Indole derivatives as dual-effective agents for the treatment of neurodegenerative diseases: Synthesis, biological evaluation, and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2013.05.044

Several indole derivatives, that were highly potent ligands of GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptor, also demonstrated antioxidant properties in ABTS method. In particular, the 2-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethanone (1) proved to be a dual-effective neuroprotective agent. With the aim to increase the antioxidant properties we added a catechol moiety onto piperidine moiety. The designed hybrid derivative 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-3-yl)-2-oxoethyl] piperidin-4-yl]benzamide (10) was the most effective antioxidant agent (>94.1 ± 0.1% of inhibition at 17 μM) and showed GluN2B/NMDA receptor affinity at low micromolar concentration (IC₅₀ 0.66 μM). By means of computational studies we explored the effect of the presence of this antioxidant fragment during the recognition process to binding pocket.

Full text of DOI:10.1016/j.bmc.2013.05.044

Bioorganic & Medicinal Chemistry 21 (2013) 4575–4580
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Indole derivatives as dual-effective agents for the treatment
of neurodegenerative diseases: Synthesis, biological evaluation,
and molecular modeling studies
b
Maria Rosa Buemi a, Laura De Luca a, Alba Chimirri a, Stefania Ferro a, Rosaria Gitto a, , Julio Alvarez-Builla ,
⇑
Ramon Alajarin b
a Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Viale Annunziata, Messina I-98168, Italy
b Departamento de Química Orgánica I, Universidad de Alcalá, Campus Universitario, 28871-Alcalá de Henares, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Several indole derivatives, that were highly potent ligands of GluN2B-subunit-containing N-methyl-D-
Received 21 March 2013
Revised 13 May 2013
Accepted 17 May 2013
Available online 2 June 2013
aspartate (NMDA) receptor, also demonstrated antioxidant properties in ABTS method. In particular,
the 2-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethanone (1) proved to be a dual-effective
neuroprotective agent. With the aim to increase the antioxidant properties we added a catechol moiety
onto piperidine moiety. The designed hybrid derivative 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-3-
yl)-2-oxoethyl]piperidin-4-yl]benzamide (10) was the most effective antioxidant agent (>94.1 0.1% of
Keywords:
Glutamate
GluN2B/NMDA
Antioxidant
Indole
inhibition at 17
(IC50 0.66 M). By means of computational studies we explored the effect of the presence of this antiox-
idant fragment during the recognition process to binding pocket.
Ó 2013 Elsevier Ltd. All rights reserved.
lM) and showed GluN2B/NMDA receptor affinity at low micromolar concentration
l
Docking
1. Introduction
used as protective agents against excitotoxic injuries.9 It is well-
known that polyphenols such as catechol derivatives are capable
of trapping radical species off the unpaired electron with the for-
mation of new low-reactive radicals and might protect against
neurodegenerative diseases.10–12
Glutamate,1 the most abundant neurotransmitter in the brain,
plays a prominent role in synaptic plasticity, learning and memory
and other cognitive functions.2 Among the glutamate receptors, the
N-methyl-
D
-aspartate (NMDA) subtype containing GluN2B sub-
In the context of discovery of new neuroprotective agents we
previously reported a successful strategy leading to the identifica-
tion of potent GluN2B subtype-selective ligands, containing an in-
dole scaffold,13–16 that were active as neuroprotective agents and
showed binding affinity similar to ifenprodil the prototype of
GluN2B/NMDA ligands. Considering that hydroxyindoles could be
efficient free radical scavengers and antioxidants,17–22 we herein
decided to study the antioxidant profile of the previously synthe-
sized GluN2B ligands showing the highest affinity (1–4, Fig. 1).
Moreover, we developed a hybridization approach incorporating
a catechol moiety to exploit antioxidant property. The evaluation
of binding affinity for displacement of [3H]ifenprodil, as well as
the antioxidant activity were carried out to describe the biological
profile. Finally, docking studies were performed to predict the
binding mode in to the GluN2B pocket.
unit3 has been intensively studied because of its implication in sev-
eral neurological conditions (Parkinson, Alzheimer, Huntington’s
chorea or amyotrophic lateral sclerosis). Excessive activation of
these receptors leads to a number of deleterious consequences,
including impairment of calcium buffering, generation of free rad-
icals, etc.4 This phenomenon is termed glutamate-related excito-
toxicity.5,6 Key mediators of excitotoxic damage are Ca ions
(Ca2+), which under physiological conditions activate a number of
Ca2+-dependent enzymes that influence a wide variety of cellular
components governing a multitude of cellular processes. In the
excitotoxicity, an excessive synaptic release of glutamate can lead
to the dysregulation of Ca2+ homeostasis and the generation of free
radicals.7 In particular the reactive oxygen species (ROS) can exert
multiple damaging reactions to proteins, lipids, carbohydrates, and
nucleic acids, thereby disrupting cellular functions.8 Therefore,
antioxidants which decrease the free radical generation could be
2. Results and discussion
With the aim to maintain the GluN2B binding affinity and im-
prove the antioxidant activity we introduced the 3,4-dihydroxy-
⇑
Corresponding author. Tel.: +39 090 6766413; fax: +39 090 6766402.
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.2013.05.044

4576
M. R. Buemi et al. / Bioorg. Med. Chem. 21 (2013) 4575–4580
X
OBn
OH
O
BnO
HO
N
i
OH
OH
OH
N
R
N
O
O
H
12
11
OH
1, R = 5-OH X = H2 (IC50 = 25 nM)
2, R = 6-OH X = H2 (IC50 = 17 nM)
3, R = 5-OH X = O (IC50 = 2.6 µM)
4, R = 6-OH X = O (IC50 = 22 nM)
ii
ifenprodil
(IC50 = 20 nM)
OBn
OBn
BnO
BnO
iii
H
H
N
N
Figure 1. Chemical structures and binding affinity data of GluN2B/NMDA ligands.
O
N-Boc
O
NH
benzoyl moiety on the piperidine portion and planned the synthe-
sis of the 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-3-yl)-2-
oxoethyl]piperidin-4-yl]benzamide (10). As depicted in Scheme 1,
we employed a new multistep synthetic route to obtain both the
intermediate 8 and the desired derivative 10. Firstly, the commer-
cially available 5-hydroxy-1H-indole (5) was readily converted
into the protected 5-(benzyloxy)-1H-indole (6) by reaction with
benzyl bromide; then the intermediate 6 was treated with chloro-
acetyl chloride to give the 1-[5-(benzyloxy)-1H-indol-3-yl]-2-chlo-
roethanone (7). Successively the intermediate 7 was coupled with
the 3,4-bis(benzyloxy)-N-piperidin-4-yl-benzamide (8) giving the
triple benzyl protected 1H-indol-3-substituted derivative 9. We at-
tempted to accomplish this coupling reaction using different con-
ditions and reagents and the best result was obtained by
refluxing the reaction mixture for 4.5 h in the presence of DIPEA.
Finally, by reaction with boron tribromide, the cleavage of protect-
ing benzyl group was successfully carried out thus giving com-
pound 10 in an overall yield of 13%.
Scheme 2 describes the synthetic pathway employed to prepare
the key reagent 8 bearing antioxidant catecholic portion. Initially,
the hydroxyl groups were protected as benzyl ethers to obtain der-
ivate 3,4-bis(benzyloxy)benzoic acid (12). In turn, by reaction with
4-amino-1-Boc-piperidine the intermediate 12 was converted into
the tert-butyl-4-(3,4-bis(benzyloxy)benzamido)piperidine-1-car-
boxylate (13). Finally, by reaction with TFA the Boc-protecting
group was removed giving key intermediate 8 with good yield.
Moreover, following previously reported procedures14,15 the 2-
(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethanone
8
13
Scheme 2. Reagents and conditions: (i) benzyl bromide, DMF, K2CO3, 60 °C, 20 h;
NaOH 2.3 M, MeOH, reflux 1 h, (yield 30%); (ii) 4-amino-1-Boc-piperidine, DMF,
HOBT, EDC, Et3N, DMAP, rt, 16 h, (yield 55%); (iii) DCM/TFA, K2CO3, 2 M, rt, 1 h,
(yield 81%).
(1), 2-(4-benzylpiperidin-1-yl)-1-(6-hydroxy-1H-indol-3-yl)etha-
none (2), 1-(4-benzylpiperidin-1-yl)-2-(5-hydroxy-1H-indol-3-yl)
ethane-1,2-dione (3), and 1-(4-benzylpiperidin-1-yl)-2-(6-hydro-
xy-1H-indol-3-yl)ethane-1,2-dione (4) have been re-synthesized.
The antioxidant ability of indole derivatives 1–4 and 10 was
determined using the ABTS method (vide infra). This is an electron
transfer-based antioxidant capacity assay23 first reported by Miller
et al.24 and later improved by Re et al.25 The base of this antioxi-
dant assay is the oxidation of 2,20-azino-bis(3-ethylbenzthiazo-
line-6-sulfonic acid) (ABTS) by potassium persulfate to generate a
radical cation ABTS.+, a soluble chromogen that is blue/green in col-
or and can be determined spectrophotometrically at 734 nm. Anti-
oxidants suppress this reaction by ABTS.+ radical scavenging in a
concentration dependent manner, and the color intensity de-
creases proportionally. The antioxidant ability was expressed as
ABTS inhibition percentage26,27 at two different fixed doses
(17 lM and 9 lM) and the results were displayed in Table 1. In
the same system, antioxidant agents such as gallic and caffeic acids
were evaluated as reference compounds. Moreover, 1H-indole, 5-
methoxy-1H-indole, 5-hydroxy-1H-indole, and 6-hydroxy-1H-in-
dole have been assayed to study the influence of hydroxyl substi-
tuent on antioxidant properties of indole nucleus (see Table 1).
As shown in Table 1 compounds 1 and 2 completely scavenged
BnO
ABTS.+ at 17
lM concentration and their antioxidant ability was
HO
i
comparable to that demonstrated by the phenols, gallic acid and
caffeic acid, two well-known antioxidant molecules. The monocar-
bonyl derivatives 1 and 2 resulted to be more antioxidant than
oxalyl-derivatives 3 and 4. In order to explain this different
N
H
N
H
6
5
ii
O
behavior, the antioxidant property at 9 lM concentration of N-for-
OBn
OBn
O
myl-piperidine and 1-(2-oxo-propyl)-piperidine were tested.
N-formyl-piperidine did not show antioxidant activity (9.0 0.3),
whereas 1-(2-oxo-propyl)-piperidine displayed a detectable anti-
oxidant capacity (28.8 0.4). Thus it seems that the presence of a
methylene bridge linking carbonyl and amine group could posi-
tively influence the activity. In fact, it is well-known that tertiary
amines are easily oxidized to N-oxides whereas amides are not.
Furthermore, the data reported in Table 1 suggested, as expected,
that hydroxyl substituent on indole nucleus was essential for anti-
oxidant effect. In fact the 5- or 6-hydroxy-1H-indoles were more
active than unsubstituted or methoxy-substituted-1H-indoles, that
Cl
BnO
+
N
H
HN
N
H
8
7
OR
OR
iii
O
O
N
N
H
RO
N
H
9, R = Bn
showed low activity when tested at 33
47.6%, respectively). The compound with the highest ABTS.+ scav-
enging measured at 9 M concentration (84.3 0.2%) was the
hybrid-derivative 10 bearing the 3,4-dihydroxyphenyl motif. In or-
der to investigate the influence of this antioxidant fragment on the
binding property on GluN2B/NMDA receptor, we evaluated its
lM concentration (25% and
iv
10, R = H
l
Scheme 1. Reagents and conditions: (i) benzyl bromide, tetrabutylammonium
chloride, DCM, NaOH 20%, rt 16 h, (yield 75%); (ii) ClCH2COCl, pyridine, toluene,
60 °C 1.5 h, (yield 50%); (iii) DIPEA, DMF, 80 °C, 4.5 h, (yield 58%); (iv) BBr3 (1.0 M
DCM), rt, 10 h, (yield 60%).

M. R. Buemi et al. / Bioorg. Med. Chem. 21 (2013) 4575–4580
4577
Table 1
ABTS inhibition percentage for compounds 1–4, 10, selected indole derivatives and reference compounds
X
N
O
Y
R 3
NH
R 1
R 2
R1
X
Y
R2,R3
Inhibitiona (% S.E.M)
17
lM
9 lM
1H-Indole
5-Methoxy-1H-indole
5-Hydroxy-1H-indole
6-Hydroxy-1H-indole
1
2
3
4
—
—
—
—
CH2
CH2
CH2
CH2
NHCO
—
—
—
—
H,H
H,H
H,H
H,H
OH,OH
NAb
NAb
NDb
NDc
>92.1 0.2
>93.9 0.3
80.1 0.3
70.7 0.9
>94.1 0.1
>94.1 0.1
71.5 2.2
NAb
NAb
79.8 1.6
83.0 1.1
69.2 0.2
66.5 0.1
57.8 0.1
54.6 1.3
88.3 0.2
61.6 0.8
NDb
5-OH
6-OH
5-OH
6-OH
5-OH
H2
H2
O
O
H2
10
Gallic acid
Caffeic acid
a
b
c
The mean of three assays was used to calculate percentage of inhibition.
NA: not active.
ND: not determined.
ability to displace [3H]ifenprodil in affinity assay.28 Derivative 10
showed IC50 value of 0.66 M displaying higher efficacy than ana-
maintains the crucial polar interaction between protonated nitro-
gen atom of piperidine with residue Q110 of GluN2B. Moreover,
compound 10 makes interactions with hydrophobic portions of
some residues on GluN1 (Y109, L135) and GluN2B (F114, F176,
l
log 3 and lower potency than the other parent compounds 1, 2 and
4 (Fig. 1).14,15
Therefore, we found that indole derivatives 1, 2 and 10 demon-
strated significant antioxidant effects combined with GluN2B-sub-
unit-containing NMDA receptors affinity. Considering that
compound 1 had also demonstrated to reduce NMDA-mediated
current in electrophysiological assay,14 the most relevant finding
of this research work was the identification of a prototype of
dual-effective agent for the treatment of neurodegenerative
diseases.
P177) and a cation-
R115 of GluN1.
p interaction between phenyl group and
In order to explore the impact of catechol on binding recogni-
tion process we performed docking studies on the hybrid-deriva-
tive 10 into ifenprodil binding pocket. It is well known that the
tetrameric NMDA receptors are composed of different subunits
GluN1, GluN2 (GluN2A–D) and GluN3 (GluN3A and B). Each sub-
unit shows a modular architecture consisting of four domains: an
amino-terminal domain (ATD), a clamshell-like ligand binding do-
main (LBD), a transmembrane domain and an intracellular car-
boxy-terminal domain (CTD). The ifenprodil binding pocket is
localized in the amino-terminal domains of GluN1/GluN2B func-
tional dimer.29 Starting from the crystal structure of GluN1/GluN2B
ATD in complex with ifenprodil (1) (PDB: 3QEL), by means of dock-
ing studies we have previously explored the binding poses of in-
dole derivatives into GluN1/GluN2B dimer interface and found
very few differences for the most efficient indoles 1 and 2.15 They
occupied the binding pocket forming specific interactions: (a) the
benzyl fragment was placed in the hydrophobic area consisting
of residues from the GluN1 (Y109 and T110) and GluN2B subunits
(I111 and F114); (b) the piperidine linker was protonated under
physiological pH and established a hydrogen bond with the oxygen
of Q110 (GluN2B); (c) the hydroxy-indole nucleus was placed in to
the pocket consisting of residues R115 and L135 from GluN1 and
Y175, F176, P177 of GluN2B; moreover we found a relevant polar
interaction between indolic hydroxyl substituent and residue
E236 of GluN2B.15,16
Figure 2. Docking pose of compound 10 (yellow) at the GluN1–GluN2B subunit
interface (PDB code 3QEL). 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 figure was prepared in PYMOL software.
The investigation of the binding pose of the new ligand 10 sug-
gested that it occupies the same binding pocket of ifenprodil such
as indoles 1 and 2. In fact, as shown in Figure 2, compound 10

4578
M. R. Buemi et al. / Bioorg. Med. Chem. 21 (2013) 4575–4580
But compound 10 is positioned upside down with respect to
1 mmol) and 1-[5-(benzyloxy)-1H-indol-3yl]-2-chloroethanone
(7) (299 mg, 1 mmol) was stirred in DMF (13 mL) at 80 °C for 4 h.
The reaction mixture was cooled, it was poured into water (5 mL)
and then it was extracted with AcOEt (3 ꢀ 5 mL). The organic phase
was washed with brine (3 ꢀ 3 mL) and dried over MgSO4. After fil-
tering, the solvent was removed under reduced pressure and the
crude compound was purified by column chromatography on silica
gel using DCM/CH3OH 94:6 as eluent. Yield 58%. Mp 198–199 °C. 1H
NMR (CDCl3), (d) 1.23–3.95 (m, 9H), 3.62 (s, 2H, CH2N), 5.10 (s, 2H,
CH2O) 5.15 (s, 2H, CH2O), 5.16 (s, 2H, CH2O), 5.95–5.99 (br s, 1H,
NH), 6.85–8.09 (m, 22H, ArH), 9.02 (br s, 1H, NH). 13C NMR (CDCl3),
166.3, 155.7, 151.5, 148.6, 137.2, 136.8, 136.6, 132.0, 130.9, 128.5,
127.9, 127.8, 127.6, 127.4, 127.1, 126.4, 120.1, 114.9, 114.0, 113.5,
112.3, 104.8, 71.3, 70.9, 70.4, 52.8, 31.6, 29.7. Anal. C43H41N3O5 (C,
H, N). HPLC-GS 98.1%. MS m/z 680 (M+1).
analogues 1 and 2. As consequence of this different orientation,
the residue E236 interacts with catecholic fragment of compound
10 rather than hydroxyl substituent on indole ring of ligands 1
and 2. In turn the 5-hydroxyl substituent of compound 10 group
makes an unexpected polar contact with residue T110 located in
the hydrophobic area. Considering that it has been reported that
the presence of hydroxyl substituted aromatic rings interacting
with the hydrophobic area could determine a reduction of binding
affinity, we can hypothesize30,31 that the different binding pose of
compound 10 could contribute to the decrease of its binding affin-
ity (IC50 value of 0.66 lM) when compared with parent compounds
1 and 2 (0.025 and 0.017 lM, respectively).
3. Conclusion
4.1.3. Synthesis of 3,4-dihydroxy-N-[1-[2-(5-hydroxy-1H-indol-
3-yl)-2-oxoethyl]piperidin-4-yl]benzamide (10)
In conclusion, the present work evidenced that compound 1
shows antioxidant effect mixed with relevant binding affinity
and antagonistic properties toward NMDA-mediated current in
functional assay. So we suggest that it could be considered a pro-
totype of ‘dual-effective’ neuroprotective agent. As expected we
also found that the presence of a catechol moiety on compound
10 positively influences the antioxidant properties, but deter-
mines the reduction of binding affinity toward GluN2B/NMDA
receptor.
Compound 9 (679 mg, 1 mmol) was dissolved in DCM (5 mL)
and BBr3 (1 M in DCM) (30 mL, 30 mmol) was added under an ar-
gon atmosphere and stirred overnight at room temperature. Then,
the reaction mixture was carefully quenched with CH3OH (7 mL) to
0 °C and the solvent removed under reduced pressure. The residue
was dissolved in AcOEt (10 mL) and consecutively washed with
H2O (3 ꢀ 10 mL) and satd. NaHCO3 (2 ꢀ 10 mL). The organic layer
was dried over Na2SO4 and the solvent was removed under re-
duced pressure. The crude compound was purified by column
chromatography on silica gel using DCM/CH3OH 90:10 as eluent.
Then the final compound was crystallized from CH3OH. Yield
60%. Mp 198–200 °C. 1H NMR (CD3OD3), (d) 1.77–3.87 (m, 9H),
3.87 (s, 2H, CH2N), 6.76–6.81 (m, 2H, ArH), 7.20–7.29 (m, 2H,
ArH), 7.70 (s, 1H, ArH), 7.90 (s, 1H, ArH), 8.20 (s, 1H, H-2). Anal.
4. Experimental section
4.1. Chemistry
All starting materials and reagents are commercially available.
Melting points were determined with a GALLENKAMP apparatus
and are uncorrected. 1H NMR and 13C NMR spectra were recorded
at 200 MHz and 50 MHz, respectively, on a Varian Gemini 200.
Chemical shifts are given in ppm relative to solvent. TLC was car-
ried out on Alugram Sil G/UV254 silica gel plates. Preparative
gravity column chromatography was performed on Merck silica
gel. Analyses indicated by the symbols of the elements were
within 0.4% of theoretical values. Purity of the hybrids and
new compounds was determined by HPLC, which indicated a pur-
ity >98% for each product. Mass spectra were recorded on a Hew-
lett-Packard 5988A or a Hewlett-Packard 1100MSD or an Agilent
6210 LC/MS TOF mass spectrometers.
C22H23N3O5 (C, H, N). HPLC: 98.2%. MS m/z 410 (M+1).
4.1.4. Synthesis of 3,4-bis(benzyloxy)benzoic acid (12)
Compound 12 was prepared according a procedure reported by
Tranchimand et al.33 and spectral data were in accordance with lit-
erature. Briefly, benzyl bromide (14 mL, 117 mmol) and K2CO3
(10.7 g, 78 mmol) were added over a solution of 3,4-dihydroxyben-
zoic acid (11) (2.00 g, 13 mmol) in dry DMF (30 mL) under an argon
atmosphere and heated to 60 °C for 20 h. The reaction mixture was
cooled to room temperature, filtered and the solvent was removed
under reduced pressure. The residue was dissolved in AcOEt
(10 mL) and washed with 1 M HCl (3 ꢀ 5 mL) and brine
(5 ꢀ 5 mL). The combined organic extracts were dried over MgSO4,
filtered and the solvent was removed under reduced pressure. The
residue was dissolved in CH3OH (38 mL), and treated with 2.3 M
NaOH (14 mL) and the mixture was heated under reflux for 1 h.
Then, solvents were evaporated under reduced pressure and the
residue was treated with H2O (5 mL) and then extracted with
Et2O (3 ꢀ 6 mL). The aqueous phase was made acidic (pH 1–2)
with 1 M HCl and extracted with AcOEt (3 ꢀ 5 mL). The organic
extracts were dried over MgSO4, filtered and the solvent was re-
moved under reduced pressure. Mp 187–188 °C as reported in
literature.33
4.1.1. Synthesis of 5-(benzyloxy)-1H-indole (6)
Compound 6 was prepared according to a previously described
procedure32 with slight modifications. Tetrabutylammonium chlo-
ride (138.9 mg, 0.5 mmol) and benzylbromide (0.130 mL,
1.1 mmol) were added to a solution of 5-hydroxy-1H-indole (5)
(133.15, 1 mmol) in DCM (12 mL) and NaOH (20%). The mixture
was stirred for 16 h at room temperature. Then, it was poured into
H2O (5 mL) and extracted with DCM (3 ꢀ 10 mL). The organic layer
was dried over dry Na2SO4 and, after evaporation of the solvent un-
der reduced pressure, the residue was purified by column chroma-
tography on silica gel using hexane/AcOEt 70:30 as eluent. Yield
75%. Mp 96–98 °C. 1H NMR (CDCl3), (d) 5.09 (s, 2H, CH2O), 3.46
(s, 1H, ArH, H-3), 6.91–7.47 (m, 10H, ArH), 8.02 (br s, 1H, NH).
13C NMR (CDCl3), 153.3, 137.6, 131.1, 128.5, 128.2, 127.7, 127.5,
124.8, 113.0, 111.6, 103.9, 102.4, 70.8. Anal. C15H13NO (C, H, N).
HPLC: 98.1%. MS: m/z 224 (M+1).
4.1.5. Synthesis of tert-butyl-4-(3,4-
bis(benzyloxy)benzamido)piperidine-1-carboxylate (13)
3,4-bis(benzyloxy)benzoic acid (12) (50.0 mg, 0.14 mmol) was
dissolved in dry DMF (2 mL), and then treated with 4-amino-1-
Boc-piperidine (34.0 mg, 0.17 mmol), HOBT (22.9 mg, 0.17 mmol),
EDC (0.030 mL, 0.17 mmol), TEA (0.058 mL, 0.42 mmol), and DMAP
(1.2 mg, 0.01 mmol). The reaction mixture was stirred at room
temperature for 16 h under an argon atmosphere and the solvent
was removed. The crude compound was purified by column chro-
matography on silica gel using hexane/AcOEt 40:60 as eluent.
4.1.2. Synthesis of 3,4-bis(benzyloxy)-N-(1-(2-(5-(benzyloxy)-
1H-indol-3-yl)-2-oxoethyl)piperidin-4-yl)benzamide (9)
A mixture of 3,4-bis(benzyloxy)-N-(piperidin-4-yl)benzamide
(8) (416 mg, 1 mmol), N-ethyldiisopropylamine (0.174 mL,

M. R. Buemi et al. / Bioorg. Med. Chem. 21 (2013) 4575–4580
4579
Yield 55%. Mp 158–160 °C. 1H NMR (CDCl3), (d) 1.46–1.58 (m,
2H), 1.50 (s, 9H, CH3), 1.94–1.99 (d, 2H, CH2, J = 10.63), 2.82–2.94
(t, 2H, J = 11.91), 4.02–4.08 (d, 3H, J = 11.90), 5.18 (s, 4H, CH2O)
5.78–5.82 (br s, NH), 6.86–7.46 (m, 13H, ArH). 13C NMR (CDCl3),
166.2, 154.7, 151.6, 148.7, 136.8, 136.6, 128.6, 128.0, 127.6,
127.5, 127.2, 119.9, 114.0, 113.6, 79.7, 71.3, 71.0, 47.2, 42.7, 32.2,
28.5; Anal. C31H36N2O5 (C, H, N). HPLC-GS 100%. MS m/z 517 (M+1).
exactly 20 min. The mean of three assays was used to calculate
percentage inhibition as in Eq. (1).
%I ¼ ðABlank ꢁ ASampleÞ=ABlank
ð1Þ
4.4. Automated molecular docking experiments
The docking methods and settings used in this study were
similar to those described previously.15 The crystal structure of
amino terminal domains of the NMDA receptor subunit GluN1
and GluN2B in complex with ifenprodil (1) was retrieved from
the RCSB Protein Data Bank (PDB: 3QEL).29 Ifenprodil and water
molecules were discarded and the hydrogen atoms were added
to protein by Discovery Studio 2.5.36 The structures of the com-
pounds were constructed using Discovery Studio 2.5.36 The con-
formational behavior of simulated compounds was investigated
by a MonteCarlo procedure (as implemented in the VEGA suite
of programs which generated 1000 conformers by randomly
rotating the rotors).37 All geometries obtained were stored and
optimized to avoid high-energy rotamers. The 1000 conformers
were clustered by similarity to discard redundancies; in this
analysis, two 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 sub-
mitted 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. GoldScore38 was chosen
as a fitness function and the standard default settings were used
in all the calculations and the ligands were submitted to 100 ge-
netic algorithm runs. The ‘allow early termination’ command
was deactivated. Results differing by less than 0.75 Å in ligand-
all atom RMSD, were clustered together. The best ranked
GOLD-calculated conformation was used for analysis and
representation.
4.1.6. Synthesis of 3,4-bis(benzyloxy)-N-(piperidin-4-
yl)benzamide (8)
TFA (0.36 mL) was added dropwise to a solution of tert-butyl-4-
(3,4-bis(benzyloxy)benzamido)piperidine-1-carboxylate 13 (516 mg,
1 mmol) in DCM (1.5 mL) at 0 °C and the reaction mixture was kept
to room temperature for 1 h. Then, the reaction mixture was cooled
to 0 °C, diluted with DCM (1.10 mL) and 2 M K2CO3 (2.37 mL) was
added carefully. The mixture was partitioned and the aqueous
phase was extracted with DCM (3 ꢀ 5 mL). The combined organic
extracts were washed with brine (3 ꢀ 5 mL), dried on MgSO4 and
the solvent was removed. Purification was performed by column
chromatography on silica gel using (DCM/MeOH 80:20).
Yield 81%. Mp 173–175 °C. 1H NMR (CDCl3), (d) 1.23 (br s, 1H,
NH), 1.55–1.97 (m, 3H), 2.77–2.89 (t, 2H, J = 11.48), 3.27–3.21 (d,
2H, J = 12.33), 4.12 (m, 1H) 5.19 (s, 4H, CH2O), 5.90–5.94 (br s,
1H, NH), 6.87–6.91 (d, 1H, ArH), 7.19–7.43 (m, 12H, ArH). 13C
NMR (CDCl3), 166.2, 151.6, 148.7, 136.8, 136.6, 128.5, 128.5,
127.9, 127.4, 127.4, 127.1, 120.0, 113.9, 113.6, 76.6, 71.3, 70.9,
46.2, 44.6, 31.6. Anal. C26H28N2O3 (C, H, N). HPLC-GS 98.3%. MS
m/z 417 (M+1).
Compounds 1–4 and compound 7 were synthesized following a
previously reported procedure and their spectral data were in
accordance with literature.14,15,34
4.2. Receptor binding studies
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).28,35 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
Acknowledgment
[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-
Financial support for this research by University of Messina
(Project number ORME09SPNC) is gratefully acknowledged.
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.
References and notes
1. Gitto, R.; De Luca, L.; De Grazia, S.; Chimirri, A. Curr. Top. Med. Chem. 2012, 12,
971.
2. Gecz, J. Nat. Genet. 2010, 42, 925.
4.3. Antioxidant activity
3. Mayer, M. L. Curr. Opin. Neurobiol. 2011, 21, 283.
4. Farooqui, T.; Farooqui, A. A. Mech. Ageing Dev. 2009, 130, 203.
5. Lau, C. G.; Zukin, R. S. Nat. Rev. Neurosci. 2007, 8, 413.
6. Lipton, S. A. Curr. Drug Targets 2007, 8, 621.
7. Vizi, E. S.; Kisfali, M.; Lorincz, T. Brain. Res. Bull. 2013, 93, 32.
8. Mehta, A.; Prabhakar, M.; Kumar, P.; Deshmukh, R.; Sharma, P. L. Eur. J.
Pharmacol. 2013, 698, 6.
9. Kumar, H.; Lim, H. W.; More, S. V.; Kim, B. W.; Koppula, S.; Kim, I. S.; Choi, D. K.
Int. J. Mol. Sci. 2012, 13, 10478.
10. Bernini, R.; Barontini, M.; Spatafora, C. Molecules 2009, 14, 4669.
11. Huvaere, K.; Olsen, K.; Skibsted, L. H. J. Org. Chem. 2009, 74, 7283.
12. Amorati, R.; Lucarini, M.; Mugnaini, V.; Pedulli, G. F. J. Org. Chem. 2003, 68,
5198.
ABTS diammonium salt and potassium persulfate were pur-
chased from Sigma. Phenols were purchased from Aldrich, Sigma
and Biosynth. UV measurements were performed in a Perkin-Elmer
Lambda 35 spectrophotometer. Methanol spectrophotometric
grade was purchased from Aldrich. ABTS diammonium salt
(96 mg) and potassium persulfate (33 mg) were dissolved in phos-
phate buffer (0.1 M, pH 7.0, 25 mL) and the mixture was allowed to
stand in the dark at room temperature for 16 h before use. The
ABTS solution was centrifuged for 1 h at 24000 rpm and then fil-
13. 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.
14. Gitto, R.; De Luca, L.; Ferro, S.; Citraro, R.; De Sarro, G.; Costa, L.; Ciranna, L.;
Chimirri, A. Bioorg. Med. Chem. 2009, 17, 1640.
15. 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.
tered through a 45 lm nylon membrane. In a UV-cuvette (4 mL)
ABTS.+ radical cation solution, and phosphate buffer were added
and absorbance of blank solution at 734 nm was adjusted (ca.
1.0 A.U.). Solutions (2 mM) of samples were freshly prepared in
methanol/phosphate buffer 1:1 and then used for radical scaveng-
ing assay (28
lL) in triplicate. Measure of inhibition percentage of
16. Gitto, R.; De Luca, L.; Ferro, S.; Buemi, M. R.; Russo, E.; De Sarro, G.; Chisari, M.;
Ciranna, L.; Chimirri, A. J. Med. Chem. 2012, 55, 10532.
the absorbance at 734 nm was taken at room temperature after

4580
M. R. Buemi et al. / Bioorg. Med. Chem. 21 (2013) 4575–4580
17. Suzen, S.; Bozkaya, P.; Coban, T.; Nebiogu, D. J. Enzyme Inhib. Med. Chem. 2006,
21, 405.
18. Mor, M.; Silva, C.; Vacondio, F.; Plazzi, P. V.; Bertoni, S.; Spadoni, G.;
Diamantini, G.; Bedini, A.; Tarzia, G.; Zusso, M.; Franceschini, D.; Giusti, P. J.
Pineal Res. 2004, 36, 95.
19. Herraiz, T.; Galisteo, J. Free Radical Res. 2004, 38, 323.
20. Cano, A.; Alcaraz, O.; Arnao, M. B. Anal. Bioanal. Chem. 2003, 376, 33.
21. Olgen, S.; Coban, T. Biol. Pharm. Bull. 2003, 26, 736.
22. Tan, D. X.; Hardeland, R.; Manchester, L. C.; Poeggeler, B.; Lopez-Burillo, S.;
Mayo, J. C.; Sainz, R. M.; Reiter, R. J. J. Pineal Res. 2003, 34, 249.
23. Huang, D.; Ou, B.; Prior, R. L. J. Agric. Food Chem. 2005, 53, 1841.
24. Miller, N. J.; Rice-Evans, C.; Davies, M. J.; Gopinathan, V.; Milner, A. Clin. Sci.
(Lond) 1993, 84, 407.
28. Eurofins Panlabs Inc., Available from http://www.eurofinspanlabs.com.
29. Karakas, E.; Simorowski, N.; Furukawa, H. Nature 2011, 475, 249.
30. 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.
31. 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.
32. Pasquini, S.; Mugnaini, C.; Brizzi, A.; Ligresti, A.; Di Marzo, V.; Ghiron, C.;
Corelli, F. J. Comb. Chem. 2009, 11, 795.
33. Tranchimand, S.; Tron, T.; Gaudin, C.; Iacazio, G. Synth. Commun. 2006, 36, 587.
34. Deng, W. P.; Nam, G.; Fan, J.; Kirk, K. L. J. Org. Chem. 2003, 68, 2798.
35. Schoemaker, H.; Allen, J.; Langer, S. Z. Eur. J. Pharmacol. 1990, 176, 249.
36. Discovery, Studio, Modeling, Environment, R. 2.5, In Accelrys Software Inc., San
Diego, 2009.
25. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free
Radical Biol. Med. 1999, 26, 1231.
26. Abdel-Wahab, B. F.; Awad, G. E.; Badria, F. A. Eur. J. Med. Chem. 2011, 46, 1505.
27. Siquet, C.; Paiva-Martins, F.; Lima, J. L.; Reis, S.; Borges, F. Free Radical Res. 2006,
40, 433.
37. Pedretti, A.; Villa, L.; Vistoli, G. J. Mol. Graphics Modell. 2002, 21, 47.
38. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.