Novel Multitarget-Directed Ligands (MTDLs) with Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4 Receptor (5-HT4R) Agonist Activities As Potential Agents against Alzheimer's Disease: The Design of Donecopride

Article abstract of DOI:10.1021/acs.jmedchem.5b00115

In this work, we describe the synthesis and in vitro evaluation of a novel series of multitarget-directed ligands (MTDL) displaying both nanomolar dual-binding site (DBS) acetylcholinesterase inhibitory effects and partial 5-HT₄R agonist activity, among which donecopride was selected for further in vivo evaluations in mice. The latter displayed procognitive and antiamnesic effects and enhanced sAPPα release, accounting for a potential symptomatic and disease-modifying therapeutic benefit in the treatment of Alzheimer's disease. (Figure Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00115

Article
pubs.acs.org/jmc
Novel Multitarget-Directed Ligands (MTDLs) with
Acetylcholinesterase (AChE) Inhibitory and Serotonergic Subtype 4
Receptor (5-HT4R) Agonist Activities As Potential Agents against
Alzheimer’s Disease: The Design of Donecopride
Christophe Rochais,*,† Cedric Lecoutey,† Florence Gaven,‡,§,∥ Patrizia Giannoni,‡,§,∥ Katia Hamidouche,⊥
́
Damien Hedou,† Emmanuelle Dubost,† David Genest,† Samir Yahiaoui,† Thomas Freret,⊥
Valentine Bouet,⊥ Francois Dauphin,⊥ Jana Sopkova de Oliveira Santos,† Celine Ballandonne,†
̧
́
Sophie Corvaisier,†,⊥ Aurelie Malzert-Freon,† Remi Legay,† Michel Boulouard,⊥ Sylvie Claeysen,‡,§,∥
́
́
and Patrick Dallemagne*,†
†UNICAEN, CERMN (Centre d’Etudes et de Recherche sur le Med
‡CNRS, UMR-5203, Institut de Gen
omique Fonctionnelle, F-34000 Montpellier, France
§Inserm, U1191, F-34000 Montpellier, France
∥Universite
de Montpellier, UMR-5203, F-34000 Montpellier, France
⊥UNICAEN, GMPc5 (Groupe Mem
oire et Plasticite comportementale), F-14032 Caen, France
́
icament de Normandie), F-14032 Caen, France
́
́
́
́
S
* Supporting Information
ABSTRACT: In this work, we describe the synthesis and in vitro
evaluation of a novel series of multitarget-directed ligands (MTDL)
displaying both nanomolar dual-binding site (DBS) acetylcholinesterase
inhibitory effects and partial 5-HT4R agonist activity, among which
donecopride was selected for further in vivo evaluations in mice. The latter
displayed procognitive and antiamnesic effects and enhanced sAPPα
release, accounting for a potential symptomatic and disease-modifying
therapeutic benefit in the treatment of Alzheimer’s disease.
ylation. Nevertheless, many of these trials recently failed, and
INTRODUCTION
■
some of these failures have been attributed to the extreme
selectivity of the tested drugs against a single target,
disregarding the fact that neurodegenerative syndromes involve
multiple pathogenic factors.5,6 Consequently, efficient treat-
ments should associate several drugs whose actions are oriented
toward several targets involved in the pathogenesis. These
drugs could be combined in a cocktail (multiple-medication
therapy) or in a single formulation (multiple-compounded
medication). However, these approaches might be disadvanta-
geous for patients with respect to compliance difficulties, drug−
drug interactions, problems of bioavailability, and metabolism.
These issues are difficult to solve when the drugs present
different pharmacokinetic profiles.7,8 Today, a new concept is
emerging; also called “hybride molecules”, “designed multiple
ligands”, or “multitarget-directed ligands” (MTDLs), it
Alzheimer’s disease (AD), the most common form of dementia
among the elderly, is a chronic, progressive and degenerative
disorder of the brain featuring impairments in cognitive
functions, especially memory processes.1 As of 2013, there
were an estimated 44.4 million people with dementia
worldwide.2 By 2050, it is projected that this figure will have
increased to over 115 million. Not only are there numerous
reasons for concern, but AD and dementia have an enormous
impact on societies.3 The total estimated worldwide costs of
dementia were U.S. $604 billion in 2010. Given that the
pathogenesis of AD is complex and related to the abnormality
and dysfunction of multisystems, the prevention and treatment
of AD have remained elusive to date. Most of the current
treatments produce only symptomatic benefits through the
catalytic inhibition of acetylcholinesterase (AChE).4 So far,
investigational new drugs that have reached clinical trials have
been targeting amyloid aggregation or neurofibrillary tangle
production linked to the abnormality of protein τ phosphor-
Received: January 21, 2015
© XXXX American Chemical Society
A
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describes those compounds that are effective in treating
complex diseases because of their ability to interact with the
multiple targets thought to be responsible for the disease
pathogenesis.9−22 Besides reducing the pharmacokinetic and
interaction problems linked to multiple-medication therapy,
MTDLs generally show a synergistic effect. They could be
particularly useful in the treatment of neurodegenerative
diseases like AD.23−34 Recently, hybrid agents with a dual
mode of action against AD have been described. One of the
most widely adopted approaches in the field has been to modify
the molecular structure of an AChE inhibitor (AChEI) in order
to complement it with additional biological activities that are
useful for treating AD, like M2 receptor antagonism,35 direct
inhibition of amyloid beta peptide (Aβ) aggregation,36−41
AChE-dependent amyloid aggregation inhibition (dual-binding
site [DBS] inhibitors),42−46 or providing it with antioxidant
properties47−50 linked to MAO-B inhibition,51−54 calcium
channel blockage,55,56 or control oxidative damage properties.57
Within this scope, we very recently described the design of 1
(donecopride), a novel drug candidate exhibiting, for the first
time, both in vitro DBS AChE inhibitory activity and a
serotonergic subtype 4 receptor (5-HT4R) partial agonist effect,
promoting the secretion of the neurotrophic protein sAPPα in
an in vitro cellular model and finally leading to an in vivo
procognitive effect in mice.58 Donecopride was conceived as a
structural compromise between donepezil (DPZ), an AChEI
drug, and 2 (RS67333),59 a 5-HT4R agonist (Figure 1).
Figure 2. Pleiotropic interest of DBS AChEI/5-HT4R agonists.
RESULTS
■
Chemistry. Donecopride was obtained according to a
previously reported multistep sequence58 starting from 4-
amino-5-chloro-2-methoxybenzoic acid (3) (Scheme 1). The
sequence starts with the synthesis of a β-ketoester (4) with a
62% yield, which took place after the carbonyldiimidazole
(CDI) activation of the carboxylic acid group of 3. The
methylpiperidine moiety was then installed through a
nucleophilic substitution using N-Boc 4-(iodomethyl)-
piperidine carboxylate at room temperature to avoid the risk
of a double substitution, immediately followed by a
saponification−decarboxylation sequence with hydroalcoholic
potassium hydroxide which gave 5 in an 86% yield. 1 was finally
obtained in 44% yield through the TFA-N-deprotection of 5,
immediately followed by another nucleophilic substitution with
bromomethylcyclohexane. Starting from 5 and using various
alkyl- and arylmethyl bromides according to these experimental
conditions, numerous other N-substituted derivatives (6−15)
were obtained. Finally, the oxime (16) and methyloxime (17)
of donecopride were selectively obtained in excellent yield
under their E form (according to their X-ray structure) under
treatment using hydroxylamine and methoxylamine, respec-
tively. The reduction of its carbonyl group, using sodium
borohydride, afforded the hydroxyl derivative (18) in an 62%
yield.
Figure 1. Structure of donecopride, DPZ, and 2.
The anisamide 19, an analog of donecopride, was issued from
a peptidic coupling between 3 and N-Boc aminomethylpiper-
idine and affording 20 which was then N-deprotected and
substituted with a methylcyclohexyl group under treatment
with bromomethylcyclohexane. Finally the ester 21 was
synthesized in 31% yield through the esterification of 3 using
the commercially available N-methylcyclohexyl-4-hydroxyme-
thylpiperidine.
In Vitro Results. All the synthesized compounds were
evaluated as potential inhibitors of human AChE and equine
BuChE as well as potential ligands for guinea pig 5-HT4R
(Table 1). In these tests, DPZ was used as an AChEI control
and 2 as both a 5-HT4R ligand and an AChEI control since we
recently reported its MTDL pharmacological profile.58
The results of these in vitro evaluations are reported in a
double-entry table where we also reported the results
concerning 2 and DPZ (Table 2). Among the 16 tested
compounds, two of them (compounds 17, 18) were found to
be almost inactive against the two designed targets. One of
them (compound 16) is highly inhibitor of AChE, but it is
almost devoid of affinity for 5-HT4R, as is the case for DPZ.
This MTDL profile seems to confer to donecopride the
ability to display both improvements in cholinergic neuro-
transmission (through the catalytic inhibition of AChE and the
5-HT4R-dependent release of ACh) and in the inhibition of Aβ
accumulation and aggregation. This second effect would be
possible through the nonamyloidogenic 5-HT4R-dependent
cleavage of the precursor of Aβ and the consequent promotion
of the neurotrophic protein, sAPPα, as well as through the
interaction of donecopride with the peripheral anionic site
(PAS) of AChE, which is able to counteract the chaperone role
played by the latter in Aβ aggregation. Finally, these
complementary activities could exert both symptomatic and
disease-modifying effects for AD treatment (Figure 2).60
The present article aims to describe the discovery of
donecopride in the context of the chemical series from which
it was selected in order to establish some structure−activity
relationships (SARs) as well as to describe additional in vivo
experiments useful for precisely determining its pharmaco-
logical profile.
B
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a
Scheme 1. Synthetic pathways for access to compounds 1 and 6−21
a
Conditions and reagents: (a) CDI, THF; (b) KO2CCH2CO2Et, MgCl2, THF; (c) N-Boc 4-iodomethylpiperidine, K2CO3, DMF; (d) KOH, EtOH/
H2O; (e) TFA, DCM; (f) RBr, K2CO3, DMF; (g) HCl/EtOH; (h) H2NOR’, HCl, pyridine; (i) NaBH4, MeOH; (j) TEA, DMF; (k) HOBT, EDCI·
HCl, N-Boc 4-aminomethylpiperidine, DMF; (l) CyHexCH2Br, K2CO3, DMF; (m) N-CyHexCH2-4-hydroxymethylpiperidine, NaH, THF.
Conversely, four compounds (6, 7, 19, 21) appear to be good
ligands for 5-HT4R, but they do not show any AChE inhibitory
effects. Finally, nine compounds (1, 8−10, 11−15) could be
considered as MTDL since they exhibit both a Ki(5‑HT4R) ≤ 20
nM and an IC50(AChE) ≤ 400 nM, as was found with 2. These
compounds further showed a good selectivity toward AChE
versus BuChE.
The most potent compounds (1, 8−10, 13) were further
evaluated to establish their affinity and their pharmacological
profile with regard to their potential agonist effects toward
human 5-HT4R and their capacity to bind to the PAS of electric
eel AChE using the propidium iodide displacement test (Table
3).
By virtue of its in vitro activities and selectivity toward AChE
and 5-HT4R as well as its good druggability parameters (water
solubility, blood-brain barrier [BBB] cross-membrane pene-
tration, P-gp inhibition, mutagenicity, cytotoxicity, hERG
affinity and so on),58 compound 1 was selected for in silico
and in vivo studies aiming to evaluate its potential in AD
treatment. We named it donecopride.
In Silico Results. Ahead of the in silico study of
donecopride, its three-dimensional structure was identified via
the X-ray diffraction technique. The analysis carried out on the
donecopride crystal showed that two molecules are present in
the asymmetric unit, which differs principally in the position of
the cyclohexane ring. As the crystal space group is a
centrosymmetric one, another conformer, related to the
asymmetric unit by a symmetric center, is present in the
crystal for both molecules of the asymmetric unit (Figure 3).
Interestingly, the carbonyl group and the methoxy substituent
of the benzene ring point in opposite directions in all solved
structures. The four conformers observed in the crystal were
used for the docking studies, and the opposite mutual
orientation of the carbonyl and methoxy groups was used as
a criterion for the selection of docking poses.
Docking studies of donecopride were then performed in a
human AChE structure (PDB ED: 4EY7)62 as well as in a
human 5-HT4R model, as previously reported by us.63
First, the four conformers of donecopride from the crystal
structure (protonated on the piperidine ring) were docked
separately into the (h)5-HT4R homology model that was built
using the β2-adrenergic receptor crystal structure as a template
(PDB ED: 2RH1).63,64 In the selected pose (Figure 4A, mean
ChemPLP fit = 78.61), the basic piperidine nitrogen interacts
with Asp100 (consistent with the constraint used during
docking), and an additional polar interaction could be engaged
through this basic nitrogen and the Tyr302 hydroxyl group. The
benzene ring is oriented toward the transmembrane helix 5
(TM5) (in yellow in Figure 4A), and the amino substituent on
the benzene ring forms a hydrogen bond with the Ser197
hydroxyl group. In this position the benzene ring is surrounded
by several aromatic residues, oriented perpendicularly to it.
During the docking of donecopride conformers into
(h)AChE, a water molecule interacting with the protonated
piperidine ring of DPZ was conserved (residue number 931).
In the selected scoring pose (Figure 4B, mean ChemPLP Score
= 109.59), the donecopride orientation was closed to DPZ and
reproduced its interactions: (1) the charged nitrogen of the
piperidine ring was oriented in a position that was suitable for
interaction with the water molecule in the proximity of Tyr337
and Tyr341; (2) the donecopride carbonyl formed a hydrogen
bond with NH of the Phe295 backbone; (3) the benzene ring
was positioned in a parallel manner to the Trp286 indole ring of
the PAS at a 3.5 Å distance to favor a π-stacking interaction
(which is in accordance with the result of the propidium
displacement test results); and (4) the cyclohexane ring
C
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Table 1. (h)AChE Inhibitory Activity and Selectivity versus (eq)BuChE and (gp)5-HT4R Affinity for DPZ, 2, and compounds 1
and 6−21
a
Results reported from Lecoutey et al.58
occupied the DPZ benzyl ring space (in the neighboring area of
Trp86).
In Vivo Results. The data concerning the preliminary
toxicological and pharmacological screening of donecopride
(Table 4) showed no symptoms at subtoxic doses (around 7-
fold less than the approximative LD50).
Thus, subsequent behavioral studies were realized at the
maximum dose of 10 mg/kg. At the tested doses, donecopride
demonstrated no effect on spontaneous locomotor activity
(Figure 5).
induced spontaneous alternation deficit, an antiamnesic effect
was observed at doses starting from 0.3 mg/kg (Figure 6).
Interestingly, a comparable effect has been obtained with the
association of 2 to DPZ and with DPZ alone (1 mg/kg).
Finally, a decreased time of immobility was observed during
the forced swimming test for the 0.3 mg/kg dose of
donecopride, suggesting a slight antidepressant-like effect
(Figure 7).
On the other hand, we have previously demonstrated that
donecopride is able to increase sAPPα release in vitro in COS-7
We already showed that donecopride exhibited a procogni-
tive effect with an improvement in memory performances,
which was observed at 0.3 and 1 mg/kg on the object
recognition test.58 Moreover, in regards to scopolamine-
cells transiently expressing 5-HT4R (EC50 = 11.3 nM, Emax =
22.5% of the maximum 5-HT response over the basal
response).58 In the present work, we administered one dose
of donecopride (1 mg/kg) IP to C57BL/6 mice and collected
D
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2
Table 2. D Representation of (gp)5-HT4R Affinity and (h)AChE Inhibitory Activity for Compounds 1, 6−21, DPZ, and 2
nM and IC50[AChE] = 403 nM) with a methyl-cyclopropyl group
results in a dramatic decrease in AChE inhibition (see
compound 6 IC50[AChE] = 937 nM), while its 5-HT4R affinity
is almost maintained (Ki[5‑HT4R] = 14.30 nM). Substituting the
piperidine moiety by a methylcyclobutyl group (compound 7)
allows us to recover the 5-HT4R affinity of 2, but it does not
totally restore its AChE inhibitory effect (IC50[AChE] = 577 nM).
A larger methylcycloalkyl group, however, highly increases the
activities of compounds 8, 1, and 9 toward the two targets
(IC50[AChE] < 100 nM; Ki[5‑HT4R] < 10 nM), thus rendering them
as excellent MTDLs according to our criteria. The apparent
degrees of freedom allowed for the nature of these alkyl groups
are however quickly exceeded since replacing them by either an
ethylcyclohexyl group (compound 11) or a methyl ortho-
methylcyclohexyl group (compound 12) results in an
important decrease in the AChE inhibitory activity, while the
5-HT4R affinity is maintained.
On the other hand, substituting the n-butyl group of 2 by a
benzyl group, similar to those of DPZ, results in a dramatic
increase in the AChE inhibition exerted by compound 13
(IC50[AChE] = 8.5 nM), while its 5-HT4R affinity is slightly
decreased (Ki[5‑HT4R] = 14.30 nM). This compound also
appears to be as good an MTDL against the considered
targets. Replacing its phenyl group by a meta (compound 14)
or para (compound 15) pyridine group, however, leads to a
decrease in 5-HT4R affinity (Ki[5‑HT4R] > 20 nM) which appears
to be particularly linked to the basic nature of the intracyclic
nitrogen, since the methylpiperidine substituent in compound 9
dramatically restores it (Ki[5‑HT4R] = 2.50 nM).
Table 3. (h)5-HT4eR Binding Assay (Displacement of 1-(2-
Methylsulfonylaminoethyl-4-piperidinyl)methyl-1-methyl-
1H-indole-3-carboxylate (22) [3[H]GR113808]61), (h)5-
HT4eR Agonist Activity, And Propidium Iodide Competition
Assay for Compounds 1, 8−10, 13
(h)5-HT4eR
control ligand
displacement
propidium iodide
%
%
% control
agonist
response
displacement from (ee)
inhibition inhibition
AChE PAS % inhibition at
at 10−6
M
at 10−8
M
10−5
M
a
DPZ
−
100
−
73
−
21.5 1.5 n = 2
a
1
48.3 0.9
n = 3
24 2 n = 2
a
2
−
−
48.7 5.2
−
a
n = 3
8
9
100
99
58
46
69
41
48
58
20
65
20
20
30
13
10
13
100
100
a
Results reported from Lecoutey et al.58
cerebrospinal fluid (CSF) 90 min after the injection.
Quantification of sAPPα in the samples showed a slight
increase in the neuroprotective APP fragment which is
nonsignificant in these conditions (Figure 8). Further experi-
ments taking into account the biodistribution of donecopride
are necessary to adjust the time point of CSF collection and to
unequivocally conclude that donecopride promotes in vivo
sAPPα secretion.
We can also point out the apparent importance of the
carbonyl group in these structures for 5-HT4R affinity since
compounds 16−18 do not bind to this target. However, the
oxime 16 remains an excellent AChEI, almost as potent as
DPZ. Conversely, the benzamide (19) and the benzoate (21)
exhibit excellent 5-HT4R affinities in a low nanomolar range
DISCUSSION
■
If we first try to establish some SAR from these results, we can
point out that replacing the n-butyl group of 2 (Ki[5‑HT4R] = 9.33
E
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Figure 3. ORTEP diagram of donecopride (A) and superposition of its four conformations in the crystal (B).
Figure 4. Donecopride positioned in the (h)5-HT4R (A) and (h)AChE (B) binding sites from docking studies. The compound and the side chains
of the binding site residues are represented by the stick-like shapes, and the protein is represented in ribbon. This figure was made with PYMOL
(DeLano Scientific, 2002, San Carlo, U.S.A.).
Table 4. Pharmacological and Toxicological Properties of
Donecopride
symptoms
doses
LD50
compound
(mg/kg) (mg/kg)
subtoxic doses
no symptoms
toxic doses
convulsions
1−10
100
donecopride
amphetamine
75
hypoactivity
passivity
2
hyperactivity
exophtalmy
irritability
chlorpromazine
10
hypoactivity ataxia
sleep
Figure 5. Effect of donecopride on spontaneous locomotor activity.
Data are expressed as the mean standard deviation (n = 9−10).
while they are both almost devoid of the AChE inhibitory
effect. The structure of 19 has been docked into the active site
of (h)AChE. While, its conformation appeared to be similar to
donecopride, the rigidity of 19 appears to be strengthened by
the mesomeric effect due to its carboxamide group.
Consequently, 19, contrary to donecopride, seems not able to
bind in the same time through its carbonyl group and its
piperidine nitrogen atom with Phe295 and Water931 of (h)AChE,
Drugs were administered intraperitoneally (IP) 30 min before placing
mice in the actimeter, except for DPZ which was administered
subcutaneously (SC) 20 min before the test. Donecopride: 0.1−1−10
mg/kg; AMP: amphetamine 2 mg/kg; CPZ: chlorpromazine 10 mg/
kg; RS + DPZ: 2 0.1 mg/kg + DPZ 0.3 mg/kg (*p < 0.05 NaCl, SNK
test).
F
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Figure 6. Effect of donecopride on scopolamine-induced impairment during the spontaneous alternation test. Data are expressed as the mean
standard deviation (n = 10−12). Drugs were administered IP 30 min before the test, except for DPZ, and scopolamine was administered SC 30 and
20 min, respectively. DC (donecopride): 0.1, 0.3, 3, and 10 mg/kg; DPZ: 1 mg/kg; RS + DPZ: 2 0.1 mg/kg + DPZ 0.3 mg/kg; scopolamine: 0.5
mg/kg (*p < 0.05 versus 50%; univariate t test).
Figure 8. Acute in vivo treatment with donecopride shows a tendency
to increase sAPPα release in the CSF of 2-month-old C57BL/6 mice.
sAPPα was quantified via enzyme-linked immunosorbent assay
(ELISA). Each dot represents an individual mouse; the horizontal
lines show the means of each group. The p value is indicated (unpaired
Figure 7. Effect of donecopride on time immobility during the forced
swimming test. Data are expressed as the mean standard deviation
(n = 10−12). Drugs were administered IP 30 min before the test.
Student’s t test).
Donecopride: 0.3, 3 mg/kg; imipramine (IMI) 16 mg/kg (* p < 0.05
NaCl, Student−Newman−Keuls [SNK] test).
agonist. This profile is in accordance with our objective,
because prolonged exposure of G protein-coupled receptors to
respectively (Figure 9). This might also explain the inactivity of
21 against AChE.
an agonist can result in receptor desensitization, which is often
dependent on the degree of intrinsic activity.66 An opposite
relationship seems to have been established between these
results and the Ki(5‑HT4R) values of the tested compounds, since
it appears that the higher their affinity, the more partial their
agonist effect. This result appears to be concordant with that
reported in the literature.67
At last, concerning the capacity of the tested compound to
displace propidium iodide from the PAS of AChE, compounds
1, 8, and 9 acted in a similar manner as DPZ (20%−24%),
while 13 displaced propidium less (13%) and 10 displaced it
slightly more (30%). The results displayed by 13 and 10 could
be correlated in an opposite relation with their IC50(AChE) values
since the most potent AChEI in the series (13) weakly
interacted with the PAS, while the less potent inhibitor (10)
interacted more strongly. The weak AChE catalytic inhibitory
effect displayed by the latter compound might appear to be
inconsistent with its docking in the enzyme-active site. Indeed,
In summary, in the structural system derived from 2 as an
MTDL hit, 5-HT4R affinity seems to be linked to the presence,
on the nitrogen atom of the piperidine ring, of a not-too-
hindering methylcycloalkyl group, eventually bearing an
intracyclic nitrogen. On the other hand, the catalytic AChE
inhibition appears to require such a methylcycloalkyl group, but
can also be displayed by a benzyl, a methylpyridine, or a
methylpiperidine substituent. It is on the basis of these results
and considering our objective that we selected compounds 1,
8−10, and 13 for additional evaluations.
All the tested compounds displayed also high human 5-HT4R
affinities at 10−8 M (Table 2) confirming the relevance of the
use (gp)5-HT4R to select potential (h)5-HT4R ligands.65
Concerning their pharmacological profile, while compounds
1, 8, 9, and 13 were found to be 5-HT4R partial agonists (in a
similar manner as 2), compound 10 acted as a very partial
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Figure 9. Two representative docking poses of compound 19 into (h)AChE. The compounds and the side chains of the binding site residues are in
stick and the protein in ribbon representation. This figure was made with PYMOL (DeLano Scientific, 2002, San Carlo, U.S.A.).
Figure 10. Two representative docking poses of compound 10 into (h)AChE. The compounds and the side chains of the binding site residues are
represented by the stick-like shapes and the protein is represented by ribbon. This figure was made with PYMOL (DeLano Scientific, 2002, San
Carlo, U.S.A.).
the cationic nitrogen atom of the middle piperidine ring of 10
(protonated at physiologic pH as well as the nitrogen atom of
the terminal piperidine) appears able to bind to the same water
molecule as donecopride (mean ChemPLP Score fit = 104.02).
At the same time, the second nitrogen atom on the terminal
piperidine ring is located near Glu202, which unusually appeared
protonated using the ProPKA software (Figure 10A).68,69
However, in these conditions, a second pose was also generated
by the docking study in which it was the terminal piperidine
that interacted with the water molecule (mean ChemPLP Score
fit = 87.61; see Figure 10B). According to this hypothesis, the
phenyl ring of compound 10 covers more Trp286 whereas its
interaction with Trp86 would be weaker. Despite the lower
score, the second pose seems to be in better agreement with
experimental results, as it features the worst binding affinity and
the greatest propidium displacement for compound 10 with
respect to donecopride.
Concerning in vivo investigations, we have shown that
donecopride has an antiamnesiant effect in a scopolamine-
induced deficit model of working memory. Together with our
previous results showing the positive effect of donecopride in
recognition memory performance in mice58 (testing recog-
nition memory), this result confirms the high therapeutic
potential of this molecule in the treatment of the cognitive
disorders associated with AD. Of note, with respect to the dose
(0.3 mg/kg) that revealed a beneficial effect on the two
memory and behavioral tests (antiamnesiant and promnesiant
effects), an interesting additional antidepressant-like effect was
demonstrated. Furthermore, these behavioral effects were
associated with an increased release of the neuroprotective
sAPPα fragment. Such observations are in agreement with data
from the literature, showing, for instance, better memory
performance in transgenic mice that overexpress MMP9, which
consequently display an increased secretion of sAPPα.70
H
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deactivated silica gel (gradient of elution: DCM to DCM/MeOH (98/
2)).
Herein, we were only able to highlight a tendency of an
increased release of the nonamyloidogenic APP fragment in the
CSF 90 min following a systemic injection. However, that
sAPPα increased upon 5-HT4R activation is transient,71 and it
is consequently highly difficult to catch the peak of sAPPα in
CSF. Kinetics studies using different collection times or
intracerebral microdialysis would be necessary to precisely
determine the acute action of donecopride in living animals.
Moreover, it should be interesting to monitor ACh levels,
sAPPα, and Aβ in the CSF or in the brain tissue at the same
time. Chronic administration of 5-HT4R agonists reduces
amyloid pathology and prevents cognitive deficits in AD mouse
models.71,72 Further experiments exploring chronic treatments
of AD transgenic mice with donecopride, in comparison with
DPZ and pure 5-HT4R agonists, would produce valuable
insights regarding the potential of donecopride to exert a
disease-modifying effect.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclohexylmethyl)-
4-piperidyl]propan-1-one (1).58 Pale yellow powder (44%): mp 153−
1
155 °C; H NMR (400 MHz, CDCl3): δ 7.79 (s, 1H), 6.48 (s, 1H),
4.44 (br s, 2H), 3.85 (s, 3H), 2.89 (m, 2H), 2.84 (m, 2H), 2.08 (d, J =
6.8 Hz, 2H), 1.68 (m, 11H), 1.47 (m, 1H), 1.20 (m, 6H), 0.85 (m,
2H); 13C NMR (100 MHz, CDCl3): δ 199.3, 159.5, 147.7, 132.2,
119.0, 111.2, 97.6, 66.3, 55.6, 54.5 (2C), 41.0, 35.8, 35.3, 32.3 (2C),
32.2 (2C), 31.4, 26.8, 26.2 (2C); LC-MS: [M + H]+ = 393.58−395.59;
IR (KBr): ν cm−1 3485, 3330, 2921, 2850, 1641, 1623, 1575, 1452,
1421, 1214, 1177; HRMS (m/z) calcd for C22H33ClN2O2 [M]+
392.22303, found 392.22399.
Ethyl 3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxopropanoate
(4). To a suspension of 4-amino-5-chloro-2-methoxybenzoic acid
(603 mg, 2.99 mmol) in dry THF (30 mL) was added portion-wise
CDI (533 mg, 3.29 mmol). The mixture was stirred at rt for 6 h and,
then, potassium 3-ethoxy-3-oxopropanoate (611 mg, 3.59 mmol) and
MgCl2 (342 mg, 3.59 mmol) were added portion-wise. The mixture
was stirred at 40 °C for 2 days. After evaporation of the solvent, the
residue was diluted with DCM, washed successively with water,
saturated aqueous NaHCO3 and brine, dried over MgSO4, and
evaporated in vacuo. The residue was then purified on silica gel
(gradient of elution: CH/AcOEt 8/2 to 7/3) to afford 603 mg of
CONCLUSION
■
In conclusion, we have developed a novel series of MTDL that
displayed both DBS AChE inhibitory effects and partial 5-
HT4R agonist activity in vitro in nanomolar ranges.
Donecopride, which appears to be the lead of this family,
further exhibited in vivo procognitive and antiamnesic effects in
NMRI mice, and it also appeared able to promote the release of
sAPPα in C57BL/6 mice in vivo. These results, which appear
to gather in a single compound both the activities of DPZ and
2, seem likely to confer a symptomatic and disease-modifying
effect to donecopride, thus designing it as a potentially
promising drug candidate in AD.
1
product. White powder (62%): mp 124 °C; H NMR (400 MHz,
CDCl3): δ 7.92 (s, 1H), 6.23 (s, 1H), 4.56 (br s, 2H), 4.17 (qd, J = 7.3
Hz, 2H), 3.87 (s, 2H), 3.82 (s, 3H), 1.24 (t, J = 7.3 Hz, 3H); 13C
NMR (100 MHz, CDCl3): δ 189.6, 168.6, 159.9, 148.7, 132.5, 117.3,
111.6, 97.0, 60.8, 55.4, 50.5, 14.2; LC-MS: [M + H]+ = 272.43−
274.43; IR (KBr): ν cm−1 3463, 3362, 3323, 2983, 1725, 1648, 1622,
1573, 1469, 1423, 1326, 1262, 1221, 1155; HRMS (m/z) calcd for
C12H15ClNO4 [M + H]+ 272.068412, found 272.068374.
tert-Butyl 4-(3-(4-amino-5-chloro-2-methoxyphenyl)-3-
oxopropyl)piperidine-1-carboxylate (5).73 To a solution of com-
pound 3 (2.3 g, 8.5 mmol) in DMF (20 mL) were added tert-butyl 4-
(iodomethyl)piperidine-1-carboxylate (3.05 g, 9.5 mmol) and K2CO3
(2.3 g, 17.1 mmol). The mixture was stirred at rt for 48 h. The reaction
mixture was diluted with water, and the product was extracted with
AcOEt. Combined organic layer was washed with water and brine and
then dried over MgSO4. Evaporation of the solvent gave a crude
mixture (4.8 g, 10.3 mmol), which was directly dissolved in EtOH
(500 mL). H2O (100 mL) and KOH (2.65 g, 47.3 mmol) were added
to the solution. The reaction mixture was refluxed for 3 h. EtOH was
then evaporated, and the resulting residue was diluted with H2O and
extracted with AcOEt. The organic layer was washed with H2O and
brine. Evaporation of the solvent provided a crude product, which was
purified by column on silica (gradient of elution: DCM to DCM/
AcOEt 6/4) to provide compound 2 (2.9 g). White powder (86%):
EXPERIMENTAL SECTION
■
Chemistry. General Material and Methods. All chemical reagents
and solvents were purchased from commercial sources and used
without further purification. Melting points were determined on a
Kofler melting point apparatus. 1H and 13C NMR spectra were
recorded on a BRUKER AVANCE III 400 MHz with chemical shifts
expressed in parts per million (in chloroform-d, methanol-d4, or
DMSO-d6) downfield from TMS as an internal standard and coupling
in Hertz. IR spectra were recorded on a PerkinElmer BX FT-IR
apparatus using KBr pellets. High-resolution mass spectra (HRMS)
were obtained by impact electronic on a Jeol JMS GCMate
spectrometer (compound 1) or by electrospray on a Bruker maXis
(compounds 4, 7, 8, 13−19) or on a Q-Tof micro Waters
(compounds 6, 9−12, 21). The purities of all tested compounds
were analyzed by LC−MS, with the purity all being higher than 95%.
Analyses were performed using a Waters Alliance 2695 as separating
module (column XBridge C18 2.5 μM/4.6 × 50 mM) using the
following gradients: A (95%)/B (5%) to A (5%)/B (95%) in 4.00 min.
This ratio was hold for 1.50 min before returning to initial conditions
in 0.50 min. Initial conditions were then maintained for 2.00 min (A =
H2O, B = CH3CN; each containing HCOOH: 0.1%). MS were
obtained on a SQ detector by positive ESI.
1
mp 135−137 °C; H NMR (400 MHz, CDCl3): δ 7.71 (s, 1H), 6.23
(s, 1H), 4.63 (br s, 2H), 4.01 (m, 2H), 3.76 (s, 3H), 2.84 (t, J = 7.5
Hz, 2H), 2.61 (m, 2H), 1.61 (m, 2H), 1.53 (m, 2H), 1.39 (s, 9H), 1.35
(m, 1H), 1.03 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 198.7, 159.5,
154.8, 148.0, 131.9, 118.3, 111.0, 97.3, 79.1, 55.5, 43.8 (2C), 40.5, 35.6,
31.9 (2C), 31.0, 28.3 (3C); LC-MS: [M + H]+ = 397.60−399.60; IR
(KBr): ν cm−1 3470, 3353, 2975, 2930, 2851, 1673, 1624, 1588, 1421,
1366, 1311, 1250, 1216.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclopropylmeth-
yl)-4-piperidyl]propan-1-one (6). Pale yellow powder (38%): mp
1
161−163 °C; H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H), 6.23 (s,
General Procedure for the Deprotection and N-Alkylation of tert-
Butyl 4-[3-(4-amino-5-chloro-2-methoxyphenyl)-3-oxo-propyl]-
piperidine-1-carboxylate (5) (1; 6−9; 11−15; 17). tert-Butyl 4-[3-
(4-amino-5-chloro-2-methoxyphenyl)-3-oxo-propyl]piperidine-1-car-
boxylate 5 was dissolved in DCM/TFA (1/1). The mixture was then
stirred 15 min at rt. After concentration under reduced pressure, the
resulting intermediate was dissolved in DMF or 1,4-dioxane (for
compounds 13-15). K2CO3 (10 equiv) and halogenoalkyle (1.3 equiv)
were added, and the mixture was warmed to 110 °C for 4 h. After
cooling, DCM was added, and the mixture was washed 4 times with
brine. The organic layer was then dried over MgSO4, filtered, and
evaporated under reduced pressure. The crude residue was purified on
1H), 4.46 (s, 2H), 3.81 (s, 3H), 3.03 (m, 2H), 2.87 (m, 2H), 2.19 (d, J
= 6.8 Hz, 2H), 1.88 (m, 2H), 1.67 (m, 2H), 1.56 (m, 2H), 1.32 (m,
1H), 1.27 (m, 2H), 0.83 (m, 1H), 0.47 (m, 2H), 0.06 (m, 2H); 13C
NMR (100 MHz, CDCl3): δ 199.2, 159.5, 147.7, 132.2, 118.9, 111.2,
97.5, 64.2, 55.6, 54.0 (2C), 40.9, 35.6, 32.2 (2C), 31.4, 8.5, 4.0 (2C);
LC-MS: [M + H]+ = 351.49−353.45; IR (KBr): ν cm−1 3466, 3350,
2924, 2849 1644, 1621, 1586, 1465, 1420, 1215, 1178; HRMS (m/z)
calcd for C19H28ClN2O2 [M + H]+ 351.1839, found 351.1841.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclobutylmethyl)-
4-piperidyl]propan-1-one (7). Yellow powder (45%): mp 142−144
1
°C; H NMR (400 MHz, CDCl3): δ 7.77 (s, 1H), 6.25 (s, 1H), 4.41
I
DOI: 10.1021/acs.jmedchem.5b00115
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(br s, 2H), 3.84 (s, 3H), 2.88 (t, J = 8.0 Hz, 2H), 2.81 (m, 2H), 2.54
(m, 1H), 2.37 (d, J = 6.6 Hz, 2H), 2.06 (m, 2H), 1.78 (m, 8H), 1.57
(m, 2H), 1.25 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 199.1, 159.4,
147.6, 132.2, 118.9, 111.2, 97.5, 65.9, 55.6, 54.0 (2C), 40.9, 35.4, 34.2,
32.2 (2C), 31.3, 28.3 (2C), 18.8; LC-MS: [M + H]+ = 365.53−367.54;
IR (KBr): ν cm−1 3485, 3327, 2923, 2852, 1642, 1624, 1575, 1453,
1421, 1214, 1175; HRMS (m/z) calcd for C20H30ClN2O2 [M + H]+
365.199032, found 365.198665.
1173; HRMS (m/z) calcd for C23H36ClN2O2 [M + H]+ 407.2465,
found 407.2468.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-[(2-
methylcyclohexyl)methyl]-4-piperidyl] propan-1-one (12). Powder
1
(49%): H NMR (400 MHz, CDCl3): δ 7.75 (s, 1H, 1H*), 6.23 (s,
1H, 1H*), 4.43 (br s, 2H, 2H*), 3.81 (s, 3H, 3H*), 2.87 (m, 2H,
3H*), 2.81 (m, 2H), 2.74 (m, 1H*), 2.32 (dd, J = 11.8 Hz, J = 3.2 Hz,
1H*), 2.06 (m, 2H), 2.00 (m, 1H*), 1.92 (m, 1H*), 1.81 (m, 3H),
1.60 (m, 7H, 11H*), 1.38 (m, 6H, 3H*), 1.21 (m, 3H, 4H*), 0.87 (d,
J = 5.8 Hz, 3H*), 0.80 (d, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 199.3 (C, C*), 159.5 (C, C*), 147.6 (C, C*), 132.2 (C,
C*), 119.0 (C, C*), 111.3 (C, C*), 97.6 (C, C*), 63.7 (C*), 62.0 (C),
56.1 (C*), 55.7 (C, C*), 54.8 (C), 54.5 (C), 53.2 (C*), 41.0 (C), 41.0
(C*), 37.2 (C, C*), 36.6 (C*), 35.9 (C), 35.9 (C*), 33.0 (C), 32.4
(2C*), 32.4 (2C), 32.3 (C*), 31.5 (C*), 31.4 (C), 31.0 (C), 26.4
(C*), 26.4 (C), 26.3 (C*), 25.6 (C), 21.9 (C, C*), 20.5 (C*), 13.6
(C); LC-MS: [M + H]+ = 407.54−409.63; HRMS (m/z) calcd for
C23H36ClN2O2 [M + H]+ 407.2465, found 407.2458. The ratio of
these 2 diastereoisomeres was obtained in proportions 3/1* according
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclopentylmeth-
yl)-4-piperidyl]propan-1-one (8). Yellow powder (35%): mp 141−
1
143 °C; H NMR (400 MHz, CDCl3): δ 7.79 (s, 1H), 6.26 (s, 1H),
4.44 (br s, 2H), 3.85 (s, 3H), 2.90 (m, 4H), 2.24 (d, J = 7.1 Hz, 2H),
2.05 (m, 1H), 1.86 (m, 2H), 1.76 (m, 2H), 1.60 (m, 6H), 1.50 (m,
2H), 1.21 (m, 5H); 13C NMR (100 MHz, CDCl3): δ 199.2, 159.4,
147.5, 132.2, 119.0, 111.2, 97.5, 65.2, 55.6, 54.3 (2C), 40.9, 37.5, 35.7,
32.2 (2C), 31.8 (2C), 31.3, 25.2 (2C); LC-MS: [M + H]+ = 379.57−
381.56; IR (KBr): ν cm−1 3486, 3332, 2924, 2860, 1644, 1623, 1574,
1453, 1422, 1214, 1175; HRMS (m/z) calcd for C21H32ClN2O2 [M +
H]+ 379.214682, found 379.214484.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cycloheptylmeth-
1
to the H NMR spectra.
yl)-4-piperidylpropan-1-one (9). Yellow powder (42%): mp 147−149
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-(1-benzyl)-4-
1
°C; H NMR (400 MHz, CDCl3): δ 7.78 (s, 1H), 6.27 (s, 1H), 4.49
1
piperidyl)propan-1-one (13). Pale yellow oil (23%); H NMR (400
(br s, 2H), 3.85 (s, 3H), 2.87 (m, 2H), 2.81 (m, 2H), 2.02 (d, J = 7.3
Hz, 2H), 1.57 (m, 17H), 1.22 (m, 3H), 1.08 (m, 2H); 13C NMR (100
MHz, CDCl3): δ 198.2, 158.5, 146.7, 131.2, 117.8, 110.2, 96.5, 65.3,
54.6, 53.3 (2C), 39.9, 35.4, 34.6, 31.9 (2C), 31.0 (2C), 30.3, 27.5
(2C), 25.5 (2C); LC-MS: [M + H]+ = 407.53−409.52; IR (KBr): ν
cm−1 3481, 3247, 2920, 2850, 1638, 1622, 1582, 1454, 1419, 1214,
1176; HRMS (m/z) calcd for C23H36ClN2O2 [M + H]+ 407.2465,
found 407.2461.
MHz, CDCl3): δ 7.71 (s, 1H), 7.19 (m, 5H), 6.18 (s, 1H), 4.39 (s,
2H), 3.76 (s, 3H), 3.45 (s, 2H), 2.81 (m, 4H), 1.90 (br t, J = 10.2 Hz,
2H), 1.61 (br d, J = 9.2 Hz, 2H), 1.52 (q, J = 6.9 Hz, 2H), 1.22 (m,
3H); 13C NMR (100 MHz, CDCl3): δ 199.2, 159.5, 147.7, 137.7,
132.3, 129.5 (2C), 128.2 (2C), 127.1, 118.9, 111.3, 97.5, 63.3, 55.6,
53.7 (2C), 40.8, 35.4, 31.9 (2C), 31.4; HRMS (m/z) calcd for
C22H28ClN2O2 [M + H]+ 387.183382, found 387.183120.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(3-pyridylmethyl)-
4-piperidyl]propan-1-one (14). Pale yellow oil (40%); 1H NMR (400
MHz, CDCl3): δ 8.51 (d, J = 2.0 Hz, 1H), 8.50 (dd, J = 1.6, 4.8 Hz,
1H), 7.78 (s, 1H), 7.69 (dt, J = 1.6, 7.6 Hz, 1H), 7.25 (dd, J = 4.8, 8.0
Hz, 1H), 6.25 (s, 1H), 4.48 (s, 2H), 3.83 (s, 3H), 2.87 (m, 4H), 1.99
(br t, J = 10.2 Hz, 2H), 1.68 (br d, J = 9.2 Hz, 2H), 1.58 (q, J = 6.9 Hz,
2H), 1.24 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 199.3, 159.7,
150.7, 148.8, 147.9, 137.2, 133.7, 132.4, 123.6, 119.0, 111.4, 97.7, 60.5,
55.7, 53.8 (2C), 40.9, 35.4, 32.0 (2C), 31.2; HRMS (m/z) calcd for
C21H27ClN3O2 [M + H]+ 388.178631, found 388.178595.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(4-pyridylmethyl)-
4-piperidyl]propan-1-one (15). Pale yellow oil (45%); 1H NMR (400
MHz, CDCl3): δ 8.52 (d, J = 6.0 Hz, 2H), 7.78 (s, 1H), 7.26 (d, J = 6.0
Hz, 2H), 6.25 (s, 1H), 4.50 (s, 2H), 3.83 (s, 3H), 3.47 (s, 2H), 2.89 (t,
J = 7.6 Hz, 2H), 2.83 (br d, J = 11.2 Hz, 2H), 1.97 (br t, J = 10.8 Hz,
2H), 1.68 (br d, J = 9.2 Hz, 2H), 1.59 (q, J = 6.9 Hz, 2H), 1.26 (m,
3H); 13C NMR (100 MHz, CDCl3): δ 199.3, 159.7, 149.8 (2C), 148.2,
147.9, 132.4, 124.2 (2C), 119.0, 111.4, 97.6, 62.2, 55.7, 54.1 (2C),
40.9, 35.4, 32.2 (2C), 31.3; HRMS (m/z) calcd for C21H27ClN3O2 [M
+ H]+ 388.178631, found 388.178502.
General Procedure for the Preparation of Oximes (16−17). To a
solution of compound 1 in pyridine was added alkoxyamine
hydrochloride (3.6 equiv) in 3 portions every 2 h. The mixture was
stirred at rt for 24 h. After dilution with water, the aqueous layer was
extracted 3 times with AcOEt. The organic layers were combined,
washed 5 times with brine, dried over MgSO4, and concentrated under
reduced pressure. The crude residue was purified on deactivated silica
gel (gradient: DCM to DCM/AcOEt (9/1)) to provide the desired
product.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(4-piperidylmethyl)-
4-piperidyl]propan-1-one dihydrochloride (10). Following the
previous general procedure, the intermediate tert-butyl 4-[[4-[3-(4-
amino-5-chloro-2-methoxyphenyl)-3-oxo-propyl]-1-piperidyl]methyl]
piperidine-1-carboxyl ate is obtained. Pale yellow powder (24%): mp
78−80 °C; 1H NMR (400 MHz, CDCl3): δ 7.76 (s, 1H), 6.23 (s, 1H),
4.44 (br s, 2H), 4.03 (m, 2H), 3.82 (s, 3H), 2.87 (t, J = 7.8 Hz, 2H),
2.80 (m, 2H), 2.65 (m, 2H), 2.10 (d, J = 6.8 Hz, 2H), 1.83 (m, 2H),
1.58 (m, 7H), 1.41 (s, 9H), 1.23 (m, 3H), 1.03 (m, 2H); 13C NMR
(100 MHz, CDCl3): δ 199.1, 159.5, 155.0, 147.6, 132.3, 119.0, 111.3,
97.6, 79.2, 65.1, 55.7, 54.6 (2C), 43.8 (2C), 40.9, 35.7, 33.8, 32.3 (2C),
31.3, 30.9 (2C), 28.4 (3C); LC-MS: [M + H]+ = 494.53−496.55, [M
+ H − BOC]+ = 394.49−396.48; IR (KBr): ν cm−1 3479, 3347, 2922,
2850, 1688, 1674, 1622, 1587, 1466, 1452, 1247, 1215, 1175; HRMS
(m/z) calcd for C26H40ClN3O4 [M + H]+ 494.2786, found 494.2766.
To a solution of tert-butyl4-[[4-[3-(4-amino-5-chloro-2-methoxyphen-
yl)-3-oxo-propyl]-1-piperidyl]methyl]piperid-ine-1-carboxylate (40
mg, 0.09 mmol) in EtOH (2 mL) was added HCl 37% (300 μL).
The mixture was stirred at rt for 3 h and then concentrated under
reduced pressure. The residue was then stirred into Et2O and filtered
to afford 30 mg of compound 9. Yellow powder (80%): mp >260 °C;
1H NMR (400 MHz, DMSO-d6): δ 10.23 (br s, 1H), 9.09 (br s, 1H),
8.96 (br s, 1H), 7.53 (s, 1H), 6.46 (s, 1H), 3.99 (s, 3H), 3.45 (m, 2H),
3.22 (m, 2H), 2.91 (m, 2H), 2.81 (m, 6H), 2.11 (m, 1H), 1.96 (m,
2H), 1.76 (m, 2H), 1.63 (m, 2H), 1.46 (m, 3H), 1.38 (m, 2H); 13C
NMR (100 MHz, DMSO-d6): δ 196.6, 159.6, 150.1, 131.1, 115.8,
109.3, 97.1, 60.4, 55.6, 52.3 (2C), 42.3 (2C), 39.7, 32.7, 30.2, 28.4
(2C), 28.3, 26.6 (2C); LC-MS: [M + H]+ = 394.38−396.42; IR
(KBr): ν cm−1 3390, 3296, 3194, 2938, 2732, 1660, 1625, 1593, 1449,
1417, 1212, 1180; HRMS (m/z) calcd for C21H33ClN3O2 [M + H]+
394.2261, found 394.2269.
(E)1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclohexylmeth-
yl)-4-piperidyl]propan-1-one oxime (16). White powder (35%): mp
194−196 °C; 1H NMR (400 MHz, CD3OD): δ 6.99 (s, 1H), 6.47 (s,
1H), 3.75 (s, 3H), 2.93 (m, 2H), 2.71 (m, 2H), 2.23 (d, J = 6.8 Hz,
2H), 2.02 (m, 2H), 1.72 (m, 8H), 1.55 (m, 1H), 1.28 (m, 7H), 0.93
(m, 2H); 13C NMR (100 MHz, CD3OD): δ 159.8, 157.3, 145.9, 129.8,
115.8, 109.4, 98.2, 65.4, 54.6, 53.9 (2C), 35.2, 34.4, 31.8, 31.6 (2C),
30.8 (2C), 26.1, 25.7 (2C), 25.3; IR (KBr): ν cm−1 3368, 2925, 2850,
2815, 1620, 1508, 1461, 1449, 1412, 1336, 1254, 1214, 992; LC-MS:
[M + H]+ = 408.53−410.51; HRMS (m/z) calcd for C22H35ClN3O2
[M + H]+ 408.241231, found 408.240998.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclohexylethyl)-4-
piperidyl]propan-1-one (11). Yellow powder (40%): mp 169−171
1
°C; H NMR (400 MHz, CDCl3): δ 7.76 (s, 1H), 6.23 (s, 1H), 4.42
(br s, 2H), 3.82 (s, 3H), 2.89 (m, 4H), 2.28 (m, 2H), 1.84 (m, 2H),
1.61 (m, 9H), 1.36 (m, 2H), 1.20 (m, 7H), 0.90 (m, 2H); 13C NMR
(100 MHz, CDCl3): δ 199.1, 159.5, 147.6, 132.2, 118.9, 111.2, 97.5,
57.0, 55.6, 54.0 (2C), 40.8, 36.5, 35.5, 34.4, 33.4 (2C), 32.0 (2C), 31.2,
26.6, 26.3 (2C); LC-MS: [M + H]+ = 407.41−409.52; IR (KBr): ν
cm−1 3486, 3324, 2918, 2847, 1637, 1621, 1571, 1453, 1304, 1212,
J
DOI: 10.1021/acs.jmedchem.5b00115
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2-Chloro-4-[(E)-C-[2-[1-(cyclohexylmethyl)-4-piperidyl]ethyl]-N-
methoxy-carbon-imidoyl]-5-methoxyaniline (17). Yellow oil (42%);
1H NMR (400 MHz, CDCl3): δ 7.12 (s, 1H), 6.25 (s, 1H), 4.10 (br s,
concentrated under reduced pressure. The residue was dissolved in
AcOEt, washed with water, dried over MgSO4, and concentrated under
in vacuo. The crude product was then purified on silica gel (gradient of
elution: DCM (100%) to AcOEt (100%)). White powder (31%): mp
2H), 3.89 (s, 3H), 3.72 (s, 3H), 2.79 (m, 2H), 2.63 (m, 2H), 2.04 (d, J
= 6.8 Hz, 2H), 1.65 (m, 9H), 1.44 (m, 1H), 1.22 (m, 8H), 0.83 (m,
2H); 13C NMR (100 MHz, CDCl3): δ 159.9, 157.1, 144.2, 130.4,
117.0, 110.5, 98.5, 66.2, 61.5, 55.5, 54.5 (2C), 36.1, 35.2, 32.4, 32.1
(2C), 32.0 (2C), 29.7, 26.5, 26.2 (2C); LC-MS: [M + H]+ = 422.54−
424.53; IR (KBr): ν cm−1 2921, 2850, 1621, 1506, 1464, 1450, 1411,
1338, 1258, 1213, 1051; HRMS (m/z) calcd for C23H38ClN3O2 [M +
H]+ 422.256882, found 422.256616.
1
128−130 °C; H NMR (400 MHz, CDCl3): δ 7.81 (s, 1H), 6.29 (s,
1H), 4.45 (br s, 2H), 4.08 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 2.88 (m,
2H), 2.10 (d, J = 7.1 Hz, 2H), 1.86 (m, 2H), 1.69 (m, 7H), 1.48 (m,
1H), 1.39 (m, 2H), 1.19 (m, 4H), 0.86 (m, 2H); 13C NMR (100 MHz,
CDCl3): δ 164.6, 160.2, 147.6, 133.2, 110.0, 109.9, 98.2, 68.9, 66.1,
56.0, 53.9 (2C), 35.6, 35.2, 32.0 (2C), 29.1 (2C), 26.8, 26.2 (2C); LC-
MS: [M + H]+ = 395.59−397.56; IR (KBr): ν cm−1 3473, 3330, 2920,
2850, 1695, 1621, 1599, 1449, 1234, 1109; HRMS (m/z) calcd for
C21H32ClN2O3 [M + H]+ 395.2101, found 395.2109.
1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-(cyclohexylmethyl)-
4-piperidyl]propan-1-ol (18). To a solution of compound 1 (33 mg,
0.084 mmol) in MeOH was added NaBH4 (25 mg, 0.673 mmol). The
mixture was stirred for 3 h. After concentration, the residue was
dissolved in DCM, washed with brine, dried over MgSO4, and then
evaporated under reduced pressure. The crude product was purified on
deactivated silica gel (gradient of elution: DCM to DCM/MeOH) to
afford 20 mg of desired product. Pale yellow powder (62%): mp: 136−
X-ray Crystallography. Single crystals of donecopride and
compound 17 suitable for X-ray crystallographic analysis were
obtained by slow evaporation from a mixture of chloroform/acetone
and ethanol, respectively. Data for crystal structures analysis were
collected at 150 K with a Bruker−Nonius Kappa CCD area detector
diffractometer with graphite-monochromatized Mo Kλ radiation (λ =
0.71073 Å). The structures were solved using direct methods and
refined by full-matrix least-squares analysis on F2.
Crystallographic Data of Donecopride. Crystal size: 0.38 × 0.25 ×
0.09 mm. Formula C22H33ClN2O2, formula weight 392.95, crystal
system triclinic, space group P-1, a = 10.5505(3) Å, b = 10.6906(4) Å,
c = 19.8075(6) Å, α = 83.6320(10)°, β = 75.3140(10)°, γ =
88.2010(10)°, V = 2147.78(12) Å3, Z = 4, calculated density = 1.215
g/cm3, μ = 0.197 mm−1, Rint = 0.041, R[F2 > 2σ(F2)] = 0.070, wR(F2)
= 0.196.
1
138 °C; H NMR (400 MHz, CDCl3): δ 7.12 (s, 1H), 6.26 (s, 1H),
4.69 (dd, J = 7.5 Hz, J = 5.8 Hz, 1H), 4.00 (br s, 2H), 3.75 (s, 3H),
2.82 (m, 2H), 2.06 (d, J = 7.0 Hz, 2H), 1.79 (m, 2H), 1.67 (m, 10H),
1.45 (m, 1H), 1.36 (m, 1H), 1.19 (m, 6H), 0.83 (m, 2H); 13C NMR
(100 MHz, CDCl3): δ 156.2, 142.5, 127.5, 124.0, 110.6, 98.8, 70.0,
66.2, 55.4, 54.5 (2C), 35.8, 35.2, 34.4, 32.9, 32.3 (2C), 32.1 (2C), 26.8,
26.2 (2C); IR (KBr): ν cm−1 3393, 2920, 2850, 1622, 1580, 1504,
1466, 1412, 1209; LC-MS: [M + H]+ = 395.64−397.64. HRMS (m/z)
calcd for C22H37ClN2O2 [M + H]+ 395.245983, found 395.245734.
4-Amino-5-chloro-N-[[1-(cyclohexylmethyl)-4-piperidyl]methyl]-
2-methoxybenzamide (19). Following the previous general procedure
used for the deprotection and N-alkylation of 5, 19 is obtained starting
from tert-butyl 4-[[(4-amino-5-chloro-2-methoxybenzoyl)amino]-
methyl] piperidine-1-carboxylate (20). White powder (38%): mp
154−156 °C; 1H NMR (400 MHz, CDCl3): δ 8.07 (s, 1H), 7.73 (t, J
= 6.0 Hz, 1H), 6.27 (s, 1H), 4.39 (br s, 2H), 3.87 (s, 3H), 3.29 (m,
2H), 2.90 (m, 2H), 2.13 (d, J = 7.0 Hz, 2H), 1.90 (m, 2H), 1.66 (m,
8H), 1.48 (m, 1H), 1.36 (m, 2H), 1.16 (m, 3H), 0.84 (m, 2H); 13C
NMR (100 MHz, CDCl3): δ 164.6, 157.4, 146.6, 133.1, 112.0, 111.6,
97.9, 65.9, 56.2, 54.0 (2C), 45.1, 36.0, 35.1, 32.0 (2C), 29.7, 26.7 (2C),
26.1 (2C); LC-MS: [M + H]+ = 394.59−396.58; IR (KBr): ν cm−1
3400, 2925, 2854, 1622, 1593, 1536, 1498, 1256; HRMS (m/z) calcd
for C21H33ClN3O2 [M + H]+ 394.225581, found 394.225427.
tert-Butyl 4-[[(4-amino-5-chloro-2-methoxybenzoyl)amino]-
methyl]piperidine-1-carboxylate (20).74 To a solution of 4-amino-
5-chloro-2-methoxybenzoic (94 mg, 0.467 mmol) in DMF (3 mL)
were added Et3N (64 μL, 0.467 mmol) and tert-butyl 4-
(aminomethyl)piperidine-1-carboxylate (100 mg, 0.467 mmol). The
mixture was cooled to −5 °C, and HOBT (63 mg, 0.467 mmol) and
EDCI·HCl (90 mg, 0.467 mmol) were then added. The mixture was
then stirred for 24 h. After dilution with water, the solution was
extracted 3 times with AcOEt. The organic layers were combined,
washed 4 times with water, dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude residue was purified
on silica gel (gradient of elution: DCM to DCM/AcOEt 7/3) to
provide 155 mg of desired product. White powder (85%): mp 106−
108 °C; 1H NMR (400 MHz, CDCl3): δ 8.08 (s, 1H), 7.77 (t, J = 5.9
Hz, NH), 6.27 (s, 1H), 4.37 (br s, 2H), 4.09 (m, 2H), 3.72 (s, 3H),
3.30 (m, 2H), 2.66 (m, 2H), 1.70 (m, 3H), 1.42 (s, 9H), 1.16 (m,
2H); 13C NMR (100 MHz, CDCl3): δ 164.7, 157.4, 154.9, 146.8,
133.1, 112.4, 111.6, 97.8, 79.4, 56.2, 45.0, 43.9 (2C), 36.5, 29.9 (2C),
28.5 (3C); LC-MS: [M + H]+ = 398.53−400.52; IR (KBr): ν cm−1
3405, 3339, 2976, 2928, 2852, 1680, 1630, 1595, 1542, 1501, 1425,
1315, 1252, 1173, 1144.
Crystallographic Data of Compound 16. Crystal size: 0.42 × 0.38
× 0.21 mm. Formula C22H34ClN3O2, formula weight 407.97, crystal
system monoclinic, space group P21/c, a = 12.0789(5) Å, b =
9.9591(5) Å, c = 19.5343(7) Å, β = 104.580(2)°, V = 2274.20(17) Å3,
Z = 4, calculated density = 1.192 g/cm3, μ = 0.189 mm−1, Rint = 0.026,
R[F2 > 2σ(F2)] = 0.040, wR(F2) = 0.113.
Program(s) Used to Solve Structures: SHELXS−97.75 Program(s)
used to refine structures: SHELXL−2014.76 Software used to prepare
material for publication: SHELXL−2014. Crystallographic data have
been deposited at the Cambridge Crystallographic Data Centre,
CCDC no. 1045006 (donecopride) and CCDC no. 1045005 (16).
Copies of this information may be obtained free of charge from the
Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (+44-
1223-336408; E-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.
cam.ac.uk).
In Vitro Biological Studies. Pharmacological Characterization
of Drugs on Guinea Pig 5-HT4R. Binding to native 5-HT4R from
guinea pig was determined using the method of Grossman.61 For
membrane preparations male guinea pigs (300−350 g, Charles River)
were subjected to euthanasia by cervical dislocation and decapitated.
Brains were rapidly removed at 4 °C, and striatal regions carefully
dissected and pooled. The tissues were then suspended in 10 volumes
of HEPES buffer 50 mM pH 7.4 at 4 °C. After homogenization at 4 °C
(Ultra-Turrax, maximal speed, 15 s) and ultracentrifugation (23,000g,
60 min, 4 °C), the pellet was resuspended in 10 volumes of HEPES
buffer 50 mM pH 7.4 at 4 °C in order to obtain a tissue concentration
of about 100 mg protein/mL. The protein concentration was
determined by the method of Lowry77 using bovine serum albumin
as standard.
For radioligand binding studies, 600 μg of membrane was incubated
in duplicate at 37 °C for 30 min with 22 (PerkinElmer), fixed
concentration of compound, and HEPES buffer 50 mM pH 7.4 at 37
°C. Incubation was terminated by rapid vacuum filtration through
0.5% polyethylenimine-presoaked Whatman GF/B filters (Alpha
Biotech) using a Brandel Cell Harvester. Filters were subsequently
washed three times with 4 mL of HEPES buffer 50 mM pH 7.4 at 4
°C.
[1-(Cyclohexylmethyl)-4-piperidyl]methyl 4-amino-5-chloro-2-
methoxybenzoate (21). To a suspension of 4-amino-5-chloro-2-
methoxybenzoic acid (101 mg, 0.5 mmol) in dry THF (3 mL) was
added CDI (89 mg, 0.55 mmol). The mixture was stirred at rt for 24 h,
and then a solution of (1-(cyclohexylmethyl)piperidin-4-yl)methanol
(106 mg, 0.50 mmol) in dry THF (2 mL) and NaH 60% (21 mg, 0.55
mmol) were added. This new mixture was stirred 3 days at rt and then
The method was validated from saturation studies: 6 concentrations
of 22 were used to give final concentrations of 0.02−0.8 nM;
nonspecific binding of 22 was defined in the presence of 30 μM
serotonin to determine the Kd and the Bmax.
K
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Article
For competition studies, 22 was used to give a final concentration of
0.1 nM. Percentages of inhibition of the binding of 22 were obtained
for concentrations of 10−6 and 10−8 M of the ligands tested. For
some of these compounds, affinity constants were calculated from 5-
point inhibition curves using the EBDA-Ligand software and expressed
as Ki SD.
First screening of AChE and BuChE activity was carried out at a
10−6 or 10−5 M concentration of compounds under study. For the
compounds with significant inhibition (≥50%), IC50 values were
determined graphically by plotting the % inhibition versus the
logarithm of six inhibitor concentrations in the assay solution using
the Origin software.
Propidium Competition Assay. Propidium exhibits an increase in
fluorescence on binding to AChE PAS, making it a useful probe for
competitive ligand binding to the enzyme.79 Fluorescence was
measured in a Tecan Infinite M200 plate reader. Measurements
were carried out in 200 μL final in 96-well plates. Five U (ee)AChE in
1 mM Tris/HCl, pH 8.0 buffer was incubated for 6 h at 25 °C, with a
150 μL 10−5 M solution of tested compounds. One micromolar
propidium iodide 50 μL solution was added 10 min before
fluorescence measurement. The excitation wavelength was 535 nm,
and that of emission, 595 nm. Each assay was repeated two different
times.
Docking Studies. For each docked compound a preliminary
calculation on its protonation state at pH 7.4 was carried out using
standard tools of the ChemAxon Package (http://www.chemaxon.
com/), and the majority microspecies protonated on one or two
nitrogen atoms at this pH was used for docking studies.
The homology model of the (h)5-HT4Rwas built using the high-
resolution (2.4 Å) crystal structure of the (h)β2 adrenergic receptor-
T4 lysozyme fusion protein bound to carazolol (PDB: 2RH1) under
the procedure described.63
Pharmacological Characterization of Drugs on Human 5-HT4R.
The method was validated from saturation studies: six concentrations
of 22 were used to give final concentrations of 0.0625−2 nM, and
nonspecific binding of 22 was defined in the presence of 30 μM
serotonin to determine the Kd and the Bmax. For competition studies,
2.5 μg of proteins (5-HT4B membrane preparations, HTS110M,
Millipore. Millipore’s 5-HT4B membrane preparations are crude
membrane preparations made from their proprietary stable recombi-
nant cell lines to ensure high level of GPCR surface expression) were
incubated in duplicate at 25 °C for 60 min in the absence or the
presence of 10−6 or 10−8 M of each drug and 0.2 nM 22 (VT 240,
ViTrax) in 25 mM Tris buffer (pH 7.4, 25 °C). At the end of the
incubation, homogenates were filtered through Whatman GF/C filters
(Alpha Biotech) presoaked with 0.5% polyethylenimine using a
Brandel cell harvester. Filters were subsequently washed three times
with 4 mL of ice-cold 25 mM Tris buffer (pH 7.4, 4 °C). Nonspecific
binding was evaluated in parallel in the presence of 30 μM serotonin.
For some of these compounds, affinity constants were calculated
from 5-point inhibition curves using the EBDA-Ligand software and
expressed as Ki SD.
Determination of cAMP Production. COS-7 cells were grown in
Dulbecco’s modified Eagle medium (DMEM) supplemented with 10%
dialyzed fetal calf serum (dFCS) and antibiotics. Cells were transiently
transfected with plasmid encoding HA-tagged 5-HT4R, then seeded in
24-well plates (70,000 cells/well). Twenty-four hrs after transfection,
cells were exposed to the indicated concentrations of 5-HT4R ligands
in the presence of 0.1 mM L-ascorbic acid and 0.1 mM of the
phosphodiesterase inhibitor RO-20−1724, at 37 °C in 250 μL of HBS
(20 mM HEPES; 150 mM NaCl; 4.2 mM KCl; 0.9 mM CaCl2; 0.5
mM MgCl2; 0.1% glucose; 0.1% BSA). After 10 min, cells were then
lysed by addition of the same volume of Triton-X100 (0.1%).
Quantification of cAMP production was performed by HTRF by using
the cAMP Dynamic kit (Cisbio Bioassays) according to the
manufacturer’s instructions.
In Vitro Tests of AChE and BuChE Biological Activity. Inhibitory
capacity of compounds on AChE biological activity was evaluated
through the use of the spectrometric method of Ellman.78 Acetyl- or
butyrylthiocholine iodide and 5,5-dithiobis(2-nitrobenzoic) acid
(DTNB) were purchased from Sigma-Aldrich. Lyophilized BuChE
from equine serum (Sigma-Aldrich) was dissolved in 0.2 M phosphate
buffer pH 7.4 such as to have enzyme solutions stock with 2.5 units/
mL enzyme activity. AChE from human erythrocytes (buffered
aqueous solution, ≥500 units/mg protein (BCA), Sigma-Aldrich)
was diluted in 20 mM HEPES buffer pH 8, 0.1% Triton X-100 such as
to have enzyme solution with 0.25 unit/mL enzyme activity. In the
procedure, 100 μL of 0.3 mM DTNB dissolved in phosphate buffer
pH 7.4 were added into the 96-well plate followed by 50 μL of test
compound solution and 50 μL of enzyme (0.05 U final). After 5 min
of preincubation at 25 °C, the reaction was then initiated by the
injection of 50 μL of 10 mM acetyl- or butyrylthiocholine iodide
solution. The hydrolysis of acetyl- or butyrylthiocholine was
monitored by the formation of yellow 5-thio-2-nitrobenzoate anion
as the result of the reaction of DTNB with thiocholine, released by the
enzymatic hydrolysis of acetyl- or butyrylthiocholine, at a wavelength
of 412 nm using a 96-well microplate plate reader (TECAN Infinite
M200, Lyon, France). Test compounds were dissolved in analytical
grade DMSO. Donepezil was used as a reference standard. The rate of
absorbance increase at 412 nm was followed every minute for 10 min.
Assays were performed with a blank containing all components except
acetyl- or butyrylthiocholine, in order to account for nonenzymatic
reaction. The reaction slopes were compared and the percent
inhibition due to the presence of test compounds was calculated by
the following expression: 100 − (vi/v0 × 100), where vi is the rate
calculated in the presence of inhibitor and v0 is the enzyme activity.
The crystallographic coordinates of (h)AChE used for docking
studies were obtained from X-ray structure of the DPZ/AChE
complex (PDB ID 4EY7, a structure refined to 2.35 Å with an R factor
of 17.7%).80
The docking of donecopride into the (h)5-HT4R was carried out
with the GOLD program (v5.0) using the default parameters.81,82 This
program applies a genetic algorithm to explore conformational spaces
and ligand binding modes. To evaluate the proposed ligand positions,
the ChemPLP fitness function was applied in these docking studies.
The binding site in the 5-HT4R model was defined as a 10 Å sphere
centered on the aspartic acid residue Asp100. Because the mutagenesis
studies have shown that the interaction between the positively
ionizable amine of ligands and Asp100 of 5-HT4R is crucial for ligand
binding, a hydrogen-bond constraint between positively ionizable
amine ligand and OD atom of Asp100 was used during the docking.
Furthermore, special attention was paid during the docking procedure
to the following amino acids in the binding site, which were kept
flexible: Arg96, Asp100, Thr104, Tyr192, Ser197, and Trp294
.
Docking of donecopride into (h)AChE was carried out also by
means of the GOLD program with the default parameters. To evaluate
the proposed ligand poses, the ChemPLP fitness function was applied.
The binding site in the AChE model was defined as a 7 Å sphere from
the cocrystallized ligand, DPZ and a water molecule interacting with
protonated piperidine ring of DPZ was conserved during the docking
(residue number 931).
In Vivo Biological Studies. Animals. Adult male NMRI mice (3
months old, weighing 35−40g) from Janvier laboratories (Le Genest-
Saint-Isle, France) were used to perform experiments. Mice were
housed by 10 in standard polycarbonate cages in standard controlled
conditions (22 2 °C, 55 10% humidity) with a reversed 12h light/
dark cycle (light on at 7 pm). Food and water were available ad libitum
in the home cage. All experiments were conducted (between 9 am and
3 pm) during the active−dark phase of the cycle and were in
agreement with the European Directives and French law on animal
experimentation (personal authorization no. 14−17 for M.B. and 14−
60 for T.F.).
CNS Activity and Acute Toxicity Test. Behavioral and neurological
changes induced by graded doses (1, 10, 100 mg/kg) of the tested
derivatives were evaluated in mice, in groups of four, by a standardized
observation technique at different times (30 min and 3 and 24 h) after
IP administration.83 Major changes of behavioral data (for example,
hypo- or hyperactivity, ataxia, tremors, convulsion, etc.) were noted in
comparison to the control group. The approximate DL50 of the
compounds were also calculated through the quantification of
L
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Journal of Medicinal Chemistry
Article
mortality after 24 h. Amphetamine (2 mg/kg) and chlorpromazine (10
mg/kg) were used as stimulant and depressive references, respectively.
Locomotor Activity. Locomotion of mice was measured using an
actimeter (Imetronic) through infrared detection. Eight individual
removable polycarbonate cages (21 cm length, 7 cm wide, and 12 cm
high), where each mouse was placed, are disposed in the actimeter.
Locomotor activity was measured by recording the number of
interruption of beams of the red light over a period of 30 min
through an attached recording system to the actimeter. Donecopride
was tested at 1, 3, and 10 mg/kg. Amphetamine (2 mg/kg),
chlorpromazine (10 mg/kg), and association 2 (0.1 mg/kg)-DPZ
(0.3 mg/kg) were used as stimulant, depressive, and pharmacological
references, respectively.84
Spatial Working Memory. Antiamnesiant activity of tested
compounds was evaluated by reversal of scopolamine (0.5 mg/kg)-
induced deficit on spontaneous alternation behavior in the Y maze
test.85 The Y maze made of gray plastic consisted of three equally
spaced arms (21 cm long, 7 cm wide with walls 15 cm high). The
mouse was placed at the end of one of the arms and allowed to move
freely through the maze during a 5 min session, while the sequence of
arm entries was recorded by an observer. An arm entry was scored
when all four feet crossed into the arm. An alternation was defined as
entries into all three arms on a consecutive occasion. The number of
possible alternation is thus the total number of arm entries minus two;
the percentage of alternation was calculated as (actual alternation/
possible alternation) 100. Donecopride was tested at 0.1, 0.3, 3, and 10
mg/kg. Different additional groups (DPZ (0.3 mg/kg) and association
2 (0.1 mg/kg) + DPZ (0.3 mg/kg)) were tested as clinical and
pharmacological references, respectively.
described in literature89 and analyzed without further freezing−
thawing cycles. ELISA kits from IBL International (Hamburg,
Germany) for the dosage of sAPPα (mouse/rat sAPPα assay kit, no.
27415) were used according to the manufacturer’s instructions.
Reactions were read at 620 and 450 nm using an Infinite 2000
luminescence counter. The sAPPα ELISA kits enable the precise and
selective quantification of sAPPα versus sAPPβ.
ASSOCIATED CONTENT
* Supporting Information
■
S
Primary binding test for Donecopride toward a broad range of
receptors. ORTEP diagram of 17. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: christophe.rochais@unicaen.fr.
*E-mail: patrick.dallemagne@unicaen.fr. Tel.: +33-2-31-56-68-
13. Fax +33-2-31-56-68-03.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
Forced Swimming Test. The FST86 was carried out in mice
individually forced to swim during 6 min in an open cylindrical
container (diameter 12 cm, height 20 cm) with a water depth of 13 cm
at 23−24 °C.87 The duration of immobility, after a delay of 2 min, was
measured during the last 4 min. Each mouse was judged to be
immobile when it ceased struggling and remained floating motionless
in the water, making only those movements necessary to keep its head
above water. Animals were not pretested. Donecopride was tested at
0.3 and 3 mg/kg. Imipramine (16 mg/kg) was used as clinical
reference.
Pharmacological Treatments. 2 and DPZ hydrochloride were
purchased from Tocris (Cookson, UK). Amphetamine (+)-α-
methylphenethylamine hemisulfate, chlorpromazine hydrochloride,
imipramine hydrochloride, and scopolamine hydrobromide were
purchased from Sigma (France). All those pharmacological com-
pounds were dissolved in NaCl 0.9% as the vehicle and were
administered IP 30 min before tests, except DPZ and scopolamine
which were SC administered 20 and 30 min, respectively, before tests.
Statistical Analysis. Results were expressed as mean SD and were
analyzed by one way analysis of variance (ANOVA), with Statview
software. In case of significance, a SNK (Student−Newman−Keuls)
post hoc test was realized. Additionally, for the spontaneous
alternation test, the percentage of alternation was compared to a
theoretical 50% value (random alternation) by an univariate t test.
Differences were considered as statistically significant if the p value was
strictly under 0.05.
Cerebrospinal Fluid (CSF) Collection and sAPPα Quantification.
To explore the effect of acute injection of donecopride on sAPPα
release in vivo, two different groups of WT C57BL/6 mice (n = 7/
group) received one IP injection of vehicle (0.9% w/v NaCl; 0.2%
dimethyl sulfoxide in water) or donecopride (1 mg/kg). 90 min
postinjection, mice were anesthetized and mounted onto a stereotaxic
instrument. The neck skin was cut, and subcutaneous tissue and
muscles separated with the help of microretractors (Fine Science
Tools, Heidelberg, Germany). Mice were then laid down so that the
head formed an angle of about 135° with the body.88 A capillary tube
(Borosilicate glass, B100-75-10, Sutter Instruments, Novato, Cal-
ifornia, U.S.A.) was used to punch the dura mater of the cisterna
magna. CSF was collected by capillary action and transferred to 0.5 mL
microtubes, immediately frozen in dry ice and stored at −80 °C until
use. Once thawed, samples were heated at 60 °C for 5 min as
ACKNOWLEDGMENTS
■
This work was supported by funding from the Conseil Reg
́
ional
de Basse Normandie, France (Dispositif de Soutien aux Projets
de Recherche Emergents), French Agence Nationale de la
Recherche Projects MALAD ANR-12-JS007-0012-01 and
́
ADAMGUARD ANR-12-BSV4-008-01, and Ligue Europeenne
Contre la Maladie d’Alzheimer Grant 12721. The authors
gratefully acknowledge the CRIHAN (Centre de Ressources
Informatiques de Haute Normandie) as well as the European
Community (FEDER) for the molecular modelling software.
English language editing of this manuscript was provided by
Journal Prep.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; ACh, acetylcholine; AChE, acetylcho-
linesterase; AChEI, acetylcholinesterase inhibitor; CAS, cata-
lytic anionic site; DBS, dual-binding site; DPZ, donepezil;
MTDL, multitarget-directed ligands; PAS, peripheral anionic
site; P-gp, P-glycoprotein
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