol) was added to a solution
of ethyl 2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]-
phenoxy}-2-methylpropanoate (2a–c, 4.2 mmol) or alkyl
2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}-
propanoate (2d–h, 4.2 mmol) in THF (10 mL). The reac-
tion mixture was stirred at rt for 1 h. Then, THF was
removed under reduced pressure and 2 N HCl was then
added to pH 6. A precipitate formed that was filtered
and washed with water. The residue was treated with
hot acetone to afford a crystalline product.
1135, 1050, 1014, 826, 733, 702 cmꢁ1
.
1H NMR
(300 MHz, CDCl3, d): 1.23 (t, J = 7.1 Hz, 3H,
OCH2CH3), 1.58 (d, J = 6.8 Hz, 3H, CHCH3), 2.66–
3.00 (m, 6H, CH2CHOH, CH2NH, CH2CH2NH), 4.20
(q, J = 7.1 Hz, 2H, OCH2CH3), 4.70 (q, J = 6.8 Hz,
2H, CHCH3), 4.86 (dd, J = 9.3 and 3.3 Hz, 1H, CHOH),
5.10–5.30 (b s, 2H, OH and NH: exchange with D2O),
6.74–6.82 (m, 2H, aromatic protons), 6.97–7.12 (m,
2H, aromatic protons), 7.24–7.35 (m, 5H, aromatic pro-
tons). 13C NMR (75 MHz, CDCl3, d): 14.35, 34.29,
50.50, 56.52, 61.47, 66.05, 72.90, 115.51, 126.03,
127.86, 128.66, 129.94, 131.84, 142.28, 156.52, 172.47.
GC–MS (70 eV) m/z (rel. int.): 339 [(M-18)+, 1], 284
(4), 250 (100), 221 (24), 207 (7), 176 (10), 150 (14), 147
(14), 132 (69), 121 (19), 107 (18), 105 (14), 104 (10),
103 (11), 91 (10), 77 (14), 43 (9). Anal. Calcd for
C21H27NO4: C, 70.55; H, 7.72; N, 3.92. Found: C,
70.58; H, 7.74; N, 3.90.
5.9.1. ( )-2-{4-[2-(2-Hydroxy-2-phenylethylamino)ethyl]-
phenoxy}-2-methylpropanoic acid (1a). Mp: 223 ꢂC (dec),
white solid (35% yield). HPLC data: k0ðRÞ ¼ 1:62,
k0ðSÞ ¼ 1:85, a ¼ 1:14; elution time: t ¼ 20:96 min,
ðRÞ
t
¼ 22:77 min. FT-IR (KBr): 3650–3200, 3000, 2987,
ðSÞ
2935, 2792, 1613, 1560, 1512, 1462, 1402, 1362, 1243,
1199, 1151, 837, 703 cmꢁ1 1H NMR (300 MHz,
DMSO-d6, d): 1.38 (s, 6H, C(CH3)2), 2.57–2.82 (m,
.

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2485
6H, CH2CHOH, CH2NH, CH2CH2NH), 3.00–4.60 (b s,
3H, OH, NH and COOH: exchange with D2O), 4.72
(dd, J = 5.8 and 2.6 Hz, 1H, CHOH), 6.69–6.72 (m,
2H, aromatic protons), 6.86–6.88 (m, 2H, aromatic pro-
tons), 7.19–7.34 (m, 5H, aromatic protons). MS-ESI m/z
(%): 344 [M+H]+ (100%). MS-ESI m/z (%): 342 [MꢁH]ꢁ
(100%). Anal. Calcd for C20H25NO4: C, 69.93; H, 7.34;
N, 4.08. Found: C, 69.97; H, 7.38; N, 4.04.
HPLC peak integrations to be a 87:13 mixture of
(S,R)/minor diastereoisomers.51 ½aꢃ ꢁ48.2 (c 1.04,
D
CH3COOH). Spectroscopic data are identical to those
ones reported for 1d.
5.9.8. (2R)-(ꢁ)-2-{4-[2-((2R)-2-Hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}propanoic acid (1h). Mp: 202 ꢂC
(dec.), white solid (48% yield), which was judged by
HPLC peak integrations to be a 90:10 mixture of (R,
5.9.2. (R)-(ꢁ)-2-{4-[2-(2-Hydroxy-2-phenylethylamino)-
ethyl]phenoxy}-2-methylpropanoic acid (1b). Mp: 232–
233 ꢂC (dec.), white solid (61% yield); eeHPLC = 97%.
R)/minor diastereoisomers.51 ½aꢃ
ꢁ0.41 (c 0.92,
D
CH3COOH). Spectroscopic data are identical to those
ones reported for 1d.
½aꢃ ꢁ21.3 (c 0.34, CH3COOH). Spectroscopic data
D
are identical to those ones reported for 1a.
6. Biological methods
6.1. General
5.9.3. (S)-(+)-2-{4-[2-(2-Hydroxy-2-phenylethylamino)-
ethyl]phenoxy}-2-methylpropanoic acid (1c). Mp: 232–
233 ꢂC (dec.), white solid (45% yield); eeHPLC = 98%.
D
identical to those ones reported for 1a.
½aꢃ +23.6 (c 0.86, CH3COOH). Spectroscopic data are
CHO cell lines transfected with human cloned b3- and
b2-ARs were kindly provided by the Institute Cochin
´ ´
´
de Genetique Moleculaire, Paris, France. The CHO cell
5.9.4. ( )-2-{4-[2-(2-Hydroxy-2-phenylethylamino)ethyl]-
phenoxy}propanoic acid (1d). Mp: 165–166 ꢂC, white
line transfected with human cloned b1-ARs was kindly
provided by Institut fur Pharmakologie und Toxikolo-
¨
gie, Universita¨t Wurzburg, Germany. For receptor
¨
solid (42% yield). HPLC elution time: t
min,
¼ 14:66
ðS;SÞ
t
¼ 15:18 min,
t
¼ 19:12 min and
binding and activity studies, compounds were dissolved
in absolute ethanol. Forskoline was purchased from To-
cris Cookson Ltd., UK; [3H]-dihydroalprenolol
(3,59TBq) was obtained from Amersham Biosciences
(Milan, Italy). Protein concentration was determined
by commercial protein determination kit based on the
Lowry method,52 using a Perkin-Elmer UV/vis LAMB-
DA BIO 20 spectrophotometer. All binding and activity
data obtained were analyzed by Graph-Pad Prism
program.53
ðR;SÞ
ðR;RÞ
t
¼ 22:40 min. FT-IR (KBr): 3600–3200, 2998,
ðS;RÞ
2807, 1613, 1585, 1511, 1453, 1423, 1227, 1138, 1099,
1037, 932, 816, 747, 698 cmꢁ1. 1H NMR (500 MHz, ace-
tic acid-d4, d): 1.61 (d, J = 6.8 Hz, 3H, CHCH3), 3.06–
3.08 (m, 2H, CH2CH2NH), 3.27 (dd, J = 12.5 and
10.7 Hz, 1H, CH2CHOH), 3.31–3.37 (m, 2H, CH2NH),
3.41 (dd, J = 12.5 and 2.5 Hz, 1H, CH2CHOH), 4.84 (q,
J = 6.8 Hz, 1H, CHCH3), 5.26 (dd, J = 10.7 and 2.5 Hz,
1H, CHOH), 6.86–6.88 (m, 2H, aromatic protons),
7.18–7.19 (m, 2H, aromatic protons), 7.28–7.31 (m,
1H, aromatic proton), 7.34–7.37 (m, 2H, aromatic pro-
tons), 7.39–7.41 (m, 2H, aromatic protons). 13C NMR
(125 MHz, CDCl3, d): 17.84, 31.24, 49.56, 54.15, 69.45,
72.18, 115.50, 126.04, 128.43, 128.78, 129.76, 130.12,
140.26, 156.91, 176.99. MS-ESI m/z (%): 330 [M+H]+
(100%). MS-ESI m/z (%): 328 [MꢁH]ꢁ (100%). Anal.
Calcd for C19H23NO4: C, 69.26; H, 7.04; N, 4.25.
Found: C, 69.28; H, 7.06, N, 4.22.
6.1.1. Cell culture and membrane preparation. Chinese
hamster ovary (CHO) cells expressing each subtype of
human cloned b1-, b2- or b3-ARs were grown in an
atmosphere of 5% CO2 in air at 37 ꢂC in Dulbecco’s
modified Eagle’s medium with nutrient mixture F12
(DMEM/F12) supplemented with 10% Foetal calf ser-
um, 2 mM L-glutamine, 100 U/mL of penicillin G and
100 lg/mL of streptomycin.
5.9.5. (2S)-(+)-2-{4-[2-((2S)-2-Hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}propanoic acid (1e). Mp: 202 ꢂC
(dec.), white solid (45% yield), which was judged by
HPLC peak integrations to be a 90:10 mixture of
Preconfluent cells were washed with ice-cold PBS,
scraped from the plate surface, collected in ice-cold lysis
buffer (10 · 106/mL; 5 mM Tris/HCl, 2 mM EDTA, pH
7.4 at 4 ꢂC) and homogenised with a Brinkman politron
(5 for 3 · 10 s). The cell membrane suspension was cen-
trifuged for 10 min at 4 ꢂC at 1000g. Supernatant was
centrifuged at 10,000g for 30 min at 4 ꢂC. The resultant
membrane pellet was resuspended in ice cold incubation
buffer (50 mM Tris/HCl, 10 mM MgCl2, pH 7.4, for b1-
AR binding experiments; 50 mM Tris/HCl, pH 7.4, for
b2-AR binding experiments) and protein content was
measured. The membrane suspension was used immedi-
ately or stored frozen at ꢁ80 ꢂC, for radioligand binding
experiments.
(S,S)/minor diastereoisomers.51 ½aꢃ +0.68 (c 1.19,
D
CH3COOH). Spectroscopic data are identical to those
ones reported for 1d.
5.9.6. (2R)-(+)-2-{4-[2-((2S)-2-Hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}propanoic acid (1f). Mp: 193–
194.5 ꢂC, white solid (65% yield), which was judged by
HPLC peak integrations to be a 87:13 mixture of
(R,S)/minor diastereoisomers.51 ½aꢃ +44.9 (c 1.00,
D
CH3COOH). Spectroscopic data are identical to those
ones reported for 1d.
6.1.2. b1- and b2-AR binding experiments. Saturation
binding experiments were performed by incubating cell
membranes (50 lg of protein) in a total volume of
500 lL incubation buffer, containing increasing
5.9.7. (2S)-(ꢁ)-2-{4-[2-((2R)-2-Hydroxy-2-phenylethyl-
amino)ethyl]phenoxy}propanoic acid (1g). Mp: 193–
194 ꢂC, white solid (41% yield); which was judged by

2486
M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
concentrations of [3H]-dihydroalprenolol (0.1 nM,
0.5 nM, 1 nM, 3 nM, 5 nM, 10 nM) (Fig. 6A). Incuba-
tions were carried out at 30 ꢂC for 30 min for b1- or
90 min for b2-AR binding assay. Non specific binding
was determined in the presence of 10 lM Alprenolol.
Reactions were terminated by rapid filtration through
Whatman GF/C glass fibre filters that had been soaked
for 60 min in 0.5% polyethyleneamine for b1- or 0.3%
polyethyleneamine for b2-AR binding assay. The filters
were washed with 3 · 1 mL of ice-cold incubation buffer.
The radioactivity bound to the filters was measured
using LS6500 Multi-Purpose scintillation Counter,
Beckman. Competition experiments were performed by
incubating 50 lg of protein with increasing amounts of
test compound (from 10ꢁ9M to 10ꢁ5 M) and 4 nM
[3H]-dihydroalprenolol for b1- or 0.4 nM for b2-AR
binding assay (Fig. 6B), in a final incubation volume
of 500 lL incubation buffer. Non specific binding was
determined in the presence of 10 lM Alprenolol.
Reactions were terminated and radioactivity quantified
as previously described.
from BD Biosciences Clontech (Palo Alto, CA).
GAL4/mousePPARaLBD receptor plasmid was pre-
pared by fusing the mouse PPARa LBD to the DBD
of yeast GAL4, and then subcloning the fusion into
the pSG5 expression vector. pCH110, that encodes the
b-galactosidase enzyme to correct for differences in
transfection efficiency, was purchased from Pharmacia
Biotech (Piscataway, NJ).
6.2.2. Transfection assay. Monkey kidney fibroblasts
(COS-7) were seeded at 1.2 · 105 cells per well, in 12-well
plates, and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% foetal bovine serum,
overnight at 37 ꢂC. Two hours before transfection, the
culture medium was replaced by fresh serum-free med-
ium and then transfection was performed with the mul-
ticomponent lipid-based FuGENE6 Transfection
Reagent (F. Hoffmann-La Roche; Basel, Switzerland)
according to the instructions of the manufacturer. The
transfection mixture containing (for each well) 0.8 lg
of the expression vector, 1.6 lg of the reporter vector,
0.8 lg of the control vector and 9 ll of FuGENE6
Transfection Reagent was added directly to the cells in
the presence of serum-free medium. After 5 h the trans-
fection medium was removed and transfected cells were
treated with three different concentrations (50, 150 and
300 lM) of the test compounds 1a–h, at 37 ꢂC for 48 h
in the complete culture medium. About 2 lM Wy,14-
643, a known PPARa ligand, was used as a positive con-
trol. Cells were then washed twice with phosphate-buf-
fered saline (PBS) and then dissolved in lysis buffer
(0.25 M Tris–HCl, pH 8) and lysed by three rapid
freeze/thaw cycles (three 5-min cycles). Cell debris was
then removed by centrifuging at 4 ꢂC, for 15 min at
15,000 rpm. Glycerol (final 10% v/v) and b-mercap-
toethanol (final 5 mM) were then added (final volume
75 ll) and the cell extracts were stored at ꢁ80 ꢂC until
assayed.
In CHO-b1 cells, Kd value for Alprenolol was 12.49 nM
and Bmax 2970 fmol/mg of protein (Fig. 6A) while in
CHO-b2-AR cells Kd value for Alprenolol was
0.50 nM and Bmax 540 fmol/mg of protein (Fig. 6B).
Kd and Bmax values have been obtained as a mean of
two experiments with samples in duplicate.
6.1.3. b3-Adrenoceptor activity by DELFIA cAMP-Eu
assay44. The DELFIA (Dissociation Enhanced Lantha-
nide Fluoro Immuno Assay) cAMP-binding assay was
performed according to technical data sheet by Perkin-
Elmer Life Science. The optimization of experimental
conditions (amount of cell, incubation times and other
parameters) is reported below.
One confluent plate of cells was trypsinised and resus-
pended in the above medium and cultured overnight at
a concentration of 50,000 cells/200 lL per well into 96-
well flat-bottomed plates. The medium was aspirated
from each well and replaced with 100 lL of preheated
(37 ꢂC) medium without serum. The plate was then
placed back in the CO2-incubator for 30 min at 37 ꢂC.
About 50 lL of 1 mM IBMX (3-isobutyl-1-methylxan-
thine), phosphodiesterase inhibitor was added to each
well. 50 lL of test compounds at different concentra-
tions (100 nM, 500 nM, 1 lM, 10 lM, 100 lM) were
then added to the wells and the plate was incubated
for 30 min at 37 ꢂC. Cells were then lysed and incubated
at room temperature for 5 min. The plate was immedi-
ately used for measuring cAMP levels or stored at
4 ꢂC until the assay was performed. Measure of the sam-
ples was carried out in Time Resolved Fluorometer
using 1420 Multilabel Counter Victor3, Perkin-Elmer.
The wavelengths were 340 nm and 615 nm in excitation
and emission, respectively.
6.2.3. Assays to determine CAT and b-galactosidase
activity. The CAT activity assay was performed as fol-
lows: 20 ll of cell lysate (prewarmed at 65 ꢂC for
10 min, to deactivate internal deacetylase enzymatic
activity) were added to 10 ll of 3.5 mg mlꢁ1 n-butyryl-
CoA, 5 ll (0.25 lCi) of [14C]-chloramphenicol and
65 ll of distilled water and incubated for 2 h at 37 ꢂC.
Reaction was blocked by adding 200 ll of the solution
xylene/2,6,10,14-tetramethylpentadecane (1:2 v/v). After
a vigorous vortexing and centrifugation for 5 min at top
speed, 150 ll of supernatant was transferred to scintilla-
tion vial in the presence of 5 ml of scintillation liquid,
and the relative radioactivity was measured by a b-coun-
ter. The b-galactosidase activity was measured as fol-
lows: 20 ll of cellular extracts were added to 750 ll of
reaction buffer consisting of 1 vol. of 2 mg mlꢁ1 ONPG
(o-nitrophenyl-b-galactopyranoside) and 3 vol. of ‘Z
buffer’ (10 mM potassium chloride, 1 mM magnesium
chloride, and 50 mM b-mercaptoethanol in phosphate
buffer). Reaction was performed at 37 ꢂC and blocked
by adding 200 ll of 1 M Na2CO3 when a typical yellow
colour became appreciable. Samples were incubated for
10 min at room temperature and then the absorbance at
420 nm (A420) was spectrophotometrically measured.
6.2. PPARa transactivation assay
6.2.1. Plasmids. The reporter construct pG5-CAT, con-
taining five copies of the high affinity binding site for
GAL4 (UAS) and used for CAT assay, was purchased

M. G. Perrone et al. / Bioorg. Med. Chem. 16 (2008) 2473–2488
2487
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Bodhankar, S. L.; Jindal, D. P.; Coumar, M. S.; Balar-
aman, R.; Bhardwaj, S. H. Pharmacology 2005, 74, 1.
17. Candelore, M. R.; Deng, L.; Tota, L.; Guan, X.-M.;
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Finally, the CAT activity was normalised to the b-galac-
tosidase activity.49
Acknowledgments
Work carried out under the framework of the Na-
tional Projects ‘Progettazione, Sintesi e Valutazione
Biologica di Nuovi Farmaci Cardiovascolari’ sup-
`
ported by the Ministero dell’Universita e della Ricerca
(MiUR, Rome), shared also with the University of
Bari and Istituto di Chimica dei Composti Organo-
Metallici (ICCOM-CNR, Bari). Thanks are also due
to Prof. Karl-Norbert Klotz (Institut fur Pharmakolo-
¨
gie und Toxikologie, Universita¨t Wurzburg, Germany)
¨
for his suggestions and supplying us CHO cells
expressing human cloned b1-adrenoceptor, and to
Prof. Susanna Cotecchia for her suggestions and
helpful text discussion.
18. Costa, T.; Cotecchia, S. Trends Pharmacol. Sci. 2005, 26,
618.
19. Strange, P. G. Trends Pharmacol. Sci. 2002, 23, 89.
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