Efficient synthesis of novel glutamate homologues and investigation of their affinity and selectivity profile at ionotropic glutamate receptors

Article abstract of DOI:10.1016/j.bmcl.2014.02.058

A convenient synthesis of four new enantiomerically pure acidic amino acids is reported and their affinity at ionotropic glutamate receptors was determined. The new compounds are higher homologues of glutamic acid in which the molecular complexity has bee

Full text of DOI:10.1016/j.bmcl.2014.02.058

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1980–1982
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of novel glutamate homologues and investigation
of their affinity and selectivity profile at ionotropic glutamate
receptors
a
a
a
b
Andrea Pinto a, , Lucia Tamborini , Federica Mastronardi , Roberta Ettari , Diego Romano ,
⇑
Birgitte Nielsen c, Carlo De Micheli a, Paola Conti a
a Dipartimento di Scienze Farmaceutiche DISFARM, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente (DeFENS), Università degli Studi di Milano, via Mangiagalli 25, 20133 Milano, Italy
c Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen OE, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient synthesis of four new enantiomerically pure acidic amino acids is reported and their affinity
at ionotropic glutamate receptors was determined. The new compounds are higher homologues of
glutamic acid in which the molecular complexity has been increased by introducing an aromatic/
heteroaromatic ring, that is a phenyl or a thiophene ring, that could give additional electronic interactions
with the receptors. The results of the present investigation indicate that the insertion of an aromatic/
heteroaromatic ring into the amino acid skeleton of glutamate higher homologues is well tolerated
and this modification could be exploited to generate a new class of NMDA antagonists.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 27 January 2014
Revised 18 February 2014
Accepted 20 February 2014
Available online 1 March 2014
Keywords:
L
-Glutamic acid
N-Methyl- -aspartate receptor
Semi-preparative chiral HPLC
-Allylglycine
Heck reaction
D
D
L
-Glutamic acid (
L
-Glu, Fig. 1) is the main excitatory neurotrans-
out that the eutomer of the majority of NMDA ligands possesses
the R configuration of the amino acid stereogenic center, at var-
iance with the endogenous ligand ( -Glu).
mitter in the central nervous system (CNS), where it is involved in
the modulation of many physiological processes such as learning,
memory, and synaptic plasticity.1 Once released from the presyn-
a
L
In the present Letter, we exploited a Heck reaction to easily gen-
erate compounds (R)-5 and (R)-6 (Fig. 2), and the corresponding
saturated derivatives (R)-7 and (R)-8, which are characterized by
the R configuration at the amino acid stereocenter and by a six-car-
bon-atom spacer between the proximal and the distal acidic
groups, therefore matching the requirements needed to generate
NMDA antagonists. The aromatic ring, for example a phenyl or a
thiophene ring, can give additional electronic interactions with
the binding pocket, which could strengthen the affinity for the tar-
get receptor. In addition, this moiety could be exploited to further
decorate the molecule, in order to increase the binding affinity.
The key step to obtain the planned compounds is the Heck
aptic neurons into the glutamatergic synaptic cleft, L-Glu activates
two main classes of receptors: G-protein-coupled metabotropic
Glu receptors (mGluRs) and ligand-gated ionotropic Glu receptors
(iGluRs). On the basis of the agonist selectivity, iGluRs have been
named N-methyl-D-aspartic acid (NMDA) receptors, (RS)-2-ami-
no-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)
receptors, and kainic acid (KA) receptors.1–5
For many years now, we have been actively involved in the
search for new selective NMDA antagonists, and we have success-
fully designed a number of ligands, some of which showed prom-
ising neuroprotective activity.6–14 NMDA antagonists are typically
characterized by an increase in the distance between the proximal
and the distal acidic groups of Glu, for example 4–6 carbon atoms.
The simplest example of a Glu higher homologue behaving as a
NMDA antagonist is (R)-aminoadipic acid [(R)-AA, Fig. 1]. The ami-
no acid skeleton may also be incorporated into a cyclic structure to
decrease the conformational freedom (Fig. 1). It is worth pointing
reaction between conveniently protected D-allyglycine (R)-9 and
the appropriate aryl halide, for example methyl 2-iodobenzoate
10 or methyl 4-bromothiophene-3-carboxylate 12. D-allyglycine
is commercially available but it is very expensive (about 700
euro/g). Therefore, we prepared the racemic alkene (RS)-9, with a
conventional procedure, by reacting allyl bromide with diethyl
acetamidomalonate,15,16 and then we developed a suitable method
for its chromatographic resolution with a semi-preparative chiral
HPLC column (Scheme 1). An excellent enantiomeric separation
⇑
Corresponding author. Tel.: +39 02 50319323; fax: +39 02 50319326.
E-mail address: andrea.pinto@unimi.it (A. Pinto).
http://dx.doi.org/10.1016/j.bmcl.2014.02.058
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

A. Pinto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1980–1982
1981
H2N
COOCH3
I
COOCH3
H2N
NHBoc
COOH
NHBoc
a
b
COOH
NH2
+
COOCH3
COOCH3
HOOC
HOOC
9
(R)-
10
11
(R)-
L-Glu
(R)-AA
H3COOC
Br
H3COOC
HOOC
HOOC
N
NHBoc
COOCH3
NH2
COOH
NHBoc
+
N
S
12
COOH
S
O
O
COOCH3
9
(R)-
13
(R)-
(5S,αR)-1
(5R,αR)-2
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, DIPEA, CH3CN,
(b) Tetrakis(triphenylphosphine)-palladium, NaOAc, DMF, 110 °C, 18 h.
l
W, 130 °C, 2 h;
HOOC
HOOC
N
NH2
COOH
NH2
COOH
N
N
Ph
N
Ph
isolated the desired product (R)-13 in only 15% yield. In this case,
the use of Tetrakis(triphenylphosphine)-palladium and sodium
acetate in DMF, under conventional heating at 110 °C for 18 h, pro-
duced intermediate (R)-13 in excellent yield (81%) (Scheme 2). The
use of the microwave heating, even at lower temperature, resulted
in a lower yield and purity.
(5S,αR)-3
(5R,αR)-4
Figure 1. Structures of
L
-Glu, (R)-AA and some NMDA antagonists.
Part of intermediates (R)-11 and (R)-13 were catalytically
hydrogenated in the presence of 5% Pd/C to yield the saturated
derivatives (R)-14 and (R)-15, respectively (Scheme 3). Final amino
acids (R)-5-8 were obtained by alkaline hydrolysis of the ester
functions followed by treatment with a 30% solution of trifluoro-
acetic acid in dichloromethane to remove the N-Boc protective
group (Scheme 3).
The new derivatives were submitted to in vitro assays, evaluat-
ing receptor binding in rat cortical membranes, using radioligands
[3H]CGP39653, [3H]AMPA, and [3H]KA for NMDA, AMPA, and KA
receptors, respectively (Table 1).18-20
COOH
HOOC
COOH
NH2
COOH
NH2
S
(R)-5
(R)-7
(R)-6
(R)-8
COOH
HOOC
S
COOH
NH2
COOH
NH2
Figure 2. Structures of the synthesized compounds.
Three out of the four tested compounds bound to NMDA recep-
tor, with (R)-8 having the best affinity (Ki = 14 lM). Interestingly,
the Ki value of (R)-8 is comparable to that of (R)-AA, our reference
NMDA antagonist. Based on their structure, we interpret these new
(a
= 4.2; Rs = 10.2) was achieved with a column containing tris-
(2-methyl-5-chloro-phenyl)carbamoyl amylose as the stationary
phase, affording (À)-9 as the first fraction and (+)-9 as the second
fraction. The absolute configuration was assigned to each enantio-
mer on the basis of the optical activity, by comparison with that
reported in the literature.17 Intermediates 10 and 12 were easily
obtained, in quantitative yield, by esterification of the correspond-
ing commercially available carboxylic acid with methanol in the
presence of SOCl2.
The Heck reaction was optimized, in terms of catalyst, base,
solvent, temperature and reaction time, for each aryl halide. Using
methyl 2-iodobenzoate 10, the best yield was achieved performing
the reaction in acetonitrile and using palladium acetate as a
catalyst and diisopropylamine (DIPEA) as a base (Scheme 2). A
good yield (75%) was obtained by refluxing the reaction mixture
for 24 h. Interestingly, a similar result can be obtained after only
two hours, by heating the reaction mixture in a microwave reactor
at 110 °C. As expected, we isolated only one stereoisomer to which
we assigned the E configuration by taking into account the high
value of the 1H NMR coupling constant (J = 16.0 Hz) between the
two olefinic protons. Unfortunately, by applying the same reaction
conditions to methyl 4-bromothiophene-3-carboxylate 12, we
COOCH3
COOCH3
a
a
NHBoc
NHBoc
COOCH3
COOCH3
(R)-11
14
(R)-
H3COOC
H3COOC
NHBoc
COOCH3
NHBoc
COOCH3
S
S
15
13
(R)-
(R)-
b,c
b,c
b,c
b,c
11
13
5
6
14
15
7
8
(R)-
(R)-
(R)-
(R)-
(R)-
(R)-
(R)-
(R)-
Scheme 3. Reagents and conditions: (a) H2, Pd/C 5%, MeOH; (b) NaOH 1 N, MeOH;
(c) 30% TFA, CH2Cl2.
Table 1
Receptor binding affinities at native rat iGluRsa
Compound [3H]AMPA IC50
M)
[3H]KAIN IC50
M)
[3H]CGP39653 Ki
M)
(l
(
l
(l
COOMe
(R)-5
>100b
>100b
>100b
>100b
nd
>100b
>100b
>100b
>100b
nd
>100
c
c
c
NHBoc
(R)-6
52 [4.29 0.04]
32 [4.49 0.04]
14 [4.86 0.05]
13
COOEt
9
(R)-7
(−)-(R)-
COOMe
EtOOC
NHAc
(R)-8
ref. 16,17
semi-preparative
(R)-AAd
NHBoc
+
chiral HPLC
COOMe
NHBoc
(RS)-9
Br
a
Data are given as mean mean pKi SEM of three independent experiments.
The compounds tested at 100 lM concentration gave 5–15% displacement of
b
the radioligand.
9
(+)-(S)-
c
Hill coefficients were 0.7 for (R)-6, 1.0 for (R)-7 and 1.1 for (R)-8.
d
Data from Ref. 21, [3H]-CPP was used as radioligand.
Scheme 1. Synthesis of the enantiopure alkenes (À)-(R)-9 and (+)-(S)-9.

1982
A. Pinto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1980–1982
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The outcome of the present investigation indicates that a reduction
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Acknowledgments
The financial support to the present research by the Italian Min-
istry of Education and Research (MIUR—Rome) is acknowledged.
14. Conti, P.; De Amici, M.; Grazioso, G.; Roda, G.; Pinto, A.; Hansen, K. B.; Nielsen,
B.; Madsen, U.; Bräuner-Osborne, H.; Egebjerg, J.; Vestri, V.; Pellegrini-
Giampietro, D. E.; Sibille, P.; Acher, F. C.; De Micheli, C. J. Med. Chem. 2005,
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Supplementary data
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Toma, L.; Micheli, C. J. Med. Chem. 2004, 47, 6740.
Supplementary data associated with this article can be found, in
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058.
17. Collet, S.; Bauchat, P.; Danion-Bougot, R.; Danion, D. Tetrahedron: Asymmetry
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