α2-adrenoreceptors profile modulation. 2.1 Biphenyline analogues as tools for selective activation of the α2C-subtype

Article abstract of DOI:10.1021/jm0408215

A series of derivatives structurally related to biphenyline (3) was designed with the aim to modulate selectivity toward the α₂-AR subtypes. The results obtained demonstrated that the presence of a correctly oriented function with positive elec

Full text of DOI:10.1021/jm0408215

6160
J. Med. Chem. 2004, 47, 6160-6173
r2-Adrenoreceptors Profile Modulation. 2.1 Biphenyline Analogues as Tools for
Selective Activation of the r2C-Subtype
Francesco Gentili,† Francesca Ghelfi,† Mario Giannella,† Alessandro Piergentili,† Maria Pigini,*,†
Wilma Quaglia,† Cristian Vesprini,† Pierre-Antoine Crassous,‡ Herve´ Paris,‡ and Antonio Carrieri§
Dipartimento di Scienze Chimiche, Universita` degli Studi di Camerino, Via S. Agostino 1, 62032 Camerino, Italy, INSERM
Unit 388, Institut Louis Bugnard, IFR31, CHU Rangueil, 1 avenue Jean Poulhes, 31403 Toulouse, France, and Dipartimento
Farmaco-Chimico, Universita` degli Studi di Bari, Via E. Orabona 4, 70125 Bari, Italy
Received March 31, 2004
A series of derivatives structurally related to biphenyline (3) was designed with the aim to
modulate selectivity toward the R2-AR subtypes. The results obtained demonstrated that the
presence of a correctly oriented function with positive electronic effect (+σ) in portion X of the
ligands is an important factor for significant R2C-subtype selectivity (imidazolines 5, 13, 16,
and 19). Homology modeling and docking studies support experimental data and highlight
the crucial role for the hydrogen bond between the pyridine nitrogen in position 3 of 5 and the
NH-indole ring of Trp6.48, which is favorably oriented in the R2C-subtype, only.
Introduction
is limited by its potent agonist activity at R1-ARs, and
guanfacine15 display a significant preference for R2A-AR,
the selectivity of the available agonists is far from being
sufficient for a complete in vivo characterization of the
three R2-AR subtypes.
Adrenoreceptors (ARs) play a crucial role in the
regulation of a large panel of biological functions and
have been considered for many years as attractive
therapeutic targets for the treatment of various dis-
eases, such as hypertension, congestive heart failure,
asthma, depression, and glaucoma.2 Chemically and
pharmacologically speaking, ARs are membrane pro-
teins belonging to the superfamily of G-protein-coupled
receptors3-5 and are classified into three classes, namely,
R1-, R2-, and â-ARs,6 which are respectively coupled to
proteins Gq, Gi/o, and Gs. As a result of the combined
development of radioligand binding assays and molec-
ular biology techniques, it became evident that each
class of AR is heterogeneous. In particular, three
distinct R2-AR subtypes (namely R2A-, R2B-, and R2C-ARs)
that are encoded by different genes have been charac-
terized in different species.7 R2-AR subtypes exhibit a
high degree of homology and share common mechanisms
of signal transduction. They are all involved in the
control of the cardiovascular and central nervous sys-
tems, although they mediate different functions.8
So far, the attribution of a physiological function to a
given R2-AR subtype has, however, been of great dif-
ficulty in vivo, due to the lack of sufficiently subtype-
selective pharmacological tools. Indeed, while somewhat
The establishment of genetically engineered mice
lacking or overexpressing R2-AR subtypes16 has yielded
important information for understanding the subtype-
specific functions. Genetic manipulation has been of
great help, but at the same time it presents limitations
insofar that significant evolutionary changes can some-
times compensate for the loss of the targeted gene.
However, as recently reviewed,17 the examination of the
phenotype of these strains of mice demonstrated that
the R2A subtype is responsible for inhibition of neu-
rotransmitter release from central and peripheral sym-
pathetic nerves and for most of the centrally mediated
effects of R2-agonists, including hypotension,18 anesthe-
sia,19 sedation,19,20 antiepileptogenesis,21 and analge-
sia.19,20,22 Further data suggested that stimulation of the
postsynaptic R2A-AR increases the blood flow in the
prefrontal cortex and enhances the working memory
functions, as treatment with the R2A agonist guanfacine
significantly improves cognitive performance.23
The R2B-AR is primarily responsible for the initial
peripheral hypertensive responses evoked by the R2-
agonists 24 and takes part in the hypertension induced
by salt.25
subtype-discriminative antagonists exist, such as BRL
44408 and BRL 48962 for R2A
and several N-pyrimidinyl-4-aminobenzenesulfonamide
9,10
;
imiloxan, ARC-239,
Clarification of the physiological role of R2C subtype
proved more difficult.16 Despite a rather wide distribu-
tion in the central nervous system, its role did not
appear critical in the mediation of the cardiovascular
effects of nonselective R2-agonists.24 Its participation has
been suggested in the hypothermia induced by dexme-
detomidine and in the hyperlocomotion induced by
D-amphetamine.26 Another potentially important re-
sponse mediated by the R2C-AR is constriction of cutane-
ous arteries, leading to a reduction in cutaneous blood
flow.27 Recent studies carried out on double knockout
mice have suggested that R2C-AR is also expressed at
the presynaptic level where, together with R2A, it
11-13
derivatives for R2B
;
and rauwolscine and MK 912
6,14
for R2C
,
although with significant limitations both
relating to poor selectivity and affinity for other neu-
rotransmitter receptors (e.g. R1-ARs for ARC-239 and
various 5-HT subtypes for rauwolscine), no strictly
selective agonists are available yet for R2B- and R2C-ARs.
Furthermore, even though oxymethazoline, whose use
* To whom correspondence should be addressed. Phone: +39 0737
402237. Fax: +39 0737 637345. E-mail: maria.pigini@unicam.it.
† Universita` degli Studi di Camerino.
‡ Institut Louis Bugnard.
§ Universita` degli Studi di Bari.
10.1021/jm0408215 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/10/2004

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6161
Chart 1
actively participates in the control of neurotransmitter
release. However, the two subtypes regulate this process
differently. Indeed, while R2A-AR is particularly efficient
at high stimulation frequencies, R2C-AR acts rather at
low stimulation frequencies.28 Moreover, it has been
suggested that R2C subtype participates in the modula-
tion of motor behavior and the memory processes.29,30
Other central effects triggered by this subtype include
also the startle reflex and aggression,31 response to
stress, and locomotion.32 Last, it was recently pointed
out that the R2C-AR may contribute to R2-agonist-
mediated spinal analgesia and adrenergic-opioid syn-
ergy.33
Previous studies from our group have demonstrated
that modest structural modifications within imidazoline
molecules resulted in significant alteration in their
affinity, both with regard to different receptor systems
and inside the same system, with resultant enhanced
selectivity.34-37
In the context of the R2-AR system, a very interesting
result was obtained by modifying the basic structure of
2-phenoxymethlylimidazoline (1) by the successive in-
troduction of a methyl substituent in the oxymethylene
bridge (compound 2) and of a phenyl group in the ortho
position of the aromatic ring (2-[1-(biphenyl-2-yloxy)-
ethyl]-4,5-dihydro-1H-imidazole, compound 3) (Chart 1).
The name of biphenyline has now been given to com-
pound 3.
very different affinity profile toward I2-imidazoline
binding sites (I2-IBS), with compound 1 having high
affinity (pKi I2 ) 9.05) and compound 2 low affinity (pKi
I2 ) 5.57).36 The methyl group on the bridge reduced
the R1-adrenergic intrinsic activity and moderately
enhanced the R2-adrenergic potency (methyl binding
pocket recognition)38 but overall markedly decreased the
I2-IBS affinity.36,37 Interestingly, compounds 1 and 2
also displayed significant R2-adrenergic selectivity to-
ward the I1-IBS.37
Since the structural characteristics of numerous
ligands of R2-ARs (such as clonidine, oxymethazoline,
idazoxan, and rilmenidine)39,40 are compatible with the
architecture of the imidazoline binding sites, the reduc-
tion of their affinity toward IBS can be useful for
improving the selectivity of drugs that can potentially
be employed for treating various diseases.
As expected, biphenyline (3) exhibited an interesting
R2-adrenergic selectivity profile [pKi R2 ) 7.91; pKi I1 )
6.90 (unpublished result); pKi I2 ) 5.79], but rather
surprisingly, it behaved as an efficient R2-agonist in in
vitro assays on isolated rat vas deferens. It is probable
that the phenyl substituent introduced in the ortho
position of the aromatic ring is able to form π-π
stacking interactions with the aromatic cluster of the
binding cavity, therefore playing an important role in
R2-AR activation. Moreover, the biphenyline eutomer
(S)-(-) displayed enhanced and long-lasting antinoci-
ceptive potency, undoubtly mediated by the R2-AR since
Imidazolines 1 and 2 both behaved as antagonists at
R2-ARs and as weak agonists at R1-ARs but exhibited a

6162 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
Scheme 1a
a Reagents: (a) CH3ONa, CH3OH/H2NCH2CH2NH2; (b) H2, Pd/C; (c) HCl(g), CH3OH/H2NCH2CH2NH2: (d) H2NCH2CH2NH2/∆; (e)
H2NCH2CH2NH3+‚pTSA/∆.
Scheme 2a
a Reagents: (f) (CH3)3Al/dry toluene: H2NCH2CH2NH2/∆; (g) NaH, N-vinyl-2-pyrrolidinone; (h) HSCH2CH2NH2, Ph3P, Et3N/∆; (i) oxalyl
chloride, Et3N/HOCH2CH2NH2; SOCl2; Et3N.
it was competitively blocked by the selective R2-
antagonist, RX 821002.36
pionic acid methyl ester36 by treatment with ethylene-
diamine in the presence of trimethylaluminum or by
treatment with 1-vinyl-2-pyrrolidinone in the presence
of NaH, respectively. Reaction of 2-(biphenyl-2-yloxy)-
propionic acid41 with 2-aminoethanethiol in the presence
of triphenylphosphine provided compound 25, and that
with ethanolamine in the presence of oxalyl chloride and
triethylamine yielded compound 24 (Scheme 2).
The intermediate nitriles were prepared according to
Scheme 3 starting from 2-chloropropionitrile and the
suitable phenols for 27-3042,43 or thiophenol for 44, from
2-(2-bromophenoxy)propionitrile and the suitable com-
mercially available boronic acids in the presence of
tetrakis(triphenylphosphine)palladium(0) for 33-42,
and from 2-(2-bromophenylsulfanyl)propionitrile (44)
and phenylboronic acid for 43. Finally, nitriles 31 and
32 were prepared as a trans/cis mixture (ratio 6:3) from
2-biphenylcarboxaldehyde and chloroacetonitrile in the
presence of t-ButOK. The two isomers 31 and 32 were
separated by flash chromatography. The stereochemical
relationship between the CN function at position 2 and
The biological activity and the pharmacological profile
of biphenyline therefore encouraged us to synthesize a
new series of derivatives with the aim to improve both
potency and R2-AR subtype selectivity. In the present
study, chemical modifications have been designed at the
level of the portions X, Y, and Z of the lead molecule 3.
The properties of these new compounds 4-19, and 21-
26 (Chart 1) were evaluated by binding and functional
studies using human R2-AR subtypes expressed in
Chinese hamster ovary (CHO) cells.
Chemistry
Compounds 4-7, 9-17, 19, and 21-23 were synthe-
sized according to standard methods by condensation
of suitable nitriles with ethylenediamine. Amino deriva-
tive 18 was obtained through catalytic hydrogenation
of 16 over Pd/C (Scheme 1).
Imidazolines 8 and 26 were obtained from 2-(2-thio-
phen-3-yl-phenoxy)- (46) and 2-(biphenyl-2-yloxy)pro-

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6163
Scheme 3a
a Reagents: (a) 2-chloropropionitrile, K2CO3, DME; (b) t-BuOK, t-BuOH/chloroacetonitrile; (c) arylboronic acid, K2CO3, Pd[(C6H5)3P]4.
the 3-biphenyl group was deduced from the coupling
constants of the hydroxy atoms at the same positions.44
Pharmacology. The pharmacological profile of com-
pounds was investigated using stable clones of Chinese
hamster ovary cells expressing the human R2-AR sub-
types individually.45 Binding experiments were carried
out on crude membrane preparations using [3H]RX
821002 as radioligand.46 The IC50 values were deter-
mined by nonlinear regression analysis of competition
data using the Graphpad Prism computer program. Ki
values were calculated from the equation of Cheng and
(∼20%), bovine rhodopsin still represents a reliable
template once a transmembrane receptor has to be
modeled; it is in fact the only eukaryotic G-protein-
coupled receptor (GPCR) whose typical folding, repre-
sented by a seven R-helix domain spanning through the
cell membrane connected by extracellular and intracel-
lular loops, has been experimentally determined; in
addition, the percentage of identity of bovine rhodopsin
with the R2-ARs rises up to ∼30% when considering only
the transmembrane domain. The homology modeling
tool MODELLER52 (ver. 6.2) was used to assemble and
refine the seven TM framework. Loops were not taken
into account, mainly since they do not directly interact
in the binding of ligands showing an agonist profile, at
least in the case of the aminergic receptors family, and
also to avoid a very hard risking phase in the homology
building procedure due to the low sequence identity with
the selected template. The Cartesian coordinates of the
CR atoms relative to the R2-ARs were copied from the
corresponding ones of the selected template (PDB code
1gzm, chain A) according to the sequence alignment
reported in Figure 1.
47
Prusoff and reported as pKi ( SEM.
Agonist potency, defined as the concentration that
produces 50% of the maximum response, is expressed
as pEC50 and was determined by use of a cytosensor
microphysiometry instrument on the above-mentioned
CHO cells. With this instrument, the rate of extracel-
lular acidification after stimulation of the receptor by
the agonist was measured.48 The intrinsic activity (ia)
of each compound is expressed as the fraction of the
maximum response elicited by (-)-noradrenaline (ia )
1).
Modeling. Numbering of Residues. Residues were
numbered according to their positions in the human R2-
AR sequences, as reported in the SwissProt sequence
database.49 Also residues were indexed according to the
numbering proposed by Ballesteros and Weinstein.50
The transmem annotation of the sequence file was taken
into account as a definition of the amino acids compris-
ing the seven transmembrane helices (TMs).
Model Building. The TM models of each R2-AR
subtype were built by starting from the X-ray structure
of the bovine rhodopsin, which has been recently solved
by Schertler et al.51 Despite low sequence identity
between the selected template and the three receptors
Five hundred different 3D structures were generated
using the slow molecular dynamic optimization schedule
refine_3. To ensure a complete investigation of the
tertiary structure, side chains were fully randomized
in a range of (180°. The models obtained were then
superposed and clustered according to their CR position
by means of the NMRCLUST algorithm;53 no predefined
root mean square threshold was applied during the
clustering procedure. For each R2-AR subtype, the model
exhibiting the lowest potential density function value,
as scored by MODELLER, was selected as input struc-
ture in the docking studies, since it was also a member
of the most populated cluster. To better assess the

6164 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
Figure 1. Pairwise alignment of the bovine rhodopsin sequence and the human R2-adrenergic receptors. The conserved residues
are in red boxes. Bars indicate R-helices and the â-sheet of the selected template. Numbering is referred to rhodopsin.
quality of the chosen structures, the stereochemical
parameters were checked by means of the PROCHECK
software;54 in all three cases, the percentage of the
residues lying in the most favored regions of the
Ramachandran plot was more than 90%.
range to the agonist structure were allowed to move
(Table 4).
Computing. MODELLER calculations were run on
a DELL Pentium IV 2.56 GHz, whereas docking studies
were carried out on a SGI Origin300 R14000.
Molecular Docking. The molecular structure of
compound 5 was built using standard bond lengths and
angle valences implemented in the latest Flo+ software
library.55 The imidazoline ring was protonated and the
absolute (S) configuration was arbitrarily selected in
agreement with the highest biological activity of the
same enantiomer of the biphenyline (3), as previously
measured.36 In fact, docking performed using the abso-
lute (R) configuration highlighted considerably different
interactions for the enantiomers, in agreement with the
measured biological data. The mixed AMBER/MM2
force field of the same software package was used for
geometry optimization and energy calculation. Flexible
docking of compound 5 to the R2-ARs binding site was
performed first by means of the DYNDOCK module of
Flo+. This algorithm operates by randomly rotating and
translating the ligand within the receptor binding site,
perturbing at the same time its internal geometry with
respect to the dihedral angles, according to the Monte
Carlo algorithm. At each step, a short dynamic run
followed by a Pollak-Ribiere conjugate gradient mini-
mization was performed on the receptor-ligand com-
plex. A single distance constraint between the proto-
nated nitrogen of the imidazoline ring and the carboxy
group of Asp3.32 was applied, because site-directed
mutagenesis experiments have previously demonstrated
that this residue is responsible for the anchoring of
natural agonists (i.e. noradrenaline) to the binding
site.56 In our case, the ligand was initially placed in the
receptor binding cavity located within TM3, -5, -6, and
-7. The receptor-ligand complex was first minimized
and then subjected to 100 steps of flexible dynamic
docking performed keeping the CR trace of the receptor
fixed to the initial position. The receptor-ligand com-
plex having the best energy of binding according to the
Flo+ scoring function was then selected for a further
3000 steps of Monte Carlo docking simulation performed
with the MCDOCK+ module. This time, only the ligand
and the amino acid residues lying in a closer distance
Results and Discussion
Tables 1-3 report the affinity values, potency, and
intrinsic activity of the new compounds and biphenyline
(3); compound 2036 was also included in the present
study for a more complete structure-activity relation-
ship investigation. None of the compounds were evalu-
ated for their antagonist activity.
The results show that biphenyline (3) behaves as a
good agonist with comparable potency and affinity
toward the R2A and R2C subtypes but as a partial agonist
with modest intrinsic activity toward the R2B-subtype.
Since the phenyl group in the ortho position of the
aromatic ring was already hypothesized as being re-
sponsible for the biphenyline R2-agonist property,36 the
main objective of the present study was to verify the
consequences of modifications affecting this portion of
the molecule. These modifications included (i) isosteric
substitution with the pyridine nucleus (compounds 4-6)
and thiophene nucleus (compounds 7-8) and (ii) intro-
duction in the ortho, meta, and para positions of the
phenyl group of substituents with different σ and π
contributions (compounds 9-19) (modifications in X,
Chart 1).57 All the new derivatives exhibited pKi values
similar to that of biphenyline and generally diplayed
higher affinity for R2A or R2C than for R2B subtype. The
most interesting point was that they showed consider-
able and selective modulation of their functional activity
toward the different subtypes (Table 1).
The introduction of the methyl substituent in ortho,
meta, and para positions (for which the -σ and +π
condition was verified) led to derivatives 9-11. Al-
though likely due to an incompatible steric hindrance,
none of the three compounds was active at R2A- or R2B
-
AR, but all three behaved as partial agonists with weak
potency at R2C-AR, suggesting agonist selectivity for this
subtype with respect to the others.
The effects produced by introducing the hydroxyl
group were more dramatic. In fact, whereas the ortho

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6165
Table 1. Potency (pEC50a), Intrinsic Activity (iab), and Binding Data (pKic) of Compounds 3-19 on Human R2-AR Subtypes
a Determined by applying the cytosensor microphysiometry system48 to study the three human R2-AR subtypes, R2A, R2B, R2C, expressed
in Chinese hamster ovary (CHO) cells and to assess its potential in the quantitative monitoring of agonist activity. The full agonist
(-)-noradrenaline was used to define agonist efficacy (ia). NA ) not active (ia < 0.3). b Intrinsic activity (ia) is the maximum effect obtained
with the agonist under study, expressed as percentage of (-)-noradrenaline taken as equal to 1. c Binding assays using [3H]RX 821002
were performed with membrane preparations from the same cell models.
and para derivatives 12 and 14 (-σ and -π) were
functionally inactive at all three R2-AR subtypes, the
m-hydroxy derivative 13 (+σ and -π) behaved as a full
agonist with intrinsic activity equivalent to that of (-)-
noradrenaline toward the R2C subtype (pEC50 R2C ) 7.33,
ia ) 1.15). It should be noted that compound 13 was
totally inactive at R2A subtype and produced only a weak
and partial activation of the R2B-AR.
Also interesting were the effects of nitro substitution
(+σ and -π). Indeed, whereas the ortho and para

6166 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
Table 2. Potency (pEC50a), Intrinsic Activity (iab), and Binding Data (pKic) of Compounds 20-23 on Human R2-AR Subtypes
a-c See Table 1.
Table 3. Potency (pEC50a), Intrinsic Activity (iab), and Binding Data (pKi c) of Compounds 24-26 on Human R2-AR Subtypes
R2A
R2B
R2C
compd
Z
pKi
5.09 ( 0.04
<5
5.09 ( 0.04
pEC50
pKi
pEC50
pKi
pEC50
24
25
26
-O-
NA
NA
NA
5.00 ( 0.09
NA
NA
NA
5.23 ( 0.01
NA
NA
NA
-S-
<5
<5
-CH2-
5.00 ( 0.02
5.23 ( 0.09
a-c See Table 1.
Table 4. Residues Defining the Binding Site in the Human R2-ARs
domain
R2A
R2B
R2C
Asp113, Val114, Phe116
Cys117, Thr118
Asp92, Val93, Phe95
Cys96, Thr97
Asp131, Val132, Phe134
Cys135, Thr136
TM3
TM5
Ser200, Ser201
Ser176, Ser177,
Ser214, Ser215
Ser204, Phe205
Ser180, Phe181
Ser218, Phe219
Phe383, Trp387, Phe390
Phe391, Phe392
Phe412, Gly415, Tyr416
Phe380, Trp384, Phe387
Phe388, Phe389
Phe412, Gly415, Tyr416
Phe391, Trp395, Phe398
Phe399, Phe400
Phe423, Gly426, Tyr427
TM6
TM7
isomers 15 and 17 were not able to activate any of the
R2-AR subtypes, the meta isomer 16 behaved as a
partial agonist with substantial activity at R2C subtype
(pEC50 R2C ) 7.00, ia ) 0.60) but was inactive toward
R2A and R2B subtypes.
Of the pyridine derivatives, the two isomers 4 and 6
were inactive at all subtypes. By contrast, the regio-
isomer 5, in which the nitrogen atom occupies a position
analogous to that of the nitro group in the meta position
of 16 (the nitrogen atom of the pyridine ring and the
nitro group of the aromatic ring possess polarity and
similar electron-withdrawing effects), was a full agonist
at the R2C-AR (pEC50 R2C ) 7.30, ia ) 1) but exhibited
lower potency and intrinsic activity toward R2A (pEC50
R2A ) 6.50, ia ) 0.50) and R2B (pEC50 R2B ) 6.00, ia )
0.50) subtypes.
The agonist potency of compounds 5 and 16 was
determined also as a function of their capacity to inhibit
forskolin-induced cAMP accumulation in the CHO cells
expressing R2A- or R2C-AR.58 The pEC50 values were
fairly similar to those obtained from the measurement
of medium acidification (Compound 5: pEC50 R2A ) 6.23,
ia 0.50; pEC50 R2C ) 7.40, ia ) 1. Compound 16: R2A
NA; pEC50 R2C ) 6.80, ia ) 0.60.).
To explain this selectivity and taking as model the
pyridine derivative 5, we hypothesized that the nitrogen
atom, equipped with a good density of negative charge,
enabled a polar interaction with an electrophilic site of
the receptor protein. In other words, the activation of
the R2C-AR was possible solely when the ligand dipole
was correctly oriented (nitrogen in position 3 but not in
2 or 4). Therefore, the introduction of chemical functions

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6167
driving force in the drug-receptor interaction, was
ensured only in the presence of the imidazoline nucleus.
In this nucleus, the conjugated acid generated as a
consequence of protonation proved to be stabilized by
two resonance structures having the same energy
content. As already known, the contribution to reso-
nance stabilization of oxygen, sulfur, or carbon atoms
to the conjugated acid analogues is more scanty. That
may explain the incapacity of the derivatives with
oxazoline (24), thiazoline (25), and pyrroline (26) nuclei
to bind any of the three R2-AR subtypes (Table 3).
Homology modeling and docking studies with com-
pound 5 were undertaken to support our hypothesis.
The 3D structure of a receptor or protein built by means
of homology modeling is always affected by the intrinsic
uncertainty deriving from the several theoretical as-
sumptions and methodological approximations that such
a kind of study determines. To assess the quality of any
model, it is then essential to examine its performance
when the binding of ligands with high affinity or efficacy
is simulated. Therefore, to validate our receptor models,
the binding of compound 5 was performed by means of
fully automated flexible docking algorithms, due to the
good R2C-selectivity that this agonist expresses toward
the R2A- and R2B-AR subtypes. It is our opinion that if
the overall spatial arrangement of a modeled receptor,
or at least of its binding site, is correct, then a docking
study should be able to detect a proper binding mode
for efficacy ligands, showing most of the experimentally
known favorable interactions, especially the ones high-
lighted by site-directed mutagenesis data. Moreover, the
better the quality of the models, the higher should be
the capability of those models to take into account and
properly score the differences, either only qualitatively
or also quantitatively, in terms of the experimental data
(i.e., affinity or efficacy). To address this topic, the
structural features of the complexes obtained by the
docking of compound 5 were carefully analyzed and
evaluated.
Most modeling studies of aminergic receptors predict
that the agonist binding site is located within the water-
accessible binding site crevice located within TM3, TM5,
TM6, and TM7. Thus our docking studies, to be valu-
able, should at least fulfill this requirement.
Relation of biologic activity to molecular orientation
is simpler when affinity, determined by radioligand
binding assays, is used, rather than intrinsic activity.
It is quite difficult to model how agonist binding is
converted into receptor activation. Nevertheless, the
receptor-ligand complexes predicted by our model were
able to reproduce some basic interactions that stabilize
binding and possibly represent the initial step of the
receptor activation process depicted in Figure 3.
Figure 2. Stimulation of extracellular acidification in CHO
cells stably expressing the human R2C-adrenergic subtype by
(-)-noradrenaline (9), biphenyline (2), 5 (1), 10 (b), 13 (3),
16 ((), 18 (0), 19 (4). Data points with error bars represent
the mean ( SEM of three to six separate experiments.
with a positive electronic effect (+σ) in position 3 of
portion X (Chart 1) can lead to efficient and selective
agonists for the R2C subtype (compounds 5, 13, and 16)
(Figure 2). Confirmation of this view was provided by
compounds 18 and 19. In fact, the m-fluoro derivative
19 (+σ and +π) proved to be a full agonist at R2C (pEC50
R2C ) 7.22, ia ) 1.15), a partial agonist at R2A (pEC50
R2A ) 6.98, ia ) 0.58), but totally inactive at R2B
-
subtype. On the other hand, the m-amino derivative 18
(-σ and -π) was only a modest partial agonist unable
to discriminate between R2A and R2C (Figure 2). Thus,
the positive or negative values of π do not appear
determinant for the biological profile of the ligands.
The thiophene nucleus, considered to be an electron-
rich heterocycle, possesses an aromaticity comparable
with that of the phenyl ring; however, its reduced size
might make more efficient the π-π aromatic stacking
interactions. Consequently, derivatives 7 and 8 behaved
similarly to biphenyline (3). They were good agonists
for both the R2A and R2C subtypes (Compound 7: pEC50
R2A ) 7.38, ia ) 0.80; pEC50 R2C ) 7.23, ia ) 0.80.
Compound 8: pEC50 R2A ) 7.12, ia ) 0.70; pEC50 R2C
7.03, ia ) 0.85.).
)
In this series of compounds, the study of the bridge
(portion Y, Chart 1) showed its critical role for the
interaction with R2-ARs. Bridge rigidity in an epoxidic
ring, a modification resulting in the blockade of free
rotation and in the alteration of the correct spatial
arrangement of aromatic portion and imidazoline nucleus
(compounds 22 and 23) (Table 2), drastically compro-
mised the affinity and efficacy of the lead. Of the two
geometric isomers, only the cis derivative 22 weakly
activated the R2A- and R2C-AR subtypes. In the same
manner, the isosteric substitution of the oxygen atom
of the bridge was rather prejudicial for activity. Indeed,
the amino derivative 2036 exhibited similar ia but lower
pKi and pEC50 than biphenyline, while the thio deriva-
tive 21 was not active at all. Moreover, we recently
verified for structurally correlated compounds that the
wholly carbon chain of the bridge tended to favor
selectivity for IBS with respect to R2-ARs.37
First of all, the salt bridge between the highly
conserved Asp3.32 and the charged nitrogen of the
agonist is observed; it is well-known that the same kind
of interaction takes place when natural ligands (i.e.,
catecholamines) bind to the receptor cavity. It has also
been proposed59,60
that the disruption of an ionic lock,
formed by the same aspartate and some negatively
charged residues located in TM6 or TM7, is a crucial
switch governing receptor activation.
In addition to this, the phenoxy ring of compound 5
is pointing toward TM5, with the carbon atoms in the
Finally, as shown by compounds with modifications
in portion Z, the ionic bond between the carboxylate
anion of an aspartic acid residue of the protein and the
protonated nitrogen of the ligand, which is likely the

6168 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
Table 5. Binding Energy for Compound 5 As Determined by
the Docking Studies
free
estimated
energy
H bond
(kcal/mol)
receptor
pI
(kcal/mol)
R2A
R2B
R2C
5.4
5.6
6.5
-7.34
-7.65
-8.84
-0.96
-1.22
-1.60
with some residues of this aromatic cluster, some
structural arrangements might occur that in turn would
lead to receptor activation. It can be observed above that
all R2C, but also R2A, subtypes present a more puckered
aromatic cluster with respect to the R2B-subtype; prob-
ably this means that π-π aromatic stacking between
portion X (provided that its chemical nature allows this)
and side chains of Phe6.51, Phe6.52, and Phe6.53 takes
place, to a large extent determining efficient R2A- and
R
2C-agonism (for example, compounds 3, 7, and 8).
Very interestingly, in the same zone the docking of
compound 5 to the three R2-AR subtypes shows the most
significant differences, giving some valuable clues that
might help interpretation of the observed selectivity of
this compound. In fact, the most interesting observation
regards the interaction between the pyridine nitrogen
in position 3 of 5 and the NH-indole ring of Trp6.48,
the latter being differently oriented in the three recep-
tor-ligand complexes; while in the case of R2A and R2B
this residue is pointing toward TM2, in that of R2C it is
interacting strongly with the ligand by means of a
hydrogen bond that is not present when the same
agonist binds to the other two subtypes. This evidence
is also supported by the free energy of binding and the
hydrogen-bond contribution, as measured by the Flo+
scoring function (Table 5). The same kind of interaction
can be hypothesized for OH, NO2, and F substituents
in the meta position of other R2C-selective agonists, 13,
16, and 19, respectively.
According to our findings, a possible explanation for
the R2C selectivity of compound 5 might be given. Since
the residues comprising the binding cavity are highly
conserved in all three receptor subtypes, a difference
in terms of accessibility and/or shape (mainly close to
the aromatic cluster controlling the activation process)
might be responsible for the observed selectivity. This
is also confirmed by the nature of the extracytoplasmatic
end of TM6, which in our model is a right-handed
R-helix for R2A- and R2B- but a left-handed one in the
case of R2C-AR subtypes.
Although our models need better refinement, it is
worth noting that some structural features, able to
justify the differences in terms of efficacy among the
three R2-AR subtypes, have been properly identified and
highlighted. This study also supports the hypothesis
that the possible interactions of portion X of the above
agonists with a residue or residues in the TM6 aromatic
cluster might be the initiating event leading to activa-
tion of the GPCRs. For this purpose, and to gain more
insight into this very interesting biological process,
exhaustive molecular dynamics simulation on the
ligand-receptor complexes is already being planned.
Figure 3. Compound 5 docked in the receptor binding site
(green, R2A; yellow, R2B; purple, R2C). Side chains of the residues
interacting with the agonist are displayed to help interpreta-
tion.
meta and para positions facing a motif made by a pair
of serines common to all the adrenoceptors (Ser5.42 and
Ser5.46). As demonstrated by mutagenesis data61,62 and
by previous docking studies performed by some of us,63
these polar residues interact through a hydrogen-bond
network with the catecholic hydroxyls of noradrenaline.
In the case of compound 5, although the same kind of
interactions cannot take place, due to the absence of
proper substituents on the phenoxy moiety, it is anyway
clear that a reliable binding mode is achieved for this
highly active agonist. As a consequence of this, portion
X (Chart 1) is therefore merged in a broad lipophilic
zone made up by Phe6.44, Trp6.48, Phe6.51, Phe6.52,
and Phe6.53 (Table 4 and Figure 3). According to the
finding of Ballesteros et al.,64 in this zone of a GPCR a
cluster of highly conserved aromatic residues faces the
binding site cavity; when an agonist binds or interacts
Conclusion
From the results of the present study, it emerges that
among the ligands bearing the basic structure of 4,5-

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6169
pressure (45 psi) using Pd/C as catalyst. Following catalyst
removal, the evaporation of the solvent gave the free base that
was transformed into the oxalate salt (Table 6).
dihydro-2-(1-phenoxyethyl)-1H-imidazole, the aromatic
nature of the portion X (Chart 1) is important for
activating the R2A- and R2C-AR subtypes (compounds 3,
7, and 8). The incompatible steric hindrance and/or the
decrease in aromaticity resulting from the introduction
of deactivating chemical functions significantly weaken
the efficacy of the interactions to the point of producing
compounds that are totally devoid of adrenergic activity.
However, a correct spatial orientation of the dipole
originated by such functions (pyridine nitrogen in
position 3 of 5 and OH, NO2, and F in the meta position
of 13, 16, and 19, respectively), allowing the formation
of a hydrogen bond with a corresponding receptor
function, results in efficient and selective activation of
the R2C-AR subtype. Modeling studies support the
experimental data and highlight this bond between the
pyridine nitrogen in position 3 of 5 and the NH-indole
Method C. 2′-[1-(4,5-Dihydro-1H-imidazol-2-yl)ethoxy]-
biphenyl-4-ol (14). HCl was bubbled through a stirred and
cooled (0 °C) solution of 40 (0.69 g, 2.88 mmol) and MeOH (0.24
mL, 5.76 mmol) in dry CHCl3 (5 mL) for 45 min. After 12 h at
0 °C, dry ether was added to the reaction mixture to give the
intermediate imidate, which was filtered. This solid (0.154 g,
0.50 mmol) was added to a cooled (0 °C) and stirred solution
of ethylenediamine (0.041 mL, 0.62 mmol) in absolute EtOH
(2.4 mL). After 1 h, concentrated HCl (a few drops) in absolute
EtOH (1.2 mL) was added to the reaction mixture, which was
stored overnight in the refrigerator. It was then diluted with
absolute EtOH (2 mL) and heated at 75 °C for 5 h. After
cooling, the solid was collected and discarded, and the filtrate
was concentrated and filtered again. The filtrate was evapo-
rated to dryness to give a residue, which was taken up in
CHCl3 (20 mL), washed with 2 N NaOH, and dried over
Na2SO4. Removal of the solvent gave a residue that was
purified by flash chromatography and then transformed into
the hydrochloride salt (Table 6).
ring of Trp6.48, which is favorably oriented in the R2C
-
subtype, only.
In conclusion, we have confirmed that it is possible,
by conservative structural modifications, to modulate
the selectivity profile of ligands inside a receptor system.
The imidazoline derivatives 5, 13, 16, and 19, which
exhibit significant selectivity for the R2C-AR subtype,
constitute an important basis for future work and may
be useful for characterizing functions specifically gov-
erned by this subtype.
Method D. 2-[1-(3′-Fluorobiphenyl-2-yloxy)ethyl]-4,5-
dihydro-1H-imidazole (19). A mixture of ethylenediamine
(3.4 mL) and 42 (1.2 g, 4.97 mmol) was heated at 115 °C for 6
h. After cooling and addition of H2O, the mixture was extracted
with CHCl3. The organic layer was washed with H2O, dried
over Na2SO4, and evaporated to give a residue that was
purified by flash chromatography and then transformed into
the oxalate salt (Table 6).
Similarly, compounds 15 and 17 (Table 6) were obtained
from the suitable nitriles.
Experimental Protocols
Method E. 2′-[1-(4,5-Dihydro-1H-imidazol-2-yl)ethoxy]-
biphenyl-3-ol (13). A mixture of 2-aminoethylammonium
toluene-p-sulfonate (11.38 g, 49 mmol) and 39 (1.17 g, 4.9
mmol) was heated at 200 °C for 2 h. After cooling and addition
of H2O, the solution was extracted with CHCl3. The organic
layer was washed with H2O, dried over Na2SO4, and evapo-
rated to give a residue that was purified by flash chromatog-
raphy and transformed into the oxalate salt (Table 6).
Method F. 2-[1-(2-Thiophen-3-ylphenoxy)ethyl]-4,5-di-
hydro-1H-imidazole (8). A solution of ethylenediamine (1.29
mL, 19.30 mmol) in dry toluene (6.7 mL) was added dropwise
to a mechanically stirred solution of 2 M trimethylaluminum
(9.6 mL, 19.30 mmol) in dry toluene (16 mL) at 0 °C in nitrogen
atmosphere. After being stirred at room temperature for 1 h,
the solution was cooled to 0 °C and a solution of 46 (2.54 g;
9.64 mmol) in dry toluene (11.5 mL) was added dropwise. The
reaction mixture was heated to 110 °C for 3 h, cooled to 0 °C,
and quenched cautiously with MeOH (4.5 mL) followed by H2O
(1 mL). After addition of CHCl3 (35 mL), the mixture was left
for 30 min at room temperature to ensure the precipitation of
the aluminum salts. The mixture was filtered and the organic
layer was extracted with 2 N HCl. The aqueous layer was
made basic with 10% NaOH and extracted with CHCl3. The
organic layer was dried over Na2SO4, filtered, and evaporated
to give the free base, which was purified by flash chromatog-
raphy and then transformed into the oxalate salt (Table 6).
Method G. 5-[1-(Biphenyl-2-yloxy)ethyl]-3,4-dihydro-
2H-pyrrole (26). Sodium hydride (0.66 g of a 50% dispersion
in mineral oil, 13.73 mmol) contained in a 100 mL flask was
washed with three 5-mL portions of hexane. The flask was
fitted with a reflux condenser, flushed with nitrogen, and
charged with 20 mL of dry THF. A solution of 2-(biphenyl-2-
yloxy)propionic acid methyl ester36 (2.64 g, 10.3 mmol) and
N-vinylpyrrolidinone (1.27 g, 11.4 mmol) in 3 mL of dry THF
was added in one portion. The mixture was stirred and, after
several minutes, an exothermic reaction with gas evolution
started. When the evolution of gas had ceased, the mixture
was heated under reflux for 1 h and then cooled to room temp-
erature. Concentrated HCl (1.72 mL), diluted with 5.72 mL of
H2O, was added, and the mixture was heated under reflux
overnight. After cooling, the solution was made basic by the
addition of concentrated aqueous NaOH (ice bath cooling). The
Chemistry. Melting points were taken in glass capillary
tubes on a Buchi SMP-20 apparatus and are uncorrected. IR
and NMR spectra were recorded on Perkin-Elmer 297 and
Varian EM-390 instruments, respectively. Chemical shifts are
reported in parts per million (ppm) relative to tetramethylsi-
lane (TMS), and spin multiplicities are given as s (singlet), d
(doublet), t (triplet), q (quartet), or m (multiplet). IR spectral
data (not shown because of the lack of unusual features) were
obtained for all compounds reported and are consistent with
the assigned structures. The microanalyses were performed
by the Microanalytical Laboratory of our department. The
elemental composition of the compounds agreed to within
(0.4% of the calculated value. Chromatographic separations
were performed on silica gel columns (Kieselgel 40, 0.040-
0.063 mm, Merck) by flash chromatography. The term “dried”
refers to the use of anhydrous sodium sulfate. Compounds
were named following IUPAC rules as applied by Beilstein-
Institut AutoNom (version 2.1), software for systematically
naming organic chemicals.
Preparation of imidazolines 4-23 and bioisosters 24-
26. Method A. 2-{2-[1-(4,5-Dihydro-1H-imidazol-2-yl)-
ethoxy]phenyl}pyridine (4). A mixture of 27 (0.67 g, 3.0
mmol), sodium methoxide (0.014 g, 0.26 mmol), and MeOH (2
mL) was stirred for 18 h. On cooling to 0-10 °C, a solution of
ethylenediamine (0.22 mL, 3.36 mmol) in MeOH (1 mL) was
added dropwise with stirring; after a few minutes a solution
of HCl in MeOH (1.043 mL of 3 N solution, 3.13 mmol) was
added dropwise and the mixture was allowed to warm to room
temperature. After 78 h, the mixture was made slightly acid
with methanolic HCl and filtered. The filtrate was evaporated
and the residue was taken up in H2O. The solution was
basified with 2 N NaOH and extracted with CHCl3. The
organic layer was dried over Na2SO4 and evaporated to give a
residue that was purified by flash chromatography (Table 6).
The free base was transformed into the oxalate salt, whose
physicochemical properties are reported in Table 6.
Similarly, compounds 5-7, 9-12, 16, and 21-23 (Table 6)
were obtained from the suitable nitriles.
Method B. 2′-[1-(4,5-Dihydro-1H-imidazol-2-yl)ethoxy]-
biphenyl-3-ylamine (18). Compound 16 (0.1 g, 0.32 mmol)
was hydrogenated in MeOH for 6 h at room temperature under

6170 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
Table 6. Physicochemical Properties of Oxalate, Sulfate, and Hydrochloride Salts of Imidazolines 4-26
eluent
yield,
%
mp, °C
(recryst solvent)
compd compositiona
1H NMR (DMSO), δ
4
5:4:1:0.1
5:4:1:0.1
5:4:1:0.1
5:3:2:0.1
5:3:2:0.1
6:4:1:0.1
6:4:1:0.1
6:4:1:0.1
5:4:2:0.1
3:7:1:0.1
6:4:1:0.1
7:3:1:0.1
4:6:1:0.1
7:3:1:0.1
-
40
37
38
36
40
31
41
42
30
60
85
35
33
40
99
74
40
50
60
25
20
58
152-155
(dry EtOH)
160-163
(dry EtOH)
143-145
(dry EtOH)
155-158
(i-PrOH/Et2O)
168-169
(i-PrOH)
158-159
(i-PrOH)
160-161
(i-PrOH)
187-188
(i-PrOH)
180
1.57 (d, 3, CH3), 3.92 (s, 4, NCH2CH2N), 5.55 (q, 1, CH), 7.18-8.72 (m, 8, ArH)
5
1.52 (d, 3, CH3), 3.86 (s, 4, NCH2CH2N), 5.35 (q, 1, CH), 7.08-8.80 (m, 8, ArH),
12.00 (br s, 1, NH, exchangeable with D2O)
6
1.50 (d, 3, CH3), 3.86 (s, 4, NCH2CH2N), 5.34 (q, 1, CH), 7.05-8.63 (m, 8, ArH),
10.52 (br s, 1, NH, exchangeable with D2O)
7
1.62 (d, 3, CH3), 3.90 (s, 4, NCH2CH2N), 5.48 (q, 1, CH), 7.03-7.80 (m, 7, ArH)
8
1.56 (d, 3, CH3), 3.86 (s, 4, NCH2CH2N), 5.31 (q, 1, CH), 7.02-7.87 (m, 7, ArH)
9b
1.30 (d, 3, CH3), 2.12 (s, 3, Ph-CH3), 3.82 (s, 4, NCH2CH2N), 4.96 (q, 1, CH),
7.10-7.42 (m, 8, ArH), 10.06 (br s, 1, NH, exchangeable with D2O)
1.45 (d, 3, CH3), 2.38 (s, 3, Ph-CH3), 3.83 (s, 4, NCH2CH2N), 5.17 (q, 1, CH),
7.03-7.40 (m, 8, ArH)
1.47 (d, 3, CH3), 2.38 (s, 3, Ph-CH3), 3.85 (s, 4, NCH2CH2N), 5.20 (q, 1, CH),
7.00-7.50 (m, 8, ArH)
10
11
12
13
14c
15c
16
17
18
19
21
22
23
24d
25c
26
1.43 (d, 3, CH3), 3.85 (m, 4, NCH2CH2N), 5.17 (q, 1, CH), 6.82-7.38 (m, 8, ArH),
10.18 (br s, 1, NH, exchangeable with D2O)
(i-PrOH/dry EtOH)
101-102
(i-PrOH)
240
(i-PrOH)
235
1.43 (d, 3, CH3), 3.82 (s, 4, NCH2CH2N), 5.05 (q, 1, CH), 6.72-7.45 (m, 8, ArH),
7.68 (br s, 1, OH, exchangeable with D2O), 10.17 (br s, 1, NH, exchangeable with D2O)
1.43 (d, 3, CH3), 3.83 (s, 4, NCH2CH2N), 5.10 (q, 1, CH), 6.80-7.41 (m, 8, ArH),
9.58 (s, 1, OH, exchangeable with D2O), 10.35 (br s, 1, NH, exchangeable with D2O)
1.32 (d, 3, CH3), 3.80 (s, 4, NCH2CH2N), 5.18 (q, 1, CH), 7.02-8.08 (m, 8, ArH),
10.25 (br s, 1, NH, exchangeable with D2O)
1.53 (d, 3, CH3), 3.88 (s, 4, NCH2CH2N), 3.93 (br s, 1, NH, exchangeable with D2O),
5.38 (q, 1, CH), 7.08-7.47 (m, 8, ArH)
1.48 (d, 3, CH3), 3.85 (s, 4, NCH2CH2N), 4.25 (br s, 1, NH, exchangeable with D2O),
5.32 (q, 1, CH), 7.03-8.33 (m, 8, ArH)
1.51 (d, 3, CH3), 3.83 (s, 4, NCH2CH2N), 5.10 (q, 1, CH), 6.55-7.37 (m, 8, ArH),
8.92 (br s, 2, exchangeable with D2O), 10.5 (br s, 1, NH, exchangeable with D2O)
1.48 (d, 3, CH3), 3.87 (s, 4, NCH2CH2N), 5.27 (q, 1, CH), 7.03-7.52 (m, 8, ArH)
(dry EtOH)
208-210
(dry EtOH)
206-208
(dry EtOH)
158-160
(dry EtOH)
102-103
(i-PrOH/Et2O)
169-170
(i-PrOH)
165-167
(dry EtOH)
158-160
(dry EtOH)
87-90
5:5:1:0.1
6:4:1:0.1
6:4:1:0.1
6:4:1:0.1
7:3e
1.38 (d, 3, CH3), 3.66 (s, 4, NCH2CH2N), 4.14 (q, 1, CH), 7.27-7.62 (m, 9, ArH),
10.06 (br s, 1, NH, exchangeable with D2O)
3.66 (m, 4, NCH2CH2N), 4.10 (br s, 1, NH, exchangeable with D2O), 4.32 (d, 1,
CH-CdN), 4.43 (d, 1, ArCH), 7.35-7.55 (m, 9, ArH)
3.84 (s, 4, NCH2CH2N), 4.04 (d, 1, CH-CdN), 4.15 (br s, 1, NH, exchangeable with
D2O), 4.27 (d, 1, ArCH), 7.30-7.55 (m, 9, ArH)
1.52 (d, 3, CH3), 3.87 (t, 2, NCH2CH2O), 4.25 (m, 2, NCH2CH2O), 4.93 (q, 1, CH),
7.03-7.65 (m, 9, ArH)
1.42 (d, 3, CH3), 3.28 (m, 2, SCH2CH2N), 4.22 (t, 2, SCH2CH2N), 4.78 (br s, 1, NH,
exchangeable with D2O), 5.30 (q, 1, CH), 7.03-7.60 (m, 9, ArH)
1.38 (d, 3, CH3), 1.72 (m, 2, NCH2CH2CH2), 2.30 (m, 2, NCH2CH2CH2),
3.73 (m, 2, NCH2CH2CH2), 5.18 (q, 1, CH), 7.02-7.78 (m, 9, ArH)
8:2e
104-105
(i-PrOH/Et2O)
94-95
5:5e
(i-PrOH)
a For flash chromatography cyclohexane/AcOEt/MeOH/33% NH4OH was eluent for the free base. b Sulfate salt. c Hydrochloride salt.
d Free base. e Cyclohexane/AcOEt was eluent for the free base.
precipitated product was extracted with CH2Cl2. The pooled
extracts were washed with H2O, dried over Na2SO4, and
evaporated. The crude product was purified by flash chroma-
tography and transformed into the oxalate salt (Table 6).
the residue was dissolved in CH2Cl2 and washed with brine.
The organic layer was dried over Na2SO4 and evaporated. Et3N
(15.1 mL, 108.7 mmol) and CHCl3 (75 mL) were added, and
the mixture was refluxed for 3 days, extracted with cold H2O,
dried over Na2SO4, filtered, and evaporated. The product was
purified by flash chromatography to give the free base 24
(Table 6).
Preparation of nitriles 27-44. Method A. 2-(2-Pyridin-
2-ylphenoxy)propionitrile (27). A mixture of 2-pyridin-2-
ylphenol 42 (0.33 g, 1.93 mmol), 2-chloropropionitrile (0.173 g,
1.93 mmol), and K2CO3 (0.27 g, 1.93 mmol) in DME was
refluxed for 8 h. The mixture was cooled and filtered. The
solvent was removed under reduced pressure to give a residue,
which was taken up in AcOEt and washed with cold 2 N
NaOH. Removal of dried solvent afforded an oil whose phys-
icochemical properties are reported in Table 7.
Similarly, compounds 28, 29, and 30 were obtained from
4-pyridin-4-ylphenol,42 2′-nitrobiphenyl-2-ol,43b and 4′-nitrobi-
phenyl-2-ol,43a respectively. Compound 44 was generated from
2-bromobenzenethiol (Table 7).
Method B. trans- and cis-3-Biphenyl-2-yloxirane-2-
carbonitrile (31 and 32). A solution of potassium tert-
butoxide (0.743 g, 6.62 mmol) in dry tert-butyl alcohol (6.5 mL)
was added dropwise to a solution of 2-biphenylcarboxaldehyde
(1.18 g, 6.47 mmol) and chloroacetonitrile (0.48 g, 6.47 mmol)
with stirring and cooling under nitrogen atmosphere. The
reaction was allowed until TLC indicated the absence of
2-biphenylcarboxaldehyde. tert-Butyl alcohol was removed in
vacuo and H2O (6 mL) was added to the residue. Extraction
with ether, drying over Na2SO4, and removal of the solvent in
Method H. 2-[1-(Biphenyl-2-yloxy)ethyl]-4,5-dihydrothi-
azole (25). A stirred solution of 2-(biphenyl-2-yloxy)propionic
acid41 (2.15 g, 8.89 mmol), 2-aminoethanethiol (0.683 g, 8.89
mmol), triphenylphosphine (6.97 g, 26.57 mmol), triethylamine
(2.7 g, 26.57 mmol), and CCl4 (13.8 g, 88.9 mmol) in CH3CN
(30 mL) was heated at 40 °C for 4 h. The solvent was removed
under reduced pressure to give a residue that was taken up
in AcOEt. The insoluble solid was filtered and washed with
AcOEt. The solvent was evaporated and the residue was
purified by flash chromatography and transformed into the
hydrochloride salt (Table 6).
Method I. 2-[1-(Biphenyl-2-yloxy)ethyl]-4,5-dihydroox-
azole (24). 2-(Biphenyl-2-yloxy)propionic acid41 (2.78 g, 11.47
mmol) was dissolved in dry CH2Cl2 under nitrogen atmo-
sphere. After addition of oxalyl chloride (1.51 mL, 16.6 mmol)
the mixture was stirred at room temperature for 2 days. Excess
oxalyl chloride was removed in vacuo. The reaction flask
containing the residue was charged with Et3N (1.96 mL, 14.04
mmol), 2-aminoethanol (1.51 mL, 24.6 mmol), and dry CH2-
Cl2 (45 mL). The mixture was stirred overnight, quenched with
saturated aqueous NH4Cl solution (15 mL) and H2O (15 mL),
and then extracted with Et2O. The organic layer was washed
with H2O, dried over Na2SO4, and evaporated. The residue was
dissolved in dry CH2Cl2 (180 mL), cooled to 0 °C, treated with
SOCl2 (3.77 mL, 51.3 mmol), and stirred at room temperature
for 24 h. After removal of the excess SOCl2 by evaporation,

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6171
1H NMR (CDCl3), δ
Table 7. Physicochemical Properties of Nitriles 27-44
eluent
yield,
%
compd
compositiona
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
-
95
94
85
93
59
32
68
46
95
95
92
50
52
51
92
90
53
58
1.62 (d, 3, CH3), 4.81 (q, 1, CH), 7.17-8.67 (m, 8, ArH)
-
1.68 (d, 3, CH3), 4.81 (q, 1, CH), 7.18-8.65 (m, 8, ArH)
5:5b
1.58 (d, 3, CH3), 4.82 (q, 1, CH), 7.08-8.03 (m, 8, ArH)
1.70 (d, 3, CH3), 4.83 (q, 1, CH), 7.08-8.33 (m, 8, ArH)
8:2b
10:0.5
10:0.5
8.5:1.5
9.5:0.5
9.5:0.5
9.5:0.5
9.5:0.5
8:2
3.40 (d, 1, J ) 1.83 Hz, CHCN), 4.22 (d, 1, J ) 1.83 Hz, ArCH), 7.18-7.52 (m, 9, ArH)
3.66 (d, 1, J ) 3.66 Hz, CHCN), 4.11 (d, 1, J ) 3.66 Hz, ArCH), 7.30-7.48 (m, 9, ArH)
1.62 (d, 3, CH3), 4.80 (q, 1, CH), 7.15-8.78 (m, 8, ArH)
1.87 (d, 3, CH3), 4.92 (q, 1, CH), 7.10-7.70 (m, 8, ArH)
1.48 (d, 3, CH3), 2.18 (s, 3, ArCH3), 4.58 (q, 1, CH), 7.17-7.47 (m, 8, ArH)
1.62 (d, 3, CH3), 2.43 (s, 3, ArCH3), 4.62 (q, 1, CH), 7.18-7.42 (m, 8, ArH)
1.65 (d, 3, CH3), 2.45 (s, 3, ArCH3), 4.63 (q, 1, CH), 7.20-7.66 (m, 8, ArH)
1.62 (d, 3, CH3), 4.63 (q, 1, CH), 4.60 (br, 1, OH, exchangeable with D2O), 7.00-7.54 (m, 8, ArH)
1.62 (d, 3, CH3), 4.63 (q, 1, CH), 6.82-7.40 (m, 8, ArH)
8:2
8:2
1.62 (d, 3, CH3), 4.63 (q, 1, CH), 5.40 (br s, 1, OH, exchangeable with D2O), 6.83-7.45 (m, 8, ArH)
1.72 (d, 3, CH3), 4.83 (q, 1, CH), 7.20-8.43 (m, 8, ArH)
8:2
6:4b
1.64 (d, 3, CH3), 4.71 (q, 1, CH), 7.02-7.45 (m, 8, ArH)
-
9:1
1.72 (d, 3, CH3), 4.30 (q, 1, CH), 7.22-7.73 (m, 4, ArH)
1.42 (d, 3, CH3), 3.53 (q, 1, CH), 7.36-7.75 (m, 9, ArH)
a For flash chromatography. Cyclohexane/AcOEt. b For flash chromatography. Cyclohexane/CH2Cl2.
vacuo gave a residue; from this, stereoisomers 31 (first
fraction) and 32 (second fraction) were obtained by flash
chromatography (Table 7).
5% fetal calf serum. The plasmids used to express R2A
-
(pR2C10Eneo), R2B- (pR2C2Eneo), or R2C-AR (pR2C4Eneo) had
been described earlier.65 Briefly, each bicistronic vector con-
tains an expression cassette comprising the human cytome-
galovirus early promoter/enhancer, the entire ORF encoding
for R2A-, R2B- or R2C-AR, an internal ribosome entry site derived
from encephalomyocarditis virus, the neomycin phosphotrans-
ferase gene, and a rabbit â-globin genomic sequence containing
an intron and a polyadenylation signal. CHO cells were
transfected using the calcium-phosphate method. At 2 days
post-transfection, cells were subcultured in the presence of
G418-sulfate (1 mg/mL) and antibiotic resistant clones were
collected individually using cloning cylinders.
Method C. 2-(2-Thiophen-2-ylphenoxy)propionitrile
(34). Na2CO3 (1.13 g, 10.7 mmol), H2O (5.35 mL), and tetrakis-
(triphenylphosphine)palladium(0) (0.255 g, 0.221 mmol) were
added to a solution of 2-(2-bromophenoxy)propionitrile (1 g,
4.42 mmol) and 2-thiophenboronic acid (0.7 g, 5.52 mmol) in
DME (8 mL). The mixture was heated at 90 °C for 14 h in the
dark under nitrogen atmosphere. Then it was cooled to room
temperature and poured into AcOEt and ice. The organic layer
was separated, washed with H2O, dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(Table 7).
Binding Experiments. Binding experiments were carried
out on crude membrane preparations using the selective
radioligand [3H]RX821002 (R2-antagonist). Frozen cell pellets
were homogenized in 5 mL of Tris-Mg buffer (50 mM Tris-
HCl, 0.5 mM MgCl2, pH 7.5) and centrifuged at 39 000g for
10 min. The particulate fraction was washed, the final crude
membrane pellet was taken up in the appropriate volume of
Tris-Mg buffer, and the protein concentration was determined
using the Coomassie Blue method.66 Total binding was mea-
sured by incubating 100 µL of cell membrane preparation with
the radioligand in a total volume of 400 µL of TM buffer. After
a 30-min period of incubation at 25 °C, bound radioligand was
separated from free by filtration through GF/C Whatman
filters using a Skatron cell harvester (Skatron, Lier, Norway).
Filters were rapidly washed with ice-cold buffer, and radio-
activity was determined by liquid scintillation spectrometry.
Specific binding was defined as the difference between total
Similarly, compounds 33 and 35-42 were obtained from
the suitable boronic acids. Compound 43 was generated by
treating 2-(2-bromophenylsulfanyl)propionitrile (44) with phen-
yl boronic acid (Table 7).
2-(2-Thiophen-3-ylphenoxy)propionic Acid Methyl Es-
ter (46). A mixture of 2-bromophenol (1.41 g, 8.15 mmol),
methyl 2-bromopropionate (1.36 g, 8.15 mmol),and K2CO3 (1.13
g, 8.15 mmol) in DME was refluxed for 8 h. The mixture was
cooled and filtered. The solvent was removed under reduced
pressure, and the remaining residue was taken up in AcOEt
and washed with cold 2 N NaOH. Removal of dried solvent
afforded an oil, which was purified by flash chromatography
using cyclohexane/AcOEt (95:5) as eluent, to give 1.9 g (yield
90%) of 2-(2-bromophenoxy)propionic acid methyl ester (45):
1H NMR (CDCl3) δ 1.72 (d, 3, CH3), 3.78 (s, 3, COOCH3) 4.88
(q, 1, CH), 6.80-7.61 (m, 4, ArH).
and nonspecific binding measured in the presence of 10-4
M
Na2CO3 (1.13 g, 10.7 mmol), H2O (5.35 mL), and tetrakis-
(triphenylphosphine)palladium(0) (0.255 g, 0.221 mmol) were
added to a solution of 45 (1.15 g, 4.42 mmol) and 3-thiophen-
boronic acid (0.7 g, 5.52 mmol) in DME (8 mL). The mixture
was heated at 90 °C for 14 h in the dark under nitrogen
atmosphere. After cooling to room temperature the mixture
was poured into AcOEt and ice and extracted with CHCl3. The
organic layer was dried over Na2SO4, filtered, and evaporated
in vacuo to give the corresponding acid which was converted
into the methyl ester by heating in CH3OH in the presence of
a catalytic amount of H2SO4. After cooling, the solvent was
evaporated to dryness to give a residue which was taken up
in CHCl3 and washed with NaHCO3 and H2O. The organic
layer was dried over Na2SO4 and evaporated to give a residue
that was purified by flash chromatography using cyclohexane/
AcOEt (95:5) as eluent. Compound 46 was obtained as an oil
(0.92 g; yield 46%): 1H NMR (CDCl3) δ 1.63 (d, 3, CH3), 3.78
(s, 3, COOCH3), 4.82 (q, 1, CH), 6.80-7.75 (m, 7, ArH).
Generation of CHO Clones Expressing R2-AR Sub-
types. CHO cells were routinely subcultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with
phentolamine. For saturation studies, the final concentrations
of [3H]RX821002 ranged from 0.1 to 15 nM. Inhibition experi-
ments were carried out at a fixed concentration of radioligand
corresponding to twice its Kd value for the considered subtype
(2 nM for R2A-AR, 8 nM for R2B-AR, and 4 nM for R2C-AR).
Competition curves were drawn using 12 different concentra-
tions of competitor ranging from 10-11 to 10-4 M.
Functional Assays. Cytosensor Microphysiometry.
Extracellular acidification was measured using an eight-
channel cytosensor microphysiometry instrument (Molecular
Devices, Menlo Park, CA). Permanent clones of CHO cells
expressing human R2-ARs individually were seeded into 12 mm
capsule cups at a density of 3 × 105 cells/cup and incubated
at 37 °C under 5% CO2 atmosphere for 24 h. The capsule cups
were loaded into the sensor chambers of the instrument and
perfused with a running medium (bicarbonate-free DMEM
containing 0.584 g/L glutamine and 2.59 g/L NaCl) at a flow
rate of 100 µL/min. Agonists were diluted into running medium
and injected through a second fluid path. Valves directed the
flow from either fluid path to the sensor chamber. For each

6172 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Gentili et al.
(14) Uhlen, S.; Lindblom, J.; Johnson, A.; Wikberg, J. E. Autorad-
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Rat Using [3H]MK912 and Subtype-Selective Drugs. Brain Res.
1997, 770, 261-266.
90 s pump cycle, the pump was on for 60 s and was then
switched off for the remaining 30 s The pH value was recorded
for 20 s (from second 68 to 88). Cells were exposed to agonists
for 240 min and consecutive agonist exposures were separated
by a 1740-min washing period. This stimulation protocol was
validated in preliminary experiments with four known ago-
nists, (-)-noradrenaline, clonidine, UK 14304, and BHT 920.
The rate of acidification of the chamber was calculated by the
Cytosoft program (Molecular Devices). Changes in the rate of
acidification were calculated as the difference between the
maximum effect after agonist addition and the average of three
measurements taken prior to agonist addition.
Statistical Analysis. The values of Ki and EC50 and the
extent of maximal response (Emax) were calculated from the
computer analysis of binding inhibition data and dose-
response curves using the program GraphPad Prism (Graph-
Pad Software, San Diego, CA). The results are expressed as
means ( SEM of three to six separate experiments.
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Acknowledgment. We thank the MIUR (Rome),
CARIMA Foundation (Macerata), and the University of
Camerino for financial support.
Supporting Information Available: Analysis of reported
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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