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ACS Chemical Neuroscience
the coꢀexpressed GluN2 subunit. We extended the series
for 1 hour at room temperature, cooled to 0 °C, and then
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4
5
6
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by exchanging the ether linkage with amide and thioether
linkers. These analogues also show varying activity. Surꢀ
prisingly, 11a and 13a showed extensive potentiation (2ꢀ
fold) of maximal current at GluN1/2C relative to the current
induced by Gly. Thus, both compounds are superagonists
at the glycine site of the GluN1/2C receptor. This observaꢀ
tion was even more pronounced with 15a and 16a showing
remarkable enhancements of agonist efficacy (398% and
308%) compared to the endogenous agonist glycine. These
levels of superagonistic activity are unprecedented among
all NMDA receptor agonists described to date.1,8,16,17 The
substituents on 15a and 16a are quite bulky and molecular
modeling support that steric effects at GluN1 F754 could be
involved in the GluN2ꢀspecific agonist activity (Figure 3).
benzylbromide (0.29 mL, 416 mg, 2.43 mmol, 1 equiv) was
added. The mixture was allowed to warm to room temperaꢀ
ture and stirred for 4 hours. The reaction was quenched
with sat. NH4Cl (1 mL) and the mixture was partioned beꢀ
tween Et2O (20 mL) and icecold 0.1 M HCl (10 mL). The
aqueous phase was extracted with Et2O (3 x 50 mL), dried
(MgSO4), concentrated and purified by column chromatogꢀ
raphy (Eluent 0ꢀ100 % EtOAc in hexane, 1 % CH3CO2H) to
afford 470 mg (66 % yield). For deprotection the product
was dissolved in dioxane (2 mL), HCl in dioxane (4 M, 1
mL) was added dropwise and the reaction stirred overnight.
The precipitate was washed with Et2O, dried (MgSO4) and
concentrated to afford 178 mg (32 % yield) of 8a as white
solid. 1H NMR (300 MHz; Methanolꢀd4): δ 7.36ꢀ7.29 (m, 5H),
4.66ꢀ4.56 (m, 2H), 4.17 (dd, J = 4.8, 3.3 Hz, 1H), 3.88 (dq, J
= 11.5, 4.1 Hz, 2H). 13C NMR (75 MHz; Methanolꢀd4): δ
169.7, 138.4, 129.3, 128.91, 128.89, 74.4, 68.2, 54.4. MS
calcd for C10H13NO3H+ [M+H]+:196.1, found: 196.1. Mp:
193.3–194.3 °C.
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Although Rꢀisomers generally are more active than S-
isomers, it is notable that the cysteine analogue 11a is the
active enantiomer since this Lꢀform has opposite geometry.
This could mean that substituents are pointing in opposite
directions in the GluN1 glycine binding site if the amino acid
(including the αꢀcarbon) moiety has an overlapping binding
(R)ꢀ3ꢀ(2ꢀNaphthamido)ꢀ2ꢀaminopropanoic acid (15a). To
BocꢀDꢀ3ꢀaminopropionic acid (500 mg, 2.4 mmol) in THF (5
mode. However, the selectivity profile of Lꢀisomers 11a, 15b
and 16b suggests that the substituents point in the same
direction towards the ABD dimer interface by overlaying of
the amino acid moieties, but not the αꢀcarbon, as can be
mL) was added DIPEA (2.1 mL, 12.2 mmol, 5 equiv) and 2ꢀ
naphthoyl chloride (460 mg, 2.4 mmol, 1 equiv). The reacꢀ
tion was stirred overnight, where after 1 M HCl was added
(until pH=2ꢀ3). The mixture was extracted with EtOAc (2 x
20 mL), washed with brine (2 x 10 mL), dried (MgSO4) and
evaporated. Purification by column chromatography (Eluent
30% EtOAc in heptane, 2% CH3CO2H) afforded 320 mg
(37% yield) as colorless oil. For deprotection the product
was stirred in 2 M HCl in Et2O (7 mL, 20 equiv) at room
temperature for 2.5 h. The precipitate was then washed with
Et2O and dried to afford 135 mg (68% yield) of 15a as a
white solid. 1H NMR (400 MHz, DMSOꢀd6) δ 13.89 (s, 1H),
8.99 (t, J = 5.8 Hz, 1H), 8.53 (s, 1H), 8.48 (brs, 3H), 8.03 –
7.94 (m, 4H), 7.70 – 7.53 (m, 2H), 4.14 (t, J = 5.8 Hz, 1H),
3.94 – 3.71 (m, 2H). 13C NMR (151 MHz, DMSO) δ 169.1,
166.9, 134.2, 132.0, 131.0, 128.8, 128.0, 127.8, 127.73
127.6, 126.8, 124.3, 52.4, 39.4. MS calcd for C14H14N2O3H+
[M+H]+: 259.1, found: 259.1. Mp: 226.3ꢀ232.4°C.
observed in the coꢀcrystal structures of
Dꢀ and LꢀGlu in the
GluN2D ABD.18
D
ꢀCycloserine (4) has been intensively studied as a GluN1
glycine site agonist with intriguing neuroactive properties.
Administration of ꢀcycloserine can enhance extinction of
fear in rodents and humans, and ꢀcycloserine has been
considered as a potential therapeutic agent in several psyꢀ
chiatric disorders.19,20 Until now,
ꢀcycloserine was the only
D
D
D
described glycine site ligand with agonist efficacy that is
highly dependent on the glutamateꢀbinding GluN2 subunits
and the only described superagonist at GluN1/2C recepꢀ
tors.21,17 Our identification of compounds 15a and 16a as
GluN2Cꢀselective superagonists provides new opportunities
for in vivo biological and behavioral studies that have previꢀ
ously relied on
tion.
Dꢀcycloserine for NMDA receptor modulaꢀ
(R)ꢀ2ꢀAminoꢀ3ꢀ(4,7ꢀdichloroꢀ1Hꢀindoleꢀ2ꢀ
carboxamido)propanoic acid (16a). 4,7ꢀdichloroꢀ1Hꢀ
indoleꢀ2ꢀcarboxylic acid (0.3 g, 1.3 mmol) in THF (5 mL)
was added 2 M oxalylchloride in CH2Cl2 (1.3 mL, 2.6 mmol,
2 equiv) and a drop of DMF. The reaction was stirred at
room temperature for 1 h and then concentrated. The crude
material was redissolved in THF (5 mL), and thenBocꢀ
aminopropanoic acid (0.27 g, 1.3 mmol, 1 equiv) and DIPEA
(1.1 mL, 6.5 mmol, 5 equiv) were added. The mixture was
stirred at room temperature for 1 h, then diluted with H2O
(10 mL) and 1 M HCl (2 mL, to pH = 2ꢀ3) and extracted with
EtOAc (2 x 20 mL). The combined organic phases were
washed with brine (2 x 10 mL), dried (MgSO4), and concenꢀ
trated. For deprotection, the purified compound was disꢀ
solved in 2 M HCl in Et2O and stirred for 2.5 h. The final
product was obtained as a white solid (yield: 9%, 3 steps for
16a). 1H NMR (600 MHz, DMSOꢀd6) δ 13.99 (brs, 1H),
12.22 (s, 1H), 9.04 (t, J = 5.8 Hz, 1H), 8.42 (brs, 3H), 7.32
(d, J = 8.1 Hz, 1H), 7.31 (d, J = 1.8 Hz, 1H), 7.18 (d, J = 8.1
Hz, 1H), 4.13 – 4.09 (m, 1H), 3.84 (dt, J = 14.3, 5.2 Hz, 1H),
3.78 – 3.72 (m, 1H). 13C NMR (151 MHz, DMSO) δ 169.0,
160.5, 134.0, 133.7, 126.8, 124.4, 124.0, 120.4, 115.8,
104.1, 52.3, 38.9. MS calcd for C12H11Cl2N3O3H+ [M+H]+:
316.0, found: 316.0. Mp: 218.7ꢀ224.8°C.
In conclusion, this study demonstrates large variation in
potency and agonist efficacy of various Ser analogues at
the glycine sites in NMDA receptors, in a GluN2 dependent
manner. Exchanging the ether linker in Ser analoges with
thioether and amide linkers yielded a series of compounds
that displayed large variation in agonist efficacy ranging
from very partial agonism (5%) to superagonism (398%) in
a GluN2ꢀspecific manner. Thus, the results of this study
suggest that differential potency, improved agonist efficacy,
and subtypeꢀselectivity at the glycine sites in NMDA recepꢀ
tors can be achieved by synthesizing novel ligands deꢀ
signed to exploit structural differences in the NMDA recepꢀ
tor ABDs.
EXPERIMENTALS
Chemistry. Representative experimental procedures for
compound 8a, 15a, and 16a. For further experimental detail
see supporting information.
OꢀBenzylꢀ
5.1 mmol, 2.1 equiv) in anhydrous DMF (2.0 mL) at 0 °C
was slowly added NꢀBocꢀ ꢀSer (500 mg, 2.43 mmol) disꢀ
solved in anhydrous DMF (4 mL). The mixture was stirred
DꢀSer (8a). To a dispersion of 60% NaH (204 mg,
D
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Maolanon, Alex R.
