From NMDA receptor antagonists to discovery of selective σ2 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2013.11.014

Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA, a small series of 1-(1H-indol-3-yl)-2-(4- phenylpiperidin-1-yl)ethanone derivatives has been synthesized by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only the 1-(5-hydroxy-1H-indol-3-yl)-2- (4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity. Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward σ₂ receptor (K_i values of 10 nM and 20 nM, respectively). Thus, in this case the discovery of new σ₂ receptor selective ligands was an unexpected result emerging from the screening of cross-activity against other CNS receptors.

Full text of DOI:10.1016/j.bmc.2013.11.014

Bioorganic & Medicinal Chemistry 22 (2014) 393–397
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
From NMDA receptor antagonists to discovery of selective r2
receptor ligands
a
a
b
c
c
Rosaria Gitto a, , Laura De Luca , Stefania Ferro , Angela Scala , Simone Ronsisvalle , Carmela Parenti ,
⇑
Orazio Prezzavento c, Maria Rosa Buemi a, , Alba Chimirri a
⇑
a Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Dipartimento di Scienze Chimiche, Viale Ferdinando Stagno d’Alcontres, I-98166 Messina, Italy
c Dipartimento di Scienze del Farmaco, Università di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2013
Revised 29 October 2013
Accepted 7 November 2013
Available online 15 November 2013
Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA,
a small series of 1-(1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives has been synthesized
by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert
off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only
the 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity.
Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward r2 receptor
Keywords:
(Ki values of 10 nM and 20 nM, respectively). Thus, in this case the discovery of new
r2 receptor selective
Glutamate
GluN2B/NMDA
Sigma receptor
Indoles
ligands was an unexpected result emerging from the screening of cross-activity against other CNS
receptors.
Ó 2013 Elsevier Ltd. All rights reserved.
Ifenprodil
1. Introduction
There are different classes of NMDAR ligands that act by differ-
ent mechanisms inhibiting or potentiating NMDA receptor func-
The ionotropic N-methyl-
D
-aspartate receptors (NMDARs) regu-
tion; they target diverse binding sites located in the NMDAR
complex.8 In fact, each subunit displays a modular architecture:
an amino terminal domain (ATD) followed by a ligand-binding do-
main (LBD), a transmembrane domain and a carboxyl terminal do-
main (CTD).1 The ATD plays a key role in subunit assembly and
exhibits a clamshell-like structure. In the case of GluN2A and
GluN2B subunits, the ATD binds allosteric inhibitors which
exert modulator effects. The prototype of noncompetitive
GluN2B-antagonists was the phenylethanolamine derivative ifen-
prodil (1, Fig. 1). It served as a template to design more selective
analogues such as traxoprodil (2) having potential as neuroprotec-
tive agents without side effects.9
late the glutamate-mediated excitatory neurotransmission which
plays a relevant role in synaptic plasticity as well as during neuro-
nal death in the mammalian central nervous system (CNS).1
NMDAR over-activation is involved in many neurological diseases
such as strokes, seizures, dementia, schizophrenia and pain.2–5
On this basis the development of NMDA agonists and antagonists
provided relevant insights about the understanding of the role of
NMDARs in physiological and pathological functions. It is well
known that NMDAR complex is a ligand gated ion channel that
consists of a combination of different subunits, GluN1, GluN2 and
more rarely GluN3 subunit (formerly known as NR1-3).6 There are
eight splice-variant isoforms of GluN1, four isoforms of GluN2
(GluN2A–D) encoded by four different genes, and two different iso-
forms of GluN3 (GluN3A–B).7 Whereas the GluN1 subunit is ex-
pressed ubiquitously in the CNS, the GluN2 subunits present a
different anatomical localization. Particularly, the expression of
GluN2B is restricted to forebrain areas and minimally in the cere-
bellum; this distribution implies that GluN2B selective antagonists
might have reduced side effects in the treatment of several neuro-
logical pathologies.8
Recent findings suggested that sigma (
clude regulatory roles on NMDA-mediated neurotransmission.10 In
particular, several receptor ligands have shown promise in phar-
r) receptor functions in-
r
macological studies of the neurological diseases and protected
against NMDAR-mediated excitotoxic brain damage.11,12 There
are two subtypes of
r receptors, named r1 and r2. The r1 receptor
exerts a modulatory role on neurotransmission and could be useful
for the treatment of CNS diseases. Increasing evidences suggest
that r2 receptor is involved in the regulation of cell proliferation
and maintenance of cell viability. This receptor is highly expressed
in several tumoral cell lines, where r2 agonists produce morpho-
logical changes and apoptosis. On this basis they might be useful
as novel anticancer and imaging agents in cancer diagnosis.13,14
⇑
Corresponding authors. Tel.: +39 090 6766413; fax: +39 090 6766402 (R.G.).
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.2013.11.014

394
R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 393–397
OH
O
O
OH
OH
N
MeO
Cl
MeO
N
N
MeO
1
ii
path A
i
R
Me
Me
N
H
HO
N
OH
N
H
H
2, Traxoprodil
1, ifenprodil
8
2
6a-f
R
9
O
O
path B
N
iii
O
iii
N
R1
R2
OH
Cl
N
Cl
O
O
a
b
c
d
e
f
H
H
H
F
4
3, haloperidol
N
Cl
OH
OH
HO
HO
O
ii
1
O
R
Cl
1
N
1
N
R
N
N
H
R
2
H
OH
CN
Br
H
R
N
H
N
H
2
R
10
7a-f
3
COMe
H
R
5a, R1 = OMe
5b, R1 = OH
6a-f, R1 = OMe; 7a-f, R1= OH
R2 = H, OH, CN, COMe
R3 = H, Br, Cl
Scheme 1. Reagents and conditions: (i) ClCH2COCl, pyridine, dioxane, MW: 5 min,
50 °C, 200 W; (ii) piperidine derivatives, K2CO3, DMF, MW: 10 min 100 °C, 200 W;
(iii) BBr3 (1.0 M DCM), rt, 10 h.
Figure 1. GluN2B-containing NMDA and sigma receptor ligands.
It is well known that 1 and several ifenprodil-like compounds
(MAOS) conditions, the 2-chloro-1-(5-methoxy-1H-indol-3-yl)eth-
anone (9) was prepared. Following pathway A the intermediate 9
was converted into the derivatives 6a–f by reaction with suitable
piperidine fragments in alkaline medium. Finally, by treatment with
boron tribromide solution, methoxy-substituted derivatives 6a–f
gave the corresponding hydroxyl analogues (7a–f). In order to in-
crease the yield an alternative synthetic procedure (pathway B)
was performed. In fact by dealkylation with boron tribromide
solution the 2-chloro-1-(5-methoxy-1H-indol-3-yl)ethanone (9)
furnished the intermediate 2-chloro-1-(5-hydroxy-1H-indol-3-
yl)ethanone (10), which led to desired compounds (7a–f). The
chemical characterization of all compounds obtained was supported
by elemental analyses and spectroscopic measurements.
display ligand promiscuity toward
r receptors. The ligand promis-
cuity towards sigma and GluN2B-containing NMDA receptors
could be explained on the basis of their very similar structural
architecture as suggested by comparison of 3D-pharmacophoric
models of NMDAR and sigma receptor ligands such as 1, 2, haloper-
idol (3) and benzoxalone derivative 4 (see Fig. 1).13,15,16 Actually,
sigma and GluN2B-containing NMDA receptor ligands share the
following common chemical features: (i) a nitrogen atom as posi-
tive ionizable feature and (ii) two hydrophobic regions (primary
and secondary).17,18 It is likely that the optimal distance between
the two hydrophobic domains as well as the presence of ancillary
or accessory functional groups controls the recognition process to-
ward sigma or GluN2B-containing NMDA receptors.
We previously disclosed a new class of GluN2B ligands19–21
(5a and 5b in Fig. 1) which contain the indole scaffold as well as
the benzylpiperidine fragment as a structural requirement that is
2.2. Binding assays
All synthesized compounds were tested to evaluate their affinity
for GluN2B-containing NMDA receptors using [3H]ifenprodil as
radioligand.25 The 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpi-
peridin-1-yl)ethanone (7a) was the most active derivative of the
series and it displays 51% of inhibition of [3H]ifenprodil binding at
shared by other well-known GluN2B and
r receptor ligands.
Interestingly, the most active compound 5b was able to bind the
GluN2B-containing NMDA receptor at nanomolar concentration
(IC50 = 25 nM and Ki = 23 nM); it showed relevant anticonvulsant
properties and reduced NMDA receptor-mediated current in patch
clamp experiments, this compound also displayed neuroprotective
effects in Human Cortical neuronal (HCN-1A) cells.20 Interestingly,
fixed dose of 10 l
M. According to our previous SAR analysis19–21
the 5-hydroxylderivative 7a proved to be more potent ligand than
5-methoxy analogue 6a. As reported in Table 1 the new compounds
6a–f and 7a–f proved to be less active than previously reported in-
doles 5a and 5b19 and reference compound ifenprodil (1, Ki value of
19 nM). These results suggest that the introduced modifications
generally gave low active GluN2B-containing NMDAR ligands. It is
likely that the loss of GluN2B affinity could be related to a reduction
of distance between the two benzene rings.26,27
the compound 5b exhibited off-target activity toward r
2 receptor.21
With this in mind our goal was (a) to improve our knowledge
about the structure activity relationships (SARs) of indole deriva-
tives as GluN2B ligands and (b) to better define the key moieties
triggering the affinity for GluN2B subunit and/or selectivity for sig-
ma receptors. Therefore we focused our interest on the introduction
of the 4-phenyl-piperidine substituent, that is a fragment of the
well-known GluN2B selective antagonist traxoprodil (2),22–24 as
well as a frequently recurring substituent of the chemical structures
of sigma ligands. On this basis we planned a facile approach to syn-
thesize a series of 1-(5-methoxy-1H-indol-3-yl)- and 1-(5-hydro-
xy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives
(6 a–f and 7 a–f, respectively) that have been evaluated for their
binding affinity towards these two receptor systems.
Given the sigma receptor off-target activity demonstrated by
several GluN2B-containing NMDA receptor ligands28–30 we also
determined the r1/r2 receptor binding affinity in competition
experiments with radioligands. Considering that the 4-hydroxy-
4-phenylpiperidin-1-yl moiety represents a structural requirement
of several sigma ligands, compounds 6b–d and 7b–d and corre-
sponding unsubstituted 4-phenylpiperidin-1-yl analogues 6a and
7a have been selected for testing. To determine
r1 and r2 receptor
affinities the competitive displacement of [3H](+)pentazocine
and [3H]DTG [1,3-di-o-tolylguanidine] in the presence of (+)-
2. Results and discussion
2.1. Chemistry
SKF10,047 (0.4 lM) were used. The Ki values are gathered in Table 2
which includes data of ifenprodil (1), haloperidol (3) and parent
compounds 5a and 5b.
The lack of the hydroxyl group at 40-position of piperidine ring
in the derivative 6a was detrimental to the r1 receptor binding
affinity (Ki = 1910 nM). Whereas, the 4-hydroxy-substituted deriv-
To obtain the designed indole derivatives 6a–f and 7a–f we em-
ployed the synthetic pathway depicted in the Scheme 1. Starting
from 5-methoxyindole (8), in Microwave Assisted Organic Synthesis

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 393–397
395
Table 1
In good agreement with the SAR information about sigma
receptor ligands we generally found that for this series of 1-(1H-in-
dol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives (6a–d)
the presence of chlorine or bromine atom in para-position of phe-
NMDA receptor binding affinity results of indoles 6a–f and 7a–f and reference
compounds (5a, 5b and 1)
R1
R2
nyl ring induced an improvement of the
r receptor affinity. Overall
O
these results were consistent with the proposed pharmacophore
n
N
R3
models for r1/r2 ligands already reported in litera-
N
H
ture.5,13,15,17,31,32 Thus the 2-[4-(4-chlorophenyl)-4-hydroxypiperi-
din-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone (6c) was the
most potent r2 receptor ligand (Ki = 7.5 nM). Whereas, the lack
of hydroxyl group at 40-position of piperidine ring and the simulta-
neous presence of the methoxy substituent at the 5-position of in-
dole ring (i.e., compound 6a) optimized r2 selectivity
R1
R2
R3
n
% Inhibition @ 10 l
Ma
5a
5b
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
MeO
OH
H
H
H
H
H
H
H
Cl
Br
H
H
H
H
Cl
Br
H
H
1
1
0
0
0
0
0
0
0
0
0
0
0
0
86 (Ki = 1.67 lM)
92 (Ki = 23 nM)
8
11
15
5
11
N.A.
51
32
26
40
16
6
MeO
MeO
MeO
MeO
MeO
MeO
OH
OH
OH
OH
OH
OH
OH
OH
CN
COMe
H
OH
OH
OH
CN
COMe
(Ki
2-(4-phenylpiperidin-1-yl)ethanone 6a show higher affinity at
2 over 1 receptor when compared to reference compounds hal-
operidol (3) and ifenprodil (1).
Based on the r1 2 receptor binding affinity data the 2-(4-ben-
r1/Kir2 = 95.5). Moreover, the 1-(5-methoxy-1H-indol-3-yl)-
r
r
/r
zylpiperidin-1-yl)-1-(5-methoxy-1H-indol-3-yl)ethanone (5a) and
1-(5-methoxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone
(6a) could be considered starting point to design new selective li-
gands at r2 receptor that is overexpressed in tumor cells and has
been proposed as a biomarker for tumor cell proliferation.14,33
OH
Ifenprodil (1)
Ki = 19 nM
a
Displacement of [3H]ifenprodil. NA: no active.
3. Conclusions
Table 2
r1 and r2 binding affinities for selected indoles 6a–d and 7a–d and reference
compounds (1 and 3)
In the course of our studies aimed to develop new GluN2B-
containing NMDA receptor ligands a small series of indole deriva-
tives has been designed and synthesized. Although all obtained
compounds show poor binding affinity towards NMDA receptors,
this study led to a serendipitous identification of new promising
selective r2 ligands. Notably, the indole derivative 5a and 6a
proved to be potent r2 ligand (Ki values of 10 nM and 20 nM,
respectively) characterized by high subtype selectivity (Kir1
>1000 nM). The key finding of this study is that subtle structural
modifications can switch a ligand selectivity between NMDA and
sigma receptors.
R1
R2
O
n
N
R3
N
H
R1
R2
R3
n
r1
r2
Kir1/Kir2
Ki SDa
(nM)
Ki SDb
(nM)
5a
5bc
6a
6b
6c
6d
7a
7b
7c
MeO
OH
H
H
H
OH
OH
OH
H
OH
OH
OH
H
H
H
H
Cl
Br
H
H
Cl
Br
1
1
0
0
0
0
0
0
0
0
>5000
10 3.0
76.5 6.5
20 1.0
21 0.8
7.5 1.0
10 1.2
224 6.5
658 17
47 2.0
57 1.8
18.8 1.0
13.0 2.0
>500
61.9
95.5
5
10.5
6
12
4.7
11.9
3.2
0.05
0.1
4734 18
1910 15
103 4.5
78.5 2.0
60 2.0
2680 65
3080 60
557 13
185 8.0
1.02 0.35
1.30 0.3
MeO
MeO
MeO
MeO
OH
OH
OH
OH
4. Experimental section
4.1. 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 Buchi melting point B-545 apparatus and are uncorrected.
Elemental analyses (C, H, N) were carried out on a Carlo Erba Model
1106 Elemental Analyzer and the results are within 0.4% of the
theoretical values. 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 CH2Cl2/CH3OH (90:10) as eluent. 1H NMR spectra
were measured in 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 protons
were confirmed by addition of D2O.
7d
Ifenprodil (1)
Haloperidol (3)
a,b
Displacement of [3H](+)pentazocine (
r
1) and [3H]DTG [1,3-di-o-tolylguanidine]
2).
in the presence of (+)-SKF10,047 (0.4 M) (
l
r
c
21
Data taken from Ref.
.
atives 6b–d and 7d showed a moderate r1 affinity (Table 2). For
compounds 7c and 7b it was also found low affinity toward r1
subtype (Ki = 557 and 3080 nM, respectively). With the exception
of compounds 7a (Ki = 224 nM) and 7b (Ki = 658 nM), the new
synthesized compounds exhibited remarkable affinity at r2
receptor showing Ki values in the range of 7.5–57 nM. We found
that the 5-methoxy-substituted derivatives 6a–d generally dis-
played a higher r2 affinity when compared to the corresponding
5-hydroxy-substituted derivatives 7a–d (Table 2). Thus, for com-
parative purpose we resynthesized the parent compound 5a20
4.1.1. Synthesis of 2-chloro-1-(5-methoxy-1H-indol-3-
yl)ethanone (9)
A mixture of chloroacetyl chloride (0.119 mL, 1.5 mmol) in
dioxane (2 mL) was added drop-wise to a solution of 5-methoxy
indole (8) (147 mg, 1 mmol) in dioxane (2 mL) and in the presence
of pyridine (0.12 mL, 1.5 mmol). The mixture was placed in a
cylindrical quartz tube (Ø2 cm), then stirred and irradiated in a
which was profiled for its r1
/r2 receptor binding affinity.
Interestingly, compound 5a proved to be more selective r2
receptor ligand (see Table 2) (Ki
analogue 5b.21
r1/Kir2 >500) than 5-methoxy

396
R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 393–397
microwave oven at 200 W for one step of 5 min at 50 °C. A solution
of water and Et2O (4:1) was added to quench the reaction and the
obtained solid product was filtered and recrystallized by treatment
with EtOH. Experimental data for 2-chloro-1-(5-methoxy-1H-in-
dol-3-yl)ethanone are in accordance with literature.19
(s, 3H, OMe), 4.92 (br s, 1H, OH), 6.83 (dd, 1H, J = 8.5, J = 2.6, 6-H,
ArH), 7.35–7.51 (m, 5H, ArH), 7.72 (d, 1H, J = 2.6, 4-H, ArH), 8.49
(d, 1H, J = 3.0, 2-H), 11.78 (br s, 1H, NH). Anal. (C22H23ClN2O3): C
66.24; H 5.81, N 7.02. Found: C 66.34; H 5.71, N 7.22.
4.1.2.5. 2-[4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl]-1-(5-
4.1.2. General methods for the synthesis of compounds 7a–f
To synthesize compounds 7a–f two different pathways (A and
B) were employed.
methoxy-1H-indol-3-yl)ethanone (6d).
Yield 52%. Mp 212–
213 °C. rf 0.16 1H NMR (DMSO-d6) (d) 1.55–1.94 (m, 4H, NCH2CH2-
C(OH)), 2.53–2.77 (m, 4H, NCH2CH2C(OH)), 3.60 (s, 2H, CH2N), 3.78
(s, 3H, OMe), 4.93 (br s, 1H, OH), 6.83 (dd, 1H, J = 8.5, J = 1.2, 6-H,
ArH), 7.36 (d, 1H, J = 8.5, ArH, 7-H, ArH), 7.42–7.51 (m, 4H, ArH),
7.72 (d, 1H, J=1.2, 4-H, ArH), 8.49 (s, 1H, 2-H), 11.78 (br s, 1H,
NH). Anal. (C22H23BrN2O3): C 59.60; H 5.23; N 6.32. Found: C
59.69; H 5.13; N 6.42.
Pathway A: To a solution of (2-chloro-1-(5-methoxy-1H-indol-
3-yl)ethanone) (9) (223 mg, 1 mmol) in DMF dry (2 mL) the appro-
priate piperidine fragments (1 mmol) and K2CO3 (70.0 mg,
0.5 mmol) were added. The mixture was placed in a cylindrical
quartz tube (Ø2 cm), then stirred and irradiated in a microwave
oven at 200 W at 100 °C for 10 min. Then the reaction mixture
was cooled, quenched with NaHCO3 saturated aqueous solution
(10 mL) and extracted with EtOAc (3 ꢀ 10 mL). The organic layer
was washed with brine (3 ꢀ 3 mL), dried over Na2SO4 and concen-
trated until dryness under reduced pressure. The crude compounds
were purified by flash chromatography DCM/CH3OH (90:10) and
they were recrystallized from ethanol to give derivatives 6a–f.
Then the appropriate derivatives 6a–f were dissolved in methylene
chloride (DCM) (5 mL), treated with BBr3 (1 M in DCM), (6 mL,
0.006 mol) and stirred overnight. Successively, methanol (7 mL)
was carefully added at 0 °C and the solvents removed under re-
duced pressure. The residue was dissolved in ethyl acetate
(10 mL) and washed with water (3 ꢀ 10 mL). The organic layer
was dried (Na2SO4), combined and concentrated in vacuo. The
crude products were purified by flash chromatography (DCM/
MeOH 90:10) and recrystallized from ethanol to give the desired
compounds (7a–f).
4.1.2.6.
1-[2-(5-Methoxy-1H-indol-3-yl)-2-oxoethyl]-4-phen-
Yield 36%. Mp 184–186 °C.
ylpiperidine-4-carbonitrile (6e).
rf 0.82 1H NMR (DMSO-d6) (d) 2.11 (m, 4H, NCH2CH2C(CN)),
2.56–3.12 (m, 4H, NCH2CH2C(CN)), 3.70 (s, 2H, CH2N), 3.78 (s, 3H,
OMe), 6.83 (d, 1H, J = 8.5, 6-H, ArH), 7.35–7.56 (m, 6H, ArH), 7.72
(s, 1H, 4-H, ArH), 8.43 (d, 1H, J = 2.1, 2-H), 11.81 (br s, 1H, NH).
Anal. (C23H23N3O2): C 73.97; H 6.21; N 11.25. Found: C 73.87; H
6.33; N 11.15.
4.1.2.7. 2-(4-Acetyl-4-phenylpiperidin-1-yl)-1-(5-methoxy-1H-
indol-3-yl)ethanone (6f).
Yield 13%. Mp 208–210 °C. rf 0.50
1H NMR (DMSO-d6) (d) 1.88 (s, 3H, COCH3), 1.99–2.72 (m, 8H),
3.60 (s, 2H, CH2N), 3.77 (s, 3H, MeO), 6.83 (d, 1H, J = 8.5, 6-H,
ArH), 7.29 (d, 1H, J = 8.5, 7-H, ArH), 7.34–7.41 (m, 5H, ArH), 7.69
(s, 1H, 4-H, ArH), 8.41 (s, 1H, 2-H), 11.75 (br s, 1H, NH). Anal.
(C24H26N2O3): C 73.82; H 6.71; N 7.17. Found: C 73.92; H 6.61; N
7.27.
Pathway B: The 2-chloro-1-(5-methoxy-1H-indol-3-yl)ethanone
(9) was de-methylated to give intermediate 10. In turn compound
10 was converted in the final products 7a–f by reaction with the
appropriate piperidine fragments in the presence of K2CO3 as pre-
viously described for compounds 6a–f.
4.1.2.8.
yl)ethanone (7a).
1-(5-Hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-
Pathway A: yield 10%. Pathway B: 42%
Mp 196–198 °C. Rf 0.24. 1H NMR (DMSO-d6) (d) 1.70 (m, 4H, NCH2-
CH2CH), 2.20 (m, 4H, NCH2CH2CH), 3.03 (m, 1H, NCH2CH2CH), 3.56
(s, 2H, CH2N), 6.68 (dd, 1H, J = 8.5, J = 3.0, 6-H, ArH), 7.18 (d, 1H,
J = 8.5, 7-H, ArH), 7.23–7.31 (m, 5H, ArH), 7.60 (d, 1H, J = 3.0, 4-H,
ArH), 8.39 (d, 1H, J = 3.4, 2-H), 8.96 (s, 1H, OH), 11.64 (br s, 1H,
NH).Anal. (C21H22N2O2): C 75.42; H 6.63; N 8.38. Found: C 75.52;
H 6.66; N 3.48.
4.1.2.1.
(10).
2-Chloro-1-(5-hydroxy-1H-indol-3-yl)ethanone
Yield 81%. Mp 275 °C dec 1H NMR (DMSO-d6) (d): 4.58
(s, 2H, CH2), 6.72 (d, 1H, J = 8.5, 6-H, ArH), 7.27 (d, 1H, J = 8.5,
7-H, ArH), 7.56 (s, 1H, 4-H, ArH), 8.33 (d, 1H, J = 2.2, 2-H), 9.07
(br s, 1H, OH), 11.90 (br s, 1H, NH). Anal. (C10H8ClNO2): C 57.30;
H 3.85; N 6.68. Trov: C 57.47; H 3.91; N 6.96.
4.1.2.9. 1-(5-Hydroxy-1H-indol-3-yl)-2-(4-hydroxy-4-phenylpi-
4.1.2.2.
yl)ethanone (6a).
1-(5-Methoxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-
Yield 25%. Mp 219 °C dec rf 0.37. 1H NMR
peridin-1-yl)ethanone (7b).
Pathway B: yield 30%. Mp
236–237 °C. Rf 0.08. 1H NMR (DMSO-d6) (d) 1.57–1.95 (m, 4H, NCH2-
CH2C(OH)), 2.59–2.73 (m, 4H, NCH2CH2C(OH)), 3.55 (s, 2H, CH2N),
4.79 (br s, 1H, OH), 6.68 (dd, 1H, J = 8.5, J = 2.1, 6-H, ArH), 7.15–
7.50 (m, 6H, ArH), 7.60 (d, 1H, J = 2.1, 4-H, ArH), 8.43 (br s, 1H, 2-
H), 8.96 (s, 1H, OH), 11.63 (br s, 1H. NH). Anal. (C21H22N2O3): C
71.98; H 6.33; N 7.99. Found: C 71.88; H 6.43; N 7.89.
(DMSO-d6) (d) 1.70 (m, 4H, NCH2CH2CH), 2.22 (m, 4H, NCH2CH2-
CH), 3.05 (m, 1H, NCH2CH2CH), 3.60 (s, 2H, CH2N), 3.75 (s, 3H,
MeO), 6.84 (d, 1H, J = 8.9, 6-H, ArH), 7.25-7.32 (m, 5H, ArH), 7.36
(d, 1H, J = 8.9, 7-H, ArH), 7.72 (s, 1H, 4-H, ArH), 8.46 (d, 1H,
J = 2.1, 2-H), 11.79 (br s, 1H, NH). Anal. (C22H24N2O2): C 75.83; H
6.94, N 8.04. Found: C 75.93; H 6.84, N 8.14.
4.1.2.10. 2-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(5-
4.1.2.3.
2-(4-Hydroxy-4-phenylpiperidin-1-yl)-1-(5-methoxy-
Yield 33%. Mp 199 °C dec rf
hydroxy-1H-indol-3yl)ethanone (7c).
Pathway B: yield
1H-indol-3-yl)ethanone (6b).
40%. Mp 240-–241 °C. Rf 0.10. 1H NMR (DMSO-d6) (d) 1.56–1.98
(m, 4H, NCH2CH2C(OH)), 2.55–2.77 (m, 4H, NCH2CH2C(OH)), 3.60
(s, 2H, CH2N), 4.92 (br s, 1H, OH), 6.69 (dd, 1H, J = 8.5, J = 2.1, 6-
H, ArH), 7.25 (d, 1H, J = 8.5, 7-H, ArH), 7.36 (d, 2H, J = 8.5, 2I, 6I,
ArH), 7.50 (d, 2H, J = 8.5, 3I, 5I, ArH), 7.60 (d, 1H, J = 2.1, 4-H,
ArH), 8.42 (d, 1H, J = 3.0, 2-H), 8.96 (br s, 1H, OH), 11.63 (br s,
1H, NH). Anal. (C21H21ClN2O3): C 65.54; H 5.50; N 7.28. Found: C
65.44; H 5.53; N 7.30.
0.13. 1H NMR (DMSO-d6) (d) 1.57–1.97 (m, 4H, NCH2CH2C(OH)),
2.56–2.78 (m, 4H, NCH2CH2C(OH)), 3.61 (s, 2H, CH2N), 3.78 (s,
3H, OMe), 4.82 (br s, 1H, OH), 6.84 (dd, J = 8.5, J = 2.4, 6-H, ArH),
7.20–7.50 (m, 6H, ArH), 7.72 (d, 1H, J = 2.4, 4-H, ArH), 8.49 (d,
1H, J = 3.0, 2-H), 11.79 (br s, 1H, NH). Anal. (C22H24N2O3): C
72.51; H 6.64, N 7.69. Found: C 72.61; H 6.74, N 7.77.
4.1.2.4. 2-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(5-
methoxy-1H-indol-3-yl)ethanone (6c).
Yield 59%. Mp 208–
4.1.2.11. 2-[4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl]-1-(5-
210 °C. rf 0.17. 1H NMR (DMSO-d6) (d) 1.55–1.97 (m, 4H, NCH2CH2-
C(OH)), 2.51–2.74 (m, 4H, NCH2CH2C(OH)), 3.59 (s, 2H, CH2N), 3.78
hydroxy-1H-indol-3yl)ethanone (7d).
Pathway B: yield
33%. Mp 241 °C dec Rf 0.10. 1H NMR (DMSO-d6) (d) 1.55–1.93 (m,

R. Gitto et al. / Bioorg. Med. Chem. 22 (2014) 393–397
397
4H, NCH2CH2C(OH)), 2.54–2.76 (m, 4H, NCH2CH2C(OH)), 3.55 (s,
2H, CH2N), 4.91 (br s, 1H, OH), 6.67 (d, 1H, J = 8.5, 6-H, ArH), 7.25
(d, 1H, J = 8.5, 7-H, ArH), 7.42–7.51 (m, 4H, ArH), 7.61 (s, 1H, 4-H,
ArH) 8.42 (s, 1H, 2-H), 8.95 (br s, 1H, OH), 11.63 (br s, 1H, NH).
Anal. (C21H21BrN2O3): C 58.75; H 4.93, N 6.53. Found: C 58.65; H
4.99, N 6.63.
bition constants (Ki values) were calculated using the EBDA/LI-
GAND program purchased from Elsevier/Biosoft.
Acknowledgments
Financial support for this research by Fondo di Ateneo per la
Ricerca (PRA 2009—Grant Number ORME09SPNC—Università di
Messina) is gratefully acknowledged.
4.1.2.12. 1-[2-(5-Hydroxy-1H-indol-3-yl)-2-oxoethyl]-4-phen-
ylpiperidine-4-carbonitrile (7e).
Pathway A: yield 31%. Path-
way B: yield 48%. Mp 230-232 °C. Rf 0.59. 1H NMR (DMSO-d6) (d)
2.11 (m, 4H, NCH2CH2C(CN)), 2.53–3.12 (m, 4H, NCH2CH2C(CN)),
3.68 (s, 2H, CH2N), 6.68 (dd, 1H, J = 8.5, J = 2.1, 6-H, ArH), 7.25 (d,
1H, J = 8.5, 7-H, ArH), 7.36–7.57 (m, 5H, ArH), 7.60 (d, 1H, J = 2.1,
4-H, ArH), 8.37 (d, 1H, J = 2.9, 2-H), 8.97 (br s, 1H, OH), 11.67 (br
s, 1H, NH). Anal. (C22H21N3O2): C 73.52; H 5.89; N 11.69. Found:
C 73.62; H 5.99; N 11.79.
References and notes
1. Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.; Vance, K. M.;
Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.; Dingledine, R. Pharmacol. Rev.
2010, 62, 405.
2. Paoletti, P.; Neyton, J. Curr. Opin. Pharmacol. 2007, 7, 39.
3. Hedegaard, M.; Hansen, K. B.; Andersen, K. T.; Brauner-Osborne, H.; Traynelis,
S. F. Neurochem. Int. 2012, 61, 601.
4. Niciu, M. J.; Kelmendi, B.; Sanacora, G. Pharmacol. Biochem. Behav. 2012, 100,
656.
5. Gitto, R.; De Luca, L.; De Grazia, S.; Chimirri, A. Curr. Top. Med. Chem. 2012, 12,
971.
6. Collingridge, G. L.; Olsen, R. W.; Peters, J.; Spedding, M. Neuropharmacology
2009, 56, 2.
7. Paoletti, P. Eur. J. Neurosci. 2011, 33, 1351.
8. Mony, L.; Kew, J. N.; Gunthorpe, M. J.; Paoletti, P. Br. J. Pharmacol. 2009, 157,
1301.
9. Beinat, C.; Banister, S.; Moussa, I.; Reynolds, A. J.; McErlean, C. S.; Kassiou, M.
Curr. Med. Chem. 2010, 17, 4166.
10. Maurice, T.; Su, T. P. Pharmacol. Ther. 2009, 124, 195.
11. Abate, C. Curr. Pharm. Des. 2012, 18, 861.
4.1.2.13.
1H-indol-3yl)ethanone (7f).
2-(4-Acetyl-4-phenylpiperidine-1-yl)-1-(5-hydroxy-
Pathway A: yield 35%. Pathway
B: yield 50%. Mp 235–236 °C. Rf 0.40. 1H NMR (DMSO-d6) (d) 1.88
(s, 3H, COCH3), 1.98–2.71 (m, 8H), 3.46 (s, 2H, CH2N), 6.67 (dd,
1H, J = 8.5, J = 3.4, 6-H, ArH), 7.22–7.38 (m, 6H, ArH), 7.57 (d, 1H,
J = 3.4, 4-H, ArH), 8.35 (s, 1H, 2-H), 8.94 (br s, 1H, OH), 11.60 (br
s, 1H, NH). Anal. (C23H24N2O3): C 73.38; H 6.43; N 7.44. Found: C
73.48; H 6.53; N 7.54.
12. Banister, S. D.; Kassiou, M. Curr. Pharm. Des. 2012, 18, 884.
13. Huang, Y. S.; Lu, H. L.; Zhang, L. J.; Wu, Z. Med Res Rev 2013, http://dx.doi.org/
10.1002/med.21297 [Epub ahead of print].
4.2. Receptor binding assays
14. Mach, R. H.; Zeng, C.; Hawkins, W. G. J Med Chem. 2013, 56, 7137–7160.
15. Laurini, E.; Zampieri, D.; Mamolo, M. G.; Vio, L.; Zanette, C.; Florio, C.; Posocco,
P.; Fermeglia, M.; Pricl, S. Bioorg. Med. Chem. Lett. 2010, 20, 2954.
16. Laurini, E.; Marson, D.; Dal Col, V.; Fermeglia, M.; Mamolo, M. G.; Zampieri, D.;
Vio, L.; Pricl, S. Mol. Pharm. 2012, 9, 3107.
17. Glennon, R. A.; Ablordeppey, S. Y.; Ismaiel, A. M.; el-Ashmawy, M. B.; Fischer, J.
B.; Howie, K. B. J. Med. Chem. 1994, 37, 1214.
18. Gund, T. M.; Floyd, J.; Jung, D. J. Mol. Graph. Model. 2004, 22, 221.
19. Gitto, R.; De Luca, L.; Ferro, S.; Citraro, R.; De Sarro, G.; Costa, L.; Ciranna, L.;
Chimirri, A. Bioorg. Med. Chem. 2009, 17, 1640.
20. Gitto, R.; De Luca, L.; Ferro, S.; Occhiuto, F.; Samperi, S.; De Sarro, G.; Russo, E.;
Ciranna, L.; Costa, L.; Chimirri, A. ChemMedChem 2008, 3, 1539.
21. Gitto, R.; De Luca, L.; Ferro, S.; Buemi, M. R.; Russo, E.; De Sarro, G.; Costa, L.;
Ciranna, L.; Prezzavento, O.; Arena, E.; Ronsisvalle, S.; Bruno, G.; Chimirri, A. J.
Med. Chem. 2011, 54, 8702.
22. Mott, D. D.; Doherty, J. J.; Zhang, S.; Washburn, M. S.; Fendley, M. J.;
Lyuboslavsky, P.; Traynelis, S. F.; Dingledine, R. Nat. Neurosci. 1998, 1, 659.
23. Burger, P. B.; Yuan, H.; Karakas, E.; Geballe, M.; Furukawa, H.; Liotta, D. C.;
Snyder, J. P.; Traynelis, S. F. Mol. Pharmacol. 2012, 82, 344–359.
24. Mutel, V.; Buchy, D.; Klingelschmidt, A.; Messer, J.; Bleuel, Z.; Kemp, J. A.;
Richards, J. G. J. Neurochem. 1998, 70, 2147.
25. Eurofins; Panlab. https://www.eurofinspanlabs.com/Panlabs.
26. Mosley, C. A.; Myers, S. J.; Murray, E. E.; Santangelo, R.; Tahirovic, Y. A.;
Kurtkaya, N.; Mullasseril, P.; Yuan, H.; Lyuboslavsky, P.; Le, P.; Wilson, L. J.;
Yepes, M.; Dingledine, R.; Traynelis, S. F.; Liotta, D. C. Bioorg. Med. Chem. 2009,
17, 6463.
27. Tahirovic, Y. A.; Geballe, M.; Gruszecka-Kowalik, E.; Myers, S. J.; Lyuboslavsky,
P.; Le, P.; French, A.; Irier, H.; Choi, W. B.; Easterling, K.; Yuan, H.; Wilson, L. J.;
Kotloski, R.; McNamara, J. O.; Dingledine, R.; Liotta, D. C.; Traynelis, S. F.;
Snyder, J. P. J. Med. Chem. 2008, 51, 5506.
The radioligand binding assays against NMDA receptor contain-
ing GluN2B-subunit were carried out using [3H]ifenprodil (Custom
Screen by Eurofins Panlab, formerly Ricerca Biosciences, LCC,
USA).25,34 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
sitive sites) for 120 min at 4 °C. Non-specific binding is estimated
lM GBR-12909 to block non-polyamine sen-
in the presence of 10 lM ifenprodil. Membranes are filtered and
washed, the filters are then counted to determine [3H]ifenprodil
specifically bound. Three concentration (in duplicate) of test com-
pounds were used in displacement assay.
r
1 binding assays were performed according to the method re-
ported by DeHaven et al.35 Briefly, guinea pig brain membranes
(500
g protein) were incubated with 3 nM [3H]-(+)-pentazocine
l
(29 Ci/mM; (Kd) was 14 0.3 nM, n = 3) and six concentrations of
tested compounds or sigma ligands (from 10ꢁ5 to 10ꢁ10 M) in
1 ml of 50 mM Tris–HCl (pH 7.4). The reaction was performed for
150 min at 37 °C and terminated by filtering the solution through
Whatman GF/B glass fiber filters which were presoaked for 1 h in
a 0.5% poly(ethylenimine) solution. Filters were washed with ice
cold buffer (2 ꢀ 4 mL). Nonspecific binding was assessed in the
presence of 10
were made according to the following protocol.36 Guinea pig brain
membranes (360
g protein) were incubated with 3 nM [3H]DTG
lM of unlabeled haloperidol. r2 binding assays
28. Tewes, B.; Frehland, B.; Schepmann, D.; Schmidtke, K. U.; Winckler, T.; Wunsch,
B. ChemMedChem 2010, 5, 687.
29. Hashimoto, K.; London, E. D. Eur. J. Pharmacol. 1995, 273, 307.
30. Wang, X.; Li, Y.; Deuther-Conrad, W.; Xie, F.; Chen, X.; Cui, M. C.; Zhang, X. J.;
Zhang, J. M.; Steinbach, J.; Brust, P.; Liu, B. L.; Jia, H. M. Bioorg. Med. Chem. 2013,
21, 215.
31. Cratteri, P.; Romanelli, M. N.; Cruciani, G.; Bonaccini, C.; Melani, F. J. Comput.
Aided Mol. Des. 2004, 18, 361.
32. Ablordeppey, S. Y.; Fischer, J. B.; Law, H.; Glennon, R. A. Bioorg. Med. Chem.
2002, 10, 2759.
l
(53.3 Ci/mM; Kd = 11 0.8 nM; n = 3) and each test compound
(from 10ꢁ5 to 10ꢁ10 M) in 0.5 mL of 50-mM Tris–HCl (pH 8.0) for
120 min at room temperature in the presence of 400 nM (+)-
SKF10,047 to mask r1 sites. Nonspecific binding was evaluated
with DTG (5 lM). Each sample was filtered through Whatman
33. Wheeler, K. T.; Wang, L. M.; Wallen, C. A.; Childers, S. R.; Cline, J. M.; Keng, P. C.;
Mach, R. H. Br. J. Cancer 2000, 82, 1223.
34. Schoemaker, H.; Allen, J.; Langer, S. Z. Eur. J. Pharmacol. 1990, 176, 249.
35. DeHaven-Hudkins, D. L.; Fleissner, L. C.; Ford-Rice, F. Y. Eur. J. Pharmacol. 1992,
227, 371.
GF/B glass fibers filters, which were presoaked for 1 h in a 0.5%
poly(ethylenimine) solution, using a Millipore filter apparatus.
Filters were washed twice with 4 mL of ice-cold buffer. Radioactiv-
ity was counted in 4 mL of ‘Ultima Gold MV’ in a 1414 Winspectral
PerkinElmer Wallac or Beckman LS6500 scintillation counter. Inhi-
36. Mach, R. H.; Smith, C. R.; Childers, S. R. Life Sci. 1995, 57, PL57.