SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist

Article abstract of DOI:10.1016/j.bmc.2009.05.040

Alpha 7 nicotinic acetylcholine receptor (α₇ nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimer's disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimer's disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the α₇ nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.

Full text of DOI:10.1016/j.bmc.2009.05.040

Bioorganic & Medicinal Chemistry 17 (2009) 5247–5258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7
nicotinic acetylcholine receptor agonist
Simon N. Haydar a, , , Chiara Ghiron b, , Laura Bettinetti b, Hendrick Bothmann b, Thomas A. Comery a,
*
John Dunlop a, Salvatore La Rosa b, Iolanda Micco b, Martina Pollastrini b, Joanna Quinn b, Renza Roncarati b,
Carla Scali b, Michela Valacchi b, Maurizio Varrone b, Riccardo Zanaletti b
a Chemical Sciences and Neuroscience Discovery Research, Wyeth Research, CN 8000, Princeton, NJ 08543-8000, USA
b Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Alpha 7 nicotinic acetylcholine receptor (a7 nAChR) agonists are promising therapeutic candidates for the
Received 3 March 2009
Revised 8 May 2009
Accepted 14 May 2009
Available online 21 May 2009
treatment of cognitive impairment associated with a variety of disorders including Alzheimer’s disease
and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function,
regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia
and Alzheimer’s disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR,
SEN12333/WAY-317538. This compound is a selective agonist of the a7 nAChR with excellent in vitro and
Keywords:
in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in
multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are
discussed.
Neuronal nicotinic acetylcholine receptors
Nicotinic ligands
Cognition
Ó 2009 Elsevier Ltd. All rights reserved.
Auditory sensory gating
1. Introduction
tor agonists have demonstrated improved cognition in animal
models and normalize sensory gating deficits, which are believed
Cognitive disorders encompass a wide range of diseases, most
notably in the psychiatric and neurology therapeutic areas. While
the positive symptomatology of schizophrenia (hallucinations,
delusions, paranoia) is addressed with current antipsychotic med-
ications that block dopamine D2 receptors, the negative symptom-
atology (cognitive impairments, reduced affect, social withdrawal,
low motivation) is largely unaffected and remains a significant
unmet medical need. Among the neurodegenerative diseases,
Alzheimer’s disease (AD) is the most common form of dementia
afflicting 10% of the population in their sixties, and approximately
half of all people in their eighties. In AD, the early and predominant
disease marker is cognitive dysfunction. Modest symptomatic
improvement in cognitive performance is provided by standard
of care agents such as cholinesterase inhibitors and NMDA receptor
antagonists.
to contribute to the cognitive fragmentation in schizophrenia.4,5
Based on these observations, selective a7 nAChR agonists are pre-
dicted to be effective for the improvement of cognition in both
schizophrenia and AD.6–8 In accordance with this hypothesis, the
prototypical nAChR agonist nicotine, as well as more selective
a7
agonists described recently, have been shown to improve cognitive
performance in both animal models and humans clinical trials.9
Herein we report the synthesis and SAR of a series of novel, potent,
small molecule agonists of the a7 nAChR.
As part of a multi-disciplinary approach to the treatment of
schizophrenia and Alzheimer’s disease, a discovery medicinal
chemistry program was initiated to identify selective a7 agonists.
Through the course of a high throughput screen, the advanced
hit N-{4-[4-(2,4-dimethoxyphenyl)-piperazin-1-yl]butyl}-4-pyri-
din-2-ylbenzamide 1, was identified as a weak partial agonist of
Several lines of experimental evidence support the involvement
of the neuronal nicotinic receptors in both schizophrenia and AD.1,2
These include reduced expression of a7 nicotinic acetylcholine
receptors (nAChR) in brain tissue from schizophrenia and AD pa-
tients, and genetic linkage studies in schizophrenia implicating
the
a7 nicotinic receptor (EC50 = 2.8 l
M).10
2. Chemistry
Starting from the original hit 1, initial efforts were made to de-
velop a new series (Fig. 1) with increased potency and improved
drug-like properties. In particular, in addition to the initial SAR
exploration, we were concerned about the number of rotatable
bonds, the molecular weight, and the relatively high number of
hydrogen bond acceptors which could impact unfavourably on
the locus of the a a7 recep-
7 receptor gene promoter.3 Prototypical
* Corresponding author. Tel.: +1 732 274 4518; fax: +1 732 274 4505.
E-mail address: haydars@wyeth.com (S.N. Haydar).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.040

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S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
An alternative synthesis of analogs 7, shown in Scheme 2, is car-
ried out in four steps from commercially available 4-aminobutanol
8. Hydroxybutyl amide 9 was obtained from the reaction of 4-ami-
nobutanol 8 and 4-bromobenzoic acid in the presence of Hunig’s
base.
These primary alcohols were then oxidized under Swern condi-
tions to afford the desired aldehydes 10 in good yields. Condensa-
tion of these aldehydes 10 with the appropriate secondary amine
and reduction of the resultant imine with sodium triacetoxyboro-
hydride gave intermediates 6.
Using a slightly modified route, analogs with a ‘reverse’ amide
linker were efficiently prepared (Scheme 3). To that end, 5-bromo-
valeryl chloride 11 was reacted with 4-bromoaniline to give amide
12 which was used in the subsequent reaction without purifica-
tion. Reaction of various secondary amines with compound 12 in
the presence of base afforded the penultimate intermediates 13.
Coupling with an aryl boronic acid or heteroaryl boronic acid under
a variety of Suzuki conditions afforded the final products 14 in
excellent yield.
N
H
N
N
N
O
O
1
O
R
"Right-hand side"
H
N
N
O
2
"Left-hand side"
H
N
Basic center Carbon chain linker
Aromatic moiety
O
Figure 1. Initially identified pharmacophore.
3. Results and discussion
the polar surface area and consequently brain permeability of the
compounds. We were also interested in exploring the possibility
of removing the arylpiperazine moiety, which could lead to poly-
pharmacology issues. The basic pharmacophore around the origi-
nal hit could be described as consisting of three constituents; a
basic moiety attached by a flexible length carbon chain bridged
to an aromatic moiety. The potential for hydrogen-bond interac-
tions also appear to impart a favourable behaviour.
All functional activities were measured in either calcium flux or
membrane potential assays using a fluorescence imaging plate
reader (FLIPRTM) system (Molecular Devices). Activity at rat
a7 nAC-
hRs was determined using a stable recombinant GH4C1 cell line
expressing the receptor.11 For the purpose of SAR discussion, the
‘left-hand side’ and the ‘right-hand side’ will be referred to as
LHS and RHS respectively as depicted in Figure 1.
Two separate synthetic routes were developed to prepare the
targeted analogs from readily available starting materials. In the
first route (Scheme 1), commercially available N-(x-bromobu-
Br
tyl)phthalimide 3 was reacted with a secondary amine to give
the desired amino-phthalimide products 4. Subsequently, com-
pounds 4 were refluxed in ethanol in the presence of hydrazine hy-
drate and upon acidic workup the desired mono-substituted
amines 5 were obtained as hydrochloride salts. The latter products
were then reacted with 4-bromobenzoyl chloride in the presence
of Et3N to give amides 6. Alternatively, activation of 4-bromoben-
zoic acid with 1,1-carbonyldiimidazole could be used to prepare
these amides 6. Finally, Suzuki coupling of these advanced inter-
mediates 6 with the appropriate substituted phenyl boronic acid
or heterocyclic boronic acid under cross-coupling conditions affor-
ded the final targeted products 7.
H
i
N
NH2
O
HO
HO
O
9
8
ii
Br
H
N
R2N
iii
Br
N
O
H
O
6
10
Scheme 2. Reagents and conditions: (i) 4-bromobenzoic acid, Hunig’s base, CH2Cl2;
(ii) (COCl)2, DMSO, Et3N; (iii) Na(AcO)3BH, DCM.
O
O
i
N
N
Br
NR2
O
O
3
4
ii
H2N
NR2
5
iii
Br
Ar(Het)
H
H
N
iv
N
R2N
R2N
O
O
7
6
Scheme 1. Reagents and conditions: (i) substituted secondary amine, K2CO3, NaI (cat.), 2-butanone, reflux; (ii) hydrazine monohydrate, ethanol, reflux; (iii) 4-bromobenzoyl
chloride, Et3N, or 4-bromobenzoic acid, 1,1-CDI, DMF; (iv) ArB(OH)2 or HetB(OH)2, Pd(PPh3)4, Na2CO3, DME/EtOH.

S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5249
Table 1
Early work on modifying the RHS of advanced Hit 1 resulted in a
Right-hand side exploration of the original Hit 1
somewhat flat SAR, as illustrated in Table 1 Synthesized com-
pounds which retained activity did not show great potency
improvement over the initial hit. However, several key qualitative
findings were notable from this study. For instance, the activity of
H
RHS
N
*
N
N
O
this series of compounds at the a7 receptor appeared to require a
biaryl moiety in the RHS as demonstrated by the poor activity of
compounds 7f and 7g. Moreover, substitution of the 2-position of
the pendant aryl ring in the biaryl RHS was important to retain po-
tency as illustrated by the poor potency of analog 7e.
O
O
Compound
RHS
EC50 SEM (n) (lM)
After extensive exploration with a large number of analogs to
support the SAR the following conclusions emerged; the amide lin-
ker could be ‘reversed’ while retaining potency at the a7 nAChR;
importantly, the aryl piperazine could be replaced with various cyc-
lic amines such as piperidine (14a), and morpholine (14b) (Table 2).
Acyclic tertiary amine substitution in the LHS, as in compound 14e,
was tolerated. Steric bulk around the basic amine in the LHS was also
tolerated, but to a lesser degree. While 6-membered ring 14a and 7-
membered ring 14h showed similar potency, additionalsubstitution
on the ring and on larger heterocycles was less desirable, as illus-
trated in compounds 14g and 14i, respectively.
Further efforts to explore the aromatic moiety on the RHS in the
‘reverse’ amide series show an increased breadth in the SAR of this
class of a7 nAChR ligands (Table 3). In general, biaryl compounds
were more potent than mono aryl derivatives as previously ob-
served in the original Hit series. For example, the potency of 14l
is about 10-fold less than that of analog 14j, although comparison
of 14j with 7f would indicate that the series is intrinsically more
potent. Interestingly, when substitution was moved to the meta
position as in compound 14o and 14s, the potency dropped dra-
matically. Replacing the biaryl moiety with a fused ring group also
led to a drop in activity as shown with compound 14m and 14n.
Compounds with hydrogen bonding (donor/acceptor) potential
on the pendant aryl ring were generally showing increased po-
tency, as exemplified by compounds 14p and 14r.
N
1
2.84 0.23 (32)
6.03 3.75 (3)
*
*
*
F
7a
7b
Cl
3.11 0.10 (2)
CH3
7c
5.77 2.26 (3)
2.91 0.37 (2)
*
*
OCH3
7d
From this emerging SAR, analog 14b was chosen to be evaluated
in in vivo efficacy models based on its generally good in vitro pro-
file, ease of synthesis, selectivity on a related panel of nicotinic (al-
pha1, alpha3, alpha4beta2) and highly homologous receptors
(5HT3A). This was confirmed in a panel of ꢀ70 binding sites includ-
ing all major classes of neurotransmitter, growth factor and pep-
tide receptors whereby no significant activity was observed
7e
7f
>30 (1)
>30 (1)
>30 (1)
*
Br
*
when the compound was tested at 10 lM concentration except
for the histamine H3 receptor where binding was observed leading
to receptor antagonism (Novascreen, Caliper Biosciences, Hopkin-
ton MA). Compound 14b also showed minimal hERG inhibition,
desirable drug-like (molecular weight, number of rotatable bonds,
7g
O2N
*
Br
O
O
i
Br
N
Br
Cl
H
12
11
ii
Br
Ar(Het)
iii
O
O
R2N
R2N
N
H
N
H
13
14
Scheme 3. Reagents and conditions: (i) Et3N, 4-bromoaniline, CH2Cl2; (ii) secondary amine, Et2N; (iii) ArB(OH)2 or HetB(OH)2, Pd(PPh3)4, Na2CO3, DME/EtOH.

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S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
Table 2
Table 3
Left-hand side exploration of the ‘reverse’ amide series
Right-hand side exploration of the ‘reverse’ amide series
O
N
RHS
*
N
N
H
O
R
N
R
N
Compound
RHS
EC50 SEM (n) (lM)
H
LHS
F
14j
0.16 0.04 (2)
Compound
LHS
EC50 SEM (n) (lM)
F
*
14a
0.53 (1)
N
O
14k
0.24 0.06 (2)
*
14b
14c
1.65 0.43 (4)
2.47 0.77 (3)
O
N
OCH3
N
14l
1.35 0.27 (2)
5.07 (1)
Br
N
*
N
14m
14n
*
H3C
O
14d
2.78 0.58 (3)
N
N
O
10.93 (1)
O
*
N
14e
14f
3.27 (1)
14o
14p
>30 (1)
N
*
NH2
3.56 0.76 (3)
N
N
O
O
0.09 0.01 (2)
*
*
N
14g
>30 (1)
O
14q
14r
14s
0.99 (1)
N
O
14h
14i
0.54 (1)
2.66 (1)
HN
N
0.12 0.04 (8)
*
O
N
Br
N
2.07 (1)
*
log P, polar surface area) and pharmacokinetic properties (Table 4).
Compound 14b, designated SEN12333/WAY-317538, is a full ago-
nist at the a7 nAChR with an EC50 of 1.6 lM as determined on its
hydrochloride salt. Further in vitro characterization of SEN12333/
WAY-317538 revealed that the compound is selective for the a7
In vivo, SEN12333/WAY-317538 improved performance in ro-
dent behavioural assays of cognitive function and perceptual
processing, producing enhancement of normal memory perfor-
mance.12 Furthermore, the ability to attenuate pharmacologically
induced deficits via either the glutamatergic or cholinergic system
(Fig. 2) was demonstrated. For example, treatment with
nAChR over other nAChRs (a1, a3) and the closely related 5-HT3
receptor.

S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5251
Table 4
addition, the compound was able to reverse both a scopolamine-
and an MK-801-induced deficit in the pharmacological models
for these recognition memory tests.
Profile of SEN12333/WAY-317538
a7
Electrophysiology
Ki
EC50
EC50
Emax
4. Conclusion
In vitro
260 nM
1.6
lM
12
lM
88%
In summary, we have identified a series of novel, potent small
molecule agonists of the a7 nAChR. One selected example,
SEN12333/WAY-317538 showed excellent brain penetrability
and a generally favourable pharmacokinetic profile. Pro-cognitive
efficacy was demonstrated in multiple behavioural cognition as-
says with this analog. These data further support the potential util-
a1
a3
5-HT3
a
4b2
hERG
Selectivity
Inactive
5.3-fold
Inactive
AUClast
45% @10
l
M
6% @ 1 lM
Cmax
T1/2
B/P
% F (po)
Pharmacokinetic (10 mg/kg, ip, male Wistar rat)
244 ng/mL 0.8–1 h 347 h ng/mL
ity of a7 agonists and SEN12333/WAY-317538 for the treatment of
neurodegenerative and psychiatric disorders with associated cog-
2.5
57%
nitive dysfunction.
5. Experimental
5.1. General methods
Unless otherwise specified, all nuclear magnetic resonance
spectra were recorded using a Varian Mercury Plus 400 MHz spec-
trometer equipped with a PFG ATB Broadband probe.
HPLC–MS analyses (5 and 10 min methods) were performed
with a Waters 2795 separation module equipped with a Waters
Micromass ZQ (ES ionization) and Waters PDA 2996, using a
Waters XTerra MS C18 3.5
l
m 2.1 ꢁ 50 mm column; alternatively,
HPLC–MS analyses were performed with an Agilent 1100 instru-
ment, using a Zorbax Eclipse XDB-C8 4.6 ꢁ 150 mm; a Zorbax CN
4.6 ꢁ 150 mm column or a Zorbax Extend C18 2.1 ꢁ 50 mm col-
umn, coupled to API-ES MS for the 2.5 min method.
Preparative HLPC 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
l
m 10 ꢁ 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 10 min unless other-
wise stated.
All column chromatography was performed following the
method of Still.13 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.
When specified for array synthesis, heating was performed on a
Buchi SyncoreÒ system.
All microwave reactions were performed in a CEM Discover
oven.
5.1.1. General procedure for aminoalkylamine synthesis
To a suspension of the required amine (1 equiv), sodium iodide
(0.5 equiv) and potassium carbonate (1.1 equiv) in 2-butanone
(4 M solution), N-(x-bromoalkyl)phthalimide (1 equiv) was added.
The resulting suspension was stirred for 18 h at 85 °C, then the
reaction was filtered and the solvent removed by vacuum distilla-
tion. The resulting oil was washed with water and recovered with
DCM. The solvent was removed under reduced pressure to yield
the amino-phthalimide products pure enough to be used in the fol-
lowing step. If necessary, a further purification was carried out by
flash-chromatography. The phthalimides thus obtained (1 equiv)
were dissolved in EtOH (2 M solution with respect to phthalimide
amount) and hydrazine monohydrate (2 equiv) was added drop-
wise. The mixture was heated at 80 °C for 4 h, after which the reac-
tion was acidified with 37% HCl and the solid which precipitated
was removed by filtration. The solution was concentrated under
vacuum and taken up with 1 N HCl. Any residual 2,3-dihydroph-
thalazine-1,4-dione was removed by filtration. The aqueous
Figure 2. In vivo efficacy of SEN12333/WAY-317538 in novel object recognition.
SEN12333/WAY-317538 (3 mg/kg, ip) minimized spontaneous de-
cay of episodic memory in a novel object recognition task in rats. In

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S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
solution was removed under reduced pressure under vacuum to
recover the pure product.
5.1.6. General procedure D for amide synthesis from
haloalkanoyl chlorides
x-
In case of acid sensitive derivatives the reaction mixture was
filtered and washed with EtOH, concentrated under vacuum and
taken up with toluene and DCM to remove excess 2,3-dihydro-
phthalazine-1,4-dione. Solvent removal under reduced pressure
afforded the pure product.
4-Bromoaniline (6 g, 0.035 mol) and 0.035 mol of Et3N
(4.87 mL) were dissolved in 120 mL of dichloromethane and cooled
at 0 °C. To this solution, 0.038 mol of
x-bromoalkanoyl chloride
(5.4 mL) were slowly added and the resulting mixture was stirred
for 1 h at 0 °C.
When all the starting material was consumed (monitoring by
LCMS) the solution was washed with 50 mL of Na2CO3 0.4 M, the
organic layer was recovered by extraction and drying over Na2SO4
and the product used without further purification: 1 equiv of the
alkylating agent was dissolved in butanone (5–10 mL/mmol sub-
strate) and to this solution 1 equiv of NaI and 1.1 equiv of the
amine were added. The mixture was stirred at 70 °C or 24 h. The
mixture was cooled to room temperature. When the products pre-
cipitated as salts, they were taken into water, free-based by addi-
tion of NaOH 10% to pH 10 and extracted with dichloromethane.
In the cases where no product precipitation occurred, the solvent
was removed under reduced pressure, the crude was taken into
dichloromethane and extracted after adjusting the pH to 10 with
NaOH 10%. If necessary, the products were further purified by flash
chromatography.
5.1.2. General procedure for acid–amine coupling method
using acid chlorides
A mixture of (4-arylpiperazin-1-yl)alkylamine (0.3 mmol), car-
boxylic acid chloride (0.3 mmol), triethylamine (0.56 mmol) and
a catalytic amount of DMAP in CH2Cl2 was stirred at 0 °C for
10 min then at room temperature for 4 h. The CH2Cl2 layer was
washed with water, dried and concentrated. The residue was puri-
fied by chromatography on silica gel with CHCl3/MeOH 95/5 as elu-
ent to give the title compound.
5.1.3. General procedure A for amide synthesis from
haloalkanoyl chlorides
x-
In a round-bottom, 2-neck flask, triethylamine (1 equiv) was
added to a solution of aryl or heteroaryl amine (1 equiv) in a vol-
ume of DCE such as to obtain a 1.2 M solution of amine; 5-bromo-
valeryl chloride (0.95 equiv) was then added dropwise as a 1.2 M
solution in DCE and the reaction was then stirred at room temper-
ature for 1 h 30 min. A 1.8 M solution of amine (3 equiv) and tri-
ethylamine (1 equiv) in DCE were then added and the reaction
mixture stirred at 55 °C for a time between 4 and 16 h, until LCMS
monitoring showed reaction completion. After this period, the
reaction mixture was partitioned between water and DCM; the or-
ganic layer was washed with saturated NaCl and dried over
5.1.7. General procedure for cross-coupling reaction with
boronic acids (microwave irradiation)
To a degassed mixture of 5-alkylpentanoic acid arylamide
(0.1 g, 1 equiv) aryl boronic acid (1.1 equiv) 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 (150 W,
pressure max) and then again other 20 min. The organic layer
was separated and concentrated, and further purified by SCX col-
umn and/or by preparative HPLC. The solvent was removed under
reduced pressure to afford the corresponding product.
Na2SO4. The crude
x-aminoalkanamides obtained after solvent
evaporation at reduced pressure were purified by trituration from
Et2O/hexane (1/1) or by flash chromatography.
5.1.8. General procedure for cross-coupling reaction with
boronic acids (thermal conditions)
To a degassed mixture of 5-alkylpentanoic acid arylamide
(0.1 g, 1 equiv) aryl boronic acid (1.1 equiv) in acetonitrile/sodium
5.1.4. General procedure B for amide synthesis from
haloalkanoyl chlorides
A solution of aniline (1 equiv) and triethylamine (1 equiv) in
dichloromethane (0.2 mmol/mL) was cooled to 0 °C under nitrogen
x-
carbonate 0.4 M solution 1/1 (4 mL),
a catalytic amount of
atmosphere. The
x-bromoalkanoyl chloride (1 equiv) in dichloro-
Pd[(PPh3)]4 (5 mmol %) was added. The reaction mixture was
heated at 90 °C for 16 h. The organic layer was separated and con-
centrated, and further purified by SCX column and/or by prepara-
tive HPLC. The solvent was removed under reduced pressure to
afford the corresponding products.
methane (0.3 mmol/mL) was slowly added. The mixture was stir-
red at room temperature for 1.5 h, after which the amine
(5 equiv) and triethylamine (1 equiv) were added all at once and
the reaction was stirred at room temperature for a further 40 h.
If necessary the reaction was heated at 50 °C to push it to comple-
tion. The organic solution was washed with brine, dried and the
solvent removed under reduced pressure. The product was tritu-
rated by hexane/diethyl ether (1/1) or purified by flash
chromatography.
5.1.9. N-{4-[4-(2,4-Dimethoxyphenyl)-piperazin-1-yl]butyl}-4-
(pyridin-2-yl)-benzamide (1)
5.1.9.1. 1-(2,4-Dimethoxyphenyl)piperazine hydrochloride. A
solution of 1.48 g (0.0097 mol) of 2,4-dimethoxyaniline, 1.89 g
(0.0160 mol) of bis-2-chloroethylamine hydrochloride and 2.00 g
of K2CO3 in 25 mL of 1-butanol was refluxed for 24 h then filtered
hot. The solvent was removed under reduced pressure and the resi-
due triturated with acetone. The resulting powder was filtered and
dried to give 1.25 g of the title compound.
5.1.5. General procedure C for amide synthesis from
haloalkanoyl chlorides (array synthesis)
To a solution of aniline (1 equiv for each molar equivalent of
amine used) and triethylamine (1 equiv for each molar equiva-
lent of amine used) in dichloromethane (0.3 mmol/mL for each
x-
1H NMR (DMSO-d6) d 9.21 (br s, 1H); 6.82 (d, 1H); 6.52 (s, 1H);
6.42 (d, 1H), 3.74 (s, 3H); 3.68 (s, 3H); 3.12 (s, 4H); 3.07 (s, 4H).
amine used) the
x-bromoalkanoyl chloride (1 equiv for each
molar equivalent of amine used) was slowly added and the mix-
ture stirred at room temperature for 2 h. The solution was then
split over as many as amines used in the array and the each
portion added to a vial containing the amine (5 equiv) and tri-
ethylamine (1 equiv). The reactions were then shaken at room
temperature for 40 h. The organic solutions were washed with
saturated brine, dried (Na2SO4), and the solvent removed under
reduced pressure. The products were purified by preparative
HPLC.
5.1.9.2. 2-{4-[4-(2,4-Dimethoxyphenyl)piperazin-1-yl]-butyl}-
isoindole-1,3-dione. A mixture of N-(4-bromobutyl)phthalimide
(0.00135 mol), 1-(20,40-dimethoxyphenyl)-piperazine hydrochlo-
ride (0.00135 mol), K2CO3 (0.00270 mol), NaI (0.00186 mol) and
methyl ethyl ketone (7 mL) was refluxed for 20 h with stirring.
After the mixture had cooled, the insoluble materials were re-
moved by filtration and washed with CHCl3. The filtrate and the
washings were concentrated to dryness in vacuo. The residue

S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5253
was purified by chromatography on silica gel with CHCl3/MeOH
95/5 as eluent. Yield: 68%.
reaction was stirred for a further 16 h. The solvent was removed
under reduced pressure and the crude mixture was treated with
5% MeOH in dichloromethane (2 mL) and washed with 10% sodium
hydroxide solution (2 mL). This mixture was passed through a col-
umn packed with 5 g of diatomaceous earth and the eluting the
product with dichloromethane. The collected organic layer, con-
taining the desired compound, was further purified using flash
chromatography eluting with 10% MeOH in dichloromethane. Frac-
tions containing the product were combined and the solvent re-
moved under reduced pressure.
1H NMR (CDCl3) d 7.73 (m, 4H), 6.82 (d, 1H), 6.40 (m, 2H), 3.79
(s, 3H), 3.73 (s, 3H), 3.65 (m, 2H), 2.98 (m, 4H), 2.61 (m, 4H), 2.41 (t,
2H), 1.66 (m, 4H).
5.1.9.3. 4-[4-(2,4-Dimethoxyphenyl)piperazin-1-yl]butyl-
amine. A solution of 2-{4-[4-(2,4-dimethoxyphenyl)piperazin-1-
yl]butyl} isoindole-1,3-dione (0.236 mmol) and hydrazine hydrate
(0.478 mmol) in ethanol (2 mL) was refluxed for 2 h with stirring.
After the solution had cooled, any insoluble materials were re-
moved by filtration and washed with EtOH. The filtrate and the
washings were concentrated in vacuo to dryness. The residue
was taken up with CHCl3. The CHCl3 layer was washed with water,
dried and concentrated to give the title amine. Yield: 50%.
1H NMR (CDCl3) d 6.85 (d, 1H), 6.41 (m, 2H), 3.81 (s, 3H), 3.75 (s,
3H); 3.01 (m, 4H), 2.63 (m, 4H), 2.40 (br t, 2H), 1.35 (m, 6H).
HRMS: calcd for C29H34ClN3O3 + H+, 508.23615; found (ESI,
[M+H]+ Obsd), 508.2362.
HPLC: column Zorbax C8 MeOH 80%/H2O 20%, 1.0 mL/min;
10 min run; tR 4.31; area = 98%.
5.1.12. 20-Methylbiphenyl-4-carboxylic acid {4-[4-(2,4-dime-
thoxyphenyl)piperazin-1-yl]butyl} amide (7c)
5.1.12.1. 20-Methylbiphenyl-4-carboxylic acid. Prepared with a
modification of the procedure outlined in Leadbeater and Marco.15
In a 10 mL glass tube were placed 4-carboxyphenyl boronic acid
5.1.9.4. N-{4-[4-(2,4-Dimethoxyphenyl)piperazin-1-yl]butyl}-4-
(pyridin-2-yl)benzamide. A mixture of (4-(2,4-dimethoxy-
phenyl)piperazin-1-yl)-alkylamine (0.3 mmol), 4-(pyridin-2-yl)-
benzoyl chloride (0.3 mmol), triethylamine (0.56 mmol) and a
catalytic amount of DMAP in CH2Cl2 was stirred at 0 °C for
10 min then at room temperature for 4 h. The CH2Cl2 layer was
washed with water, dried and concentrated. The residue was puri-
fied by chromatography on silica gel with CHCl3/MeOH 95/5 as elu-
ent to give the title compound.
(166 mg, 1.0 mmol), 2-bromotoluene (120 lL, 1.0 mmol), Na2CO3
(315 mg, 3 mmol), Pd(OAc)2 (1 mg, 0.004 mmol), 2 mL of water
and a magnetic stir bar. The vessel was sealed with a septum and
placed into the microwave cavity. Microwave irradiation (maxi-
mum emitted power 200 W) was used to increase the temperature
to 150 °C; the reaction mixture was then kept at this temperature
for 5 min.
Yield: 35%.
The mixture was allowed to cool to room temperature, and the
reaction mixture was filtered washing with little CHCl3.The aque-
ous layer was acidified, and the precipitate collected. The product
was purified by chromatography on silica gel using petroleum
ether/AcOEt 50/50 as eluent to give 67.8 mg of product, yield 32%.
1H NMR (CD3OD) d 8.05 (m, 2H, arom); 7.41 (m, 2H, arom); 7.21
(m, 4H, arom); 2.22 (s, 3H, C–CH3).
Mp 154.5–156 °C (free base), 212–216 °C (HCl salt).
1H NMR (CDCl3) d 8.66 (d, J = 4.9 Hz 1H), 8.02 (d, J = 8.3 Hz, 2H),
7.85 (d, J = 8.3 Hz, 2H), 7.75 (m, 2H), 7.23 (m, 1H), 6.85 (br t, 1H),
6.76 (d, J = 8.7 Hz, 1H), 6.42 (d, J = 2.6 Hz, 1H), 6.33 (dd, J = 8.7,
2.61 Hz, 1H), 3.79 (s, 3H), 3.72 (s, 3H), 3.47 (m, 2H), 2.97 (m, 4H),
2.62 (m, 4H), 2.47 (br t, 2H), 1.65–1.68 (m, 4H).
Mass (ES) m/z %: 475 (M+1, 100%), 497 (M+Na, 19%).
HPLC: column Zorbax C8 MeOH 80%/H2O 20%, 1.0 mL/min;
10 min run; tR 6.54; area = 99%.
Mass (ES) m/z %: 424 (2 M, 100%).
5.1.12.2. 20-Methylbiphenyl-4-carboxylic acid {4-[4-(2,4-dime-
thoxyphenyl)piperazin-1-yl]butyl} amide. Prepared from 4-[4-
(2,4-dimethoxyphenyl)piperazin-1-yl]butylamine and 20-meth-
ylbiphenyl-4-carboxylic acid following general procedure 5.1.2.
Yield: 21%.
HRMS: calcd for C28H34N4O3 + H+, 475.27037; found (ESI,
[M+H]+ Obsd), 475.2703.
5.1.10. 20-Fluoro-biphenyl-4-carboxylic acid {4-[4-(2,4-
dimethoxy-phenyl)-piperazin-1-yl]-butyl}-amide (7a)
Prepared from 4-[4-(2,4-dimethoxyphenyl)piperazin-1-yl]butyl-
amine and commercially available 20-fluorobiphenyl-4-carboxylic
acid following general procedure 5.1.2.
1H NMR (200 MHz, CDCl3) d 7.80 (d, J = 8.33 Hz, 2H); 7.35 (d,
J = 8.33 Hz, 2H); 7.2–7.4 (m, 4H); 6.88 (br s, 1H); 6.79 (d,
J = 8.6 Hz, 1H); 6.46 (d, 1H); 6.30–6.40 (m, 1H); 3.82 (s, 3H); 3.76
(s, 3H); 3.50 (m, 2H); 2.98 (m, 4H); 2.66 (m, 4H); 2.47 (m, 2H);
2.25 (s, 3H); 1.70 (m, 4H).
Yield: 20%.
Mp = 124–125.5 °C.
tR (CHCl3/MeOH 95/5) 0.21.
Mass (ES) m/z %: 488 (M+1, 100%).
HPLC: column Zorbax C8 MeCN 40%/H2O (CF3COOH pH 2.3)
60%, 1.0 mL/min; 20 min run; tR = 11.748; area 96%.
HRMS: calcd for C30H37N3O3 + H+, 488.29077; found (ESI,
[M+H]+ Obsd), 488.2907.
1H NMR (200 MHz, CDCl3) d 7.81 (d, J = 8.33 Hz, 2H), 7.56 (d,
J = 8.33 Hz, 2H); 7.11–7.41 (m, 4H); 6.99 (br s, 1H); 6.76 (d,
J = 8.6 Hz, 1H); 6.43 (d, J = 2.6 Hz, 1H); 6.33 (dd, J = 8.65, 2.6 Hz,
1H); 3.78 (s, 3H); 3.71 (s, 3H); 3.42–3.46 (m, 2H); 2.94 (m, 4H);
2.60 (m, 4H); 2.46 (br t, 2H); 1.65–1.67 (m, 4H).
Mass (ES) m/z %: 492 (M+1, 100%).
5.1.13. 20-Methoxybiphenyl-4-carboxylic acid {4-[4-(2,4-dime-
thoxyphenyl)piperazin-1-yl]butyl}amide (7d)
HPLC: column Zorbax CN AcCN 50%/H2O (CF3COOH pH 2.3) 50%,
0.4 mL/min; 20 min run; tR = 13.525; area 96%.
HRMS: calcd for C29H34FN3O3 + H+, 492.26570; found (ESI,
[M+H]+ Obsd), 492.2658.
5.1.13.1. 20-Methoxy-biphenyl-4-carboxylic acid. To a solution
of 4-carboxyphenylboronic acid (3.32 g, 20 mmol), FibrecatÒ1007
(2 g) and potassium carbonate (3.03 g, 22 mmol) in ethanol/water
(20 mL/20 mL), 1-bromo-2-methoxy-benzene was added (4.11 g,
22 mmol). The reaction mixture was heated to reflux for 3 h. After
cooling, was filtered and the solution evaporated under reduced
pressure. The residue was suspended in aq citric acid (10% w/v), fil-
tered and washed with water and diethyl ether. The resulting solid
was dried under vacuum to yield the title compound (4.02 g, 88%).
1H NMR (DMSO-d6) d 3.79 (s, 3H), 7.08 (m, 2H), 7.34 (m, 2H),
7.58 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H).
5.1.11. 20-Chlorobiphenyl-4-carboxylic acid {4-[4-(2,4-
dimethoxyphenyl)piperazin-1-yl]butyl}-amide (7b)
The commercially available 20-chlorobiphenyl-4-carboxylic acid
(0.55 mmol) was disolved in dimethylformamide (2 mL) to dis-
solve, followed by N,N0-carbonyldiimidazole (0.55 mmol). The
solution was then left to stir for 60 min before adding 4-[4-(2,4-
dimethoxyphenyl)piperazin-1-yl]butylamine (0.6 mmol) and the

5254
S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5.1.13.2. 20-Methoxybiphenyl-4-carboxylic acid {4-[4-(2,4-
dimethoxyphenyl)piperazin-1-yl]butyl}amide. To a solution of
(s, 3H), 3.78 (s, 3H), 6.36 (dd, J = 8.7, 2.6 Hz, 6.41 (d, J = 8.7 Hz, 2H),
6.70 (d, J = 8.7 Hz, 1H), 7.28 (br t, 1H), 7.88 (d, J = 8.7 Hz, 2H), 8.21
(d, J = 8.7 Hz, 2H).
4-[4-(2,4-dimethoxyphenyl)piperazin-1-yl]-butylamine
hydro-
chloride (131 mg, 0.4 mmol) in DCM (5 mL) and DMF (5 mL), tri-
ethylamine (40 mg, 0.4 mmol) was added. After stirring the
resulting mixture for 5 min, CDI (70 mg, 0.44 mmol) was added.
The mixture was stirred for a further 1 h at room temperature
and then 20-methoxybiphenyl-4-carboxylic acid (92 mg, 0.4 mmol)
was added. The resulting reaction mixture was left stirring for 40 h
at room temperature. After evaporation of the solvent, water
(5 mL) was added and the suspension sonicated for 20 min. The
resulting suspension was filtered and washed with 0.4 M Na2CO3
aqueous solution, diethyl ether and dried under vacuum to yield
the title compound (67.2 mg, 33%).
HPLC: column Zorbax C8 4.6 ꢁ 150 mm; MeOH 80%/H2O
(CF3COOH pH 6) 20%, 1.0 mL/min; 10 min run; tR = 3.05; area 99%.
Mass (M+1) e/z 444 (M+1, 100%); 465 (M+Na+, 18%).
HRMS: calcd for C23H30N4O5 + H+, 443.22890; found (ESI,
[M+H]+ Obsd), 443.2288.
5.1.17. 5-Piperidin-1-ylpentanoic acid (4-pyridin-3-
ylphenyl)amide (14a)
5.1.17.1. 5-Piperidin-1-yl-pentanoic acid (4-bromophenyl)
amide (14l). Obtained following general procedure 5.1.3. starting
from 4-bromo-aniline, 5-bromovaleryl chloride and piperidine.
The compound was purified by trituration from Et2O/hexane 1:1
to obtain 5.1 g (yield 75%).
1H NMR (DMSO-d6) d 1.55 (br s, 4H), 2.75 (br s, 2H), 2.93 (br s,
8H), 3.27 (br t, 2H), 3.64 (s, 3H), 3.68 (s, 3H), 3.69 (s, 3H),
6.41 (dd, J = 8.7, 2.6 Hz, 1H), 6.52 (d, J = 2.7 Hz, 1H), 6.81 (d,
J = 8.7 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H),
7.29–7.38 (m, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 8.3 Hz,
2H), 8.53 (s, 1H).
1H NMR (400 MHz, CDCl3) d 1.65 (m, 10H); 2.42 (m, 2H), 2.54
(m, 5H), 2.92 (m, 1H), 7.41 (m, 2H), 7.48 (m, 2H), 8.01 (br s, 1H).
Mass (calculated) [339.27]; (found) [M+H+] = [339/341].
LC tR = 2.74 (purity 99%, 10 min method).
HRMS: calcd for C16H23BrN2O + H+, 339.10665; found (ESI,
[M+H]+ Obsd), 339.1065.
HRMS: calcd for C30H37N3O4 + H+, 504.28568; found (ESI,
[M+H]+ Obsd), 504.2856.
5.1.17.2. 5-Piperidin-1-ylpentanoic acid (4-pyridin-3-ylphe-
nyl)amide. The title compound was obtained following the gen-
eral procedure 5.1.8. starting from 5-piperidin-1-ylpentanoic acid
(4-bromophenyl) amide and pyridine-3-boronic acid. The crude
was purified by flash-chromatography (DCM/MeOH 100/0 to 95/
5), giving 1.04 g of title compound (47% yield).
5.1.14. Biphenyl-4-carboxylic acid {4-[4-(2,4-dimethoxyphenyl)-
piperazin-1-yl]-butyl}amide (7e)
To a solution of 4-[4-(2,4-dimethoxyphenyl)piperazin-1-yl]-
butylamine hydrochloride (150 mg, 0.51 mmol) in DCM (10 mL),
triethylamine (85 lL, 0.61 mmol), biphenylcarboxilic acid
Mass (calculated) [337]; (found) [M+H+] = 338.
(133 mg, 0.61 mmol) and a catalytic amount of DMAP were added
at 0 °C and left stirring for 10 min. The mixture was then stirred for
4 h at room temperature. Water (10 mL) was added, the layers sep-
arated and the crude dried over sodium sulphate before solvent re-
moval and purification by flash chromatography (CHCl3/MeOH
95:5) to afford 85 mg of title product.
LC tR = 0.35–1.39 (100%, 10 min method).
1H NMR (400 MHz, DMSO-d6) d 1.46 (m, 10H), 2.27 (m, 8H),
7.43 (br dd, J = 8.0, 4.8 Hz, 1H), 7.68 (m, 4H), 8.01 (ddd, J = 8.0,
2.4, 2.4 Hz, 1H), 8.50 (dd, J = 4.8, 1.6 Hz, 1H), 8.85 (d, J = 2.4 Hz,
1H), 10.00 (s, 1H).
1H NMR (200 MHz, CDCl3) d 1.55–1.75 (m, 4H), 2.45–2.55 (br t,
2H), 2.60–2.65 (m, 4H), 2.90–3.0 (br s, 4H), 3.45–3.55 (m, 2H), 3.73
(s, 3H), 3.80 (s, 3H), 6.34 (dd, J = 8.6, 2.4 Hz, 1H), 6.45 (d, J = 2.4 Hz,
1H), 6.77 (d, J = 8.6 Hz, 1H), 6.92 (br t, 1H), 7.35–7.46 (m, 2H), 7.53–
7.65 (m, 5H), 7.82 (d, J = 8.3 Hz, 2H).
HRMS: calcd for C21H27N3O + H+, 338.22269; found (ESI, [M+H]+
Calcd), 338.2227.
5.1.18. 5-Morpholin-4-ylpentanoic acid (4-pyridin-3-ylphenyl)
amide (14b)
Mass (M+1) e/z 474 (M+1, 100%); 496 (M+Na+, 10%).
HRMS: calcd for C29H35N3O3 + H+, 474.27512; found (ESI,
[M+H]+ Calcd), 474.2750.
5.1.18.1. 5-Morpholin-4-ylpentanoic acid (4-bromophenyl)
amide. The title compound was obtained following general proce-
dure 5.1.6. starting from morpholine and 5-bromovaleryl chloride.
The reaction solution needed to be slightly heated at 50 °C for
3.5 h. The mixture was washed with brine, the organic layer was
recovered and dried. The crude material was treated with ether/
hexane (1/1) and the desired product was recovered by filtration
(6.39 g, 93% yield).
5.1.15. 4-Bromo-N-{4-[4-(2,4-dimethoxyphenyl)piperazin-1-
yl]butyl}benzamide (7f)
Prepared from 4-[4-(2,4-dimethoxyphenyl)piperazin-1-yl]butyl-
amine and 4-bromobenzoyl chloride according to general procedure
5.1.2. 60% yield.
Mass (calculated) [341.25]; (found) [M+H+] = [341–343].
LC tR = 2.50 (purity 100%, 10 min method).
1H NMR (200 MHz, CDCl3) d 1.62–1.66 (br s, 4H), 2.39–2.45 (br
t, 2H), 2.60–2.65 (m, 4H), 2.90–3.0 (m, 4H), 3.38–3.44 (m, 2H), 3.74
(s, 3H), 3.79 (s, 3H), 6.34–6.44 (m, 2H), 6.76 (d, J = 8.4 Hz, 1H), 6.92
(br t, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H).
HPLC: column Zorbax C8 4.6 ꢁ 150 mm; MeOH 80%/H2O
(CF3COOH pH 6) 20%, 0.9 mL/min; 15 min run; tR = 4.55; area 96%.
Mass (M+1) e/z 476/478 (M+1, 100%); 498/500 (M+Na+, 25%).
HRMS: calcd for C23H30BrN3O3 + H+, 476.15433; found (ESI,
[M+H]+ Obsd), 476.1543.
1H NMR (400 MHz, CDCl3) d 1.44 (m, 2H), 1.59 (m, 2H), 2.31 (m,
8H), 3.55 (m, 4H), 7.44 (d, J = 8.89 Hz, 2H), 7.54 (d, J = 8.90 Hz, 2H),
10.00 (s, 1H).
5.1.18.2. 5-Morpholin-4-ylpentanoic acid (4-pyridin-3-ylphe-
nyl) amide. The title compound was obtained following general
procedure 5.1.8. starting from 5-morpholin-4-ylpentanoic acid
(4-bromophenyl) amide and pyridine-3-boronic acid. The crude
was purified by flash-chromatography (DCM/MeOH 100/0 to 95/
5), giving 1.63 g of title compound (53% yield).
Mass (calculated) [339]; (found) [M+H+] = 340.
5.1.16. 4-Nitro-N-{4-[4-(2,4-dimethoxyphenyl)piperazin-1-
LC tR = 0.34 (100%, 10 min method).
yl]butyl}benzamide (7g)
1HNMR(400 MHz,DMSO-d6)d1.44(m,2H), 1.60(m,2H), 2.31(m,
8H), 3.54 (4H), 7.44 (m, 1H), 7.66 (d, J = 8.81 Hz, 2H), 7.71 (d,
J = 8.83 Hz, 2H), 8.02 (m, 1H), 8.51 (m, 1H), 8.86 (m, 1H), 10.07 (s, 1H).
HRMS (calculated for C20H25N3O2) (M+H+) 340.2020, found
340.2017.
Prepared
from
4-[4-(2,4-dimethoxyphenyl)piperazin-1-
yl]butylamine and 4-nitrobenzoyl chloride according to general
procedure 5.1.2. in 60% yield.
1H NMR (200 MHz, CDCl3) d 1.60–1.75 (br s, 4H), 2.39–2.45 (br t,
2H), 2.58–2.60 (m, 4H), 2.83–2.90 (m, 4H), 3.45–3.55 (m, 2H), 3.73

S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5255
5.1.19. 5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentanoic acid
5.1.23. 1-[4-(4-Pyridin-3-ylphenylcarbamoyl)butyl]piperidine-
(4-pyridin-3-ylphenyl) amide (14c)
3-carboxylic acid diethylamide (14g)
Prepared in 0.25 mmol scale from 2-methoxyphenylpiperazine,
5-bromovaleryl chloride, 4-bromoaniline and 3-pyridylboronic
acid following general procedures 5.1.3. and 5.1.8. Yield: 40%.
1H NMR (400 MHz, DMSO-d6) d 0.8–1.0 (m, 2H), 1.2–1.3 (m,
2H), 1.55–1.65 (m, 2H), 1.75–1.85 (m, 2H), 2.4–2.5 (m, 4H), 3.00–
3.15 (m, 4), 3.85 (s, 3H), 6.8–7.1 (m, 4H), 7.35 (br t, 1H), 7.5–7.6
(m, 2H), 7.6–7.7 (m, 2H), 7.85–7.90 (m, 1H), 8.15–8.20 (m, 1H),
8.55–8.60 (m, 1H), 8.80–8.85 (m, 1H).
5.1.23.1. 1-[4-(4-Bromophenylcarbamoyl)butyl]piperidine-3-
carboxylic acid diethylamide. The title compound was obtained
following general procedure 5.1.6. starting from piperidine-3-car-
boxylic acid diethylamide and 5-bromovaleryl chloride. Extraction
with DCM/H2O (pH 10) and flash-chromatography (DCM/MeOH
99/1 to 95/5) gave 2.7 g of title compound were obtained as solid
by (60% yield).
1H NMR (400 MHz, CD3OD) d 1.11 (t, J = 7.10 Hz, 3H), 1.23 (t,
J = 7.13 Hz, 3H), 1.85 (m, 8H), 2.48 (t, J = 6.74 Hz, 2H), 3.18 (m,
6H), 3.45 (m, 5H), 7.43 (d, J = 8.83 Hz, 2H), 7.52 (d, J = 8.87 Hz, 2H).
Mass (ES) m/z %: 445 (M+1, 100%).
HPLC: column Zorbax C8 MeOH 80%/H2O 20%, 1.0 mL/min;
2.5 min run; tR 1.23; area = 99%.
HRMS: calcd for C27H32N4O2 + H+, 445.25980; found (ESI,
[M+H]+ Obsd), 445.2595.
5.1.23.2. 1-[4-(4-Pyridin-3-ylphenylcarbamoyl)butyl]piperidine-
3-carboxylic acid diethylamide. The title compound was ob-
tained following general procedure 5.1.8. starting from 1-[4-(4-
bromophenylcarbamoyl)butyl]piperidine-3-carboxylic acid dieth-
ylamide and pyridine-3-boronic acid. The crude was purified by
SCX column followed by prep-HPLC, giving 56 mg of title com-
pound as formate salt (55% yield).
5.1.20. 5-(4-Acetylpiperazin-1-yl)pentanoic acid (4-pyridin-3-
ylphenyl) amide (14d)
5.1.20.1. 5-(4-Acetylpiperazin-1-yl)pentanoic acid (4-bromo-
phenyl) amide. The title compound was obtained following
general procedure 5.1.4. starting from N-acetylpiperazine and
5-bromovaleryl chloride. The reaction was stirred at room temper-
ature for 40 h. The product was purified by flash chromatography
(DCM/MeOH = 95:5) obtaining 4.0 g (yield 70%) of product.
1H NMR (400 MHz, DMSO-d6) d 1.44 (m, 2H); 1.57 (m, 2H), 1.95
(s, 3H), 2.28 (m, 8H), 3.06 (m, 1H), 3.37 (m, 1H), 7.44 (m, 2H), 7.55
(m, 2H), 9.99 (s, 1H).
Mass (calculated) [436]; (found) [M+H+] = 437; LC tR = 1.75
(100%, 10 min method).
1H NMR (400 MHz, CD3OD) d 1.11 (t, J = 7.11 Hz, 3H), 1.23 (t,
J = 7.13 Hz), 1.91 (m, 9H), 2.51 (m, 4H), 3.14 (m, 7.60), 7.51 (m,
1H), 7.64 (m, 2H), 7.72 (m, 2H), 8.09 (m, 1H), 8.49 (m, 3H), 8.79
(m, 1H).
HRMS: calcd for C26H36N4O2 + H+, 437.29110; found (ESI,
[M+H]+ Calcd), 437.2910.
5.1.20.2. 5-(4-Acetylpiperazin-1-yl)pentanoic acid (4-pyridin-3-
ylphenyl) amide. Prepared following general procedure 5.1.8. by
reaction of 5-(4-acetylpiperazin-1-yl)pentanoic acid (4-bromophe-
nyl) amide with 3-pyridylboronic acid. Yield: 35%.
Mass (ES) m/z %: 381 (M+1, 100%).
5.1.24. 5-Azepan-1-ylpentanoic acid (4-pyridin-3-yl-phenyl)
amide (14h)
5.1.24.1. 5-Azepan-1-yl-pentanoic acid (4-bromo-phenyl)-ami-
de. The title compound was obtained following general procedure
5.1.6. starting from azepane and 5-bromovaleryl chloride. The mix-
ture was stirred at 55 °C for further 24 h. The product was washed
with Et2O/hexane 1:1 obtaining 1.64 g (yield 93%) of product.
1H NMR (400 MHz, DMSO-d6) d 1.43 (m, 2H); 1.54 (m, 10H),
2.29 (m, 2H), 2.58 (m, 6H), 7.44 (m, 2H), 7.55 (m, 2H), 9.99 (s, 1H).
HPLC: column Zorbax C8 MeOH 80%/H2O 20%, 1.0 mL/min;
2.5 min run; tR 0.87; area = 93%.
5.1.21. 5-[Methyl-(2-pyridin-2-ylethyl)amino]pentanoic acid
(4-pyridin-3-yl-phenyl) amide (14e)
Prepared in 0.25 mmol scale from methyl-(2-pyridin-2-ylethyl)
amine, 5-bromovaleryl chloride, 4-bromoaniline and 3-pyridylbo-
ronic acid following general procedures 5.1.4. and 5.1.7 in 27%
yield.
5.1.24.2. 5-Azepan-1-ylpentanoic acid (4-pyridin-3-yl-phenyl)
amide. The title compound was obtained following the general
procedure 5.1.8. starting from 5-azepan-1-yl-pentanoic acid (4-
bromo-phenyl)-amide and pyridine-3-boronic acid. The crude
was purified by SCX column followed by prep-HPLC, giving
41 mg of title compound as formate salt (21% yield). Mass (calcu-
lated) [351]; (found) [M+H+] = 352; LC tR = 1.52 (100%, 10 min
method); 1H NMR (400 MHz, DMSO-d6): 1.47 (m, 12H), 2.30 (m,
3H), 2.37 (m, 1H), 2.48 (m, 1H), 2.60 (m, 3H), 7.39 (dd, J = 8.0,
4.8 Hz, 1H), 7.64 (m, 4H), 7.96 (br d, J = 8.0 Hz, 1H), 8.21 (s, 1H),
8.46 (dd, J = 4.8, 1.6 Hz,1H), 8.80 (s, 1H), 9.98 (s, 1H).
Mass (ES) m/z %: 389 (M+1, 100%).
HPLC: tR 0.32–0.46; area = 100%.
HRMS: calcd for C24H28N4O + H+, 389.23359; found (ESI, [M+H]+
Calcd), 389.2335.
5.1.22. 5-(4-Methylpiperazin-1-yl)pentanoic acid (4-pyridin-3-
ylphenyl) amide (14f)
5.1.22.1. 5-(4-Methylpiperazin-1-yl)pentanoic acid (4-bromo-
phenyl) amide. The title compound was obtained following
general procedure 5.1.6. starting from N-methylpiperazine and
5-bromovaleryl chloride. The reaction was stirred at room temper-
ature for 40 h. The product was precipitated by ether/hexane 1:1
obtaining 3.7 g (70% yield) of product. 1H NMR (400 MHz, DMSO-
d6) d 1.43 (m, 2H); 1.56 (m, 2H), 2.23 (s, 3H), 2.38 (m, 12H), 7.44
(m, 2H), 7.55 (m, 2H), 10.05 (s, 1H).
HRMS: calcd for C22H29N3O + H+, 352.23834; found (ESI, [M+H]+
Calcd), 352.2383.
5.1.25. 5-(4-Acetyl-[1,4]diazepan-1-yl)pentanoic acid (4-pyridin-
3-yl-phenyl) amide (14i)
5.1.25.1. 5-(4-Acetyl-[1,4]diazepan-1-yl)-pentanoic acid (4-
bromo-phenyl)-amide. The title compound was obtained follow-
ing the general procedure 5.1.5. and starting from 5-bromovaleryl
chloride and N-acetyl-homopiperazine. The reaction was stirred at
room temperature for further 22 h and then at 50 °C for 2 h. The
product was purified by flash chromatography (DCM/
MeOH = 95:5) obtaining 6.0 g (75% yield) of title product.
1H NMR (400 MHz, DMSO-d6) d 1.57 (m, 2H); 1.68 (m, 2H), 1.82
(m, 1H), 1.90 (m, 1H), 2.09 (s, 3H), 2.38 (m, 2H), 2.53 (m, 4H), 2.69
(m, 2H), 3.58 (m, 4H), 7.42 (m, 2H), 7.50 (m, 2H).
5.1.22.2. 5-(4-Methylpiperazin-1-yl)pentanoic acid (4-pyridin-
3-ylphenyl) amide. Prepared following general procedure 5.1.8. in
30% yield.
Mass (ES) m/z %: 353 (M+1, 100%).
HPLC: Zorbax 2.5 min run; tR 0.92; area = 100%.
HRMS: calcd for C21H28N4O + H+, 353.23359; found (ESI, [M+H]+
Obsd), 353.2332.

5256
S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5.1.25.2. 5-(4-Acetyl-[1,4]diazepan-1-yl)-pentanoic acid(4-pyri-
din-3-yl-phenyl)-amide formate salt. The title compound was
obtained following the general procedure 5.1.7. starting from 5-
(4-acetyl-[1,4]diazepan-1-yl)pentanoic acid (4-bromo-phenyl)-
amide and pyridine-3-boronic acid. The crude was purified by
SCX column followed by prep-HPLC, giving 31 mg of title com-
pound as formate salt (15% yield).
parative HPLC affords the title compound (30 mg, 17% yield) as a
white solid as formate salt.
Mass (calculated) [311]; (found) [M+H+] = 305.
LC tR = 1.24 (96%, 10 min method).
HRMS: calcd for C17H24N2O3 + H+, 305.18597; found (ESI,
[M+H]+ Calcd), 305.1859.
Mass (calculated) [394]; (found) [M+H+] = 395.
5.1.30. 5-Piperidin-1-yl-pentanoic acid m-tolylamide (14o)
3-Methylaniline (0.6 mmol, 64 mg) and triethylamine
(0.6 mmol, 60 mg) were dissolved in dimethylformamide
(0.5 mL) and 5-bromovaleryl chloride (113 mg, 0.57 mmol) in
dimethylformamide (0.5 mL) was added drop wise. After 1 h
30 min, piperidine (153 mg, 1.8 mmol) and triethylamine
(60 mg, 0.6 mmol) in dimethylformamide (0.5 mL) were
added and the reaction mixture heated at 55 °C for 4 h.
Work-up followed by preparative HPLC affords the title com-
LC tR = 0.35–0.92 (100%, 10 min method).
1H NMR (400 MHz, DMSO-d6) d 1.54 (m, 8H), 1.91–1.92 (con-
former double peak, 3H), 2.27 (m, 2H), 2.39 (m, 2H), 2.47 (m,
1H), 2.52 (m, 2H), 2.60 (m, 1H), 3.38 (m, 2H), 7.39 (ddd,
J = 8.0, 4.8, 0.4 Hz, 1H), 7.61 (m, 2H), 7.66 (m, 2H), 7.97 (ddd,
J = 8.0, 2.4, 1.6 Hz, 1H), 8.10 (s, 1H), 8.46 (dd, J = 8.0, 1.6 Hz,
1H), 8.80 (d, J = 2.4, 0.8 Hz, 1H), 9.96 (s, 1H). HRMS: calcd for
C23H30N4O2 + H+, 395.24415; found (ESI, [M+H]+ Calcd),
395.2441.
pound (74 mg, 47% yield) as
salt.
a white solid as formate
5.1.26. 5-Piperidin-1-yl-pentanoic acid (20,40-difluorobiphenyl-
4-yl) amide (14j)
Prepared using general method 5.1.7. from 5-piperidin-1-ylpen-
tanoic acid (4-bromophenyl) amide (5.1.17.1) and 2,4-dif-
luorophenylboronic acid in 30% yield.
Mass (calculated) [274]; (found) [M+H+] = 275.
LC tR = 1.24 (100%, 10 min method).
HRMS: calcd for C17H26N2O + H+, 275.21179; found (ESI, [M+H]+
Obsd), 275.2118.
Mass (ES) m/z %: 373 (M+1, 100%).
5.1.31. 40-(5-Piperidin-1-yl-pentanoylamino) biphenyl-3-car-
boxylic acid amide (14p)
Prepared using general method 5.1.7. from 5-piperidin-1-ylpen-
tanoic acid (4-bromophenyl) amide (5.1.17.1) and 3-(aminocar-
bonyl)phenylboronic acid in 15% yield.
HPLC: Zorbax 2.5 min run; tR 1.74; area = 100%.
HRMS: calcd for C22H26F2N2O + H+, 373.20859; found (ESI,
[M+H]+ Obsd), 373.2087.
Mass (ES) m/z %: 380 (M+1, 100%).
5.1.27. 5-Piperidin-1-yl-pentanoic acid (20-isopropoxybiphenyl-
4-yl) amide (14k)
Prepared using general method 5.1.7. from 5-piperidin-1-ylpen-
tanoic acid (4-bromophenyl) amide (5.1.17.1) and 2-iso-propo-
xyphenylboronic acid in 35% yield.
LC tR = 2.41 (99%, 10 min method).
HRMS: calcd for C23H29N3O2 + H+, 380.23325; found (ESI,
[M+H]+ Obsd), 380.2332.
5.1.32. 5-Piperidin-1-ylpentanoic acid (30-acetylbiphenyl-4-yl)
amide (14q)
Prepared using general method 5.1.7. from 5-piperidin-1-ylpen-
tanoic acid (4-bromophenyl) amide (5.1.17.1) and 3-acetylphenyl-
boronic acid in 17% yield.
Mass (ES) m/z %: 395 (M+1, 100%).
HPLC 10 min run; tR 3.64; area = 100%.
HRMS: calcd for C25H34N2O2 + H+, 395.26930; found (ESI,
[M+H]+ Calcd), 395.2692.
Mass (ES) m/z %: 379 (M+1, 100%).
5.1.28. 5-Piperidin-1-yl-pentanoic acid quinolin-6-ylamide
(14m)
LC tR = 2.41 (100%, 10 min method).
HRMS: calcd for C24H30N2O2 + H+, 379.23800; found (ESI,
[M+H]+ Obsd), 379.2380.
6-Aminoquinoline (0.6 mmol, 86 mg) and triethylamine
(0.6 mmol, 60 mg) were dissolved in dimethylformamide
(0.5 mL) and 5-bromovaleryl chloride (113 mg, 0.57 mmol) in
dimethylformamide (0.5 mL) was added drop wise. After 1 h
30 min, piperidine (153 mg, 1.8 mmol) and triethylamine (60 mg,
0.6 mmol) in dimethylformamide (0.5 mL) were added and the
reaction mixture heated at 55 °C for 4 h. Work-up followed by pre-
parative HPLC affords the title compound (40 mg, 23% yield) as a
white solid as formate salt.
5.1.33. 5-Piperidin-1-ylpentanoic acid (30-
acetylaminobiphenyl-4-yl) amide (14r)
Prepared using general method 5.1.7. from 5-piperidin-1-ylpen-
tanoic acid (4-bromophenyl) amide (5.1.17.1) and 3-(acetyl-
amino)phenylboronic acid in 15% yield.
Mass (ES) m/z %: 394 (M+1, 100%).
Mass (calculated) [311]; (found) [M+H+] = 312; 156 (M+2)/2.
LC tR = 0.38 (100%, 10 min method).
LC tR = 2.63 (92%, 10 min method).
HRMS: calcd for C24H31N3O2 + H+, 394.24890; found (ESI,
[M+H]+ Obsd), 394.2489.
HRMS: calcd for C19H25N3O + H+, 312.20704; found (ESI, [M+H]+
Calcd), 312.2070.
5.1.34. 5-Piperidin-1-yl-pentanoic acid (3-bromophenyl)amide
(14s)
Prepared from 3-bromoaniline and 5-bromovaleryl chloride
according to general procedure 5.1.6. to give 1.7 g (33%) of the title
compound.
C16H23N2OBr Mass (calculated) [339.28]; found [M+H+] = 339/
341 (Br), LC tR = 1.86, (98%, 10 min method).
NMR (400 MHz, DMSO-d6): 1.51–1.64 (m, 10H); 2.34 (m, 2H);
2.23 (m, 2H); 2.76 (m, 4H); 2.97 (m, 2H); 7.12–7.264 (m, 2H);
7.48 (br d, J = 8 Hz, 2H); 7.97 (s, 1H).
5.1.29. 5-Piperidin-1-yl-pentanoic acid benzo[1,3]dioxol-5-
ylamide (14n)
Benzo[1,3]dioxol-5-ylamine (0.6 mmol, 82 mg) and triethyl-
amine (0.6 mmol, 60 mg) were dissolved in dimethylformamide
(0.5 mL) and 5-bromovaleryl chloride (113 mg, 0.57 mmol) in
dimethylformamide (0.5 mL) was added drop wise. After 1 h
30 min, piperidine (153 mg, 1.8 mmol) and triethylamine (60 mg,
0.6 mmol) in dimethylformamide (0.5 mL) were added and the
reaction mixture heated at 55 °C for 4 h. Work-up followed by pre-

S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5257
5.1.35. Ca2+-flux and membrane potential measurements with
5.1.36. Electrophysiology
a FLIPRTM system
GH4C1 cells stably expressing rat
a7 nAChR were treated
The following recombinant cell lines were used as specific
sources of receptors: GH4C1 cells stably transfected with pCEP4/
with 0.5 mM Na-butyrate added to the medium for two days
before patch clamp recordings. Patch pipettes had resistances
rat
expressing human 5-HT3A receptors.11 Native neuroblastoma SH-
SY5Y cells were used as source of human ganglionic nAChRs ( 3)
and TE671 rhabdomyosarcoma cells were used as endogenous
source of muscle 1b1d receptors. GH4C1 cells expressing
a
7 nAChR as previously described,11 HEK293 cell lines stably
of ꢀ7 M
X when filled with (in mM): 5 EGTA, 120 K-Gluconate,
5 KCl, 10 HEPES, 5 K2ATP, 5 Na2-phosphocreatine, 1 CaCl2 and
2 MgCl2. Cells were voltage-clamped at ꢂ60 mV with a HEKA
EPC-9 amplifier. To measure the fast activation and desensiti-
a
a
c
a7
zation of a7 current, the Dynaflow (Cellectricon) fast perfusion
and HEK cells expressing 5-HT3A receptors were analyzed by
Ca2+-flux measurements employing a Fluorometric Imaging Plate
Reader (FLIPR, Molecular Devices) system, whereas the cells
system with 16- or 48-well chips was used. Different concen-
trations of acetylcholine or SEN12333/WAY-317538 were ap-
plied to cells in between washes with bath solution (Hanks’
Balanced Salt Solution + 10 mM HEPES). Data were acquired at
1 kHz for 2-s episodes (500 ms bath, 500 ms agonist, 1000 ms
expressing the nicotinic receptors subunits a1 and a3 were tested
in the FLIPR system with a membrane potential sensitive dye.
For Ca2+-flux analysis, cells were plated in 96-well clear-bot-
tom, poly -lysine coated black microtiter plates (Costar) at a den-
wash) with
a 10-s interval between episodes. Peak current
sity of 1 ꢁD105 cells/well for
a
7 expressing GH4C1 cells or 8 ꢁ 104
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).
cells/well for 5-HT3A expressing HEK293 cells and cultured for 24 h
prior to experiments. The medium was then replaced with 100
of Hank’s Balanced Salt Solution-HEPES 20 mM, pH 7.4 (assay buf-
fer) containing 4 M Fluo-4-AM, 0.02% pluronic acid and 5 mM
probenecid. After 40 min of incubation at 37 °C the labeling solu-
tion was replaced with 200 L assay buffer containing 2.5 mM pro-
lL
l
5.1.37. Radioligand binding assay
[3H]-epibatidine binding studies were performed as previ-
ously described (Dunlop et al., 2007). Briefly, cell membrane
preparations derived from GH4C1 cells stably expressing rat
l
benecid. Plates were then transferred to the FLIPR system.
Compounds to be tested were prepared in assay buffer as 5x-con-
centrated solutions in a separate 96-well polypropylene plate. Ba-
sal fluorescence was recorded for 30 s, followed by addition of
a
7 nAChRs were suspended in binding buffer (50 mM HEPES,
pH 7.4, 3 mM KCl, 70 mM NaCl, 10 mM MgCl2), 5 nM [3H]-epi-
batidine (GE Healthcare; S.A. = 53 Ci/mmol) and SEN12333/
50
lL of test compound (to assess agonist activity; first addition).
WAY-317538 to achieve a final volume of 200
lL in a 96-well
Measurements 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 (10 lM final
concentration) was added to each well except negative controls (to
asses positive modulation and antagonism of nicotine response).
polypropylene plate. Nicotine at 300 M was used for determi-
l
nation 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 soft-
ware package.
For testing 5-HT3A receptors CPBG (1 lM 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 con-
trol and EC50 and IC50 values were calculated using Xlfit version
4.2, with a sigmoidal concentration–response (variable slope)
equation: Y = Bottom + (Top – Bottom)/(1 + (EC50/X)Hill Slope), where
X is the concentration, Y is the response, Bottom is the bottom pla-
teau of the curve and Top the top plateau. Assay performance was
robust as reflected by a Z0 factor >0.6.
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 cells/well 24 h prior to assay. Growth media were re-
moved from the cells by flicking the plates and membrane poten-
tial dye (Molecular Devices), reconstituted in HBSS five times more
diluted compared to the manufacturer’s instructions, was added to
the wells. Plates were incubated for 60 min at rt and then directly
transferred to the FLIPR system. Compounds to be tested were pre-
pared in assay buffer as 5ꢁ-concentrated solutions in a separate
96-well polypropylene plate. Baseline fluorescence was monitored
for the first 10 s followed by the addition of compounds. For detec-
tion of antagonists or positive allosteric modulator activity, agonist
5.1.38. Receptor selectivity and hERG activity
Interaction of SEN12333/WAY-317538 with ꢀ70 binding sites
including all major classes of neurotransmitter, growth factor
and peptide receptors (Novascreen, Caliper Biosciences, Hopkinton
MA) was examined at 10 lM concentration. Activity at Histamine-
3 receptor was determined in a recombinant HEK293 cell line sta-
bly expressing the receptor. Activity at hERG ion channel was
determined employing CHO cells stably expressing the channel
and an IonWorks recording system.
5.1.39. Pharmacokinetics
Male Wistar rats (age of 6–8 weeks, body weight 200–250 g)
were administered a single dose of 5 mg/kg iv or 10 mg/kg ip of
SEN12333/WAY-317538 at time 0. Levels in plasma were deter-
mined over a time period of 8 h in the iv study and in plasma
and brain at 0, 0.5, 1 and 3 h. Concentration of SEN12333/WAY-
317538 in rat brain and plasma was measured by high perfor-
mance liquid chromatography in combination with mass spec-
trometry (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 subse-
quently centrifuged, and the supernatant was analyzed by LC–
MS/MS. Quantification was performed in a similar manner to the
plasma samples.
(epibatidine; 1 lM 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 responses were normal-
ized to the positive control (epibatidine 1
lM final concentration)
and EC50 values were calculated as described before.

5258
S. N. Haydar et al. / Bioorg. Med. Chem. 17 (2009) 5247–5258
5.1.40. Object recognition test
software. Objects, constructed with Duplo (Lego), were placed on
the arena floor in one of four locations spaced evenly around the
field approximately 10 cm from the field’s edge. Rats were habitu-
ated to the arena which contained two identical yellow cubes for a
period of 15 min 1 day prior to the T1 and T2 sessions. Following
T1 rats were returned to their home cages for a 1 h retention inter-
val and then tested in the T2 trial for recognition memory. T2 con-
sisted of a 5 min exploration of the field containing both a familiar,
previously explored, object and a novel object. The location of the
objects, counterbalanced across treatment groups, remained con-
stant for each animal during the habituation, T1 and T2 trials.
The effect of treatment on object exploration during the retention
trial was examined using a one-way ANOVA on total contact time
followed by Fisher’s LSD group mean pairwise comparisons. The
amount of time exploring the novel and familiar objects across
treatment groups was analyzed using repeated measures ANOVA
followed by Fisher’s LSD post hoc comparisons.
Object recognition was evaluated as described previously14
employing different amnesic factors. Scopolamine (0.2 mg/kg ip)
and MK-801 (0.03 mg/kg, ip) induced amnesia was provoked
30 min before acquisition trials, whereas spontaneous decay of
memory was induced by the time delay after first experience
(24 h time-delay). SEN12333/WAY-317538 (1–10 mg/kg) was
administered 30 min prior to the acquisition trial to determine
whether SEN12333/WAY-317538 could reverse the cognitive
impairments produced by the amnesic factor. In brief, for scopol-
amine and time delay protocols, Wistar rats (3 month old, ca.
250 g weight) were placed in a grey polyvinylchloride arena
(60 ꢁ 60 ꢁ 40 h cm) illuminated by
a 50 W lamp suspended
50 cm above the arena. The objects to be discriminated were
prisms, pyramids and cylinders made of plastic. The day before
testing, rats were habituated to the arena for 2 min. In the
scopolamine-induced amnesia, a session of 2 trials separated by
an inter-trial interval of 240 min was carried out. In the first trial
(acquisition trial, T1) two identical objects were presented in two
opposite corners of the arena. The rats were left in the arena until
criterion of 20 s of total exploration of the objects was reached.
Exploration was defined as directing the nose at a distance <2 cm
to the object and/or touching it with the nose. During the second
trial (retention trial, T2) one of the objects presented in T1 was re-
placed by a novel object and rats were left in the arena for 5 min.
The time spent exploring the familiar (F) and the novel object (N)
were recorded separately and the difference between the two
exploration times was taken. From one rat to the next, care was ta-
ken to avoid object and place preference by randomly changing the
role of the objects (familiar or new object) and their position in the
two opposite corners of the box during T2. In the time delay proce-
dure, T2 was performed 24 h after T1 when a spontaneous decay of
memory was present in control rats. Student paired T-test was
used to evaluated differences between exploration time. MK-801
induced novel object recognition deficit was evaluated in a proce-
dure similar to that used for the scopolamine induced deficit. Long
Evans rats (3 month old, ca. 250 g weight) were tested in a circular
field (diameter ꢀ70 cm, 30 cm high), surrounded by black curtains
masking extra-field cues, located in a dimly lit room (ꢀ10 lux at
the level of the area) in the presence of white noise (ꢀ65 dB). Ani-
mal performance was tracked by video using Noldus Ethovision XT
Acknowledgements
The authors would like to thank Eva Genesio and Ugo Zanelli for
their support in the interpretation of analytical and pharmacoki-
netic data, respectively.
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