α2-adrenoreceptors profile modulation. 4. From antagonist to agonist behavior

Article abstract of DOI:10.1021/jm800250z

The goal of the present study was to modulate the receptor interaction properties of known α₂-adrenoreceptor (AR) antagonists to obtain novel α₂-AR agonists with desirable subtype selectivity. Therefore, a phenyl group or one of its bioisosteres or aliphatic moieties with similar steric hindrance were introduced into the aromatic ring of the antagonist lead basic structure. The functional properties of the novel compounds allowed our previous observations to be confirmed. The high efficacy of 7, 12, and 13 as α₂-AR agonists and the significant α_2C-AR subtype selective activation displayed by 11 and 15 demonstrated that favorable interactions to induce α₂-AR activation were formed between the pendant groups of the ligands and the aromatic cluster present in transmembrane domain 6 of the binding site cavity of the receptors.

Full text of DOI:10.1021/jm800250z

J. Med. Chem. 2008, 51, 4289–4299
4289
r2-Adrenoreceptors Profile Modulation. 4.1 From Antagonist to Agonist Behavior†
Francesco Gentili,| Claudia Cardinaletti,| Cristian Vesprini,| Antonio Carrieri,‡ Francesca Ghelfi,| Aniket Farande,|
Mario Giannella,| Alessandro Piergentili,| Wilma Quaglia,| Jonne M. Laurila,§ Anna Huhtinen,§ Mika Scheinin,§ and
Maria Pigini*,|
Dipartimento di Scienze Chimiche, UniVersita` degli Studi di Camerino, Via S. Agostino 1, 62032 Camerino, Italy, Dipartimento
Farmaco-Chimico, UniVersita` degli Studi di Bari, Via E. Orabona 4, 70125 Bari, Italy, Department of Pharmacology and Clinical
Pharmacology, UniVersity of Turku, FI-20520 Turku, Finland
ReceiVed March 7, 2008
The goal of the present study was to modulate the receptor interaction properties of known R2-adrenoreceptor
(AR) antagonists to obtain novel R2-AR agonists with desirable subtype selectivity. Therefore, a phenyl
group or one of its bioisosteres or aliphatic moieties with similar steric hindrance were introduced into the
aromatic ring of the antagonist lead basic structure. The functional properties of the novel compounds allowed
our previous observations to be confirmed. The high efficacy of 7, 12, and 13 as R2-AR agonists and the
significant R2C-AR subtype selective activation displayed by 11 and 15 demonstrated that favorable interactions
to induce R2-AR activation were formed between the pendant groups of the ligands and the aromatic cluster
present in transmembrane domain 6 of the binding site cavity of the receptors.
Introduction
Adrenoreceptors (ARs),a belonging to the superfamily of
G-protein-coupled receptors,2 are classified into three classes
R1-, R2-, and ꢀ-ARs, and are considered attractive therapeutic
targets for the treatment of various diseases. Chemical and
biological strategies have provided evidence for their hetero-
geneity. In particular, three distinct R2-AR subtypes encoded
by different genes, namely R2A, R2B, and R2C, have been
identified and characterized in different species.3 They are
located in the central nervous system (CNS) and in peripheral
tissues. In addition to postsynaptic location, R2-ARs are also
localized presynaptically, where they act as negative modulators
of the neuronal release of catecholamines and other neurotrans-
mitters.4
Studies with knockout mice have indicated that the classical
pharmacological effects of R2-ARs, such as hypotension,
sedation, analgesia, hypothermia, antiepileptogenesis, and in-
hibition of monoamine release and metabolism in the brain, are
mainly mediated by the R2A-AR subtype. Instead, the R2B-AR
subtype plays an important role in placental angiogenesis, the
analgesic effects of NO, salt-induced hypertension and initial
peripheral hypertensive responses to R2-AR agonists. Finally,
the R2C-AR subtype appears to be involved in many CNS
processes such as the startle reflex, stress response, and
locomotion, as well as feedback inhibition of adrenal catechola-
mine release.5
Figure 1. One-dimensional nuclear Overhauser effect (1D-NOE)
between the proton H1 and the O-methyl-oxime protons of the methoxy
derivative 8.
R2-AR agonists endowed with individual subtype selectivity
appears particularly important. In fact, for example, the ben-
eficial therapeutic effects of the veterinary sedative-analgesic
drug 4-[1-(2,3-dimethyl-phenyl)ethyl]-1H-imidazole (medeto-
midine) and its (+)-enantiomer dexmedetomidine, used clini-
cally in human medicine in the intensive care setting, including
sedation, analgesia, muscle relaxation, anxiolysis, and anesthetic
sparing, are limited by cardiovascular side effects, such as
bradycardia and associated arrhythmia, hypertension or hy-
potension, and reduced cardiac output.6,7
All three R2-AR subtypes represent potential cardiovascular
drug targets. Subtype selective drugs are not currently available
for clinical trials, but lead molecules with nearly 1000-fold
selectivity margins have already been discovered. R2B- and
perhaps also R2C-AR antagonists may be useful in disorders
characterized by excess vasoconstriction.8 In contrast, activation
of R2C-AR might have beneficial sympatho-inhibitory effects
in hypertension and heart failure without the sedative side effects
accompanying current clonidine-like drugs.9
We have previously observed that very interesting modulation
of receptor interactions was obtained by introduction of a phenyl
group into the ortho position of the aromatic ring of the R2-AR
antagonist 1 (Chart 1). In fact, the compound 5 (named
biphenyline) behaved as an efficacious R2-agonist in in vitro
assays on isolated rat vas deferens [pEC50 ) 8.52, ia ) 1
compared to N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-
2-amine (clonidine)].10 Moreover, in mouse hot-plate and mouse
tail-flick tests, the eutomer (S)-(-)-5 displayed enhanced long-
lasting antinociceptive potency, undoubtedly mediated by the
R2-ARs, because this effect was competitively blocked by the
Nevertheless, the complete in vivo characterization of the
three R2-AR subtypes has been hampered by the lack of R2-
AR-subtype selective agonists. Therefore, also from the stand-
point of therapeutic interest, the discovery and development of
†
This article is dedicated to Dr. Francesco Gentili died prematurely at
the age of 39.
* To whom correspondence should be addressed. Phone: +39 0737
402257. Fax: +39 0737 637345. E-mail: maria.pigini@unicam.it.
‡
Universita` degli Studi di Bari.
University of Turku.
Universita` degli Studi di Camerino.
§
|
a Abbreviations: ARs, adrenoreceptors; CNS, central nervous system;
CHO, Chinese hamster ovary; NOE, nuclear Overhauser effect; EC50,
concentration that produces 50% of the maximum effect; Ki, dissociation
constant.
10.1021/jm800250z CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008

4290 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Gentili et al.
Chart 1. Chemical Modifications of the R2-AR Antagonists 1-4
Scheme 1a
a Reagents: (a) CH3ONa, MeOH, ethylenediamine; (b) (CH3)3Al, dry toluene, ethylenediamine, ∆; (c) NH2OX, 2N HCl pH 3-4.
selective R2-antagonist 2-(2,3-dihydro-2-methoxy-1,4-benzo-
dioxin-2-yl)-4,5-dihydro-1H-imidazole (RX 821002).10
proposed to be involved in the structural rearrangements that
participate in receptor activation.12
Subsequent studies demonstrated that 5 preferentially acti-
vated the R2A- and R2C-AR subtypes vs the R2B-subtype, whereas
in some biphenyline derivatives, the presence of correctly
oriented functions with positive electronic effect (+σ) in the
ortho phenyl pendant was an important factor for significant
R2C-subtype selective activation.11 As reported, we attributed
the modulation from antagonist to agonist activity to the
favorable interaction of the ortho pendant group with the amino
acid residues comprising the highly conserved aromatic cluster
of the sixth transmembrane domain of the predicted R2-AR
binding cavity.11 Indeed, this kind of interaction has been
Therefore, to confirm the interesting biological profile
modulation from antagonist to agonist behavior, as verified for
5, and to discover novel R2-AR agonists possibly endowed with
desirable R2-AR subtype selectivity, we introduced a phenyl
group or one of its bioisosteres or aliphatic moieties with similar
steric hindrance into the aromatic ring of known R2-AR
antagonists such as the aforementioned 110 (compounds 7-13),
213 (compound 14), 3 (idazoxan),14 and 4 (RX 821002)15
(compounds 15-18 and 19, 20, respectively) (Chart 1). Finally,
for a more complete structure-activity relationship investigation
we also prepared compound 6. In fact, 5 and 6 might be

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4291
Scheme 2a
a Reagents: (a) 2-Chloroacrylonitrile, K2CO3, acetone; (b) HClg, MeOH, ethylenediamine; (c) NBS, CCl4; (d) MeONa, ethylenediamine; (e) Flash
chromatography for 15, 18 and 19, 20, or LC-MS for 16, 17.
Scheme 3a
a Reagents: (a) Epichlorohydrin, 2N NaOH; (b) HBr; (c) NaOH, ∆; (d) KMnO4, 0.3N KOH; (e) MeOH, H2SO4; (f) (CH3)3Al, dry toluene, ethylenediamine, ∆.
considered ortho phenyl derivatives of open analogues of 3 and
4, respectively.
point of view. Compounds 1-4 were also included in the present
study for comparison
Chemistry
The receptor interaction profiles of the obtained compounds
6-20 were evaluated by receptor binding and functional studies
performed with Chinese hamster ovary (CHO) cells expressing
recombinant human R2-AR subtypes. The already known
imidazoline derivative 1316 has never been studied from this
The imidazolines 6-20 were prepared according to the
Schemes 1–3. The oximes 7-10 were obtained starting from
22 by treatment with hydroxylamine or the appropriate substi-
tuted oxylamines, at pH 3-4. The E configuration of compounds

4292 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Gentili et al.
7-10 was assigned on the basis of the chemical shift value of
the oxime proton, that, as reported in literature,17 was found
below 8 ppm. Moreover, the E configuration of 8 was confirmed
by the 1D-nuclear Overhauser effect (1D-NOE) observed
between the proton H1 and the O-methyl-oxime protons (Figure
1). Compounds 12, 13,16 and 14 were obtained starting from
the suitable methyl esters (27, 30, and 23, respectively) by
treatment with trimethylaluminum and ethylenediamine. The
esters 27 and 30 were obtained by condensation of the suitable
phenols with methyl 2-bromopropionate, whereas 23 was
prepared by condensation of biphenylphenol and methylman-
delate under Mitsunobu conditions.18 The imidazolines 6, 11,
and 22 were obtained from the corresponding nitriles 29, 28,19
and 21 by treatment with sodium methoxide and ethylenedi-
amine (Scheme 1). The nitriles 21 and 31 were obtained by
condensation of suitable phenols with 2-bromo-propionitrile.
The mixtures of imidazolines 15/18 and 16/17 were obtained
starting from the corresponding nitriles, 24 and 26, by treatment
with hydrogen chloride and ethylenediamine in methanol; the
regio-isomers of the two pairs of imidazolines 15/18 and 16/17
were separated by flash chromatography and semipreparative
HPLC, respectively (Scheme 2). The structure of 18 was
determined by stereospecific synthesis (Scheme 3). The reaction
of 2-methoxy-biphenyl-3-ol20 with epichlorohydrin afforded 32.
The cleavage of the methoxy group and following cyclization
with NaOH yielded 34, whose oxidation with potassium
permanganate in 0.3 N KOH afforded the acid 35. Further
esterification with methanol and treatment with ethylenediamine
gave the imidazoline 18. In the 1H NMR spectrum of compound
18, the imidazoline proton signal are diamagnetically shifted
by the phenyl ring magnetic anisotropy effect with respect to
the same protons of 15. Because it was impossible to obtain
suitable crystals for single-crystal X-ray diffraction analysis, the
structure of regioisomers 16 and 17 was tentatively assigned
on the basis of the same anisotropic effect shown in the
agonist.27 The intrinsic activity (ia) of each compound is
expressed as the fraction of the maximum response elicited by
(-)-noradrenaline (ia ) 1). Antagonist results are expressed as
pKb and were analyzed as the ability of the antagonist to shift
the agonist (clonidine) concentration-effect curve.28
Results and Discussion
In Tables 1–3 are reported the affinity values (pKi) of
compounds 1-20, antagonist properties (pKb) of the leads 1-4,
and the potency and intrinsic activity estimates (pEC50, ia,
respectively) of 5-20 and (-)-noradrenaline. In addition, the
antagonist activity of 11 at the R2A-AR subtype has been
included.
Since we demonstrated that the best biological profile
modulation of the structurally flexible antagonist 1 was produced
by the ortho substitution of its aromatic ring (e.g., 5)10 in the
designed new derivatives of 1 and its analogue 2 (compounds
6-13 and 14, respectively) (Tables 1 and 2), a phenyl or one
of its bioisosteres or aliphatic pendant groups have been
similarly introduced into the ortho position of their aromatic
rings.
To achieve the bioisosteric replacement for the phenyl group,
the oximino methyl moiety, which in its trans configuration
shows steric and electronic analogy with the aromatic ring,29
was selected (compound 7). Moreover, the investigation has
been extended to its alkyl derivatives 8-10.
Instead, for the antagonists 3 and 4, characterized by a
puckered and less flexible conformation due to the presence of
the benzodioxane system, we thought it useful to consider also
the other positions of their aromatic rings; therefore, the regio-
phenyl derivatives 15-20 were prepared (Table 3).
Both binding and functional data demonstrated that our leads
1-4, obviously with different potencies, did not exhibit
significant R2-AR subtype selective antagonism. The novel
derivatives 6-20 displayed comparable affinity for the three
different R2-AR subtypes, even if showed a slightly higher
affinity for the R2A- or R2C-subtypes compared to the R2B-
subtype. Nevertheless, their functional assessment highlighted
some interesting results that allowed us to confirm the afore-
mentioned biological profile modulation of the ligands. Indeed,
compound 7 behaved as a nearly full agonist, with equivalent
activity to that of (-)-noradrenaline toward the R2A- and R2C-
subtypes (pEC50 R2A ) 6.30, ia ) 0.80; pEC50 R2C ) 6.90, ia
) 0.85). Moreover, because it produced only relatively weak
partial activation of the R2B-subtype, its selectivity profile
emerged as analogous to that of 5. Therefore, as expected, the
bioisosteric analogy between the phenyl and the pseudocycle
oximinomethyl function was confirmed.
1
corresponding H NMR spectra.
The imidazolines 19 and 20 were obtained by treating the
suitable mixture of the R-brominated nitriles 25 with sodium
methoxide and ethylenediamine followed by separation through
1
flash chromatography. Their structures were attributed by H
NMR spectra analysis, analogously to the aforementioned for
the pair 15/18.
The mixtures of nitriles 24 and 26 were obtained by
condensation of biphenyl-2,3-diol and biphenyl-3,4-diol,21
respectively, with 2-chloroacrylonitrile. The mixture 25 (Scheme
2) and compound 29 (Scheme 1) were synthesized from the
corresponding nitriles 24 and 31, respectively, using NBS in
carbon tetrachloride.
The methyl and ethyl derivatives 8 and 9 selectively activated
the R2C-subtype as partial agonists and, interestingly, with
potency comparable to that of (-)-noradrenaline; they were
totally inactive at the R2A- and R2B-subtypes. Weak activation
of the R2B- and R2C-subtypes was observed for the benzyl
derivative 10.
Also 14 behaved as an agonist, and analogously to 5, it
preferentially activated the R2A- and R2C-subtypes. At the R2C-
subtype, 14 showed the same potency and only moderately lower
efficacy compared to (-)-noradrenaline (pEC50 R2C ) 6.11, ia
) 0.70), whereas at the R2A-subtype it produced less efficacious
activation.
Pharmacology
The pharmacological profiles of compounds were investigated
using CHO cell lines stably expressing cDNAs encoding human
R2A-,R2B-,andR2C-ARsubtypes.Receptorbindingexperiments22–24
were carried out on membrane preparations using [3H]RS-
79948-197 as radioligand. The IC50 values were determined by
nonlinear regression analysis of competition data using the
GraphPad Prism computer program. Ki values were calculated
from the equation of Cheng and Prusoff25 and reported as pKi
( SEM. Agonist and antagonist potencies were determined as
previously described.11,26 Agonist potency, defined as the
concentration that produces 50% of the maximum effect, is
expressed as pEC50 and was determined by use of a Cytosensor
microphysiometry instrument on CHO cells by measuring the
rate of extracellular acidification after receptor activation by the
We have previously reported that in biphenyline-related
compounds, the oxyethyl moiety of the bridge played a
significant role in favoring R2-AR potency.11 Therefore, we can
now hypothesize that both the impossibility of 14, devoid of

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4293
Table 1. Affinity (pKia), Antagonist Potency (pKbb), Agonist Potency (pEC50b), Intrinsic Activity (iab) on Human R2-AR Subtypes
a pKi values were calculated from [3H]RS-79948-197 on membrane preparations from CHO cells expressing individually each human R2-AR subtype
(R2A, R2B, R2C). b pKb, pEC50 and intrinsic activity (ia) values were determined by applying the Cytosensor microphysiometry system to the same cell
models. Intrinsic activity of the tested compounds is expressed as the fraction of that of the full agonist (-)-noradrenaline taken as equal to 1. Compounds
exhibiting ia of <0.3 were considered not active (NA). c Ref 11.
Table 2. Affinity (pKia), Antagonist Potency (pKb,b), Agonist Potency (pEC50b), Intrinsic Activity (iab) on Human R2-AR Subtypes
a pKi values were calculated from [3H]RS-79948-197 on membrane preparations from CHO cells expressing individually each human R2-AR subtype
(R2A, R2B, R2C). b pKb, pEC50 and intrinsic activity (ia) values were determined by applying the Cytosensor microphysiometry system to the same cell
models. Intrinsic activity of the tested compounds is expressed as the fraction of that of the full agonist (-)-noradrenaline taken as equal to 1. Compounds
exhibiting ia of <0.3 were considered not active (NA).
the methyl group, to interact with the receptor methyl pocket10,26
and, above all, the enhanced steric hindrance determined by
the bridge phenyl group, were responsible for its reduced activity
in comparison with 5.
Similarly, an incompatible steric hindrance, due to the
additional methoxy group in the bridge, might be responsible
for the functional inactivity of 6 at all three R2-AR subtypes.
Among the derivatives of 3, the 6- and 7-phenyl isomers (16
and 17) were inactive at all three R2-AR subtypes. On the
contrary, 5-phenyl idazoxan (15) activated the R2C-subtype with
significant selectivity and good efficacy up to an extent fairly
similar to that of (-)-noradrenaline (pEC50 R2C ) 6.10, ia )
0.75) (Figure 2). Weaker activation was produced at the R2B-
subtype. The regio-isomer 8-phenyl idazoxan (18), although
activating the same R2B- and R2C-subtypes, showed lesser
potency. To get some indications on the molecular determinants
likely affecting the agonist behavior of 15 and 18 and their
different potencies, a flexible molecular overlay of the enanti-
omers of 15 and 18 with (S)-(-)-5 was performed. Indeed, we
previously assessed the better ability of (S)-(-)-5 in producing
R2C-AR activation.26 It has to be pointed out that the potency
data for all the agonists of the present study were referred to
their racemic mixtures and, therefore, negative control on
potency might be exerted by agonist distomer. As it can be
perceived from the best molecular superposition reported in
Figure 3, valuable overlay is obtained once (R)-15 and (S)-18
are fitted on (S)-(-)-5. This might suggest that both agonists
anchor to the receptor binding site with similar topology but

4294 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Gentili et al.
Table 3. Affinity (pKia), Antagonist Potency (pKbb), Agonist Potency (pEC50b), Intrinsic Activity (iab) on Human R2-AR Subtypes
R2A
pKb
8.15 ( 0.11 7.73 ( 0.09
R2B
pKb
R2C
pKb
7.75 ( 0.08 7.92 ( 0.08
compd
R
R′
pKi
pEC50
ia
pKi
pEC50
ia
pKi
pEC50
ia
3
4
H
H
H
7.64 ( 0.09 7.16 ( 0.10
OCH3 9.44c
7.64 ( 0.10
8.79c
7.59 ( 0.09
9.10c
8.00 ( 0.08
4.90 ( 0.20 0.70 6.08 ( 0.16
NA
NA
15
16
17
18
19
20
5-C6H5
6-C6H5
7-C6H5
8-C6H5
H
H
H
H
6.14 ( 0.11
NA
5.97 ( 0.10
5.78 ( 0.14
6.00 ( 0.02
6.32 ( 0.12
6.10 ( 0.08 0.75
NA
NA
5.50 ( 0.15 0.40
NA
NA
6.55 ( 0.10
6.40 ( 0.02
6.62 ( 0.04
NA
NA
NA
NA
NA
5.80 ( 0.17
5.90 ( 0.15
4.65 ( 0.12 0.45 6.16 ( 0.10
4.80 ( 0.09 0.50
5-C6H5 OCH3 6.59 ( 0.10
8-C6H5 OCH3 6.90 ( 0.05
5.97 ( 0.08
NA
6.61 ( 0.08
(-)-Noradrenaline
6.43 ( 0.17 1.00
7.21 ( 0.25 1.00
6.10 ( 0.05 1.00
a pKi values were calculated from [3H]RS-79948-197 on membrane preparations from CHO cells expressing individually each human R2-AR subtype
(R2A, R2B, R2C). b pKb, pEC50, and intrinsic activity (ia) values were determined by applying the Cytosensor microphysiometry system to the same cell
models. Intrinsic activity of the tested compounds is expressed as the fraction of that of the full agonist (-)-noradrenaline taken as equal to 1. Compounds
exhibiting ia of <0.3 were considered not active (NA). c Ref 33.
Figure 4. Stimulation of extracellular acidification in CHO cells stably
expressing the human R2C-adrenergic subtype by clonidine (solid black
upward pointing triangle), 11 (open red upward pointing triangle), and
11 (1 µM) with clonidine (solid green downward pointing triangle).
Data points with error bars represent the mean ( SEM of three to six
separate experiments.
Figure 2. Stimulation of extracellular acidification in CHO cells stably
expressing the human R2C-adrenergic subtype by (-)-noradrenaline
(solid black upward pointing triangles), 11 (open red upward pointing
triangles), 15 (solid blue squares), and 18 (solid green circles). Data
points with error bars represent the mean ( SEM of three to six separate
experiments.
hindrance, proved inactive at the R2A- and R2B-subtypes but
showed good intrinsic activity and high potency at the R2C-
subtype, highlighting its significant and selective activation of
this receptor subtype (pEC50 R2C ) 7.30, ia ) 0.90) (Figure 2).
The lack of R2A-AR agonism and the significant R2A-AR affinity
showed by 11 prompted us to evaluate its antagonist properties
at this subtype. Interestingly, it displayed the same antagonist
character of its prototype 1 (Figure 4). The peculiar biological
profile of 11 proved to be similar to that of (R)-2-[1-(3′-
nitrobiphenyl-2-yloxy)ethyl]-4,5-dihydro-1H-imidazole [(R)-
(+)-m-nitrobiphenyline] recently reported by us;26 both com-
pounds could be considered to be of some interest for
antinociceptive drug development. While the cerebral noradr-
energic system plays an important role in the modulation of
opioid actions and R2-AR agonists have been shown to
strengthen morphine analgesia, it has been suggested that also
Figure 3. Flexible fitting of 5 (yellow), compound 15 (blue), and 18
(red). Left: superposition of (S)-(-)-5 with (R)-15 and (S)-18, super-
position energy ) -437 kJ/mol. Right: superposition of (S)-(-)-5 with
(S)-15 and (R)-18, superposition energy ) -429 kJ/mol. Molecules
are rendered with PYMOL, available at http://www.pymol.org.
reverse chirality. Similar fitting was also achieved for (S)-15
and (R)-18, but the less favorable superposition energy suggests
that these forms might represent the agonist distomers. There-
fore, the lower intrinsic activity of 18 might be ascribed to a
more negative control on potency exerted by its distomer with
respect to the distomer of 15.
R
2A-AR selective antagonists may offer a novel mechanism to
augment the antinociceptive actions of partial opioid agonists.30
Effective antagonism/agonism modulation was also produced
by the presence of the bulkier cyclopentyl and cyclohexyl
substituents (compounds 12 and 13, respectively). In these cases,
indiscriminate activation of all three R2-AR subtypes was
induced. However, the highest potencies and intrinsic activities
were obtained at the R2C-subtype (compound 12: pEC50 ) 8.00,
ia ) 0.75; compound 13: pEC50 ) 7.68, ia ) 0.80).
Among the RX 821002 derivatives 19 and 20, only the
5-phenyl isomer (19) was able to display moderate R2B-AR
activation. As already discussed for 6, this result is probably
due to steric hindrance by the methoxy substituent.
Also, aliphatic substituents proved able to induce modulation
of the biological profiles of the ligands. Indeed, the derivative
11, whose ortho substituent was endowed with moderate steric

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4295
3, CH3), 3.85 (s, 4, NCH2CH2N), 4.17 (q, 2, OCH2CH3), 5.55 (q,
1, CHCN), 7.02-7.78 (m, 4, ArH), 8.52 (s, 1, CHdN), 9.81 (br s,
1, NH, exchangeable with D2O). Anal. (C14H19N3O2 ·H2C2O4) C,
H, N.
In conclusion, the present study (i) strengthened the validity
of our design directed to induce the biological profile modulation
of some R2-AR antagonists through conservative modifications,
such as the introduction of substituents in their basic structures,
(ii) confirmed that the interaction between the pendant groups
and one or more residues in the aforementioned aromatic cluster
in transmembrane domain 6 of the binding site cavity played a
crucial role in triggering the R2-AR activation, (iii) demonstrated
that the degree of this activation and subtype selectivity were
strongly affected by the structural characteristics (such as
flexibility and steric hindrance) of the leads and by the position
and peculiar nature of the substituent, in particular, (iv) it
highlighted the good R2-AR agonist properties of 7, 12, and
13, (v) the preferential and significant R2C-subtype selective
activation of 15, and (vi) the interesting behavior of 11, which
proved to be endowed with substantial agonist and antagonist
activity at the R2C- and R2A-AR subtypes, respectively.
2-[1-(4,5-Dihydro-1H-imidazol-2-yl)-ethoxy]-benzaldehyde O-
benzyl-oxime (10). The free amine was obtained as a solid: mp
1
132-133 °C. H NMR (CDCl3): δ 1.62 (d, 3, CH3), 3.58 (m, 4,
NCH2CH2N), 5.12 (q, 1, CHCN), 5.20 (s, 2, OCH2), 6.94-7.78
(m, 9, ArH), 8.52 (s, 1, CHdN), 9.81 (br s, 1, NH, exchangeable
with D2O). Anal. (C19H21N3O2) C, H, N.
2-(2-[1,3]Dioxolan-2-yl-phenoxy)-propionitrile (21). A mixture
of 2-[1,3]dioxolan-2-yl-phenol31 (0.32 g, 1.93 mmol), 2-chloro-
propionitrile (0.173 g, 1.93 mmol), and K2CO3 (0.27 g, 1.93 mmol)
in DME was refluxed for 20 h. After cooling, the mixture was
filtered and the solvent was removed under reduced pressure to
give a residue, which was taken up in CH2Cl2 and washed with
cold 2N NaOH. Removal of dried solvent afforded an oil (0.38 g,
1.74 mmol, 90% yield). 1H NMR (CDCl3): δ 1.83 (d, 3, CH3),
4.02-4.22 (m, 4, OCH2CH2O), 4.93 (q, 1 CHCN), 6.13 (s, 1,
OCHO), 7.08-7.62 (m, 4, ArH).
Experimental Protocols
Similarly,2-(biphenyl-2-yloxy)-propionitrile (31) was obtained
from 2-phenyl-phenol. The reaction mixture was purified by flash
chromatography eluting with cyclohexane/EtOAc (95:5) to give an
oil (65% yield). 1H NMR (CDCl3): δ 1.62 (d, 3, CH3), 4.63 (q, 1,
CH), 7.08-8.18 (m, 9, ArH).
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 tetramethylsilane (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 the department of Chemical Sciences. 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.
Compounds were named following IUPAC rules as applied by
Beilstein-Institut AutoNom (version 2.1) software for systematically
naming organic chemicals. Preparative LC-MS was performed by
Waters 2767 chromatograph (detector: Waters Micromass ZQ,
Waters 2487 DAD) on a Phenomenex Gemini C18 5.0 µm 10 ×
2.1 cm. The mobile phase was water (A)-CH3CN (B) at a flow
rate of 40 mL/min. The solvent composition varied from 0 to 6.80
min: 0:100 A:B (v/v); 6.80-8.10 min: 100:0 A:B (v/v); 8.10-10.00
min: 95:5 A:B (v/v).
2-[1-(2-[1,3]Dioxolan-2-yl-phenoxy)-ethyl]-4,5-dihydro-1H-imi-
dazole (22). A solution of 2-(2-[1,3]dioxolan-2-yl-phenoxy)-pro-
pionitrile (21) (3.0 g, 13.7 mmol), sodium methoxide (0.076 g, 1.4
mmol), in MeOH (6 mL) was stirred for 18 h. After cooling to
0-10 °C, a solution of ethylenediamine (0.92 mL, 13.7 mmol) in
MeOH (6 mL) was added dropwise with stirring; after a few
minutes, a solution of HCl in MeOH (4.8 mL of 3N solution, 14.4
mmol) was added dropwise and the mixture was allowed to warm
to 60 °C for 18 h. Removal of the solvent gave a residue, which
was purified by flash chromatography eluting with cyclohexane/
EtOAc/MeOH/33% NH4OH (5:5:1:0.1) to afford 22 (1.5 g, 42%
1
yield): mp 136-137 °C. H NMR (CDCl3): δ 1.65 (d, 3, CH3),
3.42-3.68 (m, 4, NCH2CH2N), 4.03-4.22 (m, 4, OCH2CH2O), 5.13
(q, 1, CH), 5.68 (br s, 1, NH, exchangeable with D2O), 6.17 (s, 1,
OCHO), 6.94-7.52 (m, 4, ArH).
2-[(Biphenyl-2-yloxy)-phenyl-methyl]-4,5-dihydro-1H-imida-
zole (14). A solution of ethylenediamine (0.42 mL, 6.28 mmol) in
dry toluene (6 mL) was added dropwise to a mechanically stirred
solution of 2 M trimethylaluminum (3.2 mL, 6.28 mmol) in dry
toluene (4 mL) at 0 °C under a nitrogen atmosphere. After being
stirred at room temperature for 1 h, the solution was cooled to 0
°C and a solution of 23 (1 g; 3.14 mmol) in dry toluene (8 mL)
was added dropwise. The reaction mixture was heated to 110 °C
for 3 h, cooled to 0 °C, and quenched cautiously with MeOH (0.8
mL) followed by H2O (0.2 mL). After addition of CHCl3 (5 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 2N 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 an oil, which was purified by flash chromatography eluting
with cyclohexane/EtOAc/MeOH/33% NH4OH (6:4:1:0.1) to give
the free base (0.52 g, 50% yield), which was transformed into the
oxalate salt; this was recrystallized from 2-PrOH: mp 185-186
°C. 1H NMR (DMSO): δ 3.83 (s, 4, NCH2CH2N), 6.32 (s, 1, CH),
7.05-7.61 (m, 14, ArH), 9.83 (br s, 1, NH, exchangeable with D2O).
Anal. (C22H20N2O·H2C2O4) C, H, N.
2-[1-(4,5-Dihydro-1H-imidazol-2-yl)-ethoxy]-benzaldehyde Oxime
(7). A solution of hydroxylamine hydrochloride (0.53 g, 7.63 mmol)
in water was added to a solution of 22 (1.0 g, 3.81 mmol) in ethanol.
The solution was acidified with 2N HCl until pH 3-4 and then
stirred at room temperature for 3 days. After cooling, it was made
basic with 2N NaOH and extracted with CHCl3. The combined
organic layers were dried (MgSO4), filtered, and the filtrate
concentrated under reduced pressure. The free base (0.78 g, 88%
yield) was transformed into the oxalate salt, which was recrystallized
1
from 2-PrOH: mp 135-136 °C. H NMR (DMSO): δ 1.61 (d, 3,
CH3), 3.88 (s, 4, NCH2CH2N), 5.59 (q, 1, CHCN), 6.72 (br s, 1,
NH, exchangeable with D2O), 7.00-7.78 (m, 4, ArH), 8.50 (s, 1,
CHdN), 10.60 (br s, 1, OH, exchangeable with D2O). Anal.
(C12H15N3O2 ·H2C2O4) C, H, N.
Similarly, 8-10 were obtained treating 22 with the appropriate
hydroxylamine derivatives.
2-[1-(4,5-Dihydro-1H-imidazol-2-yl)-ethoxy]-benzaldehyde O-
methyl-oxime (8). The free base (90% yield) was transformed into
the oxalate salt, which was recrystallized from 2-PrOH: mp
1
173-174 °C. H NMR (DMSO): δ 1.59 (d, 3, CH3), 3.80 (s, 3,
2-[1-(2-Cyclopentyl-phenoxy)-ethyl]-4,5-dihydro-1H-imidazole
(12). Similarly, 12 was obtained from 27. The reaction mixture was
purified by flash chromatography eluting with cyclohexane/EtOAc/
MeOH/33% NH4OH (8:2:1:0.1) to give the free base (55% yield),
which was transformed into the oxalate salt; this was recrystallized
from 2-PrOH: mp 168-169 °C. H NMR (DMSO): δ 1.32 (d, 3,
CH3), 1.38-2.04 (m, 8, -(CH2)4-), 3.35 (m, 1, PhCH), 3.86 (s, 4,
OCH3), 3.85 (s, 4, NCH2CH2N), 5.53 (q, 1, CHCN), 7.02-7.74
(m, 4, ArH), 8.43 (s, 1, CHdN), 9.81 (br s, 1, NH, exchangeable
with D2O). Anal. (C13H17N3O2 ·H2C2O4) C, H, N.
2-[1-(4,5-Dihydro-1H-imidazol-2-yl)-ethoxy]-benzaldehyde O-
ethyl-oxime (9). The free base (88% yield) was transformed into
the oxalate salt, which was recrystallized from 2-PrOH: mp
178-179 °C. 1H NMR (DMSO): δ 1.25 (t, 3, OCH2CH3), 1.59 (d,
1

4296 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Gentili et al.
NCH2CH2N), 5.32 (m, 1, CH), 6.88-7.28 (m, 4, ArH), 8.52 (br s,
1, NH, exchangeable with D2O). Anal. (C16H22N2O·H2C2O4) C,
H, N.
The second fraction afforded the isomer 19 as an oil (0.23 g,
49% yield). The free base was transformed into the oxalate salt
and recrystallized from EtOH/Et2O: mp 195-196 °C. 1H NMR
(DMSO): δ 3.33 (s, 3, OCH3) 3.89 (s, 4, NCH2CH2N), 4.12 (d, 1,
OCH), 4.40 (d, 1, OCH), 7.03-7.48 (m, 8, ArH), 8.32 (br s, 1,
NH, exchangeable with D2O). Anal. (C18H18N2O3 ·H2C2O4) C, H,
N.
2-(8-Phenyl-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-
imidazole (18). Similarly, 18 was obtained from 36. The residue
was purified by flash chromatography eluting with cyclohexane/
EtOAc/MeOH/33% NH4OH (7:3:1:0.1) to give the free base (65%
yield), which was transformed into the oxalate salt; this was
recrystallized from EtOH/Et2O: mp 190-191 °C. 1H NMR
(CD3OD): δ 3.98 (s, 4, NCH2CH2N), 4.35-4.52 (m, 2, OCH2),
5.39 (m, 1, OCH), 6.93-7.56 (m, 8, ArH). Anal. (C17H16N2O2 ·
H2C2O4) C, H, N.
2-(2-Cyclopentyl-phenoxy)-propionic Acid Methyl Ester (27). A
mixture of 2-cyclopentyl-phenol (0.31 g, 1.93 mmol), methyl
2-bromopropionate (0.22 mL, 1.93 mmol), and K2CO3 (0.27 g, 1.93
mmol) in DME was refluxed for 8 h. After cooling, the mixture
was filtered and the solvent was removed under reduced pressure
to give a residue, which was taken up in CH2Cl2 and washed with
cold 2N NaOH. Removal of dried solvent afforded an oil, which
was purified by flash cromatography eluting with cyclohexane/
EtOAc (9:1) (0.35 g, 73% yield). 1H NMR (CDCl3): δ 1.64 (d, 3,
CH3), 1.47-2.14 (m, 8, -(CH2)4-), 3.42 (m, 1, PhCH), 3.76 (s, 3,
OCH3), 4.69 (q, 1, CH), 6.66-7.25 (m, 4, ArH).
2-(2-Cyclohexyl-phenoxy)-propionic Acid Methyl Ester (30).
Similarly, 30 was obtained from 2-cyclohexyl-phenol. The reaction
mixture was purified by flash chromatography eluting with cyclo-
hexane/EtOAc (9:1) to give an oil (78% yield). 1H NMR (CDCl3):
δ 1.21-1.53 (m, 6, -(CH2)3-), 1.65 (d, 3, CH3), 1.68-1.98 (m,
4, CH2CHCH2), 3.07 (m, 1, CH), 3.76 (s, 3, OCH3), 4.75 (q, 1,
OCH), 6.67-7.26 (m, 4, ArH).
(Biphenyl-2-yloxy)-phenyl-acetic Acid Methyl Ester (23). A
solution of DIAD (3.7 g, 18.31 mmol) in THF (12.5 mL) was added
dropwise to a mixture of hydroxy-phenyl-acetic acid methyl ester
(2.5 g, 15.04 mmol), biphenyl-2-ol (2.56 g, 15.04 mmol), and
triphenylphosphine (3.94 g, 15.04 mmol) in THF (25 mL). The
reaction mixture was stirred at room temperature overnight and
under a nitrogen atmosphere. The solvent was evaporated and
diethyl ether and cyclohexane were added to precipitate the formed
triphenylphosphine oxide, which was filtered off. The crude product
was purified by flash chromatography eluting with cyclohexane/
Et2O (95:5) to afford 23 (3.06 g, 64% yield). 1H NMR (CDCl3): δ
3.67 (s, 3, OCH3), 5.81 (s, 1, CH), 6.83-7.48 (m, 14, ArH).
2-[1-(Biphenyl-2-yloxy)-1-methoxy-ethyl]-4,5-dihydro-1H-imida-
zole (6). Similarly, the treatment of 2-(biphenyl-2-yloxy)-propioni-
trile (31) with N-bromosuccinimide gave 29 as an oil (80% yield)
after purification by flash cromatography eluting with cyclohexane/
Et2O (99:1). 1H NMR (CDCl3): δ 2.28 (s, 3, CH3), 7.33-7.81 (m,
9, ArH). Compound 6 was obtained starting from the intermediate
29, following the procedure described for the mixture 19/20. The
residue was purified by flash chromatography eluting with cyclo-
hexane/EtOAc/MeOH/33%NH4OH (5:4:1:0.1) to give the free base
(59% yield), which was transformed into the oxalate salt; this was
recrystallized from 2-PrOH: mp 174-175 °C. 1H NMR (DMSO):
δ 1.38 (s, 3, CH3), 3.27 (s, 3, OCH3), 3.86 (s, 4, NCH2CH2N),
5.32 (br s, 1, NH, exchangeable with D2O), 7.07-7.61 (m, 9, ArH).
Anal. (C18H20N2O2 ·H2C2O4) C, H, N.
7-Phenyl-2,3-dihydro-benzo[1,4]dioxine-2-carbonitrile and 6-Phen-
yl-2,3-dihydrobenzo[1,4]dioxin-2-carbonitrile (26). A stirred mixture
of biphenyl-3,4-diol21 (2 g, 10.7 mmol), 2-chloroacrylonitrile (0.85
mL, 10.7 mmol), and anhydrous K2CO3 (1.33 g, 9.6 mmol) in dry
acetone was heated under reflux for 18 h. The solvent was removed
in vacuo, water was added to the residue, and the mixture was
extracted with CH2Cl2. The combined extracts were washed with
brine, dried, and the solvent was evaporated. The resulting oil was
purified by flash chromatography eluting with cyclohexane/EtOAc
(9:1) to afford a mixture of the two carbonitriles 26 (1.27 g, 5.35
mmol, 50% yield), which was used without further purification.
1H NMR (CDCl3): δ 4.45 (m, 4, OCH2), 5.18 (m, 2, OCH),
7.02-7.54 (m, 16, ArH).
Similarly, mixture 24 was obtained from biphenyl-2,3-diol21 and
used without further purification. 1H NMR (CDCl3): δ 4.43 (m, 4,
OCH2), 5.11 (m, 2, OCH), 7.02-7.58 (m, 16, ArH).
2-(6-Phenyl-2,3-dihydro-1,4-benzodioxin-2-yl)-4,5-dihydro-1H-
imidazole (16) and 2-(7-Phenyl-2,3-dihydro-1,4-benzodioxin-2-yl)-
4,5-dihydro-1H-imidazole (17). Gaseous HCl was bubbled through
a stirred and cooled (0 °C) solution of 26 (1.27 g, 5.35 mmol) and
MeOH (0.43 mL, 10.7 mmol) in dry CHCl3 (9.3 mL) for 45 min.
After 12 h at 0 °C, the solvent was removed in vacuo to give an
oil (1.11 g, 3.60 mmol) that was dissolved in abs EtOH and added
to a cooled (0 °C) and stirred solution of ethylenediamine (0.3 mL,
4.50 mmol) in abs EtOH (18 mL). After 1 h, concentrated HCl
(0.15 mL) was added to the reaction mixture, which was stored
overnight in the refrigerator. The residue was then diluted with abs
EtOH (12 mL) and heated at 70 °C for 5 h. After cooling, the solid
was collected and discarded and the filtrate was concentrated and
filtered again. The filtrate was evaporated to dryness to give a
residue which was taken up in CHCl3 (20 mL), washed with 2N
NaOH, and dried over Na2SO4. Removal of the solvent gave a
residue that was purified by flash chromatography eluting with
cyclohexane/EtOAc/MeOH/33% NH4OH (7:3:1:0.1) to obtain a
mixture of two imidazolines (0.82 g, yield 55%), which were
2-(2-Methoxy-5-phenyl-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-
dihydro-1H-imidazole (19) and 2-(2-Methoxy-8-phenyl-2,3-
dihydrobenzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole (20). N-
Bromosuccinimide (3 g, 17 mmol) and catalytic amount of benzoyl
peroxide (0.02 g) were added to a mixture of 24 (4 g, 17 mmol) in
carbon tetrachloride. The resulting mixture was heated at reflux
with stirring for 12 h. After cooling, the solvent was removed in
vacuo. The residue was purified by flash chromatography eluting
with cyclohexane/EtOAc (9:1) to afford a mixture of 2-bromo-5-
phenyl-2,3-dihydro-benzo[1,4]dioxine-2-carbonitrile and 2-bromo-
8-phenyl-2,3-dihydrobenzo[1,4]dioxin-2-carbonitrile (25), which
was used without further purification (4.84 g, 15.2 mmol, 90%
1
yield). H NMR (CDCl3): δ 4.51 (two dd, 4, OCH2), 6.98-7.58
(m, 16, ArH).
A solution of 25 (4.84 g, 15.2 mmol) in methanol (3.5 mL) was
treated with sodium methoxide (0.083 g, 1.53 mmol), and the
mixture was stirred at room temperature for 4-5 h. After cooling
to 0 °C, ethylenediamine (0.11 mL, 1.63 mmol) was added,
followed by a solution of HCl in methanol (0.51 mL of 3N solution,
1.53 mmol). After 24 h, the solvent was removed in vacuo. The
two imidazolines were separated by flash chromatography eluting
with cyclohexane/EtOAc/MeOH/33% NH4OH (7:3:1:0.1): isomer
20 eluted first (0.17 g, 36% yield). The free base was transformed
into the oxalate salt, which was recrystallized from EtOH/Et2O:
mp 214-215 °C. 1H NMR (DMSO): δ 3.13 (s, 3, OCH3) 3.85 (s,
4, NCH2CH2N), 4.22 (d, 1, OCH), 4.35 (d, 1, OCH), 6.95-7.64
(m, 8, ArH), 8.46 (br s, 1, NH, exchangeable with D2O). Anal.
(C18H18N2O3 ·H2C2O4) C, H, N.
1
separated by preparative LC-MS: isomer 16 eluted first. H NMR
(DMSO): δ 3.55 (s, 4, NCH2CH2N), 4.24-4.47 (m, 2, OCH2), 5.04
(m, 1, OCH), 6.99-8.22 (m, 8, ArH), 8.67 (br s, 1, NH,
exchangeable with D2O). The free base was transformed into the
oxalate salt, which was recrystallized from EtOH/Et2O: mp
189-190 °C. Anal. (C17H16N2O2 ·H2C2O4) C, H, N.
1
The second fraction was the isomer 17. H NMR (DMSO): δ
3.51 (s, 4, NCH2CH2N), 4.22-4.45 (m, 2, OCH2), 4.97 (m, 1,
OCH), 6.94-8.24 (m, 8, ArH), 8.51 (br s, 1, NH, exchangeable
with D2O). The free base was transformed into the oxalate salt,
which was recrystallized from EtOH/Et2O: mp 193-194 °C. Anal.
(C17H16N2O2 ·H2C2O4) C, H, N.

R2-Adrenoreceptors Profile Modulation
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4297
2-(5-Phenyl-2,3-dihydrobenzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-
imidazole (15) and 2-(8-Phenyl-2,3-dihydro-benzo[1,4]dioxin-2-
yl)-4,5-dihydro-1H-imidazole (18). These were obtained similarly
to 16 and 17 from the mixture 24. Isomers 18 and 15 were separated
by flash chromatography eluting with cyclohexane/EtOAc/MeOH/
33% NH4OH (7:3:1:0.1): isomer 18 eluted first (35% yield). The
free base was transformed into the oxalate salt, which was
recrystallized from EtOH/Et2O: mp 190-191 °C. The 1H NMR
was comparable to that of the same compound obtained by
stereospecific synthesis.
The second fraction afforded the isomer 15 (45% yield). The
free base was transformed into the oxalate salt, which was
recrystallized from EtOH/Et2O: mp 194-195 °C. 1H NMR
(CD3OD): δ 4.03 (s, 4, NCH2CH2N), 4.48 (m, 2, OCH2), 5.47 (m,
1, OCH), 6.98-7.52 (m, 8, ArH). Anal. (C17H16N2O2 ·H2C2O4) C,
H, N.
for 10 h. After removing the solvent under reduced pressure, the
residue was taken up in EtOAc, and the solution was washed with
brine. Removal of dried solvent gave 36 as an oil (0.46 g, 80%
yield). H NMR (CDCl3): δ 3.82 (s, 3, CH3), 4.27-4.58 (two dd,
2, OCH2), 4.88 (m, 1, OCH), 6.84-7.66 (m, 8, ArH).
Binding Assays. Cell Culture. Chinese hamster ovary cell lines
stably expressing cDNAs encoding human R2A-, R2B-, and R2C-AR
subtypes were produced by Pohjanoksa et al.22 using the expression
vector pMAMneo (Clontech, Palo Alto, CA) that contains a
neomycin (G418) resistance gene. The stable cell lines were cultured
in R-MEM (MEM Alpha Medium) supplemented with 26 mM
NaHCO3, 50 U/mL penicillin, 50 µg/mL streptomycin, and 5% heat-
inactivated fetal bovine serum supplemented with Geneticin (200
µg/mL). Cells were grown in a humidified incubator at 37 °C/5%
CO2.
1
Membrane Preparation and Ligand Binding. Cell membranes
were prepared as previously described.23 Briefly, > 90%
confluent CHO cells were washed twice with phosphate-buffered
saline (PBS), detached with trypsin, centrifuged at 130g for 5
min at 4 °C, and washed once with PBS. Cell pellets were
suspended in ice-cold homogenization buffer (10 mM Tris, 0.1
mM EDTA, 0.32 mM sucrose, pH 7.5), followed by homogeni-
zation with an Ultra-Turrax homogenizer. Homogenates were
centrifuged at 1400g for 15 min at 4 °C, and supernatants were
collected. Pellets were rehomogenized and centrifuged as before.
The pooled supernatants were centrifuged at 23300g for 30 min
at 4 °C. Membrane pellets were washed with sucrose-free Tris-
EDTA buffer, and the centrifugation procedure was repeated.
Membranes were resuspended in sucrose-free Tris-EDTA buffer,
aliquoted, and stored at -74 °C. Protein concentrations were
determined according to the method of Bradford using bovine
serum albumin as reference.32 Receptor densities were deter-
mined with saturation binding experiments as described previ-
ously24 using the R2-antagonist radioligand [3H]RS-79948-197
(0.021-4 nM). For each cell line, saturation binding experiments
were performed in triplicate and repeated at least three times.
Equilibrium dissociation constants (Kd) and receptor densities
(Bmax) were calculated from saturation binding data using
GraphPad Prism Software (San Diego, CA). Membranes ex-
pressing receptor densities of 1-3 pmol/mg total protein were
used for all experiments.
Competition Binding Assays. The competition binding assays
were carried out using a MultiScreen vacuum manifold system
(Millipore Corporation, Bedford, MA) with Millipore MultiScreen-
FB 96-well filtration plates. Experiments were performed in 50 mM
potassium phosphate buffer, pH 7.4, using [3H]RS79948-197 at
concentrations close to its affinity constant (Kd) (human R2A, 0.20
nM; human R2B, 0.12 nM; human R2C, 0.11 nM) for each receptor,
eight serial dilutions of the competitor ligands, and cell membrane
preparations with 5-10 µg of protein per sample. After 30 min
incubation at room temperature, reactions were terminated by rapid
vacuum filtration. Filters were then washed three times with ice-
cold buffer, dried, and impregnated with Super Mix cocktail (Wallac
Oy, Turku, Finland). The incorporated radioactivity was determined
by using a Wallac 1450 Betaplate scintillation counter (Wallac Oy,
Turku, Finland). Apparent affinity (apparent Ki) of each ligand was
determined using nonlinear regression analysis (GraphPad Prism),
assuming one-site binding. For conversion of IC50 into Ki values,
the Cheng-Prusoff equation was applied.25
2-[1-(2-Allyl-phenoxy)-ethyl]-4,5-dihydro-1H-imidazole (11). Simi-
larly, 11 was obtained from 28.19 The reaction mixture was purified
by flash chromatography eluting with cyclohexane/EtOAc/MeOH/
33%NH4OH (7:3:1:0.1) to give the free base (50% yield), which
was transformed into the oxalate salt; this was recrystallized from
2-PrOH: mp 154-155 °C. 1H NMR (DMSO): δ 1.53 (d, 3, CH3),
3.42 (m, 2, CH2CH), 3.88 (s, 4, NCH2CH2N), 5.06 (dd, 2,
CHdCH2), 5.40 (q, 1, CH), 5.95 (m, 1, CHdCH2), 6.92-7.26 (m,
4, ArH), 7.81 (br s, 1, NH, exchangeable with D2O). Anal.
(C14H18N2O·H2C2O4) C, H, N.
2-(2-Methoxy-biphenyl-3-yloxymethyl)-oxirane (32). A mixture
of 2-methoxy-biphenyl-3-ol20 (1.00 g, 5.00 mmol), epichlorohydrin
(1.17 mL, 15.00 mmol), and 2N NaOH (2.5 mL, 5.00 mmol) was
vigorously stirred and heated at 100 °C for 4 h. The mixture was
cooled and extracted with Et2O. The ethereal extracts were washed
with NaOH and water, dried over Na2SO4, and evaporated under
reduced pressure. The residue was then purified by flash cromatog-
raphy eluting with cyclohexane/EtOAc (8:2) to afford 32 as an oil
(0.81 g, 63% yield). 1H NMR (CDCl3): δ 2.82-2.95 (2 dd, 2,
OCH2), 3.45 (m, 1H, OCH), 3.64 (s, 3, OCH3), 4.05-4.38 (2 dd,
2, PhOCH2), 6.92-7.58 (m, 8, ArH).
3-(3-Bromo-2-hydroxy-propoxy)-biphenyl-2-ol (33). A stirred
solution of 32 (0.81 g, 3.15 mmol) was treated with an excess of
48% hydrobromic acid solution (8.45 mL) and then was heated at
100 °C for 30 min. The solution was extracted with CHCl3 and the
solvent was removed in vacuo. The resulting oil was purified by
flash cromatography eluting with cyclohexane/Et2O/EtOAc (7:3:
0.5) to afford 33 (0.6 g, 59% yield). 1H NMR (CDCl3): δ 3.04 (br
s, 1, CHOH exchangeable with D2O), 3.58 (2, m, CH2Br), 4.22 (3,
m, OCH2CH), 6.52 (br s, 1 ArOH exchangeable with D2O),
6.86-7.64 (8, m, ArH).
(8-Phenyl-2,3-dihydro-benzo[1,4]dioxin-2-yl)-methanol (34). A
mixture of 33 (0.6 g, 1.86 mmol) and NaOH (0.074 g, 1.86 mmol)
was vigorously stirred in water and heated at 100 °C for 4 h. The
mixture was cooled and extracted with Et2O. The ethereal extracts
were washed with 2N NaOH and water, dried over Na2SO4, and
evaporated under reduced pressure. The residue was then purified
by flash chromatography eluting with cyclohexane/EtOAc (7:3) to
afford 34 (0.257 g, 57% yield). 1H NMR (CDCl3): δ 1.68 (br s, 1,
OH exchangeable with D2O), 3.83 (m, 2, CH2OH), 4.15 (m,1,
OCH), 4.32 (m, 2, OCH2), 6.88-7.54 (m, 8, ArH).
8-Phenyl-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic acid (35).
KMnO4 (1.12 g, 7.1 mmol) was added to a stirred suspension of
34 (0.86 g, 3.55 mmol) in 0.3N KOH (4.5 mmol) at 5 °C. The
mixture was stirred for 12 h at room temperature, then MeOH was
added to destroy the excess of KMnO4 and MnO2 was removed by
filtration. After removal of MeOH in vacuo, the aqueous phase was
acidified with concentrated HCl and then extracted with CHCl3.
The organic phase was evaporated under reduced pressure to afford
Functional Assays. Cell Culture. Recombinant CHO cell clones
expressing R2-AR subtypes were produced by Dr. H. Paris as
previously described.11
Cytosensor Microphysiometry. Extracellular acidification was
measured using an eight-channel Cytosensor microphysiometry
instrument (Molecular Devices, Menlo Park, CA). CHO cells
expressing human R2-ARs 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.
1
35 (0.54 g, 59% yield). H NMR (CDCl3): δ 4.34-4.58 (two dd,
2, OCH2), 4.91 (m, 1, OCH), 6.87-7.63 (m, 8, ArH), 12.52 (br s,
1, COOH exchangeable with D2O).
8-Phenyl-2,3-dihydro-benzo[1,4]dioxine-2-carboxylic Acid Meth-
yl Ester (36). A suspension of 35 (0.54 g, 2.1 mmol) in MeOH (20
mL) was treated with conc. H2SO4 (0.2 mL) and heated at reflux

4298 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Gentili et al.
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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 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 seconds 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 experi-
ments with four known agonists, (-)-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. For antagonist studies, a control concentration-
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were then exposed to antagonist for at least 30 min prior to
construction of another clonidine concentration-effect curve in
the presence of the antagonist. Each chamber therefore acted as
its own control. Antagonist data were analyzed as the ability of
the antagonist to shift the agonist concentration-effect curve
and defined as Kb.
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 (GraphPad Software, San Diego, CA).
The values of Kb were calculated as M/concentration ratio-1, where
concentration ratio is the EC50 obtained in the presence of the
antagonist divided by that obtained in the absence of the antago-
nist.28 Data were expressed as pKb [-log 10(Kb)]. The results are
expressed as means ( SEM of three to six separate experiments.
Molecular Modeling. Molecular superposition of 5, 15, and 18
was performed by means of the TFIT module implemented in the
QXP software package.34 All compounds were built in their
protonated state using the fragment library of the same software,
and the internal geometry of all the ligands was randomly perturbed
through 100000 cycles of conformational search. The TFIT
procedure is based on a mixed AMBER/MM2 force field, a
superposition force field, a Monte Carlo conformational search, and
a rigid body alignment algorithm. QXP automatically assigns short-
range attractive forces between similar atoms in different molecules.
Atoms are defined to be “similar” on the basis of their chemical
features. The typical intramolecular nonbonded energies are replaced
by the superposition energies, while internal energies (Eint) are
calculated by the normal force field by ignoring nonbonded energies.
The combined minimization of these two energies (Esup and Eint)
yielded structures with optimal superposition and relatively low
internal energy. Within a defined energy range, the program affords
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Acknowledgment. We thank the MIUR (Rome), the Uni-
versity of Camerino, and the Academy of Finland for financial
support.
Supporting Information Available: Elemental analysis of the
final compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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