Synthesis and biological characterization of 3-substituted-1h-indoles as ligands of GluN2B-containing N-methyl-D-aspartate receptors

Article abstract of DOI:10.1021/jm2008002

As an extension of our studies, novel indole derivatives were rationally designed and synthesized as ligands targeted to GluN2B/NMDA receptors. The 2-(4-benzylpiperidin-1-yl)-1-(6-hydroxy-1H-indol-3-yl)ethanone (4i) and 1-(4-benzylpiperidin-1-yl)-2-(6-hydroxy-1H-indol-3-yl)ethane-1,2-dione (6i) showed high binding affinity in [³H]ifenprodil displacement assay. By computational studies, we suggested the hypothetical interactions playing a significant role during the binding process. However, in functional and in vivo studies the most potent compound 4i did not show any activity whereas it displayed relevant affinity toward the σ₂ receptor.

Full text of DOI:10.1021/jm2008002

Brief Article
pubs.acs.org/jmc
Synthesis and Biological Characterization of 3-Substituted-1H-
indoles as Ligands of GluN2B-Containing N-Methyl-D-aspartate
Receptors
Rosaria Gitto,*,† Laura De Luca,† Stefania Ferro,† Maria Rosa Buemi,*,† Emilio Russo,‡
Giovambattista De Sarro,‡ Lara Costa,§ Lucia Ciranna,§ Orazio Prezzavento,∥ Emanuela Arena,∥
Simone Ronsisvalle,∥ Giuseppe Bruno,⊥ and Alba Chimirri†
†Dipartimento Farmaco-Chimico, Universita
̀
di Messina, Viale Annunziata, I-98168 Messina, Italy
‡Dipartimento di Medicina Sperimentale e Clinica, Universita
Italy
̀
̀
Magna Græcia, Viale Europa Localita Germaneto, I-88100 Catanzaro,
§Dipartimento di Scienze Bio-Mediche, Sezione di Fisiologia, and Dipartimento di Scienze del Farmaco, Universita
̀
di Catania, Viale
∥
Andrea Doria 6, I-95125 Catania, Italy
⊥Dipartimento di Chimica Inorganica, Chimica Analitica, e Chimica Fisica, Universita
Italy
̀
di Messina, Salita Sperone 31, I-98100 Messina,
S
* Supporting Information
ABSTRACT: As an extension of our studies, novel indole derivatives were rationally designed and synthesized as ligands
targeted to GluN2B/NMDA receptors. The 2-(4-benzylpiperidin-1-yl)-1-(6-hydroxy-1H-indol-3-yl)ethanone (4i) and 1-(4-
benzylpiperidin-1-yl)-2-(6-hydroxy-1H-indol-3-yl)ethane-1,2-dione (6i) showed high binding affinity in [3H]ifenprodil
displacement assay. By computational studies, we suggested the hypothetical interactions playing a significant role during the
binding process. However, in functional and in vivo studies the most potent compound 4i did not show any activity whereas it
displayed relevant affinity toward the σ2 receptor.
Chart 1. GluN2B/NMDA Noncompetitive Antagonists
INTRODUCTION
■
Glutamate NMDA receptors (NMDARs) are involved in many
physiological processes such as neuronal development, learning,
and memory but also in pathological states of mammalian
central nervous system, including strokes, seizures, and pain.1,2
NMDARs are multisubunit complexes containing GluN1,
GluN2, and more rarely GluN3 subunits (formerly known as
NR1−3).3−5 The GluN2 subunit exists as four subtypes
(GluN2A−D). They are organized into four modules
containing (a) two large extracellular domains, the amino-
terminal domain (ATD), and the agonist-binding domain
(ABD), (b) three transmembrane segments (M1, M3, and M4)
and a pore lining P-loop region (M2), and (c) an intracellular
carboxy terminal domain (CTD). The ATD plays a key role in
subunit assembly and exhibits a clamshell-like structure.6 In the
case of GluN2A and GluN2B subunits, the ATD binds
allosteric inhibitors exerting modulator roles. Notably, the
expression of GluN2B is restricted to forebrain areas and
minimally in the cerebellum. This distribution implies that
GluN2B selective antagonists might have reduced side effects in
the treatment of neurological pathologies.7−10 The prototypic
noncompetitive GluN2B-antagonist ifenprodil (1, Chart 1), its
more selective “prodil” analogue Ro 25-6981 (2), and some
indole-2-carboxamides (e.g. 3) have good potential as neuro-
protective agents and have been described as GluN2B
antagonists.9,10 Recently it was demonstrated11 that the
GluN1 and GluN2B ATDs form a heterodimer; in particular,
the crystal structures revealed that 1 and 2 bind at the interface
between these two subunits rather than exclusively at the
GluN2B-ATD cleft.12−15
In previous studies we reported a molecular modeling
strategy that led to the identification of some GluN2B ligands,
containing indole scaffold,16−18 structurally related to 3. The
most active derivatives 4a−c (Chart 1) were able to prevent
audiogenic seizures in DBA/2 mice, reduce NMDAR-mediated
current in patch clamp experiments, and showed neuro-
protective effects in HCN-1A cells.16 Then structure−activity
relationship (SAR) analysis highlighted some suitable structural
requirements improving the recognition process for ifenprodil
Received: April 4, 2011
Published: November 3, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1
a
Reagents and conditions: (i) (a) ClCH2COCl, pyridine, dioxane, microwave (5 min, 50 °C, 200 W); (b) ClCH2CON(CH3)2, POCl3, room temp,
2.5 h; (ii) 4-benzylpiperidine or benzylpiperazine, K2CO3, DMF, microwave (10 min, 100 °C, 200 W); (iii) ClCOCOCl, diethyl ether, room temp, 2
h; (iv) 4-benzylpiperidine, THF, TEA, room temp, 2 h; (v) BBr3 (1.0 M DCM), room temp, 10 h.
Table 1. GluN2B/NMDA Binding Affinities and Anticonvulsant Effects of Compounds 4−6
b
ED50, μmol/kg
a
compd
R
X
Z
% at 10 μM (IC50, μM)
clonus
tonus
c
d
d
c
d
d
c
4a
4b
4c
4d
4e
4f
H
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCH2
COCO
COCO
COCO
COCO
COCO
COCO
COCO
COCO
COCO
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
89% (0.769)
22.4 (13.9−36.2)
59.9 (38.7−92.7)
11.5 (7.96−16.5)
32.7 (20.0−53.6)
68.1(49.3−94.0)
80.3 (47.9−135)
>100
8.71 (5.71−13.3)
36.5 (26.1−51.0)
6.48 (4.56−9.21)
16.2(10.1−26.2)
13.7 (7.1−26.3)
35.8 (22.6−56.8)
53.8 (39.0−74.2)
30.1 (20.8−43.4)
53.7 (37.1−77.0)
d
5-OMe
5-OH
4-OMe
4-OH
7-OMe
7-OH
6-OMe
6-OH
H
86%
c
92% (0.025)
82% (0.324)
77% (6.64)
62% (2.91)
72% (7.14)
4%
4g
4h
4i
67.8 (45.9−100)
95.4 (67.5−135)
92% (0.017)
14%
c
c
5a
5b
5c
6a
6b
6c
6d
6e
6f
>100
>100
5-OMe
5-OH
H
N
ND
98.7 (72.2−113)
>100
63.8 (45.1−90.2)
44.9 (26.6−75.8)
0.95 (0.26−3.52)
N
27%
c
c
c
CH
CH
CH
CH
CH
CH
CH
CH
CH
36%
22.4 (10.9−45.9)
c
c
5-OMe
5-OH
4-OMe
4-OH
7-OMe
7-OH
6-OMe
6-OH
9%
>100
78.8 (53.7−116)
c
c
c
77% (2.66)
>100
>100
14%
>100
76.7 (65.1−90.5)
28.9 (20.9−39.8)
27.4 (18.8−39.9)
36.2 (23.0−57.1)
4.68 (3.02−7.25)
38.9 (25.1−60.5)
24%
52.1 (46.0−59.1)
51.4 (38.5−68.6)
80.5 (59.4−109)
11.9 (8.97−15.9)
84.4 (56.3−126)
33%
6g
6h
6i
79% (1.99)
ND
92% (0.022)
a
b
Displacement of [3H]ifenprodil. ND = not detectable. All data were calculated following the Litchfield and Wilcoxon method. At least 32 animals
were used to calculate each ED50. 95% confidence limits are given in parentheses. Reference 18. Reference 16
c
d
site.18 In an attempt to confirm these results and further explore
the hypothetical binding interactions, we designed novel
ligands. (i) We prepared derivatives bearing methoxy or
hydroxyl groups in the 4, 5, 6, or 7 position of indole skeleton.
(ii) We went thoroughly into the influence of a positive
ionizable feature of the piperidine nitrogen atom, introducing
an oxalyl group as linker and, on the basis of experimental
evidence, suggesting that there is a significant reduction of side
effects for GluN2B antagonists lacking a basic nitrogen (e.g., 3).
(iii) We also substituted the benzylpiperidine fragment with a
benzylpiperazine one, thus offering the possibility of an
alternative anchoring point for the electrostatic interaction by
nitrogen atom. We evaluated the effects of the structural
modifications on binding affinity, anticonvulsant properties, and
functional activity in patch-clamp experiments. Moreover,
docking simulations were carried out to explain the obtained
biological results, taking into account the recent findings
concerning the NMDAR subunit arrangement.11 Finally, the
σ1/σ2 receptor binding affinities of selected indoles were
evaluated in an attempt to clarify some biological results.
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Journal of Medicinal Chemistry
Brief Article
CHEMISTRY
■
The synthetic pathways are depicted in Scheme 1. We
synthesized 2-chloro-1-(1H-indol-3-yl)ethanone derivatives 8
following our previously reported method (pathway a)18 or an
optimized strategy (pathway b), thus improving the yields and
obtaining a cleaner reaction. Starting from intermediates 8, the
corresponding 2-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)-
ethanones (4) and 2-(4-benzylpiperazin-1-yl)-1-(1H-indol-3-
yl)ethanones 5 were obtained. In a similar way we prepared the
1-(4-benzylpiperidin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-diones
(6) through 2-(1H-indol-3-yl)-2-oxoacetyl chloride intermedi-
ates (9). All methoxyindole derivatives were readily converted
into the hydroxy analogues upon treatment with boron
tribromide. The chemical characterization of new compounds
obtained was supported by elemental analyses and spectro-
scopic measurements (see Supporting Information).
RESULTS AND DISCUSSION
■
To further study the SARs of this series of novel indole
derivatives (4d−i, 5b,c, 6d−i), we evaluated their ability to
interact with the GluN2B subunit by testing [3H]ifenprodil
binding inhibition and the results were compared with those of
previously reported 4−6 analogues and ifenprodil (1) (Table
1).16,18 The 2-(4-benzylpiperidin-1-yl)-1-(6-hydroxy-1H-indol-
3-yl)ethanone (4i), displaying the highest binding affinity (IC50
= 0.017 μM), showed a 45-fold improvement over the
unsubstituted parent 4a and a potency comparable to that of
the 5-hydroxy analogue 4c and ifenprodil (1) (IC50 = 0.020
μM). In contrast, the introduction of a 6-methoxy substituent
(e.g., 4h) led to a reduction of [3H]ifenprodil displacement.
Also, the replacement of benzylpiperidine with benzylpiper-
azine fragment reduced the affinity of 5a−c in comparison with
4a−c. The oxalyl analogues 6a−h generally demonstrated a
significant decrease in GluN2B affinity with respect to 4a−h;
however, for 6c and 6g we observed moderate inhibition
properties (IC50 of 2.66 and 1.99 μM, respectively).
Unexpectedly, 6i was extraordinarily efficacious displaying
IC50 comparable to those of 4c and 1. Overall, the lowest
IC50 values were obtained for compounds containing a 5- or 6-
hydroxyl substituent on benzene fused ring, whereas the
substitution at the 4- or 7-position led to less active ligands 4e
and 4g, and generally the optimization of affinity seems to
reside in the positive ionizable feature of piperidine nitrogen
atom.
To rationalize the obtained biological data, docking
simulations by Gold (see Supporting Information) were
performed using the structural coordinates (PDB code
3QEL) for the heterodimer GluN1b-GluN2B in complex
with ifenprodil (1). First, test docking calculations using 1 and
Ro 25-6981 (2) were carried out to compare experimental and
predicted binding modes and to validate our docking protocol.
The best 1 and 2 docking poses agreed well with the
experimental binding modes with rmsd of 0.6 and 0.65,
respectively. Figure 1A displays the superposition of the indoles
4c (magenta) and 4i (orange). They present a very similar
binding mode in the GluN1b-GluN2B subunit interface in
accordance with their comparable binding affinities (IC50 of 25
and 17 nM, respectively). We found hydrogen bonding
interactions between the hydroxyl group at C-5 (for 4c) or
C-6 (for 4i) and residue E236 of GluN2B and between residue
K131 of GluN1b and NH of indole ring. Furthermore, through
the protonated piperidine nitrogen atom, 4c and 4i make polar
Figure 1. Docking poses of 4c, 4i, and 6i at the GluN1b-Glun2B
subunit interface: (A) binding of 4c (magenta) compared to 4i
(orange); (B) binding of 6i (yellow). Important residues are drawn in
stick and colored in cyan (GluN2B) and in green (GluN1b).
Hydrogen bonds (shown as dashed yellow lines) are formed between
the compounds and GluN1b-Glun2B subunit interface. The structures
were prepared using PyMOL.28
interactions with residue Q110 of GluN2B. Finally, they form
interactions with hydrophobic portions of some residues on
GluN1b (Y109, G112, L135) and GluN2B (I111, F176, P177).
Notably, our docking results are in good agreement with the
interactions highlighted in the crystal structures of the GluN1b-
GluN2B ATD heterodimer in complex with ifenprodil (1) and
Ro 25-6981 (2).11 In particular, the major contacts of 1 and 2
were (i) polar interactions of the hydroxylphenyl group with
E236 residue and the positive ionizable nitrogen atom with
Q110 residue and (ii) hydrophobic interactions mediated by
L135 residue on GluN1b subunit and F176 and P177 residues
on the GluN2B subunit.
Considering that 4a−h generally showed lower IC50 with
respect to 6a−h, we chose to study the correlation between the
binding affinities and piperidine nitrogen atom basicity. The gas
phase proton affinity (PA) of 4c and 6c has been calculated. As
expected in the case of 2-(4-benzylpiperidin-1-yl)-1-(5-
hydroxy-1H-indol-3-yl)ethanone (4c, −243,18 kcal/mol) the
PA value was higher than that of the corresponding oxalyl
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Journal of Medicinal Chemistry
Brief Article
analogue (6c, −217.43 kcal/mol). Combining the docking
results and PA calculations, we suggested a key role of the
interaction between ionizable nitrogen atom and residue Q110,
thus generally justifying the different affinity between
compound types 4 and 6. Unexpectedly, the 6-hydroxy
derivative 6i showed a low IC50 (0.022 μM) with respect
other 6 analogues. So we docked 6i in the GluN1b-GluN2B
subunit interface (see Figure 1B). We found that 6i assumes a
binding mode similar to those of 4c and 4i except for polar
contact with Q110; however, we observed strong contacts with
hydrophobic residues of GluN1b subunit such as Y109, G112,
and L135. These last interactions could provide a reasonable
explanation for the high binding affinity of 6i even if it lacks a
positive ionizable feature.
To investigate the functional activity of 4i, we have tested its
effect on NMDA-mediated current in CA1 pyramidal neurons
from mouse hippocampal slices using whole-cell patch clamp.
Application of NMDA (50 μM, 2 s) in the presence of glycine
(0.5 μM) and tetrodotoxin (TTX, 0.5 μM) induced an inward
current with a mean amplitude of 507 ± 115 pA (mean ± SEM,
n = 7, range 251−1115 pA in different neurons, median 371
pA). In the presence of 4i (10 μM, 5 min) the amplitude of
NMDA-mediated current was not significantly different from
control (Figure 2; P = 0.27, t test, n = 7). Figure 2 illustrates
hydroxyindole derivatives 4c, 4i, 5c, 6c, and 6i and reference
compounds 1 and 2 were subjected to binding testing.
Competitive displacement of [3H](+)pentazocine and
[3H]DTG [1,3-di-o-tolylguanidine] in the presence of
(+)-SKF10,047 (0.4 μM) was used to determine σ1 and σ2
receptor affinities. The measured Ki revealed that 4c, 4i, 6c, and
6i had practically no affinity for σ1 receptor (Ki > 1000). The
N-benzylpiperazine derivative 5c displayed moderate affinity at
both σ receptors (Ki of 437 and 557 nM, respectively).
Interestingly, 4c and 4i showed nanomolar σ2 affinity (Ki of
76.5 and 43.0 nM, respectively) and a relevant selectivity over
σ1 subtype (Ki(σ1)/Ki(σ2) > 60) when compared with 1 and 2
(see Supporting Information). Therefore, we can speculate that
the effects exerted by 4i on NMDA-mediated current (Figure
2) might be due to the interaction with GluN2B site and σ2
receptor, whereas we cannot explain the biological profile of 4c.
However, we are currently investigating if each of these two
compounds behaves as agonist or antagonist of the σ2 receptor.
We also examined the anticonvulsant properties of all
synthesized compounds against audiogenic seizures in DBA/2
mice.27 The results were compared with those of the previously
reported analogues 4a−c, 5a, 6a−c (Table 1) and 2 (ED50
=
40.0 μmol/kg in clonic phase).18 The obtained data indicated
that there is no clear correlation between the binding results
and in vivo data. Disappointingly, the most in vivo potent
indole derivatives 4d, 6a, and 6h did not show any significant
[3H]ifenprodil displacement properties. In contrast, for 4i we
found an excellent binding affinity but low anticonvulsant
efficacy, in agreement with its effects on NMDA-induced
currents.
CONCLUSIONS
■
We identified new hydroxylindoles as potent GluN2B ligands
showing affinity similar to that of the ifenprodil (1). By means
of docking studies based on recently published findings11 about
the arrangement of GluN1b-GluN2B NMDA receptors, we
suggested the hypothetic binding orientation of the most
interesting compounds 4c, 4i, and 6i in the ifenprodil binding
site. This investigation also gave information concerning
similarities and differences in the binding modes of the
synthesized compounds due to the presence/absence of
positive ionizable nitrogen atom. Different from reference
compounds 1 and 2, the indole derivatives 4c and 4i were
potent and selective σ2 ligands. Further studies are in progress
to clarify if the unexpected behavior of 4i in functional and in in
vivo studies could be connected (i) to its ability to exert some
off-target activities or (ii) to its pharmacokinetic properties
influencing the brain penetration.
Figure 2. Application of NMDA (50 μM, 2 s) induced an inward
current (trace 1) that was not modified during simultaneous
application of compound 4i (10 μM, 5 min, trace 2) but was
significantly reduced by 4c (10 μM, 5 min, trace 3), a selective
antagonist of GluN2B receptors. NMDA current amplitude values
(pA) were represented on a graphic as a function of time to illustrate
the time course of effects.
representative traces from a neuron in which NMDA current
amplitude was not modified by 4i but was significantly reduced
by 4c (P < 0.01, t test, n = 7, see Supporting Information)
previously characterized as a GluN2B antagonist.18 These
results indicate that in spite of its high binding affinity, 4i did
not significantly antagonize NMDA-mediated effect. To explain
the different results observed for 4c and 4i, we turned our
attention to the possibility that our indoles could exert some
off-target activities. So we evaluated the binding affinity toward
σ receptors on the basis of the following assumptions: (i)
ifenprodil (1) and its analogues also bind σ1 and σ2
receptors;19,20 (ii) presynaptic σ receptors were shown to
modulate hippocampal glutamate release;21,22 (iii) activation of
σ receptors also modulates NMDA-mediated transmission.23,24
Furthermore, molecules containing benzylpiperidine or benzyl-
piperazine fragment bind σ1 and σ2 receptors.25,26 To
determine affinities at σ1 and σ2 receptors, some selected
EXPERIMENTAL SECTION
■
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. By combustion
analysis (C, H, N) carried out on a Carlo Erba model 1106 elemental
analyzer, we determined the purity of synthesized compounds; the
1
results confirmed a ≥95% purity. H 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.
Synthesis of 2-(4-Benzylpiperidin-1-yl)-1-(1H-indol-3-yl)-
ethanones (4d,f,h), 2-(4-Benzylpiperazin-1-yl)-1-(1H-indol-3-
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Journal of Medicinal Chemistry
Brief Article
yl)ethanone (5b), and 1-(4-Benzylpiperidin-1-yl)-2-(1H-indol-3-
yl)ethane-1,2-diones (6d,f,h). The appropriate 3-chloroacetylin-
dole derivatives (8b,d,f,h) (1 mmol), benzylpiperidine (0.176 mL, 1
mmol) or benzylpiperazine (176.3 mg, 1 mmol), and K2CO3 (147.1
mg, 0.5 mmol) in DMF (2 mL) were stirred and irradiated in a
microwave oven under the following conditions: 10 min, 100 °C, 200
W. The reaction mixture was quenched with NaHCO3 saturated
aqueous solution (10 mL) and extracted with ethyl acetate (3 × 10
mL). The organic phases were dried over dry Na2SO4. After removal
of the solvent under reduced pressure, the residue was crystallized
from ethanol to give desired 4 and 5. The appropriate indole
derivatives 7d,f,h (1 mmol) were used as starting material to
synthesize 6d,f,h following a previously reported procedure.18
Starting from methoxy substituted derivatives 4d,f,h, 5b, and 6d,f,h,
the corresponding hydroxyl derivatives 4e,g,i, 5c, and 6e,g,i were
prepared using a previously published procedure.18 Detailed analytical
data of all new synthesized compounds are in the Supporting
Information.
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ASSOCIATED CONTENT
* Supporting Information
Details for synthetic procedures and spectral data, assay
protocols, dose−response curves, binding affinity data, and
modeling parameters. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For R.G.: phone, 00390906766413; fax, 00390906766402; e-
mail, rgitto@unime.it. For M.R.B.: phone, 00390906766465; e-
mail, mbuemi@unime.it.
■
ACKNOWLEDGMENTS
Financial support for this research by MiUR (Prin2008, Grant
No. 20085HR5JK_002) is gratefully acknowledged.
■
ABBREVIATIONS USED
■
ABD, agonist-binding domain; CTD, carboxy terminal domain;
DBA, dilute brown, nonagouti; iGluR, ionotropic glutamate
receptor; HCN-1A, human cortical neuron 1A cell line;
NMDA, N-methyl-D-aspartate; NMDAR, N-methyl-D-aspartate
receptor; ATD, amino-terminal domain; PA, proton affinity;
rmsd, root mean square deviation; TTX, tetrodotoxin
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