Targeting GluN2B-containing N-methyl- D -aspartate receptors: Design, synthesis, and binding affinity evaluation of novel 3-substituted indoles

Article abstract of DOI:10.1002/ardp.201400061

In an effort to improve our knowledge about structure-affinity relationships (SARs) for a class of 3-substituted-indole derivatives as GluN2B-containing N-methyl-D-aspartate-type receptor (NMDAR) ligands, we herein describe the design, synthesis, and preliminary screening of a new series of molecules. The in vitro determination of binding affinities suggested that 5-hydroxy- and 6-hydroxyindole derivatives 12 and 13 were active ligands. Generally, the novel compounds proved to be less potent than their homologs previously reported as promising neuroprotective agents. In fact, our lead compound 3-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethan-1-one (2) was about 10-fold more active than the new propan-1-one derivative (12). To rationalize the low potency of the new analog 12, docking studies were also performed and the in silico results were consistent with the in vitro data.

Full text of DOI:10.1002/ardp.201400061

Arch. Pharm. Chem. Life Sci. 2014, 347, 533–539
533
Full Paper
Targeting GluN2B-Containing N-Methyl-D-aspartate Receptors:
Design, Synthesis, and Binding Affinity Evaluation of Novel
3-Substituted Indoles
Maria Rosa Buemi, Laura De Luca, Stefania Ferro, and Rosaria Gitto
Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Università di Messina, Messina, Italy
In an effort to improve our knowledge about structure–affinity relationships (SARs) for a class of
3-substituted-indole derivatives as GluN2B-containing N-methyl-D-aspartate-type receptor (NMDAR)
ligands, we herein describe the design, synthesis, and preliminary screening of a new series of
molecules. The in vitro determination of binding affinities suggested that 5-hydroxy- and
6-hydroxyindole derivatives 12 and 13 were active ligands. Generally, the novel compounds proved
to be less potent than their homologs previously reported as promising neuroprotective agents. In
fact, our lead compound 3-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethan-1-one (2) was about
10-fold more active than the new propan-1-one derivative (12). To rationalize the low potency of
the new analog 12, docking studies were also performed and the in silico results were consistent with
the in vitro data.
Keywords: Glutamate / GluN2B / Ifenprodil / Indoles / Molecular docking
Received: February 19, 2014; Revised: April 1, 2014; Accepted: April 2, 2014
DOI 10.1002/ardp.201400061
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
functional outputs. In particular, the different composition of
the subunits GluN2A–D and the subcellular localization of
NMDARs contribute to induce signal transduction cascade
and to promote neuronal plasticity and neuronal survival or
death [7]. While the synaptic NMDAR activation is implicated
in neuroprotection, the stimulation of extrasynaptic
NMDARs, composed of GluN2B subunits, may play a key role
in the Glu-induced excitotoxicity [8]. Thereby antagonists
targeting GluN2B-containing NMDARs are expected to show
neuroprotective effects [9]. The prototype of noncompetitive
antagonists of GluN2B-containing NMDARs is the ifenprodil
(1) [10], which binds the interface of the extracellular amino-
terminal domain of GluN1/GluN2B functional dimer thus
impairing the opening of NMDARs through the stabilization
of the twisted closed-clamshell configuration [11]. In our
efforts to identify selective GluN2B-containing NMDAR
ligands we have discovered a series of potent 3-substituted
indoles [12–19] showing high potency (IC50 values ranging
from 5.4 to 51 nM) in a [3H]ifenprodil competition binding
assay. Among them the 3-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-
1H-indol-3-yl)ethan-1-one (2, Fig. 1) reduced NMDA receptor-
mediated current in patch clamp experiments and showed
Experimental evidences demonstrated that glutamate (Glu) is
a critical transmitter for signaling neurons to degenerate
during the excitotoxic process. Glu exerts its neurotoxic
function through the activation of ionotropic receptors
(iGluRs) such as N-methyl-D-aspartate (NMDA)-type receptors
(NMDARs) [1], and the resulting elevation of intracellular
calcium can produce neuronal death [2]. So NMDARs are
involved in various neurodegenerative pathologies [3, 4], and
drugs antagonizing Glu-mediated neuronal excitation could
prevent the associated neurotoxicity. NMDA receptors are
heterotetrameric complexes composed of subunits from
seven homologous genes, GluN1, GluN2A–GluN2D, and
GluN3A–GluN3B [5, 6]. The majority of NMDARs are composed
of two GluN1 and two GluN2 subunits. It is known that
different subpopulations of NMDARs may generate different
Correspondence: Prof. Rosaria Gitto, Dipartimento di Scienze del
Farmaco e Prodotti per la Salute, Università degli Studi di Messina, Viale
Annunziata, 98168 Messina, Italy.
E-mail: rgitto@unime.it
Fax: þ39 090 6766402
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534
M. R. Buemi et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 533–539
Figure 1. Chemical structures and IC50 values of two prototypes of
GluN2B-containing NMDAR ligands: ifenprodil (1) and 3-(4-
benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethan-1-one (2).
Figure 2. Designed hydroxyindole derivatives as GluN2B-contain-
ing NMDAR ligands.
in vivo efficacy as anticonvulsant [14]. Starting from crystallo-
graphic data (PDB code 3QEL) we performed a docking study
suggesting that the compound 2 establishes crucial hydrogen
bond interactions and hydrophobic contacts within the
interface of the functional amino-terminal dimer composed
of GluN1/GluN2B subunits. In more detail the 5-hydroxyin-
dole derivative 2 engages profitable contacts both with
residues from GluN1 (Y109, G112, L135) and GluN2B (Q110,
I111, F176, P177, E236) subunits [15].
with the 4-fluorobenzylpiperidine one. Finally, in order to
reduce the distance between the nitrogen atom and aromatic
ring, the isoquinoline nucleus replaced the benzylpiperidine
fragment thus designing the corresponding 3-(3,4-dihydroi-
soquinolin-2(1H)-yl)-1-(1H-indol-3-yl)propan-1-ones.
The binding affinity for displacement of [3H]ifenprodil of
the new hydroxyindole derivatives was studied. Moreover,
docking simulations were carried out to investigate the
plausible binding mode of the title ligands.
To gain further information about SARs for this class of
GluN2B-containing receptor ligands, we herein report the
synthesis of new hydroxyindole derivatives (Fig. 2) as Results and discussion
homologs of previously reported active ligands bearing
ethanone linker. So through an extra methylene bridge, we
lengthened the linker between the indole ring and benzylpi-
peridine fragment and designed a series of 3-(4-benzylpiper-
idin-1-yl)-1-(1H-indol-3-yl)propan-1-one derivatives. Because it
is well known that the presence of a fluorine atom could
increase the metabolic stability of xenobiotic bearing a phenyl
substituent, we also replaced the benzylpiperidine fragment
The synthesis of 3-(4-benzylpiperidinyl)-1-(1H-indol-3-yl)-
propan-1-one derivatives (9–17) and 3-(3,4-dihydroisoquino-
lin-2(1H)-yl)-1-(1H-indol-3-yl)propan-1-one derivatives (18–22)
was performed as outlined in Scheme 1. Initially, the
appropriate indoles 3–5 were 3-acetylated by a Vilsmeier
Haack reaction in the presence of phosphoryl chloride and
dimethylacetamide to give intermediates 6–8 [20–23]. By
O
ii
N
A or B
R1
N
H
R2
9−13
R2 = H
R2 = F
O
R1
CH3
14−17
9, 18
H
i
R1
R1
5-OCH3
6-OCH3
10, 14, 19
N
N
H
11, 15, 20
12, 16, 21
13, 17, 22
O
H
iv
3, R1 = H
6, R1 = H
5-OH
6-OH
N
4, R1 = 5-OCH3
5, R1 = 6-OCH3
7, R1 = 5-OCH3
8, R1 = 6-OCH3
R1
N
18−22
iii
H
A or B
Scheme 1. Synthetic pathway of 3-substituted indole derivatives 9–22. Reagents and conditions: (i) POCl3, N,N-dimethylacetamide, r.t.
24 h; (ii) route A (R1 and R2 ¼ H): 4-benzylpiperidine hydrochloride, 1,3-dioxolane, HCl 37%, reflux 5 h, then r.t. 20 h; route B (R1 ¼ H, OMe,
and R2 ¼ H, F): DMF, paraformaldehyde, 4-benzylpiperidine hydrochloride or 4-fluorobenzylpiperidine hydrochloride, HCl 37%, MW 3 min
80°C; (iii) route A (R1 ¼ H): 1,2,3,4-tetrahydroisoquinoline hydrochloride, 1,3-dioxolane, HCl 37%, reflux 5 h, then r.t. 20 h; route B (R1 ¼ H,
OMe): DMF, paraformaldehyde, 1,2,3,4-tetrahydroisoquinoline hydrochloride, HCl 37%, MW 3 min 80°C; (iv) BBr3 (1.0 M DCM), r.t., 10 h.
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GluN2B-Containing N-Methyl-D-aspartate Receptor Ligands
535
reaction of ketone 6 with a suitable secondary amine
derivative and 1,3-dioxolane, we set up the synthetic
approach through the Mannich reaction conditions thus
obtaining unsubstituted indoles 9 and 18 (R1 and R2 ¼ H)
(route A) as prototypes. In an attempt to improve the yield as
well as to reduce reaction time and side products, the indoles
9 and 18 were also synthesized following an alternative
synthetic procedure that has been performed using paraform-
aldehyde under micro-assisted irradiation conditions (route
B). Therefore, this optimized synthetic route B was used to
obtain the six methoxyindoles 10–11, 14–15 and 19–20 in
good yields. Lastly, these precursors were demethylated to
give the expected hydroxyindoles 12–13, 16–17 and 21–22.
Although we carried out synthesis of a series of 14 new
indole derivatives, we chose to evaluate the affinity for
GluN2B-containing NMDA receptors only for six selected
derivatives 12–13, 16–17, and 21–22 bearing the hydroxyl
group on the indole nucleus. These selection criteria were
based on our knowledge of the highest affinity of 3-(4-
benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethan-1-one (2,
IC50 ¼ 25 nM) and its 6-hydroxy analog (IC50 ¼ 17 nM) [15] that
could establish a fruitful hydrogen bonding interaction with
the crucial residue E236 of GluN2B subunit. In addition, the
unsubstituted and 5/6-methoxy-indoles were generally inac-
tive GluN2B ligands. As reported in Table 1, a preliminary
binding assay using [3H]ifenprodil has been carried out to
measure the percentage of inhibition at fixed dose of 0.1 mM
concentration. Then, three concentrations (10 mM, 0.1 mM,
0.001 mM, in duplicate) of test compound were used to
calculate the corresponding IC50 values (see Table 1) in the
same displacement assay. The ifenprodil (1) and previously
reported indole 2 served as reference compounds to compare
the binding affinity potencies of the new indole derivatives.
The results of the in vitro assay evidenced that the new
indole derivatives were lower active ligands in comparison
with the prototype 1 and the parent compound 2. The fluorine
atom at para position of benzylpiperidine moiety decreased
the binding affinity at NMDA receptor. Moreover, compounds
21 and 22 showed no displacement of [3H]ifenprodil at 0.1 mM
concentration, thus suggesting that the rigid and bulky
1,2,3,4-tetrahydroisoquinoline system induces a remarkable
loss of affinity. Only compounds 12 and 13 were able to
produce about 50% [3H]ifenprodil displacement at 0.1 mM
concentration and consequently displayed IC50 values of 230
and 80 nM, respectively. These data could suggest that the
elongation of the linker between carbonyl group and
piperidine nitrogen atom negatively influences the binding
affinity. A noticeable reduction of affinity has been especially
observed for compound 12 (IC50 ¼ 230 nM) if compared to
parent compound 2 (IC50 ¼ 25 nM). The 6-hydroxy derivative
13 was only fourfold less active than corresponding parent
compound (IC50 ¼ 80 nM versus IC50 ¼ 17 nM).
By means of a computational approach, we attempted to
explain this significant reduction of potency due to the
presence of the extra methylene bridge for compound 12. So,
we performed a docking study using GOLD program and
the docking protocol applied in our previous work [15]. The
indole 12 was docked into the dimer GluN1/GluN2B interface
retrieved from the RCSB Protein Data Bank (entry code 3QEL)
[11]. As shown in Fig. 3 the benzylpiperidine portion of ligand
12 is surrounded by hydrophobic GluN1 (Y109 and Y114) and
GluN2B (P78, F114 and I111) residues. This ligand makes an
unexpected bidentate H bond interaction with crucial
residue Q110 of GluN2B subunit. This network involves
two pivotal functional groups of indole derivative that is both
the hydrogen atom bound to the positive ionizable piperidine
nitrogen and the oxygen atom of propanone linker.
Moreover, the ligand is also able to form a further hydrogen
bonding interaction between the hydroxyl group and E106
residue of GluN2B. This multiple H-bond interaction forces
the indole ring to occupy a novel position thus losing the
expected key interaction between hydroxyl group and E236
residue.
Table 1. GluN2B/NMDA binding affinities of indole derivatives
and reference compounds ifenprodil (1) and 2.
Inhibition %
R1
R2
(@ 0.1 mM)a)
IC50 (nM)a)
This molecular modeling investigation confirms that the
lengthening of the linker between the indole ring and
benzylpiperidine fragment determines the shift of hydroxy-
indole position that accounts for the higher IC50 value of 12
when compared to parent 5-hydroxy analog 2 (230 nM vs.
25 nM). In addition, our computational studies suggested that
there are no striking differences in the proposed binding
modes of 4-fluorobenzylpiperidine derivatives in comparison
to benzylpiperidine analogs. Thus, these experiments failed to
explain the reduction of ligand affinity measured for 4-
fluorosubstituted derivative 16 (IC50 ¼ 1130 nM) in compari-
son to unsubstituted compound 12 (IC50 ¼ 230 nM). Finally,
12
13
16
17
5-OH
6-OH
5-OH
6-OH
5-OH
6-OH
5-OH
H
H
F
47%
57%
19%
39%
<10
<10
75%
77%
230
80
1130
360
ND
ND
25.0
20.0
F
21
H
H
H
22
2ab)
Ifenprodil (1)b)
ND, not detectable.
a)
Displacement of [3H]ifenprodil, three concentrations (10 mM,
0.1 mM, 0.001 mM, in duplicate) of test compounds were used in
displacement assay.
b)
Data from reference [14].
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536
M. R. Buemi et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 533–539
the profitable contact between hydroxyl group and E236
residue, thus improving the interaction within the GluN1/
GluN2B interface.
Experimental
Chemistry
All starting materials and reagents commercially available
(Sigma–Aldrich Milan, Italy; Alfa Aesar Karlsruhe, Germany)
were used without further purification. Microwave-assisted
reactions were carried out in a focused Microwave Synthesis
System (CEM Technology Ltd Buckingham, UK). Melting points
were determined on a Buchi B-545 apparatus (BUCHI Labortech-
nik AG Flawil, Switzerland) and are uncorrected. Elemental
analyses (C, H, N) were carried out on a Carlo Erba Model 1106
Elemental Analyzer (Carlo Erba Milano, Italy); the results
confirmed a ꢀ95% purity. Merck silica gel 60 F254 plates were
used for analytical TLC (Merck KGaA, Darmstadt, Germany). Flash
Chromatography (FC) was carried out on a Biotage SP1 EXP
(Biotage AB, Uppsala, Sweden). Rf values were determined on TLC
1
plates using a mixture of DCM/MeOH (90:10) as eluent. H NMR
spectra were measured in dimethylsulfoxide-d6 (DMSO-d6) with a
Gemini 300 spectrometer (Varian Inc., Palo Alto, CA, USA);
chemical shifts are expressed in d (ppm) and coupling constants (J)
in Hz. By using a Varian Gemini 300 spectrometer the 13C NMR
spectra were measured for two selected compounds as prototypes
of this series of indoles. All exchangeable protons were confirmed
by addition of D2O.
General procedure for the synthesis of 1-(1H-indol-3-yl)
ethanone derivatives 6–8
Compounds 6–8 were prepared according to the previously
reported procedure [20–23]. In particular, phosphoryl chloride
(0.92 mL, 10 mmol) was added to ice cold dimethylacetamide
(2.79 mL, 30 mmol) and this mixture was stirred. A suitable indole
(3, 4, or 5) (1 mmol) was added and the reaction mixture was
stirred at room temperature for 24 h, then poured and basified
with a sodium hydroxide solution (4 N). The mixture was
extracted with EtOAc (3 ꢁ 10 mL) and dried over Na2SO4. After
the removal of the solvent under reduced pressure, the residue
was poured with a mixture of diethyl ether and dichloromethane
to give the desired 1-(1H-indol-3-yl)ethanone derivatives (6, 7, or 8).
The spectral data of obtained compounds 6–8 were in accordance
with literature [24, 25].
Figure 3. Docking pose of compound 12 at the GluN1-GluN2B
subunit interface (PDB code 3QEL). Crucial residues are drawn in
stick and colored in cyan (GluN2B) and in green (GluN1). Hydrogen
bonds are shown as dashed yellow lines. The figure was prepared in
PyMOL software.
we performed docking studies of inactive indoles 21 and 22
(see Supporting Information). It appears that they do not fill
well the binding pocket within dimeric GluN1/GluN2B
interface. This matter of fact could be attributed to the
shorter distance between piperidine nitrogen atom and
aromatic region of 1,2,3,4-tetrahydroisoquinoline system,
which failed to map hydrophobic region of GluN1/GluN2B
interface.
In conclusion, by structural modifications on the 3-
substituted indole moiety, new ligands targeting the
GluN2B-containing NMDA receptor were designed and
synthesized. The most potent ligands of this series exhibited
about 50% [3H]ifenprodil displacement at 0.1 mM concentra-
tion and IC50 values in the nanomolar range. This work
furnished further SAR information about the recognition
process to the GluN2B-containing NMDA receptor subtype.
Taking these findings into account, the design of new
hydroxyindole derivatives should be addressed to restore
General procedures for the synthesis of 3-(4-
benzylpiperidinyl)-1-(1H-indol-3-yl)propan-1-one
derivatives (9–17) and 3-(3,4-dihydroisoquinolin-2(1H)-yl)-
1-(1H-indol-3-yl)propan-1-one derivatives (18–22)
Route A for the synthesis of prototypes 9 and 18: The appropriate
secondary amine (4-benzylpiperidine or 1,2,3,4-tetrahydroisoqui-
noline) hydrochloride (1 mmol) was dissolved in the mixture of
1,3-dioxalane (0.22 mL, 3 mmol) and 1-(1H-indol-3-yl)ethanone 6
(1.2 mmol) in the presence of a catalytic amount of hydrochloric
acid (37%). The mixture was heated at 90°C for 5 h and then it was
stirred at room temperature for 20 h. Then, the cooled reaction
mixture was diluted with water and washed with EtOAc
(3 ꢁ 10 mL). The aqueous layer was made alkaline with sodium
hydroxide (2 N) and extracted with EtOAc (3 ꢁ 10 mL). The organic
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GluN2B-Containing N-Methyl-D-aspartate Receptor Ligands
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layer was washed with water (3 ꢁ 5 mL), dried over Na2SO4, and
concentrated in vacuo. The crude final indole derivative 9 or 18
was purified by flash chromatography (FC, DCM/CH3OH 90:10)
and recrystallized by treatment with Et2O.
H-5, J ¼ 8.8), 6.93 (s, 1H, H-7), 7.13–7.26 (m, 5H, ArH), 8.02 (d, 1H,
H-4, J ¼ 8.8), 8.21 (bs, 1H, H-2), 11.70 (bs, 1H, NH). Anal. calcd. for
C
24H28N2O2: C, 76.56; H, 7.50; N, 7.44. Found C, 76.59; H, 7.53; N,
7.47.
Route B for the synthesis of 9–11, 14–15 and 18–20: To a stirred
mixture of the appropriate 1-(1H-indol-3-yl)ethanone derivatives
6–8 (1 mmol) in DMF (3 mL), paraformaldehyde (39.0 mg,
1.3 mmol), and corresponding secondary amine (4-benzylpiper-
idine, 4-fluorobenzylpiperidine or 1,2,3,4-tetrahydroisoquino-
3-(4-Benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)
propan-1-one (12)
Colorless solid. Yield 54%. M.p.: 216–217°C. Rf ¼ 0.09. 1H NMR
(DMSO-d6) d: 1.16–2.95 (m, 15H), 6.67–7.30 (m, 7H, ArH, H-6, H-7),
7.59 (d, 1H, H-4, J ¼ 3.6), 8.22 (d, 1H, H-2, J ¼ 3.2), 8.96 (s, 1H, OH),
11.67 (bs, 1H, NH). Anal. calcd. for C23H26N2O2: C, 76.21; H, 7.23;
N, 7.73. Found C, 76.22; H, 7.24; N, 7.74.
line) hydrochloride (1.1 mmol),
a
catalitic amount of
hydrochloric acid (37%) was added. Then, the mixture was
capped in closed vessel and irradiated in a microwave oven at 80°
C for 3 min using a CEM focused Microwave Synthesis System. The
progress of the reaction was monitored by TLC using a mixture of
DCM/CH3OH (90:10) as eluent. The mixture was quenched with
water (10 mL) and extracted with EtOAc (3 ꢁ 10 mL). The aqueous
layer was made alkaline with sodium hydroxide (2 N) and
extracted with EtOAc (3 ꢁ 10 mL). The combined extracts were
dried with dry Na2SO4 and concentrated in vacuo. The crude
desired compound was purified by flash chromatography (FC,
DCM/CH3OH 90:10) and recrystallized by treatment with Et2O.
3-(4-Benzylpiperidin-1-yl)-1-(6-hydroxy-1H-indol-3-yl)
propan-1-one (13)
Colorless solid. Yield 80%. M.p.: 121–122°C. Rf ¼ 0.09. 1H NMR
(DMSO-d6) d: 1.12–2.93 (m, 15H), 6.66 (dd, 1H, H-5, J ¼ 8.5, J ¼ 1.8),
6.77 (d, 1H, H-7, J ¼ 1.8), 7.19–7.29 (m, 5H, ArH), 7.91 (d, 1H, H-4,
J ¼ 8.5), 8.13 (d, 1H, H-2, J ¼ 2.7), 9.17 (s, 1H, OH), 11.52 (bs, 1H, NH).
Anal. calcd. for C23H26N2O2: C, 76.21; H, 7.23; N, 7.73. Found C,
76.19; H, 7.21; N, 7.71.
General procedure for the synthesis of hydroxyl derivatives
12–13, 16–17, and 21–22
2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(5-methoxy-1H-
The appropriate methoxy derivative (10–11, 14–15, and 19–20)
was dissolved in DCM (5 mL), treated with BBr3 (1M in DCM) (6 mL,
6 mmol) under nitrogen atmosphere and stirred overnight at
room temperature. Successively, MeOH (7 mL) was carefully
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 solution
(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.
indol-3-yl)propan-1-one (14)
Colorless solid. Yield 47%. M.p.: 164–165°C. Rf ¼ 0.20. 1H NMR
(DMSO-d6) d: 1.11–2.98 (m, 15H), 3.76 (s, 3H, OCH3), 6.83 (dd, 1H,
H-6, J ¼ 8.8, J ¼ 2.3), 7.04–7.17 (m, 4H, ArH), 7.33 (d, 1H, H-7,
J ¼ 8.8), 7.69 (d, 1H, H-4, J ¼ 2.3), 8.28 (d, 1H, H-2, J ¼ 2.9), 11.81 (bs,
1H, NH). Anal. calcd. for C24H27FN2O2: C, 73.07; H, 6.90; N, 7.10.
Found C, 73.17; H, 6.88; N, 7.15.
2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(6-methoxy-1H-
indol-3-yl)propan-1-one (15)
Colorless solid. Yield 20%. M.p.: 207–208°C. Rf ¼ 0.20. 1H NMR
(DMSO-d6) d: 1.11–2.96 (m, 15H), 3.77 (s, 3H, OCH3), 6.80 (dd, 1H,
H-5, J ¼ 8.8, J ¼ 2.3), 6.92 (d, 1H, H-7, J ¼ 2.3), 7.04–7.20 (m, 4H,
ArH), 8.02 (d, 1H, H-4, J ¼ 8.8), 8.22 (s, 1H, H-2), 11.71 (bs, 1H, NH).
Anal. calcd. for C24H27FN2O2: C, 73.07; H, 6.90; N, 7.10. Found C,
73.15; H, 6.85; N, 7.16.
3-(4-Benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one
(9)
Colorless solid. Yield 16% (route A). Yield 40% (route B). M.p.: 191–
1
192°C. Rf ¼ 0.17. H NMR (DMSO-d6) (d) 1.12–2.96 (m, 15H), 7.07–
7.23 (m, 9H, ArH, H-6, H-7), 7.41 (m, 1H, H-5), 8.14 (m, 1H, H-4), 8.33
(d, 1H, H-2, J ¼ 3.7), 11.88 (bs, 1H, NH). Anal. calcd. for C23H26N2O:
C, 79.73; H, 7.56; N, 8.09. Found C, 79.71; H, 7.54, N 8.07.
2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(5-hydroxy-1H-
indol-3-yl)propan-1-one (16)
Colorless solid. Yield 66%. M.p.: 210–211°C. Rf ¼ 0.13. 1H NMR
(DMSO-d6) d: 1.11–2.94 (m, 15H), 6.67 (dd, 1H, H-6, J ¼ 8.8, J ¼ 2.3),
6.77 (d, 1H, H-7, J ¼ 1.8), 7.07–7.20 (m, 5H, ArH), 7.57 (d, 1H, H-4,
J ¼ 2.4), 8.21 (s, 1H, H-2), 8.96 (s, 1H, OH), 11.66 (bs, 1H, NH). Anal.
calcd. for C23H25FN2O2: C, 72.61; H, 6.62; N, 7.36. Found C, 72.71;
H, 6.50; N, 7.40.
3-(4-Benzylpiperidin-1-yl)-1-(5-methoxy-1H-indol-3-yl)
propan-1-one (10)
Colorless solid. Yield 40%. M.p.: 171–172°C. Rf ¼ 0.17. 1H NMR
(DMSO-d6) d: 1.20–3.02 (m, 15H), 3.77 (s, 3H, OCH3), 6.83 (dd, 1H,
H-6, J ¼ 2.4, J ¼ 8.8), 7.14–7.30 (m, 5H, ArH), 7.35 (d, 1H, H-7,
J ¼ 8.8), 7.69 (s, 1H, H-4), 8.29 (d, 1H, H-2, J ¼ 2.4), 11.85 (bs, 1H, NH).
13C NMR (DMSO-d6) d: 31.39, 37.13, 42.26, 53.21, 53.86, 55.27,
57.58, 103.1, 109.6, 112.7, 116.3, 126.2, 127.0, 128.2, 129.0, 131.5,
134.2, 140.3, 155.4, 193.9. Anal. calcd. for C24H28N2O2: C, 76.56; H,
7.50; N, 7.44. Found C, 76.66; H, 7.54; N, 7.34.
2-(4-(4-Fluorobenzyl)piperidin-1-yl)-1-(6-hydroxy-1H-
indol-3-yl)propan-1-one (17)
Colorless solid. Yield 40%. M.p.: 161–162°C. Rf ¼ 0.07. 1H NMR
(DMSO-d6) d: 1.11–2.93 (m, 15H), 6.65 (dd, 1H, H-5, J ¼ 8.5, J ¼ 2.1),
6.78 (s, 1H, H-7), 7.04–7.20 (m, 4H, ArH), 7.91 (d, 1H, H-4, J ¼ 8.5),
8.13 (s, 1H, H-2, J ¼ 3.2), 9.71 (s, 1H, OH), 11.51 (bs, 1H, NH). Anal.
calcd. for C23H25FN2O2: C, 72.61; H, 6.62; N, 7.36. Found C, 72.72;
H, 6.51; N, 7.42.
3-(4-Benzylpiperidin-1-yl)-1-(6-methoxy-1H-indol-3-yl)
propan-1-one (11)
Colorless solid. Yield 42%. M.p.: 187–188°C. Rf ¼ 0.18. 1H NMR
(DMSO-d6) d: 1.13–2.96 (m, 15H), 3.77 (s, 3H, OCH3), 6.81 (d, 1H,
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538
M. R. Buemi et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 533–539
3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-(1H-indol-3-yl)
propan-1-one (18)
redundancies; in this analysis, two geometries were considered
non-redundant when they differed by more than 60° in at least one
torsion angle. For each derivative, the lowest energy structure was
then submitted to docking simulations The ligands minimized in
this way were docked in their corresponding proteins by means of
Gold Suite 5.0.1. The region of interest 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. GoldScore [28] was chosen as a fitness function
and the standard default settings were used in all the calculations
and the ligands were submitted to 100 genetic algorithm runs. The
“allow early termination” command was deactivated. Results
differing by <0.75 Å in ligand-all atom RMSD, were clustered
together. The best ranked GOLD-calculated conformation was used
for analysis and representation [15].
Brown solid. Yield 20% (route A). Yield 50% (route B). M.p.: 165–
166°C. Rf ¼ 0.55. 1H NMR (DMSO-d6) d: 2.73–3.14 (m, 8H), 3.63
(s, 2H), 7.05–7.23 (m, 6H, ArH), 7.46 (m, 1H, H-5), 8.21 (m, 1H, H-4),
8.43 (s, 1H, H-2), 11.95 (bs, 1H, NH). 13C NMR (DMSO-d6) d: 28.77,
36.78, 50.50, 53.72, 55.49, 111.4, 112.2, 116.6, 121.5, 122.8, 125.5,
126.0, 126.4, 126.6, 128.4, 129.0, 134.1, 135.0, 136.7, 194.33. Anal.
calcd. for C20H20N2O: C, 78.92; H, 6.62; N, 9.20. Found C, 78.91; H,
6.61; N, 9.19.
3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-(5-methoxy-1H-
indol-3-yl)propan-1-one (19)
Brown solid. Yield 48%. M.p.: 160–161°C. Rf ¼ 0.57. 1H NMR
(DMSO-d6) d: 2.73–3.11 (m, 8H), 3.63 (s, 2H), 3.77 (s, 3H, OCH3), 6.83
(dd, 1H, H-6, J ¼ 2.2, J ¼ 8.8), 7.04–7.10 (m, 4H, ArH), 7.35 (d, 1H,
H-7, J ¼ 8.8), 7.72 (d, 1H, H-4, J ¼ 2.2), 8.36 (d, 1H, H-2, J ¼ 3.0), 11.82
(bs, 1H, NH). Anal. calcd. for C21H22N2O2: C, 75.42; H, 6.63; N, 8.38.
Found C, 75.46; H, 6.67; N, 8.42.
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) [29, 30]. Cerebral
cortices of male Wistar derived rats weighing 175 ꢂ 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 mM
GBR-12909 to block non-polyamine sensitive sites) for 120 min at
4°C. Non-specific binding is estimated in the presence of 10 mM
ifenprodil (1). Membranes are filtered and washed, the filters are
then counted to determine [3H]Ifenprodil specifically bound.
Three concentrations (10 mM, 0.1 mM, 0.001 mM, in duplicate) of
test compounds were used in displacement assay.
3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-(6-methoxy-1H-
indol-3-yl)propan-1-one (20)
Brown solid. Yield 40%. M.p.: 176–177°C. Rf ¼ 0.53. 1H NMR
(DMSO-d6) d: 2.72–3.28 (m, 8H), 3.62 (s, 2H), 3.78 (s, 3H, OCH3), 6.82
(dd, 1H, H-5, J ¼ 8.8, J ¼ 2.2), 6.93 (s, 1H, H-7, J ¼ 2.2), 7.02–7.12
(m, 4H, ArH), 8.04 (d, 1H, H-4, J ¼ 8.8), 8.29 (d, 1H, H-2, J ¼ 2.2), 11.74
(bs, 1H, NH). Anal. calcd. for C21H22N2O2: C, 75.42; H, 6.63; N, 8.38.
Found C, 75.48; H, 6.69; N, 8.45.
3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-(5-hydroxy-1H-
Financial support for this research by Fondo di Ateneo per la Ricerca
(PRA 2009 – grant number ORME09SPNC – Università di Messina).
indol-3-yl)propan-1-one (21)
Brown solid. Yield 30%. M.p.: 222–223°C. Rf ¼ 0.29. 1H NMR
(DMSO-d6) d: 2.72–3.07 (m, 8H), 3.61 (s, 2H), 6.66 (dd, 1H, H-6,
J ¼ 2.7, J ¼ 8.5), 7.02–7.10 (m, 4H, ArH), 7.22 (d, 1H, H-7, J ¼ 8.5),
7.58 (d, 1H, H-4, J ¼ 2.7), 8.25 (d, 1H, H-2, J ¼ 3.2), 8.94 (s, 1H, OH),
11.67 (bs, 1H, NH). Anal. calcd. for C20H20N2O2: C, 74.98; H, 6.29;
N, 8.74. Found C, 74.95; H, 6.26; N, 8.71.
Author contributions
M.R.B. and S.F. performed the synthesis, purification, and
chemical characterization of new molecules under the
supervision of R.G., L.D.L. carried out computational studies.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
3-(3,4-Dihydroisoquinolin-2(1H)-yl)-1-(6-hydroxy-1H-
indol-3-yl)propan-1-one (22)
Brown solid. Yield 37%. M.p.: 196–197°C. Rf ¼ 0.20. 1H NMR
(DMSO-d6) d: 2.73–3.05 (m, 8H), 3.61 (s, 2H), 6.67 (d, 1H, H-5,
J ¼ 8.8), 6.78 (bs, 1H, H-7), 7.01–7.20 (m, 4H, ArH), 7.95 (d, 1H, H-4,
J ¼ 8.8), 8.21 (bs, 1H. H-2), 9.17 (s, 1H, OH), 11.54 (bs, 1H, NH). Anal.
calcd. for C20H20N2O2: C, 74.98; H, 6.29; N, 8.74. Found C, 74.88; H,
6.33; N, 8.78.
The authors have declared no conflict of interest.
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