Discovery of diaryl imidazolidin-2-one derivatives, a novel class of muscarinic M3 selective antagonists (Part 1)

Article abstract of DOI:10.1021/jm061159a

Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives (potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds, selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound 50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor K_i = 4.8 nM and for M2 receptor K _i = 1141 nM. Functional in vitro studies on selected compounds confirmed the antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.

Full text of DOI:10.1021/jm061159a

J. Med. Chem. 2007, 50, 1571-1583
1571
Discovery of Diaryl Imidazolidin-2-one Derivatives, a Novel Class of Muscarinic M3 Selective
Antagonists (Part 1)
Ilaria Peretto,*,† Roberto Forlani,† 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,† Chiara Bigogno,† Paola Misiano,† Giulio M. Dondio,† Andrea Rizzi,‡ Elisabetta Armani,‡ Gabriele Amari,‡
Maurizio Civelli,‡ Gino Villetti,‡ Riccardo Patacchini,‡ Marco Bergamaschi,‡ Maurizio Delcanale,‡ Carolina Salcedo,§
Andre´s G. Ferna´ndez,§ and Bruno P. Imbimbo*,‡
NiKem Research, Via Zambeletti 25, 20021 Baranzate, Milan, Italy, Research and DeVelopment, Chiesi Farmaceutici S.p.A., Via Palermo 26/A,
43100 Parma, Italy, and Preclinical Research and DeVelopment, Laboratorios SALVAT, Gall 30-36, 08950-Esplugues de Llobregat,
Barcelona, Spain
ReceiVed October 6, 2006
Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class
of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility
of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two
distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the
treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives
(potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this
paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds,
selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is
mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound
50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor Ki ) 4.8 nM
and for M2 receptor Ki ) 1141 nM. Functional in vitro studies on selected compounds confirmed the
antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.
Introduction
Incontinence due to bladder hypercontractility has been
demonstrated to be mediated through increased stimulation of
M3 receptor subtype. Human bladder contains a mixed popula-
tion of M2 and M3 receptors. Although M2 receptors predomi-
nate, the contractile response is mediated through M3 recep-
tors,5,15 and the role of M2 receptors is of minor importance.6
In asthma and in chronic obstructive pulmonary disease
(COPD), inflammatory conditions lead to loss of inhibitory M2
muscarinic Ach autoreceptor function on parasympathetic nerves
supplying the pulmonary smooth muscle, causing increased Ach
release following vagal nerve stimulation. This dysfunction
results in airway hyperreactivity mediated by increased stimula-
tion of M3 receptors.7
On the other hand, it is well-known that M2 muscarinic
receptors strongly regulate vagal control of heart rate and
contraction:8 interaction with this receptor subtype could then
lead to significant adverse effects.
Even if muscarinic agonists (pilocarpine4) and antagonists
(atropine,4 oxybutynin9) have been known for over a century,
little progress has been made in the discovery of receptor
subtype selective compounds, and relatively few antimuscarinic
compounds are in use in the clinic as a result of significant
side effects. There is still, therefore, the need for highly selective
muscarinic antagonists that can interact with distinct subtypes,
thus avoiding the occurrence of adverse effects.
Acetylcholine (Ach) acts on muscarinic and nicotinic recep-
tors in central and peripheral nervous systems. Five distinct but
homologous gene sequences coding for muscarinic receptors
(m1, m2, m3, m4, and m5) have been identified and cloned.1
Muscarinic receptors are predominantly expressed in the
parasympathetic nervous system and modulate the activity of
an extraordinarily large number of physiological functions.2
Individual muscarinic receptor subtypes are expressed in a
complex, overlapping fashion in most tissues and cell types.
However, studies using knockout mice have clarified the main
role of each individual muscarinic receptor subtype in a number
of physiological functions.3 M1 receptors modulate neurotrans-
mitter signaling in cortex and hippocampus. The M2 subtype
mediates bradycardia, tremor, hypothermia, and autoinhibition
in several brain regions. M3 receptors are involved in smooth
muscle contractility, exocrine gland secretion, pupil dilatation,
food intake, and weight gain. M4 receptors modulate dopamine
activity in motor tracts and act as inhibitory autoreceptors in
striatum. The role of the M5 receptors involves modulation of
central dopamine function and the tone of cerebral blood vessels.
Muscarinic M3 receptor dysfunction has been noted in various
pathophysiological states.4
Inflammation of the gastrointestinal tract in irritable bowel
syndrome results in M3-mediated hypermotility.
The aim of our study was the identification of novel M3
antagonists selective toward M2 receptors, suitable for oral
treatment of M3-related dysfunctions.
* 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.).
Pharmacophore Generation
As for many other G-protein-coupled receptors, the structure
of the muscarinic M3 receptor is still not known. Although
several computational approaches could be pursued to identify
† NiKem Research.
‡ Chiesi Farmaceutici S.p.A.
§ Laboratorios SALVAT.
10.1021/jm061159a CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/13/2007

1572 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Peretto et al.
Scheme 1. Compounds in the Training Set Used to Build the M3 Pharmacophore Hypotheses
Scheme 2. Compounds in the Validation Set Used to Prioritize
the M3 Pharmacophore Hypotheses
Scheme 3. Compound 22a Identified as the Starting Hit
Compound
The final list of compounds was prioritized in terms of fit
score, conformer energy, and number of omitted features.
Moreover, priority was given to noncommercial compounds to
identify novel and thus patentable M3 antagonists. Considering
then chemical feasibility and tractability, some of the identified
compounds were selected for chemical synthesis and biological
testing.
Among all the compounds tested, compound 22a (Scheme
3) proved to be surprisingly active, despite missing one hydrogen
bond acceptor feature (Figure 3); it was then selected as a
starting point for further optimization.
novel, potent, and selective M3 antagonists, in this case, the
ligand-based design was chosen in view of the significant
amount of information related to the structures of M3 receptor
antagonists already available in the literature.
With the aim of defining the pharmacophoric features needed
to identify M3 antagonists, a pharmacophore hypothesis was
generated using Catalyst HipHop algorithm,10,11 starting from
the set of known muscarinic M3 receptor antagonists (training
set) reported in Scheme 1. Tiotropium (compound 1) was chosen
as the reference compound in our study, because it is a
recognized “gold standard” as anticholinergic agent in respira-
tory diseases.12 Compound 213 was chosen as a conformationally
constrained analogue of tiotropium: this compound could help
better define the geometric orientation of the two aromatic
regions. Compound 314 was selected as another analogue of
tiotropium, in which the basic feature is differently connected
to the aromatic region and differently substituted. Several
common features of pharmacophore hypotheses were generated
and prioritized on the basis of their ability to correctly map a
validation set consisting of known muscarinic antagonists
(Scheme 2).15-20
When compound 22a pharmacophore mapping (Figure 3)
from a medicinal chemistry perspective was considered, a wide
number of chemical modifications around the imidazolidin-2-
Finally, visual selection of the best pharmacophore hypothesis
was performed on the basis of the best mapping of both the
training and the validation compounds. The best pharmacophore
hypothesis presented five features: two aromatic hydrophobic
features, two hydrogen bond acceptor groups and a positive
ionizable group. Pharmacophore mapping of training and
validation compounds are reported in Figures 1 and 2.
The selected pharmacophore model was then utilized to search
for the identified M3 antagonist key pharmacophoric features
over CAP screening21 and Maybridge22 three-dimensional (3D)
databases.23 In addition, a virtual collection of compounds
designed by our medicinal chemists on the basis of structural
similarity with known M3 antagonists was included in the 3D
searching step.
Figure 1. Training set compounds mapped on the selected M3
pharmacophore model. The light blue feature is an aromatic hydro-
phobic feature. The green feature is a hydrogen bond acceptor. The
red feature is a positive ionizable group.
Following the usual Catalyst 3D database screening cascade
and allowing virtual hits to miss one of the defined pharma-
cophore features, 870 commercial compounds and 58 derivatives
from the virtual collection were extracted and filtered to satisfy
Lipinski’s rule of five test for drug likeness and Oprea’s test
for lead likeness.24,25a Furthermore, a filter to exclude molecules
containing reactive groups was also included.25b
Figure 2. Validation set compounds mapped on the selected M3
pharmacophore model.

Diaryl Imidazolidin-2-one DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1573
alkylation (with 4-mesyl-N-benzylpiperidine); we then decided
to adopt a different synthetic pathway to build the imidazolidin-
2-one moiety. Amide derivative 58 was synthesized starting
from N-Boc-protected phenylglycine by classical coupling
protocol with DCCa and HOBt. Deprotection and reduction with
a borane-dimethylsulfide complex afforded intermediate 59,
which was cyclized with CDI to give imidazolidin-2-one 60.
Eventually, deprotonation with sodium hydride and alkylation
with benzyl bromide yielded the final compound 61.
Figure 3. Compound 22a (gold) matching M3 pharmacophore model.
Biology and ADMET
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 scopolamine
was used to label the M2 and M3 binding sites, while the
nonspecific binding was determined in the presence of cold
N-methyl scopolamine (see Experimental Section for details and
methods).
In vitro ADME developability parameters of selected com-
pounds were explored by evaluating membrane permeability
in human colon adenocarcinoma (Caco-2) cell monolayers,
inhibition of major isoforms of human cytochrome P450, and
hepatic microsomial clearance (see Experimental Section for
details and methods).
one core could be devised (Scheme 4). The hydrophobic
aromatic features suggested the possibility of substituting the
aryl rings or of implementing different heterocycles (region C).
The not ideal mapping of the imidazolin-2-one carbonyl group
to the hydrogen bond accepting feature suggested that different
substitution patterns or different heterocyclic rings should be
considered (region A). Similarly, the piperidine ring can be
replaced by other rings containing positive ionizable groups
(region B). The benzyl moiety on the right-hand side was not
included in the M3 pharmacophore model. This observation
prompted the exploration of this chemical space with a chemical
array of differently substituted benzyl rings and of other
substituents (region D).
Chemistry
The functional effect of this class of compounds as M3
antagonists was studied in the in vitro models of isolated mouse
detrusor muscle. Functional selectivity for M3 over M2 receptors
was studied in isolated rat tissues (rat detrusor muscle and left
atrium).
The general synthetic pathway employed for the synthesis
of most of the compounds described in this paper is reported in
Scheme 5.
Hydantoin intermediates 14-18 were obtained by the
Bucherer-Bergs reaction of the corresponding ketones,26 per-
formed at high temperature in a stainless steel sealed tube. Only
in the case of R1 and R2 being 4-F-phenyl (hydantoin 13), the
rearrangement starting from the corresponding diketo deriva-
tive27 was used because it improved the yield and made the
purification easier. Hydantoin 12 was commercially available.
Hydantoin intermediates were then functionalized by Mit-
sunobu reaction28 with hydroxyamines 20 to afford compounds
of the general structure of 21a-l. Mitsunobu reaction did not
work with 4-hydroxytropine: in this case, the hydantoin moiety
was functionalized by alkylation29 with the mesyl derivative of
general formula 19, obtained in turn by the mesylation of
4-hydroxytropine.30 Reduction of the hydantoin moiety with
Red-Al (sodium bis-(2-methoxyethoxy)aluminum hydride) yielded
the corresponding imidazolidin-2-one final compounds 22a-l.
To introduce additional substituents on the amine substructure,
the benzyl group was removed (debenzylation by hydrogena-
tion),31 and the basic nitrogen was further functionalized to
afford compounds 24-54, mainly by alkylation32 and reductive
amination,33 arylation, or, in one instance, by a reaction with
an acyl chloride to obtain the corresponding amide. Introduction
of a methyl substituent on the imidazolidin-2-one moiety
(compound 55) was obtained by alkylation with methyl iodide.
Replacement of the imidazolidin-2-one core structure with
oxazolidin-2-one (see compound 57) required the setup of a
different synthetic pathway, which is depicted in Scheme 6.
Diphenyloxirane was reacted with 4-amino-N-benzylpiperidine
to afford the amino-alcohol intermediate 56, which was cyclized
with carbonyl-diimidazole (CDI) to give the desired compound
57.
SAR Discussion
Because the starting hit 22a was a compound endowed with
a very good M3 binding affinity (Ki ) 1.9 nM), the main
objective of the medicinal chemistry activities was to rationalize
the SARs around this chemical series to improve other properties
such as the selectivity toward the M2 receptor and some
developability parameters. The four regions A-D reported in
Scheme 4 were carefully investigated.
Region A. In Table 1, results of the investigation of the
imidazolidin-2-one scaffold are reported. Whereas replacement
of this scaffold with hydantoin (compound 21a) is not tolerated,
alkylation of the nitrogen at position 3 of the imidazolidin-2-
one ring with a methyl group (compound 55), or its replacement
with an oxygen (compound 57) caused a 10-fold decrease in
binding affinity, without significantly affecting M2 selectivity.
The good activity (although lower than for 22a) of compounds
55 and 57 is consistent with the pharmacophoric hypothesis:
in fact, no specific interactions for the NH at position 3 of the
scaffold were identified.
Movement of one of the two phenyl rings of the imidazolidin-
2-one scaffold onto the adjacent nitrogen (as a benzyl group,
see compound 61) also afforded a 14-fold less-active compound,
with a slight reduction in M2 selectivity.
Region B. Replacement of the 4-piperidinyl moiety with other
cyclic amines was then considered (Table 2). Shifting from 4-
to 3-piperidinyl substitution (compound 22b) proved to be
detrimental for M3 affinity (Ki ) 2.4 µM). Conversely, both
tropine (compound 22c) and azepine moieties (compound 22d)
allowed maintaining the same M3 binding affinity as piperidine.
Synthesis of 1,3,4 trisubstituted imidazolidin-2-one derivative
61 proceeded similarly, as shown in Scheme 7. Commercially
available 4-phenylhydantoin failed to react both in Mitsunobu
reaction conditions (with 4-hydroxy-N-benzylpiperidine) and by
a Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, 1-hydroxy-
benzotriazole; CDI, carbonyl-diimidazole; DCM, dichloromethane; DEAD,
diethyl azodicarboxylate; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; THF, tetrahydrofuran.

1574 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Peretto et al.
Scheme 4. Planned Chemical Variations on Imidazolidinone Scaffold
Scheme 5. General Synthetic Pathway for Imidazolidinone Derivatives
The selectivity toward M2 receptor was lost with azepine, while
with tropine, some selectivity was conserved (16-fold). The (R)-
pyrrolidine moiety (compound 22e) proved to be slightly less
active (Ki ) 13.3 nM) and selective (6-fold) than the hit
compound 22a, while the (S)-pyrrolidine derivative (compound
22f) was almost 40-fold less active.
Region C. SAR exploration focused then on the investigation
of the phenyl groups on the imidazolidin-2-one ring (Table 3).
Substituents at the two phenyl groups can affect both affinity
and selectivity: the presence of methoxy group at the para
position (compound 22h) caused a significant loss of affinity
(Ki ) 114 nM), while substitution with fluorine at the same
position (compound 22g) maintained good affinity (Ki ) 2.8
nM) and increased the M2 selectivity (74-fold). Replacement
of one of the phenyl groups in position 4 of the imidazolidin-
2-one ring with one cyclohexyl substituent (compound 22i) did
not significantly affect either the affinity or the M2 selectivity;
conversely, replacement of both of the phenyl groups with the
cyclohexyl moiety (compound 22j) caused a complete loss of
activity, thus indicating that at least one aromatic moiety is
necessary. Variation of the distance between the aromatic groups
and the imidazolidinone core structure, as in compound 22l
featuring the dibenzyl substitution, greatly reduced the binding
affinity (Ki ) 152 nM). Replacement of both of the phenyl
groups with the 2-thienyl moiety, as in compound 22k, preserved
the same activity and selectivity profile as the hit compound
22a.
Region D. The importance of the benzyl group on the basic
nitrogen of the piperidine moiety was then assessed (Table 3).
In fact, the inactivity of the amide derivative 24 confirmed the
role of the basic nitrogen, and the low activity or inactivity of
compounds 25, 27, and 28 proved the importance of the aromatic
ring. The best distance of this ring from the basic nitrogen is
one CH2 unit, because shorter chains (compound 26) or longer

Diaryl Imidazolidin-2-one DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1575
Scheme 6. Synthesis of Oxazolidin-2-one Derivative 57
Scheme 7. Synthesis of 1,3,4-Trisubstituted Imidazolidin-2-one Derivative 61
chains (compounds 29, 30 and 31) strongly decrease the activity.
Fixing this distance, we tried to replace the phenyl ring with
electron poor or electron rich heteroaromatic rings (see com-
pounds 32, 33, and 34); among these, only the thienyl derivative
(33) maintained a good activity and proved to be more selec-
tive than the parent compound (Ki ) 13.4 nM, 70-fold selec-
tive).
moiety were less-selective (about 60-fold) but were among the
most potent derivatives obtained.
The functional effect of this class of compounds as M3
antagonists was studied in the in vitro model of isolated mouse
detrusor muscle. Increasing concentrations of carbachol (CCh)
elicited strong force of contraction. In time-matched control
(TMC) experiments, there was no difference between the mean
EC50 from the first concentration-response curves (CRCs, 1.63
( 0.45 µM; n ) 8) and that of the second CRCs (EC50 ) 1.65
( 0.56 µM; n ) 2). The effects of compound 54 on the
cumulative CRCs for CCh in mouse detrusor are summarized
in Figure 4. Compound 54 deeply affected CCh-induced
contractions in a concentration-dependent manner, shifting the
CRCs to the right. The pA2 value calculated from Schild
regression analysis for the compound was 7.9 (CI: 7.8-8.0)
(slope ) 0.993 ( 0.060; r2 ) 0.996). The corresponding pA2
value for tolterodine was 8.8 (CI: 8.5-9.0).34
The drug effects on electric field stimulation (EFS) elicited
tension in mouse detrusor are summarized in Figure 5. Force
development remained stable for the duration of all the
experiment. Compound 54 concentration-dependently inhibited
the force of contraction, with the 1 µM concentration producing
a force reduction of 53.72 ( 6.45% of control (n ) 3). After
the wash out of the preparation, the inhibitory effects of 54 lasted
for at least 2 h, suggesting a quite slow functional dissociation
rate of the compound from the muscarinic receptors of the
mouse detrusor muscle.
Introduction of substituents on the phenyl ring was then
examined (see compounds 35-48). Both electron-releasing and
electron-withdrawing substituents afforded active compounds,
with a marked preference for the substitution in the meta
position, which resulted in the best combination of potency and
selectivity: for example, 3-chlorobenzyl derivative 36 displayed
Ki ) 3.15 nM and a >100-fold selectivity toward the M2
receptor. Surprisingly, disubstitution with chlorine at the meta
position (compound 38) afforded an inactive compound. It is
worth noting that this aromatic ring, which is so important to
modulate M3 activity and selectivity, was not included in the
initial M3 pharmacophore. The best results obtained from the
investigation of the four regions A-D were then combined to
afford the mixed derivatives 49-54 listed in Table 3. All these
compounds display good activity (Ki ranging from 1.7 nM to
6.7 nM) and good M2 selectivity (37- to 236-fold). Compounds
49 and 50 bearing the bis-(4-fluorophenyl) substitution on the
imidazolidin-2-one core structure were the most selective
compounds obtained in this class (143-fold and 236-fold,
respectively). Compounds 53 and 54 with the bis-(2-thienyl)

1576 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Peretto et al.
Table 1. Investigation of the Imidazolidin-2-one Moiety (Region A):
M3 and M2 Binding Affinities
compound. In rat detrusor muscle (M3-mediated response), both
compound 22g and tolterodine produced a rightward parallel
shift of the CRC of methacholine with pKB values of 7.77 and
8.86, respectively (Figure 6). In rat left atria (M2-mediated
response), only tolterodine caused a rightward shift of the
methacholine negative inotropic response in a concentration-
dependent manner, with a pKB value of 8.69. Conversely,
compound 22g did not affect methacholine negative inotropic
effect up to 0.1 µM.
Some of the most interesting compounds in terms of activity
and selectivity were further profiled for some in vitro ADME
parameters, such as inhibition of human CYP450 isoforms,
intrinsic clearance (Cli), and Caco-2 permeability as a model
of intestinal absorption (Table 4). Almost all the compounds
showed good permeability in the Caco-2 assay, thus indicating
the possibility of good intestinal absorption. Cli in human liver
microsomes was low for all compounds; in rat microsomes, the
compounds bearing the bis-(2-thienyl) moiety (22k and 54)
showed high Cli, while compounds bearing the bis-(4-fluo-
rophenyl) moiety (22g, 49, and 50) were more stable. None of
the compounds tested in the CYP450 assay showed significant
inhibition of any of the examined isoforms.
Conclusions
A novel class of M3 receptor antagonists was discovered35
starting from a pharmacophore model generated from known
muscarinic antagonist. Study of the structure-activity relation-
ships afforded compounds with binding affinity in the low
nanomolar range, with up to >200-fold selectivity toward the
M2 receptor. In vitro ADME parameters for selected derivatives
indicate that this class of compounds could be suitable for
systemic administration, as required for the treatment of some
pathophysiological states related to M3 disfunction, such as
bladder hypercontractility. Functional in vitro studies on selected
compounds confirmed the antagonist activity toward the M3
receptor and functional selectivity toward the M2 receptor. This
could be an important advantage for treating overactive bladder
patients.
a Inactive: Percent of control binding <50% at the concentration of 10
µM.
Table 2. Investigation of the Piperidine Basic Moiety (Region B): M3
and M2 Binding Affinities
Experimental Section
Computational Methodology. In this study, chemical-feature-
based pharmacophore hypotheses were generated automatically
using the HipHop algorithm, as implemented in Catalyst software
package (version 4.9, Accelrys, Inc., San Diego, CA) on a Silicon
Graphics Fuel computer, running the IRIX 6.5 operating system.10,11
Training set and validation set compounds were edited using the
Catalyst 2D/3D Visualizer, and, to generate a reasonable number
of conformations able to adequately cover the molecular confor-
mational space within a defined energy threshold, “poling”
algorithm was considered to generate corresponding conformers.36
On the basis of molecular structure flexibility, the conformational
analysis was carried out with the “best mode” and an optimal
number of conformations equal to 200. All remaining Catalyst
parameters were considered in their default settings.
Molecules and associated conformation models were submitted
to Catalyst hypothesis generation.
Pharmacophore Hypotheses Generation with HipHop. On the
basis of the chemical nature of the considered compounds, the
following three features were selected to represent essential
information in the hypothesis generation process: hydrogen bond
acceptor (HBA), hydrophobic aromatic (HAr), and positive ioniz-
able (PI). The objective of the HipHop run was to identify and
enumerate all possible pharmacophore configurations that are in
common to the training set. To perform this task, HipHop conducts
an exhaustive search starting with the simplest pharmacophore
configuration, that is, all possible combinations of two-feature
pharmacophores. Once all two-feature pharmacophores are ex-
a Inactive: Percent of control binding <50% at the concentration of 10
µM.
The selectivity for M3 over M2 receptor was confirmed in
vitro for compound 22g in rat isolated tissues. The well-known
muscarinic antagonist tolterodine was used as a reference

Diaryl Imidazolidin-2-one DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1577
Table 3. Investigation of Substituents on the Imidazolidin-2-one (Region C) and on the Piperidine (Region D) Moieties: M3 and M2 Binding Affinities
M2/M3
M3 affinity
(Ki, nM)
M2 affinity
(Ki, nM)
selectivity
ratio
cmpd
R1
R2
R3
22a
22g
22h
22ib
22j
22k
22l
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
Ph
Ph
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
3-Cl benzoyl
methyl
phenyl
1.9 ( 1.5
80.1 ( 13.4
207 ( 26.1
inactivea
42
74
4-F phenyl
4-OMe phenyl
phenyl
cyclohexyl
2-thienyl
benzyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
4-F phenyl
4-OMe phenyl
cyclohexyl
cyclohexyl
2-thienyl
benzyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
2.8 ( 0.5
114 ( 4.0
2.57 ( 0.47
inactivea
73.3 ( 18.9
29
2.7 ( 0.6
96.8 ( 4.5
2952 ( 524
36
19
152 ( 63
inactivea
923 ( 155
2918 ( 280
inactivea
3
101 ( 22
cyclohexyl methyl
propargyl
354 ( 56
inactivea
inactivea
phenylethyl
phenylpropyl
phenoxyethyl
3-pyridyl methyl
2-thienyl-methyl
4-imidazolyl methyl
2-Cl benzyl
117 ( 33
1630 ( 116
228 ( 33
1132 ( 104
13.4 ( 1.5
1056 ( 364
13.8 ( 0.4
3.15 ( 0.94
16.5 ( 0.6
inactivea
411 ( 98
1681 ( 176
328 ( 63
302 ( 12
927 ( 537
inactivea
4
1
1
0.3
69
inactivea
328 ( 50.5
296 ( 74
3-Cl benzyl
4-Cl benzyl
104
18
3,5-Cl benzyl
2-OMe benzyl
3-OMe benzyl
4-OMe benzyl
3-OH benzyl
3-Me benzyl
3-NH2 benzyl
3-OCF3 benzyl
3-CN benzyl
3-CF3 benzyl
3-F benzyl
3-Cl benzyl
3-F benzyl
3-Me benzyl
3-Cl benzyl
3-Me benzyl
3-F benzyl
83.7 ( 14.7
inactivea
8.8 ( 1.5
695 ( 55
490 ( 66
47.9 ( 10.4
225 ( 2.9
182 ( 12
inactivea
79
19
6
95
23
25.8 ( 0.4
7.6 ( 2.6
2.37 ( 0.93
7.9 ( 0.4
2057 ( 353
418 ( 19
inactivea
53.5 ( 25
4.32 ( 1.42
6.7 ( 3.5
inactivea
phenyl
phenyl
302 ( 12.5
955 ( 127
1141 ( 516
166 ( 9.4
515 ( 239
107 ( 20.6
187 ( 61
70
143
236
37
78
62
4-F phenyl
4-F phenyl
phenyl
phenyl
2-thienyl
2-thienyl
4-F phenyl
4-F phenyl
cyclohexyl
cyclohexyl
2-thienyl
2-thienyl
4.83 ( 2.47
4.54 ( 1.76
6.59 ( 0.38
1.72 ( 0.39
2.75 ( 1.38
68
a Inactive: Percent of control binding <50% at the concentration of 10 µM. b Racemic mixture.
hausted, it then moves to three-feature combinations. This process
continues until common pharmacophore combinations can no longer
be generated.23 All considered training molecules were required to
completely map the pharmacophore. The final HipHop output was
a user-determined number of unique pharmacophores sorted from
highest to lowest scoring.
3D Database Generation. Because the power and the efficacy
of 3D database searching is dependent on both size and diversity
of the compounds to be searched, we sought to extend our hit
identification process beyond the limitations and bias of a com-
mercial chemical series (CAP Screening Database21 and May-
bridge22) with more than 600 “in-house” designed chemical entities
built on the basis of structural similarity to known muscarininc M3
antagonists. Starting from 2D structures, the 3D database of “in-
house” developed compounds was obtained generating 250 con-
formers for each molecule with the “best” method. The overall
number of conformers decreased if it provided adequate coverage
for the conformational space. Accelrys supplied CAP Screening
and Maybridge 3D databases in version 2002 and 2001, respec-
Figure 4. Effects of 54 on cumulative CRC for CCh in mouse detrusor
strips in comparison to TMC experiments. Data are shown as means
( SEM. Responses to CCh are expressed as percentage of the maximum
effect in the first CRC. Control and TMC data are averaged values
from the first CRCs in all experiments.
tively.
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

1578 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Peretto et al.
General Procedure 2. Mitsunobu Reaction. 3-(1-Benzyl-
piperidin-4-yl)-5,5-diphenyl-imidazolidin-2,4-dione (21a). Com-
mercially available 5,5-diphenyl hydantoin 12 (phenytoin, 0.50 g,
1.98 mmol) is dissolved in dry THF (20 mL); triphenyl phosphine
(0.78 g, 2.97 mmol) and 4-hydroxy-N-benzyl piperidine (0.567 g,
2.97 mmol) are added to the reaction mixture. The resulting solution
is cooled to 0 °C and diethyl azodicarboxylate (DEAD, 0.47 mL)
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: EtOAc-
hexane 2:8 to EtOAc 100%), to yield 0.8 g of pure product as a
white solid. LC-MS (EI pos; M+) 425.13, 334.16. 1H NMR (DMSO;
343 K): 9.45 (s br, 1H, N-H), 7.53-7.32 (m, 15H, 3 × Ph), 4.25
(s br, 2H, N-CH2-Ph), 4.20 (m, 1H, N-CH-(CH2)2), 3.40 (m, 2H,
H
2,6 eq), 3.10 (m, 2H, H2,6 ax), 2.58 (m, 2H, H3,5 eq), 1.87 (m, 2H,
H3,5 ax).
Compounds 21b, 21d, 21e, 21f, 21g, 21h, 21i, 21j, 21k, and
Figure 5. Effects of compound 54 on EFS-elicited contraction of
mouse detrusor strips in comparison to experiments without any drugs
added (controls). Inhibitory activity is expressed as percent reduction
of the predrug control value. Data are expressed as mean values (
SEM.
21l were synthesized following the same procedure, starting from
the corresponding hydantoin derivatives.
3-(1-Benzyl-tropin-4-yl)-5,5-diphenyl-imidazolidine-2,4-di-
one (21c). (a) Synthesis of Methanesulfonic Acid 8-Methyl-8-
aza-bicyclo[3.2.1]oct-3-yl Ester (Tropine Mesilate). N-Benzyl-
tropin-4-ol30 (0.230 g, 1.063 mmol) is added to a solution of triethyl
amine (0.207 mL, 1.5 mmol) in dry DCM (10 mL); the resulting
mixture is cooled to 0 °C and methanesulfonyl chloride (0.099 mL,
1.276 mmol) is added. The reaction is stirred at 0 °C for 1 h, then
the solvent is evaporated under 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. Commercially available 5,5-
diphenyl hydantoin (phenytoin, 0.20 g, 0.793 mmol) is dissolved
in dry DMF (10 mL); K2CO3 (0.33 g, 2.38 mmol) and N-benzyl-
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): 451.24
(MH+).
General Procedure 3. Reduction with Red-Al. 3-(1-Benzyl-
piperidin-4-yl)-5,5-diphenyl-imidazolidin-2-one (22a). Compound
21a (1.0 g, 2.3 mmol) is dissolved in dry THF (20 mL) under a
nitrogen atmosphere. The solution is cooled to 0 °C and a 3.5 M
solution of sodium bis-(2-methoxyethoxy)aluminum hydride (Red-
Al) in toluene (5.37 mL, 18.4 mmol) is added. 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); 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 as a white
solid (0.80 g). LC-MS (ESI POS): 412.11 (MH+). 1H NMR (CDCl3
+ D2O + Na2CO3): 7.37-7.15 (m, 15H, 3 × Ph), 3.94 (s, 2H,
CON-CH2-C), 3.85 (m, 1H, N-CH-(CH2)2), 3.49 (s, 2H,
N-CH2-Ph), 2.93 (m, 2H, H2,6 eq), 2.09 (m, 2H, H2,6 ax), 1.78-
1.68 (m, 4H, H3,5).
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 CH3-
CN-H2O-TFA and solvent B is 90:10:0.05 H2O-CH3CN-TFA;
preparative HPLC purifications were performed on a Waters
SymmetryPrep C-18, using a 20 min gradient of 0-100% solvent
B, where solvent A is 99.9:0.1 H2O-TFA and solvent B is 99.9:
0.1 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);
flash chromatography was conducted using a Biotage-Quad3
apparatus and prepacked silica gel columns (KP-SIL, particle size
32-60 µm). Solid liquid extraction cartridges were purchased from
Varian (“Chem Elut”, 3 mL, unbuffered, part no. 12198003).
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
1
used in workups are aqueous solutions. The H NMR numeration
for compounds 21a, 22a, 26, 55, 57, and 61 refers to the structures
reported in the Supporting Information.
Synthetic Procedures. General Procedure 1. Synthesis of
Hydantoin Intermediates from Ketone Derivatives. 5-Phenyl-
5-cyclohexyl hydantoin (15). Cyclohexyl phenyl ketone (0.564 g,
3 mmol) is dissolved in 20 mL of a 1:1 mixture of ethanol and
water, in a stainless steel sealed tube. Potassium cyanide (0.585 g,
9 mmol) and ammonium carbonate (3.28 g, 30 mmol) are added
and the mixture is heated at 120 °C for 24 h. The reaction mixture
is then allowed to cool to room temperature, diluted with 20 mL
of water, and cooled to 0 °C; the desired product precipitates as a
white solid and is filtered to give 0.70 g of pure product. LC-MS
(ESI pos): 259.37 (MH+).
Compounds 22b, 22c, 22d, 22e, 22f, 22g, 22h, 22i, 22j, 22k,
and 22l were synthesized following the same procedure, starting
from the corresponding imidazolidin-2,4-diones.
Hydantoin derivatives 14, 16, 17, and 18 were synthesized
following the same procedure, starting from the corresponding
commercially available ketones.
General Procedure 4. Hydrogenation for the Deprotection/
Removal of the Benzyl Group. 5,5-Diphenyl-1-piperidin-4-yl-
imidazolidin-2-one (23a). Compound 22a (0.50 g, 1.2 mmol) is
dissolved in methanol (15 mL) and water (5 mL). Hydrochloric
acid (37%, 0.5 mL) and palladium on charcoal (0.20 g) are added,
and the solution is hydrogenated in a Parr apparatus (H2: 20 psi)
at room temperature for 8 h. The catalyst is filtered and the clear
solution is evaporated to yield the pure product as a white solid
(0.380 g). LC-MS (ESI POS): 322.18 (MH+). 1H NMR (CDCl3 +
D2O + Na2CO3): 7.37-7.21 (m, 10H), 3.94 (s, 2H), 3.91(m, 1H),
Synthesis of 5,5-Di-(4-fluorophenyl) Hydantoin (13). Bis-(4-
fluorophenyl)-ethandione (1.23 g, 5 mmol) is dissolved in ethanol
(20 mL); urea (0.39 g, 6.5 mmol) and potassium hydroxide (pellets,
0.476 g, 8.5 mmol) are added, and the resulting mixture is heated
at 80 °C for 24 h. The reaction is allowed to cool to room
temperature, diluted with water (40 mL), and cooled to 0 °C; the
desired product precipitates as a white solid and is filtered to give
0.62 g of pure product. LC-MS (ESI pos): 389.22 (MH+).

Diaryl Imidazolidin-2-one DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1579
Figure 6. Effect of compound 22g and tolterodine on methacholine-induced relaxation of rat detrusor muscle and rat left atrial preparations. The
concentration-response relationship for methacholine either alone or in the presence of compound 22g (0.01-0.3-0.1 µM) or tolterodine (0.01-
0.3-0.1 µM) are shown. Each point represents the mean ( SEM (n ) 6-7). Data are expressed as the % of the maximal contraction induced by
80 mM KCl (for rat detrusor muscle) and as the delta % of the basal force of contraction (for rat left atrium).
Table 4. Evaluation of Intrinsic Clearance (Rat and Human) and P450 Profile for Some Selected Compounds
M2/M3
selectivity
ratio
Cli (rat)
mL/min/g
liver
Cli (human)
mL/min/g
liver
CYP450
inhibition
IC50 (µM)
M3 affinity
(Ki, nM)
M2 affinity
(Ki, nM)
Caco-2
permeability
cmpd
22a
22g
1.9 ( 1.5
2.8 ( 0.5
80.1 ( 13.4
207 ( 26.1
42
74
high
high
4.7
2.0
1.1
0.56
1A2: >100
2C9: 7.0
2C19: 12.9
2D6: 4.7
3A4: 91.9
22k
49
2.7 ( 0.6
6.7 ( 3.5
96.8 ( 4.5
955 ( 127
36
143
high
low
23.7
1.8
2.3
0.87
1A2: >100
2C9: 4.2
2C19: 17.7
2D6: 5.3
3A4: >100
1A2: >100
2C9: 7.3
2C19: 17.4
2D6: 12.0
3A4: >100
50
4.83 ( 2.47
1141 ( 516
187 ( 61
236
high
1.4
0.78
54
2.75 ( 1.38
68
medium
13.8
0.95
1
3.11 (m, 2H), 2.69 (ddd, J ) 12.2, 2.5 Hz, 2H), 1.76 (m, 2H), 1.58
(dq, J ) 12.2, 4.1 Hz, 2H).
Compounds 23g, 23i, and 23k were synthesized following the
same procedure, starting from the corresponding N-benzyl deriva-
tives.
General Procedure 5. Alkylation of Piperidine Derivatives.
3-(1-(3-Chlorobenzyl)-piperidin-4-yl)-5,5-diphenyl-imidazolidin-
2-one (36). Compound 23a (0.100 g, 0.311 mmol) is dissolved in
dry DCM (10 mL), and solid K2CO3 (0.20 g, 1.45 mmol) is added.
The mixture is vigorously stirred at room temperature, and neat
3-chloro-benzyl chloride (0.051 g, 0.311 mol) is added. The reaction
is stirred at room temperature for 5 h, then solid K2CO3 is filtered,
and the solution is evaporated to yield a colorless oil that is
crystallized from di-isopropyl ether to afford the pure product as a
white solid (0.060 g). LC-MS (ESI pos): 446.4 (MH+). H NMR
(CDCl3): 7.37-7.20 (m, 14H), 4.89 (s br, 1H), 3.96 (s, 2H), 3.87
(m, 1H), 3.46 (s br, 2H), 2.91 (m, 2H), 2.10 (m, 2H), 1.74 (m,
4H).
Compounds 24, 25, 27, 28, 30, 31, 35, 39, 43, 47, 48, 49, 50,
51, 52, 53, and 54 were synthesized following the same procedure,
by reaction of the appropriate piperidine derivatives with the
corresponding alkylating agents.
General Procedure 6. Reductive Amination of Piperidine
Derivatives. 3-(1-(4-Chlorobenzyl)-piperidin-4-yl)-5,5-diphenyl-
imidazolidin-2-one (37). 5,5-Diphenyl-1-piperidin-4-yl-imidazo-
lidin-2-one (compound 23a; 0.100 g, 0.311 mmol) is dissolved in
methanol (10 mL) under a nitrogen atmosphere, and 4-chloroben-
zaldehyde (0.050 g, 0.341 mmol) is added. The mixture is stirred

1580 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Peretto et al.
at room temperature for 30 min, and then solid NaBH3CN (0.040
g, 0.622 mmol) is added. The reaction is stirred at room temperature
for 18 h, and then the solvent is evaporated under vacuum. The
residue is dissolved in 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 purified by flash chromatography (SiO2, 6 g; eluent, DCM/MeOH
9:1) to give the desired product as a white solid (0.074 g). LC-MS
for 2 h. 4-Amino-N-benzylpiperidine (4.18 g, 22 mmol) is added,
and the reaction is stirred at room temperature overnight. The
reaction mixture is then diluted with DCM and washed twice with
a saturated solution of potassium carbonate, water, and brine. The
organic phase is dried and concentrated under vacuum to give an
oil that is purified by flash chromatography (SiO2, 120 g; eluent,
DCM/MeOH 95:5) to give the desired product as a white solid
(6.4 g).
(b) N-2-(1-Benzylpiperidin-4-yl)-1-phenylethane-1,2-diamine
Hydrochloride (59). 2-(N-tert-Butyloxycarbonyl)-amino-2-phenyl-
N-(1-benzyl-piperidin-4-yl)-acetamide (58) is dissolved in DCM
(80 mL) and added to a saturated solution of hydrogen chloride in
diethyl ether (50 mL). The reaction mixture is stirred at room
temperature for 4 h. The primary amine (dihydrochloride salt)
precipitates as a white solid and is then filtered and dried under
vacuum (4.5 g).
To a solution of 2-amino-N-(1-benzylpiperidin-4-yl)-2-phenyl-
acetamide dihydrochloride (0.44 g, 1.15 mmol) in dry toluene (10
mL), borane-methylsulfide complex (0.22 mL, 2.3 mmol) is added
under an inert atmosphere. The mixture is refluxed for 4 h, followed
by the addition of MeOH (0.5 mL). After 1 h, the solution is cooled
and treated with 10 mL of a 10% solution of HCl in ethyl ether.
The white precipitate is filtered off and washed with ether (0.45
g).
(c) 1-(1-Benzylpiperidin-4-yl)-4-phenyl-imidazolidin-2-one (60).
To a suspension of N-2-(1-benzylpiperidin-4-yl)-1-phenylethane-
1,2-diamine hydrochloride (59; 0.4 g, 0.95 mmol) in dry 1,4-dioxane
(10 mL), CDI (0.2 g, 1.14 mmol) is added. The mixture is refluxed
for 2 h, and then the solvent removed. The crude material is treated
with water and extracted with ethyl acetate. The organic phase is
washed with brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to afford a white solid (0.22
g). LC-MS (ESI pos): 360.12 (MH+).
1
(ESI pos): 446.4 (MH+). H NMR (CDCl3 + D2O + Na2CO3):
7.37-7.20 (m, 14H), 3.95 (s, 2H), 3.85 (m, 1H), 3.44 (s, 2H), 2.90
(m, 2H), 2.08 (m, 2H), 1.78-1.68 (m, 4H).
Compounds 29, 32, 33, 34, 38, 40, 41, 42, 44, 45, and 46 were
synthesized following the same procedure, by reaction of the
appropriate piperidine derivatives with the corresponding aldehyde.
3-(1-Phenyl-piperidin-4-yl)-5,5-diphenyl-imidazolidin-2-one
(26). Compound 23a (0.1 g, 0.3 mmol) is dissolved in DMF (1
mL). Iodobenzene (0.067 mL, 0.32 mmol), copper(I) iodide (0.061
g, 0.32 mmol), and K2CO3 (0.045 g, 0.32 mmol) are added, and
the resulting mixture is heated to 150 °C under a nitrogen
atmosphere for 4 h. The mixture is then diluted with ethyl acetate
and filtered over a celite pad, and the resulting solution is dried in
vacuo. The product is purified by preparative HPLC to yield 45
mg of pure product as a white solid. LC-MS (ESI POS): 398.11
(MH+). 1H NMR (CDCl3 + Na2CO3 + D2O): 7.39-7.19 (m, 12H,
2 × Ph, H9,11), 6.93 (d, J ) 8.6 Hz, 2H, H8,12), 6.84 (dd, J ) 7.4,
7.4 Hz, 1H, H10), 4.02 (m, 1H, H4), 3.96 (s, 2H, H14), 3.74 (m, 2H,
H2,6 eq), 2.85 (m, 2H, H2,6 ax), 1.91-1.78 (m, 4H, H3,5).
1-(1-Benzyl-piperidin-4-yl)-3-methyl-4,4-diphenyl-imidazoli-
din-2-one (55). Compound 22a (0.100 g, 0.243 mmol) is dissolved
in dry THF under nitrogen atmosphere; the solution is cooled to 0
°C and solid NaH (0.010 g, 0.243 mmol) is added. The reaction is
stirred at 0 °C for 15 min, then methyl iodide (0.042 g, 0.243 mmol)
is added, and the stirring is continued at room temperature for 3 h.
The reaction 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 an oil that is purified
by preparative HPLC to give the desired product as a white solid
(0.041 g). LC-MS (ESI pos): 426.2 (MH+). 1H NMR (CDCl3, 343
K): 7.38-7.21 (m, 15H, 3 × Ph), 3.91 (m, 1H, N-CH-(CH2)2),
3.82 (s, 2H, CON-CH2-C), 3.55 (s br, 2H, N-CH2-Ph), 2.97
(m, 2H, H2,6 eq), 2.57 (s, 3H, N-CH3), 2.19 (m, 2H, H2,6 ax), 1.89-
1.71 (m, 4H, H3,5).
(d) 1-(1-Benzyl-piperidin-4-yl)-3-benzyl-4-phenyl-imidazoli-
din-2-one (61). Compound 60 (0.100 g, 0.30 mmol) is dissolved
in dry THF under a nitrogen atmosphere, the solution is cooled to
0 °C, and solid NaH (0.012 g, 0.30 mmol) is added. The reaction
is stirred at 0 °C for 15 min, then benzyl bromide (0.057 g, 0.30
mmol) is added, and the stirring is continued at room temperature
for 3 h. The reaction 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 an oil
that is purified by preparative HPLC to give the desired product as
a white solid (0.053 g). LC-MS (ESI pos): 426.1 (MH+). 1H NMR
(CDCl3 + D2O + Na2CO3): 7.39-7.06 (m, 15H, 3 × Ph), 4.89
(d, J ) 15.1 Hz, 1H, H25), 4.28 (dd, J ) 8.1, 7.8 Hz, 1H, H15),
3.89 (m, 1H, H4), 3.59 (t, J ) 8.8 Hz, 1H, H16), 3.56 (d, J ) 15.1
Hz, 1H, H25), 3.49 (s, 2H, H7), 3.13 (dd, J ) 8.8, 8.1 Hz, 1H, H15),
2.93 (m, 2H, H2,6 eq), 2.11 (m, 2H, H2,6 ax), 1.76-1.61 (m, 4H,
3-(1-Benzyl-piperidin-4-yl)-5,5-diphenyl-oxazolidin-2-one (57).
(a) 2-(1-Benzylpiperidin-4-ylamino)-1,1-diphenylethanol (56). A
mixture of 2,2-diphenyloxyrane (0.5 g, 2.55 mmol), 4-amino-N-
benzylpiperidine (1.0 g, 5.3 mmol), and water (0.05 mL, 2.8 mmol)
is heated at 100 °C for 60 h, then cooled, and passed through a
silica gel pad (methylene chloride/methanol/33% aqueous am-
monium hydroxide 95/5/0.5) to afford an oil (0.54 g). The crude
compound was employed in the next step without purification.
(b) 3-(1-Benzylpiperidin-4-yl)-5,5-diphenyloxazolidin-2-one
(57). A solution of 2-(1-benzylpiperidin-4-ylamino)-1,1-diphenyl-
ethanol (56; 0.06 g, 0.16 mmol) and CDI (0.04 g, 0.25 mmol) in
THF (3 mL) is refluxed for 24 h, cooled, and concentrated under
reduced pressure. The crude material is treated with water and
extracted with ethyl acetate. The organic phase is washed with brine,
dried over anhydrous sodium sulfate, and concentrated under
reduced pressure to afford an oil (0.07 g) that was purified by
preparative HPLC, obtaining 0.03 g of pure compound. LC-MS
H3,5).
Biology and ADMET. Cell Lines and Membrane Prepara-
tions. CHO-K1 clone cells expressing the human M2 or M3-
receptors (Swissprot P08172, P20309 respectively) were harvested
in Ca++/Mg++-free phosphate-buffered saline and collected by
centrifugation at 1500 rpm for 3 min. The pellets were resuspended
in ice cold buffer A (15 mM Tris-HCl pH 7.4, 2 mM MgCl2, 0.3
mM EDTA, 1 mM EGTA) and homogenized by a PBI politron
(setting 5 for 15 s). The crude membrane fraction was collected by
two consecutive centrifugation steps at 40 000 g for 20 min at 4
°C and separated by a washing step in buffer A. The pellets obtained
were finally resuspended in buffer B (75 mM Tris HCl pH 7.4,
12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose),
and aliquots were stored at -80 °C.
Radioligand Binding Conditions. The day of the experiment,
frozen membranes were resuspended in buffer C (50 mM Tris-
HCl pH 7.4, 2.5 mM MgCl2, 1 mM EDTA). The nonselective
muscarinic radioligand [3H]-N-methyl scopolamine37 was used to
label the M2 and M3 binding sites. Binding experiments were
performed in duplicate (10-point concentration curves) in 96-well
plates at a radioligand concentration of 0.1-0.3 nM. The nonspe-
cific binding was determined in the presence of cold N-methyl
1
(ESI pos): 413.1 (MH+). H NMR (CDCl3 + D2O + Na2CO3):
7.41-7.21 (m, 15H, 3 × Ph), 4.09 (s, 2H, CON-CH2-C), 3.78
(m, 1H, N-CH-(CH2)2), 3.48 (s, 2H, N-CH2-Ph), 2.91 (m, 2H,
H20,22 eq), 2.05 (m, 2H, H20,22 ax), 1.74-1.64 (m, 4H, H19,23).
1-(1-Benzyl-piperidin-4-yl)-3-benzyl-4-phenyl-imidazolidin-
2-one (61). (a) 2-(N-tert-Butyloxycarbonyl)-amino-2-phenyl-N-
(1-benzyl-piperidin-4-yl)-acetamide (58). Commercially available
N-Boc-protected phenyl glycine (5.0 g, 19.9 mmol) is suspended
in a mixture of acetonitrile (50 mL) and DCM (50 mL). The
suspension is vigorously stirred under a nitrogen atmosphere.
N-Hydroxybenzotriazole (2.97 g, 22 mmol) and DCC (4.53 g, 22
mmol) are added, and the mixture is stirred at room temperature

Diaryl Imidazolidin-2-one DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1581
scopolamine (10 µM). Samples (final volume 0.75 mL) were
incubated at room temperature for 60 min for M2 and 90 min for
M3 binding assay. The reaction was terminated by a rapid filtration
through GF/B Unifilter plates and two washes (0.75 mL) with cold
buffer C using a Packard Filtermate Harvester. Radioactivity on
the filters was measured by a microplate scintillation counter
TopCount NXT (Camberra Packard).
analyzed by LC-MS-MS to quantify the amount of compound.
7-Ethoxycoumarin was always tested in every experiment as the
reference compound of known clearance. Data analysis: the rate
constant, k (min-1), derived for the exponential decay equation is
utilized to calculate the rate of Cli of the compound using the
following calculation, Cli (mL/min/g liver) ) k × V × microsomal
protein yield, where V (mL/mg protein) ) incubation volume/mg
protein added and microsomal protein yield ) 52.5 mg protein/g
liver
Membrane Permeability. Intestinal drug absorption of test
compounds was estimated in vitro using human colon adenocar-
cinoma (Caco-2) cell monolayers (Areta International, Gerenzano,
Italy) as previously described.38 Cells were suspended in DMEMB
and seeded (2 × 106 cells in 0.3 mL) in 24-well plates (Transwell,
Costar, Cambridge, MA) using polycarbonate microporous cell
culture inserts. Cells were grown for 21 days. The integrity of cell
monolayers was checked by measuring transepithelial electrical
resistance (1000 Ω) and by the transport of the paracellular leakage
marker sodium fluorescein. An amount equal to 100 µL of test
compound solution (50 µM in 1% DMSO) was applied to the apical
side. After 2 h of incubation at 37 °C, the apical and basolateral
side solutions were removed and analyzed by liquid chromatography
and tandem mass spectrometry with selected reaction monitoring.
The apparent permeability (Papp) was calculated as Papp ) CbVb/
(C0‚A‚t), in which Cb ) basolateral test compound concentration
at time t (µM), Vb ) basolateral volume (cm3), C0 ) apical test
compound concentration at t ) 0 (µM), A ) filter surface area
(cm2), and t ) time (sec). The rank order of apparent permeability
of the test compounds was compared with that of known reference
compounds tested in the same experiment as internal standards
including propranolol and cimetidine. Papp values were classified
as “low” when Papp was below the limit of 10 nm/sec, “medium”
when 10 e Papp e 50 nm/sec, and “high” when Papp > 50 nm/sec.
Inhibition of Cytochrome P450 Isoenzymes. The assay was
performed in 96-well microtiter plates according to the method
described by Stresser et al.39 Microsomes from baculovirus-infected
insect cells (Supersomes, Gentest Corporation, Woburn, MA)
expressing human cytochrome P450 isoforms (CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4) were utilized. Test compounds
or standard inhibitors (dissolved in DMSO or acetonitrile) were
serially diluted in a solution containing 16.3 µM NADP+, 0.83 mM
glucose-6-phosphate, 0.83 mM MgCl2, 0.4 U/mL of glucose-6-
phosphate dehydrogenase, and 0.1 mg/mL microsomal protein
prepared from wild-type baculovirus-infected insect cells. Ap-
propriate control wells without inhibitor and without microsomes
were also added. The plates were then pre-warmed at 37 °C for 10
min, and the reaction was started by adding pre-warmed enzyme/
substrate mix (cytochrome P450 isoforms with their specific
substrates in a 0.35 M potassium phosphate buffer at pH 7.4).
Specific substrates were 3-cyano-7-ethoxycoumarin (CEC) for
CYP1A2 and CYP2C19, 7-methoxy-4-trifluoromethylcoumarin
(MFC) or dibenzylfluorescein (DBF) for CYP2C9, 3-[2(N,N-
diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC)
for CYP2D6, and 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC)
for CYP3A4. The reaction was terminated at various times,
depending on the assays, by addition of a 4:1 acetonitrile/0.5 M
Tris base solution. Known inhibitors for each isoenzyme (furafylline
for CYP1A2, sulfaphenazole for CYP2C9, tranylcypromine for
CYP2C19, quinidine for CYP2D6, and ketoconazole for CYP3A4)
were tested in all assays as positive controls. Plates were read on
a fluorometer (Fluoroskan Ascent, Thermo Electron Corporation,
Helsinki, Finland) at the appropriate emission/excitation wave-
lengths. Median inhibitory concentration (IC50) was determined by
nonlinear regression (GraFit software, Erithacus Software, Horley,
U.K.).
Isolated Mouse Detrusor Muscle. CD-1 male mice (20-30 g)
were killed by cervical dislocation, and the urinary bladder was
isolated and placed in modified Krebs’ solution (composition in
mM): NaCl, 118; KCl, 4.6; CaCl2, 1.5; MgCl2, 1.5; KH2PO4, 1.15;
NaHCO3, 25; and glucose, 11). Indomethacin (30 µM) and
hexamethonium (1 mM) were included in the Krebs’ solution to
reduce prostaglandin-induced spontaneous activity and possible
nicotinic activity, respectively. Strips of tissue (4 × 2 mm) were
cut from the posterior region of bladder body, parallel to the
longitudinal axis. Tissues were mounted in 25 mL organ baths
containing Krebs’ solution, which was maintained at 37 °C and
constantly aerated with 95% O2/5% CO2 (pH ) 7.4). Isometric
tension generated by the tissue was measured by pure isometric
transducers (Cibertec, Spain) and recorded using the PowerLab
system (ADInstruments, Australia). Tissues were maintained at a
resting tension of 0.5 g during an equilibration period of 60 min.
Tension adjustments were made as necessary. Tissues were washed
every 15 min. The viability of each tissue was assessed by
determining the contractile response to KCl (120 mM) at the start
of the experimental protocol. After washing, tissues were re-
equilibrated for 15 min and allowed to regain baseline tension.
Repetitive CCh (3 µM) contractions were then induced as stimulus
to obtain three consecutive contractions with less than 10%
difference. After tissue equilibration, cumulative consecutive CRCs
to CCh were then constructed in each bladder preparation (3 nM-
30µM, 5 min exposure time each). The antagonist was incubated
for a 60 min period between curves. The pA2 was calculated for
each antagonist and tissue using CCh as agonist (cumulative curve).
TMC experiments were run. Peak increase in force of contraction
induced by the individual CCh concentrations was expressed as
percentage of the maximum effect observed during the first CRC.
In the EFS experiments, mouse strips were challenged twice with
KCl (120 mM), with a 15 min washout period between exposures.
After 15 min of stabilization, muscle strips were subjected to EFS.
The parameter for EFS (Cibertec Stimulator CS-220, Cibertec,
Spain) was pulse duration 0.3 ms at 40 Hz with 30 mA. Stimuli
trains lasted 1 s at 60-s intervals. The compounds under investiga-
tion were added at fixed concentrations. Average values for the
EFS-induced muscle contraction amplitudes were obtained from
the last five contractions. The magnitude of drug effect is given in
percent inhibition of the electrically evoked contraction amplitude
before any substance addition. The functional rate of dissociation
of the tested compounds from the muscarinic receptor was estimated
by measuring force contraction after continuous wash out prepara-
tion (25 min after compound application). All data are expressed
as the mean ( SEM. Cumulative CRC were analyzed by nonlinear
regression of each individual experiment using GraphPad Prism
4.02 (GraphPad Software Inc., San Diego, U.S.A.). Mean EC50
values for CCh (molar concentration producing 50% of the
maximum contraction response) were calculated for CRC before
and after test drug addition. The pA2 values for the antagonistic
effect of test compounds on CRC for CCh were estimated from
Schild plot analysis.
Intrinsic Clearance in Microsomes.40 Rat and human mi-
crosomes (Xenotech), at the final concentration of 0.5 mg/mL, were
preincubated with test compound (0.5 µM) in phosphate buffer pH
7.4 for 10 min at 37 °C. Reaction was then started by adding the
cofactor mixture solution (NADP, glucose-6-phosphate, glucose-
6-phosphate dehydrogenase). Samples were taken at time 3, 6, 9,
12, 15, 18, 24, and 30 min and added to acetonitrile to stop the
reaction. Samples were then centrifuged and the supernatant was
Isolated Rat Detrusor Muscle and Atria. Detrusor smooth
muscle strips were excised from the bladder of female rats (250-
350 g body weight) and set up in organ baths in normal Krebs-
Henseleit solution at 37 °C under a resting tension of 1 g. Motor
activity was recorded isometrically. All experiments were performed
in the presence of eserine (1 µM). CRCs to methacholine were
constructed in the absence or presence of tolterodine or compound
22g (0.01, 0.03, and 0.1 µM; 60 min before, each). Contractile

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responses produced by methacholine were expressed as the percent
of maximal contraction produced by KCl (80 mM).
One left atrial preparation was taken from each rat, and set up
occurred as described for the rat urinary bladder, under a resting
tension of 0.5 g and at 33 °C. EFS was applied to preparations at
a basal rate of 1 Hz, 5 ms pulse duration, and 1.5 times the threshold
voltage to obtain a stabilization of contractions (basal force).
Electrodes were connected to asynchronous EMKA stimulators
(model STM-B01). After that, isoprenaline at 30 nM was tested,
and strips having a positive inotropic response less than 10% of
the basal force were discarded. CRCs to methacholine on EFS-
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45 min before, each). The negative inotropic effects produced by
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contraction. Antagonist potency of tolterodine or compound 22g
was estimated as the ability of the compounds to rightward shift
CRCs to methacholine in both the rat urinary bladder and the left
atrium. Competitive antagonism was checked by the Schild plot
method by plotting the log of antagonist concentrations versus log-
(DR-1): a plot with linear regression line and slope not significantly
different from unity was considered as proof of simple reversible
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Acknowledgment. This study was supported by Chiesi
Farmaceutici, Parma, Italy. The authors thank Dr. Alberto Cerri,
Dr. Sergio Menegon, and Dr. Samuele Pedraglio (NiKem
Research) for the analytical support and Dr. Stefano Palea
(Urosphere) for the functional in vitro studies on rat isolated
tissues.
Supporting Information Available: Spectroscopic data and
elemental analyses for compounds 14, 16-18, 21b, 21d-l, 22b-
l, 23g, 23i, 23k, 24, 25, 27-35, 38-54. This material is available
free of charge via the Internet at http://pubs.acs.org.
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