Synthesis and biological characterization of 3-substituted 1 H -indoles as ligands of GluN2B-containing N-methyl-d-aspartate receptors. Part 2

Article abstract of DOI:10.1021/jm301508d

In the course of the identification of new indole derivatives targeting GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptor, the (N-1H-indol-6-methanesulfonamide-3-yl)-2-(4-benzylpiperidin-1-yl)ethanone (10b) was identified as a potent ligand for this NMDA receptor subunit. It displays very high binding affinity (IC₅₀ of 8.9 nmol) for displacement of [³H]ifenprodil, thus showing improved potency with respect to the previously reported analogues as confirmed by functional assay. This finding was consistent with the docking pose of compound 10b within the binding pocket localized in the GluN1-GluN2B subunit interface of NMDA receptor tetraheteromeric complex.

Full text of DOI:10.1021/jm301508d

Article
pubs.acs.org/jmc
Synthesis and Biological Characterization of 3‑Substituted
1H‑Indoles as Ligands of GluN2B-Containing N‑Methyl‑D‑aspartate
Receptors. Part 2
Rosaria Gitto,*,† Laura De Luca,† Stefania Ferro,† Maria R. Buemi,† Emilio Russo,‡
Giovambattista De Sarro,‡ Mariangela Chisari,§ Lucia Ciranna,∥ and Alba Chimirri†
†Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Universita
‡Dipartimento di Science of Health, Universita
Magna Graecia, Viale Europa Localita
̀
di Messina, Viale Annunziata, I-98168 Messina, Italy
Germaneto, I-88100 Catanzaro, Italy
̀
̀
§Dipartimento di Biomedicina Clinica e Molecolare, Sezione di Farmacologia e Biochimica, Universita
6, I-95125 Catania, Italy
̀
di Catania, Viale Andrea Doria
∥Dipartimento di Scienze Bio-Mediche, Sezione di Fisiologia, Universita
̀
di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy
S
* Supporting Information
ABSTRACT: In the course of the identification of new indole
derivatives targeting GluN2B-subunit-containing N-methyl-D-
aspartate (NMDA) receptor, the (N-1H-indol-6-methanesulfo-
namide-3-yl)-2-(4-benzylpiperidin-1-yl)ethanone (10b) was
identified as a potent ligand for this NMDA receptor subunit.
It displays very high binding affinity (IC50 of 8.9 nmol) for
displacement of [3H]ifenprodil, thus showing improved potency
with respect to the previously reported analogues as confirmed
by functional assay. This finding was consistent with the docking
pose of compound 10b within the binding pocket localized in
the GluN1-GluN2B subunit interface of NMDA receptor
tetraheteromeric complex.
has been made in the identification of different classes of
GluN2B subunit selective antagonists as allosteric modulators
that interact with the extracellular amino-terminal domain
(ATD).11 The prototype of this class of ligands was the
phenylethanolamine derivative 1 (ifenprodil),12 which provided
the starting point for developing new selective ligands. During
the optimization process other ifenprodil-like compounds have
been identified such as 2 (Ro 25-6981)13 and 3 (CP-
101,606),14 which demonstrated more selectivity and wider
safety profile.15 Notably, 3 showed efficacy in treatment of
refractory major depressive disorder (phase 2 trials).16,17
Subsequently extensive studies led to the identification of
new chemical entities with a high degree of subunit selectivity.
In this context, we reported a molecular modeling strategy
useful for identifying several indole derivatives (4, 5; Chart 1)
as GluN2B ligands with IC50 values from 17 nM to 2.66
μM.18−21 Interestingly, one of the most potent ligand (4a,
Scheme 1) was able to prevent audiogenic seizures in DBA/2
mice and reduce NMDA receptor-mediated current recorded in
patch clamp experiments from hippocampal neurons and also
showed neuroprotective effects in HCN-1A cells.18,20
INTRODUCTION
■
Most of the excitatory neurotransmission in the mammalian
central nervous system is mediated by glutamate (Glu). Three
different classes of postsynaptic ionotropic glutamate receptors
(iGluRs) have been identified.1−5 Among them the N-methyl-
D-aspartate (NMDA) receptors have gained much attention
because of their implication in acute and chronic neurological
diseases (among which are stroke, traumatic brain injury,
neuropsychiatric disorders, dementia, and epilepsy).6−8 The
tetraheteromeric structure of NMDA receptor contains two
GluN1 subunits and two of the four possible GluN2 subunits
(GluN2A−D) encoded by different genes.9 The four possible
GluN2 subunits (GluN2A−D) are differently distributed
among brain regions and contribute to functional diversity of
NMDA receptors.10,11 It was demonstrated that antagonists
targeting GluN2B-containing receptors could provide an
opportunity to pharmacologically modulate NMDA receptor
function for selected group of neurons, thus avoiding side
effects that have so far limited the therapeutic utility of broad-
spectrum NMDAR antagonists as neurotherapeutic agents.11
GluN2B subunit shows a modular architecture consisting of
four discrete domains: an amino-terminal domain (ATD), a
clamshell-like ligand binding domain (LBD), a transmembrane
domain forming the ion pore, and an intracellular carboxy-
terminal domain (CTD).1 In the past years, significant progress
Received: July 31, 2012
© XXXX American Chemical Society
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Chart 1. GluN2B Subunit-Containing NMDA Receptor Ligands
a
Scheme 1
a
Reagents and conditions: (i) Boc2O, diethyl ether, NaOH, rt 18 h; (ii) ClCH2COCl, pyridine, dioxane, microwave 5 min, 50 °C, 200 W; (iii) 4-
benzylpiperidine, K2CO3, DMF, microwave 10 min 50 °C, 200 W; (iv) DCM/TFA, rt 1 h; (v) MsCl, DCM, pyridine, rt 20 h; (vi) ClCOCOCl,
diethyl ether, rt 2 h; (vii) 4-benzylpiperidine, HBTU, TEA, DMF, rt 2 h.
Then structure−activity relationship (SAR) analysis and
molecular docking studies highlighted several structural
requirements improving the recognition process into binding
site. The obtained results were in good agreement with X-ray
data that more recently demonstrated that the GluN1/GluN2B
dimer interface represents the binding site for noncompetitive
GluN2B subunit-selective NMDA receptor antagonists.22,23
Herein we report the development of new indole derivatives
that were designed to optimize the binding affinity and
pharmacological activity of this class of ligands. The evaluation
of binding affinity for displacement of [3H]ifenprodil, patch
clamp experiments to test functional activity, and anticonvul-
sant screening were carried out to describe the biological profile
of the new synthesized compounds. Moreover, to further
rationalize the biological results, molecular docking studies have
been performed.
Chart 2. Designed Ligands
GluN1-GluN2B ATD in complex with 1 and 2 (Protein Data
Bank with accession codes 3QEL and 3QEM)22 and on our
docking experiments.21 These studies revealed that the most
GluN2B-selective antagonists interact with the binding site
through specific aminoacidic residues in GluN1 and GluN2B
subunits.21
We attempted the isosteric and bioisosteric replacement of
hydroxyl group of previously reported compounds 4a,b and
5a,b to investigate the role of H-bonding interaction and/or
polar contacts with residue E236 (GluN2B) during the
recognition process into the binding site. To this end, amino
or methanesulfonamide substituent (R = NH2 and NHMs,
Chart 2) has been inserted at the C-5 or C-6 position.
Moreover we substituted the ethanone linker with a 2-
oxoethanone one, thus inducing the lowering of nitrogen
RESULTS AND DISCUSSION
■
To enhance binding affinity and improve our understanding of
SARs of this class of NMDA antagonists, we planned to
synthesize eight new indoles (Chart 2). This structural
optimization program started taking into account the binding
poses in the ATD-binding site and SARs of other GluN2B-
selective antagonists.15 Particularly, we focused on X-ray data of
B
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basicity to assess the role of the electrostatic interaction
between the positive ionizable nitrogen atom (piperidine
nucleus) and the carbonyl group of residue Q110 (GluN2B)
within ifenprodil binding pocket.22
As shown in Table 1 the replacement of the hydroxyl
substituent with an amino group gave derivatives 9a,b and
13a,b displaying significant decrease of binding affinity. We
observed a similar behavior for the C-5 methanesulfonamide
derivatives 10a and 14a (R = NHMs) when compared to
compounds 4a and 5a, respectively, whereas 5-NHMs
derivative 10a was a more potent ligand than amino-substituted
analogue 9a. Notably, the presence of a methanesulfonamide
substituent at the C-6 position gave better results. In fact the 6-
NHMs-derivative 10b showed the best IC50 of 0.0089 μM that
was about 2-fold more potent than that of the 6-hydroxyl
analogue 4b and about 250-fold more potent than that of the 6-
NH2-derivative 9b. Among new 2-oxoethanones 13 and 14 (X
= O) it was observed that the 6-NHMs derivative 14b was the
most active ligand, showing IC50 of 0.063 μM, which was in
good agreement with the result obtained for 6-substituted
derivative 10b. However, compound 14b showed lower
potency than 6-hydroxyl 2-oxoethanone analogue 5b (see
Table 1).
Overall the results of the binding affinity evaluation
suggested that the optimization of binding affinity has been
reached by introducing a methanesulfonamide substituent at C-
6 position. In fact, among NHMs derivatives the 6-substituted
compounds were the most active ligands (10b and 14b). This
behavior was in apparent discrepancy with previously reported
SAR data, displaying very similar potency for the two ethanone
derivatives C-5 and C-6 hydroxy-substituted (4a and 4b, X =
H2), but it was in accordance with data obtained for 2-
oxoethanone compounds (X = O), among which we found that
C-5 substituted derivative 5a was less active than C-6 analogue
5b. So we can speculate that the substituent at C5/C6 position
could play a relevant role during the molecular recognition to
binding pocket. We postulate that this substituent could be able
to make H-bonding interaction as donor moiety; however, its
position seems very crucial for the optimization of binding
affinity as explored through docking studies (vide infra).
In order to assess the role of lipophilicity for this class of
GluN2B ligands, we calculated their ligand efficiency (LE) and
lipophilic efficiency (LipE or LLE). In recent years LE and
LLE26,27,29−31 have been shown to be useful tools in the lead
optimization process. LE emerged as an approach to mean-
ingfully interpret molecular size by balancing the size of the
molecule against its potency. On the other hand LLE relates
potency to lipophilicity, providing an estimate of the binding
efficiency in the context of lipophilicity; therefore it has been
used as an index of lipophilicity per unit of potency. LLE
measures the extent to which binding is driven by specific
interactions between ligand and protein as opposed to simple
hydrophobic effects (binding in a protein cavity as way to
escape from solvent). Notably, an LE ranging from 0.3 to 0.5
characterizes a good drug candidate and compounds with LLE
above 5 are usually considered highly optimized. As shown in
Table 1, the most active indole derivatives exhibited promising
LE and LLE values compared to compound 1. In particular,
compound 5b exhibits significant LE (0.40) and excellent LLE
(5.12) combined with cLogD7.4 of 2.54 considered optimal for
drug developability. All these values are very similar to those of
drug prototype 1. Although the most potent ligand 10b shows
good LE (0.39), it displays high cLogD7.4 (3.70), thus
generating lower LLE when compared to compounds 5b and
14b bearing the 2-oxoethanone linker. Interestingly, for
compound 14b we observed a good compromise between
binding affinity (IC50 = 63nM) and LLE (5.18). Among other
Chemistry. The synthesis of target indole derivatives 9, 10,
13, and 14 was accomplished as outlined in Scheme 1. The
synthesis started from indoles as Boc-protected amine
derivatives 6a,b. By reaction of indoles 6a,b with chloroacetyl
chloride, we obtained intermediates 7a,b which were coupled
with benzylpiperidine to afford compounds 8a,b. By treatment
with DCM/TFA, compounds 8a,b gave 1-(5-amino-1H-indol-
3-yl)-2-(4-benzylpiperidin-1-yl)ethanone (9a) and 1-(6-amino-
1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)ethanone (9b), which
were converted into methanesulfonamide derivatives 10a,b.
The reaction of indoles 6a,b with oxalylchloride yielded 2-(1H-
indol-3-yl)-2-oxoacetic acids 11a,b. In turn they were used as
crude products for the coupling with benzylpiperidine using a
standard amide coupling reaction to furnish Boc-protected
aminoindoles 12a,b. Finally, we prepared the desired 1-(amino-
1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)-2-oxoethanones
(13a,b) and (N-1H-indolmethanesulfonamide-3-yl)-2-(4-ben-
zylpiperidin-1-yl)-2-oxoethanones (14a,b) by using the same
procedure previously employed to obtain final compounds 9a,b
and 10a,b. The chemical characterization of new indoles was
supported by elemental analyses and spectroscopic measure-
ments (see Experimental Section).
Biological Characterization. All synthesized compounds
were evaluated for their ability to interact with the GluN2B
subunit by testing [3H]ifenprodil binding inhibition,24 and
results were compared with those previously reported for 4a,b,
5a,b, and 1 (Table 1).
Table 1. GluN2B/NMDA Binding Affinities and
Physicochemical Properties of Indole Derivatives (4, 5, 9,
10, 13, 14) and Reference Compound 1
a
IC50, μM
(inhibition at
b
c
d
compd
R
X
10 μM)
LE
LLE
cLogD7.4
e
4a
5-OH
H2
H2
O
0.025
0.41
0.42
0.29
0.40
0.33
0.32
0.31
0.39
3.79
3.53
3.47
5.12
2.42
1.97
2.78
4.35
3.81
4.24
2.11
2.54
3.51
3.67
3.71
3.70
1.84
2.00
2.01
2.02
2.33
e
4b
6-OH
0.017
e
5a
5-OH
2.66
e
5b
6-OH
O
0.022
9a
5-NH2
H2
H2
H2
H2
O
1.180
2.270
0.320
0.0089
40%
9b
6-NH2
10a
10b
13a
13b
14a
14b
1
5-NHMs
6-NHMs
5-NH2
6-NH2
O
32%
5-NHMs
6-NHMs
ifenprodil
O
42%
O
0.063
0.020
0.34
0.45
5.18
5.37
a
b
Displacement of [3H]ifenprodil.24 LE = −1.4 log IC50/number of
heavy atoms.25,26 LLE = −log IC50 − cLogD7.4.27 cLogD7.4 data at
c
d
pH 7.4 are predicted using a commercially available program (ACD/
Labs).28 Data from ref 19.
e
C
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Figure 1. Compound 10b behaves as an antagonist of GluN2B-containing NMDA receptors. Excitatory postsynaptic currents mediated by NMDA
receptors (EPSCNMDA) were recorded in mouse CA1 pyramidal neurons in Mg2+-free ACSF in the presence of CNQX (10 μM), glycine (10 μM),
and bicuculline (5 μM). (A) EPSCNMDA was inhibited by 2 (Ro 25-6981) (6 μM, 5 min), a selective antagonist of NMDA receptors containing the
GluN2B subunit (EPSCNMDA amplitude of 29 11% of control (mean SEM, n = 5, P < 0.001). (B) Application of 10b (100 nM, 5 min) reduced
the amplitude of EPSCNMDA to 23.3 9% of control (mean SEM, n = 4, P < 0.0001). EPSC amplitude values are expressed as % of control
amplitude. Pooled data of % EPSC values are plotted vs time. Insets (traces 1 and 2) show individual EPSCs from representative experiments.
derivatives, potent ligands 4a and 4b show excellent LE values
(0.41 and 0.42, respectively); however, the high lipophilicity
(cLogD7.4 > 3.5) reduces LLE values, thus compromising their
drug developability.
of 10b was fully reversible. A similar result was observed in rat
CA1 neurons, in which application of 10b (100 nM, 5 min)
reversibly reduced EPSCNMDA amplitude to 7.9 and 50% of
control (n = 2).
On the basis of the observations described, we selected
compound 10b among other derivatives as a candidate for
functional investigations on biological models (Figure 1). To
evaluate the functional activity of the most potent ligand, 10b,
we used the patch clamp technique to record NMDA-mediated
synaptic currents in mouse hippocampus, where GluN2B-
containing NMDA receptors are expressed at high levels at
early postnatal age.32 Excitatory postsynaptic currents (EPSCs)
were recorded from CA1 neurons at a holding potential of −60
mV in Mg2+-free ACSF, in the presence of 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX) (10 μM), glycine (10
μM), and bicuculline (5 μM). Application of D-AP5 (50 μM, 5
min) reduced EPSC amplitude to 28 14% of control (n = 3,
P < 0.0001), indicating that EPSCs were mediated by NMDA
receptors (EPSCNMDA). Application of 1 (10 μM, 5 min)
induced a biphasic effect on EPSCNMDA amplitude, with a
transient increase (EPSC amplitude of 153 24% of control,
To evaluate in vivo effects, all synthesized compounds were
tested for their ability to prevent audiogenic seizures in DBA/2
mice.35 In contrast to previous findings indicating that GluN2B
subunit-containing receptor ligands such as 1, 2, and 4a exhibit
anticonvulsant effects,18,36 the new synthesized compounds are
not able to demonstrate any efficacy (see Supporting
Information). To understand the lack of in vivo efficacy, we
calculated37 the topological polar surface (TPSA), which is a
relevant parameter to estimate compound polarity and to
predict blood−brain barrier penetration.38,39 Drugs or
candidates acting on CNS generally have optimal TPSA values
2
́
estimated in the range 60−70 Å ; thus, the high TPSA value for
2
́
the most active ligand 10b (82.27 Å ) could explain the lack of
in vivo efficacy due to its low capability to permeate the blood−
brain barrier.
Computational Studies. To understand the effect of some
of these modifications on GluN2B/NMDA receptor inter-
actions, the docking poses within the GluN1/GluN2B interface
for selected 6-substituted ligands (e.g., 4b, 9b, and 10b) were
examined (see Experimental Section). As shown in Figure 2A,
the most active ligand 10b (IC50 of 8.9 nM) establishes a polar
interaction with residue Q110 of GluN2B through the positive
ionizable piperidine nitrogen atom. This ligand is also able to
form hydrogen bonding interactions between the methane-
sulfonamide group at C-6 and residue E236 of GluN2B as well
as between residue K131 of GluN1b and NH of the indole ring.
Moreover, by means of the hydrogen bonding interaction
between the oxygen atom and the backbone NH of M207, the
mesyl functionality of 10b occupies a further subpocket located
at the bottom of dimeric interface (see Figure 2A). Finally, the
benzylpiperidine portion and indole nucleus of ligand 10b form
hydrophobic interactions with some crucial residues on GluN1
(Y109, G112, L135) and GluN2B (I111, F176, P177). As seen
in Figure 2B, we further examined the alignment of docking
poses of the most potent ligand 10b and other 6-substituted
derivatives 4b and 9b. In spite of their different affinities in
binding to GluN2B/NMDA receptor (see Table 1), we found
mean
SEM, n = 3, P < 0.0001) followed by a reduction
(EPSC of 79 4% of control, mean SEM, n = 3, P < 0.001).
Similar results were observed in previous studies showing that
compound 1, besides behaving as an antagonist of NMDA
receptors, also increases NMDA receptor affinity for glutamate-
site agonists and thus can either enhance or suppress NMDA-
mediated effects.33,34 We next tested compound 2, described as
a high-affinity and selective blocker of GluN2B-containing
NMDA receptors, with IC50 values of 0.009 and 0.017 μM,
respectively, for NR1C/NR2B and NR1F/NR2B subunit
combination of NMDA receptors expressed in Xenopus
oocytes.13 Application of 2 (6 μM, 5 min) reduced EPSCNMDA
amplitude to 29 11% of control (mean SEM, n = 5, P <
0.001, Figure 1A), confirming that in our preparation the
largest fraction of EPSCNMDA amplitude was mediated by
NMDA receptors containing the GluN2B subunit. Application
of compound 10b (100 nM, 5 min) reduced the amplitude of
EPSCNMDA to 23.3 9% of control (mean SEM, n = 4, P <
0.0001; Figure 1B), which is comparable to the amount of
inhibition induced by 2 at a maximally effective dose. The effect
D
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bonding donor interaction. Finally, the improvement of the
potency of methanesulfonamide compound 10b with respect to
hydroxyl analogous 4b could be explained considering the
additional interaction with M207 which has been recently
highlighted23 as a very crucial residue during the recognition
process into the GluN1/GluN2B interface of NMDA ATD.
CONCLUSIONS
■
As a continuation of our previous research, we designed and
synthesized new 3-substituted-1H-indoles that proved to be
highly potent ligands of GluN2B subunit-containing NMDA
receptors. We considered as starting point the X-ray data of
GluN1-GluN2B ATD in complex with 1 and 2 and our docking
experiments. All synthesized compounds were evaluated for
their ability to interact with the GluN2B subunit by testing
[3H]ifenprodil binding inhibition. The best results were
obtained with the 6-NHMs-derivative 10b displaying an IC50
of 0.0089 μM, better than 1 and previously synthesized
analogues (e.g., 4b). Also functional assays confirmed this
behavior, thus suggesting that the nature and position of the
substituents could play a relevant role for the modulation of
biological effects. Docking studies allowed us to postulate that
the methanesulfonamide substituent at the C-6 position (10b)
could be able to make H-bonding interaction as donor moiety.
Moreover, the position of this substituent furnishes an
additional interaction with M207 which has been recently
highlighted as a very crucial residue during the recognition
process into the GluN1/GluN2B interface of NMDA ATD. We
show that compound 10b displays high affinity and selective
binding properties as well as a potent antagonist activity on
GluN2B-containing NMDA receptors. Although 10b does not
exert any significant effect against audiogenic seizures in vivo
(probably because of poor brain penetration, as estimated by
TPSA calculation), this potent ligand might be a starting point
for the design and development of new compounds showing
more optimized physicochemical properties in CNS drug space.
EXPERIMENTAL SECTION
Figure 2. Docking poses of compounds 4b, 9b, and 10b at the GluN1-
GluN2B subunit interface (PDB code 3QEL): (A) binding of
compound 10b (magenta); (B) alignments of compounds 4b
(orange), 9b (yellow), and 10b (magenta). Important residues are
drawn in stick and colored in cyan (GluN2B) and in green (GluN1).
Hydrogen bonds (shown as dashed yellow lines) are formed between
the compounds and GluN1-GluN2B subunit interface. The structures
were prepared in PyMOL software.40
■
Chemistry. All starting materials and reagents commercially
available (Sigma-Aldrich, Milan, Italy) were used without further
purification. Microwave-assisted reactions were carried out in a CEM
focused microwave synthesis system. Melting points were determined
on a Stuart SMP10 apparatus and are uncorrected. Elemental analyses
(C, H, N) were carried out on a Carlo Erba model 1106 elemental
analyzer; the results confirmed a ≥95% purity. Merck silica gel 60
F254 plates were used for analytical TLC; column chromatography
was performed on Merck silica gel 60 (70−230 mesh). Flash
chromatography (FC) was carried out on a Biotage SP1 EXP. Rf
values were determined on TLC plates using a mixture of CH2Cl2/
CH3OH (9:1) as eluent. 1H NMR spectra were measured in CDCl3 or
dimethylsulfoxide-d6 (DMSO-d6) with a Varian Gemini 300
spectrometer. Chemical shifts are expressed in δ (ppm) and coupling
constants (J) in Hz. All exchangeable protons were confirmed by
addition of D2O.
General Procedures for the Preparation of tert-Butyl 1H-
Indolylcarbamates (6a,b). A solution of appropriate aminoindoles
(396.0 mg, 3 mmol) with NaOH (7.5 mL, 1.6 M) was added to a
solution of di-tert-butyl pyrocarbonate (Boc2O) (720.0 mg, 3.3 mmol)
in Et2O (7.5 mL). The resulting mixture was stirred at room
temperature overnight. The layers were separated and the organic
phase was dried using dry Na2SO4, filtered, and concentrated under
reduced pressure. The final compounds were purified by crystallization
with Et2O.
that these three compounds share a very similar orientation and
network of interactions within the GluN1/GluN2B interface.
Particularly they seem to differ exclusively from the strength of
hydrogen bonding interaction to the crucial residue E236
(GluN2B). Overall we can hypothesize that the lower potency
of the 6-amino derivative 9b (IC50 of 2.270 μM) compared to
ligands 4b (IC50 of 0.017 μM) and 10b (IC50 of 0.0089 μM)
could be related to the lack of a suitable chemical feature
capable of being a good H-bond donor group to make a
relevant interaction with E236. These findings were in good
agreement with the pKa values (calculated with ACD/Labs)28
of the most active ligands. Thus, 6-hydroxyl derivative 4b and
6-methanesulfonamide derivative 10b possessed similar weak
acid sites with pKa values of 9.63 and 9.97, respectively. On the
contrary the medium basicity of the 6-amino substituent of
compound 9b probably reduced the strength of the hydrogen
General Procedure for the Preparation of tert-Butyl [3-
(Chloroacetyl)-1H-indolyl]carbamates (7a,b). The synthesis of
E
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compounds 7a,b has been carried out following a previously reported
procedure20 that has been optimized by means of the use of
microwave irradiation. A mixture of chloroacetyl chloride (0.121 mL,
1.5 mmol) in dioxane (2 mL) was added dropwise to a solution of the
appropriate Boc-protected amine derivatives (6a,b) (232 mg, 1 mmol)
and pyridine (0.126 mL, 1.5 mmol) in dioxane (2 mL). The reaction
mixture was placed in a cylindrical quartz tube (⌀, 2 cm), then stirred
and irradiated in a microwave oven under the following conditions: 5
min, 50 °C, 200 W. Then the reaction was quenched with water (5
mL), and extraction was with EtOAc (3 × 10 mL). The combined
organic layers were dried using Na2SO4, filtered, and concentrated to
dryness in vacuo. The crude material was then subjected to silica gel
chromatography by using cyclohexane/EtOAc (60:40).
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 extracts were dried with
Na2SO4 and concentrated in vacuo. A mixture of the crude
compounds (1 mmol) and (HBTU) (379 mg, 1 mmol) in DMF (2
mL) was stirred for 30 min at room temperature. Successively, 4-
benzylpiperidine (0.351 mL, 2 mmol) and TEA (0.139 mL, 1 mmol)
were added. The reaction mixture was stirred for 2 h at room
temperature. Then this 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 compound
was purified by flash chromatography (FC) (DCM/CH3OH, 96:4)
and recrystallized from Et2O.
General Procedure for the Preparation of Carbamates 8a,b.
A DMF (2 mL) solution of the appropriate 3-chloroacetylindole
derivative 7a,b (308.4 mg, 1 mmol) and 4-benzylpiperidine (0.263 mL,
1.5 mmol) in alcaline medium by K2CO3 (70.0 mg, 0.5 mmol) was
stirred and irradiated in a microwave oven under the following
conditions: 10 min, 50 °C, 200 W. The reaction mixture was quenched
with NaHCO3 saturated aqueous solution (10 mL) and extracted with
EtOAc (3 × 10 mL). The combined organic phases were washed with
brine, dried over dry Na2SO4, filtered, and concentrated to dryness in
vacuo. The crude material was purified by flash chromatography (FC)
(DCM/CH3OH, 9:1) and then crystallized by treatment with EtOH
to give the desired products 8a,b.
General Procedure for the Preparation of 1-(Amino-1H-
indol-3-yl)-2-(4-benzylpiperidin-1-yl)ethanones (9a,b). A solu-
tion of TFA and DCM (1:1, 2 mL) was slowly added to cooled (0 °C)
carbamates 8a,b (447 mg, 1 mmol), and the mixture was stirred for 1 h
at room temperature. Successively, the reaction mixture was quenched
with H2O (3 mL) and saturated aqueous NaHCO3 solution (5 mL)
and extracted with EtOAc (3 × 10 mL). The organic layer was dried
(Na2SO4), combined, and concentrated in vacuo. The crude product
was purified by flash chromatography (FC) (DCM/CH3OH 9:1) and
recrystallized by treatment with Et2O to give the desired final products.
1-(5-Amino-1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)-
ethanone (9a). Yield 47%. Mp 148−150 °C, dec. Rf = 0.15. 1H NMR
(DMSO-d6) (δ) 1.25−3.52 (m, 13H), 4.96 (bs, 2H, NH2), 6.51−6.54
(dd, 1H, ArH, J = 8.1), 7.10−7.25 (m, 6H, ArH,), 7.37 (s, 1H, ArH),
8.23 (s, 1H, H-2), 11.49 (bs, 1H, NH).
1-(6-Amino-1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)-
ethanone (9b). Yield 90%. Mp 142−144 °C, dec. Rf = 0.11. 1H NMR
(DMSO-d6) (δ) 1.22−3.61 (m, 13H), 4.88 (bs, 2H, NH2), 6.50−6.57
(d, 2H, ArH), 6.58 (s, 1H, ArH), 7.13- 7.25 (m, 5H, ArH), 7.76−7.79
(dd, 2H, ArH), 8.14 (s, 1H, H-2), 11.29 (bs, 1H, NH).
General Procedure for the Preparation of (N-1H-Indoleme-
thanesulfonamide-3-yl)-2-(4-benzylpiperidin-1-yl)ethanones
(10a,b). Methanesulfonyl chloride (0.080 mL, 1.1 mmol) was added
dropwise at 0 °C to a solution of appropriate amino derivatives 9a,b (1
mmol) in DCM (3 mL) and in the presence of pyridine (0.089 mL,
1.1 mmol), under N2 atmosphere. The reaction mixture was stirred
overnight at room temperature, and the reaction 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 with CH3OH.
General Procedure for the Preparation of 1-(Amino-1H-
indol-3-yl)-2-(4-benzylpiperidin-1-yl)-2-oxoethanones (13a,b)
and (N-1H-Indolmethanesulfonamide-3-yl)-2-(4-benzylpiperi-
din-1-yl)-2-oxoethanone (14a,b). By use of the same synthetic
procedure previously described to obtain derivatives 9a,b and 10a,b,
the desired compounds 13a,b and 14a,b have been synthesized.
1-(5-Amino-1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)-2-ox-
1
oethanone (13a). Yield 70%. Mp 147−150 °C, dec. Rf = 0.56. H
NMR (CDCl3) (δ) 1.25−4.65 (m, 9H), 2.54−2.57 (d, 2H, J = 6.69),
6.65−7.65 (m, 7H, ArH), 7.72 (s, 1H, ArH), 7.73 (s, 1H, H-2), 10.10
(bs, 1H, NH).
1-(6-Amino-1H-indol-3-yl)-2-(4-benzylpiperidin-1-yl)-2-ox-
1
oethanone (13b). Yield 75%. Mp 128−130 °C, dec. Rf = 0.50. H
NMR (CDCl3) (δ) 1.28−4.64 (m, 9H), 2.54−2.56 (d, 2H, J = 6.87),
6.52 (s, 1H, ArH), 6.68−6.71 (dd, 1H, ArH), 7.11−7.31 (m, 5H,
ArH), 7.58−7.59 (s, 1H, H-2), 8.04−8.07 (dd, 1H, ArH), 9.34 (bs,
1H, NH).
(N-1H-Indol-5-methanesulfonamide-3-yl)-2-(4-benzylpiperi-
din-1-yl)-2-oxoethanone (14a). Yield 41%. Mp 121−123 °C, dec.
Rf = 0.66. 1H NMR (DMSO-d6) (δ) 1.05−4.40 (m, 11H), 2.91 (s, 3H,
Me), 7.15−7.51 (m, 7H, ArH), 8.01 (s, 1H, ArH), 8.11 (s, 1H, H-2),
9.54 (s, 1H, NH), 12.31 (bs, 1H, NH).
(N-1H-Indol-6-methanesulfonamide-3-yl)-2-(4-benzylpiperi-
din-1-yl)-2-oxoethanone (14b). Yield 35%. Mp 240−242 °C, dec.
Rf = 0.54. 1H NMR (DMSO-d6) (δ) 1.05−4.39 (m, 11H), 2.91 (s, 3H,
Me), 7.10−7.28 (m, 6H, ArH), 7.43 (s, 1H, ArH), 7.98−8.01 (dd, 1H,
ArH, J = 8.5), 8.08 (s, 1H, H-2), 9.68 (bs, 1H, NH), 12.21 (bs, 1H,
NH).
Receptor Binding Studies. The radioligand binding assays
against NMDA receptor containing the GluN2B subunit were carried
out using [3H]ifenprodil (Custom Screen by Ricerca Biosciences,
U.S.).24,41 Cerebral cortices of male Wistar derived rats weighing 175
25 g are used to prepare glutamate NMDA receptors in Tris-HCl
buffer, pH 7.4. A 5 mg aliquot is incubated with 2 nM [3H]Ifenprodil
(plus 5 μM GBR-12909 to block nonpolyamine sensitive sites) for 120
min at 4 °C. Nonspecific binding is estimated in the presence of 1 (10
μM). Membranes are filtered and washed, and the filters are then
counted to determine [3H]Ifenprodil specifically bound. Three
concentrations (in duplicate) of test compounds were used in the
displacement assay.
Electrophysiology. Hippocampal slices were prepared from FVB
mice and Wistar rats (postnatal PN age 10−15 days) as previously
described,42 in accordance with the European Communities Council
Directive of November 24, 1986 (86/609/EEC). The brains were
removed, placed in oxygenated ice-cold artificial cerebrospinal fluid
(ACSF; in mM: NaCl 124; KCl 3.0; NaH2PO4 1.2; MgSO4 1.2; CaCl2
2.0; NaHCO3 26; D-glucose 10; pH 7.3), and cut into 300 μm slices.
Slices were continually perfused with oxygenated ACSF and viewed
with infrared microscopy (Leica DMLFS). Schaffer collaterals were
stimulated with negative current pulses (duration of 0.3 ms, delivered
every 15 s by A310 Accupulser, WPI, U.S.). Evoked excitatory
postsynaptic currents (EPSCs) were recorded under whole-cell
configuration from CA1 pyramidal neurons (holding potential of
−60 mV; EPC7-plus amplifier HEKA, Germany). Immediately after
the beginning of recording, the slice was perfused with a Mg2+-free
ACSF containing CNQX (10 μM), glycine (10 μM), and bicuculline
(5 μM) to isolate NMDA-mediated EPSCs. The recording electrode
(N-1H-Indol-5-methanesulfonamide-3-yl)-2-(4-benzylpiperi-
din-1-yl)ethanone (10a). Yield 65%. Mp 212−214 °C, dec. Rf =
1
0.23. H NMR (DMSO-d6) (δ) 1.20−2.91 (m, 11H), 2.56 (s, 3H,
CH3), 3.44 (s, 2H, CH2CO), 6.78−6.81 (dd, 1H, ArH, J = 8.3), 7.09−
7.29 (m, 6H, ArH), 7.71 (s, 1H, ArH), 8.19 (s, 1H, H-2).
(N-1H-Indol-6-methanesulfonamide-3-yl)-2-(4-benzylpiperi-
din-1-yl)ethanone (10b). Yield 36%. Mp 160−162 °C, dec. Rf =
1
0.17. H NMR (DMSO-d6) (δ) 1.21−3.18 (m, 13H), 2.93 (s, 3H,
Me), 7.10−7.30 (m, 6H, ArH), 7.42 (s, 1H, ArH), 8.07−8.09 (dd, 1H,
ArH), 8.44 (s, 1H, H-2), 9.65 (bs, 1H, NH), 12.09 (bs, 1H, NH).
General Procedure for the Preparation of Carbamates
12a,b. Oxalyl chloride (0.175 mL, 2 mmol) was added dropwise at
0 °C to a solution of appropriate Boc-protected aminoindoles 6a,b
(232 mg, 1 mmol) in Et2O (5 mL). The reaction mixture was stirred at
F
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Journal of Medicinal Chemistry
Article
was a glass micropipet (resistance, 1.5−3 MΩ) filled 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). NMDA-mediated EPSC amplitude was
measured as the difference between peak current and baseline. For
each neuron studied, EPSC amplitude was measured during 5 min in
control conditions and in the presence of compound 10b and the two
sets of values were compared using Student’s unpaired t test (Graph
Pad software).
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ASSOCIATED CONTENT
* Supporting Information
Physicochemical and spectral data for intermediates 6a,b, 7a,b,
8a,b, 12a,b; anticonvulsant effects against sound-induced
seizures in DBA/2 mice; [3H]ifenprodil binding curves of
ligands 10b and 14b. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 090 6766413. Fax: +39 090 6766402. E-mail:
rgitto@unime.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this research by MiUR (Prin2008, Grant
20085HR5JK_002 and International Promotion of Young
Researchers “Montalcini Program”) is gratefully acknowledged.
ABBREVIATIONS USED
■
ATD, amino-terminal domain; CTD, carboxy-terminal domain;
DBA, dilute brown non-agouti; EPSC, evoked excitatory
postsynaptic current; iGluR, ionotropic glutamate receptor;
LBD, ligand binding domain; LE, ligand efficiency; LLE,
lipophilic efficiency; TPSA, topological polar surface area
G
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