Enantiopure indolo[2,3-a]quinolizidines: Synthesis and evaluation as NMDA receptor antagonists

Article abstract of DOI:10.3390/molecules21081027

Enantiopure tryptophanol is easily obtained from the reduction of its parent natural amino acid trypthophan (available from the chiral pool), and can be used as chiral auxiliary/inductor to control the stereochemical course of a diastereoselective reaction. Furthermore, enantiopure tryptophanol is useful for the syntheses of natural products or biological active molecules containing the aminoalcohol functionality. In this communication, we report the development of a small library of indolo[2,3-a]quinolizidines and evaluation of their activity as N-Methyl D-Aspartate (NMDA) receptor antagonists. The indolo[2,3-a]quinolizidine scaffold was obtained using the following key steps: (i) a stereoselective cyclocondensation of (S)- or (R)-tryptophanol with appropriate racemic δ-oxoesters; (ii) a stereocontrolled cyclization on the indole nucleus. The synthesized enantiopure indolo[2,3-a]quinolizidines were evaluated as NMDA receptor antagonists and one compound was identified to be 2.9-fold more potent as NMDA receptor blocker than amantadine (used in the clinic for Parkinson's disease). This compound represents a hit compound for the development of novel NMDA receptor antagonists with potential applications in neurodegenerative disorders associated with overactivation of NMDA receptors.

Full text of DOI:10.3390/molecules21081027

molecules
Communication
Enantiopure Indolo[2,3-a]quinolizidines: Synthesis
and Evaluation as NMDA Receptor Antagonists
Nuno A. L. Pereira 1, Francesc X. Sureda 2, Maria Pérez 3, Mercedes Amat 3 and
Maria M. M. Santos 1,
1
*
Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa,
Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; nalpereira@ff.ulisboa.pt
Pharmacology Unit, Faculty of Medicine and Health Sciences, Universitat Rovira i Virgili, C./St. Llorenç 21,
43201 Reus (Tarragona), Spain; francesc.sureda@urv.cat
Laboratory of Organic Chemistry, Faculty of Pharmacy and Institute of Biomedicine (IBUB),
University of Barcelona, Av. Joan XXIII, s/n, 08028 Barcelona, Spain;
2
3
mariaperez@ub.edu (M.P.); amat@ub.edu (M.A.)
*
Correspondence: mariasantos@ff.ulisboa.pt; Tel.: +351-217-946-451; Fax: +351-217-946-470
Academic Editors: Carlo Siciliano and Constantinos M. Athanassopoulos
Received: 31 May 2016; Accepted: 2 August 2016; Published: 6 August 2016
Abstract: Enantiopure tryptophanol is easily obtained from the reduction of its parent natural amino
acid trypthophan (available from the chiral pool), and can be used as chiral auxiliary/inductor
to control the stereochemical course of a diastereoselective reaction. Furthermore, enantiopure
tryptophanol is useful for the syntheses of natural products or biological active molecules containing
the aminoalcohol functionality. In this communication, we report the development of a small library
of indolo[2,3-a]quinolizidines and evaluation of their activity as N-Methyl D-Aspartate (NMDA)
receptor antagonists. The indolo[2,3-a]quinolizidine scaffold was obtained using the following key
steps: (i) a stereoselective cyclocondensation of (S)- or (R)-tryptophanol with appropriate racemic
δ-oxoesters; (ii) a stereocontrolled cyclization on the indole nucleus. The synthesized enantiopure
indolo[2,3-a]quinolizidines were evaluated as NMDA receptor antagonists and one compound was
identified to be 2.9-fold more potent as NMDA receptor blocker than amantadine (used in the clinic
for Parkinson’s disease). This compound represents a hit compound for the development of novel
NMDA receptor antagonists with potential applications in neurodegenerative disorders associated
with overactivation of NMDA receptors.
Keywords: indoloquinolizidines; 1,2-aminoalcohols; tryptophanol; NMDA receptor; antagonists
1. Introduction
Chiral pool synthesis uses chiral natural products by incorporating part of them into the target
structure. As both enantiomers of the desired final product can be potentially generated, compounds
from the chiral pool are extremely valuable and versatile in asymmetric synthesis.
A wide range of enantiopure amino acids, isolated from natural sources, have been used in
academia and pharmaceutical companies as chiral auxiliaries/inductors to synthesize biologically
active enantiopure compounds [
molecules can also be achieved using enantiopure 1,2-aminoalcohols containing a stereogenic centre
(which can be obtained by reduction of the parent natural amino acids) as chiral inductors [ ]. In the last
1]. The asymmetric syntheses of natural products, or biological active
2
few years we have developed several novel bioactive compounds in this area of research starting from
the enantiopure 1,2-aminoalcohols tryptophanol and phenylalaninol. Using this versatile synthetic
approach we have developed libraries of enantiopure phenylalaninol-derived oxazolopyrrolidone
lactams [
3], tryptophanol-derived oxazolopiperidone lactams [4,5], oxazoloisoindolinones [6,7],
and indolizinoindolones [8] designed to act on important therapeutic targets (Scheme 1).
Molecules 2016, 21, 1027; doi:10.3390/molecules21081027
www.mdpi.com/journal/molecules

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Scheme 1. Synthesis of biological active small molecules starting from enantiopure 1,2-aminoalcohols
(tryptophanol and phenylalaninol).
One area of research that we are particularly interested is in the development of small molecules
that control exacerbated N-methyl-D-aspartate (NMDA) receptor activity. This area of research
is incredibly challenging, as these ligand-gated ion channels exhibit a complex pharmacology
and molecular architecture, which renders most antagonists unsuitable for therapeutic use [9].
Besides playing a crucial role in the biochemical cascade signaling behind the development of
neurodegenerative disorders such as Alzheimer’s and Parkison’s diseases, NMDA receptors are also
extremely important in sustaining healthy memory, learning and cognition processes [10]. Therefore,
compounds that can suppress NMDA receptor activity during glutamate-induced excitotoxicity
episodes, but leave its normal physiological role unharmed, are of great interest [11]. These constraints
are quite difficult to overcome and to date only a handful of clinically tolerated molecules exist, such as
amantadine (1) and memantine (2) (Figure 1) [12,13].
In the last few years, starting from chiral 1,2-aminoalcohols, we have developed libraries of
novel enantiopure bicyclic lactams which were screened for activity as NMDA receptor antagonists.
From our previous screenings, two hit compounds (3 and 4) were identified to block NMDA receptor
activity, presenting IC50 values of around 60 µM (Figure 1) [3,4].
Figure 1. Chemical structure of N-methyl-D-aspartate receptor antagonists and natural products
hirsutine, hirsuteine, and geissoschizine methyl ether.
Following this work, we were interested in studying the indolo[2,3-a]quinolizidine scaffold
obtained by cyclization of tryptophanol-derived oxazolopiperidones (Figure 2). This interest
resulted from reports that several indolo[2,3-a]quinolizidine natural products protect neurons from
NMDAR-mediated death. In fact, some alternatives for the treatment of mental dementia-associated
symptoms come from long-used traditional medicines such as extracts from Uncaria plant species.
These were found to contain biologically active compounds protecting neurons from NMDAR-mediated
death [14]. In particular, the indolo[2,3-a]quinolizidines hirsutine and hirsuteine (Figure 1), present
in choto-san and yokukansan extracts, reduce NMDAR-mediated Ca2+ neural overload [15
,16].
Another indolo[2,3-a]quinolizidine present in these plant extracts is geissoschizine methyl ether,
which besides presenting neuroprotective activity similar to that observed with hirsutine and hirsuteine
is able to cross the blood-brain barrier by oral administration [17]. Although some of the fundamental

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pharmacological mechanisms through which these natural products exert their biological activity
have been demonstrated [18], to the best of our knowledge, no studies have been performed with the
indolo[2,3-a]quinolizidine nucleus reported herein. In this communication, we present the synthesis of
a series of enantiopure indolo[2,3-a]quinolizidine derivatives, and their evaluation as NMDA receptor
antagonists. In order to perform an initial structure-activity relationship (SAR) study, particularly
directed to understand the structural features for optimal inhibition of NMDA receptor activity, we
synthesized: (i) compounds containing different substituents at the piperidinone ring; (ii) pairs of
enantiomers; (iii) compounds with different ring size of the lactam; (iv) compounds with the indole
nitrogen free and protected (Figure 2).
Figure 2. Compounds to be synthesized.
2. Results and Discussion
2.1. Chemistry
The asymmetric synthesis of indoloquinolizidines was achieved starting from enantiopure
tryptophanol, a methodology used previously by the groups of Allin [19,20] and Amat-Bosch [21–27]
for the synthesis of several indole alkaloids. In this synthetic strategy, tryptophanol is used not only
as the source of chirality, but is also used to incorporate the tryptamine moiety present in the target
alkaloids [28,29].
In order to explore the potential of the indolo[2,3-a]quinolizidine scaffold for the development
of novel NMDA receptor antagonists, we synthesized a series of compounds containing different
substituents (H, Et and CH2CO2Et) around the indolo[2,3-a]quinolizidine skeleton, starting from
enantiopure tryptophanol (Scheme 2).
Scheme 2. Synthesis of indolo[2,3-a]quinolizidines 7a–c, and 8a–b.

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Reaction of (S)-tryptophanol with
δ
-oxoacid derivatives 9a
–
c
afforded bicyclic lactams 5a
,
5b and
5c, along with minor amounts of the respective diastereoisomers at the 8a position, 8,8a positions
and 7,8a positions, respectively. The relative stereochemistry between H-3 and H-8a in the major
lactams 5a
with 1.25M HCl, led to the formation of indolo[2,3-a]quinolizidine 7a as a single diastereoisomer
(Scheme 2) [19 20]. Cyclization of bicyclic lactam 5b by intramolecular -amidoalkylation on the indole
2-position using HCl led to 6,12b-trans indoloquinolizidine 7b 22]. Not surprisingly, the cyclization of
, 5b, and 5c is cis (Scheme 2) [19,22,23]. Stereocontrolled cyclization of bicyclic lactam 5a
,
α
[
bicyclic lactam 5c in the presence of HCl-EtOH caused transesterification of the ester moiety and led to
indoloquinolizidine 7c with very good yield (97%).
Bearing in mind the importance of the absolute stereochemistry of biologically active compounds,
we have also prepared enantiomers 8a
–
b
starting from enantiopure (R)-tryptophanol-derived
-oxoacid derivative 9a afforded bicyclic
oxazolopiperidones 6a . Reaction of (R)-tryptophanol with δ
–
b
lactam 6a and the 8,8a-diastereoisomer 6a’ in 60% yield as a 5:1 mixture of separable diastereoisomers.
Similar results were obtained with racemic oxoester 9b affording enantiopure lactam 6b in 58% yield,
along with minor amounts of the 8,8a-diastereoisomer 6b’ (12%), in a process involving a dynamic
kinetic resolution with epimerization of the stereogenic center
(Scheme 2) [ ]. Cyclization of bicyclic lactams 6a and 6b with 1.25M HCl, led to the formation of
indolo[2,3-a]quinolizidines 8a and 8b, respectively.
α to the aldehyde carbonyl group
4
We then decided to synthesize compounds 11 and 13, N-indole protected derivatives of
indolo[2,3-a]quinolizidines 7a and 8a, respectively, to better understand the structural requisites
for NMDA receptor antagonistic activity. Compound 11 and the enantiomer 13 were obtained in
80% yield via intramolecular amidoalkylation of intermediates 10 and 12, respectively (Scheme 3).
Taking advantage of the N-H and O-H polarized bonds present in the scaffold of our target molecules,
compound 14 was easily synthesized, by reaction of indoloquinolizidine 7b with methylene iodide,
with 74% yield.
Scheme 3. Synthesis of compounds 11, 13, and 14.
Moreover, in order to explore the fused piperidone ring contraction effect on NMDA
receptor antagonism activity, we synthesized compound 16 containing a fused pyrrolidone ring
by intramolecular amidoalkylation of bicyclic lactam 15 (obtained by reaction of (S)-tryptophanol with
methyl 4-oxobutanoate 9d, Scheme 4).

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MeO2C
CHO
9d
OH
O
HCl 1.25M, EtOH
N
N
N
H
OH
O
N
H
N
H
H2N
O
H
H
16
15
(S)-Tryptophanol
Scheme 4. Synthesis of compound 16.
2.2. Biological Activity
The NMDA receptor blocking activity of compounds 7
by measuring the ability of the compounds to inhibit the intracellular calcium increase, induced by
NMDA, in in vitro cultures of cerebellar granule neurons. Addition of NMDA (100 M) in the presence
of glycine (10 M) produced a robust and stable increase in intracellular calcium, which was challenged
a–c, 8a–b, 11, 13, 14 and 16 was evaluated
µ
µ
with the compounds to be tested (Figure 3). Compounds 7a and 8a, without any substituent in the
piperidone ring, were more active than amantadine. By contrast, compounds with an ethyl group
at C-1 (compounds 7b and 8b) or a CH2CO2Et at C-2 (compound 7c) inhibited less than 30% the
NMDA-induced intracellular calcium increase. Except for compound 7a, the compounds derived from
(R)-tryptophanol were more active than the corresponding enantiomers, derived from (S)-tryptophanol
(7b and 11 versus 8b and 13, respectively). Compounds 11 and 13, which have the N-indole protected
with a benzyl group, were less active than the corresponding unprotected indoloquinolizidines 7a and
8a, respectively, suggesting that the indole N-H can be important for the NMDA receptor antagonist
activity or that the benzyl group is too bulky for the binding pocket in the NMDA receptor. The same
result was observed for compound 14, which was less active than the unprotected indoloquinolizidine
7b. Compound 16, with a pyrrolidone ring, lost activity compared with the piperidone 7a counterpart,
which further supports the importance of the piperidone ring for NMDA receptor inhibitory activity,
previously observed with tryptophanol-derived oxazolopiperidone lactams [4].
100
30 μM
100 μM
80
60
40
20
0
AM 7a 7b 7c 8a 8b 11 13 14 16
Figure 3. Inhibitory effect of compounds 7a
(NMDA)-induced intracellular calcium increase in cultured cerebellar granule neurons are shown at
30 M (black) and 100 M (dark grey) of each compound. Amantadine (AM) at 30 M and 100
M due to
–c, 8a–b, 11, 13, 14, 16 on N-Methyl D-Aspartate
µ
µ
µ
µM
was included in the assay as a positive control. Compound 8a was not evaluated at 100
µ
solubility issues.
The IC50 value was determined for the most active compound (compound 7a, Figure 4). The IC50
value obtained for indoloquinolizidine was 30.4 M (Table 1), representing a 2.9-fold increase of
M) and a 2-fold increase of activity compared with
µ
activity compared with amantadine (IC50 = 88.5
µ
tryptophanol-derived lactam 4 (IC50 = 63.4 µM).

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Amantadine
100
80
60
40
20
0
4
7a
-7
-6
-5
-4
log [drug] (M)
Figure 4. Inhibitory effect on NMDA-induced intracellular calcium increase in cultured cerebellar
granule neurons treated with increasing doses of amantadine, 4 and 7a.
Table 1. IC50 values obtained for compounds 4, 7a, and amantadine.
NMDA (100 µM)
Compound
IC50 µM a
4
7a
63.4 ˘ 9.0
30.4 ˘ 2.5
88.5 ˘ 11.8
Amantadine
a
Data are expressed as the mean of three independent experiments.
3. Materials and Methods
3.1. General Information
(L)-tryptophanol [(S)-tryptophanol] was bought from Sigma-Aldrich (Schnelldorf, Bavaria,
Germany). Bicyclic lactams 5a
in the literature. Methyl 4-oxobutanoate (9d) was synthesized using the method described for
the synthesis of methyl 5-oxopentanoate (9a) but starting from -butyrolactone. Evaporation of
–c [19,22] and δ-oxo-esters 9a–c [30,31] were synthesized as described
γ
solvents was accomplished with a rotatory evaporator. Thin-layer chromatography was done on
SiO2 (silica gel 60 F254), and the spots were located by UV. For column chromatography silica gel
1
200–400 mesh was used. H- and 13C-NMR spectra were recorded on a Bruker 400 MHz Ultra-Shield
(Wissembourg, Bas-Rhin, France). 1H- and 13C-NMR chemical shifts are reported as
δ values, in
parts per million (ppm) referenced to the solvent used. Data are reported in the following manner:
chemical shift, multiplicity, coupling constant (J) in hertz (Hz), integrated intensity, and assignment
(when possible). Multiplicities are reported using the following abbreviations: s, singlet; d, doublet;
t, triplet; q, quartet; dd, doublet of doublets; tt, triplet of triplets; m, multiplet. Spectra were assigned
using appropriate COSY, DEPT and HMQC sequences. Microanalysis were performed in a Thermo
ScientificTM FLASH 2000 Series CHNS/O analyser (Waltham, MA, USA) and are within
theoretical values.
˘0.5% of
3.2. Synthesis of (R)-Tryptophanol
LiAlH4 (5 g, 134.64 mmol) was slowly added to a suspension of (D)-tryptophan (5 g, 24.48 mmol)
˝
in THF (200 mL) at 0 C. After 30 min, the mixture was heated at reflux overnight. The resulting mixture
˝
was cooled to 0 C and a saturated aqueous solution of Na2SO4 was added. The suspension was
filtered, and the filtrate extracted with EtOAc (250 mL). The aqueous phase was washed with EtOAc
(3
ˆ
100 mL), and the combined organic phases were dried, and concentrated to give (R)-tryptophanol
(4.58 g, 98%).

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3.3. General Procedure for the Synthesis of Compounds 6a–b, and 15
A solution of (R)- or (S)-tryptophanol (1 equiv), and
γ
-oxo-ester (1.1 equiv) in toluene was
heated at reflux, under Dean-Stark conditions, till consumption of the tryptophanol. The solvent was
removed under reduced pressure and the residue obtained was purified by flash chromatography on
silica gel [4,22].
[(3R,8aR)-3-((1H-Indol-3-yl)methyl)tetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one) (6a). Following the
general procedure, starting from (R)-tryptophanol (1.17 g, 6.15 mmol), toluene (40 mL) and methyl
5-oxopentanoate (9a) (0.88 g, 6.77 mmol). Eluent for flash chromatography: EtOAc/n-Hex (1:1).
20
1
Compound 6a (0.63 g, 76%): rαsD = +35.6˝ (c = 1.9, CH2Cl2); H-NMR spectra was found to be
identical to that obtained for compound 5a [4,19].
[(3R,8S,8aR)]-8-Ethyl-3-(3-indolylmethyl)-5-oxo-2,3,6,7,8,8a-hexahydro-5H-oxazolo[3,2-a]pyridine
(6b).
Following the general procedure, starting from (R)-tryptophanol (0.54 g, 2.84 mmol), toluene
(20 mL) and methyl 4-formylhexanoate (9b) (0.49 g, 3.12 mmol). Eluent for flash chromatography:
20
EtOAc/n-Hex (2:1). Compound 6b (0.54 g, 58%): rαsD
=
´
18.2˝ (c = 2.2, CH2Cl2); 1H-NMR spectra
was found to be identical to that obtained for compound 5b [4,22].
(3S,7aR)-3-((1H-Indol-3-yl)methyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (15). Following the general
procedure, starting from (S)-tryptophanol (1 g, 5.26 mmol), toluene (80 mL) and methyl 4-oxobutanoate
(
9d) (0.67 g, 5.78 mmol). Eluent for flash chromatography: EtOAc/n-Hex (1:1). Compound 15 was
20
obtained after recrystallization in EtOAc (0.45 g, 33%): rαsD = +67.8˝ (c = 1.6, CH2Cl2); 1H-NMR was
found to be identical to that obtained for the enantiomer previously described [
4]; Anal. calcd. for
C15H16N2O2: C 70.29, H 6.31, N 10.93, found: C 70.28, H 6.35, N 10.94.
3.4. General Procedure for the Synthesis of Compounds 10 and 12
To a solution of the starting lactam (0.26 mmol, 1.0 equiv.) in anhydrous DMF (3 mL) was added
NaH 60% (0.39 mmol, 1.5 equiv.). The mixture was allowed to stir for 15 min and then benzyl bromide
(0.05 mL, 1.5 equiv.) was added dropwise. The mixture was stirred at room temperature for 1 h.
Water was added, followed by extraction with EtOAc. The combined organic extracts were washed
with brine, dried, and concentrated. The crude residue was purified by flash chromatography with the
eluent EtOAc/n-hexane (2:1) to give the product as a white solid.
(3S,8aS)-3-[(1-Benzyl-1H-indol-3-yl)methyl]tetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one (10). Following
1
the general procedure, starting from lactam 5a (0.07 g, 0.26 mmol). 10 (0.08 g, 86%): H NMR (CDCl3)
δ
7.69 (d, J = 7.8 Hz, 1H, ar), 7.29 (dd, J = 13.8, 6.5 Hz, 5H, ar), 7.18 (t, J = 7.2 Hz, 1H, ar), 7.11 (dd,
J = 13.3, 6.9 Hz, 2H, ar), 6.93 (s, 1H, H-2-indole), 5.29 (m, 2H, N-CH2), 4.61 (qd, J = 7.8, 3.2 Hz, 1H, H-3),
4.42 (dd, J = 8.9, 4.4 Hz, 1H, H-8a), 4.06 (m, 1H, H-2), 3.68 (m, 1H, H-2), 3.30 (dd, J = 14.3, 3.2 Hz, 1H,
CH2-indole), 3.06 (dd, J = 14.3, 8.5 Hz, 1H, CH2-indole), 2.49 (m, 1H, H-6), 2.30 (m, 1H, H-alkyl, H-6),
2.14 (m, 1H, H-alkyl), 1.84 (m, 1H, H-alkyl), 1.43 (m, 2H, H-alkyl).
(3R,8aR)-3-[(1-Benzyl-1H-indol-3-yl)methyl]tetrahydro-2H-oxazolo[3,2-a]pyridin-5(3H)-one (12). Following
1
the general procedure, starting from lactam 6a (0.09 g, 0.33 mmol). 12 (0.11 g, 92%): H NMR spectra
was found to be identical to that obtained for compound 10.
3.5. General Procedure for the Synthesis of Compounds 7a–c, 8a–b, 11, 13 and 16
In EtOH, 1.25 M HCl was added to the proper starting lactam and the reaction mixture was stirred at
room temperature till consumption of the starting material. The solvent was evaporated and the resulting
mixture was dissolved in EtOAc and washed with saturated aqueous NaHCO3. After extraction with

Molecules 2016, 21, 1027
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EtOAc, the combined organic extracts were washed with H2O, dried, and concentrated to give a precipitate.
The precipitate was washed with cold EtOAc and recrystallized from the adequate solvent.
(6S,12bR)-6-(Hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one (7a). Following
the general procedure, starting from lactam 5a (0.18 g, 0.66 mmol) and 1.25 M HCl in EtOH (2.6 mL).
Reaction time: 24 h. Recrystallized from EtOAc/n-hexane to yield a yellow solid 7a (0.146 g, 80%):
20
˝
rαsD = +143.6 (c = 2.1, MeOH); 1H-NMR spectra was identical to that described previously [20]; Anal.
calcd. C16H18N2O2: C 71.08, H 6.73, N 10.37, found: C 70.91, H 6.81, N 10.26.
(1R,6S,12bR)-1-Ethyl-6-(hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one (7b).
Following the general procedure, starting from lactam 5b (0.2 g, 0.67 mmol) and 1.25 M HCl in
EtOH (2.7 mL). Reaction time: 24 h. Recrystallized from EtOH as a white solid 7b (0.138g, 69%):
1H-NMR was found to be identical to that described previously [22]; Anal. Calcd for C18H22N2O2: C,
72.46; H, 7.43; N, 9.39. Found: C, 72.19; H, 7.39; N, 9.24.
Ethyl 2-((2R,6S,12bR)-6-(hydroxymethyl)-4-oxo-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]-quinolizin-2-yl)acetate
(7c). Following the general procedure and starting from lactam 5c (0.05 g, 0.15 mmol) and 1.25 M HCl
in EtOH (3.5 mL). Reaction time: 24 h. Recrystallized from CHCl3 as a white solid (0.047 g, 97%); mp
˝
˝
109.5 C–112 C; IR (KBr) 3256 (NH), 1730 (C=O, acid), 1618 (C=O, amide) cm´1; 1H-NMR (400 MHz,
CDCl3 with a drop of CD3OD) 7.42 (d, J = 7.8 Hz, 1H, H-ar), 7.29 (d, J = 8.0 Hz, 1H, H-ar), 7.13
δ
(t, J = 7.5 Hz, 1H, H-ar), 7.05 (t, J = 7.4 Hz, 1H, H-ar), 5.43–5.32 (m, 1H, H-6), 4.73 (d, J = 10.6 Hz, 1H,
H-12b), 4.14 (q, J = 7.1 Hz, 2H, CH2CH3), 3.64–3.49 (m, 2H, OCH2), 2.92 (dd, J = 15.9, 4.9 Hz, 1H,
H-7), 2.68 (m, 2H, H-7 & H-alkyl), 2.60 (d, J = 12.6 Hz, 1H, H-alkyl), 2.45 (m, 1H, H-2), 2.31 (m, 2H,
CH2CO2Et), 2.11 (dd, J = 17.3, 12.2 Hz, 1H, H-alkyl), 1.43 (m, 1H, H-alkyl), 1.26 (t, J = 7.1 Hz, 3H,
CH2C
(C-q), 121.92 (CH-ar), 119.36 (CH-ar), 118.10 (CH-ar), 111.03 (CH-ar), 105.88 (C-q), 61.55 (OCH2), 60.87
H2CH3), 50.20 (C-12b), 49.04 (C-6), 39.97 ( H2CO2CH2CH3), 38.38 (CH2-alkyl), 34.72 (CH2-alkyl),
28.17 (C-2), 21.30 (C-7), 14.11 (CH2 H3); Anal. calcd. for: C20H24N2O4 0.75H2O: C 64.92, H 6.69,
N 7.57, found: C 64.62, H 6.73, N 7.41.
H δ 172.16 (C=O), 170.42 (C=O), 136.52 (C-q), 131.47 (C-q), 126.79
3); 13C-NMR (100 MHz, CDCl3)
(C
C
C
¨
(6R,12bS)-6-(Hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one (8a). Following
the general procedure, and starting from lactam 6a (0.1 g, 0.37 mmol) and 1.25 M HCl in EtOH
(3.5 mL). Reaction time: 24 h. Recrystallized from EtOAc/n-hexane as a white solid 8a (0.075 g, 75%):
20
˝
rαsD
=
´
143.3 (c = 1.9, MeOH); 1H-NMR spectra was found to be identical to that obtained for compound
7a; Anal. Calcd. C16H18N2O2¨0.25H2O: C 69.92, H 6.80, N 10.20, found: C 69.80, H 6.62, N 10.01.
(1S,6R,12bS)-1-Ethyl-6-(hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one (8b).
Following the general procedure, and starting from lactam 6b (0.05 g, 0.17 mmol) and 1.25 M HCl in
EtOH (3.5 mL). Reaction time: 20 h. Recrystallized from EtOAc/n-hexane as a white solid 8b (0.046 g,
92%): The 1H-NMR spectra was found to be identical to that to that obtained for compound 7b.
(6S,12bR)-12-Benzyl-6-(hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one
(11).
Following the general procedure, and starting from lactam 10 (0.07 g, 0.19 mmol) and 1.25 M HCl in
EtOH (1.3 mL). Reaction time: 20 h. Recrystallized from EtOAc/n-hexane to yield a white solid 11
1
(0.056 g, 80%): H-NMR was found to be identical to that described in the literature [30]; Anal. calcd.
for: C23H24N2O2¨0.25H2O: C 75.69, H 6.78, N 7.68, found: C 75.65, H 6.50, N 7.71.
(6R,12bS)-12-Benzyl-6-(hydroxymethyl)-1,2,3,6,7,12b-hexahydroindolo[2,3-a]quinolizin-4(12H)-one
(13).
Following the general procedure, and starting from lactam 12 (0.11 g, 0.32 mmol) and 1.25 M HCl
in EtOH (2 mL). Reaction time: 20 h. Recrystallized from EtOAc/n-hexane to yield a white solid 13

Molecules 2016, 21, 1027
9 of 12
(0.092 g, 80%): The 1H-NMR spectra was found to be identical to that to that obtained for compound 11
;
Anal. calcd. for: C23H24N2O2: C 76.64, H 6.71, N 7.77, found: C 76.55, H 6.58, N 7.84.
(5S,11bR)-5-(Hydroxymethyl)-5,6,11,11b-tetrahydro-1H-indolizino[8,7-b]indol-3(2H)-one (16). Following the
general procedure, and starting from lactam 15 (0.14 g, 0.55 mmol) and 1.25 M HCl in EtOH (4 mL).
1
Reaction time: 18 h. Recrystallized from EtOAc to yield a white solid 16 (0.05 g, 36%): H-NMR was
found to be identical to that described in the literature [32]; Anal. calcd. for C15H16N2O2
C 69.56, H 6.36, N 10.93, found: C 69.40, H 6.32, N 10.43.
¨0.15H2O:
3.6. Synthesis of (1R,6S,12bR)-1-Ethyl-6-(methoxymethyl)-12-methyl-1,2,3,6,7,12b-hexahydroindolo
[2,3-a]quinolizin-4(12H)-one (14)
NaH (3 eq., 60% dispersion in mineral oil) was added to a solution of the indoloquinolizidine 7b
(0.11 g, 0.37 mmol) in DMF (5 mL). After stirring for 30 min, MeI (3 eq., 0.08 mL) was added dropwise
whilst maitaining the solution under inert atmosphere and an ice bath. The reaction mixture was
allowed to stir at room temperature for 16 h and after this period was quenched with ice cold deionized
water (20 mL). The mixture was then extracted three times with EtOAc (3
ˆ
20 mL). The gathered
extracts were washed with brine (30 mL) and dried over Na2SO4 before evaporating the solvent to
dryness. The crude compound was then purified by flash chromatography using EtOAc/n-Hex (3:1).
The precipitate was recrystallized from EtOAc/n-Hex. 14 (0.089 g, 74%); mp 141 ˝C–143 ˝C; IR (KBr)
1638 (C=O) cm´1; 1H-NMR (400 MHz, CDCl3)
δ 7.54 (d, J = 8.0 Hz, 1H, ar), 7.32 (d, J = 8.0 Hz, 1H,
ar), 7.28–7.22 (m, 1H, ar), 7.15 (m, 1H, ar), 5.32 (m, 1H, H-6), 4.54 (d, J = 6.6 Hz, 1H, H-12b), 3.70
(s, 3H, NCH3), 3.30–3.16 (m, 4H, OCH3 and CH2OH), 3.03 (dd, J = 9.5, 8.2 Hz, 1H, CH2OH), 2.94
(d, J = 2.8 Hz, 2H), 2.68–2.43 (m, 2H), 2.10–1.89 (m, 2H), 1.72–1.41 (m, 3H), 0.92 (t, J = 7.3 Hz, 3H,
CH2CH δ 172.03 (C=O), 138.15 (C-q), 133.16 (C-q), 127.03 (C-q), 121.84
3); 13C-NMR (100 MHz, CDCl3)
(CH-ar), 119.54 (CH-ar), 118.40 (CH-ar), 109.25 (CH-ar), 108.50(C-q), 71.21 (CH2OH), 58.70 (OCH3),
54.61 (C-12b), 47.64 (C-6), 42.99 (C-1), 31.57 (NCH3), 29.71 (CH2), 25.42 (CH2), 22.91 (CH2), 21.61 (CH2),
11.97 (CH2CH3).
3.7. NMDA Receptor Antagonist Activity
The activity of the synthesized compounds as NMDA receptor antagonists was evaluated
using primary cultures of rat cerebellar neurons. Cultures were prepared from 7–8 day-old Wistar
rats (Charles River, Saint-Germain-sur-l’Arbresle, France). Cerebella were dissected, minced and
trypsinized, and after several sedimentations, cells were plated on poly-lysinized coverslips placed in
24-well plates at a density of 1
Gunma, Japan). After 16–18 h, 10
ˆ
106 cells/mL. Plates were kept at 37 ˝C in a cell incubator (Sanyo,
M cytosine arabinoside (Sigma-Aldrich, USA) was added to
µ
avoid excessive proliferation of astrocytes. Cultures prepared in this manner are ready to be used
in the NMDA receptor activity assays from the 6th to the 10th day in vitro. Activity at the NMDA
receptor was assessed using the calcium-sensitive probe Fura-2 (Molecular Probes-ThermoFisher,
Eugene, OR, USA). After incubation with 6 µM Fura-2 acetoxymethyl ester (Fura-2 AM) for 30–45 min
˝
at 37 C, a coverslip was transferred to a plastic holder that was inserted in a quartz cuvette for
fluorescence measurements. Recordings of Fura-2 fluorescence were performed using a PerkinElmer
LS55 luminiscence spectrometer, both at 340 and 380 nm excitation wavelengths, and at 510 nm of
emission. The rati˝o of F340/F380 (R) is proportional to intracellular calcium. All the measurements
were made at 37 C and under mild stirring. Once the recording was started, NMDA (100 µM,
in the presence of 10 µM glycine) was added to the cuvette. This produced a sustained increase in R,
indicating the activation of the NMDA receptors and that the intracellular calcium concentration was
high. After 400 s, this intracellular calcium increase was challenged with cumulative concentrations of
the compounds under investigation, (from 1
would act as antagonists at the NMDA receptor this would be detected as a decrease in the R value.
Experiments were repeated 3 to 5 times, using different batches of cultures. Amantadine was used as
ˆ
10´7 M up to up to 3
ˆ
10´4 M). If the compounds

Molecules 2016, 21, 1027
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a positive control. When a minimum of 50% of inhibition was reached, the IC50 value was calculated
using non-linear regression with GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).
4. Conclusions
Previous works have described that some indoloquinolizidines alkaloids act as NMDA receptor
antagonists. However, the study of the indoloquinolizidine nucleous for the NMDA receptor
antagonism has not been previously studied. The present work aimed at screening a small series of
enantiopure indoloquinolizidines for their in vitro activity as NMDA receptor blockers. To achieve
this objective, we have synthesized a series of enantiopure indolo[2,3-a]quinolizidines starting from
(S)- or (R)-trypthophanol. The compounds were screened for NMDA receptor antagonistic activity
using cerebellar granule neurons and one compound was identified to be 2.9 times more potent than
the positive control amantadine and 2 times more active than the trypthophanol-derived lactam
4
previously identified by us as NMDA receptor antagonist. Besides its potential applicability in
neurodegenerative diseases where NMDA receptor activity is exacerbated, compound 7a is a promising
starting point for the development of more potent derivatives targeting NMDA receptors.
Acknowledgments: This work was supported by FCT (Fundação para a Ciência e a Tecnologia) through
iMed.ULisboa (Pest-OE/SAU/UI4013/2014), by grant 2009/SGR00853 from the Generalitat de Catalunya
(autonomous government of Catalonia), and the research project PTDC/QUI-QUI/111664/2009. M.M.M.S.
would like to acknowledge FCT, “Programa Operacional Potencial Humano” and the European Social Fund for
the IF Program (IF/00732/2013).
Author Contributions: M.M.M.S. conceived and designed the experiments; M.A. and M.M.M.S. coordinated the
task of synthesis; N.A.L.P., M.P. and F.X.S. performed the experiments; All authors read and approved the final
version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples are not available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).