Discovery of diaryl imidazolidin-2-one derivatives, a novel class of muscarinic M3 selective antagonists (Part 2)

Article abstract of DOI:10.1021/jm061160+

Synthesis and biological activity of a novel class of quaternary ammonium salt muscarinic M3 receptor antagonists, showing high selectivity versus the M2 receptor, are described. Selected compounds exhibited potent anticholinergic properties, in isolated guinea-pig trachea, and good functional selectivity for trachea over atria. In vivo, the same compounds potently inhibited acetylcholine-induced bronchoconstriction after intratracheal administration in the guinea pig.

Full text of DOI:10.1021/jm061160+

J. Med. Chem. 2007, 50, 1693-1697
1693
Brief Articles
Discovery of Diaryl Imidazolidin-2-one Derivatives, a Novel Class of Muscarinic M3 Selective
Antagonists (Part 2)
Ilaria Peretto,*,† Claudia Fossati,† Giuseppe A. M. Giardina,† Alessandra Giardini,† Matilde Guala,† Elena La Porta,†
Paola Petrillo,† Stefano Radaelli,† Luigi Radice,† Luca F. Raveglia,† Enza Santoro,† Roberta Scudellaro,† Francesca Scarpitta,†
Alberto Cerri,† Sergio Menegon,† Giulio M. Dondio,† Andrea Rizzi,‡ Elisabetta Armani,‡ Gabriele Amari,‡ Maurizio Civelli,‡
Gino Villetti,‡ Riccardo Patacchini,‡ Marco Bergamaschi,‡ Franco Bassani,‡ Maurizio Delcanale,‡ and Bruno P. Imbimbo*,‡
NiKem Research, Via Zambeletti 25, 20021 Baranzate, Milan, Italy, and Research and DeVelopment, Chiesi Farmaceutici S.p.A.,
Via Palermo 26/A, 43100 Parma, Italy
ReceiVed October 6, 2006
Synthesis and biological activity of a novel class of quaternary ammonium salt muscarinic M3 receptor
antagonists, showing high selectivity versus the M2 receptor, are described. Selected compounds exhibited
potent anticholinergic properties, in isolated guinea-pig trachea, and good functional selectivity for trachea
over atria. In vivo, the same compounds potently inhibited acetylcholine-induced bronchoconstriction after
intratracheal administration in the guinea pig.
Introduction
compound with subnanomolar M3 affinity and long-lasting
bronchodilating activity. Unfortunately, tiotropium bromide
binds with similar subnanomolar affinity to the human musca-
rinic M2 receptor subtype (although it displays kinetic selectivity
toward the M2 receptor).4 Thus, new quaternary ammonium
muscarinic antagonists with selectivity for M3 over M2 receptors
may provide more ideal anticholinergic therapy for both asthma5
and COPD.6
Muscarinic receptors modulate the activity of an extraordinar-
ily large number of physiological functions. Individual musca-
rinic receptor subtypes are expressed in a complex, overlapping
fashion in most tissues and cell types.1 In human airways, in
particular, M2 and M3 receptor subtypes are expressed and
differentially distributed. M2 receptors are localized on the
postganglionic cholinergic nerve terminals and provide a
functional negative feedback of acetylcholine (Ach) release. M3
receptors are localized on effector cells, smooth muscle, mucosal
glands, and blood vessels and mediate bronchoconstriction,
mucus secretion, and vasodilatation.2 In asthma and chronic
obstructive pulmonary disease (COPD), bronchoconstriction and
mucus secretion are increased and the airways are hyper-
responsive to contractile agents as a result of increased
parasympathetic nerve activity. The number and function of
post-junctional M3 muscarinic receptors in the airways are
unchanged; rather, it is the supply of Ach to the post-junctional
effector cells (smooth muscle and submucosal gland) that is
increased. The increase in Ach release occurs because prejunc-
tional, inhibitory M2 muscarinic receptors on the parasympa-
thetic nerves are dysfunctional.3 Therefore, while inhibition of
M3 receptors is useful to limit bronchoconstriction and mucus
secretion, inhibition of M2 muscarinic receptors at the airways
level may potentiate bronchoconstriction.
Chemistry
Following the discovery of a class of tertiary amine deriva-
tives as selective M3 antagonists,1 we considered the possibility
of derivatization of these tertiary amines as quaternary am-
monium salts to obtain a class of compounds more suitable for
the topical treatment of pulmonary diseases. Starting from the
imidazolidinone core structure previously identified (Scheme
1), several possible structural modifications were considered and
implemented.
The general synthetic pathway employed for the synthesis
of all the derivatives described in this paper is outlined in
Scheme 2.
Intermediates 3a-i were synthesized utilizing procedures
previously described for analogue compounds.1 Reduction of
the hydantoin moiety with Red-Al (sodium bis-(2-methoxy-
ethoxy)aluminum hydride), followed by reaction with the
appropriate alkylating agent, allowed obtaining the imidazolidin-
2-one final compounds 4-34. Substitution at the basic nitrogen
with two different groups (R3 and R4) resulted in the formation
of isomers that were not separated. If another stereogenic center
was already present in the molecule, the final compounds were
obtained as a mixture of diastereoisomers that could not be easily
separated by chromatography and were tested as such.
Quaternary ammonium muscarinic M3 antagonists have been
successfully used by inhalation to antagonize bronchoconstric-
tion in asthma and COPD, because their fixed positive charge
assures a poor absorption in the systemic circulation, thus
limiting the side effects. The clinically most effective quaternary
ammonium muscarinic antagonist is tiotropium bromide, a
* To whom correspondence should be addressed. Bruno P. Imbimbo for
editorial communication and pharmacology and Ilaria Peretto for medicinal
chemistry. Phone: +39 0521 279278 (B.P.I.); +39 02 356947587 (I.P.).
Fax: +39 0521 279579 (B.P.I.); +39 02 356947606 (I.P.). E-mail:
b.imbimbo@chiesigroup.com (B.P.I.); ilaria.peretto@nikemresearch.com
(I.P.).
Biology
The binding affinity of the compounds toward the muscarinic
M3 and M2 receptors was evaluated in membranes of
CHO-K1 clone cells expressing the human M2 or M3 receptors.
The nonselective muscarinic radioligand [3H]-N-methyl scopo-
† NiKem Research.
‡ Chiesi Farmaceutici S.p.A.
10.1021/jm061160+ CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/13/2007

1694 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Brief Articles
Scheme 1. Planned Chemical Variations on Imidazolidinone Scaffold
Scheme 2. General Synthetic Pathway for Imidazolidinone Derivatives
Scheme 3. Compound 35 Selected as Initial Hit
were also considered, such as in compounds 12-15; none of
these compounds displayed a significant improvement of activity
if compared to the cyclic derivatives.
We then investigated in more detail the substitution patterns
at the quaternary nitrogen atom, selecting the pyrrolidine
scaffold as template. In fact, among the compounds reported in
Table 1, pyrrolidine derivatives maintained an appreciable
affinity both in the presence of the dimethyl- and the methyl-
benzyl substitution (compounds 9 and 10); thus, these derivatives
might better tolerate diverse substituents on the quaternary
nitrogen atom.
lamine was used to label the M2 and M3 binding sites, while
the nonspecific binding was determined in the presence of cold
N-methyl scopolamine.
As a first attempt, we considered the phenethyl-methyl
derivative 16 (Table 2), which surprisingly displayed a signifi-
cant improvement of activity (M3 Ki ) 0.67 nM) if compared
to the corresponding benzyl-methyl derivative 8 (M3 Ki ) 727
nM). We further investigated the phenethyl moiety by insertion
of additional carbon or oxygen atoms in the side chain
(compounds 17-20), but neither affinity nor selectivity were
improved. The importance of the aromatic ring in the side chain
was assessed with compound 21, featuring the 3,3-dimethyl-
butan-2-one moiety, which proved to be completely inactive.
On the other hand, the corresponding acetophenone derivative
22, maintaining the phenyl moiety, displayed a good affinity
for the M3 receptor (Ki ) 1.89 nM) and the best selectivity
observed so far in this class of quaternary ammonium salts (16.7-
fold). Other compounds (23 and 24) featuring the phenyl moiety
and a hydrogen-bond acceptor group in a different substitution
pattern within the side chain did not result in significant
improvement in the activity or the selectivity.
Replacement of the methyl group on the quaternary nitrogen
atom with cyanomethyl or methoxyethyl moieties was detri-
mental for affinity, as shown by compounds 25 and 26 (M3 Ki
) 541 nM and 356 nM, respectively).
To confirm the importance of the stereochemistry at the
pyrrolidine C3 stereogenic center, compounds 27 and 28,
consisting of (R)- and (S)-isomers, were synthesized and tested.
The (S)-isomer 28 displayed M3 Ki ) 7.60 nM and 20-fold
selectivity toward the M2 receptor, while the (R)-isomer 27 was
the more active (M3 Ki ) 1.04 nM) although poorly selective
(only 2.3-fold); the (R)-isomer was then considered as privileged
scaffold due to its higher binding affinity.
In vitro functional activity of selected test compounds against
M3 and M2 receptors was investigated in isolated guinea-pig
trachea and atria, respectively. Ach-induced bronchoconstriction
in anaesthetized guinea pig was used to investigate in vivo
potency of the most interesting compounds.
SAR Discussion
In a previous paper1 we described the discovery of a class of
tertiary amines as selective M3 antagonists. The hit com-
pound 35 displayed good M3 binding affinity (Ki ) 1.9 nM)
and also quite good selectivity toward the M2 receptor (40-
fold; Scheme 3).
We considered the possibility of derivatization of these
tertiary amines as quaternary ammonium salts. Compound 4
(Table 1), unfortunately, proved to be only weakly active (M3
Ki ) 612 nM), and the dimethyl quaternary ammonium salt 5
was only slightly more active (M3 Ki ) 249 nM). We then
considered different cycle sizes and different substitution
patterns on the basic moiety. Introduction of tropine did not
significantly improve binding affinity: derivative 6, bearing the
methyl-benzyl substitution, was almost inactive, while the
corresponding dimethyl derivative 7 displayed a better affinity
for the M3 receptor (Ki ) 153 nM). Pyrrolidinyl derivative 8,
despite being a mixture of four diastereoisomers, was almost
as active as compound 4 (Ki ) 727 nM). Fixing the stereo-
chemistry at the pyrrolidine stereogenic center resulted in a
significant separation of activity: compound 10 (R stereochem-
istry at this center) showed M3 Ki ) 282 nM, while compound
11 (S stereochemistry) was almost inactive. Linear basic moieties

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1695
Table 1. Modifications of the Basic Moiety and of the Substituents on
Table 2. Investigation of Substitution on the Basic Nitrogen:
the Basic Nitrogen
Pyrrolidine Derivatives
a When the final compounds were obtained as a mixture of stereoisomers,
they were not separated and were tested as such. b Inactive: Percent of
control binding <50% at the concentration of 10 µM.
Table 3. Difluorophenyl Imidazolidin-2-one Derivatives: Investigation
of the Substituents on the Basic Nitrogen
a When the final compounds were obtained as a mixture of stereoisomers,
they were not separated and were tested as such. b Inactive: Percent of
control binding <50% at the concentration of 10 µM.
Introduction of 4-fluoro substituents on the phenyl rings of
the imidazolidinone scaffold as in compound 29 (Table 3),
featuring the methyl-phenethyl substitution on the basic nitrogen,
maintained good binding affinity (M3 Ki ) 0.75 nM) and
significantly improved the selectivity toward the M2 receptor
(almost 30-fold selective). This finding suggested combining
the same difluoro substitution pattern with the best substituent
on the quaternary nitrogen atom in terms of selectivity, that is,
the acetophenone side chain; the resulting compound 30
displayed M3 Ki ) 1.84 nM and 51-fold selectivity toward the
M2 receptor.
Introduction of 3-fluoro substitution on the acetophenone
moiety (compound 31) did not improve activity or selectivity.
Replacement of the phenyl ring of acetophenone with the
heterocycles was then considered. Thienyl derivative 32 was
as active and selective as the corresponding acetophenone
derivative 30. Conversely, pyridyl derivatives proved to be more
interesting, and the position of the basic pyridine nitrogen proved
a When the final compounds were obtained as a mixture of stereoisomers,
they were not separated and were tested as such.
to be crucial for affinity and selectivity. In fact, 2-pyridyl
derivative 34 maintained good activity (M3 Ki ) 2.61 nM) and
displayed excellent selectivity toward the M2 receptor (100-

1696 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Brief Articles
Table 4. In Vitro Functional Selectivity and In Vivo Potency of
Selected Compounds in Guinea Pig
thermore, compounds 29, 31, and 32, after intratracheal
administration, potently inhibited Ach-induced bronchocon-
striction in guinea pigs, suggesting that these compounds could
provide efficient bronchodilator activity with minimal side
effects for the treatment of obstructive airway diseases.
isolated trachea
(IC50, nM)
left atria
(IC50, nM)
selectivity in vivo potencya
cmpd
(M2/M3)
(ID50 nmol/kg)
29
31
32
4.5
7.8
7.6
140
1000
880
31.1
128.2
115.8
1.04
3.86
5.76
Experimental Section
a The ID50 is the dose producing 50% inhibition of Ach-induced
bronchoconstriction.
1
Chemistry. H NMR spectra were recorded on a Bruker ARX
300 (300 MHz); chemical shifts are reported downfield in parts
per million (ppm) relative to TMS, utilizing the solvent peaks as
the reference. EI mass spectra were recorded on a Thermo Finnigan
TSQ700 spectrometer; ESI-LCMS analysis was performed on a
Phenomex Luna C-18, 3 µm, 4.6 × 50 mm column, with an AQA
Thermo Finnigan single quadrupole instrument or with a Waters
Micromass ZQ2000 instrument; high performance liquid chroma-
tography (HPLC) analysis was performed on a Shimadzu SCL-
10A equipped with an SIL-10AD injector and an SPD-M10A
detector normally operating in a 200-360 nm range, with a Waters
Symmetry C-18, 3.5 µm, 4.6 × 75 mm column, using a 10 min
gradient of 0-100% solvent B, where solvent A is 90:10:0.05
CH3CN-H2O-TFA and solvent B is 90:10:0.05 H2O-CH3CN-
TFA. Reactions were monitored by TLC using 0.25 mm Merck
silica gel plates (60 F254); column chromatography was performed
on Merck silica gel 60 (particle size 0.063-0.2 mm); and flash
chromatography was conducted using a Biotage-Quad3 apparatus
and prepacked silica gel columns (KP-SIL, particle size 32-60 µm).
Anhydrous solvents were purchased from Aldrich and used as
received. “Brine” refers to a saturated aqueous solution of NaCl.
Unless otherwise specified, solutions of common inorganic salts
used in workups are aqueous solutions.
Synthetic Procedures. General Procedure 1. Mitsunobu
Reaction. 3-(1-Methyl-piperidin-4-yl)-5,5-diphenyl-imidazolidin-
2,4-dione (3a). Commercially available 5,5-diphenyl hydantoin
(phenytoin, 0.50 g, 1.98 mmoles) is dissolved in dry THFa (20 mL);
triphenyl phosphine (0.78 g, 2.97 mmoles) and 4-hydroxy-N-methyl
piperidine (0.342 g, 2.97 mmoles) are added to the reaction mixture.
The resulting solution is cooled to 0 °C and DEAD (0.47 mL, 2.97
mmoles) is added dropwise. The reaction is then stirred at room
temperature for 24 h. The solvent is evaporated under vacuum, and
the product is purified by chromatography on silica gel (500 g,
eluent: AcOEt/hexane ) 2:8 to AcOEt) to yield 0.8 g of pure
product as a white solid. LC-MS (ESI pos): 350.52 (MH+).
Figure 1. Effect of compounds 29, 31, and 32 in the isolated guinea-
pig trachea assay. Isoprenaline-induced relaxation was used as the
maximal inhibitory reference effect.
fold selectivity), while 3-pyridyl derivative 33 showed a
significant loss of activity (M3 Ki ) 22.9 nM).
The functional selectivity of selected compounds was inves-
tigated in vitro by studying their ability to inhibit carbachol-
induced contractions in guinea-pig trachea and methacholine-
induced bradycardia in the guinea-pig left atria (Table 4).
Compounds 29, 31, and 32 showed concentration-dependent
inhibition of carbachol-induced contraction in guinea-pig tra-
chea, with IC50 values of 4.5, 7.8, and 7.6 nM, respectively
(Figure 1). Furthermore, the same compounds showed good to
high functional selectivity toward the M2 receptor, determined
in the in vitro assay on guinea-pig isolated atria, thus confirming
that these quaternary ammonium salts act as potent and selective
functional antagonists at the M3 receptor.
Finally, compounds 29, 31, and 32 were tested in vivo in
anesthetized guinea pig for their ability to inhibit bronchocon-
striction produced by i.v. injection of Ach after intratracheal
administration (Table 4). All the compounds showed a potent
bronchodilator activity, with ID50 values of 1.04, 3.86, and 5.76
nmol/kg, respectively.
Compounds 3c-i were synthesized, following the same proce-
dure, starting from the corresponding hydantoin derivatives.
3-(1-Methyl-tropin-4-yl)-5,5-diphenyl-imidazolidine-2,4-di-
one (3b). (a) Synthesis of Methanesulfonic Acid 8-Methyl-8-
aza-bicyclo[3.2.1]oct-3-yl Ester (Tropine Mesilate). Commercially
available tropine (0.150 g, 1.063 mmoles) is added to a solution of
triethyl amine (0.207 mL, 1.5 mmoles) in dry DCM (10 mL); the
resulting mixture is cooled to 0 °C and mesyl chloride (0.099 mL,
1.276 mmoles) is added. The reaction is stirred at 0 °C for 1 h,
then the solvent is evaporated in vacuum and the product obtained
is employed in the next step without purification.
(b) Synthesis of 3-(8-Methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-5,5-
diphenyl-imidazolidin-2,4-dione (3b). Commercially available 5,5-
diphenyl hydantoin (phenytoin, 0.20 g, 0.793 mmoles) is dissolved
in dry DMF (10 mL), and K2CO3 (0.33 g, 2.38 mmoles) and
N-methyl-tropine mesilate (prepared in the previous step) dissolved
in dry DMF (3 mL) are added. The reaction is then stirred at 80
°C for 18 h. The reaction mixture is diluted with water and extracted
with ethyl acetate; the organic phase is washed with water, then
with brine, and finally dried and concentrated under vacuum to
give a yellowish solid that is crystallized from ethyl ether to give
the desired product as a white solid (0.18 g). LC-MS (ESI pos):
Conclusions
A novel class of M3 receptor antagonists was discovered,7
characterized by the moiety of a quaternary ammonium salt,
suitable for the topical treatment of pulmonary diseases.
Compounds with binding affinity in the low nanomolar range
were obtained, with up to 100-fold selectivity toward the M2
receptor. Functional in vitro studies on selected compounds
demonstrated high subtype selectivity for tracheal M3 over
cardiac M2 receptors in the isolated guinea-pig tissues. Fur-
1
376.10 (MH+). H NMR (CDCl3): 7.41-7.29 (m, 10H), 5.89 (s
a Abbreviations: DCM, dichloromethane; DEAD, diethyl azodicarboxy-
late; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; THF,
tetrahydrofuran.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1697
br, 1H), 4.45 (m, 2H), 3.53 (m, 2H), 2.72 (s, 3H), 2.33 (m, 2H),
1.82 (m, 2H), 1.27 (m, 2H).
counteract bronchoconstriction produced by i.v. injection of Ach,
20 µg kg-1. Bronchoconstriction, quantified as a reduction of tidal
volume, was evaluated according to the method of Konzett and
Roessler.9 Ach challenge was applied at 5, 15, and 30 min and
then every 30 min up to 180 min from the intratracheal administra-
tion of test compounds. The effect of test compounds was expressed
as percentage of inhibition of Ach-evoked bronchoconstriction, as
compared to basal response obtained in control animals. Ethical
approval of the experimental protocols with animals (in vitro and
in vivo) was obtained from the local Ethics Committee.
General Procedure 2. Reduction with Red-Al and Alkylation.
Synthesis of 1-Benzyl-1-methyl-4-(2-oxo-4,4-diphenyl-imidazo-
lidin-1-yl)-piperidinium (4). (1) 3-(1-Methyl-piperidin-4-yl)-5,5-
diphenyl-imidazolidin-2-one. A 3.5 M solution of sodium bis-(2-
methoxyethoxy)aluminum hydride (Red-Al) in toluene (5.37 mL,
18.4 mmoles) is dissolved in dry THF (15 mL) under nitrogen
atmosphere and cooled to 0 °C. A solution of compound 3a (0.803
g, 2.3 mmoles) in dry THF (20 mL) is added dropwise. The mixture
is then heated to 85 °C for 4 h. The reaction is cooled again to 0
°C and quenched with water (5 mL); then 2 M sodium hydroxide
is added (10 mL) and the mixture is extracted with ethyl acetate;
and the organic phase is washed with water, then with brine, and
finally dried and concentrated under vacuum to give a yellowish
solid that is crystallized from acetone to give the desired product
Acknowledgment. This study was supported by Chiesi
Farmaceutici, Parma, Italy. The authors thank Dr. Samuele
Pedraglio and Dr. Chiara Bigogno (NiKem Research) for the
analytical support.
1
as a white solid (0.65 g). LC-MS (ESI POS): 336.20 (MH+). H
Supporting Information Available: Spectroscopic data and
elemental analyses for compounds 3a-i and 4-34. This material
is available free of charge via the Internet at http://pubs.acs.org.
NMR (DMSO; 343 K): 7.80 (s br, 1H), 7.40-7.21 (m, 10H), 3.91
(s, 2H), 3.80 (m, 1H), 3.41 (m, 2H), 3.09 (m, 2H), 2.72 (s, 3H),
2.01 (m, 2H), 1.80 (m, 2H).
References
(2) 1-Benzyl-1-methyl-4-(2-oxo-4,4-diphenyl-imidazolidin-1-
yl)-piperidinium (4). 3-(1-Methyl-piperidin-4-yl)-5,5-diphenyl-
imidazolidin-2-one (0.08 g, 0.24 mmoles) is dissolved in CH3CN
(4 mL) in a microwave tube. Benzyl bromide (0.164 g, 0.96 mmol)
is added, the tube is sealed, and the solution is heated at 120 °C in
a microwave oven for 2 h. The solvent is evaporated and the crude
compound is triturated with i-Pr2O/DCM. The desired product 4 is
obtained as a pale yellow solid (0.08 g). LC-MS (ESI pos): 426.18
(1) Peretto, I.; Forlani, F.; Fossati, C.; Giardina, G. A. M.; Giardini, A.;
Guala, M.; La Porta, E.; Petrillo, P.; Radaelli, S.; Radice, L.; Raveglia,
L. F.; Santoro, E.; Scudellaro, R.; Scarpitta, F.; Bigogno, C.; Misiano,
P.; Dondio, G. M.; Rizzi, A.; Armani, E.; Amari, G.; Civelli, M.;
Villetti, G.; Patacchini. R.; Bergamaschi, M.; Delcanale, M.; Salcedo,
C.; Ferna´ndez, A. G.; Imbimbo, B. P. Discovery of diaryl imidazo-
lidin-2-one derivatives, a novel class of muscarinic M3 selective
antagonists (Part 1) J. Med. Chem. 2007, 50, 1571-1583 and
references therein.
(2) Minette, P. A.; Barnes, P. J. Muscarinic receptor subtypes in lung.
Clinical implications. Am. ReV. Respir. Dis. 1990, 141, S162-S165.
(3) Coulson, F. R.; Fryer, A. D. Muscarinic acetylcholine receptors and
airway diseases. Pharmacol. Ther. 2003, 98, 59-69.
(4) Saberi, F.; O’Donnell, D. E. The role of tiotropium bromide, a long-
acting anticholinergic bronchodilator, in the management of COPD.
Treat. Respir. Med. 2005, 4, 275-281.
(5) Bowerfind, W. M.; Fryer, A. D.; Jacoby, D. B. Double-stranded RNA
causes airway hyperreactivity and neuronal M2 muscarinic receptor
dysfunction. J. Appl. Physiol. 2002, 92, 1417-1422.
(6) On, L. S.; Boonyongsunchai, P.; Webb, S.; Davies, L.; Calverley, P.
M.; Costello, R. W. Function of pulmonary neuronal M2 muscarinic
receptors in stable chronic obstructive pulmonary disease. Am. J.
Respir. Crit. Care Med. 2001, 163, 1320-1325.
(7) Peretto, I., Scarpitta, F., La Porta, E., Raveglia, L. F., Giardina, G.
A. M., Imbimbo, B. P., Rizzi, A., Villetti, G. Preparation of
imidazolone derivatives and heteroanalogs with antimuscarinic activ-
ity. International patent WO 06066924, June 29, 2006.
(8) Villetti, G.; Bergamaschi, M.; Bassani, F.; Bolzoni, P. T.; Harrison,
S.; Gigli, P. M.; Janni, A.; Geppetti, P.; Civelli, M.; Patacchini, R.
Pharmacological assessment of the duration of action of glycopyr-
rolate vs tiotropium and ipratropium in guinea-pig and human
airways. Br. J. Pharmacol. 2006, 148, 291-298.
(9) (a) Konzett, H.; Roessler, R. Versuchanornungzu untersuchungen an
der bronchialmuskulatur. Arch. Exp. Path. Pharmak. 1940, 195, 71-
74. (b) Salcedo, C.; Davalillo, S.; Catena, J.; Enrich, E.; Ferna´ndez-
Serrat, A.; Miquel, I.; Sanagust´ın, J.; Farrerons, C.; Lagunas, C.;
Balsa, D.; Ferna´ndez, A. G. Functional characterization of SVT
40776, a novel and potent M3 receptor antagonist, on mice detrusor
and atria isolated preparations using in vitro and ex vivo protocols.
Br. J. Pharmacol. 2002, 136 (suppl), 45P. (c) Lambrecht, G.; Feiferi,
R.; Wagner-Roder, M.; Strohmann, C.; Zilch, H.; Tacke, R.;
Wallbroeck, M.; Christophe, J.; Boddeke, H.; Mutschler, E. Affinity
profiles of hexahydro-siladefenidol analogues at muscarinic receptor
subtypes. Eur. J. Pharmacol. 1989, 273, 959-966.
1
(MH+). H NMR (CDCl3): 7.66-7.21 (m, 15H), 5.00 (s br, 1H),
4.13 (m, 2H), 4.09 (s, 2H), 3.92 (m, 2H), 3.56 (m, 2H), 3.23 (s,
3H), 2.59 (m, 2H), 2.00 (m, 2H).
All the quaternary ammonium salts reported in Tables 1-3 were
synthesized following the same procedure starting from the corre-
sponding tertiary amine and the appropriate alkylating agent.
Biology. Cell lines and membrane preparations and radioligand
binding conditions are as described previously.1
Isolated Guinea-Pig Trachea. Tracheal zigzag preparations were
obtained from tracheal segments excised from male albino Dunkin-
Hartley guinea pigs (450-550 g, Charles River Laboratories Italia,
Calco, Italy) and set up as described previously.8 Test compounds
were assayed for their ability to relax a smooth muscle tone raised
by a submaximally effective concentration (0.3 µM) of carbachol.
The antagonist concentration producing a 50% reversal of carba-
chol-induced tonic contraction (IC50) was taken as a measure of
antagonist potency. Isoprenaline-induced relaxation was used as
the maximal inhibitory reference effect.
Isolated Guinea-Pig Left Atrium. The hearts were rapidly
removed from male albino Dunkin-Hartley guinea pigs (325 (
25 g), and the left and right atria were separately excised. The left
atria were mounted with a preload of 0.5 g in McEwens, pH 7.4.
The solution was maintained at 32 °C, and tissues were stimulated
by the application of methacholine 1 µM. Negative ionotropic
responses to the addition of methacholine were recorded as changes
in isometric tension, as previously reported.9 Dose-response curves
were obtained for selected test compounds.
In Vivo Bronchoconstriction in Guinea Pig. All in vivo
experiments were performed on male albino Dunkin-Hartley
guinea pigs (450-550 g, Charles River Laboratories Italia, Calco,
Italy), as described previously.8 Test compounds (given intratra-
cheally to anesthetized animals) were assayed for their ability to
JM061160+