Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors of recombinant and native N-methyl-d-aspartate receptors

Article abstract of DOI:10.1016/j.neuint.2011.12.020

N-Methyl-d-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neurodegenerative disorders and consequently the development of compounds that modulate the function of these receptors has been the subject of intense interest. We have recently reported that 6-bromocoumarin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-containing NMDARs. In the present study, a series of commercially available and newly synthesized coumarin derivatives have been evaluated in a structure-activity relationship (SAR) study as modulators of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the coumarin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excellent starting points for the development of more potent and GluN2 subunit selective inhibitors, which may have application in the treatment of a range of neurological disorders such as neuropathic pain, epilepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors. UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is therefore a novel template for the development of potent and subunit selective NMDAR potentiators that may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.

Full text of DOI:10.1016/j.neuint.2011.12.020

Neurochemistry International 61 (2012) 593–600
Contents lists available at SciVerse ScienceDirect
Neurochemistry International
journal homepage: www.elsevier.com/locate/nci
Coumarin-3-carboxylic acid derivatives as potentiators and inhibitors
of recombinant and native N-methyl-D-aspartate receptors
Mark W. Irvine a,1, Blaise M. Costa b,1, Arturas Volianskis a, Guangyu Fang a, Laura Ceolin a,
Graham L. Collingridge a, Daniel T. Monaghan b,1, David E. Jane a,
⇑
,1
a MRC Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK
b Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 13 January 2012
N-Methyl-D-aspartate receptors (NMDARs) are known to be involved in a range of neurological and neu-
rodegenerative disorders and consequently the development of compounds that modulate the function
of these receptors has been the subject of intense interest. We have recently reported that 6-bromocouma-
rin-3-carboxylic acid (UBP608) is a negative allosteric modulator with weak selectivity for GluN2A-con-
taining NMDARs. In the present study, a series of commercially available and newly synthesized
coumarin derivatives have been evaluated in a structure–activity relationship (SAR) study as modulators
of recombinant NMDAR activity. The main conclusions from this SAR study were that substituents as large
as iodo were accommodated at the 6-position and that 6,8-dibromo or 6,8-diiodo substitution of the cou-
marin ring enhanced the inhibitory activity at NMDARs. These coumarin derivatives are therefore excel-
lent starting points for the development of more potent and GluN2 subunit selective inhibitors, which
may have application in the treatment of a range of neurological disorders such as neuropathic pain, epi-
lepsy and depression. Surprisingly, 4-methyl substitution of UBP608 to give UBP714, led to conversion of
the inhibitory activity of UBP608 into potentiating activity at recombinant GluN1/GluN2 receptors.
UBP714 also enhanced NMDAR mediated field EPSPs in the CA1 region of the hippocampus. UBP714 is
therefore a novel template for the development of potent and subunit selective NMDAR potentiators that
may have therapeutic applicability in the treatment of patients with cognitive deficits or schizophrenia.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
NMDA receptors
Glutamate
Allosteric modulator
Antagonist
Oocyte
Hippocampus
1. Introduction
ligand-gated ion channels belonging to three families of receptors,
the N-methyl-D-aspartate (NMDA), kainate, and (S)-2-amino-3-hy-
The primary excitatory neurotransmitter of the vertebrate CNS,
-glutamate, generates fast synaptic responses by the activation of
droxy-5-methylisoxazole-4-propionic acid (AMPA) receptors
(Traynelis et al., 2010; Watkins and Jane, 2006). Considerable
interest has focused on NMDA receptors because of their special
role underlying synaptic plasticity (Bliss and Collingridge, 1993),
memory formation and other aspects of CNS function as well as
their central role in various neuropathological and psychiatric con-
ditions. NMDA receptor signaling contributes to the expression of
epilepsy, schizophrenia, drug addiction, mood disorders, post-trau-
matic stress disorder and neuropathic pain (Lindsley et al., 2006;
Sanacora et al., 2008; Wasterlain and Chen, 2008). Excessive NMDA
receptor activation may also be a common mechanism causing
neuronal cell death in stroke, traumatic brain injury and various
neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Hun-
tington’s, amyotrophic lateral sclerosis (ALS), and Creutzfeldt–
Jakob disease (Kalia et al., 2008; Khosravani et al., 2008; Villmann
and Becker, 2007).
L
Abbreviations: AMPA, (S)-2-amino-3-hydroxy-5-methylisoxazole-4-propionic
acid; D-AP5, D-2-amino-5-phosphonopentanoic acid; CPP, (RS)-4-(3-phosphono-
propyl)piperazine-2-carboxylic acid; EPSP, excitatory postsynaptic potential; L-
689,560, trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tet-
rahydroquinoline; NAM, negative allosteric modulator; NBQX, 2,3-dihydroxy-6-
nitro-7-sulphamoyl-benzo(f)quinoxaline; NMDA, N-methyl-D-aspartate; PAM, posi-
tive allosteric modulator; UBP512, 9-iodophenanthrene-3-carboxylic acid; UBP552,
4,7-dibromo-3-hydroxy-2-naphthoic acid; UBP574, 7-bromo-3-hydroxy-2-naph-
thoic acid; UBP608, 6-bromocoumarin-3-carboxylic acid; UBP649, coumarin-3-
carboxylic acid; UBP651, 6,8-dibromocoumarin-3-carboxylic acid; UBP652, 7-
hydroxycoumarin-3-carboxylic acid; UBP653, 7-methoxycoumarin-3-carboxylic
acid; UBP654, 7-(diethylamino)coumarin-3-carboxylic acid; UBP656, 6-(2-carboxy-
vinyl)coumarin-3-carboxylic acid; UBP657, 6-iodocoumarin-3-carboxylic acid;
UBP658, 6,8-diiodocoumarin-3-carboxylic acid; UBP659, benzo[5,6]coumarin-3-
carboxylic acid; UBP714, 6-bromo-4-methylcoumarin-3-carboxylic acid.
NMDA receptors are heterotetrameric complexes composed of
two glycine-activated GluN1 subunits, of which there are 8 alterna-
⇑
Corresponding author. Tel.: +44 (0)117 3311409; fax: +44 (0)117 3312288.
E-mail address: david.jane@bristol.ac.uk (D.E. Jane).
MI and BMC contributed equally as first authors and DEJ and DTM as senior
1
tively spliced forms, and two
L-glutamate-activated GluN2 subunits
of which there are 4 distinct gene products (GluN2A–GluN2D). In
authors.
0197-0186/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2011.12.020

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M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
limited cases, NMDA receptors may also contain a GluN3 subunit.
The functional heterogeneity of NMDA receptors is largely due to
the presence of different GluN2 subunits. These have different spa-
tial and temporal expression patterns in the brain and have distinct
physiological properties. They differ significantly in their decay
moderate degree of subtype selectivity for GluN2A-containing
NMDA receptors. The purpose of the present study was to evaluate
a series of structurally-related coumarins for their activity at
NMDA receptors. In addition to defining structural features under-
lying inhibitory activity at NMDA receptors, we report a modifica-
tion to the coumarin structure that confers potentiating activity.
times, desensitization, Mg++-sensitivity,
L-glutamate and glycine
affinity, synaptic/extrasynaptic localization, and downstream sig-
naling (Traynelis et al., 2010).
2. Materials and methods
The discovery that NMDA receptors participate in diverse neuro-
pathological and psychiatric conditions led to high expectations for
clinical studies of NMDA receptor antagonists. Unfortunately, the
results from these studies have been largely disappointing due to
adverse effects and limited therapeutic efficacy (Kalia et al., 2008;
Villmann and Becker, 2007). Despite these clinical failures, there re-
mains considerable untapped potential for NMDA receptor thera-
peutic agents. Overall, there are at least three general approaches
that may significantly improve effectiveness, or expand the func-
tional repertoire, of NMDA receptor therapeutic agents. Identify
agents that (1) have greater subtype selectivity, (2) are allosteric
modulators, or (3) can potentiate NMDA receptor responses.
Considerable improvements could be made in terms of subtype
selectivity. To date, the NMDA receptor pharmacological agents that
have been tested in the clinic are either non-selective compounds
that cannot distinguish among NMDA receptor subtypes or com-
pounds that have only one pattern of selectivity (GluN2B-selective).
Thus it has not been possible to selectively block the most therapeu-
tically relevant subpopulation of NMDA receptors while minimizing
adverse effects of blocking other NMDA receptor populations.
Development of allosteric modulators represents another
approach for potentially improving NMDA receptor therapeutics.
NMDA receptor compounds that have been clinically tested have
been NMDA receptor channel blockers, competitive antagonists
2.1. Synthesis of coumarin derivatives
Structures of compounds synthesized and tested for this report
are presented in Fig. 1. 6-Iodocoumarin-3-carboxylic acid
(UBP657), 6,8-diiodocoumarin-3-carboxylic acid (UBP658), benzo
[5,6]coumarin-3-carboxylic acid (UBP659), 6-bromo-4-methyl-
coumarin-3-carboxylic acid (UBP714), and 6-(2-carboxyethen-1-
yl)coumarin-3-carboxylic acid (UBP656) were synthesized as
shown in Fig. 2. With the exception of UBP656, the coumarin deriv-
atives were synthesized via a Knoevenagel condensation reaction
between Meldrum’s acid (1) and the appropriate aldehyde or ke-
tone (Song et al., 2003). Due to its lower reactivity toward nucleo-
philic attack, 5-bromo-2-hydroxyacetophenone (2) was first
reacted with ammonia to form a ketimine intermediate which
was then reacted with 1 to afford UBP714 (Song et al., 2003).
UBP656 was synthesized from ethyl-6-bromocoumarin-3-carbox-
ylate (3) in two steps. Firstly, Heck coupling of 3 with ethyl acrylate
afforded di-ester 4 which was then hydrolyzed using base to yield
UBP656. After synthesis and purification, compound structure
was verified by 1H NMR and mass spectroscopy. All novel com-
pounds had elemental analyses in which the determined percent-
ages for C, H, and N were less than 0.4% different from theoretical
values. 7-Bromo-3-hydroxy-2-naphthoic acid (UBP574) and 4,7-
dibromo-3-hydroxy-2-naphthoic acid (UBP552) were synthesized
according to literature procedure (Murphy et al., 1990). The
remaining compounds were purchased from Sigma–Aldrich
(coumarin-3-carboxylic acid (UBP649), 6-bromocoumarin-3-
carboxylic acid (UBP608), 7-hydroxycoumarin-3-carboxylic acid
(UBP652), 7-methoxycoumarin-3-carboxylic acid (UBP653), 7-
(diethylamino)coumarin-3-carboxylic acid (UBP654)) and Alfa
Aesar (6,8-dibromocoumarin-3-carboxylic acid (UBP651)).
at either the L-glutamate or glycine binding site or ifenprodil-like
ligands that bind at the N-terminal regulatory domain selectively
inhibiting GluN2B-containing receptors (Jane et al., 2000). By
analogy to G-protein coupled receptors, as well as to AMPA and
GABA-A receptors, allosteric modulators can offer therapeutic
advantages in having noncompetitive or uncompetitive activity
(Arai and Kessler, 2007). Negative allosteric modulators (NAMs)
have no intrinsic activity and bind to a different site than
L-glutamate to inhibit receptor function (Conn et al., 2009). There
can be advantages to modulating the gain of receptor signaling
rather than indiscriminately turning the receptor on or off. Based
upon the structural homology to AMPA receptors, NMDA receptors
are likely to be amenable to allosteric modulators that bind in sites
homologous to those by which inhibitors such as GYKI53655
and potentiators such as cyclothiazide bind in AMPA receptors
(Sobolevsky et al., 2009; Sun et al., 2002).
A third area of significant potential for new drug discovery is in
the identification of compounds that potentiate NMDA receptor re-
sponses. Positive allosteric modulators have no intrinsic activity
2.1.1. General condensation procedure
A mixture of the appropriate aldehyde (8.06 mmol), Meldrum’s
acid (1) (1.16 g, 8.06 mmol) and piperidinium acetate (29 mg,
0.2 mmol) in ethanol (25 mL) was stirred at room temperature
for 20 mins and then heated under reflux for 2 h. The reaction mix-
ture was allowed to cool to room temperature before being stirred
at 0 °C for another hour. The solid which precipitated out of solu-
tion was filtered off, washed thoroughly with ethanol and dried
in vacuo to afford the desired product.
and bind to a different site than L-glutamate to increase its potency
2.1.2. 6-Iodocoumarin-3-carboxylic acid (UBP657)
(Conn et al., 2009). To date, no NMDA receptor positive allosteric
modulators (PAMs) have been developed for clinical study, and
yet some clinical indications are predicted to benefit from an
NMDA receptor potentiator rather than an inhibitor (e.g. schizo-
phrenia or cognitive enhancement).
Recently, we and others have identified allosteric modulators
that include both inhibitors and potentiators with improved
subtype-selectivity (Bettini et al., 2010; Costa et al., 2010; Mosley
et al., 2010; Mullasseril et al., 2010). In our study, we identified a
series of naphthoic and phenanthroic acids that displayed positive
and negative allosteric modulatory activity at NMDA receptors
(Costa et al., 2010). Intriguingly, these compounds displayed varied
patterns of subunit selectivity. For example, one related structure
Following the general procedure, 2-hydroxy-5-iodo-benzalde-
hyde (2.00 g, 8.06 mmol) afforded UBP657 as a yellow solid
(1.97 g, 77%); mp: 204–206 °C; 1H NMR (400 MHz, DMSO-d6) d
7.25 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 8.8 and 2.0 Hz, 1H), 8.29 (d,
J = 2.0 Hz, 1H), 8.65 (s, 1H), 13.34 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6) d 88.2, 118.3, 119.2, 120.1, 137.8, 141.9, 146.8, 154.0,
156.0, 163.6; HRMS-CI calcd. for C10H5IO4 [M+H]+ 316.9311; found
316.9303; analysis (calcd., found for C10H5IO4): C (38.00, 38.16), H
(1.59, 1.94).
2.1.3. 6,8-Diiodocoumarin-3-carboxylic acid (UBP658)
Following the general procedure, 3,5-diiodo-2-hydroxybenzal-
dehyde (3.01 g, 8.06 mmol) afforded UBP658 as a light yellow solid
we reported, the coumarin derivative UBP608 displayed
a

M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
595
2
2
Br
CO2H
OH
Br
CO2H
OH
3
CO2H
7
7
3
9
3
I
4
Br
UBP552
UBP574
UBP512
5
4
CO2H
O
Br
Br
CO2H
O
I
CO2H
O
6
3
7
2
O
O
O
8
UBP608
UBP649
UBP657
CO2H
O
I
CO2H
O
CO2H
O
O
O
O
Br
I
UBP658
UBP659
UBP651
HO2C
CO2H
O
Br
CO2H
O
CO2H
O
O
O
HO
O
UBP656
UBP714
UBP652
CO2H
O
CO2H
O
MeO
O
N
O
UBP653
UBP654
Fig. 1. Structures of recently reported NMDAR allosteric modulators and coumarin derivatives.
(1.85 g, 52%); mp: >250 °C (lit. 273–274 °C, Bonsignore et al.,
1999); 1H NMR (400 MHz, DMSO-d6) d 8.26 (d, J = 2.0 Hz, 1H),
8.40 (d, J = 2.0 Hz, 1H), 8.56 (s, 1H), 13.39 (br s, 1H); 13C NMR
(100 MHz, DMSO-d6) d 86.2, 89.2, 119.7, 120.5, 138.0, 146.8,
149.1, 153.5, 155.6, 163.4.
1H); 13C NMR (100 MHz, DMSO-d6) d 15.5, 116.0, 118.2, 122.5,
127.4, 130.8, 132.7, 139.5, 150.6, 158.0, 166.4.
2.1.6. Ethyl 6-(2-ethoxycarbonylethen-1-yl)coumarin-3-carboxylate
(4)
A flame dried flask was charged with ethyl-6-bromocoumarin-
3-carboxylate (3) (1.50 g, 5.05 mmol), palladium acetate
(11.5 mg, 1 mol%), and tri-o-tolylphosphine (62.1 mg, 4 mol%).
The flask was then briefly evacuated and backfilled with argon
three times. Degassed anhydrous dimethylformamide (25 mL)
was then added followed by ethyl acrylate (0.69 mL, 6.31 mmol)
and triethylamine (1.76 mL, 12.62 mmol). The resultant mixture
was heated at 100 °C overnight. After being allowed to cool to
room temperature the reaction mixture was filtered through a cel-
ite pad to remove any precipitated Pd(0) and then poured into a
stirred solution of ethyl acetate (100 mL), water (100 mL) and
aqueous 1 M HCl (10 mL). The organic layer was subsequently iso-
lated and the aqueous phase further extracted with ethyl acetate
(2 Â 30 mL). The organic extracts were pooled, washed with water
(5 Â 100 mL), brine (100 mL) and dried over MgSO4. Concentration
in vacuo afforded a light orange solid which was re-crystallized
from ethanol to afford 4 as an off-white solid (935 mg, 58%); mp:
168–170 °C; 1H NMR (400 MHz, CDCl3) d 1.35 (t, J = 7.2 Hz, 3H),
1.42 (t, J = 7.2 Hz, 3H), 4.29 (q, J = 7.2 Hz, 2H), 4.43 (q, J = 7.2 Hz,
2H), 6.46 (d, J = 16.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.69 (d,
J = 16.0 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.81 (dd, J = 8.4 and
1.6 Hz, 1H), 8.52 (s, 1H); 13C NMR (100 MHz, CDCl3) d 14.2, 14.3,
60.8, 62.2, 117.6, 118.2, 119.2, 119.8, 129.1, 131.5, 133.0, 141.9,
148.0, 155.9, 156.1, 162.8, 166.4; HRMS-CI calcd. for C17H16O6
[M+H]+ 317.1025; found 317.1030; analysis (calcd., found for
2.1.4. Benzo[5,6]coumarin-3-carboxylic acid (UBP659)
Following the general procedure, 2-hydroxy-1-naphthaldehyde
(1.39 g, 8.06 mmol) afforded UBP659 as a yellow solid (1.76 g,
91%); mp: 234–237 °C (lit. 236–237 °C, Song et al., 2003); 1H
NMR (400 MHz, DMSO-d6) d 7.56 (d, J = 8.8 Hz, 1H), 7.63 (t,
J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.27
(d, J = 8.8 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 9.32 (s, 1H), 13.31 (br s,
1H); 13C NMR (100 MHz, DMSO-d6) d 112.0, 116.4, 117.1, 122.2,
126.3, 128.9, 128.9, 128.9, 129.7, 135.7, 143.6, 154.9, 156.7, 164.2.
2.1.5. 6-Bromo-4-methylcoumarin-3-carboxylic acid (UBP714)
A stirring solution of 5-bromo-2-hydroxyacetophenone (2)
(9.95 g, 46 mmol) in methanol (80 mL) was cooled to 0 °C and
ammonia gas bubbled through the solution until it was saturated.
The resultant mixture was allowed to warm to room temperature,
stirred for 18 h, and then concentrated in vacuo. Meldrum’s acid
(7.95 g, 55.2 mmol) and ethanol (150 mL) were added to the resi-
due and the resultant mixture heated under reflux for 5 h. The
solution was then allowed to cool to room temperature during
which time a solid precipitated out of solution. This was filtered
off, washed thoroughly with ethanol, and dried in vacuo to afford
UBP714 as a light yellow solid (7.55 g, 58%); mp: 201–203 °C; 1H
NMR (400 MHz, DMSO-d6) d 2.30 (s, 3H), 7.30 (d, J = 8.8 Hz, 1H),
7.67 (d, J = 8.8 and 2.4 Hz, 1H), 7.85 (d, J = 2.4 Hz, 1H), 13.32 (br s,
C17H16O6): C (64.55, 64.60), H (5.10, 5.35).

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M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
(GRIN1a, GRIN2A, GRIN2C, and GRIN2D) and SP6 (GRIN2B) RNA
polymerase using the mMessage mMachine transcription kits
(Ambion, Austin, TX, USA).
X
CHO
OH
X
CO H
2
O
O
Y
a
Y
2.3. GluN subunit expression and electrophysiology in Xenopus oocytes
X = I, Y = H UBP657
Oocytes from mature female Xenopus laevis (Xenopus One, Ann
Arbor, MI, USA) were removed and isolated using procedures ap-
proved by the University of Nebraska Medical Center’s Institutional
Animal Care and Use Committee in compliance with the National
Institutes of Health guidelines. NMDA receptor subunit RNAs were
dissolved in sterile distilled H2O. GluN1a and GluN2 RNAs were
mixed in a molar ratio of 1:1–3. 50 nl of the final RNA mixture
was microinjected (15–30 ng total) into the oocyte cytoplasm. Oo-
cytes were incubated in ND-96 solution for 1–5 days at 17 °C prior
to electrophysiological assay.
X = I, Y = I UBP658
O
O
O
O
H C
CH
3
3
CHO
1
OH
CO H
2
O
O
a
UBP659
Electrophysiological responses were measured using a standard
two-microelectrode voltage clamp model OC-725B (Warner Instru-
ments, Hamden, CT, USA) designed to provide fast clamp of large
cells. The recording buffer contained 116 mM NaCl, 2 mM KCl,
0.3 mM BaCl2 and 5 mM HEPES, pH 7.4. Response magnitude was
determined by the steady plateau response elicited by bath appli-
CH
O
3
Br
CO H
2
Br
b
CH
3
O
O
OH
2
UBP714
cation of 10
membrane potential of À60 mV. Response amplitudes for the four
heteromeric complexes were generally between 0.1 and 3 A. After
lM L-glutamate plus 10 lM glycine and held at a
l
O
O
O
X
obtaining a steady-state response to agonist application, test com-
pounds were bath applied (Automate Scientific 16-channel perfu-
sion system) and the responses were digitized for analysis
(Digidata 1440A and pClamp-10, Molecular Devices). Dose–re-
sponse relationships were fit to a single-site with variable slope
(GraphPad Prism, ISI Software), using a nonlinear regression to cal-
culate IC50 and % maximal inhibition. All experiments were per-
formed at least 4 times. IC50 and % maximal inhibition values
were compared between drugs using ANOVA followed by a Bonfer-
roni test.
Br
X
c
OEt
O
O
O
3
4
X = CO Et
2
d
X = CO H UBP656
2
Fig. 2. Schemes for the synthesis of coumarin derivatives. (a) Piperidinium acetate,
EtOH, reflux, 2 h then 0 °C, 1 h; (b) (i) MeOH/NH3, 18 h, rt, (ii) 1, EtOH, reflux, 5 h; (c)
ethyl acrylate, P(o-tolyl)3, TEA, Pd(OAc)2, DMF, 100 °C, 18 h; (d) (i) 10% NaOH (aq),
EtOH, reflux, 2 h, (ii) 2 M HCl (aq).
2.4. Electophysiological studies on NMDAR- and AMPAR mediated
EPSPs in the CA1 region of the hippocampus
2.1.7. 6-(2-Carboxyethen-1-yl)coumarin-3-carboxylic acid (UBP656)
To a stirring suspension of 4 (450 mg, 1.42 mmol) in aqueous
10% NaOH (30 mL) was added ethanol (30 mL) to aid dissolution.
The resultant solution was refluxed for 2 h before being allowed
to cool to room temperature. Acidification to pH 1 using aqueous
2 M HCl led to precipitation of a light yellow solid. This suspension
was stirred at 0 °C for 45 mins and then filtered to afford UBP656
as a light yellow solid which was dried over P2O5 (362.5 mg,
98%); mp: >250 °C; 1H NMR (400 MHz, D2O/NaOD, pH 11) d 6.03
(d, J = 16.0 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 16.0 Hz,
1H), 7.25 (dd, J = 8.4 and 2.4 Hz, 1H), 7.28 (s, 1H), 7.57 (d,
J = 2.4 Hz, 1H); 13C NMR (100 MHz, D2O/NaOD, pH 11) d 117.7,
120.5, 120.8, 124.8, 128.8, 129.1, 130.1, 135.2, 142.4, 169.1,
174.0, 176.9, 178.2; MS (ESIÀ) m/z: 259 (M–H, 100); Analysis
(calcd., found for C13H8O6Á1.0H2O): C (56.12, 56.05), H (3.62, 3.24).
Experiments were performed according to national and EU
guidelines for animal care on hippocampal slices from adult male
Wistar rats (272 20 g, mean SD), as described previously
(Volianskis and Jensen, 2003). Briefly, transverse hippocampal
slices (400 lm) were prepared using a McIllwain tissue chopper.
The slices were pre-incubated submerged at room temperature
(ꢀ20 °C) for at least 2 h before starting the experiments. During
the experiments the slices were kept submerged at ꢀ31 °C and
superfused at a rate of 3 ml/min with saline solution (in mM:
124 NaCl, 3.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgSO4
and 10 glucose), which was saturated with 95% O2 and 5% CO2.
AMPAR-mediated field excitatory postsynaptic potentials
(f-EPSPs) were recorded in the CA1-B area of stratum radiatum
using glass electrodes filled with saline solution in response to
electrical stimulation (100 ls duration) of the Schaffer collaterals.
f-EPSPs were evoked at a frequency of 0.067 Hz and the stimula-
tion current was fixed to three times the threshold for evoking
the f-EPSPs. After a stable period of at least 30 min (baseline)
2.2. NMDA receptor constructs
GRIN1a cDNA encoding the NMDAR1a subunit (GluN1a) was a
generous gift of Dr. Shigetada Nakanishi (Kyoto, Japan) (Moriyoshi
et al., 1991). cDNA encoding the GluN2A, GluN2C and GluN2D
subunits (GRIN2A, GRIN2C, and GRIN2D) were kindly provided
by Dr. Peter Seeburg (Heidelberg, Germany) (Monyer et al., 1992)
and the GRIN2B cDNA was the generous gift of Drs. Dolan Pritchett
and David Lynch (Philadelphia, USA) (Lynch et al., 1995). Plasmids
were linearized with Not I (GRIN1a, GRIN2C, and GRIN2D), EcoR I
(GRIN2A) or Sal I (GRIN2B) and transcribed in vitro with T7
NMDAR antagonist
L-689,560 (5 lM) was applied for 30 min
followed by 100 M UBP714 to examine its effects on AMPAR-
l
mediated responses. To quantify the effects of the compounds on
AMPAR-mediated synaptic transmission the initial (ꢀ0.8 ms) slopes
of AMPAR f-EPSPs were measured and normalised to baseline.
NMDAR-mediated f-EPSPs were recorded after a baseline period
of AMPAR responses, which were then blocked with 10 lM NBQX.
To evoke NMDAR f-EPSPs the divalent cation ratio in the saline
solution was increased (3 CaCl2, 1 MgSO4) and so was the

M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
597
stimulation intensity (2–5 times). After a further stable period of
NMDAR mediated f-EPSPs UBP714 was applied, followed by an
The introduction of an 8-bromo substituent to UBP608 or an 8-
iodo group to UBP657 enhanced antagonist activity at GluN2A-,
GluN2B- and GluN2D-containing receptors with UBP651 and
UBP658 inhibiting >85% of the receptor responses (Figs. 3 and 4).
Introduction of either a 7-hydroxy, 7-methoxy, or 7-diethylamino
substituent to UBP649 (UBP652, UBP653, and UBP654, respec-
tively) had little effect on compound activity (Fig. 3). At a concen-
application of 30
cacy of NMDAR-mediated synaptic transmission was assessed by
subtracting the mean waveform after application of 30 M D-AP5
lM D-AP5 at the end of the experiments. The effi-
l
from each preceding waveform in the individual experiments,
measuring the amplitudes of NMDAR f-EPSPs and normalising
them to baseline. Data are presented both as single experiments
and as mean values of experimental groups ( S.E.M., n = 10 for ef-
fect of UBP714 on NMDAR mediated f-EPSPs and n = 4 for effect on
AMPAR mediated f-EPSPs).
tration of 100 lM UBP652 inhibited approximately 16, 11, 11, and
7% of GluN2A-D receptor responses. UBP653 and UBP654 displayed
activities that were generally similar to UBP652 and the parent
compound UBP649.
Interestingly, the introduction of a methyl substituent to the 4-
position of UBP608 altered activity from inhibition to weak poten-
tiation. UBP714 (100 lM) displayed approximately 17, 14, and 4%
3. Results
potentiation of GluN2A (Fig. 4), GluN2B and GluN2D responses,
respectively (Fig. 3). The difference between the potentiating effect
of UBP714 on GluN2A and GluN2B versus GluN2D was statistically
significant (Table 1).
3.1. Characterization of coumarin derivatives on recombinant GluN1/
GluN2A-D receptors
A series of coumarin-3-carboxylic acid derivatives were evalu-
ated for their ability to inhibit the activation of GluN1/GluN2A-D
NMDA receptor responses expressed in Xenopus oocytes using
two-electrode voltage clamp at À60 mV. After obtaining a stea-
3.2. Characterization of UBP714 on NMDAR mediated field EPSPs in the
CA1 region of the hippocampus
dy-state response to 10 lM L-glutamate and 10 lM glycine, the
Application of UBP714 in acute hippocampal slices resulted in a
potentiation of NMDAR-mediated f-EPSPs, which were evoked in
the presence of the AMPA and kainate receptor antagonist NBQX
(Fig. 5a). More specifically, UBP714 increased the peak of the
NMDA response and slowed down its decay without much of an
effect on the slope of the response (see inset Fig. 5a). Such poten-
tiation of NMDAR-mediated f-EPSPs was very reliable in that it was
seen in 10 out of 10 slices and, on average, the amplitudes of
NMDA f-EPSPs increased to 118.6 1.5% of their initial values after
1 h period of UBP714 application (Fig. 5b). In contrast, UBP714 did
not potentiate the amplitude and did not change the decay of
AMPAR-mediated f-EPSPs, which were recorded in the presence
individual test compounds were then co-applied with the agonists.
The structures of the compounds tested are shown in Fig. 1. In
initial studies, the ability of a 100 lM concentration of each com-
pound to inhibit NMDA receptor responses was evaluated (Table
1, Figs. 3 and 4). The parent compound of the series, coumarin-3-
carboxylic acid (UBP649) displayed very weak antagonist activity
at GluN2A and GluN2C containing receptors (approximately 15–
20% inhibition) and was effectively inactive at GluN2B and GluN2D
containing receptors (0–5% inhibition). The introduction of either a
bromo or iodo substituent at the 6-position of UBP649 (UBP608
and UBP657, respectively) enhanced inhibitory activity, especially
at GluN2A-containing receptors (Fig. 3). At a concentration of
of NMDAR antagonist L-689,560 (Fig. 5c). Thus, after 1 h period of
UBP714 application the slope of AMPAR mediated f-EPSPs was
100 lM, the reference compound UBP608 (data taken from Costa
et al., 2010) inhibited approximately 89, 63, 56, and 24% of the Glu-
N2A-D responses. The 6-iodo derivative, UBP657, had similar
antagonist activity, inhibiting 93, 55 and 23% of the GluN2A
(Fig. 4), GluN2B and GluN2D receptor responses, respectively
(Fig. 3). The difference in inhibitory activity at GluN2A versus
GluN2B and GluN2D observed for UBP608 and UBP657 was
statistically significant (Table 1). Replacement of the 6-bromo sub-
stituent in UBP608 with a more polar 2-carboxyvinyl moiety to
give UBP656 reduced activity at GluN2A-D-containing receptors
(0–10% inhibition). Linking the 5- and 6-positions of UBP649
through a phenyl ring to give UBP659, did not lead to enhanced
antagonist activity at GluN2 subunits (Fig. 3).
97.2 3.4% (n = 4) of baseline values.
In summary, UBP714 at a concentration of 100 lM is selective
between native NMDA and AMPA receptors; it potentiates NMDAR
responses and has no effect on AMPARs.
4. Discussion
The development of competitive NMDAR antagonists such as D-
AP5 (Evans et al., 1982) and CPP (Aebischer et al., 1989) allowed
studies into the functions of these receptors in the CNS and her-
alded an explosion of interest in the development of such com-
Table 1
Effect of coumarin derivatives on NMDAR subtypes expressed in Xenopus oocytes.a
Compound
GluN1/GluN2A
GluN1/GluN2B
GluN1/GluN2C
GluN1/GluN2D
UBP649
UBP608
UBP657
UBP659
UBP651
UBP658
UBP714
UBP656
UBP652
UBP653
UBP654
18.3 0.4BD
89.3 0.4BCD
93.3 1.5BD
2.9 3.7B
0.2 4.7C
63.5 0.5CD
54.5 13.5D
14.3 7.9
97.5 0.9d
92.6 0.8
À14.1 1.0d
9.6 8.5
13.2 3.2D
56.1 0.8D
ND
ND
95.2 0.9
ND
ND
ND
11.1 6.0
8.5 2.5
30.5 7.1D
4.4 1.5
23.6 0.6
23.1 2.4
6.9 1.4
91.3 3.1
86.6 5.9
À4.4 1.2
5.3 1.5
6.8 1.1
5.0 0.2
8.1 0.1
98.6 0.5d
98.7 0.5D
À17.0 2.2D
2.1 3.1
15.6 4.9D
22.4 6.7BCD
18.9 3.9BCD
10.7 2.3
4.3 0.8
4.8 0.7C
Statistical significance: b,B,B indicate values that are statistically different from values for GluN1/GluN2B receptor responses, c,C,C are values different from those for GluN1/
GluN2C receptors, and d,D,D are values different from those for GluN1/GluN2D receptors; b,c,d: p < 0.05; B,C,D: p < 0.01; B,C,D: p < 0.001.
a % Antagonism or potentiation by 100
lM of the compound of responses induced by 10 lM concentrations of both L-glutamate and glycine on the NMDAR subtype indicated.
Data represents mean values S.E.M., n = 4; UBP608 included as a reference compound, data taken from Costa et al., 2010; ND = not determined; A negative value indicates
that the compound potentiated the NMDAR response.

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M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
Fig. 3. Activity of coumarin derivatives on NMDA receptor responses. After obtaining a steady-state response to 10
tested at 100 M for the inhibition or potentiation of responses by GluN1/GluN2A (2A), GluN1/GluN2B (2B), GluN1/GluN2D (2D) and in some cases GluN1/GluN2C (2C)
receptors. At a concentration of 1 M UBP658 inhibited agonist stimulated responses on GluN1/GluN2A and GluN1/GluN2B by 26.3 5.9 and 2.3 3.5%, respectively. Values
lM L-glutamate plus 10 lM glycine, each compound was
l
l
represent mean % inhibition S.E.M., n P 4.
Fig. 4. Representative recordings of inhibition or potentiation of GluN1/GluN2A NMDA receptor responses in Xenopus laevis oocytes. Responses were evoked by 10 lM L-
glutamate plus 10 lM glycine (Agonist, black bar) and then 100 lM UBP657, UBP658, or UBP714 was applied as indicated (gray bar).
pounds for the treatment of neurological (Lindsley et al., 2006;
Sanacora et al., 2008; Wasterlain and Chen, 2008) and neurodegen-
erative disorders (Kalia et al., 2008; Villmann and Becker, 2007).
Other sites on the NMDAR were later targeted for drug develop-
ment leading to the discovery of uncompetitive channel blockers
(Lodge and Johnson, 1990), antagonists binding to the glycine site
(Leeson and Iversen, 1994), and antagonists that selectively bind to
the N-terminal domain of GluN2B such as ifenprodil (Williams,
1993). Few drugs that target these sites have made it into the
clinic, though the low affinity ion channel blocker memantine
has been licensed for the treatment of moderate-severe Alzhei-
mer’s disease (Kalia et al., 2008).
Competitive NMDAR antagonists based on the structure of
D-AP5 and CPP were later shown to have the following rank order
of affinity when tested on NMDAR subtypes: GluN2A > GluN2B > -
GluN2C > GluN2D (Buller et al., 1994; Feng et al., 2005). Later,
NVP-AAM007 was discovered which was initially reported to show
100-fold selectivity for human GluN2A versus GluN2B (Auberson
et al., 2002) but shows 6–10-fold selectivity for rat GluN2A versus
GluN2B (Bartlett et al., 2007; Feng et al., 2004). Two competitive
antagonists, UBP141 and UBP145, were shown to be 10–17-fold
selective for GluN2C/2D versus GluN2A and GluN2B (Costa et al.,
2009; Morley et al., 2005).
While developing the competitive GluN2C/GluN2D selective
antagonists such as UBP145 we noticed that some of the phenan-
threne-3-carboxylic acids used in their synthesis also had NMDAR
antagonist activity (Costa et al., 2010). This led to a new class of
compounds that were either negative allosteric modulators
(NAMs) or positive allosteric modulators (PAMs) of recombinant
NMDARs, with different sites of action to previous generations of
antagonists (Costa et al., 2010). Structure–activity relationship
(SAR) studies revealed that 9-iodophenanthrene-3-carboxylic acid
(UBP512) (Fig. 1) was a GluN2C/GluN2D antagonist with no activ-
ity on GluN2B and weak potentiating activity on GluN2A (Costa
et al., 2010). Further SAR studies identified the 6-bromo substi-
tuted coumarin derivative, UBP608, as a NAM with weak selectiv-
ity for GluN2A versus GluN2B (Costa et al., 2010). Thus, UBP608
had an IC50 value of 19
receptors and IC50 values of 90, 68 and 426
l
M for blocking GluN2A-containing
M for blocking
l
NMDARs containing GluN2B, GluN2C or GluN2D, respectively
(Costa et al., 2010).
We therefore decided to conduct SAR studies around the
coumarin nucleus, using commercially available and newly
synthesized derivatives, to identify the structural requirements
for antagonism of NMDARs. In the present study we have shown
that hydrophobic substituents as large as iodo (UBP657) are well

M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
599
GluN2 subunit selectivity (Costa et al., 2010). One possible expla-
nation for the weak GluN2A versus GluN2B and GluN2D selectivity
of UBP608 and UBP657 is that a hydrogen bond donor such as the
2-hydroxy group of UBP552 is required for optimal GluN2B and
GluN2D antagonist activity and replacement with a hydrogen bond
acceptor, such as the carbonyl group at the 2-position of the cou-
marin ring is detrimental to binding to these two subunits but is
accommodated by the binding site on GluN2A.
100 µM UBP714
a
b
c
30 µM AP5
150
100
50
(b)
Baseline (a)
We have determined that the 8-position on the coumarin ring
can be substituted with groups as large as iodo to enhance NMDAR
antagonist activity, as 6,8-dibromo- (UBP651) and 6,8-diiodo-
(UBP658) substituted coumarins have greater NMDAR antagonist
potency than the mono substituted 6-bromo (UBP608) or 6-iodo
(UBP657) derivatives. However, though addition of a bromo group
at the 8-position of UBP608 increases NMDAR antagonist potency,
it reduces selectivity for GluN2A. Addition of polar substituents
such as hydroxyl (UBP652), methoxy (UBP653) or diethylamino
(UBP654) groups at the 7-position of the coumarin ring did not im-
prove NMDAR antagonist activity beyond that observed with the
parent compound UBP649. We have reported previously that
UBP608 is a negative allosteric modulator (Costa et al., 2010) and
it is likely that the UBP608 derivatives described here have the
same mode of action, though further experiments will be required
to confirm this. Fusing a phenyl ring to the 5- and 6-positions of
the coumarin ring to give UBP659 led to weaker NMDAR antago-
nist activity than that observed for UBP608, perhaps suggesting
that substituents at the 5-position are not accommodated by the
binding site of the NMDAR. This will need to be confirmed in fur-
ther SAR studies on 5-substituted coumarin derivatives.
One surprising observation was that UBP714, the 4-methyl-
substituted derivative of UBP608, potentiated activity at recombi-
nant NMDARs containing GluN2A or GluN2B and less so at receptors
containing GluN2D subunits. We have reported that replacement of
the 9-iodo group of UBP512 (Fig. 1) with a cyclopropyl or long chain
alkyl group leads to compounds with NMDAR potentiating activity
(Costa et al., 2010). However, the 4-methyl group on the coumarin
ring does not correspond to the 9-position of the phenanthrene ring
and so it appears to be a novel site for addition of substituents to
produce NMDAR potentiators. The potentiating effect of UBP714
is likely to be due to binding to an allosteric site on the NMDAR;
however, further studies will be required to confirm this.
We have shown in previous studies using chimeric GluN2 sub-
units that the S1 domain rather than the S2 domain of the gluta-
mate binding site has the most influence on the negative
allosteric modulatory activity of UBP608 (Costa et al., 2010). In
contrast, the GluN2A potentiating activity of UBP512 is influenced
mostly by the S2 domain. These studies do not distinguish between
the possibilities that the NAMs and PAMs are binding directly to
the S1 or S2 domains, respectively, or that these domains are in-
volved in the transduction of their effects. Nonetheless, it is possi-
ble that there may be separate binding sites for these NMDAR
NAMs and PAMs and this may be the basis of an explanation of
the difference in activity of UBP608 and its 4-methyl derivative.
Thus, the binding site responsible for the potentiating effect of
UBP714 appears to require a hydrophobic substituent at the 4-po-
sition of the coumarin ring, whereas this substituent seems not to
be accommodated by the binding site, that when occupied leads to
inhibitory activity.
0.2 mV
20 ms
0
NMDAR f-EPSP
10 µM NBQX
0
0
0
30
60
90
120
120
120
Time (min)
100 µM UBP714
30 µM AP5
150
100
50
(b)
Baseline (a)
0.2 mV
20 ms
0
NMDAR f-EPSP (n = 10)
10 µM NBQX
30
60
90
Time (min)
100 µM UBP714
150
100
50
Baseline (a)
(b)
0.2 mV
5 ms
0
AMPAR f-EPSP (n = 4)
5 µM L-689,560
30
60
Time (min)
90
Fig. 5. UBP714 potentiates native NMDAR responses and has no effect on AMPAR-
mediated f-EPSPs. (a) A single experiment showing that application of 100
UBP714 increased the amplitude of NMDAR response (filled circles) to 117.4% of
baseline value whereas subsequent application of 30 M D-AP5 blocked it. Thick
lines indicate the time of compound application. Inset shows f-EPSPs before (black)
and 1 h after application of UBP714 (red) together with the calibration bar. Please
note that UBP714 changes the amplitude of NMDAR f-EPSPs but not their slope. (b)
Pooled data from ten experiments showing potentiation of NMDAR f-EPSPs by
UBP714 (n = 10, mean S.E.M.). Inset shows f-EPSPs from another experiment than
that shown in panel ‘‘a’’. (c) A group of experiments, which show that application of
lM
l
100 lM UBP714 has no effect on AMPAR-mediated f-EPSPs (n = 4, mean S.E.M.).
Inset shows AMPAR f-EPSPs before (black) and 1 h after (red) application of UBP714.
It is important to show that UBP714 will not only have a poten-
tiating effect on recombinant NMDARs but also those expressed in
neurones. We have shown in this study that application of UBP714
can potentiate field EPSPs generated by NMDARs expressed in the
CA1 region of the hippocampus but has little effect on AMPAR-
mediated EPSPs in the same brain region. This is an important
observation given the role of NMDARs in the hippocampus in
mechanisms that are thought to underlie learning and memory
tolerated at the 6-position of the coumarin ring, though a 6-substi-
tuent bearing a negatively charged carboxylate group (UBP656)
was detrimental to NMDAR antagonist activity. This is consistent
with the observation that bromo substitution at the equivalent po-
sition on the naphthalene ring of derivatives UBP574 and UBP552
(Fig. 1) leads to enhanced NMDAR antagonist activity, though no

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M.W. Irvine et al. / Neurochemistry International 61 (2012) 593–600
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6,8-positions are moderately potent NMDAR antagonists, which
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subunit selective NAMs that may have applicability for the treat-
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a NMDAR potentiator
(UBP714), showing some selectivity for GluN2A/GluN2B versus
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NMDA receptor cDNA constructs. This work was supported by
grants from the National Institutes of Health (MH60252) and the
UK Medical Research Council (MRC Grant Number G0601812).
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