Synthesis, modelling and biological characterization of 3-substituted-1H-indoles as ligands of GluN2B-containing N-methyl-d-aspartate receptors

Article abstract of DOI:10.1016/j.bmc.2013.12.040

A three-step synthetic pathway has been employed to synthesize a small library of 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,2-dione derivatives that have been screened in [³H]ifenp

Full text of DOI:10.1016/j.bmc.2013.12.040

Bioorganic & Medicinal Chemistry 22 (2014) 1040–1048
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, modelling and biological characterization of
3-substituted-1H-indoles as ligands of GluN2B-containing
N-methyl-D-aspartate receptors
a
a
b
b
Rosaria Gitto a, , Laura De Luca , Stefania Ferro , Emilio Russo , Giovambattista De Sarro ,
Mariangela Chisari c, Lucia Ciranna d, Julio Alvarez-Builla e, Ramon Alajarin e, Maria Rosa Buemi a,
Alba Chimirri a
⇑
a Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Dipartimento di Scienze della Salute, Università Magna Graecia, Viale Europa Località Germaneto, I-88100 Catanzaro, Italy
c Dipartimento di Biomedicina Clinica e Molecolare, Sezione di Farmacologia e Biochimica, Università di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy
d Dipartimento di Scienze Bio-Mediche, Sezione di Fisiologia, Università di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy
e Departamento de Química Orgánica I, Universidad de Alcalá, Campus Universitario, 28871 Alcalá de Henares, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A three-step synthetic pathway has been employed to synthesize a small library of 2-(4-arylpiperidin-1-yl)-
1-(1H-indol-3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,2-dione derivatives
that have been screened in [3H]ifenprodil competition binding assay. Some compounds exhibited
significant binding affinity at nanomolar concentration, the most active being ligand 35 (IC50 = 5.5 nM).
Docking experiments suggested the main interactions between 35 and GluN2B-containing NMDA
receptors. Notably, the compound 35 reduced NMDA-mediated excitatory post-synaptic currents
recorded in mouse hippocampal slices indicating antagonistic effects (50 nM). Moreover, the compound
35 has shown antioxidant effects in a preliminary screening, thus suggesting that it might be considered
prototype for future drug development of novel ‘dual target’ neuroprotective agents.
Received 1 October 2013
Revised 10 December 2013
Accepted 17 December 2013
Available online 30 December 2013
Keywords:
Glutamate
GluN2B
Indoles
Ifenprodil
Molecular docking
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
(LBD), transmembrane domain forming an ion pore, and intracellu-
lar C-terminal domain (CTD).
The N-methyl-
D
-aspartate receptors (NMDARs) belong to the
Functional NMDARs contain two essential binding sites: (a) the
glycine-binding site, localized on GluN1 subunit; (b) the gluta-
mate-binding site localized on GluN2 subunit.7,8 It has been
recently demonstrated that the different composition of GluN2A–
D subunits and the subcellular localization of NMDARs contribute
to induce signal transduction cascade and to promote neuronal
plasticity and neuronal survival or death. While the synaptic
NMDAR activation is implicated in neuroprotection, the stimula-
tion of extrasynaptic NMDARs triggers cell death signaling
pathways and may play a key role in the neurodegeneration asso-
ciated with Glu-induced excitotoxicity.9,10 Experimental evidence
suggests that GluN2B subunits are present in extrasynaptic
NMDAR tetrameric assemblies and are mainly involved in the
promotion of pro-death gene expression, calcium overload and
activation of cell death signaling pathways.9 Thereby antagonists
targeting GluN2B-containing NMDARs are predicted to show neu-
roprotective effects. Ifenprodil (1) is the prototype of a wide class
of noncompetitive antagonists of GluN2B-containing NMDARs.
Compound 1 and its analogues bind the interface of extracellular
ATD of the GluN1/GluN2B dimer thus impairing the opening of
ionotropic glutamate receptor (iGluR) family. NMDA receptors
are widely expressed in the central nervous system (CNS); due to
their peculiar gating properties and Ca2+ permeability, these
receptors mediate synaptic plasticity and contribute to neuronal
development and behavioral learning.1,2 However, overstimulation
of NMDARs results in activation of excitotoxic pathways thus
inducing neuronal death.3 In fact, NMDARs are implicated in a
wide range of neurodegenerative processes and represent an ideal
molecular target for the treatment of neuropathological conditions,
as brain injury, ischemia, epilepsy, Parkinson and Alzheimer dis-
eases4 as well as for the therapeutic treatment of major depressive
diseases.5,6
NMDARs are hetero-tetrameric complexes consisting of two
GluN1 subunits joined with two GluN2A–D or GluN3 subunits.
Each subunit has a modular structure, consisting of four separate
areas: amino-terminal domain (ATD), ligand-binding domain
⇑
Corresponding author. Tel.: +39 0906766413; fax: +39 0906766402.
E-mail address: rgitto@unime.it (R. Gitto).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.040

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
1041
Hydrophobic contacts
NMDARs through the stabilization of the twisted closed-clamshell
configuration.11
F176
P177
Q110
H bond interaction
By computational approaches we have disclosed the 2-(4-ben-
zylpiperidin-1-yl)-1-(1H-indol-3-yl)ethanone 2 (Fig. 1) as promis-
ing ‘hit structure’ to identify new GluN2B-containing NMDAR
ligands.12 We subsequently synthesized several analogs and dis-
covered the ligands 3–7 (Fig. 1) showing IC50 values ranging from
8.9 to 63 nM in [3H]ifenprodil competition binding assay.13–15 In
particular, the most potent ligand N-1H-indol-6-methanesulfon-
amide-3-yl)-2-(4-benzylpiperidin-1-yl) ethanone 5 (Fig. 1) was
also effective NMDA receptor antagonist. It proved to significantly
reduce NMDA receptor-mediated current recorded from hippo-
campal neurons in patch clamp experiments.15 For this class of li-
gands the structure activity relationships (SARs) were in good
agreement with previous works16–19 and highlighted several struc-
tural requirements controlling the optimization of binding affinity.
We successively rationalized SAR findings through docking studies
which showed that the hydrogen bond interactions and hydropho-
bic contacts could improve the recognition process within ifenpro-
dil binding-pocket localized in to the GluN1/GluN2B dimer
interface.
H
N+
O
M207
Y109
N
MsNH
H
H bond interactions
I111
Hydrophobic contacts
E236
G112
L135
Hydrophobic contacts
Figure 2. The main interactions between the active ligand
5 and the crucial
aminoacid residues of GluN1/Glu2B dimer interface.
binding affinity evaluation of this new series of indoles. The most
active compound was further evaluated in in silico, in vitro and
in vivo experiments.
2. Results and discussion
Following previously reported procedure13–15 and using Micro-
wave-assisted approach the synthesis of the two series of the
Figure 2 outlines the main interactions between the most active
ligand 5 and aminoacid residues of the GluN1 subunit (Y109, G112
and L135) and GluN2B subunit (Q110, I111, P177, F176, E236, and
M207).15 Notably, these crucial contacts were consistent with the
pattern of interactions that have been revealed by crystallization
of compound 1 with GluN1/GluN2B dimer interface (PDB code
3QEL).11
O
Cl
R 1
R 2
R 1
R 2
i
The aim of the current work was an improvement of our
knowledge about SARs for indoles as GluN2B ligands. Thus it was
synthesized a new series of 2-(4-arylpiperidin-1-yl)-1-(1H-indol-
3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,
2-dione derivatives strictly related to previously reported indoles
3–7. Considering that the hydroxyl group at C-5 or C-6 position
of hit compounds 3 and 4 had been identified as a crucial struc-
tural requirement, we planned to synthesize the corresponding
5,6-dihydroxy-derivative. To further improve the receptor
affinity we attempted to strengthen the hydrophobic contacts
within GluN1/GluN2B dimer interface by introducing of a fluorine
atom at para position of the benzyl substituent. Moreover, the
presence of this substituent at para position could increase meta-
bolic stability of new synthesized compounds. Finally, to explore
the role of the contact between the positive ionizable nitrogen
atom of the piperidine nucleus and the residue Q110, it was syn-
thesized a novel series of indoles bearing ethane-1,2-dione rather
ethanone linker between the indole and the benzylpiperidine frag-
ment. Actually, this modification results in the conversion of the
tertiary amine into the amide functionality through the introduc-
tion of an additional carbonyl group that reduces the proton affin-
ity of the nitrogen atom of piperidine ring. Herein we described the
N
H
N
H
13-17
8-12
iii
ii
O
O
O
X
R 1
R 2
N
R 1
R 2
R 3
N
N
H
H
18-19 X= Cl
20-22 X=OH
v
35-38
23-26
vi
27-28
39-40
39-40
47-48
iv
vii
O
N
O
R 1
R 2
R 3
N
H
R1
8, 13, 18 MeO
v
R2
29-32
33-34
45-46
41-44
45-46
49-50
vi
H
9, 14, 19
H
MeO
MeO
H
vii
10, 15, 20 MeO
11, 16, 21 NHBoc
12, 17, 22
H
NHBoc
O
HO
R1
R2
R3
R1
R2
R3
N
OH
N
35, 41
OH
OH
H
OH
H
H
F
F
F
F
23 29
24, 30
25, 31
26, 32
MeO
MeO
H
MeO
H
H
F
F
F
F
F
36, 42
37, 43
38, 44
39, 45
N
H
Me
OH
OH
H
MeO
MeO
H
OH
MeO
NH2
2
1, ifenprodil
27, 33 NHBoc
40, 46
H
NH2
F
GluN2B/NMDA IC50=0.769 µM
GluN2B/NMDA IC50=20nM
28, 34
H
NHBoc
47, 49
NHMs
H
F
48, 50
H
NHMs
F
X
O
3, R = 5-OH
4, R = 6-OH
5, R = 6-MsNH X = H2
6, R = 6-OH X = O
7, R = 6-MsNH X = O
X = H2
X = H2
N
Scheme 1. Reagents and conditions: (i) ClCH2COCl, pyridine, dioxane, MW: 5 min,
50 °C, 200 W, or ClCH2CON(CH3)2, POCl3, room temperature, 2.5 h; (ii) 4-fluor-
obenzylpiperidine or benzylpiperidine, K2CO3, DMF, MW: 10 min 50 °C, 200 W; (iii)
ClCOCOCl, diethyl ether, rt, 2 h; (iv) 4-fluorobenzylpiperidine, or benzylpiperidine,
DMF, TEA, HBTU, rt, 2 h; or: THF, 4-fluorobenzylpiperidine, TEA, rt, 2 h; (v) BBr3
(1.0 M DCM), rt, 10 h; (vi) DCM/TFA, rt, 1 h; (vii) MsCl, DCM, pyridine, rt, 20 h.
R
N
H
Figure 1. GluN2B-containing NMDAR ligands.

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R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
target compounds was carried out. As reported in Scheme 1, the in-
doles 8–12 were converted in the corresponding key intermediates
13–22 which reacted with benzylpiperidine or 4-fluorobenzylpi-
peridine to give the title compounds 23–34. Dealkylation of the
two series of methyl ethers 23–26 and 29–32 gave the final com-
pounds 35–38 and 41–44 in good yields. Whereas, the NHBoc
derivatives 27–28 and 33–34 were deprotected to provide the
indoles 39–40 and 45–46, afterwards converted into the methane-
sulfonamide derivatives 47–48 and 49–50.
very similar potency than unsubstituted analog 5. The 5-methane-
sulfonamide fluoro-derivative 47 was inefficacious ligand and this
result was in good agreement with previously obtained data.15
As shown in Table 1 compounds bearing ethane-1,2-dione lin-
ker were generally less active ligands than corresponding ethanone
analogues. Among this second series of indoles the most active
derivatives were compounds 43 and 50 bearing hydroxyl or meth-
ansulfonamide substituent at C-6 position. They displayed the
same potency of reference compound ifenprodil (1).
The two series of the new synthesized compounds were evalu-
ated for their ability to interact with the GluN2B subunit. The ob-
tained results have been compared with previously reported
data13–15 for the most active indoles 3–7 and ifenprodil (1) as
reference compounds. Table 1 reports the binding affinity data of
several indoles that have been selected among all synthesized com-
pounds taking into account previously reported SARs. The biological
results for remaining compounds were reported as Supplementary
X
O
N
R 1
R 2
R3
N
H
To rationalize the biological data reported in Table 1, the docking
pose within GluN1/GluN2B interface for the most active antagonist
35 (IC50 value of 5.5 nM) was examined and the results were re-
ported in Figure 3. The compound 35 establishes interaction with
residue Q110 of GluN2B through the hydrogen atom of positive
ionizable piperidine ring. The ligand is also able to form a network
of hydrogen bonding interactions between the catechol moiety and
residue E236 of GluN2B. Moreover we observed another hydrogen
bonding interaction between residue K131 of GluN1 and NH of in-
dole ring. Finally, the benzylpiperidine portion and indole nucleus
of ligand 35 form several hydrophobic interactions with some cru-
cial residues on GluN1 (Y109, G112, L135) and GluN2B (I111, F176,
P177). Additionally, the docking study suggests cation-pi interac-
tions between the indole ring and side-chain of residue R115 of
GluN1 subunit (see Supplementary data).
Overall the compound 35 retains all interactions highlighted for
the two potent previously studied 5- or 6-hydroxyindole deriva-
tives 3 and 413 that demonstrated significant affinity towards
GluN2B-containing NMDARs (IC50 value of 25.0 and 17.3 nM,
respectively). As shown in Table 1 the 5,6-dihydroxyindole deriva-
tive 35 showed up to 5-fold lower IC50 value in comparison to the
monohydroxy-derivatives 3 and 4. Analyzing the proposed docking
pose we can speculate that the high affinity binding properties of
compound 35 could be mainly related to the strength of bidentate
Data. Data presented in the Table 1 show that a 0.1 lM concentra-
tion of seven of the examined indoles (35–38, 43, 48, and 50) pro-
duced greater than 50% displacement of [3H]ifenprodil suggesting
nanomolar affinity (IC50). For the seven selected more active com-
pounds the IC50 value determination (Table 1) highlighted that
the most potent ligand was the 2-(4-benzylpiperidin-1-yl)-1-(5,6-
dihydroxy-1H-indol-3-yl)ethanone (35). This compound was able
to produce 91% displacement of [3H]ifenprodil and displayed the
IC50 value of 5.5 nM. On the basis of this finding we hypothesized
that the presence of two hydroxyl groups at 5 and 6 positions of
indole ring significantly improved the binding affinity of the
corresponding monohydroxyl-substituted analogues 3 and 4.
Within the first series of compounds bearing the ethanone lin-
ker between the indole system and the benzylpiperidine fragment,
the effect of fluorine atom on the benzyl group has been also
explored. As shown in Table 1, the introduction of a fluorine atom
improved the potency of 4-benzylpiperidin-1-yl derivative 4 (IC50
value of 17.3 nM) so the corresponding 2-[4-(4-fluorobenzyl)piper-
idin-1-yl]-1-(6-hydroxy-1H-indol-3-yl)ethanone (37) exhibited
the IC50 value of 9.1 nM. The 5-hydroxy-derivative 36 resulted less
active than unsubstituted analog 3. The fluoro-derivative 38 was
about 5.5-fold less active than the corresponding unsubstituted
analog 35. On the contrary, the presence of fluorine atom was
tolerated for 6-methanesulfonamide derivative 48 that showed a
Table 1
GluN2B/NMDA binding affinities of indole derivatives and reference compound
ifenprodil (1)
a
R1
R2
R3
X
% Inhibitiona (@ 0.1
lM)
IC50 nM
3b
4b
35
36
37
38
5b
47
48
6b
41
42
43
44
7b
49
50
1
OH
H
OH
OH
H
OH
H
NHMs
H
H
H
H
H
F
F
F
H
F
F
H
H
F
F
F
H
F
F
H2
H2
H2
H2
H2
H2
H2
H2
H2
O
O
O
O
O
O
O
O
—
75
77
91
60
82
84
85
10
81
72
40
11
61
38
74
4
25.0
17.3
5.5
51.0
9.1
30.0
8.9
ND
9.4
22.2
ND
ND
20.0
ND
OH
OH
H
OH
OH
NHMs
H
NHMs
OH
OH
H
OH
OH
NHMs
H
H
OH
OH
H
OH
H
NHMs
H
—
63.0
ND
18.0
20.0
NHMs
—
67
ND
—
Figure 3. Docking pose of indole 35 (magenta) at the GluN1–GluN2B subunit
interface (PDB code 3QEL). Important residues are drawn in stick and coloured in
cyan (GluN2B) and in green (GluN1). Hydrogen bonds (shown as dashed yellow
lines) are formed between 35 and GluN1–GluN2B subunit interface. The figure was
prepared in PyMOL software.
ND = not determined.
a
Displacement of [3H]ifenprodil. Three concentration (10, 0.1, 0.001
lM, in dupli-
cate) of test compounds were used in the displacement assay.
b
Data from Ref. 15.

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
1043
hydrogen bonding interactions with the crucial residue E236
(GluN2B subunit) mediated by the two hydroxyl groups of catechol
moiety. Preliminary docking studies have been carried to explore
the binding poses of the new synthesized 4-fluorobenzylpiperi-
dine-derivatives in to the GluN1/GluN2B interface. As expected
these studies highlighted that there are not striking differences
in the proposed binding modes of 4-fluorobenzylpiperidine deriv-
atives in comparison to benzylpiperidine analogues. Thus these
experiments failed to explain the reduction of ligand affinity mea-
sured for the fluorosubstituted compound 38 in comparison to the
unsubstituted analogue 35.
Considering that the Topological Polar Surface (TPSA) is a parame-
ter to estimate compound polarity and predict blood–brain barrier
(BBB) penetration22,23 we decided to calculate the TPSA values of
the most active ligands before to study the in vivo activity. It is well
known that drugs or candidates acting on CNS should display low
polarity thus having ‘optimal’ TPSA values estimated in the range
of 60–70 Å2. However, given a cutoff of 90 Å2 for CNS penetration24
we chose to study compound 35 having the highest0binding affinity
associated with a calculated TPSA value of 76.56 ÅA.2 Actually, the
compound 35 showed low in vivo efficacy: the determined ED50
values for compound 35 were 89.43 and 76.72 lmol/kg in clonus
The most potent ligand 35 was selected for functional studies of
its binding at GluN2B-containing NMDAR. To evaluate the func-
tional activity of the ‘hit compound’ 35 on NMDA receptors, we
used patch clamp techniques to record from mouse hippocampal
slices. GluN2B-containing NMDA receptors were found to be ex-
pressed at high levels at early postnatal days, 6–20.20 Therefore
we performed experiments with mice at postnatal days 10–15.
Excitatory post-synaptic currents mediated by activation of NMDA
receptors (EPSCNMDA) were recorded from CA1 neurons at a holding
potential of ꢀ60 mV in Mg2+-free ACSF, in the presence of CNQX
and tonus phases, respectively. Considering that 35 failed to pro-
duce significant in vivo efficacy, we can hypothesize that under
physiological conditions, other factors, including solubility,
absorption, and metabolic issues, might affect the CNS bioavailabil-
ity of compound 35.
Moreover, considering that hydroxyindole derivatives could also
be efficient free radical scavengers and antioxidants,25–31 we decided
to study the antioxidant profile of the active GluN2B ligand 35 bear-
ing a catechol moiety. Our idea was to explore the development of a
hypothetical ‘dual target’ neuroprotective agent, which might con-
trast glutamate-mediated excitotoxic cascade. The antioxidant
activity was determined using the ABTS method, which is and elec-
tron transfer-based antioxidant capacity assay32 first reported by
Miller and Rice-Evans33 and later improved by Re et al.34 The antiox-
idant ability was expressed as ABTS inhibition percentage35,36 at two
(10
previously,15 in these recording conditions EPSCs were blocked
by the NMDA antagonist D-AP5 (50 M) and by Ro 25-6981
(6 M), a selective antagonist of GluN2B-containing NMDA recep-
lM), glycine (10 lM) and bicuculline (5 lM). As we reported
l
l
tors. Application of the compound 35 (50 nM, 5 min) reversibly
reduced the amplitude of EPSCNMDA (Fig. 4A) and the amount of
reduction was statistically significant (EPSC amplitude 57 18%
of control, mean SEM, n = 4, P <0.05, Fig. 4B). Given these data
demonstrating significant antagonistic effects toward NMDA-med-
iated neurotransmission, we chose to further study pharmacologi-
cal effects of the compound 35. Previous findings indicated that
GluN2B subunit-containing receptor ligands such as 1 and 3 exhib-
ited anticonvulsant effects.1,14 So we planned the evaluation of
in vivo property to prevent audiogenic seizures in DBA/2 mice.21
different fixed doses (17 and 9 lM). In the same system, antioxidant
agents such as gallic and caffeic acids were evaluated as reference
compounds (see Supplementary data). Dihydroxyindole 35 com-
pletely scavenged ABTS+ at 17
lM concentration (>93.6 0.3 per-
centage of inhibition) and its antioxidant ability was comparable
to that demonstrated by the indoles and phenols, gallic acid and
caffeic acid, two well-known antioxidant molecules. Compound 35
displayed antioxidant effect (64.3 2.4 percentage of inhibition) at
fixed dose of 9 lM concentration.
In summary a series of 2-(4-arylmethylpiperidin-1-yl)-1-(1H-
indol-3-yl)ethanones and 1-(4-arylmethylpiperidin-1-yl)-2-(1H-
indol-3-yl)ethane-1,2-diones has been synthesized to improve our
knowledge about SARs of this class of GluN2B-containing NMDA
receptor ligands. We verified that two hydroxyl groups on the in-
dole nucleus significantly improved binding affinity at the ifenpro-
dil pocket of NMDA receptor located in the interface between GluN1
and GluN2B subunits. The most active ligand 2-(4-benzylpiperidin-
1-yl)-1-(5,6-dihydroxy-1H-indol-3-yl)ethanone (35) displayed
very low IC50 value (5.5 nM) and antagonistic effects against
NMDA-evoked current (at 50 nM of concentration). Computational
studies provided insights about the docking pose of compound 35 at
GluN1/GluN2B subunit interface and rationalized the SAR for this
class of ligands. Nevertheless, the lack of effects in vivo by com-
pound 35 might limit its use in clinical investigations. However,
based on its high affinity on GluN2B–NMDA containing receptors,
compound 35 is a valid alternative to compound 1 for in vitro stud-
ied to better understand the functional role of GluN2B-containing
NMDARs. Moreover, data on this study suggest that the high bind-
ing affinity of compound 35 was combined with antioxidant effects.
Taken together, these evidences suggest that compound 35 might
be considered a prototype of ‘dual target’ neuroprotective activity
for future drug development.
3. Experimental section
3.1. Chemistry
Figure 4. (A) Application of compound 35 (50 nM, 5 min) reversibly reduced the
amplitude of NMDA receptor-mediated excitatory post synaptic currents
(EPSCNMDA). EPSC amplitude (pA) values were plotted versus time; insets (1 and
2) show individual EPSC traces from the same recording. (B) Pooled data of %
EPSCNMDA values from different neurons (mean SEM, n = 4): the amplitude of
EPSCNMDA was significantly reduced by application of 35.
All starting materials and reagents commercially available
(Sigma–Aldrich Milan Italy and Alfa Aesar Karlsruhe Germany)
were used without further purification. Microwave-assisted

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R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
reactions were carried out in a CEM focused Microwave Synthesis
System. Melting points were determined on a Buchi B-545 appara-
tus and are uncorrected. Elemental analyses (C, H, N) were carried
out on a Carlo Erba Model 1106 Elemental Analyzer; the results
confirmed a P95% purity. Merck silica gel 60 F254 plates were
used for analytical TLC. Flash chromatography (FC) was carried
out on a Biotage SP1 EXP. Rf values were determined on TLC plates
using a mixture of DCM/MeOH (90:10) as eluent. 1H NMR spectra
were measured in CD3OD or dimethylsulfoxide-d6 (DMSO-d6) with
a Varian Gemini 300 spectrometer; chemical shifts are expressed
in d (ppm) and coupling constants (J) in Hz. All exchangeable pro-
tons were confirmed by addition of D2O.
3.1.2.3. 2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(6-methoxy-1H-
indol-3-yl)ethanone (25). Yield 57%. Mp 199–201 °C. Rf 0.30. 1H
NMR (DMSO-d6) (d) 1.19–2.91 (m, 11H), 3.50 (s, 2H, CH2CO), 3.78
(s, 3H, OMe), 6.81 (d, 1H, H-5, J = 8.8, ArH), 6.95 (s, 1H, H-7, ArH),
7.05-7.22 (m, 5H, ArH), 8.02 (d, 1H, H-4, J = 8.8, ArH), 8.35 (s, 1H,
H-2), 11.65 (br s, 1H, NH). Anal. (C23H25FN2O2): C 72.61; H 6.62;
N 7.36. Found: C 72.72; H 6.54; N 7.49.
3.1.2.4. 1-[5,6-Dimethoxy-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)
piperidin-1-yl]ethanone (26). Yield 35%. Mp 166–168 °C. Rf 0.30.
1H NMR (DMSO-d6) (d) 1.15–2.89 (m, 11H), 3.47 (s, 2H, CH2CO),
3.75 (s, 3H, OMe), 3.76 (s, 3H, OMe), 6.96 (s, 1H, H-7, ArH), 7.03–
7.19 (m, 4H, ArH), 7.65 (s, 1H, H-4, ArH), 8.26 (d, 1H, H-2,
J = 2.7), 11.57 (br s, 1H, NH). Anal. (C24H27FN2O3): C 70.22; H
6.63; N 6.82. Found: C 70.42; H 6.53; N 6.92.
3.1.1. General procedure for the synthesis of intermediates
13–15
Phoshorus oxychloride (0.913 mL, 10 mmol) was added drop-
wise at 0 °C to a solution of the appropriate indole (8–10)
(177 mg, 1 mmol) in dimethylamide of chloroacetic acid
(3.08 mL, 30 mmol) and the mixture was stirred for 2.5 h at room
temperature. The reaction mixture was neutralized by NaOH 2 N
aqueous solution (4 mL), extracted with EtOAc (3 ꢁ 10 mL) and
washed with NaHCO3 saturated aqueous solution (2 ꢁ 10 mL).
The organic layer was dried over dry Na2SO4 and after evaporation
of the solvent under reduced pressure, the residue was crystallized
from DCM. Spectral data for compounds 13–14 were in accordance
with literature.13,14
3.1.2.5. tert-Butyl 3-[2-(4-(4-fluorobenzyl)piperidin-1-yl)ace-
tyl]-1H-indol-5-ylcarbamate (27). Yield 52%. Mp 135–136 °C.
1H NMR (DMSO-d6) (d) 1.19-2.92 (m, 11H), 1.48 (s, 9H), 3.52
(s, 2H, CH2CO) 7.08–7.24 (m, 6H, ArH),7.32 (d, 1H, H-6, J = 8.8,
ArH), 8.37 (d, 1H, H-7, J = 8.8, ArH), 9.17 (br s, 1H, NH), 11.75 (br
s, 1H, NH). Anal. (C27H32FN3O3): C 69.66; H 6.93; N 9.03. Found:
C 69.76; H 7.00; N 9.20.
3.1.2.6. tert-Butyl 3-[2-(4-(4-fluorobenzyl)piperidin-1-yl)ace-
tyl]-1H-indol-6-ylcarbamate (28). Yield 29%. Mp 189–190 °C.
1H NMR (DMSO-d6) (d) 1.19–2.91 (m, 11H), 1.49 (s, 9H), 3.49
(s, 2H, CH2CO), 7.05–7.22 (m, 5H, ArH), 7.22 (s, 1H, ArH), 7.77 (s,
1H, H-7, ArH), 7.96 (d, 1H, H-4, J = 8.2, ArH) 8.36 (d, 1H, H-2,
J = 2.8), 9.33 (br s, 1H, NH), 11.69 (br s,1H, NH). Anal. (C27H32FN3
O3): C 69.66; H 6.93; N 9.03. Found: C 69.76; H 6.80; N 9.10.
3.1.1.1. 2-Chloro-1-(5,6-dimethoxy-1H-indol-3-yl)ethanone
(15). Yield 40%. Mp 221–223 °C. 1H NMR (DMSO-d6) (d) 3.79 (s,
6H, OMe), 4.82 (s, 2H, CH2CO), 7.00 (s, 1H, H-7, ArH), 7.63 (s, 1H,
H-4, ArH), 8.24 (s,1H, H-2), 11.86 (br s, 1H, NH). Anal. (C12H12
ClNO3): C 56.82; H 4.77; N 5.52. Found: C 56.42; H 4.79; N 5.82.
Compounds 16–17 were synthesized following a previously re-
ported procedure and their spectral data were in accordance with
literature.15
3.1.3. General procedure for the synthesis of compounds 29 and
32–34
To a solution of appropriate intermediates 10–12 (1 mmol) in
diethyl ether (5 mL) the oxalyl chloride (0.173 mL, 2 mmol) was
added slowly at 0 °C under nitrogen atmosphere. The reaction mix-
ture was stirred at room temperature for 2 h. A saturated aqueous
NaHCO3 solution (5 mL) was added to quench the reaction and the
mixture was extracted with EtOAc (3 ꢁ 10 mL). The combined ex-
tracts were dried with dry Na2SO4 and concentrated in vacuo. A
mixture of crude compounds (20–22) (1 mmol) and N,N,N,N-tetra-
3.1.2. General procedure for the synthesis of compounds 23–28
To a microwave tube containing the appropriate intermediates
13–17 (1 mmol) dissolved in DMF, K2CO3 (69.1 mg, 0.5 mmol), 4-
benzylpiperidine (0.176 mL, 1 mmol) or 4-fluorobenzylpiperidine
(193.3 mg, 1 mmol) were added. The reaction mixture was placed
in the CEM Discover microwave for 5 min at 50 °C (200 W), then
was quenched with NaHCO3 saturated aqueous solution (10 mL)
and extracted with EtOAc (3 ꢁ 10 mL). The organic phases were
washed with brine (3 ꢁ 10 mL) and then dried over Na2SO4. After
the removal of the solvent under reduced pressure, the desired
final compounds were obtained by purification with by flash chro-
matography (FC) (DCM/MeOH 90:10) and recrystallization with
EtOH.
methyl-O-(1H-benzotriazol-1-yl)uronium
hexafluorophosphate
(HBTU) (379 mg, 1 mmol) in dimethylformamide (2 mL) was stir-
red for 30 min at room temperature. Successively, 4-benzylpiperi-
dine (0.175 mL, 1 mmol) or 4-fluorobenzylpiperidine (193.3 mg,
1 mmol) and TEA (0.139 mL, 1 mmol) were added, the reaction
mixture was stirred for 2 h at room temperature. Then the reaction
mixture was quenched with H2O (10 mL) and extracted with EtOAc
(3 ꢁ 10 mL). The combined extracts were dried with dry Na2SO4
and concentrated in vacuo. The crude compounds were purified
by flash chromatography (FC) (DCM/MeOH 96:4) and recrystal-
lized by treatment with Et2O to give the desired final products
29, 32–34.
3.1.2.1. 2-(4-Benzylpiperidin-1-yl)-1-(5,6-dimethoxy-1H-indol-
3-yl)ethanone (23). Yield 50%. Mp 192–194 °C. Rf 0.52. 1H NMR
(DMSO-d6) (d) 1.19–2.91 (m, 11H), 3.33 (s, 2H, CH2CO), 3.77
(s, 3H, OMe), 3.78 (s, 3H, OMe), 6.97 (s, 1H, H-7, ArH), 7.14–7.29
(m, 5H, ArH), 7.66 (s, 1H, H-4, ArH), 8.28 (s, 1H, H-2), 11.59 (br s,
1H, NH). Anal. (C24H28N2O3): C 73.44; H 7.19; N 7.14. Found: C
73.64; H 7.09; N 7.19.
3.1.3.1. 1-(4-Benzylpiperidin-1-yl)-2-(5,6-dimethoxy-1H-indol-
3-yl)ethane-1,2-dione (29). Yield 80%. Mp 221–223 °C. Rf 0.92.
1H NMR (DMSO-d6) (d) 1.03–4.39 (m, 11H), 3.78 (s, 6H,
OMe), 7.02 (s, 1H, H-7, ArH), 7.10–7.28 (m, 4H, ArH), 7.55 (s, 1H,
H-4, ArH), 7.87 (s, 1H, H-2), 11.97 (br s, 1H, NH). Anal.
(C24H26N2O4): C 70.92; H 6.45; N 6.89. Found: C 70.82; H 6.50; N
6.94.
3.1.2.2. 2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(5-methoxy-1H-
indol-3-yl)ethanone (24). Yield 50%. Mp 200–202 °C. Rf 0.33. 1H
NMR (DMSO-d6) (d) 1.18–2.91 (m, 11H), 3.50 (s, 2H, CH2CO), 3.77
(s, 3H, OMe), 6.83 (dd, 1H, H-6, J = 2.2, J = 8.8, ArH), 7.05–7.27 (m,
4H, ArH), 7.35 (d, 1H, H-7, J = 8.8, ArH), 7.69 (d, 1H, H-4, J = 2.2,
ArH), 8.45(s, 1H, H-2), 11.76 (br s, 1H, NH). Anal. (C23H25FN2O2):
C 72.61; H 6.62; N 7.36. Found: C 72.71; H 6.52; N 7.46.
3.1.3.2. 1-(5,6-Dimethoxy-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)
piperidin-1-yl)ethane-1,2-dione (32). Yield 32%. Mp 177–179 °C.
Rf 0.81. 1H NMR (CD3OD) (d) 1.17–4.58 (m, 11H), 3.89 (s, 3H,

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
1045
OMe), 3.90 (s, 3H, OMe), 6.96–7.19 (m, 4H, ArH), 7.08 (s, 1H, ArH),
7.72 (s, 1H, ArH), 7.85 (s, 1H, H-2). Anal. (C24H25FN2O4): C 67.91; H
5.94; N 6.60. Found: C 67.81; H 5.84; N 6.64.
3.1.5.1. 2-(4-Benzylpiperidin-1-yl)-1-(5,6-dihydroxy-1H-indol-
3-yl)ethanone (35). Yield 50%. Mp 175–177 °C. Rf 0.09. 1H NMR
(DMSO-d6) (d) 1.20–2.92 (m, 11H), 3.47 (s, 2H, CH2CO), 6.81 (s, 1H,
H-7, ArH), 7.16–7.31 (m, 5H, ArH), 7.55 (s, 1H, H-4, ArH), 8.19
(s,1H, H-2), 8.72 (br s, 2H, OH), 11.35 (br s, 1H, NH). Anal.
(C22H24N2O3):C 72.51;H 6.64; N 7.69. Found:C 72.59;H 6.60;N 7.80.
3.1.3.3. tert-Butyl 3-(2-(4-(4-fluorobenzyl)piperidin-1-yl)-2-
oxoacetyl)-1H-indol-5-ylcarbamate (33). Yield 17%. Mp 245–
246 °C. 1H NMR (DMSO-d6) (d) 1.49 (m, 9H), 1.07–3.55 (m, 11H),
7.20–7.28 (m, 4H, ArH), 7.08 (d, 1H, H-6, J = 8.5, ArH), 7.39 (d,
1H, H-7, J = 8.5, ArH) 8.01 (s, 1H, H-4, ArH), 8.35 (s, 1H, H-2),
9.29 (br s, 1H, NH), 12.16 (br s, 1H,NH). Anal. (C27H30FN3O4): C
67.63; H 6.31; N 8.76. Found: C 67.59; H 6.40; N 8.73.
3.1.5.2. 2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(5-hydroxy-1H-
indol-3-yl)ethanone (36). Yield 30%. Mp 192–194 °C. Rf 0.19.
1H NMR (DMSO-d6) (d) 1.16–2.88 (m, 11H), 3.44 (s, 2H, CH2CO),
6.66 (dd, 1H, H-6, J = 8.5, J = 2.2, ArH), 7.22 (d, 1H, H-7, J = 8.5,
ArH), 7.03–7.19 (m, 4H, ArH), 7.55 (d, 1H, H-4, J = 2.2, ArH), 8.33
(d, 1H, H-2, J = 2.7), 8.94 (s, 1H, OH), 11.60 (br s, 1H, NH). Anal. (C22
H23FN2O2): C 72.11; H 6.33; N 7.64. Found: C 72.19; H 6.43; N 7.74.
3.1.3.4. tert-Butyl 3-(2-(4-(4-fluorobenzyl)piperidin-1-yl)-2-
oxoacetyl)-1H-indol-6-ylcarbamate (34). Yield 34%. Mp 219–
221 °C. 1H NMR (DMSO-d6) (d) 1.03–4.40 (m, 11H), 1.49 (m, 9H),
7.07 (d, 1H, J = 8.3, ArH,), 7.12–7.97 (m, 6H, ArH), 8.00 (s, 1H,
H-2) 9.44 (br s, 1H, NH), 12.14 (br s, 1H, NH). Anal. (C27H30FN3O4):
C 67.63; H 6.31; N 8.76. Found: C 67.70; H 6.37; N 8.80.
3.1.5.3. 2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(6-hydroxy-1H-
indol-3-yl)ethanone (37). Yield 40%. Mp 144–146 °C. Rf 0.12.
1H NMR (DMSO-d6) (d) 1.18–2.90 (m, 11H), 3.47 (s, 2H, CH2CO),
6.66 (d, 1H, H-5, J = 8.3, ArH), 6.80 (s, 1H, H-7, ArH), 7.05–7.21
(m, 4H, ArH), 7.91 (d, 1H, H-4, J = 8.3, ArH), 8.28 (s, 1H, H-2),
9.19 (br s, 1H, OH), 11.50 (br s, 1H, NH). Anal. (C22H23FN2O2): C
72.11; H 6.33; N 7.64. Found: C 72.31; H 6.63; N 7.54.
3.1.4. General procedure for the synthesis of compounds 30 and
31
Appropriate indoles 8 and 9 (1 mmol) were dissolved in diethyl
ether (5 mL) and oxalyl chloride (0.173 mL, 2 mmol) was added
slowly at 0 °C under nitrogen atmosphere. The reaction mixture
was stirred at room temperature for 2 h, this was followed by con-
centration in vacuo to remove the diethyl ether. The crude material
obtained (intermediates 18–19) was dissolved in THF (5 mL), the
4-fluorobenzylpiperidine (193.3 mg, 1 mmol) and catalytic amount
of TEA were added. The mixture was stirred for 2 h at room tem-
perature. A saturated aqueous NaHCO3 solution (5 mL) was added
to quench the reaction and the mixture was extracted EtOAc. The
combined extracts were dried with dry Na2SO4 and concentrated
in vacuo. The crude compounds were purified by flash chromatog-
raphy (FC) (DCM/MeOH, 90:10) and recrystallized by treatment
with Et2O to give the desired final products 30 and 31.
3.1.5.4. 1-(5,6-Dihydroxy-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)
piperidin-1-yl)ethanone (38). Yield 65%. Mp 221–223 °C. Rf 0.06.
1H NMR (DMSO-d6) (d) 1.15–2.88 (m, 11H), 3.44 (s, 2H, CH2CO),
6.77 (s, 1H, H-7, ArH), 7.03–7.17 (m, 5H, ArH), 7.51 (s, 1H, H-4,
ArH), 8.14 (s, 1H, H-2, J = 2.6), 8.68 (br s, 1H, OH), 8.71 (br s, 1H,
OH), 11.31 (br s, 1H, NH). Anal. (C22H23FN2O3): C 69.09; H 6.06;
N 7.32. Found: C 69.19; H 6.20; N 7.34.
3.1.5.5. 1-(4-Benzylpiperidin-1-yl)-2-(5,6-dihydroxy-1H-indol-
3-yl)ethane-1,2-dione (41). Yield 45%. Mp 251–253 °C. Rf 0.24.
1H NMR (DMSO-d6) (d) 1.02–4.38 (m, 11H), 6.84 (s, 1H, H-7, ArH),
7.13–7.28 (m, 5H, ArH), 7.43 (s, 1H, H-4, ArH), 7.75 (s, 1H, H-2),
8.84 (s, 1H, OH), 8.87 (s, 1H, OH), 11.70 (br s, 1H, NH). Anal.
(C22H22N2O4): C 69.83; H 5.86; N 7.40. Found: C 69.93, H 5.70; N 7.50.
3.1.4.1. 1-(4-(4-Fluorobenzyl)piperidin-1-yl)-2-(5-methoxy-1H-
indol-3-yl)ethane-1,2-dione (30). Yield 60%. Mp 97–99 °C. Rf
0.85. 1H NMR (DMSO-d6) (d) 1.07–4.41 (m, 11H), 3.80 (s, 3H,
OMe), 6.90 (d, 1H, H-6, J = 8.8, ArH), 7.06–7.23 (m, 4H, ArH), 7.42
(d, 1H, H-7, J = 8.8, ArH), 7.60 (s, 1H, H-4, ArH), 8.02 (d, 1H, H-2,
J = 2.8), 12.16 (br s, 1H, NH). Anal. (C23H23FN2O3): C 70.04; H
5.88; N 7.10. Found: C 70.24; H 5.99; N 7.20.
3.1.5.6. 1-(4-(4-Fluorobenzyl)piperidin-1-yl)-2-(5-hydroxy-1H-
indol-3-yl)ethane-1,2-dione (42). Yield 80%. Mp 214–216 °C. Rf
0.62. 1H NMR (DMSO-d6) (d) 1.05–4.38 (m, 11H), 6.72 (d, 1H,
H-6, J = 8.5, ArH), 7.04–7.20 (m, 4H, ArH), 7.29 (d, 1H, H-7, J = 8.5,
ArH), 7.46 (s, 1H, H-4, ArH), 7.92 (d, 1H, H-2, J = 3.2), 9.13 (s, 1H,
OH), 12.02 (br s, 1H, NH). Anal. (C22H21FN2O3): C 69.46; H 5.56;
N 7.36. Found: C 69.66; H 5.76; N 7.66.
3.1.4.2. 1-(4-(4-Fluorobenzyl)piperidin-1-yl)-2-(6-methoxy-1H-
indol-3-yl)ethane-1,2-dione (31). Yield 22%. Mp 145-147 °C. Rf
0.84. 1H NMR (DMSO-d6) (d) 1.04–4.38 (m, 11H), 3.78 (s, 3H,
OMe), 6.88 (d, 1H, H-5, J = 8.9, ArH), 7.05–7.19 (m, 4H, ArH), 7.40
(d, 1H, H-4, J = 8.9, ArH), 7.58 (s, 1H, H-7, ArH), 8.00 (s, 1H, H-2),
12.14 (br s, 1H, NH). Anal. (C23H23FN2O3): C 70.04; H 5.88; N
7.10. Found: C 70.34; H 5.97; N 7.15.
3.1.5.7. 1-(4-(4-Fluorobenzyl)piperidin-1-yl)-2-(6-hydroxy-1H-
indol-3-yl)ethane-1,2-dione (43). Yield 70%. Mp 145–147 °C. Rf
0.53. 1H NMR (DMSO-d6) (d) 1.05–4.38 (m, 11H), 6.72 (dd, 1H,
H-5, J = 8.5, J = 2.1, ArH), 6.84 (d, 1H, H-7, J = 2.1, ArH), 7.04–7.21
(m, 4H, ArH), 7.82 (d, 1H, H-4, J = 8.5, ArH), 7.88 (d, 1H, H-2,
J = 3.2), 9.33 (br s, 1H, OH), 11.88 (br s, 1H, NH). (C22H21FN2O3):
C 69.46; H 5.56; N 7.36. Found: C 69.56; H 5.46; N 7.30.
3.1.5. General procedure for the synthesis of hydroxyindoles
35–38 and 41–44
The appropriate methoxy-derivatives 23–26, 29–32 were dis-
solved in methylene chloride (5 mL), treated with BBr3 (1 M in
DCM) (6 mL, 6 mmol) under nitrogen atmosphere and stirred over-
night at room temperature. Successively, MeOH (7 mL) was care-
fully added at 0 °C and the solvent removed under reduced
pressure. The residue was dissolved in EtOAc (10 mL) and washed
with H2O (3 ꢁ 10 mL) and with NaHCO3 saturated aqueous solu-
tion (2 ꢁ 10 mL). The organic layer was dried over dry Na2SO4
and after the solvent was removed under reduced pressure. The
crude compound was purified by flash chromatography (FC)
(DCM/MeOH, 90:10) and recrystallized by treatment with EtOH
and Et2O to give the desired final products.
3.1.5.8. 1-(5,6-Dihydroxy-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)
piperidin-1-yl)ethane-1,2-dione (44). Yield 67%. Mp 118–120 °C.
Rf 0.47. 1H NMR (DMSO-d6) (d) 1.05–4.37 (m, 11H), 6.84 (d, 1H,
H-7, J = 4.2, ArH), 7.04–7.60 (m, 4H, ArH), 7.43 (s, 1H, H-4, ArH),
7.74 (d, 1H, H-2, J = 2.2), 8.86 (br s, 2H, OH), 11.71 (br s, 1H, NH).
Anal. (C22H21FN2O4): C 66.66; H 5.34; N 7.07. Found: C 66.76; H
5.44; N 7.17.
3.1.6. General procedure for the synthesis of aminoindoles 39–
40 and 45–46
A mixture of TFA and DCM (1:1, 2 mL) was slowly added to car-
bamates 27–28, 33–34 and the reaction was stirred 1 h at room

1046
R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
temperature. Successively, the mixture was quenched with H2O
(3 mL) and saturated aqueous NaHCO3 solution (5 mL) and ex-
tracted with EtOAc (3 ꢁ 10 mL). The organic layer was dried (Na2-
SO4), combined and concentrated in vacuo. The crude products
were purified by flash chromatography (FC) (DCM/MeOH 90:10)
and recrystallized by treatment with Et2O to give the desired final
products.
CH3), 3.53 (s, 2H, CH2CO), 7.05–7.21 (m, 5H, ArH), 7.38 (d, 1H, H-
7, J = 1.6, ArH), 8.07 (d, 1H, H-4, J = 8.5, ArH), 8.45 (d, 1H, H-2,
J = 3.2), 9.50 (br s, 1H, NH), 11.85 (br s, 1H, NH). Anal. (C23H26FN3-
O3S): C 62.28; H 5.91; N 9.47. Found: C 62.32; H 5.80; N 9.57.
3.1.7.3.
N-(3-(2-(4-(4-Fluorobenzyl)piperidin-1-yl)-2-oxoace-
tyl)-1H-indol-5-yl)methanesulfonamide (49). Yield 27%. Mp
199–200 °C. Rf 0.72. 1H NMR (DMSO-d6) (d) 1.04–4.40 (m, 11H),
2.90 (s, 3H. CH3), 7.06–7.21 (m, 4H, ArH), 7.07 (d, 1H, H-6,
J = 8.2, ArH), 7.49 (d, 1H, H-7, J = 8.2, ArH), 8.01 (s, 1H, H-4, ArH),
8.10 (s, 1H, H-2), 9.50 (br s, 1H, NH), 12.03 (br s, 1H, NH). Anal.
(C23H24FN3O4S): C 60.38; H 5.29; N 9.18. Found: C 60.48; H 5.20;
N 9.11.
3.1.6.1. 1-(5-Amino-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)piperi-
din-1-yl)ethanone (39). Yield 84%. Mp 98–99 °C. Rf 0.18. 1H NMR
(DMSO-d6) (d) 1.11–2.91 (m, 11H), 3.49 (s, 2H, CH2CO), 4.82 (br s,
2H, NH2), 6.54 (dd, 1H, H-6, J = 8.9, J = 2.2, ArH), 7.05–7.21 (m, 5H,
ArH), 7.39 (d, 1H, H-4, J = 2.2, ArH), 8.23 (d, 1H, H-2, J = 3.3), 11.47
(br s, 1H, NH). Anal. (C22H24FN3O): C 72.31; H 6.62; N 11.50. Found:
C 72.40; H 6.50; N 11.60.
3.1.7.4. N-(3-(2-(4-(4-fluorobenzyl)piperidin-1-yl)-2-oxoacetyl)-
1H-indol-6-yl)methanesulfonamide (50). Yield 62%. Mp 147–
148 °C. Rf 0.70. 1H NMR (DMSO-d6) (d) 1.05–4.39 (m, 11H), 2.92
(s, 3H, CH3), 7.05 (d, 1H, H-5, J = 8.0, ArH), 7.10–7.20 (m, 5H,
ArH), 7.43 (s, 1H, H-7, ArH), 7.99 (d, 1H, H-4, J = 8.0, ArH), 8.07 (s,
1H, H-2), 9.68 (br s, 1H, NH), 12.20 (br s, 1H, NH). Anal. (C23H24FN3-
O4S): C 60.38; H 5.29; N 9.18. Found: C 60.48; H 5.31; N 9.10.
3.1.6.2. 1-(6-Amino-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)piperi-
din-1-yl)ethanone (40). Yield 96%. Mp 148–150 °C. Rf 0.17. 1H
NMR (DMSO-d6) (d) 1.16 –2.89 (m, 11H), 3.45 (s, 2H, CH2CO),
4.89 (br s, 2H, NH2) 6.49 (dd, 1H, H-5, J = 8.5, J = 2.1, ArH), 6.57
(d, 1H, H-7, J = 2.1, ArH), 7.03–7.20 (m, 4H, ArH), 7.76 (d, 1H, H-
4, J = 8.5, ArH), 8.12 (d, 1H, H-2, J = 2.7), 11.28 (br s, 1H, NH). Anal.
(C22H24FN3O): C 72.31; H 6.62; N 11.50. Found: C 72.42; H 6.59; N
11.60.
3.2. Receptor binding studies
The radioligand binding assays against NMDA receptor contain-
ing GluN2B-subunit were carried out using [3H]ifenprodil (Custom
Screen by Eurofin Panlab, LCC, USA).37,38 Cerebral cortices of male
Wistar derived rats weighing 175 25 g are used to prepare gluta-
mate NMDA receptors in Tris–HCl buffer pH 7.4. A 5 mg aliquot is
3.1.6.3. 1-(5-Amino-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)piperi-
din-1-yl)ethane-1,2-dione (45). Yield 98%. Mp 124–125 °C. Rf
0.70. 1H NMR (DMSO-d6) (d) 1.03–4.40 (m, 11H), 4.90 (s, 2H,
NH2), 6.59 (d, 1H, H-6, J = 8.9, ArH), 7.06–7.23 (m, 5H, ArH), 7.31
(s, 1H, H-4, ArH), 7.81 (s, 1H, H-2), 11.92 (br s, 1H, NH). Anal. (C22-
H22FN3O2): C 69.64; H 5.84; N 11.07. Found: C 69.60; H 5.90; N
11.12.
incubated with 2 nM [3H]Ifenprodil (plus 5
l
M GBR-12909 to
block non-polyamine sensitive sites) for 120 min at 4 °C. Non-spe-
cific binding is estimated in the presence of 10 M ifenprodil (1).
l
Membranes are filtered and washed, the filters are then counted
3.1.6.4. 1-(6-Amino-1H-indol-3-yl)-2-(4-(4-fluorobenzyl)piperi-
din-1-yl)ethane-1,2-dione (46). Yield 65%. Mp 86–87 °C. Rf 0.65.
1H NMR (DMSO-d6) (d) 1.02–4.39 (m, 11H), 5.06 (s, 2H, NH2), 6.57
(d, 1H, H-5, J = 8.0, ArH), 6.64 (s, 1H, H-7, ArH), 7.06–7.20 (m, 4H,
ArH), 7.71 (d, 1H, H-4, J = 8.0, ArH), 7.76 (s, 1H, H-2), 11.72 (br s,
1H, NH). Anal. (C22H22FN3O2): C 69.64; H 5.84; N 11.07. Found: C
69.60; H 5.89; N 11.17.
to determine [3H]Ifenprodil specifically bound. Three concentra-
tion (10, 0.1, 0.001 lM, in duplicate) of test compounds were used
in displacement assay.
3.3. Electrophysiology
Hippocampal slices were prepared from FVB mice (post natal
PN age 10–15 days) as previously described,39 in accordance with
the European Communities Council Directive of 24 November
1986 (86/609/EEC). Brains were removed, placed in oxygenated
ice-cold artificial cerebrospinal fluid (ACSF; in mM NaCl 124; KCl
3.1.7. General procedure for the synthesis of indole-
methanesulfonamides 47–48 and 49–50
Methanesulfonyl chloride (0.107 mL, 1.1 mmol) was added
dropwise at 0 °C to a solution of appropriate amino-derivatives
39–40 or 45–46 (1 mmol) in DCM (3 mL) and in the presence of
pyridine (0.058 mL, 1.1 mmol), under N2 atmoshere. The reaction
mixture was stirred overnight at room temperature, and it was
quenched with NaOH (6 N, 5 mL) and H2O (3 mL). The layers were
separated and the aqueous phase was washed with DCM
(3 ꢁ 5 mL). The aqueous layer was cooled (0 °C), and acidified to
pH = 2.0 by using 18% aqueous HCl. The obtained solid product
was filtered and recrystallized by treatment EtOH.
3.0; NaH2PO4 1.2; MgSO4 1.2; CaCl2 2.0; NaHCO3 26;
D-glucose
10, pH 7.3) and cut into 300 m slices. Slices were continually per-
l
fused with oxygenated ACSF and CA1 pyramidal neurons in hippo-
campus were visualized with infrared microscopy (Leica DMLFS).
Schaffer collaterals were stimulated with negative current pulses
(duration 0.3 ms, delivered every 15 s by A310 Accupulser, WPI,
USA). Evoked excitatory post synaptic currents (EPSCs) were
recorded under whole-cell configuration (holding potential
ꢀ60 mV; EPC7-plus amplifier HEKA, Germany). Immediately after
the beginning of recording, the slice was perfused with a Mg++-free
3.1.7.1.
N-(3-(2-(4-(4-Fluorobenzyl)piperidin-1-yl)acetyl)-1H-
ACSF containing CNQX (10
(5 M) to isolate NMDA-mediated EPSCs (EPSCNMDA). The record-
ing electrode was a glass micropipette (resistance 1.5–3 M ) filled
lM), glycine (10 lM) and bicuculline
indol-5-yl)methanesulfonamide (47). Yield 55%. Mp 295–296 °C.
Rf 0.24. 1H NMR (DMSO-d6) (d) 1.16–2.89 (m, 11H), 3.49 (s, 2H,
CH2CO), 2.86 (s, 3H, CH3), 7.06 (d, 1H, H-6, J = 8.8, ArH), 7.10–
7.20 (m, 4H, ArH), 7.41 (d, 1H, H-7, J = 8.8, ArH), 8.07 (d, 1H, H-4,
J = 2.0, ArH), 8.48 (s, 1H, H-2), 9.42 (br s, 1H,NH), 11.88 (br s, 1H,
NH). Anal. (C23H26FN3O3S): C 62.28; H 5.91; N 9.47. Found: C
62.38; H 5.80; N 9.50.
l
X
with intracellular solution (composition in mM: K-gluconate 140;
HEPES 10; NaCl 10; MgCl2 2; EGTA 0.2; QX-314 1; Mg-ATP 3.5;
Na-GTP 1; pH 7.3). Data were acquired and analyzed with Signal
software (Cambridge Electronic Design, England). EPSCNMDA ampli-
tude was measured as the difference between peak current and
baseline. For each neuron studied, EPSCNMDA amplitude was
measured during 5 min in control conditions and in the presence
of compound 35, respectively, and the two sets of values were
compared using Student’s unpaired t test (Graph Pad software).
3.1.7.2.
N-(3-(2-(4-(4-Fluorobenzyl)piperidin-1-yl)acetyl)-1H-
indol-6-yl)methanesulfonamide (48). Yield 45%. Mp 187–189 °C.
Rf 0.21. 1H NMR (DMSO-d6) (d) 1.19–2.88 (m, 11H), 2.91 (s, 3H,

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 1040–1048
1047
3.4. Antioxidant activity
rotors).43 All geometries obtained were stored and optimized to
avoid high-energy rotamers. The 1000 conformers were clustered
by similarity to discard redundancies; in this analysis, two geome-
tries were considered non-redundant when they differed by more
than 60° in at least one torsion angle. For each derivative, the low-
est energy structure was then submitted to docking simulations
The ligands minimized in this way were docked in their corre-
sponding proteins by means of Gold Suite 5.0.1. The region of inter-
est used by Gold was defined in order to contain the residues
within 15 Å from the original position of the ligand in the X-ray
structure. The side chain of residue Leu135 was allowed to rotate
according to the internal rotamer libraries in GOLD Suite 5.0.1.
GoldScore44 was chosen as a fitness function and the standard de-
fault settings were used in all the calculations and the ligands were
submitted to 100 genetic algorithm runs. The ‘allow early termina-
tion’ command was deactivated. Results differing by less than
0.75 Å in ligand-all atom RMSD, were clustered together. The best
ranked GOLD-calculated conformation was used for analysis and
representation. Minimization process. The GluN2B/ligand complex
obtained by docking studies was minimized using 1000 iterations
of SD and 1000 interaction of Polak-Ribiere Conjugate Gradient.
Minimization of complexes was performed using OPLS-200545
force field and implicit GB/SA water model46 as solvation treat-
ment using Macromodel.47
ABTS diammonium salt and potassium persulfate were pur-
chased from Sigma. Phenols were purchased from Aldrich, Sigma
and Biosynth. UV measurements were performed in a Perkin–El-
mer Lambda 35 spectrophotometer. MeOH spectrophotometric
grade was purchased from Aldrich. ABTS diammonium salt
(96 mg) and potassium persulfate (33 mg) were dissolved in phos-
phate buffer (0.1 M, pH 7.0, 25 mL) and the mixture was allowed to
stand in the dark at room temperature for 16 h before use. The
ABTS solution was centrifuged for 1 h at 24,000 rpm and then fil-
tered through a 45 lm nylon membrane. In a UV-cuvette (4 mL)
ABTS+ radical cation solution, and phosphate buffer were added
and absorbance of blank solution at 734 nm was adjusted (ca. 1.0
A.U.). Solutions (2 mM) of samples were freshly prepared in
MeOH/phosphate buffer 1:1 and then used for radical scavenging
assay (28
lL) in triplicate. Measure of inhibition percentage of
the absorbance at 734 nm was taken at room temperature after
exactly 20 min. The mean of 3 assays was used to calculate
percentage inhibition as in Eq. 1.
%I ¼ ðABlank ꢀ ASampleÞ=ABlank
ð1Þ
3.5. Anticonvulsant activity in DBA/2 mice
All experiments were performed with DBA/2 mice which are
genetically susceptible to sound-induced seizures.40 DBA/2 mice
(8–12 g; 22–25-days-old) were purchased from Harlan Italy
(Corezzano, Italy). Groups of 10 mice of either sex were exposed
to auditory stimulation 30 min following administration of either
the vehicle or each dose of the drugs studied. The compound was
administered intraperitoneally (ip) (0.1 mL/10 g of mouse body
weight) as a freshly-prepared solution in 50% dimethylsulfoxide
(DMSO) and 50% sterile saline (0.9% NaCl). Individual mice were
placed under a hemispheric perspex dome (diameter 58 cm), and
60 s were allowed for habituation and assessment of locomotor
activity. Auditory stimulation (12–16 kHz, 109 dB) was applied
for 60 s or until tonic extension occurred, and induced a sequential
seizure response in control DBA/2 mice, consisting of an early wild
running phase, followed by generalized myoclonus and tonic flex-
ion and extension sometimes followed by respiratory arrest. The
control and drug-treated mice were scored for latency to and
incidence of the different phases of the seizures. The experimental
protocol and all the procedures involving animals and their care
were conducted in conformity with the institutional guidelines
and the European Council Directive of laws and policies. Statistical
comparisons between groups of control and drug-treated animals
were made using Fisher’s exact probability test (incidence of the
seizure phases). The ED50 values of each phase of audiogenic
seizures was determined for each dose of the compound adminis-
tered, and dose-response curves were fitted using a computer
program by Litchfield and Wilcoxon’s method.41
Acknowledgments
Financial support for this research by Fondo di Ateneo per la
Ricerca (PRA 2009 – grant number ORME09SPNC – Università di
Messina) and International Promotion of Young Researchers ‘Mon-
talcini Program’ are gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.040.
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