Synthesis and preliminary biological evaluation at the glycineB site of (+)- and (-)-3-oxetanylglycine, novel non-proteinogenic amino acids

Article abstract of DOI:10.1016/S0014-827X(01)01101-6

Two novel non-proteinogenic amino acids, (+)- and (-)-3-oxetanylglycine were synthesized and evaluated for their ability to diplace [³H]-glycine from the glycine site of the NMDA receptor complex. The lack of activity of these compounds at conc

Full text of DOI:10.1016/S0014-827X(01)01101-6

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 609–613
Synthesis and preliminary biological evaluation at the glycine site
B
of (+)- and (−)-3-oxetanylglycine, novel non-proteinogenic
amino acids
Maria del Carmen Ter a´ n Moldes, Gabriele Costantino, Maura Marinozzi,
Roberto Pellicciari *
Dipartimento di Chimica e Tecnologia del Farmaco, Uni6ersit a` degli Studi di Perugia, Via del Liceo 1, 06123 Perugia, Italy
Received 31 October 2000; accepted 5 February 2001
Abstract
Two novel non-proteinogenic amino acids, (+)- and (−)-3-oxetanylglycine were synthesized and evaluated for their ability to
3
diplace [ H]-glycine from the glycine site of the NMDA receptor complex. The lack of activity of these compounds at
concentrations up to 100 mM may help in understanding the topological requirements of the glycine site of the NMDA receptor
complex © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Novel non-proteinogenic amino acids; Glycine site; NMDA receptor complex
1. Introduction
receptor functions, thus avoiding many of the side-ef-
fects associated with competitive NMDA antagonists.
The simplest natural amino acid glycine (1, Chart 1)
plays a fundamental role in the mammalian central
nervous system, interacting with either inhibitory
(
strychnine-sensitive) or excitatory (strychnine-insensi-
tive) receptors. More than a decade has elapsed since
the first connection between the strychnine-insensitive
glycine sites and the NMDA receptor complex [1], and
glycine (1) is now recognized to be essential for NMDA
activation and is considered a co-agonist of glutamate.
The considerable interest in the modulation of the
glycine site of the NMDA receptor complex (glycineB
site, according to the most recent nomenclature) is
motivated by the intriguing possibility of achieving
pharmacological control of the fundamental functions
mediated by this class of ionotropic receptors [2].
Central to the development of glycine site modula-
B
tors is the notion that a partial blockage of the glycineB
site is insufficient to completely switch-off the NMDA
*
Corresponding author.
E-mail address: rp@unipg.it (R. Pellicciari).
Chart 1.
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01101-6

610
M. del Carmen Ter a´ n Moldes et al. / Il Farmaco 56 (2001) 609–613
(
R)-HA-966 (6) [6] or -687,414 (7) [7] which have been
L
under clinical trials as cognitive enhancers but now
abandoned.
Minor chemical modifications have been shown to
dramatically alter the binding and the functional prop-
erties of glycine analogs, thus making the understand-
ing of the topological requirements of the glycine
agonist site of the NMDA receptor an intriguing prob-
lem. In 1991, Kozikoski et al. reported the synthesis
and the biological evaluation of a series of four-mem-
bered ring amino acids as rigid glycine or glycine-
bioisoster analogs (8–10) [8]. Despite their structural
similarity with known glycine partial agonists, the
above derivatives showed no effect at the glycine site.
B
Aiming at further elucidating the topology of the
glycine agonist site of the NMDA receptor and in the
frame of a research project devoted to the design and
synthesis of non proteinogenic amino acids, we report
here the synthesis of (+) and (−)-3-oxetanylglycine
(
11a and 11b, respectively), a cyclic derivative of ho-
moserine endowed with a size-limited side chain and
with a hydrogen bond accepting atom. The incorpora-
tion of the oxygen atom into a strained four membered
ring may allow to mimic the disposition of the polar
Scheme 1. (a) Z-a-Phosphonoglycine, trimethyl ester, TMG. (b) H2,
Pearlman’s catalyst. (c) (1) -(+)-Tartrate/ -(−)-tartrate, PhCHO;
2) NaHCO . (d) NaOH.
L
D
(
3
atom in the
D
-serine, although compounds 11a and 11b
are formally analogs of the inactive homoserine.
On the other hand, the NMDA receptor complex is
involved in many aspects of neuronal plasticity and in
the long term potentiation (LTP) of the synaptic activ-
2
. Results and discussion
ity, so that administration of glycine agonists or par-
B
tial agonists may results in improved brain functions,
such as memory, learning and information storage,
without reaching those levels of NMDA activation that
result in excitotoxicity [3].
Enantiomerically pure (+) and (−)-3-oxetanyl-
glycine were prepared according to Scheme 1 starting
from the known 3-oxetanone (12) [8].
A Wittig–Horner type reaction [9] involving the con-
densation of the heterocyle ketone 12 with Z-a-phos-
phonoglycine-trimethyl ester [10] in the presence of
tetramethylguanidine (THF, −78°C) afforded the pro-
tected didehydro-a-amino ester 13 in 32% isolated
yield. Catalytic hydrogenation (Pearlman’s catalyst,
MeOH–CH Cl , r.t., 1 atm) of 13 gave the correspond-
A great deal of work has been done over the last
years on glycine antagonists, starting from the endoge-
B
nous glycineB antagonist kynurenic acid. Carboxyte-
trahydroquinolines, hydroxyquinolones, quinoxaline-
diones and carboxy-indole have provided a valuable
source of extremely potent glycine antagonists, many
B
2
2
of which are featured with physico-chemical parameters
suitable for in vivo testing. Currently, carboxy-indoles
derivatives are under clinical trials for stroke [4].
ing racemic 3-oxetanylglycine methyl ester (14) with
7% yield. Asymmetric resolution of the chiral center
was achieved through the initial reaction with
7
L
-
Glycine agonists and partial agonists have been pro-
B
(+)tartaric acid followed by treatment with benzalde-
posed as potential cognitive enhancers and a wide
variety of simple amino acid derivatives have been
evaluated in a number of pharmacological models.
hyde to give, after 3 days, a colorless solid constituted
by (+)-3-oxetanlglycine metyl ester-L-hemitartrate
(20% yield), readily deprotected (sat. NaHCO ) to (+)-
3
D
-serine (2) and
D
-alanine (3) are among the most
3-oxetanylglycine methyl ester (15, 73% yield). Alkaline
potent glycineB agonists, together with the cyclic ho-
hydrolysis (0.3 N NaOH, H O–MeOH, r.t.) of the ester
2
mologous 1-aminocyclopropanecarboxylic acid (ACPC,
15, followed by ion exchange chromatography on
Dowex 50X2-200 resin and elution with 10% pyridine
afforded (+)-3-oxetanylglycine (11a) in 50% yield.
Analogously, treatment of the racemic methyl ester 14
4). An increase in ring size usually decreases both
affinity and efficacy, resulting in partial agonism. -Cy-
D
closerine (5), a widely used antitubercolotic, is endowed
with activity at the NMDA receptors and is the proto-
with
benzaldehyde afforded, after 5 days, the corresponding
(−)-3-oxetanylglycine methyl ester- -hemitartrate
D-(−)-tartaric acid followed by reaction with
type of a glycine partial agonist [5]. Manipulation of
B
D
-cycloserine has resulted in other derivatives, such as
D

M. del Carmen Ter a´ n Moldes et al. / Il Farmaco 56 (2001) 609–613
611
(
(
52% yield) which was deprotected (sat. NaHCO ) to
−)-3-oxetanylglycine methyl ester (16, 91% yield). Al-
3.1. N-Benzyloxycarbonyl-h,i-didehydro-(3-oxetanyl)-
glycine, methyl ester (13)
3
kaline hydrolysis (0.3 N NaOH, H O–MeOH, r.t.) of
2
the ester 16, followed by ion exchange chromatography
on Dowex 50X2-200 resin and elution with 10% pyri-
dine afforded (−)-3-oxetanylglycine (11b) in 54% yield.
The absolute configuration of compounds 11a and
Tetramethylguanidine (0.63 ml, 5.00 mmol) was
added to a magnetically stirred solution of N-benzy-
loxycarbonyl-(phosphonoglycine-trimethyl ester (1.72 g,
5.20 mmol) in dry THF (8 ml) at −70°C in a nitrogen
atmosphere. Stirring was continued for 1 h after which
3-oxetanone (12, 0.360 g, 5.00 mmol) was added and
the resulting mixture was maintained under stirring at
room temperature overnight. The reaction mixture was
then diluted with AcOEt (10 ml), filtered and the solid
residue was carefully washed with AcOEt (60 ml). The
combined organic phases were washed with 1% citric
acid (2×20 ml), brine (20 ml), and dried over Na SO .
11b remains to be determined and will be communi-
cated in the due course.
Compounds 11a and 11b were preliminarily evalu-
3
ated for their capacity to displace [ H]-glycine from rat
cortical membranes. Briefly, synaptosomal membranes
from rat cerebral cortex were washed five times with
pH 7.4 buffer and brine and were preincubated with
3
[
H]-glycine 20 nM. Non-specific binding was evaluated
2
4
by adding glycine (1) 1 mM. Neither compound 11a
nor 11b showed significant binding at concentrations up
to 100 mM.
Evaporation of the solvent yielded an oily residue (0.6
g) which was submitted to flash chromatography: elu-
tion with light petroleum–AcOEt 8:2 afforded 13 as a
1
The complete lack of activity of the two oxetanyl-
glycine derivatives 11a and 11b disproves our working
hypothesis on the possibility that the four-membered
ring could orient the endocyclic oxygen in such a way
to productively interact with hydrogen bonding donor
site(s) of the receptor. Alternatively, the strain intro-
duced by the small ring may unfavorably alter the
electronic character of the oxygen atom, thus prevent-
ing it from interact with the receptor.
colorless solid (0.440 g, 32%), mp 121–123°C; H NMR
(CDCl ) l 3.75 (3H, s, CH O), 5.10 (2H, s, PhCH ),
3
3
2
5.32–5.45 (4H, m, 2×CH ), 6.80 (1H, br s, NH), 7.33
2
13
(5H, s, aromatic’s); C NMR (CDCl ) l 52.53, 67.32,
3
78.53, 115.84, 128.12, 128.31, 128.51, 135.65, 139.87,
152.66, 163.48.
3.2. (9)-3-Oxetanylglycine, methyl ester (14)
Despite their lack of activity at the glycine site of the
NMDA receptor complex, derivatives 11a and 11b may
serve as novel building block for peptidomimetics. In-
deed, the need of reducing many of the limitations
associated with the therapeutic use of peptides is moti-
vating a search of suitable non-proteinogenic replace-
ments for coded amino acids [11,12]. In light of their
peculiar steric characteristic such as a size limited ring
and a reduced conformational mobility, as well as the
presence of a hydrogen acceptor atom, amino acids 11a
and 11b may be considered as potential surrogate for
polar, coded amino acids such as threonine, serine, and
asparagine [13].
Hydrogen was bubbled into a suspension of Pearl-
man’s catalyst (0.020 g) and 13 (0.190 g, 0.68 mmol) in
CH Cl –MeOH (30 ml, 1:1) at room temperature for 2
h under vigorous magnetic stirring. After filtration and
evaporation of the solvent, the residue (0.15 g) was
submitted to flash chromatography: elution with
2
2
CHCl –MeOH (95:5) gave 14 as a pale yellow oil
3
1
(0.076 g, 77%); H NMR (CDCl ) l 1.65 (2H, s, NH ),
3
2
3.05–3.25 (1H, m, CH CH), 3.72 (3H, s, CH ), 3.75
2
3
13
(1H, d, CHNH ), 4.55–4.85 (4H, m, 2xCH ); C NMR
2
2
(CDCl ) l 38.76, 51.97, 56.36, 74.04, 74.24, 174.60.
3
3.3. (+)-3-Oxetanylglycine, methyl ester (15)
3. Experimental
A solution of -(+)-tartaric acid (0.497 g, 3.31
L
Melting points were determined by the capillary
mmol) in dry EtOH (8 ml) was added to a magnetically
1
method on a B u¨ chi 535 electrothermal apparatus. H
stirred solution of 14 (0.480 g, 3.31 mmol) in dry Et O
2
1
3
NMR and C NMR spectra were taken on a Brucker
(10 ml). The white precipitate readily formed was then
AC 200 spectrometer as solutions in CDCl , unless
washed with EtOH (20 ml), Et O (20 ml) and dried
3
2
otherwise indicated. Proton chemical shifts are reported
under vacuum to give (9)-3-oxetanylglycine, methyl
in p.p.m. downfield from tetramethylsilane, except with
ester-L-hemitartrate (0.5 g). This compound was then
D O which was also used as internal standard. Carbon
suspended in dry EtOH (5 ml) containing benzaldehyde
(0.35 ml, 0.33 mmol) and refluxed for 5 min. The
resulting clear solution was allowed to cool to room
temperature and was magnetically stirred for 3 days. A
colorless solid was then collected by filtration and
washed with EtOH (20 ml) to give (+)-3-oxetanyl-
2
chemical shifts are reported in ppm using MeOH as
internal standard. (l 49.0). Flash chromatography was
performed on Merck silica gel (0.040–0.063 mm). Spe-
cific rotations were recorded on a Jasco Dip-360 digital
polarimeter.

612
M. del Carmen Ter a´ n Moldes et al. / Il Farmaco 56 (2001) 609–613
glycine, methyl ester-
Treatment of the tartrate salt with saturated NaHCO3
10 ml) was followed by extraction with AcOEt (4×
0 ml); the combined organic phases were dried over
Na SO and the solvent evaporated off to give the
L
-hemitartrate (0.197 g, 20%).
elution with 10% pyridine afforded 11b as a colorless
solid (0.062 g, 54%), mp 158.5–160.5°C; H NMR
1
(
1
(D O) l 3.20–3.50 (1H, m, CH CH), 3.70–3.90 (1H,
2
2
13
d, CHNH ), 4.50–4.80 (4H, m, 2×CH ); C NMR
2
2
2
5
2
4
(D O) l 35.62, 55.97, 74.38, 74.46, 172.46; [h] =
2
D
1
ester 15 (0.070 g, 73%); H NMR (CDCl ) l 1.63 (2H,
3
−17.6 (c 1, H O).
2
s, NH ), 3.15 (1H, m, CH CH), 3.72 (3H, s, CH ),
2
2
3
3
.75 (1H, d, CHNH ), 4.70 (4H, m, 2×CH ).
2
2
3.4. (+)-3-Oxetanylglycine (11a)
Acknowledgements
1
N NaOH (0.5 ml) was added to a solution of 15
We gratefully acknowledge Professor Alessandro
Galli, Universit a` of Firenze, for testing derivatives 11a
and 11b.
(
0.055 g, 0.38 mmol) in MeOH (1 ml) and the result-
ing mixture was magnetically stirred at room tempera-
ture for 4 h. The reaction mixture was neutralized
with 3 N HCl, concentrated under vacuum to reduce
the volume (2 ml) and submitted to ion exchange
chromatography on Dowex 50x2-200 resin: elution
with 10% pyridine afforded 11a as white crystals
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25
(
0.025 g, 50%), m.p. 159–161°C; [h] = +19.8 (c 1,
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2
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etanylglycine, methyl ester-D-hemitartrate (1.068 g,
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1
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[