Novel alpha-7 nicotinic acetylcholine receptor agonists containing a urea moiety: Identification and characterization of the potent, selective, and orally efficacious agonist 1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1- ylbutyl) Urea (SEN34625/WYE-103914)

Article abstract of DOI:10.1021/jm901692q

Alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the α7 nAChR deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the α7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

Full text of DOI:10.1021/jm901692q

J. Med. Chem. 2010, 53, 4379–4389 4379
DOI: 10.1021/jm901692q
Novel Alpha-7 Nicotinic Acetylcholine Receptor Agonists Containing a Urea Moiety:
Identification and Characterization of the Potent, Selective, and Orally Efficacious Agonist
1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) Urea (SEN34625/WYE-103914)
Chiara Ghiron,*,†,§ Simon N. Haydar,‡,§ Suzan Aschmies,‡ Hendrick Bothmann,† Cristiana Castaldo,† Giuseppe Cocconcelli,†
Thomas A. Comery,‡ Li Di,‡ John Dunlop,‡ Tim Lock,‡ Angela Kramer,‡ Dianne Kowal,‡ Flora Jow,‡ Steve Grauer,‡
Boyd Harrison,‡ Salvatore La Rosa,† Laura Maccari,† Karen L. Marquis,‡ Iolanda Micco,† Arianna Nencini,† Joanna Quinn,†
Albert J. Robichaud,‡ Renza Roncarati,† Carla Scali,† Georg C. Terstappen,† Elisa Turlizzi,† Michela Valacchi,†
Maurizio Varrone,† Riccardo Zanaletti,† and Ugo Zanelli†
†Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy, and ‡Chemical Sciences and Neuroscience Discovery Research,
Wyeth Research, CN 8000, Princeton, New Jersey 08543-8000. §These authors contributed equally to this work.
Received November 16, 2009
Alpha-7 nicotinic acetylcholine receptor (R7 nAChR) agonists are promising therapeutic candidates for
the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists
(4-18) of the R7 nAChR deriving from our continuing efforts in the areas of Alzheimer’s disease and
schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be
a selective agonist of the R7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and
oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models.
Structural modifications leading to the improved selectivity profile and the biological evaluation of
this series of compounds are discussed.
Introduction
acetylcholine receptors (nAChR) in brain tissue from schizo-
phrenia and AD patients, as well as genetic linkage studies in
schizophrenia, implicating the locus of the R7 receptor gene
promoter.5 Prototypical R7 receptor agonists have demon-
strated improved cognition in animal models and normalized
sensory gating deficits, which are believed to contribute to the
cognitive fragmentation in schizophrenia.6,7 On the basis of
these observations, there is strong hope that selective R7nAChR
agonists will prove effective in the improvement of cognition in
both schizophrenia and AD.8-10 In accordance with this hy-
pothesis, the prototypical nAChR agonist nicotine, as well as
more selective R7 agonists such as 2-methyl-5-[6-phenylpyrida-
zin-3-yl]octahydropyrrolo[3,4-c]pyrrole (A-582941) described
recently, have been shown to improve cognitive performance
in both animal models and human clinical trials.11
As part of a multidisciplinary approach toward the treat-
ment of schizophrenia and Alzheimer’s disease, a medicinal
chemistry project was initiated to develop selective R7 ago-
nists. In the initial phase of the program, structure-activity
relationship work around the initial amide “hit” N-{4-[4-(2,4-
dimethoxyphenyl)-piperazin-1-yl]butyl}-4-pyridin-2-ylbenza-
mide (1, Figure 1)12 allowed the identification of the agonist
“lead” 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-
amide SEN12333/WAY-31753813,14 (2, Figure 1).
Cognitive disorders encompass a wide range of diseases and
represent a still unmet medical need in the psychiatric and
neurology therapeutic area. In particular, the negative sympto-
matology (cognitive impairments, reduced affect, social with-
drawal, low motivation) of schizophrenia is largely unaffected
by current antipsychotic medications that block dopamine
D2 receptors, and in Alzheimer’s disease, the most common
form of dementia, only modest symptomatic improvement in
cognitive performance is provided by standard of care agents
such as acetyl cholinesterase inhibitors and 3,5-dimethyla-
damantan-1-ylamine (memantine, a NMDAa receptor antag-
onist).
Several lines of experimental evidence support the involve-
ment of the neuronal nicotinic receptors in both schizophrenia
and AD.1-4 These include reduced expression of R7 nicotinic
*To whom correspondence should be addressed. Phone: (þ39)
0577381408. Fax: (þ39) 0577381303. E-mail: cghiron@sienabiotech.it.
aAbbreviations: ACh, acetylcholine; AChBP, acetylcholine binding
protein, AD, Alzheimer’s disease; AMMC, 3-[2-(N,N-diethyl-N-
methylamino)ethyl]-7-methoxy-4-methylcoumarin; BFC, 7-benzyloxy-
4-trifluoromethylcoumarin; ACN, acetonitrile; CHO, Chinese hamster
ovary; DCM, dichloromethane; ES, electrospray; FA, formic acid;
FLIPR, fluorescence imaging plate reader; HBSS, Hank’s balanced salt
solution; HEK, human embryo kidney; hERG, human ether-a-go-go-
related gene; HPLC-MS, high pressure liquid chromatography-mass
spectrometry; HR-MS, high resolution mass spectrometry; HTS, high-
throughput screening; LTQ, linear trap quadrupole; MED, minimum
efficacious dose; MFC, 7-methoxy-4-trifluoromethylcoumarin; NMDA,
N-methyl-D-aspartate; NOR, novel object recognition; P450, cytochrome
P450; PAMPA, parallel artificial membrane permeability assay; PDA,
photodiode array; PPI, prepulse inhibition; SAR, structure-activity rela-
tionship; SCX, strong cation exchanger; TLC, thin layer chromatography;
TOF, time-of-flight; UPLC, ultra performance liquid chromatography.
Although displaying only moderate potency against the
R7 nicotinic receptor (EC50 = 1.65 μM), “reverse amide” 2
showed promising overall selectivity and pharmacokinetic
profiles, displayed in vivo activity in various animal models
and was deemed worthy of further optimization. Starting
from 2, medicinal chemistry efforts were focused to further
improve both the potency and the selectivity from the initial
r
2010 American Chemical Society
Published on Web 05/14/2010
pubs.acs.org/jmc

4380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ghiron et al.
series. Molecular modeling studies were also applied to in-
vestigate the structural basis of ligand-receptor binding and
to define the pharmacophoric features of described R7 ago-
nists. Herein we report the synthesis and SAR of novel
derivatives which are potent and selective agonists of the R7
nAChR.
molecules. Similarly, we previously found that the presence of
a pyridine ring in the right-hand side biaryl system was
beneficial, so we initially kept this feature unchanged.13
For the present study, we decided to primarily explore a
carbon chain length of four carbon atoms. Having observed
thatincreasing the chainlength tofivecarbons resulted inonly
small functional potency changes (Table 1, compounds 3 and
4), we investigated the impact of replacing the amide moiety
with a urea in the four-carbon chain series. We noted that
potency in the urea series was maintained if not improved
(Table 1, compare results for compounds 4 and 5). This can at
least in part be explained by assuming the urea derivatives to
be a “combination” of the initial amide hit series (as repre-
sented by 1) and the longer chain “reverse amides” series to
which lead molecule 2 belongs. We were also pleased to see
that amine basicity impacted on potency with the same
general trend (Table 1, compare results for compounds 5
and 6 to 3 and 2, respectively), thus confirming this part of
the SAR.
The R7 receptor comprises five homologous subunits,
each consisting of extracellular and transmembrane main
domains.18 Although the R7 X-ray crystal structure has not
been determined to date, several crystals of R7 agonists bound
to the extracellular domain of the homologous acetylcholine
binding protein (AChBP) are available19 and commonly used
as templates to build homology models. The agonist binding
site is located at the interface between two adjacent subunits in
the extracellular domain of the receptor and is mainly defined
by an aromatic cage composed of W53, Y89, W143, Y164,
and Y185 and a disulfide-bridged loop (Cys-loop). Cation-π
or hydrogen bond interaction to W143 are reported to be key
pharmacophoric features for R7 agonists.20 To determine the
structural components of the urea series potentially respon-
sible for the biological activityobserved, we applied molecular
modeling methods, docking representative compounds into
the orthosteric binding site of a human R7 homology model.
In the putative binding mode of compound 5 into the
theoretical model for human R7 receptor, the protonated
nitrogen of piperidine makes hydrogen bond contact to the
backbone carbonyl of W143. The urea moiety is placed within
Results and Discussion
During the initial phase of structure-activity exploration
on the amide series, we noticed that the basicity of the left-
hand side amine was crucial to the modulation of potency at
the R7 receptor.13 The presence of a more basic amine was
usually associated with higher potency. This can be related to
stronger cation-π interactions, reportedly15 important in the
ligand-receptor association.
We chose piperidine as the “reference” amine around which
to explore further structural variations as piperidine was
foundto confer a minimal potency level (compare for example
compound 3 to morpholine-containing 2 in Table 1), thus
facilitating the impact of modifications in other areas of the
Figure 1. Initial hit (1) and lead (2) molecules, with schematic
pharmacophore features.
Table 1. Effect of the Amine Basicity, Chain Length, and Amide Moiety Replacement on Activity16
a All functional activities were measured in a calcium flux assay using a fluorescence imaging plate reader. Activity at rat R7-nAChRs was determined
using a stable recombinant GH4C1 cell line expressing the receptor.17 Reported EC50/IC50 values were determined in a single experiment and obtained
from triplicate data points generated within the same experimental session. Data are averaged from multiple experiments (n) and reported as the average
( SEM (n). Nicotine under the same conditions had EC50 = 1.34 μM ( 0.02 (n = 62). Compounds with Emax >70% of nicotine were considered to be
full agonists. All reported compounds gave values >90%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4381
the aromatic cage, with the delocalized electronic system
stacking over Y89 (Figure 2). The four-carbon chain serves
asspacerbetweenthe piperidine andthe urea pharmacophoric
centers. In our studies, the right-hand side biaryl system
appears to reach an area of the agonist binding site rich in
hydrophilic residues (highlighted in blue in Figure 2), making
this region prone to hydrogen bonding contacts to the pyr-
idine nitrogen of compound 5.
With this information in hand, and considering that a urea
moiety would be intrinsically more stable than an amide, we
set out to explore the urea class in more detail. A representa-
tive selectionof themoleculeswhichwere synthesizedisshown
in Table 2 with their R7 activity, selectivity over homologous
receptor subtypes, and early profiling data. The selection of
Table 2 aims to explore the effects of ring substitution in terms
ofsterichindranceandtorsional angle ofthe biarylsystemand
electronic effects on the aromatic rings. In our selection of
substituents, we selected moieties that would allow for further
exploration of the hydrophilic region pointed out by the
docking studies without introducing additional hydrogen-
bond donors beyond the ones brought by the urea linker itself
to avoid increasing the polar surface area and thus potentially
depressing brain penetration.21
As can be observed from the data reported in Table 2, all
compounds maintained an excellent level of activity including
unsubstituted compound 7, showing a relative breadth of
modifications possible for the second aryl ring and demon-
strating the impact of the urea moiety on receptor affinity.
With the exception of 10, solubility and passive permeability
were also excellent, showing potential for further progression.
Although no clear indications of a strong SAR was ob-
tained with regards to R7 potency, all compounds with the
exception of 12 presented relatively high activity against the
homologous ganglionic receptor (R3) as well as substantial
cytochrome P450 inhibition, especially the 2D6 subtype.
Considering that SAR analysis takes into account the impact
of modifications on the main target of interest as well as the
effects on the overall compound profile, we favored those
substituents showing an acceptable balance of properties.
Given that the presence of a pyridyl ring appeared to
improve selectivity against R3 (compound 12) and to lower
2D6 inhibition (compound 11 and 12), and that lipophilicity
tends to be one of the major factors in molecular recognition
bythe cytochromefamily of enzymes,24 wepreparedanumber
of analogues where pyridine replaced the first phenyl ring of
the right-hand side biaryl system. We maintained the pyridine
nitrogen potentially pointing to the same binding area as in
compound 12, hoping that the reasons for the observed
improvement in selectivity could be the result of unfavorable
interactions with the R3 receptor. Results are reported in
Table 3, where compounds 13-18 are exact analogues of
7-12, respectively.
Figure 2. Compound 5 docked into the orthosteric site of the
homology model for human R7 nAChR. Residues involved in key
interactions with the ligand are highlighted in orange. For sake of
clarity, part of the Cys-loop has been removed from the image.
Amino acid numbering refers to the 1UW6 sequence which X-ray
structure was used as a template for homology modeling.
While again with almost no exceptions the molecules
showed excellent solubility, passive diffusion permeability,
and metabolic stability, we were pleased to see that their
profile in terms of selectivity was further improved over the
Table 2. Activity, Selectivity (EC50 for Agonist Behavior, IC50 for Antagonist Behavior, μM), Solubility (Sol, μM), Permeability Class (Perm),
Metabolic Stability (Met Stab, % Remaining), P450 Subtype Percent Inhibition at 3 μM, for a Representative Selection of Phenyl Urea Derivatives
aAll functional activities were measured in either calcium flux or membrane potential assays using a fluorescence imaging plate reader. Compounds
with Emax >70% of nicotine were considered to be full agonists. The reported compounds gave values >90%. b Solubilities were determined at pH 7.4 at
pseudothermodynamic equilibrium. c Permeability is based on measuring the permeation rate of the test article through an artificial membrane. The
assay uses a mixtureof porcine pig brain lipids in dodecane(2% w/v).22 d Metabolic stabilitywas determined as percentage remaining after incubation for
1 h with recombinant hCYP3A4. e Percent cytochrome inhibition23 values <15-20% were considered low. See Experimental Section for further details
on all assays.

4382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ghiron et al.
Table 3. Activity, Selectivity (EC50 for Agonist Behavior, IC50 for Antagonist Behavior, μM), Solubility (Sol, μM), Permeability Class (Perm),
Metabolic Stability (Met Stab, % Remaining), P450 Subtype Percent Inhibition at 3 μM, for a Representative Selection of Pyridyl Urea Derivativesa
a Notes as in Table 2: All functional activities were measured in either calcium flux or membrane potential assays using a fluorescence imaging plate
reader. Compounds with Emax >70% of nicotine were considered to be full agonists. The reported compounds gave values >90%. Solubilities were
determined at pH 7.4 at pseudothermodynamic equilibrium. Permeability is based on measuring the permeation rate of the test article through an
artificial membrane. The assay uses a mixture of porcine pig brain lipids in dodecane (2% w/v).22 Metabolic stability was determined as percentage
remaining after incubation for 1 h with recombinant hCYP3A4. Percent cytochrome inhibition23 values <15-20% were considered low. See
Experimental Section for further details on all assays.
Table 4. Pharmacological Characterization and Rat PK Parameters for
Compound 16
additional pharmacology
R7 Ki (nM)
44
R7 e-physa EC50 (nM)
R7 e-physa Emax (%)
R4β2 IC50,EC50 (μM)
hERGb IC50 (μM)
490
70
>30
1.3
PK
Cmax (ng/mL)c
40
T
max (h) c
5.3
318
100
0.3
AUClast (h ng/mL) c
3
%Fd
B/Pe
a Whole cell patch clamp recordings were performed on GH4C1 cells
expressing rat R7 receptors. Each cell was exposed to at least one
concentration of acetylcholine and a full range of concentrations of
16. b hERG response was obtained from CHO cells stably expressing the
channel using an IonWorks system. c Pharmacokinetic parameters were
determined following a single 3 mg/kg po dose in male Long Evans rats.
d Oral bioavailability (%F) was determined in a separate experiment as
the ratio of AUC0-inf normalized for the dose, obtained following single
2 mg/kg iv and 10 mg/kg po doses in male Han Wistar rats. In the same
experiment, the determined T1/2 was 3.5 h. e The brain to plasma (B/P)
ratio was determined as the ratio of AUC0-inf following a single 2 mg/kg
po dose in male Han Wistar rats. In this experiment, AUC0-inf for
plasma and brain were 768 and 215 h ng/mL, respectively.
3
Figure 3. (a) In vivo results for compound 16. MK-801-induced
deficit in the novel object recognition test. Compound 16 blocks the
MK-801-induced deficit in the novel object recognition test (1 h
delay) in Long Evans rats. (b) In vivo results for compound 16. MK-
801-disrupted prepulse inhibition test. Compound 16 blocks the
MK-801-disrupted prepulse inhibition in both acute and chronic
studies in Long Evans rats.
phenyl analogues. This was observed not only when compar-
ing cytochrome inhibition, thus removing some concerns
about potential for drug-drug interactions, but selectivity
was also improved over the homologous nicotinic muscle R1
and ganglionic R3 receptors.
Considering the overall excellent spectrum of activity and
selectivity shown, we selected the para-fluoro substituted
compound 16 for further studies, trying to pre-empt potential
problems related to broader metabolism and particularly
oxidative activation of unsubstituted compounds like 13.
Indeed, wefound that while 13 hadmedium intrinsic clearance
in rat liver microsomes, with a value of 18 μL/min/mg, 16 had
a low clearance value (below 5 μL/min/mg), indicating a more
favorable elimination profile.25,26
Table 4 shows further characterization of compound 16,
both in vitro and in vivo, showing high selectivity also against
the R4β2 nicotinic receptor, and a good pharmacokinetic
profile with excellent oral absorption, and initial plasma levels
in line with the binding affinity. Compared with previously
reported compound 2,13 16 displayed improved binding affi-
nity and its increased potency was also confirmed in the
electrophysiology experiment. Longer half-life and improved

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4383
oral bioavailability were also observed, potentially linked to
increased stability of the urea moiety compared to the amide
present in compound 2. A lower brain-to-plasma ratio was
measured for 16, and unfortunately moderate hERG activity
was observed, although a level of differentiation over the
affinity for the R7 receptor was reached, especially when
considering the pharmacokinetic parameters and a protein
binding of 75%.27
tolerance to the compound. Although the brain-to-plasma
ratio suggests brain exposure levels one order of magnitude
lower than the in vitro EC50 value (181 ng/mL for the electro-
physiology experiment), our results are in line with recent
findings that brain levels of agonists required to elicit in vivo
effects may lie at the low end of the concentration-response
curve defined in vitro using rapid agonist application.11
Compound 16 was also characterized in several in vivo
behavioral models, displaying efficacy in assays of cognitive
function and perceptual processing, showing ability to attenu-
ate pharmacologically induced deficits via the glutamatergic
system. Treatment with 16 reversed a (þ)-5-methyl-10,11-
dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine,
MK-801) induced deficits in both the novel object recognition
memory (Figure 3a) and prepulse inhibition models (Figure 3b)
with a minimum efficacious dose of 3 mg/kg in spite of the
relatively moderate brain to plasma ratio. Differently from the
initial lead 2 where efficacy was observed after ip administra-
tion, activity of 16 was observed in both models after oral
administration.
Chemistry
The compounds investigated were synthesized using two
modifications of the same general route.
The reverse amide compounds were synthesized as already
described13 and shown in Scheme 1 in a one-pot reaction
between the appropriate ω-bromoalkanoyl chloride (19a,b)
and 4-bromoaniline, followed by treatment with piperidine or
morpholine and Suzuki coupling with 3-pyridylboronic acid.
Ureas 5-11 were synthesized using standard methodology,
starting from 4-bromophenylisocyanate reaction with ami-
noalkylamines 20 or 21, followed by cross-coupling reaction
(Scheme 2).
In the case of compound 12, we failed to obtain the desired
product through reaction of 22 with 2-pyridylboronic acid.
We therefore reversed the cross-coupling partners after
synthesizing boronic esters 24 and 25 as shown in Scheme 3.
Boronic ester 25 was then submitted to standard coupling
conditions to afford 12.
Importantly, in the prepulse inhibition test 16 was active
both in the acute and in the chronic studies, showing no
Scheme 1. Synthesis of Aminoalkanoic Acid Amidesa
For compounds 13-18, where the first ring in the right-
hand side system is a pyridine, we activated 6-bromo-pyridin-
3-ylaminewithtriphosgeneand reacted the transiently formed
isocyanate in situ with amine 20, as shown in Scheme 4. For
large-scale reactions, we found more advantageous to prepare
intermediate 26 activating 6-bromo-pyridin-3-ylamine via the
more stable isopropenyl carbamate derivative which could be
a (a) Et3N, 4-bromoaniline, CH2Cl2; (b) piperidine or morpholine,
Et3N; (c) 3-pyridylboronic acid, Pd(PPh3)4, Na2CO3, MeCN/H2O.
Scheme 2. Synthesis of Aminoalkyl Phenyl Ureasa
a (a) Et3N, 4-bromophenylisocyanate, CH2Cl2; (b) aryl or heteroaryl boronic acid, Pd(PPh3)4, Na2CO3, MeCN/H2O.
Scheme 3. Alternative Route to Aminoalkyl Phenyl Ureasa
a Reagents and conditions: (a) N-methylpyrrolidine (cat.), THF, 55 °C; (b) 2-bromopyridine, Pd(PPh3)4, Na2CO3, MeCN/H2O.
Scheme 4. Synthesis of Aminoalkyl Pyridyl Ureasa
a Reagents and conditions: (i) Et3N, 6-bromo-pyridin-3-ylamine, triphosgene, or isopropenyl chloroformate, CH2Cl2; (ii) aryl or heteroaryl boronic
acid, Pd(PPh3)4, Na2CO3, MeCN/H2O.

4384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ghiron et al.
isolated and reacted more conveniently and safely (see Experi-
mental Section).
enough to be used in the following step. If necessary, a further
purification was carried out by flash-chromatography. The phtha-
limides thus obtained (1 mol equiv) were dissolved in EtOH to
obtain a 2 M solution and hydrazine monohydrate (2 mol equiv)
was added dropwise. The mixture was heated at 80 °C for 4 h, after
which the reaction was acidified with 37% HCl and the solid which
precipitated was removed by filtration. The solution was concen-
trated under vacuum and taken up with 1N HCl. Any residual 2,3-
dihydrophthalazine-1,4-dione was removed by filtration. The aqu-
eous solution was removed under reduced pressure under vacuum
to recover the pure product.
Conclusions
In summary, we have identified a series of novel derivatives
containing a urea moiety, potent agonists of the R7 nAChR
with improved profile over the initial lead molecule 2. In
particular, compound 16 (SEN34625/WYE-103914) showed
potency, selectivity, and exposures leading to excellent in vivo
efficacy and was selected for further exploratory studies. This
series is in our opinion of particular interest as it represents
further development of a novel structural motif in the nico-
tinic agonists area compared to the more explored azabicyclic
derivatives.28
4-Piperidin-1-yl-butylamine (20). The general procedure for
aminoalkylamine synthesis was followed, starting with piper-
idine (1.98 mL, 20 mmol). The title product was purified by SCX
cartridge eluting with MeOH:DCM 1:1, followed by 2 M NH3
in MeOH, to obtain 410 mg of compound as a colorless oil
(35% yield).
1H NMR (400 MHz, CDCl3): δ (ppm): 1.39-1.62 (m, 9H),
1.91-2.08 (m, 3H), 2.27-2.33 (m, 2H), 2.33-2.44 (m, 3H), 2.71
(t, J = 6.8, 2H). Mass (ES) m/z %: 157 (M þ 1, 100%). HPLC:
Experimental Section
1. Chemistry. General Methods. Unless otherwise specified,
all nuclear magnetic resonance spectra were recorded using a
Varian Mercury Plus 400 MHz spectrometer equipped with a
PFG ATB broadband probe. HPLC-MS analyses (5 and 10 min
methods) were performed with a Waters 2795 separation mod-
ule equipped with a Waters Micromass ZQ (ES ionization)
and Waters PDA 2996, using a Waters XTerra MS C18 3.5 μm
2.1 mm ꢀ 50 mm column. Preparative HPLC was run using a
Waters 2767 system with a binary gradient module Waters 2525
pump and coupled to a Waters Micromass ZQ (ES) or Waters
2487 DAD, using a Supelco Discovery HS C18 5.0 μm 10 mm ꢀ
21.2 mm column. Gradients were run using 0.1% formic acid/
water and 0.1% formic acid/acetonitrile with gradient 5/95 to
95/5 with a runtime of 5 or 10 min as stated in the examples.
Retention times are expressed in minutes. The purity of com-
pounds submitted for screening were >95% as determined by
integrating at 215 nm the peak area of the LC chromatograms.
To further support the purity statement, all compounds were
also analyzed at a different wavelength (254 nm), and total ion
current (TIC) chromatogram and NMR spectra were used to
further substantiate results. HRMS data were obtained through
(data with five-decimal place precision) a LTQ-Orbitrap mass
spectrometer or (data with four-decimal place precision) through
an Agilent 6220 TOF, using the manufacturer’s MassHunter soft-
ware, via direct infusion of a 1 μM sample in ACN 30%/FA
0.1% (flow: 5 μL/min). The LTQ-Orbitrap instrument was cali-
brated prior to analysis with a H3PO4 1% solution in mass range
100-800 m/z. Each determination was the combination of
10 successive scans acquired at 60.000 fwhm resolution. For
the Agilent 6220 TOF, tuning and calibration parameters were
set using the manufacturer’s auto tune and auto calibrate
solutions and software. Elemental composition calculations
were executed using the specific tool included in the QulBrowser
module of Xcalibur (Termofisher Scientific, release 2.0.7) soft-
ware, using a tolerance of 5 ppm. All column chromatography
was performed following small modifications of the original
method of Still.29 All TLC analyses were performed on silica
gel (Merck 60 F254) and spots revealed by UV visualization at
254 nm and KMnO4 or ninhydrin stain. All microwave reactions
were performed in a CEM Discover oven. The Isolute SCX
cartridges (1, 2, and 10 g sorbent weight) were purchased from
Biotage.
t
R = 0.31, area 100%; (5 min method).
4-Morpholin-4-yl-butylamine (21). The general procedure for
aminoalkylamine synthesis was followed, starting from mor-
pholine (2.07 g, 23.8 mmol). The crude was treated with HCl
2 M, the solid was filtered off and the aqueous solution was
evaporated to dryness. The residue was taken in isopropanol
and filtered to give 3.10 g of solid title compound as hydro-
chloride salt (67% yield). 1H NMR (400 MHz, CDCl3) δ (ppm):
1.18-1.34 (m, 2H), 1.42-1.57 (m, 4H), 2.35 (t, J = 7.7, 2H),
2.39-2.50 (m, 4H), 2.71 (t, J = 6.2, 2H), 3.68-3.75 (m, 4H).
General Procedure for Urea Synthesis. WARNING: isocya-
nates and phosgene derivatives (e.g., triphosgene) are highly
toxic and should only be handled in a fume hood while wearing
appropriate personal protective equipment. To a cooled solution
of amine (1 mol equiv) in dichloromethane, the required isocyanate
(1 mol equiv) was added. The mixture was left stirring at 0 °C for
1-4 h. The p-bromophenyl ureas generally precipitated out of
solution as a white solid, recovered by filtration, and if necessary
purified further by washing with Et2O or by flash chromatography.
1-(4-Bromophenyl)-3-(4-piperidin-1-ylbutyl) urea (22). Follow-
ing the general procedure for urea synthesis, 4-piperidin-1-yl-
butylamine (2.34 g, 15.0 mmol) was dissolved in 60 mL of DCM
and 4-bromophenylisocyanate (2.97 g, 15.0 mmol) in 15 mL of
DCM to give 3.98 g of title compound obtained as a white solid
(75% yield) after solvent removal and trituration with Et2O,
which was used without further characterization. 1H NMR (400
MHz, DMSO-d6): δ 1.40 (m, 10H), 2.19 (m, 2H), 2.26 (m, 4H),
3.05; (m, 2H), 6.16 (s, 1H), 7.35 (m, 4H), 8.52 (s, 1H). Mass (ES)
m/z %: 354.30, 356.25 (M þ 1, 100%, Br pattern). HPLC tR
=
1.73; area 100% (10 min method)
1-(4-Bromophenyl)-3-(4-morpholin-4-ylbutyl) urea (23). Follow-
ing the general procedure for urea synthesis and starting from
4-morpholin-4-ylbutylamine (0.72 g, 4.58 mmol), 1.4 g of title
compound were obtained as solid by filtration from the reaction
solution (86% yield). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.40
(m, 4H), 2.30 (m, 6H), 3.06 (m, 2H), 3.53 (m, 4H), 6.16 (m, 1H),
7.34 (s, 4H), 8.51 (s, 1H).
1-(6-Bromopyridin-3-yl)-3-(4-piperidin-1-ylbutyl)-urea (26).
To a solution of 6-bromopyridin-3-ylamine (3.46 g, 20 mmol)
in dichloromethane (70 mL), triphosgene (1.96 g, 6.6 mmol) and
triethylamine (2.2 g, 22 mmol) were added under N2 at 0 °C.
After 15 min, a solution of 4-piperidin-1-ylbutylamine (3.12 g,
20 mmol) in DCM (10 mL) was added dropwise at 0 °C. The
reaction mixture was allowed to warm to room temperature and
stirred for 2 h. Dichloromethane was removed in vacuo and the
residue dissolved in EtOAc and washed with H2O. The aqueous
layer was basified with solid Na2CO3 to pH 9-10 and extracted
with EtOAc. The combined organic layers were dried over
Na2SO4 and evaporated in vacuo to give 5 g of the desired
product as a solid (yield: 70%). 1H NMR (400 MHz, CDCl3): δ
2. Synthesis. General Procedure for Aminoalkylamine Synthesis.
To a suspension of the required amine (1 mol equiv), sodium
iodide (0.5 mol equiv), and potassium carbonate (1.1 mol equiv)
in 2-butanone (4 M solution) N-(ω-bromoalkyl) phthalimide
(1 mol equiv) was added. The resulting suspension was stirred for
18 h at 85 °C, and then the reaction was filtered and the solvent
removed by vacuum distillation; the resulting oil was washed with
water and recovered with DCM. The solvent was removed under
reduced pressure to yield the amino-phthalimide derivative pure

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4385
1.45-1.62 (m, 8H), 2.29-2.48 (m, 8H), 3.22-3.24 (m, 2H), 6.19
(m, 1H), 7.35 (d, J = 8.7, 1H), 7.54 (bs, 1H), 7.95 (dd, J = 8.7,
2.8, 1H), 8.17 (d, J = 2.8, 1H).
the product were combined and dried under reduced pressure.
The title compound was obtained in 43% yield (36 mg). 1H NMR
(400 MHz, MeOD): δ 1.43 (m, 4H), 2.28 (m, 6H), 3.08 (m, 2H),
3.54 (m, 4H), 6.20 (m, 1H), 7.41 (m, 1H), 7.50 (d, J = 7.4 Hz, 2H),
7.58 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 7.7 Hz, 1H), 8.48 (s, 1H),
8.57 (m, 1H), 8.83 (s, 1H). Mass (ES) m/z %: 355 (M þ 1, 100%).
HPLC tR = 0.58; area 100%; (10 min method). HRMS: calcd for
C20H26N4O2 þ Hþ, 355.2129; found (ESI, [M þ H]þ obsd),
355.2125.
6-Piperidin-1-ylhexanoic acid (4-pyridin-3-yl-phenyl)-amide
(4). (a) 6-Piperidin-1-yl-hexanoic acid (4-bromophenyl)-amide:
A 0.2 mmol/mL solution of 4-bromoaniline (697 mg, 4.1 mmol)
and triethylamine (415 mg, 4.1 mmol) in dichloromethane was
cooled at 0 °C under nitrogen atmosphere. A 0.3 mmol/mL
solution of 6-bromohexanoyl chloride (19b, 869 mg, 4.1 mmol)
in dichloromethane was slowly added. The mixture was stirred
at room temperature for 1.5 h, after which piperidine (1.74 g,
16.8 mmol) and triethylamine (415 mg, 4.1 mmol) were added.
The mixture was stirred at room temperature for 18 h and then
at reflux temperature for further 20 h. The mixture was then
cooled, the organic solution was washed with brine and dried,
and the solvent removed under reduced pressure. Trituration
from Et2O:hexane 1:1 gave 1.02 g of title compound (yield 71%).
1H NMR (400 MHz, DMSO-d6): δ 1.40 (m, 12H), 2.23 (m, 8H),
7.43 (d, J = 8.9 Hz, 2H,), 7.54 (d, J = 8.9 Hz, 2H), 9.99 (s, 1H).
(b) 6-Piperidin-1-yl-hexanoic acid (4-pyridin-3-ylphenyl)-amide:
To a degassed mixture of 6-piperidin-1-ylhexanoic acid (4-bro-
mophenyl)amide (120 mg, 0.34 mmol), 3-pyridyl boronic acid
(46 mg, 0.37 mmol) in acetonitrile/sodium carbonate 0.4 M solution
1/1 (4 mL), a catalytic amount of Pd(PPh3)4 (5 mmol %) was
added. The reaction mixture was heated at 90 °C for 20 min under
microwave irradiation (power set at 150 W) and then again other
20 min. The organic layer was separated and concentrated, and the
solvent was removed under reduced pressure to afford the crude
product which was purified on a SCX cartridge followed by
preparative HPLC column, giving 42 mg of title compound
1-(4-Piperidin-1-ylbutyl)-3-(4-pyridin-2-ylphenyl) urea (12).
(a) 1-(4-Piperidin-1-ylbutyl)-3-[4-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)phenyl] urea (25): 4-(4,4,5,5-Tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline (2.0 g, 9.1 mmol) and N-methyl-
morpholine (1.22 mL, 10.9 mmol) were dissolved in 15 mL of
THF, and the resulting solution was cooled down to 0 °C.
Isopropenyl chloroformate (1.19 mL, 10.9 mmol) was added
dropwise with stirring, and the mixture was allowed to reach
ambient temperature and stirred for additional 3 h. The solvent
was removed under reduced pressure and the crude product
dissolved in DCM, washed twice with 10 mL of brine, and dried
over MgSO4. The solution was filtered and the solvent removed
under reduced pressure to give [4-(4,4,5,5-tetramethyl-[1,3,2]-
dioxaborolan-2-yl)-phenyl]carbamic acid isopropenyl ester 24
as a white powder, which was used in the next step without
further purification (2.49 g, yield 90%). A solution of 24
(14.5 mmol) and 4-piperidin-1-ylbutylamine (2.24 g, 14.5 mmol)
in THF (5 mL) was heated to 55 °C. To this solution, 1-methyl-
pyrrolidine was added (0.16 mL, 1.45 mmol) and the reaction
was stirred at 55 °C for 24 h. Upon reaction completion, as
monitored by carbamic ester disappearance (LCMS analysis),
the reaction was cooled to room temperature. The solvent and
residual reagents were removed under reduced pressure to afford
25 in 92% yield (5.38 g, 13.4 mmol). 1H NMR (400 MHz, CDCl3):
1.32 (m, 12H); 1.55 (m, 10H); 2.37 (m, 6H); 3.25 (s, 2H); 6.80 (s, 1H),
6.55 (s, 1H), 7.30 (d, 2H), 7.75 (d, 2H). Mass (ES) m/z %: 402.23
(M þ 1, 100%. HPLC: tR = 2.20; area 100%; (10 min method).
(b) 1-(4-Piperidin-1-yl-butyl)-3-(4-pyridin-2-yl-phenyl)-urea (12):
A suspension of 2-bromopyridine (31 mg, 0.20 mmol), 25 (96 mg,
0.24 mmol), and Pd(PPh3)4 (23 mg, 0.02 mmol) in a mixture of
1 mL of sodium carbonate 0.4 M and 1 mL of acetonitrile was
stirred at 75 °C for 12 h. The acetonitrile was removed in vacuum
and the title compound purified by crystallization from ethyl
acetate. Pd traces were removed by dissolving the compound in
methanol and passing the solution through a 500 mg PL-Thiol MP
SPE (Polymer Laboratories, now part of Varian Inc.) cartridge
eluting with methanol. The solvent was evaporated to give 12 mg
of the title compound (17% yield). 1H NMR (400 MHz, DMSO):
δ 1.33-1.49 (m, 10H), 2.19-2.27 (m, 6H), 3.07-3.09 (m, 2H),
6.25 (t, J = 8.0, 1H), 7.22-7.26 (m, 1H), 7.47-7.49 (m, 2H),
7.77-7.85 (m, 2H), 7.93-7.96 (m, 2H), 8.58-8.59 (m, 1H), 9.57
1
(35% yield) as the formate salt. H NMR (400 MHz, DMSO-
d6): δ 1.44 (m, 12H), 2.32 (t, J = 7.4 Hz, 2H), 2.56 (m, 6H), 7.44
(dd, J = 7.9, 4.8 Hz, 1H), 7.66 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8
Hz, 2H), 8.02 (ddd, J = 8.0, 2.1, 1.7 Hz, 1H), 8.25 (s, 1H), 8.51 (dd,
J = 4.7, 1.5 Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H), 10.05 (s, 1H). Mass
(ES) m/z %: 352 (M þ 1, 100%). HPLC tR = 0.28; area 100%;
(10 min method). HRMS: calcd for C22H29N3O þ Hþ, 352.2383;
found (ESI, [M þ H]þ obsd), 352.2382.
1-(4-Piperidin-1-yl)butyl-3-(4-pyridin-3-ylphenyl) urea (5). 1-(4-
Bromophenyl)-3-(4-(piperidin-1-yl)butyl) urea (3.0g, 8.45 mmol),
pyridine-3-boronic acid (1.76 g, 14.4 mmol), and cesium carbo-
nate (5.51 g, 16.9 mmol) were suspended in 60 mL of toluene and
30 mL of ethanol and the mixture degassed with a nitrogen stream.
Tetrakis (triphenylphosphine) palladium (0.29 g, 0.25 mmol) was
added and the mixture heated at 85 °C for 15 h. The mixture was
then filtered warm on celite and the solvent removed under
reduced pressure. HCl 1 M was added and the impurities extracted
with EtOAc. The aqueous phase was then basified by NaOH 10%
and extracted with DCM. The precipitate formed at the interface
was filtered and recrystallized from acetonitrile affording 1.56 g of
pure product (yield: 52%).
(bs, 1H). Mass (ES) m/z %: 353 (M þ 1, 100%). HPLC: tR
=
1H NMR (400 MHz, DMSO-d6): δ 1.41 (m, 10H), 2.23 (m,
6H), 3.07 (m, 2H), 6.17 (t, J = 5.6 Hz, 1H), 7.42 (dd, J = 8.0, 3.2
Hz, 1H,), 7.50 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.99
(ddd, J = 7.9, 2.4, 1.7 Hz, 1H), 8.48 (dd, J = 4.7, 1.6 Hz, 1H),
8.54 (s, 1H), 8.83 (dd, J = 2.4, 0.7 Hz, 1H). Mass (ES) m/z %:
353 (M þ 1, 100%). HPLC: tR = 0.27; area 100%; (10 min
method). HRMS: calcd for C21H28N4O þ Hþ, 353.23359; found
(ESI, [M þ H]þ obsd), 353.23357.
double peak at solvent front observed at 0.35, 0.84; total area
100% (10 min method). HRMS: calcd for C21H28N4O þ Hþ,
353.23359; found (ESI, [M þ H]þ obsd), 353.23370.
1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) urea
(16). (a) (6-Bromo-pyridin-3-yl)-carbamic acid isopropenyl ester:
To a solution of NaOH (1.13 g, 28.3 mmol) in 56 mL of water,
a solution of 6-bromo-pyridin-3-ylamine (3.26 g, 18.8 mmol) in
112 mL of DCM was added. The mixture was cooled at 0 °C, and
isopropenyl chloroformate (3.16 g, 26.4 mmol) was added in one
hour dissolved in 15 mL of DCM maintaining the solution at 0 °C.
The mixture was then allowed to reach room temperature and
stirred overnight. The organic phase was separated and evaporated
at reduced pressure, maintaining the temperature of the evaporator
bath below 25 °C. The crude product obtained was used in the next
reaction without further purification. Mass (ES) m/z %: 257-259
(M þ 1, 100%, Br pattern). HPLC tR = 1.88; area 84% (5 min
method). 1-(6-Bromopyridin-3-yl)-3-(4-piperidin-1-ylbutyl) urea
(26): The crude product obtained in the previous reaction
was dissolved in 80 mL of THF, and 4-piperidin-1-ylbutylamine
1-(4-(Morpholin-4-yl)butyl)-3-(4-pyridin-3-ylphenyl) urea (6).
To a degassed solution of 1-(4-bromophenyl)-3-(4-morpholin-
4-ylbutyl) urea (82 mg, 0.23 mmol), pyridine-3-boronic acid
(37 mg, 0.3 mmol) was added dissolved in acetonitrile/0.4N
aqueous Na2CO3 1/1 (1 mL/g substrate). After addition of
Pd(PPh3)4 (5-10% mol), the reaction mixture was heated at
90 °C for 20 min under microwave irradiation (power set at 150 W).
The acetonitrile layer was separated, the solvent was removed under
reduced pressure, and the crude material was purified through a
SCX cartridge (eluting with a gradient of DCM/MeOH, followed
by MeOH, and finally NH3/MeOH). The fractions containing

4386 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ghiron et al.
(2.94 g, 18.8 mmol) was added. The solution was refluxed under
nitrogen atmosphere for 2.5 h. After evaporation of the solvent,
the product was dissolved in DCM. The organic phase was
washed with brine, evaporated, and dried to give 6.22 g of title
product (yield: 93%). Mass (ES) m/z %: 355-357 (M þ 1, 100%,
Br pattern). HPLC tR = 1.08; area 100% (5 min method). (b)
1-[6-(4-Fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) urea:
1-(6-Bromopyridin-3-yl)-3-(4-piperidin-1-ylbutyl) urea (6.22 g,
17.5 mmol), 4-fluorophenylboronic acid (3.68 g, 26.3 mmol), and
cesium carbonate (11.4 g, 35.0 mmol) were dissolved in 143 mL of
toluene and 72 mL of EtOH and the mixture degassed with a
nitrogen stream. Tetrakis(triphenylphosphine) palladium (0.61 g,
0.52 mmol) was added and the mixture heated at 90 °C for 2 h. The
warm reaction mixture was then filtered on celite and the solvent
evaporated. The product was purified by SiO2 column (gradient
from 100% DCM to DCM-2N methanolic ammonia 8:2). The
product obtained was crystallized from ethyl acetate, affording
with a sigmoidal concentration-response (variable slope) equa-
tion: Y = Bottom þ (Top - Bottom)/(1 þ (EC50/X)Hill Slope),
where X is the concentration, Y is the response, Bottom is the
bottom plateau of the curve, and Top the top plateau.
Activity of compounds at the muscle and ganglionic type
nAChR receptors was determined using a membrane potential
sensitive fluorescent dye. TE671 and SHSY5Y cells were plated
at a density of 5 ꢀ 104 and 1 ꢀ 105 cells/well, respectively, 24 h
prior to assay. Growth media were removed from the cells by
flicking the plates, and membrane potential dye (Molecular
Devices), reconstituted in HBSS five times more diluted com-
pared to the manufacturer’s instructions, was added to the wells.
Plates were incubated for 60 min at room temperature and then
directly transferred to the FLIPR system. Compounds to be
tested were prepared in assay buffer as 5ꢀ-concentrated solu-
tions in a separate 96-well polypropylene plate. Baseline fluore-
scence was monitored for the first 10 s followed by the addition
of compounds. For detection of antagonists activity, agonist
(epibatidine; EC80 final concentration) was added to every well
except negative controls. Signal recordings were performed as
above. Results were exported from the FLIPR raw data as SUM
of fluorescence signal intensity for the first addition and
MAX-MIN for the second addition of compounds. The re-
sponses were normalized to the positive control (epibatidine
1 μM final concentration). The compounds tested were found to
display antagonist activity, and IC50 values were calculated as
described before.
Solubility Assay. Standard and sample solutions were pre-
pared from a 10 mM DMSO stock solution using an automated
dilution procedure. For each compound, three solutions were
prepared; one to be used as standard and the other two as test
solutions. Standard: 250 μM standard solution in acetonitrile/
buffer, with a final DMSO content of 2.5% (v/v). Test sample
for pH 3.0: 250 μM sample solution in acetic acid 50 mM, pH =
3, with a final DMSO content of 2.5% (v/v). Test sample for pH
7.4: a 250 μM sample solution in ammonium acetate buffer
50 mM, pH = 7.4, with a final DMSO content of 2.5% (v/v).
The 250 μM product suspensions/solutions in the aqueous
buffers were prepared directly in Millipore MultiScreen-96 filter
plates (0.4 μm PTCE membrane) and sealed. Plates were left for
24 h at room temperature under orbital shaking to achieve
“pseudo-thermodynamic equilibrium” and to presaturate the
membrane filter. Product suspensions/solutions were then fil-
tered using centrifugation, diluted 1:2 with the same buffer
solution, and analyzed by UPLC/UV/TOF-MS, using UV-
detection at 254 nm for quantitation. Solubility was calculated
1
2.14 g (yield: 33%) of the title product. H NMR (400 MHz,
DMSO): δ 1.31-1.47 (m, 10H), 2.19-2.34 (m, 6H), 3.07-3.09 (m,
2H), 6.31 (t, J = 5.6, 1H), 7.25 (t, J = 8.9, 2H), 7.81 (d, J = 8.7,
1H), 7.95-8.04 (m, 3H), 8.56 (d, J = 2.5, 1H), 8.68 (s, 1H). Mass
(ES) m/z %: 371 (M þ 1, 100%). HPLC: tR = 1.75; area 99%
(10 min method). HRMS: calcd for C21H27FN4O þ Hþ 371.22417;
found (ESI, [M þ H]þ obsd), 371.22421.
3. Biology. Ca2þ-Flux and Membrane Potential Measure-
ments with a Fluorescence Imaging Plate Reader. The following
recombinant cell lines were used as specific sources of receptors:
GH4C1 cells stably transfected with pCEP4/rat R7 nAChR as
previously described,17 HEK293 cell lines stably expressing
human 5-HT3A receptors.17 Native neuroblastoma SH-SY5Y
cells were used as source of human ganglionic nAChRs (R3), and
TE671 rhabdomyosarcoma cells were used as endogenous
source of muscle R1β1δγ receptors. GH4C1 cells expressing
R7 and HEK cells expressing 5-HT3A receptors were analyzed by
Ca2þ-flux measurements employing a Fluorometric Imaging
Plate Reader (FLIPR, Molecular Devices) system, whereas the
cells expressing the nicotinic receptor subunits R1 and R3 were
tested in the FLIPR system with a membrane potential sensitive
dye. For Ca2þ-flux analysis, cells were plated in 96-well clear-
bottom, poly D-lysine coated black microtiter plates (Costar) at
a density of 1 ꢀ 105 cells/well for R7 expressing GH4C1 cells or
8 ꢀ 104 cells/well for 5-HT3A expressing HEK293 cells and
cultured for 24 h prior to experiments. The medium was then
replaced with 100 μL of Hank’s Balanced Salt Solution-HEPES
20 mM, pH 7.4 (assay buffer) containing 4 μM Fluo-4-AM,
0.02% pluronic acid, and 5 mM probenecid. After 40 min
of incubation at 37 °C, the labeling solution was replaced with
200 μL of assay buffer containing 2.5 mM probenecid. Plates
were then transferred to the FLIPR system. Compounds to be
tested were prepared in assay buffer as 5ꢀ-concentrated solu-
tions in a separate 96-well polypropylene plate. Basal fluore-
scence was recorded for 30 s, followed by addition of 50 μL of
test compound (to assess agonist activity; first addition). Mea-
surements were made at 1 s intervals for 1 min, followed by
measurements every 30 s for 10 min. Subsequently, for the
second addition for alpha-7 expressing GH4C1 cells, nicotine
was added to each well except negative controls at EC80 final
concentration to assess antagonism of nicotine response or at
EC20 final concentration to assess positive modulation of nico-
tine response. Assay performance was robust as reflected by a Z0
factor >0.6.
by comparing the sample and standard UV areas: S = (Asmp
ꢀ
FD ꢀ Cst)/Ast, where S is the solubility of the compound (μM),
Asmp is the UV area of the sample solution, FD is the dilution
factor (2), Cst is the standard concentration (250 μM), and Ast is
the UV area of the standard solution.
Cytochrome Inhibition Assay.23,30 The fluorescent P450 in-
hibition assay was performed using the Gentest method (http://
www.gentest.com). Test compounds were dissolved in DMSO
at 1.5 mM. The stock solution of 12 μL was added by a robotic
system to 1488 μL of 0.1 M phosphate buffer at pH 7.4. The
solution was mixed, and 50 μL of the diluted samples was added
to a 1 mL 96-well polypropylene plate. Then 50 μL of cofactor
with NADPH regenerating system were added to the wells. The
plate was incubated at 37 °C for at 10 min. Enzyme-substrate
mix was prepared by prewarming the buffer at 37 °C for at least
10 min, and the enzymes and substrates were added right before
addition to the reaction plate. Enzyme-substrate mix (100 μL)
was added to the wells to start the reaction. The final substrate
and isozyme concentrations and incubation time were: BFC
(50 μM)/CYP3A4 (5 pmol/mL) for 30 min, AMMC (1.5 μM)/
CYP2D6 (7.5 pmol/mL) for 30 min, and MFC (75 μM)/2C9
(20 pmol/mL) for 45 min. The reactions were stopped with 80%
ACN/20% 0.5 M Tris buffer. The signals were quantified using
a fluorescent plate reader. The final DMSO concentration was
For testing 5-HT3A receptor activity, m-chlorophenylbigua-
nide (CPBG, EC80 final concentration) was added to each well
except negative controls. Measurements were made at 1 s
intervals for 1 min after the second addition and at 3 s intervals
for the remaining 3 min. Results were exported from the FLIPR
raw data as MAX-MIN of fluorescence signal intensity in two
intervals corresponding to the first and the second addition of
compounds. The responses were normalized to the positive control
and EC50 and IC50 values were calculated using XlFit version 4.2,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4387
0.2%. Compounds were tested in duplicates. Percent inhibition
was determined at 3 μM compound concentration. High nega-
tive values are usually indicative of fluorescent interference from
the test compounds or metabolites. The % CV obtained was
typically within 10%.
Electrophysiology. GH4C1 cells stably expressing rat R7-nAChR
were treated with 0.5 mM sodium butyrate added to the medium
for two days before patch clamp recordings. Patch pipettes
had resistances of ∼7 MΩ when filled with (in mM): 5 EGTA,
120 K-gluconate, 5 KCl, 10 HEPES, 5 K2ATP, 5 Na2-phospho-
creatine, 1 CaCl2, and 2 MgCl2. Cells were voltage-clamped at
-60 mV with a HEKA EPC-9 amplifier. To measure the fast
activation and desensitization of R7 current, the Dynaflow
(Cellectricon) fast perfusion system with 16- or 48-well chips
was used. Different concentrations of acetylcholine or 16 were
applied to cells in between washes with bath solution (Hanks’
Balanced Salt Solution þ 10 mM HEPES). Data were acquired
at 1 kHz for 2 s episodes (500 ms bath, 500 ms agonist, 1000 ms
wash) with a 10 s interval between episodes. Peak current
amplitude and total charge (area under the curve) were mea-
sured with the HEKA Pulse program. Concentration-response
curves and EC50 values were plotted and calculated with Origin
(MicroCal).
Metabolic Stability Assay. Compounds in 10 mM DMSO
solution were added to an incubation mixture in a 96-well
microplate containing 20 pmol/mL of hCYP3A4 (0.1-0.2 mg/mL
protein). The mixture was split in two aliquots: one receiving a
NADPH regenerating system, the other an equal amount of
phosphate buffer. The final substrate concentration was 1 μM
along with 0.25% of organic solvent. Incubation proceeded for 1 h
at 37 °C and was stopped by addition of acetonitrile to precipitate
proteins. Metabolic stability is given as the percent remaining
following incubation with cofactor (NADPH) with reference to
the incubation mixture without NADPH: % remaining = Area
NADPH ꢀ 100/Area ctrl where Area ctrl is the MS peak area of
the sample solution without NADPH and AreaNADPH is the MS
area of the sample solution with NADPH. The % CV obtained
was typically within 10%.
Radioligand Binding Assay. [3H]-Epibatidine binding studies
were performed as previously described.17 Briefly, cell mem-
brane preparations derived from GH4C1 cells stably expressing
rat R7 nAChRs were suspended in binding buffer (50 mM
HEPES, pH 7.4, 3 mM KCl, 70 mM NaCl, 10 mM MgCl2),
5 nM [3H]-epibatidine (GE Healthcare; SA = 53 Ci/mmol) and
16 to achieve a final volume of 200 μL in a 96-well polypropylene
plate. Nicotine at 300 μM was used for determination of
nonspecific binding. Following incubation at room temperature
(23 °C) for 1 h, samples were rapidly filtered through Unifilter
GF/B filters using a Filtermate (Perkin-Elmer) and washed five
times with ice-cold binding buffer. Samples were processed and
counted for radioactivity using a TopCount NXT (Perkin-
Elmer). Competition binding curves were fitted with a four
parameter logistic model. Ki values were calculated by the
Cheng-Prusoff equation using the GraphPad Prism software
package.
Permeability Assay. The assay was run in a PAMPA filter plate,
and compounds (10 μM in HBSS þ Hepes buffer) were added to
the donor chamber and incubated for 4 h at 37 °C and 80%
humidity. Warfarin was used as reference control. Concentrations
of reference, donor, and acceptor solutions were measured by
UPLC-MS-TOF (references, donor, and acceptor were injected in
UPLC-MS in this order to compare the MS quantitative signal).
The passive permeability is calculated according to a modified
version of the following expression31
ꢂ
ꢃ
ꢀ
ꢁ
ꢀ
ꢁ
1
1
þ
t
M
M
V
V
D
A
CAAðtÞ ¼
þ
Cað0Þ -
e - PeA
VD þ VA
VD þ VA
where M refers to total amount of drug in the system minus the
amount of sample lost in membrane (and surfaces), CA(t) is the
concentration of the drug in the acceptor well at time t, CA(0) is the
concentration of the drug in the acceptor well at time 0, VA is the
volume of the acceptor well, VD is the volume of the donor well, Pe is
the effective permeability, A is the membrane area, and t is the
permeation time. Compounds are defined as low, medium, or highly
permeable following the following classification: >10 ꢀ 10-6 cm/s,
high (passive permeability is unlikely to be limiting for passive
diffusion); between 2 and 10 ꢀ 10-6 cm/s, medium (permeability
may be limiting in case of low solubility, high metabolic turnover
rate or active secretion); between 0 and 2 ꢀ 10-6 cm/s, Low (high
risk that permeability is limiting for passive diffusion).
Rat Microsomal Stability. DMSO stock solutions of test
compounds were prepared at 0.5 mM concentration. Diluted
solutions of test compounds were prepared by adding 50 μL of
each DMSO stock solution to 200 μL of acetonitrile to make
0.1 mM solutions in 20% DMSO/80% acetonitrile. Rat liver
microsomal solution was prepared by adding 1.58 mL of concen-
trated rat liver microsomes (20 mg/mL protein concentration) to
48.29 mL of prewarmed (to 37 °C) 0.1 M potassium phosphate
buffer (pH 7.4) containing 127 μL of 0.5 M EDTA to make a
0.633 mg/mL (protein) microsomal solution. Then 11.2 μL of each
test compound diluted solution was each added directly to 885 μL
of rat liver microsomal solution (allowing direct binding of drugs
to microsomal proteins and lipids to minimize precipitation and
nonspecific binding to the plasticware). This solution was mixed,
and 180 μL was transferred to time 0 and time 15 min plates (each
in duplicate wells). For the time 15 min plate, NADPH regenerat-
ing agent (45 μL) was added to each well to initiate the reaction, the
plate was incubated at 37 °C for 15 min, followed by quenching of
the reaction by adding 450 μL of cold acetonitrile to each well.
For the time 0 plate, 450 μL of cold acetonitrile was added to each
well, followed by addition of NADPH regenerating agent (45 μL)
and no incubation. All of the plates were centrifuged at 3000 rpm
for 15 min, and the supernatants were transferred to other well
plates for analysis by LC-MS.25,26
Receptor Selectivity and hERG Activity. Interaction of 16 with
∼70 binding sites including all major classes of neurotransmitter,
growth factor, and peptide receptors (Novascreen, Caliper Bio-
sciences, Hopkinton MA) was examined at 10 μM concentration.
Detectable activity was only observed for 5HT4 (Ki = 6 μM):
adrenergic R-2A, (IC50 = 7.5 μM), adrenergic R-2b (IC50
=
35 μM). Activity at hERG ion channel was determined employing
CHO cells stably expressing the channel and an IonWorks record-
ing system.
Pharmacokinetics. Long Evans rats (age of 6-8 weeks, body
weight 250-550 g) were administered compound 16 as a single
dose of 3 mg/kg po at time 0 as a solution in 2% Tween, 0.5%
methocell in water (volume 2.5 mL/kg). Levels in plasma were
determined over a time period of 6 h in the po study and in
plasma and brain at 0, 0.5, 1, and 3 h. Concentration of 16 in rat
brain and plasma was measured by high performance liquid
chromatography in combination with mass spectrometry
(LC-MS/MS) with a limit of detection of 1 ng/mL in plasma and
10 ng/g in brain. Plasma samples were prepared by protein
precipitation with acetonitrile containing 250 ng/mL of internal
standard, centrifugation, and analysis of the supernatant by
LC-MS/MS. Brain samples were prepared by homogenization and
extraction with methanol. The homogenates were subsequently
centrifuged, and the supernatant was analyzed by LC-MS/MS.
Quantification was performed in a similar manner to the plasma
samples.
Object Recognition Test. Male Long Evans rats (ca. 250 g)
were individually housed with ad lib access to food and water
and provided with nestlets for environmental enrichment. All
habituation, training, and testing was performed under low
illumination (approximately 10 lx) in a circular arena (70 cm
diameter, 30 cm height) constructed of plastic surrounded by a
black mesh curtain and containing corncob bedding. Fecal
pellets were removed and bedding mixed after each trial. The
novel object recognition (NOR) task is composed of three

4388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Ghiron et al.
sessions: habituation, a sample trial, and a choice trial. During
habituation, rats were placed into the arena in the presence of
two identical yellow blocks (constructed with Duplos plastic
blocks) and allowed to explore freely for 10 min. On the
following day, the sample trial was initiated in which the animals
were returned to the arena for 5 min exposure to two identical
stimuli (objects constructed of Duplos) located at opposing com-
pass points approximately 10 cm in from the walls of the arena.
After a predefined interval of time, the choice trial was initiated in
which animals were again returned to the arena for 5 min, this time
with one familiar stimulus (the object the animal was exposed to
during the sample trial) and one novel stimulus. To evaluate the
effect of 16 in the MK-801-induced deficit model of NOR, the
choice trial was carried out 1 h after the sample trial, and animals
were treated with vehicle or 16 (0.3-30 mg/kg, po) 60 min prior to
the sample trial plus vehicle or MK-801 (0.03 mg/kg, ip) 30 min
prior to the sample trial (N = 10 per treatment group).
Stefano Gotta for generation of HRMS, Martina Pollastrini
and Laura Bettinetti, for the synthesis of previously reported
compounds and Silvia Papini for in vitro testing of some of the
reported compounds, and Nicola Caradonna for useful dis-
cussions on the pharmacokinetics data.
Supporting Information Available: Experimental details for
compounds 7-11, 13-15, 17-18 and FLIPR functional data
assessing compound 16 in ‘positive modulator’ assay mode in
comparison with data obtained for reported positive modulator
PNU120596. This material is available free of charge via the
Internet at http://pubs.acs.org.
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