Design, synthesis, and biological evaluation of new 5-ht4 receptor agonists: Application as amyloid cascade modulators and potential therapeutic utility in alzheimer's disease

Article abstract of DOI:10.1021/jm801327q

Serotonin 5-HT₄ receptor (5-HT₄R) agonists are of particular interest for the treatment of Alzheimer's disease because of their ability to ameliorate cognitive deficits and to modulate production of amyloid β-protein (Aβ). However, d

Full text of DOI:10.1021/jm801327q

2214
J. Med. Chem. 2009, 52, 2214–2225
Design, Synthesis, and Biological Evaluation of New 5-HT4 Receptor Agonists: Application as
Amyloid Cascade Modulators and Potential Therapeutic Utility in Alzheimer’s Disease
Olivier Russo,†,| Marthe Cachard-Chastel,§,| Ce´line Rivie`re,# Mireille Giner,†,| Jean-Louis Soulier,†,| Magali Berthouze,‡,|,
,O
Tristan Richard,# Jean-Pierre Monti,# Sames Sicsic,†,| Frank Lezoualc’h,‡,|, and Isabelle Berque-Bestel*,
CNRS UMR C 8076 (BioCIS), Mole´cules Fluore´es et Chimie Me´dicinale, UMR-S769, EA3544, Se´rotonine et Neuropharmacologie, IFR-141,
Faculte´ de Pharmacie, UniVersite´ Paris-Sud, F- 92296 Chaˆtenay-Malabry, France, INSERM U769, Signalisation et Physiopathologie
Cardiaque, F-92296 Chaˆtenay-Malabry, France, Laboratoire de Physique et Biophysique, GESVAB, EA3675, ISVV, UniVersite´ Victor Segalen,
F-33076 Bordeaux, France, INSERM U869, UniVersite´ Victor Segalen, Bordeaux 2, F-33076 Bordeaux, France, ARNA, UniVersite´ Victor
Segalen, F-33076 Bordeaux, France
ReceiVed October 20, 2008
Serotonin 5-HT4 receptor (5-HT4R) agonists are of particular interest for the treatment of Alzheimer’s disease
because of their ability to ameliorate cognitive deficits and to modulate production of amyloid ꢀ-protein
(Aꢀ). However, despite the range of 5-HT4R agonists synthesized to date, potent and selective 5-HT4R
agonists are still lacking. In the present study, two libraries of molecules based on the scaffold of ML10302,
a highly specific and partial 5-HT4R agonist, were efficiently prepared by parallel supported synthesis and
their binding affinities and agonist activities evaluated. Furthermore, we showed that, in vivo, the two best
candidates exhibited neuroprotective activity by increasing the level of the soluble form of the amyloid
precursor protein (sAPPR) in the cortex and hippocampus of mice. Interestingly, one of these compounds
could also inhibit Aꢀ fibril formation in vitro.
Introduction
a
The serotonin 5-HT4 receptor (5-HT4R) is one of the seven
subtypes of serotonin receptors that constitute the largest single
family of G-protein coupled receptors (GPCR). Their widespread
activity is mediated by several splice variants that differ in their
intracellular C-terminal ends and are largely distributed through-
out central and peripheral systems.1,2
Besides important effects on the gastrointestinal and urinary
tracts,3 5-HT4R has gained a lot of attention for its physiological
role in the brain. To date, 5-HT4R has been described as
modulating mood,4 feeding,5 memory, and cognition,2 and these
Figure 1. Design of 5-HT4 agonists.
features could be exploited for therapeutic purposes. Alzheimer’s
disease (AD), for instance, could benefit from the positive effects
of 5-HT4R on learning and memory performances.6 Moreover,
5-HT4R regulates also the production of the neurotoxic amyloid
ꢀ-peptide (Aꢀ), which represents one of the major AD
pathogenic pathways.7 Indeed, 5-HT4R agonists can stimulate
the nonamyloidogenic pathway, leading to the release of the
soluble form of the amyloid precursor protein (sAPPR), which,
in contrast to Aꢀ, has putative neurotrophic and neuroprotective
properties.6,8,9 The role played by 5-HT4R in amyloid precursor
protein (APP) processing as well as in cognitive processes
clearly elucidates the great hope that 5-HT4R agonists represent
for the treatment of AD.
Despite the number of described 5-HT4R agonists belonging
to the benzamide, indole, or benzimidazoline families,1 only
two partial agonists SL 65.0155 (Sanofi-Aventis)10 and PRX-
03140 (Epix Pharmaceuticals)11 have reached phase II clinical
trials for the treatment of AD. Therefore, more work needs to
be done to design and synthesize more effective 5-HT4R
agonists.
* To whom correspondence should be addressed. Phone: +33 5 57 57
10 16. Fax: +33 5 57 57 10 15. E-mail: isabelle.bestel@inserm.fr.
In a recent study, we demonstrated that ML10302,12 a 5-HT4R
partial agonist with a benzoate-based structure, could selectively
enhance sAPPR levels in the hippocampus and cortex of mice.13
We therefore decided to design new, potential 5-HT4R agonists
by tuning the basal structure of ML10302 to favor possible
interactions with Asp3.32, a highly conserved amino acid in
the GPCR family, which is involved both in ligand recognition
and activation of 5-HT4R.14,15 Two libraries of molecules
differing by the absence (library a) or the presence (library b)
of a methylene unit linking a substituted amide group to the
ML10302 piperidine ring were thus prepared by parallel
supported synthesis (Figure 1). One original additional molecule
constituted by two ML10302 pharmacophores linked by a spacer
(compound 14)16 and its monovalent counterpart (molecule 19)
were also prepared in order to highlight the potential benefit of
†
CNRS UMR C 8076 (BioCIS), Mole´cules Fluore´es et Chimie Me´-
dicinale.
‡
UMR-S769.
EA3544, Se´rotonine et Neuropharmacologie.
IFR-141, Faculte´ de Pharmacie, Universite´ Paris-Sud.
§
|
INSERM U769, Signalisation et Physiopathologie Cardiaque.
Laboratoire de Physique et Biophysique, GESVAB, EA3675, ISVV,
#
Universite´ Victor Segalen.
INSERM U869, Universite´ Victor Segalen.
ARNA, Universite´ Victor Segalen.
O
a Abbreviations: 5-HT4R, serotonin 5-HT4 receptor; cAMP, cyclic
adenosine monophosphate; Aꢀ, amyloid ꢀ-protein; AD, Alzheimer’s disease;
(s)APPa, (soluble form of) amyloid precursor protein; BAL, backbone amide
linker; GPCR, G-protein coupled receptor; ML10302, 4-amino-5-chloro-
2-methoxy-benzoic acid 2-piperidin-1-yl-ethyl ester; GR113808, 1-methyl-
1H-indole-3-carboxylic acid 1-(2-methanesulfonylamino-ethyl)-piperidin-
4-ylmethyl ester.
10.1021/jm801327q CCC: $40.75
2009 American Chemical Society
Published on Web 03/31/2009

5-HT4 Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2215
Scheme 1. Synthesis of Building Blocks Aa
We then produced the two amide libraries by solid-phase
synthesis (Scheme 2). The chosen protocol allowed us to
introduce three points of diversity: the aminopiperidines A{a}
and A{b}, the carboxylic acids B{a-x} and a halide C, which
are presented in Figure 2. Specifically, the primary amines A{a}
(hydrochloride salts) and A{b} (hemifumarate salts) were loaded
onto the BAL resin 4 by reductive amination using 3 equiv of
amine salts and 5 equiv of NaBH(OAc)3 in DMF (Scheme 2).
Using this procedure, the loading of resins 5{a} and 5{b} was
quantitative for both aminopiperidines as determined by picric
acid assay.25
a Legend: (i) NsCl, NEt3, CH2Cl2, 0 °C to RT; (ii) HCl/MeOH 4 N, RT;
(iii) PhCHO, toluene, reflux, Dean-Stark trap, 1.5 h; (iv) NsCl, NEt3, 0
°C to RT; (v) KHSO4 aq 1 M; (vi) NaOH, extraction; (vii) fumaric acid,
EtOH, RT.
The acylation step was optimized in parallel. The best results
were obtained with 6 equiv of carboxylic acid (130 mM, final
concentration), 6 equiv of HBTU (130 mM, final concentration),
and 7 equiv of DIEA in a 7/3 mixture of DMF and dichlo-
romethane at room temperature for 18 h. This procedure led to
compounds 6{a; a-x} and 6{b; a-x}. Deprotection of the nosyl
amine (7{a; a-x} and 7{b; a-x}) was realized by treatment
with a DMF solution containing 0.5 M of thiophenol and 1 M
of potassium carbonate in 2 cycles of 30 min/each. The
N-alkylation step to prepare compounds 8{a; a-x} and 8{b;
a-x} was difficult to optimize. Finally, the best conditions
obtained were the use of 5 equiv of bromoester 7 (150 mM,
final concentration) and 7 equiv of DIEA in DMF in 2 cycles
of 20 h/each at 40 °C. Products 9{a; a-x} and 9{b; a-x} were
cleaved from the BAL resin with a 1/4 mixture of TFA and
dichloromethane and air-dried. Purification of the crude products
was done by ion-exchange chromatography on a sulfonic resin
(DSC-SCX). All compounds (48 molecules in total, distributed
between the two libraries) were then analyzed by LC-MS, and
a selection of compounds (i.e., half of the library) was analyzed
by 1H NMR. Overall yields, HPLC purities, and mass data are
presented in Table 1.
the “bivalent-ligand” approach on agonist activity.17-19 Because
molecules 14 and 19 do not have a methylene unit linking the
ML10302 piperidine ring to the amide group, these molecules
were classified as belonging to library a. Binding affinity and
functional activity of all synthesized molecules were then
evaluated in C6 glial cells that stably express 5-HT4R. In
addition, the possible neuroprotective effects of the best
candidates were investigated in vivo (i.e., APP processing in
the hippocampus and cortex of C57BL/6j mice) and in vitro
(i.e., inhibition of Aꢀ peptide polymerization).
Results
Design and Chemistry. Design. With the aim of synthesizing
new, more active 5-HT4R agonists, we decided to design
molecules structurally based on the scaffold of ML10302, a
specific 5-HT4R partial agonist.12 The observation that mutations
in Asp3.32 (Asp3.32Ala14,20 or Asp3.32Asn21) had no impact
on ML10302 affinity for the receptor suggests that ML10302
interacts very weakly with Asp3.32. Therefore, to develop other
interactions with Asp3.32, we decided to introduce in position
four of ML10302 piperidine ring a substituted amide group.
Preliminary molecular modeling studies (data not shown)
indicated that Asp3.32 could be equidistant from the basic
piperidine amino group and from the amide function of
ML10302, thus allowing the formation of an ionic interaction
with the first and a hydrogen bond with the second. Furthermore,
the substituent could be located in a large hydrophobic pocket
of the 5-HT4R (also called secondary binding site)22 and develop
supplementary interactions potentially favorable to the activation
of the receptor (Figure 1).
Compounds 9{a;b}, 9{a;d}, 9{a;n}, 9{a;t}, 9{a;u}, 9{a;x},
9{b;m}, and 9{b;n} were not further tested due to their low
purity.
Synthesis in Solution. On the basis of the pharmacological
results, compounds 9{a;q} and 9{b;w} were also synthesized
in solution using the same protocol, purified, and fully analyzed
(NMR, LC-MS, elementary analysis).
Conversely, molecules 9{a;y}, 14, and 19 were prepared only
in solution as they were difficult to obtain by solid-phase
synthesis.
Parallel Solid-Phase Synthesis. Two libraries of compounds
that used ML10302 as a scaffold were thus prepared by a parallel
solid-phase synthesis using a BAL resin. Following our synthetic
strategy, first, we synthesized two piperidine moieties (i.e., A{a}
and A{b}) that possessed a protected cyclic nitrogen and a free
exocyclic amine (Scheme 1). Aminopiperidine A{a} was easily
synthesized from the commercially available 4-N-tert-butoxy-
carbonylaminopiperidine 1 by introducing a nosyl group in
dichloromethane and triethylamine.23 The Boc group of the fully
protected compound 2 was then selectively removed with HCl
in MeOH to obtain the monoprotected molecule A{a} in the
form of hydrochloride salt.
The protected aminomethylpiperidine A{b} was prepared
from piperidin-4-ylmethyl-amine 3 (Scheme 1) following a
modified version of an existing protocol for selective protection
of secondary amines in the presence of a primary amine.24 The
primary amine was temporarily neutralized with benzaldehyde,
followed by protection of the intracyclic amine with nosyl
chloride. After hydrolysis of the imine and extraction, the free
primary amine was converted into its hemifumarate salt A{b}.
To prepare molecule 9{a;y} (Scheme 3), amine 1026 was
coupled with 4-nitro-1,8-naphthalimido-N-caproic acid27 using
a general procedure (EDC, HOBt, and NEt3 in dry DMF) to
obtain the nitro compound 11 with very moderate yield. The
nitro group of 11 was then reduced by catalytic hydrogenation
with hydrogen under atmospheric pressure and Raney Ni.
Compound 9{a;y} was thus easily obtained, avoiding the
sensitive reducible chlorine group of the aryl moiety.
Preparation of the bivalent molecule 14 started from the
previously described compound 12.28 Boc protecting groups
were removed by HCl in MeOH, and the resulting, very
hygroscopic hydrochloride salt 13 was converted to the more
stable tetrafumarate salt 14 (Scheme 4).
Tosynthesizeligand19(Scheme5),aSonogashira-Linstrumelle
coupling reaction was performed at room temperature between
the propargylic acid 1528 and iodobenzene, leading to the aryl
propargyl compound 16. After classical hydrogenation on Pd/C
(molecule 17), a condensation with amine 10 using EDC, HOBt,
and ET3N allowed preparation of molecule 18. A Boc protecting

2216 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 2. Solid-Phase Synthesis of Amides 9a
Russo et al.
a Legend: (i) A{a-x} (3 equiv), NaBH(OAc)3 (5 equiv), DMF, RT, 46 h; (ii) barboxylic acids B{a-x} (6 equiv), HBTU (6 equiv), DIEA (7 equiv),
DMF/CH2Cl2 (7/3), RT, 18 h; (iii) PhSH 0.5 M, K2CO3 1 M, DMF, RT, 2 × 30 min; (iv) halide C (5 equiv), DIEA (7 equiv), DMF, 40 °C, 2 × 20 h; (v)
TFA/CH2Cl2 (1/4), RT, 2 h.
Figure 2. Building blocks used for the solid-phase synthesis of amides 9.
group was then removed by HCL in MeOH, and as for the
bivalent molecule 14, the obtained hydrochloride salt was
converted to the more stable fumarate salt 19.
Pharmacological and Functional Properties of the
Synthesized Molecules. Binding affinities of the synthesized
ligands (10 nM/each) were analyzed in C6 glial cells that stably

5-HT4 Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2217
Table 1. Overall Yields, Physicochemical Data, and Purity of Amides 9 Synthesized on Solid-Phase
overall
yield, %
tR
(min)
LC purity,
%
molecular
weight
mass
overall
yield, %
tR
(min)
LC purity,
%
molecular
weight
mass
compd
compd
[M + H]+
[M + H]+
9{a;a}
9{a;b}
9{a;c}
9{a;d}
9{a;e}
9{a;f}
9{a;g}
9{a;h}
9{a;i}
9{a;j}
9{a;k}
9{a;l}
9{a;m}
9{a;n}
9{a;o}
9{a;p}
9{a;q}
9{a;r}
9{a;s}
9{a;t}
9{a;u}
9{a;v}
9{a;w}
9{a;x}
98
41
42
24
38
59
64
27
55
35
28
22
21
30
53
9
66
93
63
24
24
41
18
20
12.26
13.52
14.28
16.15
14.13
13.98
14.26
18.18
16.48
12.78
15.88
18.48
13.82
11.83
17.94
16.63
16.38
19.00
15.09
16.03
18.32
17.93
16.75
18.00
76
28
90
40
81
78
73
93
82
57
79
84
48
35
82
77
75
78
60
25
41
76
58
41
397.18
397.18
411.19
425.21
437.12
437.12
449.15
451.22
459.19
462.17
465.15
465.24
466.13
470.17
473.21
477.15
479.14
487.22
490.16
505.20
517.20
523.09
566.20
628.21
398
398
412
426
438
438
450
452
460
463
466
466
467
471
474
478
480
488
491
506
518
524
567
629
9{b;a}
9{b;b}
9{b;c}
9{b;d}
9{b;e}
9{b;f}
9{b;g}
9{b;h}
9{b;i}
9{b;j}
9{b;k}
9{b;l}
9{b;m}
9{b;n}
9{b;o}
9{b;p}
9{b;q}
9{b;r}
9{b;s}
9{b;t}
9{b;u}
9{b;v}
9{b;w}
9{b;x}
35
36
36
32
37
41
43
36
42
20
21
34
36
19
44
39
44
44
37
35
27
37
11
30
12.64
12.52
14.20
16.02
14.20
14.23
14.53
18.12
15.98
13.28
15.52
20.09
13.79
15.73
18.14
16.17
16.64
19.23
15.46
16.14
18.14
18.05
19.80
22.89
82
83
84
83
68
65
65
77
68
60
58
66
28
18
68
61
57
66
65
58
51
60
55
64
411.19
411.19
425.21
439.22
451.13
451.13
463.17
465.24
473.21
476.18
479.16
479.26
480.14
484.19
487.22
491.16
493.15
501.24
504.18
519.21
531.21
537.10
580.21
642.23
412
412
426
440
452
452
464
466
474
477
480
480
544
485
488
492
494
502
505
520
532
538
581
643
Scheme 3. Synthesis of Amide 9{a;y}a
a Legend: (i) 4-nitro-1,8-naphthalimido-N-caproic acid, EDC, HOBt, NEt3, CH2Cl2, 0 °C to RT, 20 h; (ii) Raney Ni, H2, MeOH, atmospheric pressure,
RT, 14 h.
Scheme 4. Synthesis of Bivalent Ligand 14a
a Legend: (i) HCl/MeOH 4 N, RT, 30 min; (ii) aq sat. Na2CO3, extraction; (iii) fumaric acid, EtOH, RT.
express the human 5-HT4(e/g)R isoform29 (Table 2) using [3H]-
GR113808, a 5-HT4R specific antagonist, as radioligand for the
binding assays. Displacement results revealed that compounds
9{a;a-y} from library a, in which the amide function is directly
attached to the piperidine ring, had weaker binding affinities
than ML10302. At 10 nM, compounds with the highest
displacements (9{a;e}, 9{a;i}, 9{a;o}, 9{a;p}, and 9{a;s}) did
not exceed 30% of the bound radioligand compared to 40-50%
of displacement for ML10302 (Table 2). Conversely, agonists
9{b;a-x} of library b, where the amide function is linked to
the piperidine ring through a methylene unit, showed binding
affinities that were globally better than those of library a, with
9{b;w} and 9{b;q} displacing more than 50% of the bound
radioligand (Table 2).
Then, their functional properties were assessed by measuring
their ability to induce cAMP production at a concentration of
10 nM using a previously reported radioimmunoassay tech-
nique29 (Table 2). In library a, compounds 9{a;q}, 9{a,y}, and
14 induced higher cAMP production than ML10302 (more than
50%). In library b, four molecules (9{b;c}, 9{b;l}, 9{b;s}, and
9{b;w}) also were found to stimulate cAMP production better
than ML10302.
A particular attention was devoted to identify the influence
of the different chemical substituents used on the pharmacologi-

2218 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 5. Synthesis of Molecule 19a
Russo et al.
and sacrificed 90 min after the injection. The level of sAPPR
was then determined by Western blot analysis in extracts from
hippocampus and cortex. Both agonists (5 mg/kg) increased
sAPPR levels in hippocampus (Figure 3A,B) and cortex (Figure
3D,E) as compared to vehicle alone (0 mg/kg). When we
quantified our results in hippocampus and cortex (Figure 3C,F),
we observed that compound 9{a,y} had a stronger effect than
agonist 14 in both. Moreover, the effect of 9{a,y} and 14, at 5
mg/kg, on sAPPR concentration were in the same range or
higher than that previously reported of ML10302, but at 20 mg/
kg,13 confirming the real potential of the proposed molecules.
Effects of 5-HT4 Receptor Agonists on Aꢀ Fibril
Formation. Because compounds 9{a,y} and 14 present struc-
tural similarities with the antiamyloidogenic molecules curcumin
and polyphenols, which possess two phenolic rings linked by a
spacer, we decided to test whether the two 5-HT4R agonists (at
the concentration of 10 µM) could inhibit Aꢀ fibril formation
by using an in vitro assay based on UV-visible measurements.30
Specifically, this assay examines the effects of candidate
molecules on the ꢀ amyloid peptide 25-35 (Aꢀ25-35) that
presents neurotoxic and aggregation properties comparable to
those of Aꢀ.31,32 Curcumin, which has been shown to possess
in vitro33 and in vivo34 antiamyloidogenic activities and to
a Legend: (i) iodobenzene, PdCl2(PPh3)2 5 mol%, CuI 10 mol%, NEt3,
DMF, 80 °C. (ii) H2 (1 bar), Pd/C 10%, MeOH, RT. (iii) 10, HBTU, NEt3,
DMF, RT. (iv) (a) HCl/MeOH 4 N, RT, 30 min; (b) aq sat. Na2CO3,
extraction; (c) fumaric acid, EtOH, RT.
cal properties of the agonists we synthesized. Specifically,
among the agonist with aliphatic substituents, the best activities
were observed with molecules 9{b;c} and 9{b;l} (library b),
which are lipophilic and conformationally flexible. When
aromatic substituents were used, the presence of an aromatic
ring not directly bound to the amide functional group could be
advantageous for agonist activity independently from the
absence/presence of a methylene unit and the size of the aryl
ring (five or six) (see Table 2, agonists 9{a;o}, 9{a;q}, 9{a;s},
9{a;v}, and 9{b;i}, 9{b;k}, 9{b;q}, and 9{b;s}). Among the
agonists with phenyl substituents, 9{b;s} (library b), which has
an electron-withdrawing group, was a better agonist compared
to 9{b;q}, 9{b;t}, and 9{b;v} whose aryl rings were substituted
by electron-donating groups. Conversely, in library a, the
presence of electron-donating groups on the aryl ring, as in
9{a;q}, led to a better agonist activity. Furthermore, in library
b, a polar group, such as a sulfonamide, gave the best agonist
compound (9{b;w}) of this series. However, a bulkier substitu-
ent on the sulfonamide group (9{b;x}) prevented both binding
and activity.
On the basis of these general results, we selected the four
best agonist molecules (i.e., 9{a;q}, 9{a,y}, 14, and 9{b;w})
to determine their Ki, EC50, and Emax values on purified samples
(Table 3). [3H]-GR113808 competition curves of pure 9{a;q}
and 9{b;w} were monophasic, and their affinity values (5 ( 2
nM and 2 ( 1 nM, respectively) were in the same range as that
of ML10302. The dose-response cAMP production measure-
ments revealed EC50 of 18 ( 5 nM for 9{a;q} and 68 ( 12
nM for 9{b;w} and Emax respectively of 29% of 5-HT and 65%
of 5-HT. Although 9{a,y} and 14 displayed lower affinities than
9{a;q} and 9{b;w} (Ki values respectively of 111 ( 16 nM for
9{a,y} and 14 ( 7 nM for 14), their EC50 (4 ( 3 nM for 9{a;y}
and 9 ( 4 nM for 14) and Emax values (63% for 9{a;y} and
70% for 14) were very similar to those of 5-HT (EC50 of 12 (
2 nM and Emax of 100%).29 Interestingly, the bivalent molecule
14, despite a similar 5-HT4R affinity, presented a better Emax
than its monovalent counterpart 19.
33
30
inhibit fibril formation using Aꢀ1-40 or Aꢀ25-35 fragments,
was used as reference.
Compound 9{a,y} did not show any important inhibitory
activity compared to curcumin, whereas the bivalent compound
14 inhibited Aꢀ fibril formation (EC50 value of 9 µM) as well
as curcumin (Table 4).
Discussion
During the last decades, many 5-HT4R agonists have been
synthesized, but so far, only two (SL65.0155 and PRX-03140)
manifest a promising future for the treatment of AD. The aim
of this study was to discover new 5-HT4R agonists to be used
as drug candidates for brain disorders. We therefore prepared
two libraries in which ML10302,12 was used as a scaffold.
Compounds were designed to improve the agonist properties
of ML10302 by introducing a substituted amide group on the
piperidine moiety (position four) of the molecule to facilitate
interactions both with Asp3.32 and with residues of the 5-HT4R
hydrophobic pocket. In addition, one original 5-HT4R bivalent
ligand (14) and its monovalent counterpart (19) were added to
the series of potential agonists to be assessed.
The introduction of a substituted amide function onto the
piperidine ring of ML10302 allowed us to obtain four molecules
with comparable or higher agonist activity than ML10302:
9{a,y}, 14, 9{b,w}, and to a lesser extent 9{a,q} (Figure 4).
The presence of the amide group has been shown to be essential
for the molecules to exhibit agonist properties. For instance, an
analogue of molecule 9{a,y} that lacks the amide group
displayed an antagonist profile.26 Nevertheless, it is worth noting
that for several molecules, the presence of the amide group is
necessary but not sufficient to activate 5-HT4R. Therefore, the
development of supplementary interactions with residues of the
hydrophobic pocket of the 5-HT4R binding site could improve
their agonist activity. This idea is supported by the observation
that three of the best candidates possess a polar group that is
located four or five atoms away from their amido piperidine
ring and can interact with the hydrophobic pocket: a naphtal-
imide group for 9{a,y}, a basic amino group for the bivalent
molecule 14, and a sulfonamide functional group for 9{b,w}
(Figure 4). Interestingly, when this amino group is Boc-
protected, it looses its agonist properties.28 This further high-
According to these data, compound 9{a,y} and the bivalent
molecule 14 were considered the best candidates for in vivo
and in vitro biological experiments to test their neuroprotective
effects.
Effects of 5 HT4 Receptor Agonists on sAPPr Levels in
Mice. To measure in vivo the neuroprotective effects of agonists
9{a,y} and 14, mice were subcutaneously injected with a single
dose of these molecules (5 mg/kg) or vehicle alone (5 mL/kg)

5-HT4 Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2219
Table 2. Binding Affinities to 5-HT4R and Effect on cAMP Production of Amides 9d
b
a Mean of at least two independent determinations made in triplicate. Percentage of displacement of [3H]GR113808 by agonists at 10 nM. c cAMP
production induced by compounds at a 10 nM concentration. Results were normalized to the maximal response induced by 5-HT. All experiments were
performed in C6 glial cells. d n.d.: not determined.

2220 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Russo et al.
Table 3. Ki, EC50, and Emax of Agonists 9{a;q}, 9{a;y}, 14, 19, and 9{b;w}
a Mean ( SEM of at least two or three independent determinations made in triplicate. b Ki values were calculated using the Cheng-Prussof equation from
the IC50 values with the PRISM program. c EC50 values correspond to the concentration of agonist required to obtain half-maximal stimulation of adenylyl
cyclase. d The maximum response produced by each molecule was normalized to the maximum response induced by 5-HT (Emax).
lights the crucial role not only of the amide group but also of
the nature of the amide substituent for 5-HT4R activation.
Finally, the presence of flexibility around the amide group seems
secondary for the agonist activity.
Concerning bivalent molecule 14, it has been previously
reported that bimultivalent ligands could show enhanced binding
and better activity because of entropic considerations.20-22 This
is indeed the case for molecule 14 that presented the best agonist
profile of all synthesized molecules. This result was corroborated
by the fact that its monovalent counterpart 19, constituted by
one ML10302 unit and a truncated spacer, displayed lower
agonist properties.
hippocampus and cortex, the best candidates 14 and 9{a;y} seem
to cross the blood--brain barrier and act on APP processing.
Interestingly, the neuroprotective effects were not always linked
to best biological activities. For instance, the bivalent molecule
14 showed better Ki and Emax than 9{a,y}, but the latter had a
stronger effect on APP processing.
Nevertheless, during in vitro inhibition assays of fibril
formation, bivalent ligand 14 only exhibited an inhibition level
higher than the reference curcumin. The naphtalimide group of
9{a,y}, which we think is important to explain its strong agonist
activity, could be the cause of its weak inhibition of Aꢀ fibril
formation through steric hindrance and rigidity of the naphtal-
imide extremity. On the other hand, the long size of the spacer
of the bivalent compound 14, compared to curcumin and
As previously reported for ML10302 and prucalopride, which
selectively enhanced sAPPR level in vivo in adult mice

5-HT4 Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2221
Figure 3. Study of the effect of compounds 9{a;y} and 14 on sAPPR level in the hippocampus (A, B, and C) and cortex (D, E, and F) in 8 week
old male C57BL/6j mice. The mice (7-9 mice per group) were treated with a single dose of ligand (5 mg/kg for compounds 9{a;y} and 14) or
saline (5 mL/kg). sAPPR and ꢀ-actin levels (used as internal control) were measured in brain homogenates 90 min after the injection. A, B, D, and
E, representative immunoblots illustrating the effects of 5-HT4 agonists (5 mg/kg) or saline (0 mg/kg) on sAPPR levels. C and F, the mean densitometric
values ((SEM) of sAPPR, relatively to ꢀ-actin (used as internal control), are expressed as percentages of the values observed in untreated control
mice. Values for ML10302 were retrieved from a previous work13 in which the agonist was injected subcutaneously at a dose of 20 mg/kg, mice
were sacrificed after 90 min and hippocampus and cortex processed in the same way as for the present study. Data show results of a typical
experiment. One-way ANOVA, F(1,13) ) 19.53; ***p < 0.001 in the hippocampus and F(1,13) ) 10.25; *p0.05 in the cortex for compound 14
as compared to saline treated animals (white bar). One-way ANOVA, F(1,15) ) 6.25; *p0.05 in the hippocampus and F(1,17) ) 26.37; ***p <
0.001 in the cortex for 9{a;y} as compared to saline treated animals (white bar).
Table 4. Inhibition of Aꢀ Fibril Formation
nols and certainly orients the molecule in the same manner to
interact with the Aꢀ25-35 fragment.
Conclusions
We have prepared new 5-HT4R agonists mainly synthesized
by a rapid and efficient parallel solid-phase strategy. ML10302,
a specific 5-HT4R partial agonist, was used as a scaffold, and a
substituted amide group was introduced on the piperidine moiety
of the molecule to allow interaction with Asp3.32 and with
residues of the 5-HT4R hydrophobic pocket. After preliminary
pharmacological screenings to test the binding and activity
properties of the synthesized agonists, two (9{a,y} and the
bivalent molecule 14) were selected for in vivo and in vitro
biological tests (APP processing and Aꢀ fibril formation). They
both increased sAPPR concentration in the hippocampus and
cortex of mice, suggesting that they could be useful for the
treatment of AD. Moreover, molecule 14 displayed an inhibitory
effect on Aꢀ fibril formation. To our knowledge, this is the
first report showing that a 5-HT4 ligand can interfere in the
process of Aꢀ fibril formation. Further experiments are needed
to test the potential neuroprotective properties of these new
5-HT4 agonists in animal models of AD.
a For agonists exhibiting inhibitory activity at least equal to that of
curcumin, IC50 was calculated by using a least-squares fitting technique to
match the experimental data with a sigmoidal curve. IC50 was the effective
concentration of agonist inhibiting the formation of Aꢀ fibrils to 50% of
the control value. Three independent measurements were made for all cases.
polyphenols,30,35 did not prevent inhibition of Aꢀ fibril forma-
tion. The strong inhibitory effect of this molecule more likely
resides in the nature of the aromatic ring (methoxy and ester
substituents), which presents structural similarities with polyphe-
Experimental Section
Chemistry. Melting points were determined on a Kofler melting
point apparatus. NMR spectra were performed on a Bruker AMX

2222 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Russo et al.
(1-(2-Nitrophenylsulfonyl)piperidin-4-yl)methylamine Fuma-
rate A{b}. To a solution of piperidin-4-ylmethylamine 3 (2.00 g,
17.51 mmol, 1.00 equiv) in toluene (30 mL) was added all at once
benzaldehyde (1.78 mL, 17.51 mmol, 1.00 equiv), and the mixture
was refluxed under argon with a Dean-Stark trap for 1.5 h. After
cooling to 0 °C, NEt3 (2.81 mL, 20.14 mmol, 1.15 equiv) was
added, followed by slow addition of nitrobenzenesulfonyl chloride
(4.08 g, 18.39 mmol, 1.05 equiv). The solution was allowed to reach
room temperature and stirred overnight. After removal of the
solvent, the residue was stirred vigorously with aqueous KHSO4 1
M (25 mL) for 1.5 h and diluted with brine (25 mL). This aqueous
solution was extracted with Et2O (1 × 50 and 2 × 25 mL) and
made strongly basic with NaOH 2 N, followed by extraction with
CH2Cl2 (1 × 50 and 2 × 25 mL). The combined organic layers
were dried (Na2SO4), filtered, and concentrated. The residue was
dissolved in EtOH (25 mL), treated with a solution of fumaric acid
(2.03 g, 17.51 mmol, 1.00 equiv) in EtOH (50 mL), and the resulting
mixture was refluxed for 15 min. After cooling, the obtained
precipitate was filtered, washed with cold EtOH, and dried.
Recrystallization from MeOH gave 6.99 g (96%) of A{b} as a white
1
solid. H NMR (300 MHz, CD3OD/DMSO-d6) δ 7.29 (m, 1H),
7.13 (m, 3H), 5.94 (s, 2H), 3.16 (d, J ) 10.3 Hz, 2H), 2.08 (m,
4H), 1.17 (m, 3H), 0.63 (m, 2H). 13H NMR (75 MHz, CD3OD/
DMSO-d6) δ 170.6, 149.4, 136.0, 135.3, 132.9, 131.4, 131.3, 125.1,
46.4, 44.7, 34.4, 29.8; mp 197-199 °C. Anal. (C12H17N3O4S ·
C4H4O4), C, H, N.
Figure 4. Best 5-HT4 synthesized agonists.
Resin 5{a}. BAL resin 4 (3.65 g, 0.79 mmol/g, 1 equiv) was
suspended in DMF (3 × 65 mL for 5 min). Following addition of
DMF (39 mL), the amine A{a} (2.78 g, 8.65 mmol, 3 equiv) and
NaBH(OAc)3 (3.06 g, 14.42 mmol, 5 equiv) were added. The
reactor was purged with argon, sealed, and agitated on an orbital
shaker at room temperature for 46 h. After a negative DNPH test
and a positive Kaiser test, the mixture was diluted with MeOH (30
mL), agitated for 5 min, and filtered. The resin was successively
washed with MeOH (2 × 65 mL for 5 min), DMF (3 × 65 mL for
5 min), MeOH (3 × 65 mL for 5 min), CH2Cl2 (3 × 65 mL for 5
min), and dried overnight to give 4.45 g of resin 5{a}. The loading
level was determined to 0.67 mmol/g (100%) by picric acid assay.
Resin 5{b}. Resin 5{b} was prepared by following the same
procedure described for 5{a}, using BAL resin 4 (4.00 g, 0.79
mmol/g, 1 equiv), amine A{b} (3.94 g, 9.48 mmol, 3 equiv), and
NaBH(OAc)3 (3.35 g, 15.80 mmol, 5 equiv) to obtain 4.96 g of
resin 5{b}. The loading level was determined to 0.66 mmol/g
(100%) by picric acid assay.
Amide Library 9. Acylation Step (6{a-x} and 6{b-x}). Resin
5{a} (approximately 100 mg, 0.067 mmol, 1 equiv) or resin 5{b}
(approximately 100 mg, 0.066 mmol, 1 equiv) was distributed into
Teflon tubes, swelled in CH2Cl2 (3 × 3 mL for 3 min), followed
by a 9/1 mixture of CH2Cl2/DIEA (3 × 3 mL for 3 min) and a 7/3
mixture of DMF/CH2Cl2 (3 × 3 mL for 3 min). A solution of each
acid B{a-x} (0.402 mmol for resin 5{a}, 0.396 mmol for resin
5{b}, 6 equiv) in a 7/3 mixture of DMF/CH2Cl2 (1.50 mL) was
added to the resin, followed by 1.50 mL of a stock solution
containing HBTU (4.03 g, 10.61 mmol) and DIEA (2.06 mL, 12.38
mmol) in a 7/3 mixture of DMF/CH2Cl2 (40 mL), and the resulting
mixtures were agitated at 25 °C for 18 h (the final concentration of
the acid and HBTU is 130 mM). The resins were filtered, washed
alternatively with DMF, MeOH, CH2Cl2 (5 × (3 mL of DMF for
3 min followed by 3 mL of MeOH for 3 min, followed by 3 mL
of CH2Cl2 for 3 min)), and CH2Cl2 (2 × 3 mL) and air-dried for
10 min.
200 (1H, 200 MHz; 13C, 50 MHz) or Bruker AVANCE 400 (1H,
400 MHz; 13C, 100 MHz). Unless otherwise stated, CDCl3 was
used as solvent. Chemical shifts δ are in ppm, and the following
abbreviations are used: singlet (s), broad singlet (bs), doublet (d),
triplet (t), and multiplet (m). Elemental analyses (C, H, N) were
performed at the Microanalyses Service of the Faculty of Pharmacy
at Chaˆtenay-Malabry (France) and were within 0.4% of the theorical
values unless otherwise stated. Mass spectra were obtained using
a Bruker Esquire electrospray ionization apparatus.
Materials. DMF distilled from CaSO4, CH2Cl2 distilled from
calcium hydride, and standard solvents were purchased from VWR
International (Fontenay-sous-Bois, France). Liquid chromatography
was performed on Merck silica gel 60 (70/30 mesh), and TLC was
performed on silica gel, 60F-254 (0.26 mm thickness) plates.
Visualization was achieved with UV light and Dragendorff reagent
unless otherwise stated.
Synthesis of N-tert-Butoxycarbonyl-1-(2-nitrophenylsulfo-
nyl)piperidin-4-amine 2. To a solution of N-tert-butoxycarbon-
ylpiperidin-4-amine 1 (1.83 g, 9.13 mmol, 1 equiv) and NEt3 (1.46
mL, 10.50 mmol, 1.15 equiv) in CH2Cl2 (35 mL) at 0 °C was added
dropwise 2-nitrobenzenesulfonyl chloride (2.13 g, 9.59 mmol, 1.05
equiv) in CH2Cl2 (8.5 mL). The resulting solution was stirred and
allowed to reach room temperature. After 2.5 h, the solution was
diluted with H2O (25 mL), the organic phase was separated,
successively washed with citric acid 10% (25 mL), NaHCO3 1 M
(25 mL), and brine (25 mL) and dried (Na2SO4) to give 3.46 g
(98%) of 2 as a white solid. 1H NMR (300 MHz, CDCl3) δ
8.03-7.87 (m, 1H), 7.74-7.66 (m, 2H), 7.63-7.58 (m, 1H), 4.50
(bs, 1H), 3.77 (d, J ) 12.9 Hz, 2H), 3.53 (bs, 1H), 3.01-2.76 (m,
2H), 1.99 (dd, J ) 12.9 and 2.9 Hz, 2H), 1.48 (m, 11H). 13H NMR
(75 MHz, CDCl3) δ 155.0, 148.2, 133.7, 131.7, 131.6, 130.8, 124.1,
79.6, 47.0, 44.9, 32.0, 28.3; mp 150-152 °C. Anal. (C16H23N3O6S),
C, H, N.
Synthesis of 1-(2-Nitrophenylsulfonyl)piperidin-4-amine Hy-
drochloride A{a}. To a solution of 2 (3.41 g, 8.84 mmol) in MeOH
(50 mL) was added HCl/MeOH 4 N (50 mL). After stirring at room
temperature for 3 h, the solvent was removed to give 2.84 g (100%)
of A{a} as a white solid. 1H NMR (300 MHz, CD3OD) δ
8.12-7.97 (m, 4H), 3.96 (d, J ) 11.2 Hz, 2H), 3.30 (m, 1H), 2.92
(dt, J ) 11.2 and 2.2 Hz, 2H), 2.07 (m, 2H), 1.65 (dd, J ) 12.1
and 4.0 Hz, 2H). 13C NMR (75 MHz, CD3OD) δ 149.4, 135.3,
132.8, 131.9, 131.5, 125.1, 48.6, 45.1, 30.6; mp 246-248 °C. Anal.
(C11H15N3O4S·HCl), C, H, N.
Deprotection Step (7{a-x} and 7{b-x}). Resins 6{a-x} and
6{b-x} were swelled in DMF (2 × 3 mL for 3 min) and 3 mL of
a stock solution containing thiophenol 0.5 M, and K2CO3 1 M in
DMF was added to each resin. The resulting mixtures were agitated
at 25 °C for 30 min. Each reaction was diluted with a 1/1 mixture
of THF/H2O (2 mL), agitated for 1 min, and then the resins were
filtered, washed with a 1/1 mixture of THF/H2O (2 × 3 mL for 3
min) and DMF (2 × 3 mL for 3 min). Resins were replaced in 3
mL of a stock solution containing thiophenol 0.5 M and K2CO3 1
M in DMF, and the resulting mixtures were agitated at 25 °C for

5-HT4 Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2223
30 min. Each reaction was diluted with a 1/1 mixture of THF/H2O
(2 mL), agitated for 1 min, and then the resins were filtered, washed
successively with a 1/1 mixture of THF/H2O (2 × 3 mL for 3 min),
DMF (4 × 3 mL for 3 min), and CH2Cl2 (2 × 3 mL for 3 min) and
air-dried for 10 min.
tography on silica. Eluting first with (CH2Cl2/MeOH 80:20) and
second with (CH2Cl2/toluene/MeOH 50:30:20) gave 65 mg (91%)
of 9{a;y} as an orange foam. Rf (CH2Cl2/toluene/MeOH 50:30:
1
20) 0.57. H NMR (400 MHz, CD3OD) δ 8.55 (dd, 1H, J ) 8.3
Hz and J ) 1.2 Hz), 8.46 (dd, 1H, J ) 7.3 Hz and J ) 1.2 Hz),
8.33 (d, 1H, J ) 8.3 Hz), 7.73 (s, 1H), 7.63 (dd, 1H, J ) 8.3 Hz
and J ) 7.4 Hz), 6.98 (d, 1H, J ) 8.3 Hz), 6.43 (s, 1H), 4.37 (t,
2H, J ) 5.4), 4.10 (t, 2H, J ) 7.3 Hz), 3.78 (s, 3H), 3.67 (m, 1H),
3.06 (m, 2H), 2.88 (t, 2H, J ) 5.4 Hz), 2.39 (m, 2H), 2.18, (t, 2H,
J ) 7.2 Hz), 1.83 (m, 2H), 1.65 (m, 4H), 1.54 (m, 2H), 1.39 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 175.5, 166.4, 166.2, 165.6,
164.2, 162.0, 151.6, 135.3, 134.2, 132.6, 131.3, 130.8, 126.0, 124.7,
123.1, 113.0, 111.3, 110.3, 108.2, 98.7, 62.2, 57.6, 56.2, 53.6, 49.4,
49.2, 49.0, 48.8, 48.6, 48.4, 47.1, 40.8, 36.8, 31.9, 28.8, 27.5, 26.7.
ESI m/z 637 (M + H+), 659 (M + Na); mp 150 °C; Anal.
(C33H38ClN5O5 ·3H2O), C, H, N.
N-Alkylation Step (8{a-x} and 8{b-x}). Resins 7{a-x} and
7{b-x} were swelled in a 9/1 mixture of DMF/DIEA (3 mL for 3
min), DMF (2 × 3 mL for 3 min), and 2.33 mL of a stock solution
containing C (2.73 g, 8.84 mmol) and DIEA (2.16 mL, 12.38 mmol)
in 57 mL of DMF were added to each resin (the final concentration
of the bromoester C is 150 mM). The mixtures were agitated at 40
°C for 20 h, cooled to room temperature, filtered, and washed three
times sequentially with DMF, MeOH, and CH2Cl2 (3 mL of DMF
for 2 min, followed by 3 mL of MeOH for 2 min, followed by 3
mL of CH2Cl2 for 3 min) and then with DMF (3 × 3 mL for 2
min). Then, 2.33 mL of the C/DIEA stock solution were added to
each resin. Mixtures were agitated at 40 °C for 20 h, cooled to
room temperature, filtered, and washed three times sequentially with
DMF, MeOH, and CH2Cl2 (3 mL of DMF for 2 min, followed by
3 mL of MeOH for 2 min, followed by 3 mL of CH2Cl2 for 3
min), three times sequentially with CH2Cl2 and MeOH (3 mL of
CH2Cl2 for 3 min, followed 3 mL of MeOH for 3 min) and finally
with CH2Cl2 (3 × 3 mL for 2 min).
Bivalent Ligand 13. A solution of 1228 (301 mg, 0.25 mmol)
in HCl/MeOH 4 N (15 mL) was stirred at room temperature for 30
min, and the solvent was removed to give 285 mg (99%) of 11 as
1
a beige hygroscopic foam. H NMR (200 MHz, CD3OD) δ 7.71
(s, 2H), 7.06 (s, 4H), 6.52 (s, 2H), 4.51 (bs, 4H), 3.76-3.64 (m,
8H), 3.59-3.32 (m, 8H) 2.92 (m, 8H), 2.57 (m, 4H), 2.28 (m, 4H),
2.15-1.65 (m, 20H). 13C NMR (50 MHz, CD3OD) δ 174.1, 165.7,
161.7, 149.5, 139.7, 134.4, 129.6, 111.8, 109.4, 98.4, 59.9, 56.9,
56.7, 53.6, 45.6, 33.8, 33.1, 30.1, 29.0, 27.6, 23.1. m/z ) 984 [M
+ H]+. Anal. (C50H72Cl2N8O8 ·4HCl + 9.5H2O), C, H, N.
Cleavage (9{a-x} and 9{b-x}). Resins 8{a-x} and 8{b-x}
were swelled in CH2Cl2 (2 × 3 mL for 3 min) and cleaved with a
1/4 mixture of TFA/CH2Cl2 (2 mL) for 2 h at 25 °C. Resins were
filtered, and filtrates were concentrated.
Bivalent Ligand 14. Compound 13 (200 mg, 0.18 mmol, 1
equiv) was treated with a saturated aqueous solution of Na2CO3 (5
mL), and the aqueous phase was extracted twice with CH2Cl2 (1
× 15 mL and 2 × 10 mL). The two organic phases were pooled,
dried (Na2SO4), filtered, and concentrated. The resulting foam was
dissolved in absolute EtOH (10 mL) and treated with a solution of
fumaric acid (82 mg, 0.71 mmol, 4 equiv) in absolute EtOH (10
mL). The solution was stirred 10 min at room temperature, and
the solvent was removed. The residue was solubilized in hot EtOH,
and a resin was formed upon cooling. The supernatant was removed,
and the procedure was repeated. The resin was finally taken in hot
EtOH, concentrated, and dried to give 187 mg (73%) of 14 a white
Purification by Solid-Phase Extraction (SPE). SPE cartridges
(Supelco, DSC-SCX, 1 g/6 mL) were conditioned in MeOH (2 ×
3 mL) and charged with the crude products dissolved in MeOH (1
mL). Columns were washed with MeOH (4 × 4 mL), and the final
products were eluted with a solution of NH4OH 2 M in MeOH (2
× 3 mL). Filtrates were concentrated to give amides 9.
Analytical Control of Library 9. Amides 9 were dissolved in
MeOH (1.50 mL, HPLC grade), and 100 µL of this solution were
diluted with 900 µL of H2O milliQ + 0.025% TFA. The solutions
were analyzed by HPLC using the standard conditions.
A selection of compounds (9{a;a}, 9{a;c}, 9{a;e}, 9{a;h},
9{a;i}, 9{a;o}, 9{a;q}, 9{a;r}, 9{a;s}, 9{a;v}, 9{b;a}, 9{b;c},
9{b;e}, 9{b;g}, 9{b;i}, 9{b;j}, 9{b;l}, 9{b;p}, 9{b;q}, 9{b;r}, and
9{b;w}) was analyzed by 1H NMR (and 13C NMR for 9{a;g} and
9{b;w}). Elemental analyses were carried out on pure samples of
9{a;q} and 9{b;w}. See Supporting Information for full details.
2-[4-({6-[6-Nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl]hexanoyl}amino)piperidino]ethyl 4-Amino-5-chloro-2-meth-
oxybenzoate 11. To a solution of 4-nitro-1,8-naphthalimido-N-
caproic acid27 (500 mg, 1.4 mmol, 1 equiv) in dry DMF (40 mL)
and cooled at 0 °C were added 1-hydroxbenzotriazole hydrate
(HOBT; 586 mg, 4.3 mmol, 3.1 equiv), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC; 0.665 g, 3.4 mmol, 2.5
equiv), and triethylamine (641 mg, 6.3 mmol, 4.5 equiv). The
reaction mixture was stirred at this temperature for 5-10 min, and
then 2-(4-aminopiperidin-1-yl)ethyl 4 amino-5-chloro-2-methoxy-
benzoate hydrochloride 1026 (562 mg, 1.4 mol, 1 equiv) was added.
The reaction was then stirred at room temperature for 20 h before
it was evaporated to dryness. The residue was purified twice by
chromatography on silica. Elution first with (CH2Cl2/MeOH 90:
10) and then with (CH2Cl2/iPrOH 60:40) gave 80 mg (8.5%) of 11
(C33H36ClN5O8) as an ochre foam. Rf (CH2Cl2/iPrOH 60:40) 0.36.
1H NMR (200 MHz) δ 8.69 (m,1H), 8.63 (m,1H), 8.50 (m, 1H),
8.35 (m, 1H), 7.94 (s, 1H), 7.76 (m, 1H), 6.23 (s, 1H), 5.94 (bd,
1H), 4.46 (m, 4H), 4.16 (m, 2H), 3.89 (m, 1H), 3.80 (s, 3H), 3.29
(m, 2H), 3.02 (t, J ) 5.4 Hz, 2H), 2.55 (m, 2H), 2.21 (t, J ) 7.3
Hz, 2H), 1.95 (m, 2H), 1.76 (m, 6H), 1.43 (m, 2H).
1
foam. H NMR (200 MHz, CD3OD) δ 7.80 (s, 2H), 7.18 (s, 4H),
6.73 (s, 8H), 6.49 (s, 2H), 4.61 (bs, 4H), 4.08-3.77 (m, 8H), 3.66
(m, 4H), 3.51 (m, 4H), 3.30-2.92 (m, 8H), 2.71 (m, 4H), 2.40 (m,
4H), 2.25-1.77 (m, 20H). 13C NMR (50 MHz, CD3OD) δ 174.2,
170.8, 165.9, 162.0, 151.9, 139.7, 136.2, 134.4, 129.6, 110.5, 107.6,
98.7, 59.8, 56.6, 56.4, 52.8, 45.4, 33.8, 33.1, 29.7, 29.0, 27.2, 23.5.
Anal. (C50H72Cl2N8O8 ·4C4H4O4 + 4.5 H2O), C, H, N.
4-(N-tert-Butoxycarbonyl(3-phenylprop-2-ynyl)amino)butano-
ic Acid 16. To a stirred solution of iodobenzene (612 mg, 3.00
mmol, 1.00 equiv), PdCl2(PPh3)2 (106 mg, 0.15 mmol, 0.05 equiv),
and CuI (56 mg, 0.30 mmol, 0.10 equiv) in a 1/1 mixture of freshly
distilled NEt3 and anhydrous DMF (20 mL) at room temperature
was added dropwise a solution of 1528 (763 mg, 3.15 mmol, 1.05
equiv) in anhydrous DMF (10 mL). The solution was stirred at 80
°C for 3.5 h and cooled to room temperature. After removal of the
solvents, the crude mixture was purified by chromatography on
silica gel, using AcOEt/cHex (2/8) + 0.5% AcOH followed by
AcOEt/cHex (3/7) + 0.5% AcOH, to give 780 mg (82%)
1
(C18H23NO4) of 16 as a yellow oil. H NMR (300 MHz) δ 7.41
(dd, J ) 6.7 and 3.0 Hz, 2H), 7.35-7.27 (m, 3H), 4.26 (s, 2H),
3.46 (t, J ) 7.2 Hz, 2H), 2.42 (t, J ) 7.2 Hz, 2H), 1.97 (m, J )
7.2 Hz, 2H), 1.49 (s, 9H). 13C NMR (75 MHz) δ 176.3, 155.5,
131.8, 128.4, 123.0, 85.1, 80.2, 45.9, 31.3, 28.5, 27.1, 23.4.
4-(N-tert-Butoxycarbonyl(3-phenylpropyl)amino)butanoic Acid
17. To a stirred solution of 16 (500 mg, 1.58 mmol) in MeOH (15
mL) at room temperature was added Pd/C 10% (47 mg). The
mixture was hydrogenated at atmospheric pressure overnight and
filtered through a pad of celite. After evaporation of the solvent, a
purification by chromatography on silica gel, using CH2Cl2 + 0.5%
AcOH followed by CH2Cl2/MeOH (95/5) + 0.5% AcOH, gave 478
mg (94%) of 17 (C18H27NO4) as a colorless oil. 1H NMR (300 MHz)
δ 10.40 (bs, 1H), 7.52 (m, 5H), 3.26 (bs, 4H), 2.62 (t, J ) 7.7 Hz,
2H), 2.37 (t, J ) 7.2 Hz, 2H), 1.88 (m, 4H), 1.46 (s, 9H). 13C
2-[4-({6-[6-Amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl]hexanoyl}amino)piperidino]ethyl 4-Amino-5-chloro-2-meth-
oxybenzoate 9{a;y}. To a solution of 11 (75 mg, 0.113 mmol) in
methanol (6 mL) was added Raney nickel. The reaction mixture
was stirred under atmospheric pressure at room temperature for
14 h and filtered through celite. The solvent was removed under
reduced pressure, and the residue was purified twice by chroma-

2224 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Russo et al.
NMR (75 MHz) δ 177.2, 155.6, 141.8, 128.5, 128.4, 126.0, 79.9,
47.1, 46.3, 33.4, 31.4, 30.2, 28.5, 23.7.
counter. Binding data are expressed as a percentage of displacement
for the screening of the library compounds. For selected compounds,
Ki were analyzed by computer-assisted nonlinear regression analysis
(Prism, Graphpad Software, San Diego, CA). The data are the
results of two or three independent determinations performed in
triplicate.
2-(4-(4-(N-tert-Butoxycarbonyl(3-phenylpropyl)amino)butyl-
amido)piperidin-1-yl)ethyl 4-Amino-5-chloro-2-methoxyben-
zoate 18. To solution of 17 (506 mg, 1.24 mmol, 1.0 equiv) in dry
DMF (10 mL) at room temperature was added 1026 (403 mg, 1.24
mmol, 1.0 equiv), HBTU (520 mg, 1.37 mmol, 1.1 equiv), and
NEt3 (694 µL, 4.98 mmol, 4.0 equiv), and the mixture was stirred
overnight at room temperature. After solvent removal, the crude
product was dissolved in AcOEt (30 mL), washed with saturated
aqueous Na2CO3 (20 mL), and brine (20 mL). The organic phase
was dried (Na2SO4) and concentrated. Purification by chromatog-
raphy on silica gel, using AcOEt/MeOH (90/10) followed by
AcOEt/MeOH/NH4OHaq 20% (87/10/3), afforded 540 mg (68%)
of 18 as white foam. 1H NMR (300 MHz) δ 7.90 (s, 1H), 7.34 (m,
5H), 6.83 (bs, 1H), 6.39 (s, 1H, H3), 4.61 (bs, 2H), 4.47 (t, J )
5.8 Hz, 2H), 3.93 (m, 4H, 12), 3.33 (m, 4H), 3.07 (m, 2H), 2.89 (t,
J ) 5.8 Hz, 2H), 2.69 (t, J ) 7.7 Hz, 2H), 2.44 (m, 2H), 2.23 (t,
J ) 6.8 Hz, 2H), 2.10-1.84 (m, 6H), 1.65 (m, 2H), 1.54 (s, 9H).
13C NMR (75 MHz) δ 172.2, 164.6, 160.4, 156.5, 148.0, 141.7,
133.5, 128.5, 128.4, 126.4, 110.1, 109.7, 98.4, 79.8, 62.2, 56.8,
56.2, 52.8, 47.0, 46.4, 45.8, 33.7, 33.4, 32.0, 30.2, 28.6, 24.7. m/z
) 632 [M + H]+. Anal. (C33H47N4O6 + 2H2O), C, H, N.
2-(4-(4-(3-Phenylpropylamino)butylamido)piperidin-1-yl)eth-
yl 4-Amino-5-chloro-2-methoxybenzoate, Fumarate 19. A solu-
tion of 18 (495 mg, 0.78 mmol) in MeOH (10 mL) and HCl/MeOH
4N (10 mL) was stirred at room temperature for 30 min, and the
solvent was removed to give 467 mg (99%) of a white foam. This
foam was treated with a saturated aqueous solution of Na2CO3 (5
mL), and the aqueous phase was extracted with AcOEt (10 mL).
The organic phase was washed with brine (5 mL), dried (Na2SO4),
filtered, and concentrated. The resulting foam was dissolved in
absolute EtOH (10 mL) and treated with a solution of fumaric acid
(165 mg, 1.42 mmol, 2 equiv) in absolute EtOH (10 mL). The
solution was stirred 10 min at room temperature, and the solvent
was removed. The residue was solubilized in hot iPrOH, and a resin
was formed upon cooling. The supernatant was removed, and the
processus was repeated. The resin was finally taken in hot iPrOH,
concentrated, and dried to give 331 mg (89%) of 19 as a white
foam. 1H NMR (300 MHz, CD3OD) δ 7.80 (s, 1H), 7.24 (m, 5H),
6.70 (s, 4H), 6.48 (s, 1H), 4.53 (t, J ) 5.0 Hz, 2H), 3.89 (m, 1H),
3.82 (s, 3H), 3.52 (m, 2H), 3.34 (m, 2H), 3.10-2.92 (m, 6H), 2.73
(t, J ) 7.6 Hz, 2H), 2.37 (t, J ) 6.9 Hz, 2H), 2.15-1.90 (m, 6H),
1.82 (m, 2H). 13C NMR (75 MHz, CD3OD) δ 174.1, 171.4, 166.1,
162.1, 151.9, 141.6, 136.2, 134.4, 129.6, 129.4, 127.4, 110.5, 107.7,
98.6, 60.4, 56.8, 56.3, 53.1, 45.9, 48.3, 33.6, 33.5, 30.3, 29.1, 23.1.
m/z ) 531 [M + H]+. Anal. (C28H39ClN4O4 ·2C4H4O4 + H2O),
C, H, N.
Biology. Membrane Preparation and Radioligand Binding
Assays. C6 glial cells, which were stably transfected with h5-HT4(e)
receptor, were grown to confluence and incubated with serum-free
medium for 4 h, washed twice with phosphate-buffered-saline
(PBS), and centrifuged at 300g for 5 min. Pellets were used
immediately or stored at -80 °C. The pellet was resuspended in
10 volumes of ice-cold HEPES buffer (50 mM, pH 7.4) and
centrifuged at 40000g for 20 min at 4 °C. The pellet was then
suspended in 15 volumes of HEPES (50 mM, pH 7.4), and protein
concentration was determined by the method of Bradford using
bovine serum albumin as the standard. Radioligand binding studies
were performed in 250 µL of HEPES buffer (50 mM, pH 7.4), 20
µL of the studied ligand (at 10 nM for the screening of the libraries
or 7 concentrations for Ki determination), and 20 µL of [3H]-
GR113808 at a concentration of 0.2 nM and 50 µL of membranes
preparation (100-200 µg of protein). Nonspecific binding was
determined with 10 µM GR113808. Tubes were incubated at 25
°C for 30 min, and the reaction was terminated by filtration through
Whatman GF/B Filter paper using the Brandel 48R cell harvester.
Filters were presoaked in a 0.1% solution of polyethylenimine,
washed with ice-cold buffer (50 mM Tris-HCl, pH 7.4), and placed
overnight in 4 mL of ready-safe scintillation cocktail. Radioactivity
was measured using a Beckman model LS6500C liquid scintillation
cAMP Accumulation. C6 glial cells stably transfected with the
h5-HT4(e)R were grown to confluence and incubated with serum-
free medium for 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 and 10 µM pargyline. 5-HT
(1 µM) and/or compounds (10 nM) were added and incubated for
an additional 15 min at 37 °C in 5% CO2. The reaction was stopped
by aspiration of the medium and addition of 50 µL of ice-cold
perchloric acid (20%). After 30 min, neutralization buffer was added
(HEPES 25 mM, KOH 2 N), supernatant was eliminated by 5 min
centrifugation at 2000g and cAMP was quantified using a radio-
immunoassay kit (cAMP competitive radioimmunoassay, Beckman,
France). The ligand concentration-effect curves were calculated
using seven concentrations (10-10-10-5). EC50 values cor-
respond to the concentration of agonists required to obtain half-
maximal stimulation of adenylyl cyclase. The maximum response
produced by each molecule was normalized to the 5-HT induced
maximum response (Emax). Values are the means ( SEM of two
or three experiments performed in triplicate.
Effects of Agonists on sAPPr Levels in Mice. Adult male
C57BL/6j wild-type mice (8 week/old, weighing 23-27 g) from
Janvier (Le Genest-Saint-Isle, France) were used in this study. Mice
were housed in a room with a 12 h light-dark cycle with food and
water ad libitum. They received a single subcutaneous injection of
0.9% NaCl (control), or 12 or 9{a;y} at 5 mg/kg dissolved in 0.9%
NaCl. Mice were sacrificed 90 min after the injection by cervical
dislocation, and their brains were then treated as previously
described.13 Hippocampus and cortex were homogenized by
sonication in 150 and 300 µL of 50 mM Tris, pH 7.4, 150 mM
NaCl, and proteinase inhibitors (Sigma, France), respectively, and
cleared by centrifugation (100000g, 45 min, 4 °C). Only the
supernatant was used for Western blot experiments (polyclonal
antiserum R1736, 1:4000 dilution (a gift from Dr. Selkoe), goat
antirabbit immunoglobulin antibody, 1:15000, Amersham Pharma-
cia Biotech, (Orsay, France). Immunoreactive bands were then
visualized with the ECL plus detection kit (Amersham Pharmacia
Biotech, Orsay, France) on Kodak ML lights films. The membrane
was stripped and blocked again before being probed with an anti-
ꢀ-actin antibody (monoclonal anti-ꢀ-actin, 1:5000 dilution; Abcam,
Cambridge, UK), used as internal control. After 1 h incubation with
a horseradish peroxidase-linked goat antimouse immunoglobulin
antibody (Cell Signaling, Ozyme, Saint-Quentin-en-Yvelines, France)
at 1:2000 dilution, immunoreactive bands were visualized with the
ECL detection kit (Amersham Pharmacia Biotech, Orsay, France)
on Kodak ML lights films. After digitization, densitometric values
were quantified by using an image analysis. Statistical analyses were
performed using the computer software StatView 4.02 (Abacus
Concepts Inc., Berkeley, CA). Data are means ( SEM of sAPPR
levels expressed as percentage of control values (mice which
received a saline injection). Values were compared between the
different groups (7-9 mice/group) by using a one-way analysis of
variance (ANOVA) followed by protected least significant differ-
ence tests (PLSD). The significance level was set at p < 0.05.
Measurement of Inhibitory Activity on Aꢀ25-35 Polymer-
ization. Initial screening for inhibition was carried out using a
reaction mixture containing 80 µL of phosphate buffer (10 mM
final concentration), 10 µL of MeOH containing 10 µM final
concentration of agonist and 10 µL of Aꢀ25-35 (100 µM final
concentration), pH 7.2. All steps were carried out at 4 °C to prevent
Aꢀ25-35 polymerization. UV-visible spectroscopy was performed
on a Cary 300 bio UV-visible spectrophotometer. First, the
UV-visible spectra of agonist alone were recorded by using the
spectrometer in the range of 190-600 nm at 15 °C. Optimal
measurements of Aꢀ25-35 were then recorded by using the
spectrometer in the range of 190-600 nm at 15 °C to control for

5-HT4 Receptor Agonists
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any artifacts due to agonist aggregation. Finally, polymerization
kinetics were monitored at 200 nm, this wavelength corresponding
to the absorption of peptide bonds. Data were collected 6 h after
incubation. For each inhibitory experiment, one sample containing
Aꢀ25-35 alone and another containing the agonist alone were used
in parallel as control in the same experimental conditions. Moreover,
to rule out any influence due to agonist absorbance, their UV-visible
spectra were subtracted from the Aꢀ25-35 absorption spectra. At
least three independent measurements were recorded for all cases.
All results are presented with means and standard deviation.
For agonists exhibiting inhibitory activity at least equal to that
of curcumin, IC50 was calculated by using a least-squares fitting
technique to match the experimental data with a sigmoidal curve.
IC50 was the effective concentration of agonist inhibiting the
formation of Aꢀ fibrils to 50% of the control value.
Acknowledgment. This work was supported by grants from
INSERM and the Ministe`re de la Recherche et de l’Enseignement
Supe´rieur.
Supporting Information Available: Chemistry experimental,
spectroscopic data, and results from elemental analyses of all the
listed compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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