Novel multitarget-directed ligands targeting acetylcholinesterase and σ1 receptors as lead compounds for treatment of Alzheimer's disease: Synthesis, evaluation, and structural characterization of their complexes with acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2018.10.064

Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT₄ receptor, which displays promising activities in vivo. Pharmacomodulation of donecopride allowed us to develop a novel series of indole derivatives possessing interesting in vitro activities toward AChE and the σ₁ receptor. The crystal structures of complexes of the most promising compounds with Torpedo californica AChE were solved in order to further understand their mode of inhibition.

Full text of DOI:10.1016/j.ejmech.2018.10.064

European Journal of Medicinal Chemistry 162 (2019) 234e248
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel multitarget-directed ligands targeting acetylcholinesterase and
s1 receptors as lead compounds for treatment of Alzheimer's disease:
Synthesis, evaluation, and structural characterization of their
complexes with acetylcholinesterase
,
,
c
,
d
,
1
a
a
Julien Lalut a 1, Gianluca Santoni b
, Delphine Karila , Cedric Lecoutey ,
ꢀ
Audrey Davis a, Florian Nachon e, Israel Silman f, Joel Sussman g, Martin Weik b
,
, c, d
Tangui Maurice h, Patrick Dallemagne a **, Christophe Rochais a
,
, *
a
ꢀ
Centre d’Etudes et de Recherche sur le Medicament de Normandie, Normandie Univ, UNICAEN, CERMN, 14000, Caen, France
b Univ Grenoble Alpes, IBS, F-38027, Grenoble, France
c CNRS, IBS, F-38027, Grenoble, France
d CEA, IBS, F-38027, Grenoble, France
e
ꢀ
ꢀ
ꢀ
ꢀ
Departement de Toxicologie et Risques Chimiques, Institut de Recherche Biomedicale des Armees, 91220, Bretigny-sur-Orge, France
f Department of Neurobiology, Weizmann Institute of Science, 76100, Rehovot, Israel
g Department of Structural Biology, Weizmann Institute of Science, 76100, Rehovot, Israel
h Molecular Mechanisms in Neurodegenerative Diseases, MMDN Laboratory, University of Montpellier, Ecole Pratique des Hautes Etudes, Institut National
ꢀ
ꢀ
de la Recherche et de la Sante Medicale, UMR-S1198, 34095, Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial
diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical
entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target
Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously
inhibit acetylcholinesterase and act as an agonist of the 5-HT4 receptor, which displays promising ac-
tivities in vivo. Pharmacomodulation of donecopride allowed us to develop a novel series of indole de-
rivatives possessing interesting in vitro activities toward AChE and the s1 receptor. The crystal structures
of complexes of the most promising compounds with Torpedo californica AChE were solved in order to
further understand their mode of inhibition.
Received 2 October 2018
Received in revised form
24 October 2018
Accepted 29 October 2018
Available online 8 November 2018
Keywords:
Alzheimer's disease
Acetylcholinesterase
Sigma 1 receptors
MTDL
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
the cholinergic hypothesis this decline is linked to a decrease in the
levels of acetylcholine released at cholinergic synapses in certain
Along with the observed increase of life expectancy, senile de-
mentia is becoming increasingly prevalent. As many of 46 million
cases of dementia were reported worldwide in 2015 [1]. The most
common dementia, Alzheimer's disease (AD), is characterized by a
progressive and irreversible decline in memory and cognitive
function as a consequence of neuronal dysfunction. According to
areas of the brain. Indeed, until now, with one exception, the drugs
authorized by the FDA for treatment of AD are acetylcholinesterase
(AChE) inhibitors that provide symptomatic relief in the early
stages of the disease [2]. However, with progression of AD, these
drugs, including 1 [donepezil (DPZ)], rivastigmine and galanth-
amine, lose their efficacy. One of the greatest challenges facing
pharmacologists and medicinal chemists is the discovery and
development of treatments that could exert a disease-modifying
effect on AD [3]. To this end many studies have been directed to-
wards clarifying the molecular origins of the disease. AD is princi-
pally characterized by the formation in the brain of amyloid plaques
* Corresponding author.
** Corresponding author.
E-mail addresses: patrick.dallemagne@unicaen.fr (P. Dallemagne), christophe.
rochais@unicaen.fr (C. Rochais).
whose main component is the Ab peptide, and of neurofibrillary
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2018.10.064
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
235
tangles (NFTs), which are aggregates of hyperphosphorylated tau
protein [4]. Since the multifactorial origin of AD has been clearly
established, most efforts have been directed towards a search for
potent and agents that could interfere with the formation of the
amyloid plaques and the NFTs, or limit the inflammation of the
brain associated with the disease [5]. However, to date, these efforts
have failed to identify novel therapeutics. These failures have been
ascribed to difficulties in patient stratification or selection, as well
as to lack of appropriate transgenic mice models [6]. It has also been
suggested that the clinical ineffectiveness of the drug candidates is
due to their high selectivity for a single target, implying that
pleiotropic intervention will be needed due to the multifactorial
origin of AD [7].
Pleiotropic intervention could of course be realized through use
of a cocktail of drugs, as is currently the case in some ongoing
clinical trials, in which AChE inhibitors (AChEIs), including DPZ and
rivastigmine, are associated with other candidates [8,9]. Another
strategy involves development of single compounds capable of
simultaneously acting on several targets, which are known as
Multi-Target Directed Ligands (MTDLs) [10e12]. Several activities
have been associated in single compounds in order to act on several
pathogenic pathways mainly those targeting the formation of the
the Y-maze spontaneous alternation test [34]. 3 appears to be se-
lective for its targets, though it also displays lower affinity for 5-
HT2BR and for
s receptors; among the latter s1R appears to be an
interesting off-target due to its implication in the pathogenesis of
AD [35]. Several s1R ligands have recently been disclosed that show
promise for the treatment of AD, whether alone [36] or combined
with additional drugs [37]. 1 itself has nanomolar affinity for s1
R
[38] and was proved to possess anti-amnesic and neuroprotective
effects involving interaction with s1R [39], which could explain the
affinity of its structural analog 3 for this target. Combining AChEI
and s1R affinities shows promise for AD treatment, so we decided
to continue our modulation of 3 in order to improve its affinity for
this novel target. To this end, we first docked 3 into the active-site
gorge of hAChE [34]. In this study the pose of 3 was seen to be
similar to that of 1, involving interaction of the charged nitrogen of
the piperidine ring with Tyr337, an H-bond between the carbonyl of
3 and the main-chain NH of Phe295, and, more interestingly, a
p-
stacking interaction between the aromatic ring of 3 positioned and
the indole ring of Trp286 in the PAS (Fig. 2).
We postulated that replacement of the benzene ring of 3 by an
indole residue (Fig. 1) should increase the interaction of the ligand
with the PAS, thus resulting in increased inhibition of
gation. In addition, the influence of this substitution on other tar-
gets, such as 5-HT4R and -1R, as well as upon bioavailability, could
bA aggre-
A
b
plaques, the NFTs or the neuroinflammation associated with AD.
Recent examples include compounds able to simultaneously act on
MAO-B inhibition and oxidative stress [13], GSK-3 and -secretase
inhibition [14], or to inhibit A aggregation, act as metal chelators
s
b
b
thus be assessed. Moreover, it has been demonstrated that
haloperidol-inspired molecules with an indole ring and a piperi-
dine chain could act as potent s1R ligands with neuroprotective
effects [40].
b
and serve as antioxidant [15]. Due to the diversity of chemical
scaffolds among the AChEIs, and to their clinical efficacy, one of the
most commonly adapted strategies has been to graft on to an AChEI
a moiety displaying another pharmacological activity useful for
treating AD. Several examples have been recently published asso-
ciating cholinesterase inhibition properties with MAO-B inhibition
[16], antioxidant properties [17] or combining several activities
[18]. Based on the amyloid hypothesis, several leads with promising
in vivo activities have been developed that both inhibit AChE and
limit the formation of senile plaques [19,20]. These studies took
advantage of the demonstration by Inestrosa and coworkers that
ligands binding at the peripheral anionic site (PAS) [21] of AChE can
2. Results
2.1. Chemistry
A first assessment of our strategy was obtained by synthesis of
the indole analog of 3 bearing the same methoxy and chlorine
substituents. The supplementary pyrrole cycle was obtained using a
Larock heteroannulation synthesis starting from ortho-
halogenated aniline (Scheme 1). An iodine atom was introduced
at position 3 of the aromatic using iodine in AcOH at room tem-
perature to afford 4a in quantitative yield. 4a was engaged in a
Sonogashira cross-coupling with ethynyltrimethylsilane and a
mixture of Pd(PPh3)4, XPhos and K2CO3 in refluxing THF [41]. The
cleavage of the trimethylsilyl group of the resulting intermediate 5a
under acidic conditions yielded the target compound 6a in two
steps with a 71% yield.
On the basis of the promising in vitro activities displayed by 6a
we decided to develop a more convergent route to obtain structural
analogs. Indolic derivatives 6a-g were synthesized by the synthetic
route shown in Scheme 2, from acids 7a-c (see Supporting Infor-
mation). A convergent synthesis was designed with late stage
construction of the indole ring, except for 6h.
retard aggregation of Ab [22]. Many studies have then been devoted
to the development of Dual Binding Site (DBS) AChEIs, which can
bind simultaneously to the anionic subsite of the active site and to
the PAS [23e25]. In addition to the classical targets considered for
treatment of AD, the serotonergic system has also shown promise
in this context, in particular modulation of 5-HT4 and 5-HT6 re-
ceptors [26,27]. 5-HT4 receptors (5-HT4R) control brain functions
such as learning and memory, feeding and mood behavior. In the
context of AD, activation of 5-HT4R can promote the non-
amyloidogenic cleavage of Amyloid Protein Precursor (APP) lead-
ing to the formation of a neurotrophic protein, sAPP
a [28,29].
Indeed, administration of a specific 5-HT4R agonist, RS67333, in a
transgenic mouse model of AD retarded amyloidogenesis and
reduced behavioral deficits [30].
Compounds 9a-c were synthesized in a three-step sequence
In this context we have initiated a drug discovery program to
assess the pharmacological benefits of generating an MTDL by
combining RS67333 (2) and DPZ (1), and testing the activity of this
MTDL in an object recognition test that had been used earlier to
assess the synergy of RS67333 and DPZ administered together [31].
Several chemical series [32] were developed with the objectives of
combining the two functions in a single compound which resulted
in the recent identification of a promising MTDL candidate for AD
treatment: 3 (donecopride), a 5-HT4R agonist (Ki for h5-HT4R
10.4 nM; IC50 for hAChE 16 nM) (Fig. 1) [33]. Thanks to its double
mechanism of action and to its good bioavailability 3 was able to
improve the memory performance of mice in an object recognition
test, and to reverse the memory deficit induced by scopolamine in
through formation of b-keto ester derivatives by reaction between
acids 7a-c, activated in situ with CDI, and potassium 3-ethoxy-3-
oxopropanoate [42,43]. Subsequent alkylation with a previously
synthesized piperidine chain, and followed by reaction with KOH in
an EtOH/H2O mixture provided a saponification-decarboxylation
reaction to afford the corresponding derivatives 9a-c [44]. Then
compounds 4a-g were obtained by deprotection of the Boc group
before alkylating the nitrogen of piperidine by various alkyl chains.
Finally, the indolic ring system was constructed via a Larock
palladium-catalyzed annulation using ethynyltrimethylsilane, a
terminal alkyne, and a mixture of Pd(PPh3)4, XPhos and K2CO3 in
refluxing THF [41]. Then a Sonogashira cross coupling-cyclization
reaction, and cleavage of the trimethylsilyl group under acidic

236
J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
Fig. 1. Chemical structure of Donepezil 1, RS67,333 2 and Donecopride 3.
the insertion of palladium in the carbon-halogen bond. We decided
to synthesize the indole ring directly from ester 11 (see the Sup-
porting Information) under the same conditions as previously
described to obtain compounds 6a-g from 4a-g. Compound 13 was
obtained by dehalogenation of chlorinated compound 12 in the
presence of ammonium formate and Pd/C in refluxing MeOH, and
was then reacted with NaOH in EtOH to give acid 14. Finally, the
target compound 6h was obtained in a five-step sequence by the
same method used to prepare 4a-g from 7a-c.
2.2. In vitro evaluation
All the synthesized compounds were evaluated for their ca-
pacity to inhibit hAChE and to bind to guinea pig (gp)5-HT4R
(Table 1). The most promising one were further tested for their
capacity to bind to s1R. In these tests, DPZ was used as both an
AChEI and a control ligand for s1R, and 3 was used both as a ligand
for 5-HT4R and as an AChEI control [33].
Finally, kinetic measurements were performed at three con-
centrations of 6a in order to determine its mode of inhibition of
hAChE (Fig. 3). The kinetic data clearly show that 6a acts as a non-
competitive inhibitor of the enzyme.
Fig. 2. Docking of 3, donecopride, within the active-site gorge of hAChE [34].
2.3. In vivo evaluation
conditions yielded the target compounds 6a-g. In order to study
their influence on the biological results, different groups with
various electronic effects have been introduced on the nitrogen of
the indole moiety. Consequently, compounds 10a-d were obtained
by alkylation of the nitrogen with a methyl and a benzyl (alkyl- and
aromatic electron donating groups) or a benzenesulfonyl (electron
withdrawing group), and compound 10c, obtained as an oil, was
reacted with fumaric acid in isopropanol at 50 ꢀC to give the cor-
responding fumarate salt for biological evaluation.
To synthesize compound 6h, dehalogenation of chlorinated 6a
was attempted with ammonium formate and palladium, but the
reaction did not lead to the expected product, probably due to the
presence of the basic nitrogen of piperidine which does not permit
s1R Agonists are potent anti-amnesic drugs. In particular, they
significantly prevent the amnesia induced in rodents by blockade of
the NMDA receptor [45,46]. Compound 6c was therefore chosen on
the basis of its in vitro activities towards the three targets for a
preliminary in vivo confirmation of its s1R activity. Animals were
tested for spontaneous alternation performance in the Y-maze, an
index of spatial working memory, and for passive avoidance
response, an index of long-term non-spatial memory [37,45,46]. As
shown in Fig. 4, compound 6c failed to attenuate dizocilpine-
induced spontaneous alternation deficit in the dose range tested,
0.1e1 mg/kg IP (Fig. 4a), but the lowest dose very significantly
attenuated dizocilpine-induced alteration in passive avoidance
response (Fig. 4b).
Scheme 1. Synthesis of indole analog 6a
aReagents, conditions and yields: (a) I2, AcOH, rt, 3 h, quant.; (b) ethynyltrimethylsilane, Pd(PPh3)4, XPhos, K2CO3, dry THF, reflux, 18 h, 71%; (c) TFA, DCM, rt, 2 h, quant.

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
237
Scheme 2. Synthetic route for target compounds 6a-h and 10a-ca
aReagents, conditions and yields: (a) CDI, dry THF, rt, 5e7 h, then potassium 3-ethoxy-3-oxopropanoate, MgCl2, 40 ꢀC, 14e48 h, 57e70%; (b) tert-butyl 4-(iodomethyl)piperidine-1-
carboxylate, K2CO3, DMF, rt, 24e48 h; (c) KOH, EtOH/H2O (5:1), reflux, 3e5 h, 37e76% over two steps; (d) TFA, DCM, rt, 30min; (e) alkyl-halogenated derivatives, K2CO3, DMF, 110 ꢀC,
5e15 h, 52e75% over two steps; (f) ethynyltrimethylsilane, Pd(PPh3)4, XPhos, K2CO3, dry THF, reflux, 17e24 h; (g) TFA, DCM, rt, 1e2 h, 26e73%; (h) CH3I, K2CO3, DMF, rt, overnight,
77%; (i) BnBr, K2CO3, DMF, rt, overnight, 83%; (j) PhSO2Cl, NaOH aq., (nBu)4NHSO4, rt, 2 h, 84%; (k) ammonium formate, Pd/C, MeOH, reflux, overnight, 82%; (l) 1N NaOH aq., EtOH, rt,
overnight, 83%.
3. Discussion
to what was seen for the donecopride series, a N-benzyl substituent
on the piperidine ring greatly enhanced inhibition of AChE relative
to a cycloalkyl or an alkyl substituent. Once again, the chloro and
methoxy substituents on the indole ring have little influence on
affinity, whereas an N-substituent on the aromatic ring dramati-
cally decreases it.
As suggested by the kinetic study performed on 6a, these
compounds could behave as non-competitive AChEIs, suggesting
that they could interact with the PAS, as well as with the anionic
subsite of the active site (Fig. 3). This potential dual binding site
inhibition of AChE was confirmed by solution of the crystal struc-
tures of complexes obtained by soaking into TcAChE crystals (Fig. 5a
and b). The structures were solved at resolutions of 2.6 Å and 2.0 Å,
respectively, for the 6a and 6b complexes (see Supplementary
Table S1), and show how both compounds bind in a very similar
way, as was to be expected from their chemical structures.
Both 6a and 6b span the whole length of the active-site gorge.
The indole moiety interacts with Trp279 in the PAS. Neither the
methoxy nor the chlorine substituents on the indole group seem to
Relative to donecopride (Ki ¼ 9.5 nM), its indole analogs showed
overall a decrease in affinity for 5-HT4R affinity, 6f being the most
potent (Ki ¼ 25 nM). As already observed for the donecopride series
[33], a cycloalkyl or an alkyl substituent on the piperidine ring
appears favor affinity for 5-HT4R activity relative to an N-benzyl
ring. The chloro and methoxy substituents on the indole ring do not
influence the activity. However, the introduction of the novel aro-
matic ring on the scaffold has a strong influence on the profile of
the ligand. Indeed tested in a cellular functional assay using cAMP
quantification, compound 6a appears to be a potent antagonist for
the 5-HT4R (see Supporting Information) whereas donecopride is a
partial agonist for this receptor.
On the contrary, in most cases, the inhibitory capacity towards
AChE was maintained and compounds 6b-e and 6h are strong
AChEI (IC50 ¼ 13e51 nM) similar in affinity to donecopride
(IC50 ¼ 16 nM) and, for 6d particularly, as DPZ (IC50 ¼ 6 nM).
Opposite to what was seen for affinity for the 5-HT4R, and similarly

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J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
Table 1
Inhibition of hAChE, and binding to gp5-HT4R and s1R of indole derivatives.
Compound
Structure
hAChE
%inh 10ꢁ6 M
e
(gp)5-HT4R
s1R
IC50 (nM)
%inh 10ꢁ6
M
Ki (nM)
Ki (nM)
Haloperidol
e
e
e
1.6 0.7 n ¼ 2
RS67,333
Donepezil
e
e
100%
10%
9.33 5 n ¼ 3
e
e
98%
6.0 0.6, n ¼ 5
e
Donecopride
96%
95%
78%
16 5, n ¼ 2*
100%
100%
62%
9.5 5 n ¼ 2*
40.9 4 n ¼ 3
e
51 6.6, n ¼ 2
5.1 1.7, n ¼ 2
e
6a
5b
91.4 12.7, n ¼ 2
275 12, n ¼ 2
6b
6c
97%
95%
98%
93%
92%
55%
96%
97%
87%
20.4 0.8, n ¼ 2
28.8 2.4, n ¼ 2
13.3 0.4, n ¼ 2
51 1.9, n ¼ 2
94 2.6, n ¼ 2
716 28, n ¼ 2
40 1, n ¼ 2
96%
100%
90%
100%
100%
97%
100%
e
150 45 n ¼ 3
37.5 15 n ¼ 3
169 23 n ¼ 3
37.5 15 n ¼ 3
25 1.6 n ¼ 3
49.6 23 n ¼ 3
51 15 n ¼ 3
e
5.7 1.4, n ¼ 2
5.1 1.8, n ¼ 2
3.3 0.7, n ¼ 2
6d
6e
6f
e
e
e
e
e
6g
6h
10a
10b
84.4 13.4, n ¼ 2
260.6 31.5, n ¼ 2
e
e
10c
10d
74%
93%
559 8.4, n ¼ 2
133 22, n ¼ 2
62%
42%
e
e
e
e
The inhibitory effect against hAChE was established according to the Ellman's method. *IC50 obtained from Rochais et al. [34].
-: not tested.

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
239
evaluated in vivo in the dizocilpine-induced amnesia assay in mice
[45,46]. Dizocilpine-treated mice showed highly significant mem-
ory impairments in both spontaneous alternation performance and
passive avoidance tests. Compound 6c was initially tested in the
0.1e1 mg/kg doseerange and did not attenuated the dizocilpine-
induced spontaneous alternation performance deficit. However,
6c at the lower dose tested dose significantly enhanced attenuated
the passive avoidance deficit (40% protection). The compound
therefore presented an in vivo efficacy coherent with its pharma-
cological profile and further studies must address lower doses and
analyses in presence of NE-100.
Fig. 3. Lineweaver Burk plots for inhibition of hAChE by 6a (Donepezil has been used
as control).
4. Conclusion
Starting from donecopride, a dual AChEI/5-HT4R agonist, phar-
macomodulations led to a series of thirteen novel indole de-
rivatives. Some of them displayed potent nanomolar inhibition of
contribute to the binding of the ligands to the enzyme. Conversely,
the cyclic moiety at the other extremity of both molecules, either a
benzyl or a methylcyclohexyl group, is well suited to stack against
Trp84 in the active-site of the enzyme. The benzyl of 6b, in partic-
hAChE, and affinity for the
s-1R affinity associated with a lower
affinity for the 5-HT4R. This pharmacological profile suggested a
potential therapeutic approach to the treatment of AD. This, in turn,
has been supported by preliminary in vivo experiments in which 6c
displayed a protecting effect against dizocilpine-induced impair-
ment in the passive avoidance test in mice that correlated with its
ular, allows formation of a
gorge, the piperidine moiety is stabilized by a cation-
p
-
p
interaction. Midway down the
interaction
p
between the nitrogen atom and residue Phe330, already described
in the literature for other AChEIs [47].
Comparison of the complexes of 6a and 6b with that of the
TcAChE/DPZ complex (pdb acces code 1eve), reveals that 6a and 6b
bind higher up the gorge, as can be seen from their mode of
interaction at the PAS. Other than that, the three compounds
occupy the gorge in a very similar fashion.
in vitro potent s-1R affinity. These results have to be strengthened
by additional in vivo experiments in order to precise the pharma-
cological potential of 6c.
5. Experimental section
Concerning the
s-1R, the affinity of some members of the 6
series was dramatically enhanced; thus, compounds 6a-d are very
potent ligands with nanomolar affinities (Ki ¼ 3.3e5.7 nM)
compared to donecopride (Ki ¼ 51 nM), to DPZ (IC50 ¼ 14.6 nM).
5.1. Chemistry
All commercially available compounds were used without
further purification. Melting points were determined on a Kofler
apparatus. Analytical thin-layer chromatography (TLC) was per-
formed on silica gel 60 F254 on aluminium plates (Merck) and
visualized with UV light (254 nm). Flash chromatography was
conducted on a VWR SPOT II Essential instrument with silica gel 60
Presence of the indole ring appears crucial for interaction with s-1R
€
activity, whatever the substituents on the aromatic ring or the
piperidine moiety.
Based on its global pharmacological profile, compound 6c (IC50
¼ 28.8 nM, Ki[5-HT4R] ¼ 37.5 nM, Ki[s1R] ¼ 5.1 nM) was
[AChE]
Fig. 4. Effect of 6c on dizocilpine-induced learning impairments in mice. (a) spontaneous alternation performance and (b) Step-through latency. Animals received 6c (0.1e1 mg/kg
ip), 10 min before dizocilpine (Dizo, 0.15 mg/kg ip), 20 min before the Y-maze test session. In (a), data show mean SEM of n ¼ 12e16 per group; ANOVA: F(4,66) ¼ 13.8, p < 0.0001. In
(b), data show median and interquartile range of n ¼ 12e15 per group; Kruskal-Wallis ANOVA: H ¼ 40.9, p < 0.0001, in (b). ***p < 0.001 vs. (V þ V)-treated group; ##p < 0.01 vs.
(V þ Dizo)-treated group; Dunnett's test in (a), Dunn's test in (b).

240
J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
Fig. 5. Feature-enhanced electron density maps at 1s level for ligands inside the gorge of acetylcholinesterase. (A) View of the active-site gorge in the crystal structure of the
TcAChE-6a complex (pdb access code 6EZH), at a resolution of 2.6 Å (B) View of the active-site gorge in the crystal structure of the TcAChE-6b complex (pdb access code 6EZG) at a
resolution of 2.0 Å, (C) superimposition between the crystal structure of the TcAChE-6a complex and the crystal structure of the TcAChE-DPZ complex (1eve). Donepezil is shown in
cyan. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(40e63
m
m). Column size and flow rate followed the manufac-
yield); mp 87 ꢀC; 1H NMR (CDCl3, 400 MHz)
d 7.75 (s, 1H), 5.18 (br s,
turer's recommendations. NMR spectra were recorded at 295 K, at
400 or 500 MHz (Bruker Avance III 400/500 MHz) for 1H NMR and
at 100 or 126 MHz for 13C NMR in chloroform-d, methanol-d4 or
2H), 4.17 (q, 3J ¼ 7.2 Hz, 2H), 3.95 (s, 2H), 3.78 (s, 3H), 1.22 (t,
3J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 190.5 (CO), 168.1 (CO),
160.2 (Cq), 149.1 (Cq), 131.8 (CH), 121.1 (Cq), 113.5 (Cq), 81.3 (Cq), 62.7
DMSO-d6 with chemical shifts (
d) given in parts per million (ppm)
(CH3), 61.3 (CH2), 48.2 (CH2), 14.1 (CH3); IR (neat, cmꢁ1
) n 3431,
relative to TMS as internal standard and recorded. The following
abbreviations are used to describe peak splitting patterns when
appropriate: br ¼ broad, s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet, dd ¼ doublet of doublet, dt ¼ doublet of
triplet. Coupling constants J are reported in hertz units (Hz).
Infrared spectra (IR) were obtained on a PERKIN-ELMER FT-IR
spectrometer and are reported in terms of frequency of absorption
(cmꢁ1) using KBr discs. High-resolution mass spectra (HRMS) were
obtained by electronic impact (HRMS/EI), or by electrospray
(HRMS/ESI) on a Bruker maXis mass spectrometer. LC-MS (ESI)
analyses were realized with Waters Alliance 2695 as separating
module using the following gradients: A (95%)/B (5%) to A (5%)/B
(95%) in 4.00min. This ratio was hold during 1.50 min before return
to initial conditions in 0.50 min. Initial conditions were then
maintained for 2.00 min (A ¼ H2O, B ¼ CH3CN; each containing
3335, 2983, 2939, 1723, 1653, 1615, 1567, 1391, 1150; HRMS (ESI)
calcd. for C12H14ClINO4 [MþH]þ 397.9651, found 397.9648.
Ethyl
3-(4-amino-3-chloro-5-iodo-phenyl)-3-oxo-prop-
anoate (8b). The compound was prepared from 4-amino-3-chloro-
5-iodo-benzoic acid 7b (860 mg, 2.90 mmol) according to method
A, stirring the reaction for 6 h at room temperature and then for
14 h at 40 ꢀC. The residue was purified by flash chromatography on
silica gel (cyclohexane/EtOAc, gradient 100:0 to 80:20), to give 8b
as a white solid (66% yield); mp 84 ꢀC; 1H NMR (CDCl3, 400 MHz)
d
8.15 (d, 4J ¼ 2.0 Hz,1H), 7.86 (d, 4J ¼ 2.0 Hz,1H), 5.10 (br s, 2H), 4.21
(q, 3J ¼ 7.3 Hz, 2H), 3.86 (s, 2H), 1.26 (t, 3J ¼ 7.3 Hz, 3H); 13C NMR
(CDCl3, 100 MHz)
d 188.8 (CO), 167.6 (CO), 148.0 (Cq), 139.0 (CH),
130.6 (CH), 128.0 (Cq), 117.1 (Cq), 82.0 (Cq), 61.7 (CH2), 45.5 (CH2),
14.2 (CH3); IR (neat, cmꢁ1
)
n
3455, 3351, 2986, 2938, 2638, 1723,
1603.5, 1319, 1275, 1181; HRMS (ESI) calcd. for C11H12ClINO3
[MþH]þ 367.9545, found 367.9542.
HCOOH: 0.1%; column XBridge C18 2.5
m
m/4.6 ꢂ 50 mm; flow rate
0.8 mL/min). MS were obtained on a SQ detector by positive ESI.
Mass spectrum data are reported as m/z.
Ethyl 3-(4-amino-3-iodo-phenyl)-3-oxo-propanoate (8c). The
compound was prepared from 4-amino-3-iodo-benzoic acid 7c
(3.30 g, 12.5 mmol) according to method A, stirring the reaction for
7 h at room temperature and then for 14 h at 40 ꢀC. The residue was
purified by flash chromatography on silica gel (cyclohexane/EtOAC,
gradient 100:0 to 80:20) to give 8c as a yellow solid (70% yield); mp
Representative procedure of method A for the synthesis of
8a-c and 15. To a solution of benzoic acid derivatives (1.0 eq.) in dry
THF (10 mL/mmol) was added CDI (1.1 eq.) and the resulting
mixture was stirred at room temperature for 5e7 h. Then potas-
sium 3-ethoxy-3-oxopropanoate (1.2 eq.) and MgCl2 (1.2 eq.) were
added portion-wise. The reaction mixture was stirred at 40 ꢀC for
14e48 h. After removal of the solvent, the residue was dissolved
with EtOAc, and washed with a saturated aqueous NaHCO3 fol-
lowed by brine. The organic layer was dried over MgSO4 and
concentrated in vacuo. Chromatographic separation gave the title
compounds.
88 ꢀC; 1H NMR (CDCl3, 400 MHz)
d
8.25 (d, 4J ¼ 2.0 Hz, 1H), 7.73 (dd,
3
3J ¼ 8.5 Hz, 4J ¼ 2.0 Hz, 1H), 6.70 (d, J ¼ 8.3 Hz, 1H), 4.71 (br s, 2H),
4.20 (q, 3J ¼ 7.1 Hz, 2H), 3.87 (s, 2H),1.25 (t, 3J ¼ 7.1 Hz, 3H); 13C NMR
(CDCl3, 100 MHz)
d 189.6 (CO), 168.0 (CO), 151.7 (Cq), 140.8 (CH),
130.8 (CH), 128.0 (Cq), 113.3 (CH), 82.7 (Cq), 61.6 (CH2), 45.6 (CH2),
14.2 (CH3); IR (neat, cmꢁ1
)
n
3451, 3459, 2976, 2934, 2905, 2642,
1736, 1611, 1584, 1330, 1204, 1029. HRMS (ESI) calcd. for C11H13INO3
Ethyl
oxo-propanoate (8a). The compound was prepared from 4-
amino-5-chloro-3-iodo-2-methoxy-benzoic acid 7a (2.5 g,
3-(4-amino-5-chloro-3-iodo-2-methoxy-phenyl)-3-
[MþH]þ 333.9935, found 333.9931.
Ethyl 3-(4-methoxy-1H-indol-5-yl)-3-oxo-propanoate (15).
The compound was prepared from 4-methoxy-1H-indole-5-
carboxylic acid 14 (320 mg, 1.67 mmol) according to method A,
stirring the reaction for 6 h at room temperature and then for
17 h at 40 ꢀC. The residue was purified by flash chromatography on
silica gel (cyclohexane/EtOAc, gradient 100:0 to 70:30), to give 15 as
7.63 mmol) according to method A, stirring the reaction for 5 h at
room temperature and then for 48 h at 40 ꢀC. The residue was pu-
rified by flash chromatography on silica gel (cyclohexane/EtOAc,
gradient 100:0 to 80:20), to give 8a as a pale yellow solid (63%

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
241
a colorless oil (57% yield); 1H NMR (CDCl3, 400 MHz)
d
8.56 (br s,
1H), 6.69 (d, 3J ¼ 8.5 Hz, 1H), 4.72 (br s, 2H), 4.05 (m, 2H), 2.83 (t,
3J ¼ 7.6 Hz, 2H), 2.64 (m, 2H), 1.67e1.58 (m, 4H), 1.42 (m, 10H), 1.09
1H), 7.75 (d, 3J ¼ 8.6 Hz, 1H), 7.20 (m, 1H), 7.08 (d, 3J ¼ 8.6 Hz, 1H),
6.79 (m, 1H), 4.21 (q, 3J ¼ 7.2 Hz, 2H), 4.21 (s, 3H), 4.03 (s, 2H), 1.26
(m, 2H); 13C NMR (CDCl3, 100 MHz)
d 197.4 (CO), 155.0 (CO), 151.2
(t, 3J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
193.4 (CO), 169.1
(Cq), 140.1 (CH), 130.1 (CH), 128.7 (Cq), 113.2 (CH), 82.7 (Cq), 79.3
(Cq), 44.2 (CH2), 43.6 (CH2), 35.6 (CH2), 34.9 (CH2), 32.0 (2*CH2),
(CO), 155.9 (Cq), 141.6 (Cq), 125.0 (CH), 124.4 (CH), 120.1 (Cq), 118.8
(Cq), 106.7 (CH), 103.0 (CH), 61.1 (CH3), 60.6 (CH2), 50.7 (CH2), 14.2
30.9 (CH), 28.5 (3*CH3); IR (neat, cmꢁ1
) n 3459, 3336, 2976, 2927,
(CH3); IR (neat, cmꢁ1
)
n
3348, 2984, 2941, 2844, 1735, 1650, 1603,
2857, 1662, 1625, 1584, 1430, 1283, 1162; HRMS (ESI) calcd. for
1359, 1231, 1081, 730; HRMS (ESI) calcd. for C14H16NO4 [MþH]þ
C
19H28IN2O3 [MþH]þ 459.1139, found 459.1134.
262.1074, found 262.1074.
Tert-butyl
4-[3-(4-methoxy-1H-indol-5-yl)-3-oxo-propyl]
Representative procedure of method B for the synthesis of
9a-c, 16. To a solution of b-keto ester derivatives 8a-c and 15 (1.0
piperidine-1-carboxylate (16). The compound was prepared from
ethyl 3-(4-methoxy-1H-indol-5-yl)-3-oxo-propanoate 15 (370 mg,
1.42 mmol) by method B, stirring for 24 h under argon atmosphere
at room temperature for the first step, and refluxing for 5 h for the
second step. The residue was purified by chromatography on silica
gel (CH2Cl2/EtOAc, gradient 100:0 to 90:10) to give 16 as a white
solid (55% yield over 2 steps); mp 149 ꢀC; 1H NMR (CDCl3, 400 MHz)
eq.) in DMF (10 mL/mmol) were added K2CO3 (2.0 eq.) and tert-
butyl 4-(iodomethyl)piperidine-1-carboxylate (1.2 eq.). The
resulting mixture was stirred at room temperature for 24e48 h
then concentrated in vacuo. The residue was dissolved with EtOAc
and washed with brine. The organic layer was dried (MgSO4) and
concentrated in vacuo. To a stirred solution of residue (1.0 eq.), used
without any purification, in a mixture of EtOH/H2O 5:1 (24 mL/
mmol) was added KOH (4.5 eq.) and the resulting mixture was
refluxed for 3e5 h. After removal of the solvent, EtOAc was added.
The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by chromatography
on silica gel to give the title compounds 9a-c and 16.
d
8.84 (br s, 1H), 7.58 (d, 3J ¼ 8.7 Hz, 1H), 7.20 (m, 1H), 7.10 (d,
3J ¼ 8.6 Hz, 1H), 6.76 (m, 1H), 4.16 (s, 3H), 4.09 (m, 2H), 3.08 (m, 2H),
2.68 (m, 2H),1.73e1.65 (m, 4H),1.45 (m,10H),1.12 (m, 2H); 13C NMR
(CDCl3, 100 MHz)
d 203.1 (CO), 155.1 (CO), 154.6 (Cq), 140.7 (Cq),
124.5 (CH), 124.3 (CH), 122.1 (Cq), 119.8 (Cq), 106.9 (CH), 102.2 (CH),
79.4 (Cq), 61.2 (CH3), 44.5 (CH2), 43.9 (CH2), 40.7 (CH2), 35.9 (CH2),
32.2 (2*CH2), 31.4 (CH), 28.5 (3*CH3); IR (neat, cmꢁ1
) n 3263, 2990,
Tert-butyl
4-[3-(4-amino-5-chloro-3-iodo-2-methoxy-
2971, 2934, 2842, 1655, 1432, 1288, 1161, 966, 858, 740; HRMS (ESI)
m/z calcd. for C22H31N2O4 [MþH]þ 387.2278, found 387.2278.
Representative procedure of method C for the synthesis of
4a-g, 6h. To a stirred solution of tert-butyl piperidine-1-carboxylate
derivatives 9a-c and 16 (1.0 eq.) in CH2Cl2 (20 mL/mmol) was added
TFA (2 mL/mmol). The resulting mixture was stirred at room tem-
perature for 30 min. Removal of the solvent under vacuum afforded
the crude product, which was directly engaged in the next step. The
residue obtained (1.0 eq.) was dissolved in DMF (10 mL/mmol) and
alkyl-halogenated derivatives (1.1e1.25 eq.) and K2CO3 (10.0 eq.)
were added. The resulting mixture was stirred at 110 ꢀC for 5e15 h,
and then concentrated in vacuo. EtOAc was added, the organic layer
was washed several times with brine, dried over MgSO4 and
concentrated in vacuo. The crude was purified by chromatography
on silica gel column and concentrated under reduced pressure to
afford the corresponding alkylated compounds 4a-g and 6h.
1-(4-Amino-5-chloro-3-iodo-2-methoxy-phenyl)-3-(1-
phenyl)-3-oxo-propyl]piperidine-1-carboxylate (9a). The com-
pound was prepared from ethyl 3-(4-amino-5-chloro-3-iodo-2-
methoxy-phenyl)-3-oxo-propanoate 8a (1.63 g, 4.10 mmol) ac-
cording to method B, stirring for 48 h under argon atmosphere at
room temperature for the first step, and then refluxing for 5 h for
the second step. The residue was purified by chromatography on
silica gel (cyclohexane/EtOAc, gradient 100:0 to 80:20) to give 9a as
a yellow oil (37% yield over 2 steps); 1H NMR (CDCl3, 400 MHz)
d
7.67 (s, 1H), 5.05 (br s, 2H), 4.07 (m, 2H), 3.78 (s, 3H), 2.96 (m, 2H),
2.65 (m, 2H), 1.68e1.59 (m, 4H), 1.44 (s, 9H), 1.41 (m, 1H), 1.10 (m,
2H); 13C NMR (CDCl3, 100 MHz)
199.2 (CO), 159.5 (CO), 155.1 (Cq),
d
148.2 (Cq), 131.4 (CH), 122.8 (Cq), 113.4 (Cq), 82.2 (Cq), 79.4 (Cq), 62.7
(CH3), 44.0 (2*CH2), 38.9 (CH2), 35.7 (CH2), 32.1 (2*CH2), 31.1 (CH),
28.5 (3*CH3); IR (neat, cmꢁ1
) n 3461, 3369, 2929, 2852, 1682, 1603,
1408, 1160; HRMS (ESI) calcd. for C20H29ClIN2O4 [MþH]þ 523.0855,
found 523.0854.
Tert-butyl
4-[3-(4-amino-3-chloro-5-iodo-phenyl)-3-oxo-
benzyl-4-piperidyl)propan-1-one (4a). The compound was pre-
pared from tert-butyl 4-[3-(4-amino-5-chloro-3-iodo-2-methoxy-
propyl]piperidine-1-carboxylate (9b). The compound was pre-
pared from ethyl 3-(4-amino-3-chloro-5-iodo-phenyl)-3-oxo-
propanoate 8b (690 mg, 1.88 mmol), by method B, stirring for 48 h
under argon atmosphere at room temperature for the first step, and
then refluxing for 3 h for the second step. The residue was purified
by chromatography on silica gel (cyclohexane/EtOAc, gradient
100:0 to 90:10) to give 9b as a yellow oil (48% yield over 2 steps); 1H
phenyl)-3-oxo-propyl]piperidine-1-carboxylate
0.96 mmol) and bromomethylbenzene (126
9a
(500 mg,
m
L, 1.06 mmol) ac-
cording to method C, with stirring for 6 h at 110 ꢀC. The residue was
purified by flash chromatography on silica gel column (cyclo-
hexane/EtOAc, gradient 60:40 to 0:100) to give 4a as a yellow oil
(62% yield over 2 steps); 1H NMR (CDCl3, 400 MHz)
d
7.66 (s, 1H),
7.31e7.23 (m, 5H), 5.04 (br s, 2H), 3.77 (s, 3H), 3.48 (s, 2H), 2.95 (m,
2H), 2.87 (m, 2H), 1.91 (m, 2H), 1.67e1.58 (m, 5H), 1.27 (m, 2H); 13
NMR (CDCl3,100 MHz) 199.6 (CO),159.4 (Cq),148.1 (Cq),138.7 (Cq),
NMR (CDCl3, 400 MHz)
d
8.16 (d, 4J ¼ 1.9 Hz, 1H), 7.87 (d, 4J ¼ 1.9 Hz,
1H), 5.01 (br s, 2H), 4.09 (m, 2H), 2.86 (t, 3J ¼ 7.6 Hz, 2H), 2.67 (m,
C
2H), 1.70e1.63 (m, 4H), 1.45 (m, 10H), 1.13 (m, 2H); 13C NMR (CDCl3,
d
100 MHz)
d
196.5 (CO), 155.1 (CO), 147.4 (Cq), 138.4 (CH), 130.1 (CH),
131.4 (CH), 129.5 (2*CH2), 128.3 (2*CH2), 127.1 (CH), 122.9 (Cq), 113.4
(Cq), 82.2 (Cq), 63.6 (CH2), 62.7 (CH), 53.9 (2*CH2), 39.2 (CH2), 35.5
129.0 (Cq), 117.1 (Cq), 82.1 (Cq), 79.5 (Cq), 44.4 (CH2), 43.6 (CH2), 35.7
(CH2), 35.1 (CH2), 32.0 (2*CH2), 30.8 (CH), 28.6 (3*CH3); IR (neat,
(CH), 32.3 (2*CH2), 31.3 (CH2); IR (neat, cmꢁ1
) n 3471, 3373, 3026,
cmꢁ1
)
n
3471, 3366, 2976, 2924, 2846, 1681, 1607, 1276, 1161; HRMS
2923, 1670, 1602, 1387, 699; HRMS (ESI) m/z calcd. for
(ESI) calcd. for C19H27ClIN2O3 [MþH]þ 493.0749, found 493.0746.
C
22H27ClIN2O2 [MþH]þ 513.0800, found 513.0795.
Tert-butyl
4-[3-(4-amino-3-iodo-phenyl)-3-oxo-propyl]
1-(4-Amino-5-chloro-3-iodo-2-methoxy-phenyl)-3-[1-
piperidine-1-carboxylate (9c). The compound was prepared from
ethyl 3-(4-amino-3-iodo-phenyl)-3-oxo-propanoate 8c (1.50 g,
4.5 mmol) by method B, stirring for 48 h under argon atmosphere
at room temperature for the first step, and refluxing for 5 h for the
second step. The residue was purified by flash chromatography on
silica gel (CH2Cl2/EtOAc, gradient 100:0 to 95:5) to give 9c as a
yellow solid (76% yield over 2 steps); mp 129 ꢀC; 1H NMR (CDCl3,
(cyclohexylmethyl)-4-piperidyl] propan-1-one (4b). The com-
pound was prepared from tert-butyl 4-[3-(4-amino-5-chloro-3-
iodo-2-methoxy-phenyl)-3-oxo-propyl]piperidine-1-carboxylate
9a (255 mg, 0.488 mmol) and bromomethylcyclohexane (84 mL,
0.61 mmol) according to method C, with stirring for 12 h at 110 ꢀC.
The residue was purified by chromatography on deactivated silica
gel column (cyclohexane/EtOAc, gradient 100:0 to 80:20) to give 4b
as a brown oil (68% yield over 2 steps); 1H NMR (CDCl3, 500 MHz)
400 MHz)
d
8.23 (d, 4J ¼ 1.9 Hz, 1H), 7.72 (dd, 3J ¼ 8.3 Hz, 4J ¼ 1.9 Hz,

242
J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
d
7.66 (s, 1H), 5.05 (br s, 2H), 3.77 (s, 3H), 2.99 (m, 2H), 2.95 (m, 2H),
tert-butyl 4-[3-(4-amino-3-iodo-phenyl)-3-oxo-propyl]piperidine-
1-carboxylate 9c (350 mg, 0.76 mmol) and iodomethylcyclo-
2.22 (d, 3J ¼ 6.6 Hz, 2H), 1.99 (m, 2H), 1.76 (m, 2H), 1.70e1.60 (m,
7H), 1.54 (m, 1H), 1.40 (m, 2H), 1.31 (m, 1H), 1.24e1.10 (m, 3H), 0.89
pentane (110 mL, 0.84 mmol) by method C, with stirring for 6 h at
(m, 2H); 13C NMR (CDCl3, 126 MHz)
d
199.2 (CO), 159.3 (Cq), 148.1
110 ꢀC. The residue was purified by flash chromatography on silica
gel (cyclohexane/EtOAc, gradient 100:0 to 60:40) to give 4f as a pale
yellow solid (54% yield over 2 steps); mp 99 ꢀC; 1H NMR (CDCl3,
(Cq), 131.3 (CH), 122.8 (Cq), 113.3 (Cq), 82.2 (Cq), 65.6 (CH2), 62.7
(CH3), 54.2 (2*CH2), 39.1 (CH2), 35.2 (CH), 34.9 (CH), 32.2 (2*CH2),
31.4 (2*CH2), 31.0 (CH2), 26.7 (CH2), 26.2 (2*CH2); IR (neat, cmꢁ1
)
n
500 MHz)
d
8.27 (d, 4J ¼ 2.0 Hz, 1H), 7.76 (dd, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz,
3470, 3359, 2924, 2851, 1666, 1602, 1568, 1444, 1390, 1193, 961;
HRMS (EI) m/z calcd for C22H32ClIN2O2 [M]þ. 518.1197, found
518.1218.
1-(4-Amino-3-chloro-5-iodo-phenyl)-3-[1-(cyclo-
hexylmethyl)-4-piperidyl]propan-1-one (4c). The compound was
prepared from tert-butyl 4-[3-(4-amino-3-chloro-5-iodo-phenyl)-
3-oxo-propyl]piperidine-1-carboxylate 9b (450 mg, 0.91 mmol)
1H), 6.71 (d, 3J ¼ 8.5 Hz, 1H), 4.56 (br s, 2H), 2.91 (m, 2H), 2.85 (m,
2H), 2.23 (m, 2H), 2.04 (m, 1H), 1.86 (m, 2H), 1.77e1.71 (m, 2H),
1.69e1.61 (m, 4H), 1.60e1.55 (m, 2H), 1.52e1.48 (m, 2H), 1.29 (m,
3H), 1.20e1.13 (m, 2H); 13C NMR (CDCl3, 126 MHz)
d 197.7 (CO),
150.8 (Cq), 140.2 (CH), 130.1 (CH), 129.2 (Cq), 113.3 (CH), 82.8 (Cq),
65.3 (CH2), 54.4 (2*CH2), 37.7 (CH), 35.7 (CH), 35.4 (CH2), 32.4
(2*CH2), 31.9 (2*CH2), 31.3 (CH2), 25.4 (2*CH2); IR (neat, cmꢁ1
) n
and bromomethylcyclohexane (140
m
L, 1.0 mmol) by method C,
3454, 3341, 3219, 2929, 2862, 1665, 1618, 1582, 1333, 1205, 1188,
666; HRMS (ESI) m/z calcd. for C20H30IN2O [MþH]þ 441.1397, found
441.1393.
1-(4-Amino-3-iodo-phenyl)-3-(1-butyl-4-piperidyl)propan-
1-one (4g). The compound was prepared from tert-butyl 4-[3-(4-
amino-3-iodo-phenyl)-3-oxo-propyl]piperidine-1-carboxylate 9c
with stirring for 5 h at 110 ꢀC. The residue was purified by flash
chromatography on silica gel column (cyclohexane/EtOAc, gradient
100:0 to 60:40) to give 4c as a pale yellow oil (70% yield over 2
steps); 1H NMR (CDCl3, 500 MHz)
d
8.14 (d, 4J ¼ 1.9 Hz, 1H), 7.84 (d,
4J ¼ 1.9 Hz, 1H), 4.99 (br s, 2H), 2.82 (m, 4H), 2.05 (d, 3J ¼ 7.1 Hz, 2H),
1.79 (m, 2H), 1.74e1.60 (m, 9H), 1.45 (m, 1H), 1.28e1.11 (m, 6H), 0.83
(350 mg, 0.76 mmol) and 1-iodobutane (96 mL, 0.84 mmol) by
(m, 2H); 13C NMR (CDCl3, 126 MHz)
d
196.6 (CO), 147.1 (Cq), 138.2
method C, with stirring for 5 h at 110 ꢀC. The residue was purified by
flash chromatography on silica gel column (cyclohexane/EtOAc,
gradient 100:0 to 60:40) to give 4g as a pale yellow solid (60% yield
(CH), 130.0 (CH), 128.9 (Cq), 117.0 (Cq), 82.1 (Cq), 66.3 (CH2), 54.5
(2*CH2), 35.6 (CH), 35.4 (CH2), 35.3 (CH), 32.3 (2*CH2), 32.2 (2*CH2),
31.1 (CH2), 26.9 (CH2), 26.3 (2*CH2); IR (neat, cmꢁ1
)
n
3463, 3368,
over 2 steps); mp 69 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 8.26 (d,
2924, 2851, 1669, 1606, 1447, 1396, 1266, 1199; HRMS (ESI) m/z
calcd. for C21H31ClIN2O [MþH]þ 489.1164, found 489.1159.
1-(4-Amino-3-iodo-phenyl)-3-(1-benzyl-4-piperidyl)
4J ¼ 1.9 Hz, 1H), 7.76 (dd, 3J ¼ 8.5 Hz, 4J ¼ 1.9 Hz, 1H), 6.70 (d,
3J ¼ 8.5 Hz, 1H), 4.57 (br s, 2H), 2.92 (m, 2H), 2.86 (m, 2H), 2.28 (m,
2H), 1.86 (m, 2H), 1.72e1.62 (m, 4H), 1.50e1.43 (m, 2H), 1.34e1.25
propan-1-one (4d). The compound was prepared from tert-butyl
4-[3-(4-amino-3-iodo-phenyl)-3-oxo-propyl]piperidine-1-
carboxylate 9c (350 mg, 0.76 mmol) and bromomethylbenzene
(m, 5H), 0.90 (m, 3H); 13C NMR (CDCl3, 126 MHz)
d 197.6 (CO), 150.9
(Cq), 140.2 (CH), 130.1 (CH), 129.2 (Cq), 113.3 (CH), 82.8 (Cq), 59.1
(CH2), 54.1 (2*CH2), 35.6 (CH), 35.3 (CH2), 32.3 (2*CH2), 31.2 (CH2),
(100
m
L, 0.84 mmol) by method C, with stirring for 5 h at 110 ꢀC. The
29.4 (CH2), 21.1 (CH2),14.2 (CH3); IR (neat, cmꢁ1
) n 3460, 3341, 3223,
residue was purified by flash chromatography on silica gel (cyclo-
hexane/EtOAc, gradient 100:0 to 60:40) to give 4d as a pale yellow
solid (75% yield over 2 steps); mp 106 ꢀC; 1H NMR (CDCl3, 500 MHz)
2919, 2845, 1665, 1619, 1582, 1331, 1199, 667; HRMS (ESI) m/z calcd.
for C18H28IN2O [MþH]þ 415.1241, found 415.1237.
3-[1-(Cyclohexylmethyl)-4-piperidyl]-1-(4-methoxy-1H-
indol-5-yl)propan-1-one (6h). The compound was prepared from
tert-butyl 4-[3-(4-methoxy-1H-indol-5-yl)-3-oxo-propyl]piperi-
dine-1-carboxylate 16 (255 mg, 0.66 mmol) and benzyl bromide
d
8.26 (d, 4J ¼ 2.0 Hz, 1H), 7.75 (dd, 3J ¼ 8.5 Hz, 4J ¼ 2.0 Hz, 1H), 7.31
(m, 4H), 7.26e7.22 (m, 1H), 6.70 (d, 3J ¼ 8.5 Hz, 1H), 4.58 (br s, 2H),
3.48 (s, 2H), 2.87 (m, 2H), 2.84 (m, 2H), 1.93 (m, 2H), 1.69e1.62 (m,
4H), 1.31e1.25 (m, 3H); 13C NMR (CDCl3, 126 MHz)
d
197.6 (CO),
(101 mL, 0.73 mmol) by method C. The residue was purified by flash
150.9 (Cq),140.2 (CH),138.6 (Cq),130.1 (CH),129.4 (2*CH),129.1 (Cq),
128.2 (2*CH), 127.0 (CH), 113.3 (CH), 82.8 (Cq), 63.6 (CH2), 53.9
(2*CH2), 35.5 (CH), 35.4 (CH2), 32.3 (2*CH2), 31.3 (CH2); IR (neat,
chromatography on silica gel column (CH2Cl2/MeOH, gradient
100:0 to 95:5) to give 6h as a yellow oil (52% yield over 2 steps); 1H
NMR (CDCl3, 500 MHz)
d
8.48 (br s,1H), 7.58 (d, 3J ¼ 8.6 Hz,1H), 7.20
cmꢁ1
)
n
3458, 3341, 3219, 3028, 2921, 2858, 2797, 2750, 1663, 1616,
(m, 1H), 7.11 (d, 3J ¼ 8.6 Hz, 1H), 6.76 (m, 1H), 4.16 (s, 3H), 3.06 (t,
3J ¼ 7.6 Hz, 2H), 2.94 (m, 2H), 2.18 (m, 2H), 1.92 (m, 2H), 1.77e1.64
(m, 9H), 1.52 (m, 1H), 1.35e1.12 (m, 6H), 0.88 (m, 2H); 13C NMR
1581, 1330, 1196, 698; HRMS (ESI) calcd. for C21H26IN2O [MþH]þ
449.1084, found 449.1080.
1-(4-Amino-3-iodo-phenyl)-3-[1-(cyclohexylmethyl)-4-
piperidyl]propan-1-one (4e). The compound was prepared from
tert-butyl 4-[3-(4-amino-3-iodo-phenyl)-3-oxo-propyl]piperidine-
1-carboxylate 9c (410 mg, 0.90 mmol) and bromomethylcyclohex-
(CDCl3, 126 MHz) d 203.0 (CO), 154.6 (Cq), 140.5 (Cq), 124.5 (CH),
124.3 (CH), 122.4 (Cq), 119.9 (Cq), 106.7 (CH), 102.3 (CH), 66.0 (CH2),
61.3 (CH3), 54.5 (2*CH2), 40.9 (CH2), 35.6 (CH), 35.1 (CH), 32.1
(4*CH2), 31.4 (CH2), 26.8 (CH2), 26.3 (2*CH2); IR (neat, cmꢁ1
) n 3301,
ane (140
m
L, 0.99 mmol), by method C, with stirring for 15 h at
2922, 2849, 1656, 1606, 1449, 1355, 1218, 1066, 733; LC-MS (ESI)
110 ꢀC. The residue was purified by chromatography on silica gel
(cyclohexane/EtOAc, gradient 100:0 to 60:40) to give 4e as a pale
yellow solid (61% yield over 2 steps); mp 116 ꢀC; 1H NMR (CDCl3,
tR ¼ 3.48 min; m/z [MþH]þ 383.62; HRMS (ESI) m/z calcd. for
C
24H35N2O2 [MþH]þ 383.2693, found 383.2694.
Representative procedure of method D for the synthesis of
400 MHz)
d
8.27 (d, 4J ¼ 2.0 Hz, 1H), 7.77 (dd, 3J ¼ 8.4 Hz, 4J ¼ 2.0 Hz,
6a-g. To a stirred solution of iodo-aniline derivatives 4a-g (1.0 eq.),
Pd(PPh3)4 (10 mol%), XPhos (20 mol%) and K2CO3 (2.0 eq.) in dry
THF (15 mL/mmol) was added alkyne (3.0 eq.) under argon atmo-
sphere and the reaction mixture was refluxed for 17e24 h. After
evaporation in vacuo to remove THF, the residue was dissolved in
EtOAc and washed with brine. The organic layer was dried over
MgSO4, and concentrated under reduced pressure. To a stirred so-
lution of previous crude 5a-g (1.0 eq.) (except the compound 5b
which was isolated by flash chromatography on silica gel column) in
CH2Cl2 (12 mL/mmol) was added TFA (1.25 mL/mmol). The result-
ing mixture was stirred at room temperature for 1e2 h, and then
concentrated in vacuo. The crude was purified by flash
1H), 6.71 (d, 3J ¼ 8.4 Hz, 1H), 4.56 (br s, 2H), 2.85 (m, 4H), 2.07 (d,
3J ¼ 7.0 Hz, 2H), 1.83e1.60 (m, 11H), 1.47 (m, 1H), 1.28e1.12 (m, 6H),
0.85 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 197.9 (CO), 150.9 (Cq),
140.3 (CH), 130.2 (CH), 129.3 (Cq), 113.3 (CH), 82.8 (Cq), 66.4 (CH2),
54.6 (2*CH2), 35.7 (CH), 35.4 (CH2), 35.3 (CH), 32.3 (2*CH2), 32.2
(2*CH2), 31.3 (CH2), 26.9 (CH2), 26.3 (2*CH2); IR (neat, cmꢁ1
) n 3538,
3388, 3314, 3202, 2939, 2844, 1659, 1635, 1585, 1403, 1211, 1144,
670; HRMS (ESI) calcd. for C21H32IN2O [MþH]þ 455.1554, found
455.1552.
1-(4-Amino-3-iodo-phenyl)-3-[1-(cyclopentylmethyl)-4-
piperidyl]propan-1-one (4f). The compound was prepared from

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
243
chromatography on silica gel column and concentrated under
reduced pressure to afford the corresponding derivatives 6a-g
(26e73% isolated yields).
1-(7-Chloro-4-methoxy-1H-indol-5-yl)-3-[1-(cyclo-
hexylmethyl)-4-piperidyl]propan-1-one (6a). The compound was
prepared from 1-(4-amino-5-chloro-3-iodo-2-methoxy-phenyl)-3-
(CH), 31.4 (CH2), 30.4 (2*CH2); IR (neat, cmꢁ1
)
n
3428, 2953, 2930,
2852, 1677, 1602, 1202, 1134, 721; LC-MS (ESI) tR ¼ 3.59 min; m/z
[MþH]þ 411.53/413.55; HRMS (ESI) m/z calcd. for C24H28ClN2O2
[MþH]þ 411.1834, found 411.1831.
1-(7-Chloro-1H-indol-5-yl)-3-[1-(cyclohexylmethyl)-4-
piperidyl]propan-1-one (6c). The compound was prepared from
1-(4-amino-3-chloro-5-iodo-phenyl)-3-[1-(cyclohexylmethyl)-4-
piperidyl]propan-1-one 4c (275 mg, 0.56 mmol) and ethynyl-
[1-(cyclohexylmethyl)-4-piperidyl]propan-1-one
4a
(65 mg,
0.13 mmol) according to method D, refluxing for 18 h under argon
atmosphere for the first step, and then stirring for 2 h at room
temperature for the second step. The residue was purified by
chromatography on silica gel column (CH2Cl2/MeOH, gradient
100:0 to 90:10) to give 6a as a pale yellow solid (52% yield); mp
trimethylsilane (237 mL, 1.68 mmol) according to method D,
refluxing for 17 h under argon atmosphere for the first step, and
then stirring for 1 h at room temperature for the second step. The
residue was purified by flash chromatography on silica gel column
(cyclohexane/EtOAc 50:50 to CH2Cl2/MeOH 95:5) to give 6c as a
white solid (64% yield); mp 172 ꢀC; 1H NMR (MeOD-d4, 500 MHz)
156 ꢀC; 1H NMR (MeOD-d4, 400 MHz)
d 7.49 (s, 1H), 7.35 (d,
3J ¼ 3.3 Hz, 1H), 6.86 (d, 3J ¼ 3.3 Hz, 1H), 4.19 (s, 3H), 3.51 (m, 2H),
3.12 (t, 3J ¼ 7.0 Hz, 2H), 2.91 (m, 4H), 1.99 (m, 2H), 1.82 (m, 1H),
1.80e1.70 (m, 7H), 1.65 (m, 1H), 1.52 (m, 2H), 1.40e1.18 (m, 3H), 1.03
d
8.29 (d, 4J ¼ 1.4 Hz, 1H), 7.79 (d, 4J ¼ 1.3 Hz, 1H), 7.42 (d, 3J ¼ 3.2 Hz,
1H), 6.70 (d, 3J ¼ 3.2 Hz, 1H), 3.56 (m, 2H), 3.14 (t, 3J ¼ 7.2 Hz, 2H),
2.92e2.86 (m, 4H), 2.02 (m, 2H), 1.83 (m, 1H), 1.80e1.69 (m, 7H),
1.65 (m, 1H), 1.52 (m, 2H), 1.38e1.30 (m, 2H), 1.26e1.21 (m, 1H), 1.03
(m, 2H); 13C NMR (MeOD-d4, 100 MHz)
d 202.8 (CO), 155.2 (Cq),
138.9 (Cq), 127.1 (CH), 123.0 (CH), 122.7 (Cq), 122.4 (Cq), 112.8 (Cq),
103.8 (CH), 64.4 (CH2), 61.7 (CH3), 54.4 (2*CH2), 41.3 (CH2), 34.6
(CH), 34.3 (CH), 31.9 (2*CH2), 31.5 (CH2), 30.4 (2*CH2), 27.0 (CH2),
(m, 2H); 13C NMR (MeOD-d4, 126 MHz)
d 201.1 (CO), 137.2 (Cq), 131.1
(Cq), 130.6 (Cq), 128.6 (CH), 122.5 (CH), 121.4 (CH), 118.3 (Cq), 105.6
(CH), 64.6 (CH2), 54.6 (2*CH2), 36.1 (CH2), 34.4 (CH), 34.0 (CH), 31.7
(2*CH2), 31.4 (CH2), 30.5 (2*CH2), 26.9 (CH2), 26.5 (2*CH2); IR (neat,
26.6 (2*CH2); IR (neat, cmꢁ1
) n 3436, 2927, 2852, 1651, 1604, 1484,
1404, 1351, 1204, 1134, 728; LC-MS (ESI) tR ¼ 3.71 min; m/z [MþH]þ
417.58/419.60; HRMS (ESI) m/z calcd. for C24H34ClN2O2 [MþH]þ
417.2303, found 417.2304.
cmꢁ1
) n 3382, 3202, 2933, 2854, 1675, 1452, 1202, 1131, 719; LC-MS
(ESI) tR ¼ 3.74 min; m/z [MþH]þ 387.61/389.58; HRMS (ESI) m/z
calcd. for C23H32ClN2O [MþH]þ 387.2198, found 387.2194.
3-(1-Benzyl-4-piperidyl)-1-(1H-indol-5-yl)propan-1-one
(6d). The compound was prepared from 1-(4-amino-3-iodo-
3-(1-Benzyl-4-piperidyl)-1-(7-chloro-4-methoxy-2-
trimethylsilyl-1H-indol-5-yl)propan-1-one (5b). To a stirred so-
lution of 1-(4-amino-5-chloro-3-iodo-2-methoxy-phenyl)-3-(1-
benzyl-4-piperidyl)propan-1-one 4b (260 mg, 0.51 mmol, 1.0 eq.),
Pd(PPh3)4 (59 mg, 0.051 mmol, 10 mol%), XPhos (48 mg, 0.10 mmol,
20 mol%) and K2CO3 (141 mg, 1.02 mmol, 2.0 eq.) in dry THF (8 mL)
phenyl)-3-(1-benzyl-4-piperidyl)propan-1-one
0.55 mmol) and ethynyltrimethylsilane (233
4d
(245 mg,
m
L, 1.65 mmol) ac-
cording to method D, refluxing for 18 h under argon atmosphere for
the first step, and then stirring for 1 h at room temperature for the
second step. The residue was purified by flash chromatography on
silica gel column (cyclohexane 100% to CH2Cl2/MeOH 95:5) to give
6d as a white solid (53% yield); mp 165 ꢀC; 1H NMR (MeOD-d4,
was added ethynyltrimethylsilane (215 mL, 1.52 mmol, 3.0 eq.) un-
der argon atmosphere and the reaction mixture was refluxed for
23 h. After evaporation in vacuo to remove THF, the residue was
purified by flash chromatography on silica gel column (cyclo-
hexane/EtOAc, gradient 100:0 to 90:10) and concentrated under
reduced pressure to give 5b as a pale yellow solid (45% yield); mp
3
4
500 MHz)
d
8.33 (m, 1H), 7.80 (dd, J ¼ 8.7 Hz, J ¼ 1.6 Hz, 1H), 7.50
(br s, 5H), 7.44 (d, 3J ¼ 8.7 Hz, 1H), 7.34 (d, 3J ¼ 3.2 Hz, 1H), 6.60 (dd,
3J ¼ 3.1 Hz, 4J ¼ 0.6 Hz, 1H), 4.28 (s, 2H), 3.49 (m, 2H), 3.14 (t,
3J ¼ 7.1 Hz, 2H), 2.98 (m, 2H), 2.05 (m, 2H),1.73 (m, 2H),1.68 (m,1H),
162 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 8.37 (br s, 1H), 7.58 (s, 1H), 7.31
(m, 4H), 7.24 (m, 1H), 6.91 (d, 4J ¼ 2.3 Hz, 1H), 4.15 (s, 3H), 3.49 (s,
2H), 3.03 (t, 3J ¼ 7.6 Hz, 2H), 2.88 (m, 2H), 1.94 (m, 2H), 1.69e1.63
1.45 (m, 2H); 13C NMR (MeOD-d4, 126 MHz)
d 202.6 (CO), 140.5 (Cq),
(m, 4H),1.28 (m, 3H), 0.38 (s, 9H); 13C NMR (CDCl3,126 MHz)
d
201.8
132.3 (2*CH), 131.3 (CH), 130.4 (Cq, 2*CH), 129.9 (Cq), 129.1 (Cq),
127.6 (CH), 123.7 (CH), 122.4 (CH), 112.2 (CH), 104.3 (CH), 61.8 (CH2),
53.8 (2*CH2), 36.1 (CH2), 34.5 (CH), 31.7 (CH2), 30.6 (2*CH2); IR
(CO), 153.2 (Cq), 139.9 (2*Cq), 138.7 (Cq), 129.4 (2*CH), 128.3 (2*CH),
127.0 (CH), 123.4 (CH), 122.9 (Cq), 122.0 (Cq), 111.9 (CH), 111.4 (Cq),
63.7 (CH2), 61.5 (CH3), 54.0 (2*CH2), 41.0 (CH2), 35.7 (CH), 32.4
(neat, cmꢁ1
) n 3308, 2996, 2945, 2745, 2575, 1690, 1666, 1607, 1200,
(3*CH2), ꢁ1.0 (3*CH3); IR (neat, cmꢁ1
)
n
3323, 3031, 2938, 2926,
1126, 718; LC-MS (ESI) tR ¼ 3.35 min; m/z [MþH]þ 347.56; HRMS
(ESI) m/z calcd. for C23H27N2O [MþH]þ 347.2118, found 347.2117.
3-[1-(Cyclohexylmethyl)-4-piperidyl]-1-(1H-indol-5-yl)
2854, 2757, 1648, 1596, 1301, 838; LC-MS (ESI) tR ¼ 4.23 min; m/z
[MþH]þ 483.57/485.59; HRMS (ESI) m/z calcd. for C27H36ClN2O2Si
[MþH]þ 483.2229, found 483.2226.
propan-1-one (6e). The compound was prepared from 1-(4-
amino-3-iodo-phenyl)-3-[1-(cyclohexylmethyl)-4-piperidyl]
propan-1-one 4e (160 mg, 0.35 mmol) and ethynyltrimethylsilane
3-(1-Benzyl-4-piperidyl)-1-(7-chloro-4-methoxy-1H-indol-
5-yl)propan-1-one (6b). To a stirred solution of 3-(1-benzyl-4-
piperidyl)-1-(7-chloro-4-methoxy-2-trimethylsilyl-1H-indol-5-yl)
propan-1-one 5b (85 mg, 0.18 mmol, 1.0 eq.) in DCM (2.5 mL) was
(148 mL,1.05 mmol) according to method D, refluxing for 23 h under
argon atmosphere for the first step, and then stirring for 1 h at room
temperature for the second step. The residue was purified by flash
chromatography on silica gel column (cyclohexane 100% to EtOAc
100%) to give 6e as a colorless oil (73% yield); 1H NMR (CDCl3,
added TFA (250 mL). The resulting mixture was stirred at room
temperature for 1 h and then concentrated in vacuo. The residue
was dissolved with EtOAc, and washed with a saturated NaHCO3
solution and brine. The organic layer was dried over MgSO4. The
crude was purified by flash chromatography on silica gel column
(cyclohexane/EtOAc, gradient 50:50 to 80:20) and concentrated
under reduced pressure to give 6b as a pale brown solid (81% yield);
500 MHz)
d
8.61 (br s, 1H), 8.32 (m, 1H), 7.87 (dd, 3J ¼ 8.6 Hz,
4J ¼ 1.6 Hz, 1H), 7.41 (d, 3J ¼ 8.6 Hz, 1H), 7.28 (m, 1H), 6.66 (m, 1H),
3.06 (t, 3J ¼ 7.4 Hz, 2H), 2.90 (m, 2H), 2.12 (d, 3J ¼ 7.0 Hz, 2H), 1.87
(m, 2H), 1.77e1.64 (m, 9H), 1.50 (m, 1H), 1.34 (m, 3H), 1.28e1.11 (m,
mp 148 ꢀC; 1H NMR (MeOD-d4, 500 MHz)
d
7.48 (br s, 6H), 7.34 (d,
3H), 0.87 (m, 2H); 13C NMR (CDCl3, 126 MHz)
d 200.9 (CO), 138.6
3J ¼ 3.3 Hz, 1H), 6.85 (d, 3J ¼ 3.3 Hz, 1H), 4.24 (s, 2H), 4.18 (s, 3H),
3.42 (m, 2H), 3.09 (t, 3J ¼ 7.1 Hz, 2H), 2.94 (m, 2H), 1.98 (m, 2H), 1.69
(m, 2H), 1.64 (m, 1H), 1.46 (m, 2H); 13C NMR (MeOD-d4, 126 MHz)
(Cq), 129.7 (Cq), 127.6 (Cq), 125.9 (CH), 122.7 (CH), 122.2 (CH), 111.2
(CH), 104.3 (CH), 65.8 (CH2), 54.3 (2*CH2), 35.8 (CH2), 35.5 (CH),
35.0 (CH), 32.2 (2*CH2), 31.8 (2*CH2), 31.4 (CH2), 26.8 (CH2), 26.3
d
202.7 (CO), 155.2 (Cq), 138.9 (Cq), 132.2 (2*CH), 131.1 (CH), 131.0
(2*CH2); IR (neat, cmꢁ1
) n 3428, 2922, 2851, 1660, 1612, 1449, 730;
(Cq), 130.3 (2*CH),127.1 (CH), 123.0 (CH), 122.6 (Cq), 122.4 (Cq), 112.8
(Cq), 103.8 (CH), 61.6 (CH3), 61.5 (CH2), 53.5 (2*CH2), 41.3 (CH2), 34.5
LC-MS (ESI) tR ¼ 3.56 min; m/z [MþH]þ 353.60; HRMS (ESI) m/z
calcd. for C23H33N2O [MþH]þ 353.2587, found 353.2585.

244
J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
3-[1-(Cyclopentylmethyl)-4-piperidyl]-1-(1H-indol-5-yl)
bromide (20 mL, 0.17 mmol, 1.1 eq.) and K2CO3 (83 mg, 0.60 mmol,
propan-1-one (6f). The compound was prepared from 1-(4-amino-
3-iodo-phenyl)-3-[1-(cyclopentylmethyl)-4-piperidyl]propan-1-one
4f (160 mg, 0.36 mmol) and ethynyltrimethylsilane (155 mL,
1.1 mmol) according to method D, refluxing for 17 h under argon
atmosphere for the first step, and then stirring for 1 h at room
temperature for the second step. The residue was purified by flash
chromatography on silica gel column (cyclohexane 100% to EtOAc
100%) to give 6f as a pale yellow solid (26% yield); mp 151 ꢀC,1H NMR
4.0 eq.) and the reaction mixture was stirred at room temperature
overnight. After removal of DMF, the residue was dissolved with
EtOAc and washed with brine. The organic layer was dried over
MgSO4, concentrated in vacuo and the residue was purified by
chromatography on silica gel (EtOAc/MeOH gradient 100:0 to
90:10), to give 10b as a white solid (83% yield); mp 83 ꢀC; 1H NMR
(CDCl3, 400 MHz)
d 7.46 (s, 1H), 7.28e7.17 (m, 3H), 7.01 (d,
3J ¼ 3.3 Hz, 1H), 6.99e6.94 (m, 2H), 6.69 (d, 3J ¼ 3.3 Hz, 1H), 5.69 (s,
2H), 4.02 (s, 3H), 2.98 (t, 3J ¼ 7.5 Hz, 2H), 2.93e2.83 (m, 2H),
2.17e2.07 (m, 2H), 1.95e1.83 (m, 2H), 1.75e1.56 (m, 10H), 1.52e1.43
(m, 1H), 1.33e1.25 (m, 2H), 1.16e1.07 (m, 3H), 0.89e0.76 (m, 2H);
(MeOD-d4, 500 MHz)
d
8.33 (m, 1H), 7.80 (dd, 3J ¼ 8.7 Hz, 4J ¼ 1.7 Hz,
1H), 7.44 (d, 3J ¼ 8.7 Hz, 1H), 7.34 (d, 3J ¼ 3.2 Hz, 1H), 6.61 (dd,
3J ¼ 3.2 Hz, 4J ¼ 0.9 Hz, 1H), 3.10 (m, 2H), 3.03 (m, 2H), 2.40 (d,
3J ¼ 7.0 Hz, 2H), 2.13e2.05 (m, 3H), 1.85e1.79 (m, 4H), 1.69 (m, 2H),
1.66e1.61 (m, 2H),1.58e1.54 (m, 2H),1.41 (m,1H),1.38e1.28 (m, 2H),
13C NMR (CDCl3, 100 MHz)
d 201.0 (CO), 153.2 (Cq), 138.2 (Cq), 135.0
(Cq), 131.0 (CH), 128.8 (2*CH), 127.7 (CH), 126.3 (2*CH), 124.9 (CH),
124.4 (Cq), 123.0 (Cq), 112.2 (Cq), 101.9 (CH), 65.7 (CH2), 62.0 (CH3),
54.3 (2*CH2), 51.7 (CH2), 40.4 (CH2), 35.3 (CH), 34.9 (CH), 32.1
(2*CH2), 31.6 (CH2), 31.0 (2*CH2), 26.6 (CH2), 26.1 (2*CH2); IR (neat,
1.23e1.16 (m, 2H); 13C NMR (MeOD-d4, 126 MHz)
d 203.4 (CO), 140.5
(Cq), 130.0 (Cq), 129.0 (Cq), 127.5 (CH), 123.7 (CH), 122.5 (CH), 112.2
(CH), 104.3 (CH), 65.9 (CH2), 55.2 (2*CH2), 38.3 (CH), 36.6 (CH2), 36.4
(CH), 32.9 (2*CH2), 32.7 (CH2), 32.5 (2*CH2), 26.1 (2*CH2); IR (neat,
cmꢁ1
) n 2918, 2851, 2804, 2763, 1656, 1589, 1481, 1449, 1329, 1288,
cmꢁ1
)
n
3301, 2924, 2863, 1663, 1610,1574,1453,1328,1126, 729; LC-
732; LC-MS (ESI) tR ¼ 4.27 min; m/z [MþH]þ 507.63/509.60; HRMS
MS (ESI) tR ¼ 3.38 min; m/z [MþH]þ 339.58; HRMS (ESI) m/z calcd.
(ESI) m/z calcd. for
507.2780.
C
31H40ClN2O2 [MþH]þ 507.2773, found
for C22H31N2O [MþH]þ 339.2431, found 339.2430.
3-(1-Butyl-4-piperidyl)-1-(1H-indol-5-yl)propan-1-one (6g).
The compound was prepared from 1-(4-amino-3-iodo-phenyl)-3-
(1-butyl-4-piperidyl)propan-1-one 4g (175 mg, 0.42 mmol) and
1-[1-(Benzenesulfonyl)-7-chloro-4-methoxy-indol-5-yl]-3-
[1-(cyclohexylmethyl)-4-piperidyl]propan-1-one (10c). To a so-
lution of 6a (28 mg, 0.068 mmol, 1.0 eq.) in CH2Cl2 (1 mL) were
ethynyltrimethylsilane (178
mL, 1.26 mmol) according to method D,
added phenylsulfonyl chloride (14 mL, 0.074 mmol, 1.1 eq.), an
refluxing for 18 h under argon atmosphere for the first step, and
then stirring for 1 h at room temperature for the second step. The
residue was purified by flash chromatography on silica gel column
(cyclohexane 100% to EtOAc 100%) to give 6g as a yellow oil (33%
aqueous solution of NaOH (60% weight, 0.6 mL) and (nBu)4NHSO4
(1 mg, 0.0034 mmol, 5 mol%). The reaction mixture was stirred at
room temperature for 2 h then diluted with water. The organic
layer was separated, dried over MgSO4 and concentrated in vacuo.
The residue was purified by chromatography on silica gel (EtOAc/
MeOH, gradient 100:0 to 90:10), to give 7c as a yellow oil (84%
yield); 1H NMR (MeOD-d4, 500 MHz)
d 8.34 (m, 1H), 7.80 (dd,
3J ¼ 8.7 Hz, 4J ¼ 1.7 Hz, 1H), 7.44 (d, 3J ¼ 8.6 Hz, 1H), 7.34 (d,
3J ¼ 3.2 Hz, 1H), 6.61 (dd, 3J ¼ 3.2 Hz, 4J ¼ 0.8 Hz, 1H), 3.56 (m, 2H),
3.15 (t, 3J ¼ 7.2 Hz, 2H), 3.05 (m, 2H), 2.91 (m, 2H), 2.05 (m, 2H),
1.76e1.67 (m, 5H), 1.51e1.37 (m, 4H), 0.99 (t, 3J ¼ 7.4 Hz, 3H); 13C
yield); 1H NMR (CDCl3, 500 MHz)
d
7.87 (d, 3J ¼ 3.8 Hz, 1H),
7.77e7.74 (m, 2H), 7.57e7.54 (m, 1H), 7.47e7.43 (m, 2H), 7.44 (s,
1H), 6.84 (d, 3J ¼ 3.8 Hz, 1H), 3.96 (s, 3H), 3.32e3.22 (m, 4H), 2.95 (t,
3J ¼ 7.3 Hz, 2H), 2.58e2.50 (m, 2H), 1.80e1.72 (m, 3H), 1.71e1.63 (m,
7H), 1.62e1.56 (m, 2H), 1.49e1.43 (m, 1H), 1.22e1.16 (m, 3H),
NMR (MeOD-d4, 126 MHz) d 202.6 (CO), 140.5 (Cq), 129.9 (Cq), 129.0
(Cq), 127.6 (CH), 123.7 (CH), 122.4 (CH), 112.2 (CH), 104.3 (CH), 58.1
(CH2), 54.1 (2*CH2), 36.1 (CH2), 34.6 (CH), 31.7 (CH2), 30.8 (2*CH2),
1.01e0.91 (m, 2H); 13C NMR (CDCl3, 126 MHz)
d 200.0 (CO), 152.0
27.1 (CH2), 20.9 (CH2), 13.9 (CH3); IR (neat, cmꢁ1
)
n
3435, 2959,
(Cq), 139.7 (Cq), 134.9 (Cq), 133.9 (CH), 130.4 (CH), 129.3 (2*CH),
128.0 (Cq), 127.8 (CH), 127.1 (2*CH), 126.7 (Cq), 114.4 (Cq), 105.2 (CH),
70.5 (CH2), 62.7 (CH3), 53.4 (2*CH2), 39.7 (CH2), 34.0 (CH), 33.6 (CH),
31.9 (2*CH2), 29.9 (CH2), 28.9 (2*CH2), 25.9 (CH2), 25.6 (2*CH2); IR
2927, 2857, 1679, 1206, 1130, 736; LC-MS (ESI) tR ¼ 3.22 min; m/z
[MþH]þ 313.55; HRMS (ESI) m/z calcd. for C20H29N2O [MþH]þ
313.2274, found 313.2272.
1-(7-Chloro-4-methoxy-1-methyl-indol-5-yl)-3-[1-(cyclo-
hexylmethyl)-4-piperidyl]propan-1-one (10a). To a solution of 6a
(neat, cmꢁ1
) n 3455, 3351, 2986, 2938, 2638, 1723, 1603.5, 1319,
1275, 1181; LC-MS (ESI) tR ¼ 4.25 min; m/z [MþH]þ 557.59/559.60;
HRMS (ESI) calcd. for C30H38ClN2O4S [MþH]þ 557.2234, found
557.2235.
(56 mg, 0.13 mmol, 1.0 eq.) in DMF (1.8 mL) were added CH3I (10 mL,
0.15 mmol, 1.1 eq.) and K2CO3 (74 mg, 0.54 mmol, 4 eq.) and the re-
action mixture was stirred at room temperature overnight. After
removal of DMF, the residue was dissolved with EtOAc and washed
with brine. The organic layer was dried over MgSO4, concentrated in
vacuo and the residue was purified by chromatography on silica gel
(EtOAc/MeOH gradient 100:0 to 90:10) to give 10a as a brown solid
1-(1-Benzylindol-5-yl)-3-[1-(cyclohexylmethyl)-4-piperidyl]
propan-1-one (10d). To a solution of 6e (100 mg, 0.28 mmol, 1.0
eq.) in DMF (4 mL) were added benzyl bromide (38 mL, 0.31 mmol,
1.1 eq.) and K2CO3 (155 mg, 1.12 mmol, 4.0 eq.) and the reaction
mixture was stirred at room temperature overnight. After removal
of DMF, the residue was dissolved with EtOAc and washed with
brine. The organic layer was dried over MgSO4, concentrated in
vacuo and the residue was purified by chromatography on silica gel
(EtOAc/MeOH gradient 100:0 to 60:40), to give 10d as a white solid
(77% yield); mp 90 ꢀC; 1H NMR (CDCl3, 400 MHz)
d 7.55 (s, 1H), 6.98
(d, 3J ¼ 3.2 Hz, 1H), 6.67 (d, 3J ¼ 3.2 Hz, 1H), 4.15 (s, 3H), 4.08 (s, 3H),
3.11e3.00 (m, 2H), 3.06 (t, 3J ¼ 7.4 Hz, 2H), 2.35e2.23 (m, 2H),
2.16e2.01 (m, 2H), 1.86e1.62 (m, 10H), 1.59e1.48 (m, 2H), 1.45e1.35
(m, 1H), 1.31e1.10 (m, 3H), 1.01e0.86 (m, 2H); 13C NMR (CDCl3,
(86% yield); mp 98 ꢀC; 1H NMR (CDCl3, 500 MHz)
d 8.32 (m, 1H),
100 MHz)
d
200.9 (CO), 153.3 (Cq), 135.5 (Cq), 131.6 (CH), 124.3 (CH),
7.85 (dd, 3J ¼ 8.7 Hz, 4J ¼ 1.7 Hz, 1H), 7.33e7.27 (m, 4H), 7.19 (d,
3J ¼ 3.2 Hz, 1H), 7.10 (m, 2H), 6.66 (dd, 3J ¼ 3.2 Hz, 4J ¼ 0.8 Hz, 1H),
5.35 (s, 2H), 3.04 (m, 2H), 2.86 (m, 2H), 2.08 (d, 3J ¼ 7.0 Hz, 2H), 1.84
(m, 2H), 1.77e1.64 (m, 9H), 1.48 (m, 1H), 1.34e1.11 (m, 6H), 0.86 (m,
124.1 (Cq), 122.6 (Cq), 112.5 (Cq), 100.9 (CH), 65.4 (CH2), 61.9 (CH3),
54.1 (2*CH2), 40.3 (CH2), 36.7 (CH3), 35.0 (CH), 34.7 (CH), 32.0
(2*CH2), 31.1 (CH2), 30.9 (2*CH2), 26.5 (CH2), 26.0 (2*CH2); IR (neat,
cmꢁ1
)
n
2924, 2848, 2804, 2766, 1662, 1589, 1487, 1452, 1284, 1011,
2H); 13C NMR (CDCl3, 126 MHz)
d 200.7 (CO), 138.9 (Cq), 137.0 (Cq),
729; LC-MS (ESI) tR ¼ 4.04 min; m/z [MþH]þ 431.60/433.62; HRMS
(ESI) m/z calcd. for C25H36ClN2O2 [MþH]þ 431.2460, found 431.2458.
1-(1-Benzyl-7-chloro-4-methoxy-indol-5-yl)-3-[1-(cyclo-
hexylmethyl)-4-piperidyl]propan-1-one (10b). To a solution of 6a
(63 mg, 0.15 mmol, 1.0 eq.) in DMF (2 mL) were added benzyl
129.9 (CH), 129.7 (Cq), 129.0 (2*CH), 128.4 (Cq), 128.0 (CH), 126.9
(2*CH), 123.0 (CH), 122.2 (CH), 109.7 (CH), 103.8 (CH), 66.4 (CH2),
54.7 (2*CH2), 50.5 (CH2), 36.0 (CH2), 35.9 (CH), 35.5 (CH), 32.5
(2*CH2), 32.3 (2*CH2), 31.7 (CH2), 27.0 (CH2), 26.4 (2*CH2); IR (neat,
cmꢁ1
) n 3031, 2919, 2850, 1675, 1452, 1133, 730, 718; LC-MS (ESI)

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
245
tR ¼ 4.16 min; m/z [MþH]þ 443.75; HRMS (ESI) m/z calcd. for
concentrations of [3H]GR113808 were used to give final concen-
trations of 0.0625e2 nM, and nonspecific binding of [3H]GR113808
C
30H39N2O [MþH]þ 443.3057, found 443.3054.
Methyl 7-chloro-4-methoxy-1H-indole-5-carboxylate (12).
was defined in the presence of 30
mM serotonin to determine the Kd
To
a
stirred solution of methyl 4-amino-5-chloro-3-iodo-2-
and the Bmax. For competition studies, 2.5 mg of proteins (5-HT4B
methoxy-benzoate 11 (1.5 g, 4.4 mmol, 1.0 eq.), Pd(PPh3)4
(508 mg, 0.44 mmol, 10 mol%), XPhos (420 mg, 0.88 mmol, 20 mol
%) and K2CO3 (1.22 g, 8.8 mmol, 2.0 eq.) in dry THF (60 mL) was
added ethynyltrimethylsilane (1.87 mL, 13.2 mmol, 3.0 eq.) under
argon atmosphere and the reaction mixture was refluxed for 24 h.
After evaporation in vacuo to remove THF, the residue was dis-
solved with EtOAc and washed with brine. The organic layer was
dried over MgSO4, and concentrated under reduced pressure. To a
stirred solution of previous crude in CH2Cl2 (60 mL) was added
TFA (6 mL). The resulting mixture was stirred at room temperature
for 1 h, and then concentrated in vacuo. The crude was purified by
flash chromatography on silica gel column (cyclohexane/DCM
50:50 to DCM/EtOAc 95:5) and concentrated under reduced
pressure to afford 12, as a brown solid (45% yield); mp 109 ꢀC; 1H
membrane preparations, HTS110M, Millipore. Millipore's 5-HT4B
membrane preparations are crude membrane preparations made
by use of their proprietary stable recombinant 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 10ꢁ6 or 10ꢁ8M
of each compound together with 0.2 nM [3H]-GR 113808 (VT 240,
ViTrax) in 25 mM Tris buffer (pH 7.4, 25 ꢀC). At the end of the in-
cubation, 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). Non-
specific binding was evaluated in parallel, in the presence of
30
mM serotonin.
For some of these compounds, affinity constants were calculated
NMR (CDCl3, 400 MHz)
6.80 (m, 1H), 4.10 (s, 3H), 3.92 (s, 3H); 13C NMR (CDCl3, 100 MHz)
166.6 (CO), 154.5 (Cq), 137.1 (Cq), 125.1 (CH), 124.1 (CH), 122.7
(Cq), 114.6 (Cq), 111.4 (Cq), 103.2 (CH), 62.1 (CH3), 52.2 (CH3); IR
(neat, cmꢁ1
3366, 3019, 2993, 2948, 2841, 1690, 1606, 1435,
d
8.75 (br s, 1H), 7.71 (s, 1H), 7.24 (m, 1H),
from five-point inhibition curves using the EBDA-Ligand software
and expressed as Ki SD.
d
5.2.2. Functional assay: cyclic AMP radioimmunoassay
)
n
For measurement of intracellular cyclic AMP accumulation,
stably transfected cells were grown to confluence and were incu-
bated with serum-free medium 4 h before the beginning of the
assay. Then, the cells were preincubated for 15 min with serum-free
medium supplemented with 5 mM theophylline, 10 mM pargyline
and 1 mM GR127935 in CHO cells to block the activity of endoge-
nous 5-HT1B receptors. 5-HT or 6a were then added for an addi-
tional 15 min. The reaction was stopped by aspiration of the
medium and addition of 500 ml of ice-cold ethanol. After 30 min
incubation at room temperature, the ethanol fraction was collected
and evaporated under vacuum. The pellet was reconstituted and
cyclic AMP was quantified using a radio-immunoassay kit (cyclic
AMP competitive radioimmunoassay, Immunotech, Marseille,
France). Student's t-tests were performed using the QuickTTest
software according to the CEREP cellular functional assay [48].
1312, 1242, 974, 770; HRMS (ESI) m/z calcd. for C11H11ClNO3
[MþH]þ 240.0422, found 240.0421.
Methyl 4-methoxy-1H-indole-5-carboxylate (13). To a stirred
solution of methyl 7-chloro-4-methoxy-1H-indole-5-carboxylate 12
(290 mg,1.21 mmol,1.0 eq.) in MeOH (15 mL) was added ammonium
formate (763 mg, 12.1 mmol, 10.0 eq.) and then 10% Pd/C (129 mg,
10 mol%) was added under nitrogen. The resulting mixture was
refluxed overnight, the catalyst was filtered through celite and the
filtrate was concentrated under vacuum. The residue was dissolved
with ethyl acetate, and washed with brine. The organic layer was
dried over MgSO4 and concentrated under reduced pressure to
afford 13, as a white solid (82% yield); mp 150 ꢀC; 1H NMR (CDCl3,
400 MHz)
d
8.51 (br s, 1H), 7.70 (d, 3J ¼ 8.6 Hz, 1H), 7.20 (m, 1H), 7.13
3
(d, J ¼ 8.5 Hz, 1H), 6.77 (m, 1H), 4.12 (s, 3H), 3.92 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d 167.7 (CO), 155.6 (Cq), 140.4 (Cq),125.5 (CH),124.5
(CH), 121.6 (Cq), 113.7 (Cq), 106.7 (CH), 101.9 (CH), 61.9 (CH3), 52.0
5.2.3. In vitro measurement of AChE activity
(CH3); IR (neat, cmꢁ1
)
n
3335, 3127, 2990, 2943, 2832, 1705, 1607,
Inhibitory capacity of the novel ligands synthesized towards
AChE biological activity was evaluated spectrometrically by the
Ellman method [49]. Acetylthiocholine-iodide (ATC) and 5,5-
dithiobis-(2-nitrobenzoic) acid (DTNB) were purchased from
Sigma Aldrich. AChE purified from human erythrocytes (buffered
aqueous solution, ꢃ500 units/mg protein (BCA), Sigma Aldrich) was
diluted in 0.1% Triton X-100/20 mM HEPES, pH 8, to yield an
enzyme stock solution with 0.25 unit/mL activity. In the procedure,
1431, 1349, 1277, 1192, 1055, 763; HRMS (ESI) m/z calcd. for
C
11H12NO3 [MþH]þ 206.0812, found 206.0812.
4-Methoxy-1H-indole-5-carboxylic acid (14). To a stirred so-
lution of methyl 4-methoxy-1H-indole-5-carboxylate 13 (430 mg,
2.10 mmol, 1.0 eq.) in EtOH (20 mL) was added a 1N NaOH solution
(21 mL, 21 mmol, 10.0 eq.) under nitrogen atmosphere at room
temperature. The resulting mixture was stirred overnight at room
temperature, and then concentrated in vacuo to remove EtOH. The
residue was diluted with water. The aqueous layer was acidified by
addition of a HCl solution until acidic pH, and extracted several
times with AcOEt. The combined organic extract was washed with
brine, dried over MgSO4 and concentrated under reduced pressure
to give 14 as a pale yellow solid (83% yield); mp 163 ꢀC; 1H NMR
100 mL aliquots of 0.3 mM DTNB dissolved in phosphate buffer pH
7.4 were dispended into a 96 wells plate followed by 50
appropriate dilution of the test compound solution and 50
m
L of an
mL of the
enzyme stock. After 5 min preincubation at 25 ꢀC, the reaction was
initiated by the injection of 50 mL of 10 mM ATC solution. The hy-
drolysis of ATC 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 ATC, at 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 absorbance
at 412 nm was monitored at 1-min intervals for 10 min. Assays were
performed with a blank containing all components except ace-
tylthiocholine, in order to correct for non-enzymatic hydrolysis.
The percent inhibition due to the presence of the test compounds
was calculated by the following expression: 100 - (vi/v0 x 100)
where vi is the rate calculated in the presence of the inhibitor, and
v0 is the activity in its absence.
(MeOD-d4, 400 MHz)
d
7.69 (d, 3J ¼ 8.8 Hz, 1H), 7.29 (d, 3J ¼ 3.2 Hz,
1H), 7.18 (d, 3J ¼ 8.8 Hz, 1H), 6.74 (d, 3J ¼ 3.3 Hz, 1H), 4.17 (s, 3H); 13
C
NMR (MeOD-d4, 100 MHz) d 170.6 (CO), 156.3 (Cq), 142.9 (Cq), 126.6
(CH), 125.9 (CH), 121.5 (Cq), 112.5 (Cq), 108.2 (CH), 101.8 (CH), 62.1
(CH3); IR (neat, cmꢁ1
) n 3211, 3156, 3016, 2964, 2847, 1706, 1606,
1362, 1223, 720; HRMS (ESI) m/z calcd. for C10H10NO3 [MþH]þ
192.0655, found 192.0651.
5.2. In vitro evaluation
5.2.1. Pharmacological characterization of drugs on human 5-HT4R
The method was validated by saturation studies: six

246
J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
Initial screening for inhibitory capacity was performed at 10ꢁ6
and fitted into the density using Phenix.ligand_fit. Crystal struc-
tures could be obtained from the pdb for both structure 6a (pdb
entry 6EZH) and 6b (pdb entry 6EZH).
or 10ꢁ5 M concentrations of compounds studied. For those dis-
playing ꢃ50% inhibition, IC50 values were determined graphically,
by plotting % inhibition versus the logarithms of six inhibitor con-
centrations tested, using Origin software.
5.4. In vivo evaluation
5.2.4. In vitro tests of hAChE kinetic studies
5.4.1. Animals
Kinetic studies of compounds on AChE biological activity was
evaluated through the use of the spectrometric method of Ellman.
Acetylthiocholine iodide, lyophilized human Acetylcholinesterase
and 5,5-dithiobis-(2-nitrobenzoic) acid (DTNB) were purchased
from Sigma Aldrich. AChE was dissolved in 0.2 M phosphate buffer
pH 7.4 such as to have enzyme solutions stock with 0.25 units/ml
Male Swiss mice, 6 weeks old and weighing 32 2 g, from
JANVIER (Saint Berthevin, France), were kept for housing and ex-
periments taken place within the animal facility building of the
University of Montpellier (CECEMA, Office of Veterinary Services
agreement # B-34-172-23). Animals were housed in groups with
access to food and water ad libitum, except during behavioral ex-
AChE activity. In the procedure, 100
phosphate buffer pH 7.4 were added into the 96 wells plate fol-
lowed by 50 L of test compound solution and 50 L of enzyme
solution. After 5 min of preincubation, the reaction was then initi-
ated by the addition of 50 L of different concentrations of ace-
tylthiocholine iodide solution (from 0.02 to 0.2 mM). The hydrolysis
of acetylthiocholine 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 ace-
tylthiocholine, at a wavelength of 412 nm every minute for 10 min
using a 96-well microplate plate reader (TECAN Infinite M200,
Lyon, France). Test compounds were dissolved in analytical grade
DMSO.
m
L of 0.3 mM DTNB dissolved in
periments. They were kept in a temperature and humidity
controlled animal facility on a 12 h/12 h light/dark cycle (lights off
at 07:00 p.m.). Mice were numbered by marking their tail using
permanent markers. All animal procedures were conducted in
strict adherence to the European Union Directive of September 22,
2010 (2010/63/UE).
m
m
m
5.4.2. Experimental design
Groups of 12e16 mice each were included in this study. Com-
pounds were solubilized in DMSO 10% in saline at the stock solution
concentration (5 mg/ml), then diluted in saline. Control vehicle
solution was DMSO 2% in saline. Compounds or the vehicle solution
was injected intraperitoneally (IP), 10 min before dizocilpine
(0.15 mg/kg) or physiological saline, IP, 20 min before the behav-
ioral test (Y-maze test session or passive avoidance training
session).
The kinetic studies were performed using four concentrations of
inhibitor (0e1 mM).
5.2.5. Sigma-1 receptor affinity
The binding assays were performed by CEREP (Poitiers, France),
according to Ganapathy [50]. The Sigma-1 receptor binding assay
was carried out by incubating Jurkat cell membranes (10e20 mg
protein per tube) with [3H](þ)-pentazocine (15 nM) and a range of
concentrations of test ligands, at 37 ꢀC for 2 h, in 5 mM Tris/HCl
buffer (pH ¼ 7.4). Bound radioactivity was measured using liquid
scintillation counting. Inhibition constants (Ki) were calculated
from the IC50 values according to the method of Cheng and Prusoff
[51].
5.4.3. Spontaneous alternation performances
All animals were tested for spontaneous alternation perfor-
mance in the Y-maze, an index of spatial working memory. The Y-
maze is made of grey polyvinylchloride. Each arm is 40 cm long,
13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and
converging at an equal angle. Each mouse was placed at the end of
one arm and allowed to move freely through the maze during an
8 min session. The series of arm entries, including possible returns
into the same arm, were checked visually. An alternation is defined
as entries into all three arms on consecutive occasions. The number
of maximum alternations is therefore the total number of arm
entries minus two and the percentage of alternation was calculated
as (actual alternations/maximum alternations) x 100. Parameters
included the percentage of alternation (memory index) and total
number of arm entries (exploration index) [45,54]. Animals that
showed an extreme behavior (Alternation percentage <20% or
>90% or number of arm entries < 10) were discarded. In the present
study, 8 animals were discarded accordingly, corresponding to 8.3%
attrition. Data were analyzed using a parametric ANOVA, followed
by a Dunnett's post-hoc test for multiple comparison.
5.3. X-ray crystallography
Torpedo californica acetylcholinesterase (TcAChE) was purified
by Lilly Toker in the Silman laboratory, as described [52].
Cholinesterase inhibitors 6a and 6b were synthesized by the
CERMN in Caen (France).
5.3.1. Crystallization of TcAChE and soaking procedure
Orthorhombic crystals (spacegroup P212121) of TcAChE were
grown at 4 ꢀC with the hanging drop vapor diffusion method using
36% PEG200/150 mM MES pH 5.8, and a protein concentration of
10 mg/ml. After 5e10 days, orthorhombic crystal plates had grown
5.4.4. Passive avoidance test
to a size of around 300 ꢂ 50
m
m.
The apparatus is a two-compartments (15 ꢂ 20 ꢂ 15 cm high)
box with one illuminated with white polyvinylchloride walls and
the other darkened with black polyvinylchloride walls and a grid
floor. A guillotine door separates each compartment. A 60 W lamp
positioned 40 cm above the apparatus lights up the white
compartment during the experiment. Scrambled footshocks
(0.3 mA for 3 s) could be delivered to the grid floor using a shock
generator scrambler (Lafayette Instruments, Lafayette, USA). The
guillotine door was initially closed during the training session. Each
mouse was placed into the white compartment. After 5 s, the door
was raised. When the mouse entered the darkened compartment
and placed all its paws on the grid floor, the door was closed and the
footshock delivered for 3 s. The step-through latency, that is, the
Crystals were soaked for 24e36 h in the mother liquor con-
taining 5 mM 6a or 6b, with a final concentration of 10% DMSO.
They were then mounted on a standard cryo-loop and flash cooled
in liquid nitrogen.
Data collection was performed at the ESRF in Grenoble on
beamlines ID14-4 and id23-1. Data collection information and
statistics are displayed in Table S1. Data were processed using XDS/
XSCALE, and amplitude factors were generated using XDSCONV.
Molecular replacement was performed with PhaserMR, taking as a
model the crystal structure of native TcAChE in the orthorhombic
spacegroup (pdb entry 1W75). The ligand was modeled using
Phenix.elbow [53] with the corresponding SMILES string as input

J. Lalut et al. / European Journal of Medicinal Chemistry 162 (2019) 234e248
247
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Funding sources
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This work was supported by funding from the French Agence
Nationale de la Recherche Projects MALAD ANR-12-JS007-0012-01
ꢀ
and Ligue Europeenne Contre la Maladie d’Alzheimer Grant 12721
ꢀ
ꢀ
ꢀ
ꢀ
[17] M.I. Fernandez-Bachiller, C. Perez, N.E. Campillo, J.A. Paez, G.C. Gonzalez-
and the Interreg program AI-Chem Channel. The authors are
grateful for financial support provided by the Regional Council of
Normandy, FEDER.
~
ꢀ
ꢀ
Munoz, P. Usan, E. García-Palomero, M.G. Lopez, M. Villarroya, A.G. García,
A. Martínez, M.I. Rodríguez-Franco, Tacrine-melatonin hybrids as multifunc-
tional agents for Alzheimer's disease, with cholinergic, antioxidant, and
neuroprotective properties, ChemMedChem 4 (2009) 828e841. https://doi.
org/10.1002/cmdc.200800414.
ꢀ
Abbreviations
[18] O.M. Bautista-Aguilera, S. Hagenow, A. Palomino-Antolin, V. Farre-Alins,
ꢁ
ꢀ
L. Ismaili, P.L. Joffrin, M.L. Jimeno, O. Soukup, J. Janockova, L. Kalinowsky,
ꢀ
E. Proschak, I. Iriepa, I. Moraleda, J.S. Schwed, A. Romero Martínez, F. Lopez-
AD, Alzheimer's disease; ACh, acetylcholine; AChE, acetylcho-
linesterase; AChEI, acetylcholinesterase inhibitor; CAS, catalytic
anionic site; DBS, dual-binding site; DPZ, donepezil; MTDL, multi-
target directed ligands; PAS, peripheral anionic site.
~
Munoz, M. Chioua, J. Egea, R.R. Ramsay, J. Marco-Contelles, H. Stark, Multi-
target-directed ligands combining cholinesterase and monoamine oxidase
inhibition with histamine H3R antagonism for neurodegenerative diseases,
Angew. Chem., Int. Ed. Engl. 56 (2017) 12765e12769. https://doi.org/10.1002/
anie.201706072.
~
[19] E. Nepovimova, E. Uliassi, J. Korabecny, L.E. Pena-Altamira, S. Samez,
A. Pesaresi, G.E. Garcia, M. Bartolini, V. Andrisano, C. Bergamini, R. Fato,
D. Lamba, M. Roberti, K. Kuca, B. Monti, M.L. Bolognesi, Multitarget drug
design strategy: quinoneetacrine hybrids designed to block Amyloid-В ag-
gregation and to exert anticholinesterase and antioxidant effects, J. Med.
Chem. 57 (2014) 8576e8589. https://doi.org/10.1021/jm5010804.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.10.064.
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