NMDA receptors are tetrameric complexes composed of GluN1 and GluN2A-D subunits that mediate a slow Ca(2+)-permeable component of excitatory synaptic transmission. NMDA receptors have been implicated in a wide range of neurological diseases and thus represent an important therapeutic target. We herein describe a novel series of pyrrolidinones that selectively potentiate only NMDA receptors that contain the GluN2C subunit. The most active analogues tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors as well as AMPA and kainate receptors. This series represents the first class of allosteric potentiators that are selective for diheteromeric GluN2C-containing NMDA receptors.
NMDA receptors are tetrameric complexes composed of GluN1 and GluN2A-D subunits that mediate a slow Ca(2+)-permeable component of excitatory synaptic transmission. NMDA receptors have been implicated in a wide range of neurological diseases and thus represent an important therapeutic target. We herein describe a novel series of pyrrolidinones that selectively potentiate only NMDA receptors that contain the GluN2C subunit. The most active analogues tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors as well as AMPA and kainate receptors. This series represents the first class of allosteric potentiators that are selective for diheteromeric GluN2C-containing NMDA receptors.
N-Methyl-d-aspartate
(NMDA) receptors are members of the family of ionotropic glutamate
receptors that mediate excitatory neurotransmission. NMDA receptors
are tetrameric assemblies of two GluN1 subunits, which bind the coagonist
glycine, and two GluN2 subunits, which bind glutamate.[1] Both GluN1 and GluN2 subunits share a similar architecture,
comprised of an extracellular amino-terminal domain (ATD), an extracellular
ligand-binding domain (LBD), a transmembrane domain (TMD), and an
intracellular carboxyl-terminal domain (CTD).[2] The GluN2 subunit is encoded by four distinct gene products (GluN2A-D),
which have temporally and spatially distinct expression patterns in
the brain.[3] The GluN2 subunit controls
pharmacological characteristics such as agonist sensitivity, deactivation
time course, mean open time, and open probability.[2,3b,4]The distinct anatomical locations
of the GluN2 subunits could allow subunit-selective modulators (either
potentiators or inhibitors) to target specific brain regions for therapeutic
gain. NMDA receptors are thought to play a role in neuronal development,
learning, and memory formation,[5] as well
as being implicated in ischemia,[6] dementia,[7] schizophrenia,[8] treatment
resistant depression,[9] and Parkinson’s
disease.[10] Recently discovered modulators
have demonstrated selectivity for GluN2A, 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulfonamide
(TCN201); GluN2A/GluN2B, 9-cyclopropylphenanthrene-3-carboxylic acid
(UBP710); and GluN2C/GluN2D, (3-chlorophenyl) [3,4-dihydro-6,7-dimethoxy-1-[(4-methoxyphenoxy)methyl]-2(1H)-isoquinolinyl]methanone (CIQ), 4-[6-methoxy-2-[(1E)-2-(3-nitrophen yl)ethenyl]-4-oxo-3(4H)quinazolinyl]benzoic acid (QNZ46), 5-(4-bromophenyl)-3-(1,2-dihydro-6-
methyl-2-oxo-4-phenyl-3-quinolinyl)-4,5-dihydro-g-oxo-1H-pyrazole-1-butanoic acid (DQP1105), and (2R,3S)-1-(phenanthrenyl-3-carbonyl)piperazine-2,3-dicarboxylic
acid (UBP141).[11] Here, we describe the
development of the first class of positive allosteric modulators that
are selective for GluN2C-containing NMDA receptors over GluN2A-, GluN2B-,
and GluN2D-containing receptors.To identify this class of ligands,
a GluN1/GluN2C cell line and multiwell fluorescence-based assay were
developed to enable screening of compound libraries for NMDA receptor
modulators. We screened two commercial diversity libraries to identify
several compounds that modulate GluN2C-containing NMDA receptors.
One of these screening hits established a novel class of subunit-selective
potentiators for recombinant GluN1/GluN2CNMDA receptors, exemplified
by compound 1 (Figure 1). Optimization
of the initial lead pyrrolidinone scaffold involved the development
of a structure–activity relationship, which led to the identification
of a novel series of compounds with potency in the low micromolar
range and high selectivity for recombinant GluN2C-containing receptors
over GluN2A/B/D-containing NMDA receptors. In addition, no detectable
potentiation was observed at recombinant AMPA, kainate, GABA, glycine,
serotonin, nicotinic, or purinergic receptors (data not shown). These
analogues represent a novel class of NMDA receptor modulators that
are highly selective for diheteromeric GluN1/GluN2C receptor subtypes
and provide a useful tool with which to evaluate the physiological
role of GluN2C in normal and neuropathological conditions.
Figure 1
Structure of
screening hit. Chemical structure of methyl 4-(1-(2-(1H-indol-3-yl)ethyl)-3-acetyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate (compound 1) that was
identified as a positive modulator using a fluorescence-based screen
of compound libraries in a cell line expressing diheteromeric GluN1/GluN2C
NMDA receptors.
Structure of
screening hit. Chemical structure of methyl 4-(1-(2-(1H-indol-3-yl)ethyl)-3-acetyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate (compound 1) that was
identified as a positive modulator using a fluorescence-based screen
of compound libraries in a cell line expressing diheteromeric GluN1/GluN2CNMDA receptors.
Results
Chemistry
We used bioinformatic searches and medicinal chemistry to obtain
analogues for our initial screening hit, compound 1 (see
below). Both commercially available analogues and compounds synthesized
via a mi-component Biginelli-like reaction (Scheme 1) were assessed at 30 μM. We determined the EC50 and maximal potentiation from concentration–effect curves
for compounds that showed potentiation of more than 120% of control
at 30 μM. No compounds in this class potentiated GluN2A-, GluN2B-,
or GluN2D-containing receptors, suggesting remarkable selectivity
for this class (see below). Modifications were made at either R1, the A-ring, or the B-ring using alternative methodologies
to access the appropriate precursor.
Scheme 1
Synthesis of 1H-Pyrrol-2(5H)-ones
Reaction
conditions: PPTS, rt, 1–24 h, 2% to >99% (procedure I).
Final compounds 161–180, in which
either the A or B ring is replaced, were also prepared using these
conditions.
Synthesis of 1H-Pyrrol-2(5H)-ones
Reaction
conditions: PPTS, rt, 1–24 h, 2% to >99% (procedure I).
Final compounds 161–180, in which
either the A or B ring is replaced, were also prepared using these
conditions.Addition of diethyl oxalate and
sodium ethoxide to a methyl ketone generated a series of pyruvate
analogues (3–20) containing modifications
at R1 (Scheme 2). Only when R1 was a phenol was it necessary to first protect the hydroxyl
group with triisopropyl chloride (TIPSCl) before the addition of diethyl
oxalate. Standard deprotection afforded the target pyruvate (21).
Scheme 2
Route for the Synthesis of Pyruvate Derivatives
Reaction conditions: (a) diethyl
oxalate, NaOEt, EtOH, 0 °C to rt, 4 h, 15% to >99% (procedure
II); (b) TIPSCl, imidazole, rt, 6 h, >99%; (c) diethyl oxalate,
NaOEt, EtOH, 0 °C to rt, 4 h, 28% (procedure II); (d) TBAF, 0
°C to rt, 1 h, 43%.
Route for the Synthesis of Pyruvate Derivatives
Reaction conditions: (a) diethyl
oxalate, NaOEt, EtOH, 0 °C to rt, 4 h, 15% to >99% (procedure
II); (b) TIPSCl, imidazole, rt, 6 h, >99%; (c) diethyl oxalate,
NaOEt, EtOH, 0 °C to rt, 4 h, 28% (procedure II); (d) TBAF, 0
°C to rt, 1 h, 43%.Analogues containing
disubstituted A-rings were synthesized using several procedures based
on the commercially available precursors (Scheme 3). Benzaldehydes 32–41 were
prepared from methyl esters 145–154. Dibromination and hydrolysis afforded analogues 32 and 33.[12] Suzuki coupling
between dibutyl vinylboronate and the appropriately substituted methyl
4-iodobenzoate, followed by ozonolysis, gave phenols 34 and 35. Alternatively, addition of a Grignard reagent
and N,N-dimethylformamide (DMF)
led to isolation of benzaldehyde 36. A palladium-catalyzed
formylation was used to access benzaldehydes 37–40.[13] Finally, anisole 41 was prepared via a dialkylation of both the hydroxyl and carboxylic
acid functional groups.
Scheme 3
Synthetic Routes to Access Substituted Benzaldehydes 32–41
Reaction
conditions: (a) 2.0 equiv NBS, (PhCOO)2, reflux, 4 h, then
AgNO3, rt, 3 h, 38–63%; (b) dibutyl vinylboronate,
5 mol % (PPh3)2PdCl2, NaCO3, reflux, 2 h, 68–80%; (c) O3; then (CH3)2S, −78 °C to rt, 12 h, 60–87%; (d) i-PrMgCl, DMF, −15 °C to rt, 3 h, 70%; (e) CO(g), (PPh3)2PdCl2, NaCO3, 110 °C, 8–24% (procedure IV); (f) CH3I, K2CO3, rt, 3 h, 58%.
Synthetic Routes to Access Substituted Benzaldehydes 32–41
Reaction
conditions: (a) 2.0 equiv NBS, (PhCOO)2, reflux, 4 h, then
AgNO3, rt, 3 h, 38–63%; (b) dibutyl vinylboronate,
5 mol % (PPh3)2PdCl2, NaCO3, reflux, 2 h, 68–80%; (c) O3; then (CH3)2S, −78 °C to rt, 12 h, 60–87%; (d) i-PrMgCl, DMF, −15 °C to rt, 3 h, 70%; (e) CO(g), (PPh3)2PdCl2, NaCO3, 110 °C, 8–24% (procedure IV); (f) CH3I, K2CO3, rt, 3 h, 58%.Benzaldehydes containing a para-amide (42–44) or para-ester (45–47) substituent were synthesized as illustrated
in Scheme 4. Primary amide 42 was
synthesized from carboxylic acid 155 by generating the
acid chloride in situ. Standard amide coupling conditions were employed
for the preparation of amides 43 and 44.
Alkylation of carboxylic acid 155 with the appropriate
alkyl iodide afforded esters 45 and 46,
while t-butyl ester 47 was prepared
using a method previously described.[14]
Scheme 4
Routes for the Synthesis of Amides 42–44 and Esters 45–47
Reaction conditions: (a) Vilsmeier reagent, aq NH3,
0 °C, 16 h, 30%; (b) R2aR2bN where R2a = H and R2b = Me or where R2a = R2b = Me, DMAP, EDCI, 0 °C to rt, 24 h, 14–51%;
(c) R2I where R2 = Et or R2 = i-Pr, K2CO3, rt, 4 h, 24–87%;
(d) (CH3)2NCH(Ot-Bu)2, reflux, 11/2 h, 81%.
Routes for the Synthesis of Amides 42–44 and Esters 45–47
Reaction conditions: (a) Vilsmeier reagent, aq NH3,
0 °C, 16 h, 30%; (b) R2aR2bN where R2a = H and R2b = Me or where R2a = R2b = Me, DMAP, EDCI, 0 °C to rt, 24 h, 14–51%;
(c) R2I where R2 = Et or R2 = i-Pr, K2CO3, rt, 4 h, 24–87%;
(d) (CH3)2NCH(Ot-Bu)2, reflux, 11/2 h, 81%.An alternative strategy was used to synthesize analogues containing
a modification at R11 starting from pyrrolidinones 1 and 106 (Scheme 5).
Protection of analogue 1 with trimethylsilyl diazomethane
afforded methoxy 156. Amine 157 was generated
by reaction with ammonium formate. Esterification of 1 with acetic anhydride gave acetate 158. Alternatively,
esters 159 and 160 were synthesized from
enol 106 using the appropriate acyl chloride and triethylamine.
A fluorescence-based
screen of 57504 compounds obtained from Asinex and ChemDiv libraries
was performed in BHK cells with inducible expression of GluN1/GluN2C
receptors. Hits were defined as compounds that produced changes that
were 2.5 standard deviations away from the average response to maximally
effective agonist (i.e., glutamate and glycine) application. In this
primary screen, 1% of the compounds met these criteria. Compounds
that showed potentiation were further evaluated for their ability
to produce responses in cells with no NMDA receptor expression (in
uninduced cells) in order to identify false positive hits. False positive
results can occur when the compounds directly release Ca2+ from intracellular stores, enhance Ca2+ channel function,
possess fluorescent properties in the excitation/emission range of
Fluo-4, or otherwise produce an increase in intracellular Ca2+ signal independent of NMDA receptor activation. Compounds that showed
potentiation of glutamate responses in induced cells and did not produce
responses in uninduced cells were subsequently studied by two-electrode
voltage-clamp recording of NMDA receptor responses.A single
compound was found to selectively potentiate the GluN1/GluN2C receptors
and did not show any activity at GluN2A/B/D-containing NMDA receptors
expressed in Xenopus laevis oocytes
(Figure 2A). Compound 1, which
contains a pyrrolidinone core motif, potentiated GluN1/GluN2C responses
to 238 ± 8.2% of control at 100 μM with an EC50 of 24 ± 2.4 μM (n = 12) (Figure 2B). Compound 1 had no agonist activity
on its own in that it did not induce current responses in oocytes
expressing GluN1/GluN2C in the absence of glutamate and glycine (n = 4). In addition, 30 μM of compound 1 did not potentiate homomeric recombinant GluA1 AMPA receptor responses
(97 ± 1.1% control, n = 16). In addition, 120
μM of compound 1 did not potentiate homomeric GluK2
recombinant kainate receptors (95 ± 2.3% of control, n = 5).
Figure 2
Compound 1 selectively potentiates the GluN1/GluN2C
response. (A) Current traces for 1 at the GluN1/GluN2A,
GluN1/GluN2B, GluN1/GluN2C, and the GluN1/GluN2D receptors. (B) Compound 1 selectively potentiates the GluN1/GluN2C receptor to a fitted
maximum of 275 ± 10% with an EC50 of 24 ± 2.4, n = 12. (C) The EC50 for glycine in the absence
and presence of 1 is 0.20 ± 0.01 μM (n = 6) and 0.16 ± 0.02 μM (n = 4), respectively. The EC50 for glutamate in the absence
and presence of 1 is 0.8 ± 0.07 μM (n = 8) and 1.2 ± 0.04 μM (n =
6), respectively. The presence of 1 did not shift the
glycine or glutamate EC50 values significantly. (D) The
reversal potential is −5.1 ± 0.8 mV when activated by
coagonists (100 μM glutamate and 30 μM glycine) and is
−5.0 ± 1.2 mV (n = 6) when the GluN1/GluN2C
receptor is potentiated by 1. The reversal potential
was not significantly shifted in the presence of 1, suggesting
that potentiation is independent of membrane potential.
Compound 1 selectively potentiates the GluN1/GluN2C
response. (A) Current traces for 1 at the GluN1/GluN2A,
GluN1/GluN2B, GluN1/GluN2C, and the GluN1/GluN2D receptors. (B) Compound 1 selectively potentiates the GluN1/GluN2C receptor to a fitted
maximum of 275 ± 10% with an EC50 of 24 ± 2.4, n = 12. (C) The EC50 for glycine in the absence
and presence of 1 is 0.20 ± 0.01 μM (n = 6) and 0.16 ± 0.02 μM (n = 4), respectively. The EC50 for glutamate in the absence
and presence of 1 is 0.8 ± 0.07 μM (n = 8) and 1.2 ± 0.04 μM (n =
6), respectively. The presence of 1 did not shift the
glycine or glutamate EC50 values significantly. (D) The
reversal potential is −5.1 ± 0.8 mV when activated by
coagonists (100 μM glutamate and 30 μM glycine) and is
−5.0 ± 1.2 mV (n = 6) when the GluN1/GluN2C
receptor is potentiated by 1. The reversal potential
was not significantly shifted in the presence of 1, suggesting
that potentiation is independent of membrane potential.Compound 1 (68 μM) did not detectably
alter the EC50 of glycine or glutamate (n = 4–6; Figure 2C). Additionally, the
reversal potential of glutamate and glycine induced current responses
was unchanged in the presence (−5.0 + 1.2 mV, n = 6) or absence (−5.1 + 0.8 mV, n = 6) of
compound 1. Potentiation was not significantly different
at −40 mV (202 ± 11%) compared to +30 mV (180 ± 12%; p = 0.2679; paired t test), indicating
that potentiation of GluN2C-containing receptors by compound 1 at 20 μM was voltage-independent (n = 6; Figure 2D).
Effect of Modifications
to R1 on Potency at GluN2C-Containing Receptors
We subsequently evaluated the response to 30 μM of all pyrrolidinone
analogues at GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, and GluN1/GluN2D
and proceeded to determine the concentration–effect curve when
potentiation exceeded 120% of control. Exploration of the effects
of keto-linked R1 (Scheme 1; see Chemistry section) substitutions on potentiation
of GluN2C-containing receptors in oocytes revealed that additional
steric bulk was tolerated, with only minimal improvements in potency
(Table 1, 62–65). For example, replacement of R1 with a phenyl group,
as in 65, produced a small increase in potency (EC50 = 17 ± 2.3) accompanied by a modest decrease in maximal
potentiation compared to compound 1 (Table 1). Analogues containing m-substituted phenyl
rings (66–70) offered variable potentiation,
while analogues with o- and p-substituted
phenyl rings were inactive (data not shown). Notably, 66, with a meta-hydroxyl group, displayed a considerably
higher potency at GluN2C-containing receptors (7.0 ± 0.9 μM)
but caused significant inhibition of GluN2A-, GluN2B-, and GluN2D-containing
receptors at 100 μM (responses were 76 ± 2.0%, 42 ±
1.6%, and 48 ± 2.4% of control, respectively, normalized to agonist
activated current). Such mixed-action modulators that potentiate one
subunit while inhibiting another are intriguing but of little utility
as pharmacological probes. Two compounds containing a pyridine ring
at R1 potentiated responses up to ∼200% with EC50 values of 12 ± 1.9 μM (72) and 8.9
± 1.3 μM (73). Interestingly, 71, which contains a 2-substituted pyridine ring, was inactive at all
receptor subunits. These initial experiments confirmed the ability
of derivatives within this class to selectively potentiate GluN2C-containing
receptors compared to other NMDA receptor subtypes.
Table 1
Optimization of Potency through Evaluation of Keto-Linked Substituents
Fitted EC50 values are shown for GluN1/GluN2C to two significant figures when
potentiation at 30 μM of the test compound exceeded 120% of
control; values in parentheses are the fitted maximum response as
a percentage of the initial glutamate (100 μM) and glycine (30
μM) response. Hill slopes varied between 1.2 and 2.0. Data for
active compounds at GluN1/GluN2C are from between 6 and 12 oocytes
from 2–3 frogs for each compound. When no effect was found
(n = 3–15 oocytes), the lack of effect was
confirmed by testing at 100 μM (data not shown, n ≥ 3 oocytes all compounds). For all tables, GluN2 subunits
were coexpressed with GluN1 in Xenopus oocytes and evaluated using two-electrode voltage-clamp recordings.
The response to 100 μM
of test compound was greater than 140% of control.
Fitted EC50 values are shown for GluN1/GluN2C to two significant figures when
potentiation at 30 μM of the test compound exceeded 120% of
control; values in parentheses are the fitted maximum response as
a percentage of the initial glutamate (100 μM) and glycine (30
μM) response. Hill slopes varied between 1.2 and 2.0. Data for
active compounds at GluN1/GluN2C are from between 6 and 12 oocytes
from 2–3 frogs for each compound. When no effect was found
(n = 3–15 oocytes), the lack of effect was
confirmed by testing at 100 μM (data not shown, n ≥ 3 oocytes all compounds). For all tables, GluN2 subunits
were coexpressed with GluN1 in Xenopus oocytes and evaluated using two-electrode voltage-clamp recordings.The response to 100 μM
of test compound was greater than 140% of control.
Effect of A-Ring Modifications
Next,
we evaluated the effects of various A-ring substituents (Table 2) utilizing R1 substitutions shown to
offer the desired activity. Positional isomer analogues 81 and 82 were inactive at GluN1/GluN2C. One compound, 84, which contains an ethyl ester at ring position R2, displayed comparable potency compared to screening hit 1. Analogues containing bulkier ester substituents (e.g., 85 with an iso-propyl ester and 86 with
a tert-butyl ester) led to inactivity. A series of
compounds containing ester isosteres including a nitrile (87), nitro (88), amide (89–91), and sulfonamide (92 and 93) were also
evaluated for their ability to potentiate GluN2C-containing NMDA receptors.
Unfortunately, none of these analogues exhibited any activity.
Table 2
Optimization of A-Ring Substituents
Fitted EC50 values are shown for GluN1/GluN2C
to two significant figures when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. Hill slopes varied
between 1.3 and 1.7. Data for active compounds at GluN1/GluN2C are
from between 8 and 12 oocytes from 2–3 frogs for each compound.
When no effect was found (n = 3–11 oocytes),
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 3 oocytes all compounds).
Fitted EC50 values are shown for GluN1/GluN2C
to two significant figures when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. Hill slopes varied
between 1.3 and 1.7. Data for active compounds at GluN1/GluN2C are
from between 8 and 12 oocytes from 2–3 frogs for each compound.
When no effect was found (n = 3–11 oocytes),
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 3 oocytes all compounds).A variety of substituents at
A-ring positions R3 and R4 were systematically
tested while holding the para-methyl ester constant
at R2 (Table 3). Substitution at
the meta position (R3) revealed either
a reduction in potency (95) or complete inactivity (96–99). Evaluation of a series of ortho (R4) ring substituents demonstrated a preference
for electron donating groups. For example, analogues containing an ortho-hydroxyl (100) exhibited potentiation
with a modest increase in potency, whereas ortho-chloro
(103) or -fluoro (104) substituents were
slightly less active.
Table 3
Evaluation of Combinations
of A-Ring Substituents
I30 μM/Icontrol (mean ± SEM %)
EC50 (max) μM (%)a
#
R3
R4
GluN2A
GluN2B
GluN2C
GluN2D
GluN2C
95
OH
H
81 ± 1.7
79 ± 1.2
123 ± 2.5
90 ± 3.0
29 ± 2.8 (151)
96
OMe
H
98 ± 3.3
80 ± 1.8
92 ± 2.4
86 ± 1.4
97
Me
H
108 ± 3.4
91 ± 2.5
88 ± 2.1
86 ± 0.4
98
Cl
H
88 ± 4.3
95 ± 5.1
113 ± 4.2
83 ± 1.5
99
F
H
99 ± 4.1
83 ± 2.2
114 ± 3.3
93 ± 5.3
100
H
OH
107 ± 3.9
86 ± 3.8
173 ± 3.0
88 ± 1.9
15 ± 0.6 (202)
101
H
OMe
102 ± 6.1
83 ± 0.3
132 ± 3.3
100 ± 3.1
46 ± 19 (183)
102
H
Me
101 ± 1.8
95 ± 4.2
129 ± 3.6
85 ± 1.9
35 ± 1.4 (165)
103
H
Cl
103 ± 3.9
87 ± 1.7
139 ± 2.8
90 ± 0.6
36 ± 3.0 (191)
104
H
F
93 ± 2.5
96 ± 2.0
123 ± 3.4
91 ± 1.1
37 ± 2.6 (155)
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response; Hill slopes ranged
between 1.3 and 1.8. Data for active compounds at GluN1/GluN2C are
from between 3 and 12 oocytes from 2–3 frogs for each compound.
When no effect was found (n = 3–15 oocytes),
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 5 oocytes for all compounds).
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response; Hill slopes ranged
between 1.3 and 1.8. Data for active compounds at GluN1/GluN2C are
from between 3 and 12 oocytes from 2–3 frogs for each compound.
When no effect was found (n = 3–15 oocytes),
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 5 oocytes for all compounds).
Effect of B-Ring Modifications
Replacement of the B-ring with an assortment of acyclic, cyclic,
and heterocyclic systems generated a series of compounds that were
evaluated for potency and subunit selectivity while retaining optimal
R1 and A-ring substitutions (Table 4). Interestingly, substitution with a napthyl derivative, as in 162, led to strong inhibition at all four subunits. Replacement
of the indole NH with an oxygen atom led only to weak activity (163), suggesting the presence of a hydrogen bond in the binding
pocket. In all other instances, removal of the indole led to complete
inactivity (i.e., 164 and 165). These data
suggest that the indole functionality is preferred for activity.
Table 4
Effect of Replacing the B-Ring
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. All data are from
3–14 oocytes from 2–3 frogs. When no effect was found,
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 3 oocytes for all compounds).
Inhibited at GluN1/GluN2A with
an IC50 of 18 μM, at GluN1/GluN2B with an IC50 of 7.2 μM, at GluN1/GluN2C with an IC50 of 11 μM, and GluN1/GluN2D with an IC50 of 5.7
μM.
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. All data are from
3–14 oocytes from 2–3 frogs. When no effect was found,
the lack of effect was confirmed by testing at 100 μM (data
not shown, n ≥ 3 oocytes for all compounds).Inhibited at GluN1/GluN2A with
an IC50 of 18 μM, at GluN1/GluN2B with an IC50 of 7.2 μM, at GluN1/GluN2C with an IC50 of 11 μM, and GluN1/GluN2D with an IC50 of 5.7
μM.This led us to
examine B-ring substituents as an alternative strategy to access increased
potency. The data describing these compounds is summarized in Table 5. Methylation of the indolenitrogen led to inactivity
(105), further suggesting the importance of a hydrogen
atom at this position in the binding pocket. The best potency was
obtained for analogues with substitutions at B-ring position R9. Compound 111 demonstrated an ability to selectively
potentiate GluN2C-containing NMDA receptors up to 218% with an EC50 value of 4.3 ± 0.3 μM. It is unclear whether
the increase in potency observed for 111 can be ascribed
to a steric effect or, alternatively, to a mildly electropositive
effect. Consistent with a steric effect, analogues which contain larger
R9 substituents such as R9 = OMe (112) revealed a loss of potency compared to 111. Analogues
containing strongly electron withdrawing R9 substituents
such as R9 = F (109) also decreased the observed
activity.
Table 5
Optimization of B-Ring Substituents
Fitted EC50 values are shown for GluN1/GluN2C to two significant digits when
potentiation at 30 μM of the test compound exceeded 120% of
control; values in parentheses are the fitted maximum response as
a percentage of the initial glutamate (100 μM) and glycine (30
μM) response. Hill slopes were between 1.3 and 1.9. Data for
active compounds at GluN1/GluN2C are from between 6 and 27 oocytes
from 2–3 frogs for each compound. When no effect was found
(n = 4–11 oocytes), the lack of effect was
confirmed by testing at 100 μM (data not shown, n ≥ 4 oocytes for all compounds).
Fitted EC50 values are shown for GluN1/GluN2C to two significant digits when
potentiation at 30 μM of the test compound exceeded 120% of
control; values in parentheses are the fitted maximum response as
a percentage of the initial glutamate (100 μM) and glycine (30
μM) response. Hill slopes were between 1.3 and 1.9. Data for
active compounds at GluN1/GluN2C are from between 6 and 27 oocytes
from 2–3 frogs for each compound. When no effect was found
(n = 4–11 oocytes), the lack of effect was
confirmed by testing at 100 μM (data not shown, n ≥ 4 oocytes for all compounds).
Effect of Combinatorial Modifications on Potency at GluN2C-Containing
Receptors
We subsequently evaluated the effect of combining
modifications at R1, the A-ring and the B-ring that had
previously demonstrated an improvement in potency (Table 6). Substitution with either a meta- or para-substituted pyridine ring at R1 and a para-ethyl ester at R2 revealed
potentiation of GluN1/GluN2C responses with EC50 values
of 8.2 ± 0.9 μM (116) and 9.7 ± 0.6 μM
(117), respectively. Modification of the B-ring and either
R1 (R1 = m-pyridine) or R2 (R2 = p-CO2Et) exhibited
a similar increase in on-target potency. For example, substitution
with a methyl group at R6 and a para-ethyl
ester at R2, as in analogue 119, resulted
in a 2-fold potency enhancement.
Table 6
Optimization of Potency
though Additional Modifications
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. Data for active
compounds at GluN1/GluN2C are from between 8 and 14 oocytes from 2
frogs for each compound; the Hill slope varied between 1.2 and 1.5.
When no effect was found at 30 μM (n = 3–6
oocytes), the lack of effect was confirmed by testing at 100 μM
(data not shown, n ≥ 7 oocytes for all compounds).
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120% of control; values in parentheses are
the fitted maximum response as a percentage of the initial glutamate
(100 μM) and glycine (30 μM) response. Data for active
compounds at GluN1/GluN2C are from between 8 and 14 oocytes from 2
frogs for each compound; the Hill slope varied between 1.2 and 1.5.
When no effect was found at 30 μM (n = 3–6
oocytes), the lack of effect was confirmed by testing at 100 μM
(data not shown, n ≥ 7 oocytes for all compounds).
Effect of Linker Modifications
on Potency of 1616-Series
The original screening hit, 1, contains a two carbon region linking the B-ring with the
core pyrrolidinone. The linker modifications explored are illustrated
in Table 7. Both shortening (121) and extending (122) the linker eliminated all activity,
suggesting that the potency of pyrrolidinone analogues is highly dependent
on the length of the carbon linkage.
Table 7
Optimization
of Potency though Linker Modifications
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120%; values in parentheses are the fitted
maximum response as a percentage of the initial glutamate (100 μM)
and glycine (30 μM) response. Data for active compounds at GluN1/GluN2C
are from between 7 and 8 oocytes from 2 frogs for each compound tested;
the Hill slope varied between 1.3 and 1.4. When no effect was found
at 30 μM (n = 3–11 oocytes), the lack
of effect was confirmed by testing at 100 μM (data not shown, n ≥ 4 oocytes for all compounds).
Fitted EC50 values are shown for GluN1/GluN2C
to two significant digits when potentiation at 30 μM of the
test compound exceeded 120%; values in parentheses are the fitted
maximum response as a percentage of the initial glutamate (100 μM)
and glycine (30 μM) response. Data for active compounds at GluN1/GluN2C
are from between 7 and 8 oocytes from 2 frogs for each compound tested;
the Hill slope varied between 1.3 and 1.4. When no effect was found
at 30 μM (n = 3–11 oocytes), the lack
of effect was confirmed by testing at 100 μM (data not shown, n ≥ 4 oocytes for all compounds).
Effect of Modifications to R11 on Potency at GluN2C-Containing Receptors
Several modifications
were made at R11 to determine the significance of the enol
in controlling potency and selectivity (Table 8). Replacement with an amine, as in 157, led to a complete
loss of potentiation at concentrations up to 100 μM. In most
instances, compounds containing a protected alcohol led to less potent
analogues. For example, a 2-fold decrease in potency was observed
for acetate 158. In contrast, propyl ester 159 maintained activity comparable to lead analogue 1,
with an EC50 of 17 ± 1.8 μM. These data suggest
that enhancements in potency cannot be gained though modifications
of the enol.
Table 8
Optimization of Potency though Evaluation
of Vinyl Substituents
Fitted
EC50 values are shown for GluN1/GluN2C to two significant
digits when potentiation at 30 μM of the test compound exceeded
120% of control; values in parentheses are the fitted maximum response
as a percentage of the initial glutamate (100 μM) and glycine
(30 μM) response. Data for active compounds at GluN1/GluN2C
are from between 5 and 9 oocytes from 2–3 frogs for each compound.
The Hill slope varied between 1.2 and 1.8 and was fixed to be 1.5
for less potent analogues (157, 159). When
no effect was found (n = 3–9 oocytes), the
lack of effect was confirmed by testing at 100 μM (data not
shown, n ≥ 4 oocytes for all compounds).
Fitted
EC50 values are shown for GluN1/GluN2C to two significant
digits when potentiation at 30 μM of the test compound exceeded
120% of control; values in parentheses are the fitted maximum response
as a percentage of the initial glutamate (100 μM) and glycine
(30 μM) response. Data for active compounds at GluN1/GluN2C
are from between 5 and 9 oocytes from 2–3 frogs for each compound.
The Hill slope varied between 1.2 and 1.8 and was fixed to be 1.5
for less potent analogues (157, 159). When
no effect was found (n = 3–9 oocytes), the
lack of effect was confirmed by testing at 100 μM (data not
shown, n ≥ 4 oocytes for all compounds).
Effect of Absolute Configuration
on Potency at GluN2C-Containing Receptors
To enable evaluation
of potential stereoselectivity for pyrrolidinone analogues at GluN1/GluN2C,
we separated the enantiomers of 106 using a semipreparatory
OD-RH chiral HPLC column (see Chemistry Experimentals). Each enantiomer was subjected to two-electrode voltage clamp analysis
in Xenopus laevis oocytes. The results,
illustrated in Figure 3, indicate that only
one enantiomer (106a) is active and may account for the
activity of 106. Compound 106a potentiated
GluN2C response by 259 ± 7.8% with an estimated EC50 value of 18 ± 0.6 μM (n = 6). In contrast,
no activity was observed for the other enantiomer (106b) (n = 6). The active analogue demonstrated weak
inhibition at GluN2D-containing receptors and had no effect at GluN2A-
or GluN2B-containing receptors. Compound 106b was inactive
at all other subunits. These data suggest that the activity of pyrrolidinone
analogues may rely on a single enantiomer and that the binding pocket
can distinguish between the enantiomers.
Figure 3
Composite concentration–effect
curves for 106 enantiomers. Concentration–effect
curves for the enantiomers of 106 demonstrate that only
one enantiomer, 106a, is active, potentiating the GluN1/GluN2C
receptor to a fitted maximum of 259 ± 8% of control with an EC50 of 18 ± 0.6 μM (n = 6).
Composite concentration–effect
curves for 106 enantiomers. Concentration–effect
curves for the enantiomers of 106 demonstrate that only
one enantiomer, 106a, is active, potentiating the GluN1/GluN2C
receptor to a fitted maximum of 259 ± 8% of control with an EC50 of 18 ± 0.6 μM (n = 6).
Conclusion
The
incorporation of a methyl group at the C-7 position of the indole
of initial screening hit 1 afforded 111,
which selectively potentiates GluN2C-containing NMDA receptors with
a potency of 4.3 ± 0.3 μM. In addition, the activity of
this series appears to originate from one enantiomer. These compounds
represent the first class of allosteric potentiators selective for
diheteromeric GluN1/GluN2C receptors over receptors containing GluN2A-,
GluN2B-, and GluN2D subunits. Future studies will address the activity
of this series of modulators on triheteromeric GluN2C-containing NMDA
receptors containing two different GluN2 subunits (e.g., GluN1/GluN2A/GluN2C).
This series of molecules may serve as a pharmacological tool to evaluate
the role of the GluN2C subunit in normal and neuropathological function.
Experimental Methods
Biology Experimentals
All protocols involving Xenopus laevis were approved by the Emory University Institutional Animal Care
and Use Committee. Two-electrode voltage-clamp recordings were made
from Xenopus laevis oocytes expressing
recombinant GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, GluN1/GluN2D,
GluA1, or GluK2 receptors following injection of cRNA. cDNAs for ratGluN1–1a (GenBank accession numbers U11418 and U08261; hereafter
GluN1), GluN2A (D13211), GluN2B (U11419), GluN2C (M91563), GluN2D
(L31611), GluA1 (X17184), and GluK2 (Z11548) were provided by Drs. S. Heinemann (Salk Institute), S. Nakanishi
(Kyoto University), and P. Seeburg (University of Heidelberg). Oocyte
isolation, cRNA synthesis, and cRNA injection have been previously
described;[15] some experiments were performed
with oocytes obtained from Ecocyte (Austin, TX). Voltage-clamp recordings
from oocytes were made during perfusion with recording solution containing
90 mM NaCl, 1.0 mM KCl, 0.5 mM BaCl2, 0.005 mM EDTA, and
10 mM HEPES at pH 7.4 (23 °C). Glass microelectrodes had resistances
of 0.3–1.0 MΩ and were filled with 0.3–3.0 M KCl;
the membrane potential was held at −40 mV for all recordings.
Compounds were made as 20 mM stock solutions in DMSO and diluted to
the final concentration in recording solution; final DMSO content
was 0.05–0.5% (v/v). Oocytes expressing GluK2 receptors were
pretreated with 10 μM concanavalin A for 10 min. NMDA receptors
were activated by 100 μM glutamate plus 30 μM glycine;
GluA1 and GluK2 receptors were activated by 100 μM glutamate.
To prevent a gradual increase in current response over the course
of the experiment of GluN1/GluN2A receptor responses in oocytes, some
oocytes expressing GluN1/GluN2A were injected with 20–50 nL
of 2 mM K-BAPTA (potassium 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). When the response to agonist
in the presence of 30 μM of a test compound exceeded 120% of
control, the response to glutamate and glycine in the absence and
presence of 5–7 concentrations of active analogues were recorded
in multiple oocytes obtained from two or more different frogs for
all experiments. The EC50 (half-maximally effective concentration
of potentiator) was determined by fitting the equationto the concentration–response data normalized to the current
in the absence of potentiator (100%) for each oocyte, and the mean
(±SEM) presented. N is the Hill slope, which
ranged between 1 and 2 and is not reported; maximum is the fitted maximal response expressed as a percent of control
to a saturating concentration of potentiator. When responses were
inhibited by test compound at 30 μM to less than 60% of control,
the IC50 value was determined by fitting the equationto the concentration–response data
normalized to the current. For some compounds, visual detection of
precipitation led to inclusion of 1–10 mM 2-hydroxypropyl-β-cyclodextrin
in the recording solution to enhance solubility and enable generation
of the full concentration–response data.To generate
a cell line with inducible NMDA receptor expression, we used a previously
described Tet-On (tetracycline-inducible promoter; Clontech, Mountain
View, CA) baby hamster kidney (BHK-21, ATCC CCL-10) cell line.[16] The BHK-21 Tet-On cell line was maintained at
37 °C, 5% CO2, and 95% relative humidity in culture
medium composed of Dulbecco’s Modified Eagle Medium (DMEM)
containing GlutaMAX-I, 4500 mg/L glucose, and 110 mg/L sodium pyruvate
(Invitrogen, Carlsbad, CA) supplemented with penicillin (100 units/mL),
streptomycin (100 μg/mL), (Invitrogen, Carlsbad, CA), 10% dialyzed
fetal bovine serum (Invitrogen, Carlsbad, CA), and 1 mg/mL G418 (Invitrogen,
Carlsbad, CA). The selection marker G418 was always included to provide
continuous selection of Tet-On-compatible BHK-21 cells. The cells
were cotransfected with ratGluN1-1a (GenBank accession no. U11418) in the
inducible pTRE2 vector and ratGluN2C (GenBank accession no. D13212) in the
pCI-IRES-bla vector (see ref (16) for details on this vector) using Fugene 6 transfection
reagent (Promega, Madison, WI). The ratio of GluN1 and GluN2C DNA
used for transfection was 10:1. The NMDA receptor antagonists dl-2-amino-5-phosphonopentanoate (AP5) (200 μM; Abcam,
Cambridge, MA) and 7-chloro-kynurenate (7-CKA) (200 μM; Abcam,
Cambridge, MA) were added to the culture medium to prevent NMDA receptor-mediated
cell death. The following day, the cells were diluted 1:1000 and 1:10,000
and seeded in 144 mm dishes. The next day (e.g., two days after transfection),
10 μg/mL blasticidin S (Invivogen, San Diego, CA) was added
to the culture medium to select for transfected cells. Unless otherwise
stated, the culture medium for the cell lines always contained 1 mg/mL
G418 and 10 μg/mL blasticidin S for selection as well as 200
μM AP5 and 200 μM DCKA to prevent NMDA receptor-mediated
cell death. The media was changed every 2–3 days, and blasticidin
S-resistant clones were isolated 10–20 days after transfection
and evaluated for their response properties. Fluorescence-based assays
were conducted as previously described,[17] and test compounds were screened at 10 μM.
Chemistry Experimentals
Compounds for which synthesis is not described were purchased from
commercial vendors. Purity of purchased compounds was greater than
90%, as determined by the suppliers, via HPLC or NMR.All dry
solvents were obtained from a Glass Contour System. Reagents used
were acquired from commercial suppliers and utilized without additional
purification. Precoated glass plates (silica gel 60 F254, 0.25 mm)
were used to monitor the progress of reactions by thin layer chromatography
(TLC). Purification by flash column chromatography was performed on
a Teledyne ISCO Combiflash Companion using prepackaged Teledyne RediSep
disposable normal phase silica columns. Melting temperatures were
determined on a Mel-Temp apparatus and are uncorrected. 1H and 13C NMR experiments were each carried out on an
INOVA-400 (400 MHz), VNMR 400 (400 MHz), INOVA-600 (600 MHz), Unity-600
(600 MHz), or Mercury 300 Vx (300 MHz). All chemical shifts are reported
in parts per million and referenced to the residual solvent peak.
All coupling constants are reported in hertz (Hz). The IR spectra
were acquired with a Nicolet Avatar 370 DTGS. Mass spectra were performed
by the Emory University Mass Spectrometry Center on a VG 70-S Nier
Johnson or JEOL instrument. Purity of all final compounds was found
to be ≥95% by LC/MS analysis unless otherwise noted.
Separation
of Enantiomers
The separation of the enantiomers of 106 was obtained using a ChiralPak OD-RH 30 mm × 250
mm, 5 μm column with the following conditions: flow rate 10
mL/min, injection volume 1–2 mL (5 mg/mL), 44% ACN/66% water
with 0.1% formic acid; 106atR = 121.3 min; 106btR =
129.3 min. Enantiomeric excess (ee) of both enantiomers 106a and 106b was determined using a ChiralPak
OD-RH 4.6 mm × 150 mm, 5 μm column with the following conditions:
flow rate 0.5 mL/min, injection volume 10 μL, 44% ACN/66% water
with 0.1% formic acid; 106a [α]20 −18
(c = 0.10, methanol), tR = 26.1 min, 98% ee; 106b [α]20 + 9 (c = 0.10, methanol), tR = 29.1 min, 96% ee. A Perkin-Elmer 314 instrument
was used to obtain optical rotation data.
General Procedure for Synthesis
of Pyrrolidinone Compounds (Procedure I: 1, 62–82, 84–122, 161–180)
To a stirred solution
of aldehyde (1.0 mmol) in dioxane (1.0 M) was added tryptamine (1.0
equiv) and 10 mol % pyridinium 4-methylbenzenesulfonate. Upon the
formation of a slurry, methyl acetopyruvate (1.0 equiv) was added.
The resulting mixture was allowed to stir at rt for up to 12 h. In
most instances, a precipitate was visible, which was collected via
filtration and washed with Et2O. The solid was dissolved
in an appropriate solvent and washed with saturated ammonium chloride
and brine before being dried over MgSO4, filtered, and
concentrated in vacuo. If a precipitate did not form, the mixture
was concentrated in vacuo before being subjected to the workup as
described above. Purification was achieved via flash column chromatography
on SiO2 (MeOH/DCM) to afford the desired pyrrolidinone.
Additional purification was obtained by HPLC (85% ACN/15% water with
0.1% formic acid) as needed.
General Preparation of Pyruvate Compounds
(Procedure II: 3–20, 144)
To a solution of sodium ethanolate (1.0 equiv) in EtOH
(0.72 M) at 0 °C was added a mixture of diethyl oxalate (1.0
equiv) and ethanone (1.0 mmol) over 20 min. The mixture was allowed
to stir at rt for 4 h. In most instances, a precipitate had formed
which was collected via filtration and washed with absolute EtOH.
If no precipitate was evident, a minimal amount of water was added
and the mixture was concentrated in vacuo. The residue was dissolved
in water, neutralized with acetic acid, and extracted with Et2O (3×). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. Purification was achieved
as needed via flash column chromatography on SiO2 (hexanes/EtOAc:
4/1) to obtain the product.
General Preparation of Methyl Benzoate Compounds
(Procedure III: 184–186)
To a solution of 4-bromobenzoic acid (1.0 mmol) in THF:MeOH (4:1,
0.3 M) at 0 °C was added (diazomethyl)trimethylsilane (2.4 equiv).
The reaction was allowed to warm to rt over the period of 1 h. At
this time, the mixture was concentrated in vacuo and 1.0 M HCl was
added. The mixture was extracted with EtOAc (2×), dried over
MgSO4, filtered, and concentrated in vacuo to afford the
product.
General Preparation of Methyl 4-Formylbenzoate Compounds (Procedure
IV: 37–40)
To a solution
of methyl 4-bromobenzoate (1.0 mmol) in DMF (0.6 M) was added 17 mol
% bis(triphenylphosphine)palladium(II) dichloride and sodium formate
(1.5 equiv). The reaction mixture was stirred at 110 °C under
a steady stream of CO(g) for 2 h. At this time, the mixture
was cooled to rt, diluted with saturated sodium carbonate, and extracted
with EtOAc (2×). The combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated in
vacuo. Purification was achieved via flash column chromatography on
SiO2 (hexanes/EtOAc: 3/1) to yield the desired product,
which was taken on without further purification.
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Figure 1
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Figure 2
Figure 3