Systematic variation of the benzenesulfonamide part of the GluN2A selective NMDA receptor antagonist TCN-201

Article abstract of DOI:10.1016/j.ejmech.2017.02.018

GluN2A subunit containing N-methyl-D-aspartate receptors (NMDARs) are highly involved in various physiological processes in the central nervous system, but also in some diseases, such as anxiety, depression and schizophrenia. However, the role of GluN2A subunit containing NMDARs in pathological processes is not exactly elucidated. In order to obtain potent and selective inhibitors of GluN2A subunit containing NMDARs, the selective negative allosteric modulator 2 was systematically modified at the benzenesulfonamide part. The activity of the test compounds was recorded in two electrode voltage clamp experiments using Xenopus laevis oocytes expressing exclusively NMDARs with GluN1a and GluN2A subunits. It was found that halogen atoms in 3-position of the benzenesulfonamide part result in high GluN2A antagonistic activity. With an IC₅₀ value of 204?nM the 3-bromo derivative 5i (N-{4-[(2-benzoylhydrazino)carbonyl]benzyl}-3-bromobenzenesulfonamide) has 2.5-fold higher antagonistic activity than the lead compound 2 and represents our new lead compound.

Full text of DOI:10.1016/j.ejmech.2017.02.018

European Journal of Medicinal Chemistry 129 (2017) 124e134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Systematic variation of the benzenesulfonamide part of the GluN2A
selective NMDA receptor antagonist TCN-201
,
,
Sebastian L. Müller a 1, Julian A. Schreiber a 1, Dirk Schepmann a,
Nathalie Strutz-Seebohm b, Guiscard Seebohm b, Bernhard Wünsch a
, c, *
a Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, D-48149 Münster, Germany
b Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Muenster, D-48149 Muenster, Germany
c Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), University Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
GluN2A subunit containing N-methyl-D-aspartate receptors (NMDARs) are highly involved in various
Received 14 December 2016
Received in revised form
20 January 2017
Accepted 7 February 2017
Available online 14 February 2017
physiological processes in the central nervous system, but also in some diseases, such as anxiety,
depression and schizophrenia. However, the role of GluN2A subunit containing NMDARs in pathological
processes is not exactly elucidated. In order to obtain potent and selective inhibitors of GluN2A subunit
containing NMDARs, the selective negative allosteric modulator 2 was systematically modified at the
benzenesulfonamide part. The activity of the test compounds was recorded in two electrode voltage
clamp experiments using Xenopus laevis oocytes expressing exclusively NMDARs with GluN1a and
GluN2A subunits. It was found that halogen atoms in 3-position of the benzenesulfonamide part result in
high GluN2A antagonistic activity. With an IC50 value of 204 nM the 3-bromo derivative 5i (N-{4-[(2-
benzoylhydrazino)carbonyl]benzyl}-3-bromobenzenesulfonamide) has 2.5-fold higher antagonistic ac-
tivity than the lead compound 2 and represents our new lead compound.
Keywords:
NMDA receptor
GluN2A selective antagonists
TCN-201
Synthesis
Electrophysiology
Two electrode voltage clamp
Structure activity relationships
Antagonistic activity
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
Both endogenous agonists glycine and (S)-glutamate are required
to activate the NMDA receptor [1,2]. With these three subtype
N-Methyl-
D
-aspartate receptors (NMDARs) are glutamate and
families diheteromeric receptors, containing GluN1 and one type of
GluN2 subunits can be formed. Additionally, triheteromeric re-
ceptors, containing two different GluN2 subunits or one GluN2 and
one GluN3 subunit and, furthermore, glycine activated NMDARs
containing two GluN1 and two GluN3 subunits are known. The high
number of possible combinations of GluN subunits and the influ-
ence of the different subunits on the properties of the NMDAR lead
to highly diverse functional activity of the NMDA system in the CNS
[1,3].
The subunit composition differs depending on the develop-
mental stage of the CNS, especially for the GluN2A subunit.
Whereas the neonatal GluN2A subunit expression level is quite low
in rat brain, in the adult brain the GluN2A subunit represents one of
the predominant GluN2 subunits [4]. Furthermore, GluN2A con-
taining NMDARs are involved in synaptic plasticity and learning
processes [5]. It was found that GluN2A knockout mice show
decreased anxiety-like behavior across multiple tests and less
depressant-like behavior in the forced swimming test, which im-
plicates a possible involvement of GluN2A subunit containing
glycine gated ion channels, which are highly expressed in the
central nervous system (CNS). The heterotetrameric receptor can be
comprised of seven different subunits, which are subclassified into
three subfamilies GluN1, GluN2 and GluN3 [1]. Whereas eight
different splice variants of the GluN1 subunit (GluN1a-h) are
encoded by a single gene, four different types of GluN2 subunits
(GluN2A-D) and two different types of GluN3 subunits (GluN3A, B)
are encoded by different genes [2]. A functional NMDAR is
comprised of two GluN1 subunits containing the binding site for
the co-agonist glycine and two additional subunits from the GluN2
or GluN3 family. Whilst GluN3 subunits also have a binding site for
glycine, the GluN2 subunits are responsible for glutamate binding.
* Corresponding author. Institute of Pharmaceutical and Medicinal Chemistry,
University of Münster, Corrensstr. 48, D-48149 Münster, Germany.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
1
Both authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2017.02.018
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
125
NMDARs in the pathophysiology of these diseases [6]. Different
studies demonstrated the involvement of GluN2A subunit hypo-
function in the pathophysiology of chronic schizophrenia [7,8].
Moreover, GluN2A subunit containing NMDARs are also correlated
to other neurological diseases like cerebral ischemia, seizure dis-
order, Huntington's, Parkinson's and Alzheimer's disease. However,
the role of the GluN2A subunit is not fully understood so far, due to
contrary results in different studies [9]. Thus, GluN2A subunit se-
lective ligands are highly needed as tools to investigate the role of
GluN2A subunits on physiological and pathophysiological
processes.
5-Phosphonomethylquinoxalinedione PEAQX and its active
diastereomer NVP-AAM077 (1) belong to the first described com-
pounds selectively targeting GluN2A containing NMDARs (Fig. 1).
Both compounds compete with glutamate for its binding site at the
GluN2A subunit [10]. However, the selectivity towards NMDARs
with other GluN2 subunits, in particular the GluN2B subunit, is
rather low [11].
substitution pattern of 2. Since 2 was the result of a high-
throughput screening [12e14]. systematic structure activity re-
lationships are not yet reported. Very recently, MPX-007 (4, Fig. 1),
which is derived from 2, was reported to have 13-fold higher
GluN2A activity and better solubility in H2O than 2 [17]. The main
structural differences between 2 and 4 are the replacement of the
central benzene ring by a pyrazine ring and the exchange of the
hydrazide moiety by a N-methylcarboxamido group [18].
In order to identify the structural elements, which are respon-
sible for high GluN2A antagonistic activity, single point variations
of 2 were performed systematically herein and the blockade of
GluN2A subunit containing NMDARs was recorded. In this work, we
focused on the systematic variation of the 3-chloro-4-fluorophenyl
moiety. The substitution pattern of the phenyl ring should be
modified (compounds 5) and the phenyl ring should be replaced
either by other aromatic (compounds 6) or aliphatic substituents
(compounds 7). (Fig. 2).
In 2010, Bettini et al. reported the most promising selective
GluN2A negative allosteric modulator (NAM) TCN-201 (2, Fig. 1),
which shows moderate antagonistic activity at GluN2A containing
NMDARs (IC50 ¼ 109 nM, HEK cells) and high selectivity over all
other GluN2 subunits [12e14]. Based on biological tests, a new
binding site was postulated for 2, which was confirmed in 2012 by
mutagenesis of GluN2A containing NMDARs. The new binding site
of 2 is located at the interface of the GluN1 and GluN2A subunits.
Hansen et al. also showed that binding of 2 resulted in a noncom-
petitive potency reduction of glycine at the GluN1 subunit [14].
Recently this hypothesis of the mechanism was confirmed by
crystallization of 2 with the ligand binding domain of the GluN1/
GluN2A containing receptor [15].
In 2016 Hackos et al. described some positive allosteric modu-
lators (PAMs) binding at the same or overlapping binding site as the
NAM 2. One of the most active compounds with sufficient selec-
tivity for GluN2A over other GluN2 subunits was the thiazolopyr-
imidine GNE-0723 (3, Fig. 1) with an EC50-value of 21 nM. NAMs
(e.g. 2) and PAMs (e.g. 3) would represent valuable tools for the
analysis of the effects resulting from inhibition or overactivation of
GluN2A containing NMDARs [16]. However, the activity of the NAM
2 (IC50 ¼ 109 nM) is still too low to serve as versatile tool
compound.
2. Synthesis
For the synthesis of novel NAMs 5e7 three reaction paths were
pursued, which used the common educt 4-(aminomethyl)benzoic
acid (8) (Scheme 1). Path A was preferred, since it allowed the
introduction of diverse substituents in the last reaction step of the
synthesis (late stage diversification strategy). Therefore, most of the
compounds were synthesized according to Path A.
Path A started with the introduction of the Boc-protective group
by reaction of 8 with di-tert-butyl dicarbonate [19]. The resulting
BOC-protected amino acid 9 was treated with benzoylhydrazine
and COMU® as coupling agent [20] to form the diacylhydrazine 10
in 90% yield. Subsequent deprotection of 10 with trifluoroacetic
acid resulted in the building block 11 as trifluoroacetate salt, which
was transformed into the free amine by treatment with NaHCO3.
The free amine 11 and also the trifluoroacetate salt were used in the
last step of the synthesis to obtain diverse sulfonamides 5e7.
The amine 11 was reacted with the corresponding sulfonyl
chloride in H2O under similar reaction conditions as reported by
Deng et al. [21]. According to this report, the pH value was
constantly kept at 8 by addition of Na2CO3 using a syringe pump
equipped with a pH-meter. The careful control of the pH value is
essential, as hydrolysis of sulfonyl chlorides occurs at pH > 10 and
protonation (i.e. deactivation) of the amine at pH < 7. To simplify
the method a Na2B4O7 buffer keeping the pH value constant at 9
was used instead of pH-meter and syringe pump. Most of the
Therefore, we aim at the development of novel NAMs for
GluN2A subunit containing NMDARs with high activity. An
approach to increase the activity is the systematic variation of the
Br
H
H2
PO3
N
O O
S
Cl
H
N
N
O
O
O
H
3C
H
H
N
F
N
H
N
H
O
NVP-AAM077
TCN-201
(2)
(1)
NC
H
H
O
O O
S
CH3
N
CF3
F
F
N
N
N
S
H
N
N
H
H
N
F
3C
3C
N
S
N
O
Cl
GNE-0723
MPX-007
(4)
(3)
Fig. 1. GluN2A selective NMDAR ligands.

126
S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
2
O O
S
O O
S
N
N
O
O
Alkyl
H
H
H
H
N
N
N
N
X
H
H
O
O
5
7
O O
S
Ar
N
O
H
H
N
N
H
O
6
Fig. 2. Design of novel GluN2A selective NMDAR antagonists by systematic variation of lead compound 2.
Path A
BOC
BOC
H2N
N
O
N
H
H
H
a)
b)
N
OH
OH
N
H
O
O
10
O
8
9
d)
c)
O O
S
O O
S
Path B
e)
H2N
O
H
R
N
O
R
N
d)
H
N
N
H
H
N
OH
N
H
H
O
O
O
12
f)
5-7
11
Path C
h)
O O
O O
S
S
R
N
R
N
g)
H
N
H
H
H
N
BOC
N
NH2
H
O
O
13
14
Scheme 1. Reagents and reaction conditions: (a) (Boc)2O, NaOH, H2O, 0 ꢀC, 1 h, rt, 16 h, 92%;19 (b) PhCONHNH2, COMU®, DIPEA, THF, rt, 16 h, 90%; (c) 1. TFA, CH2Cl2, rt, 1 h; 2.
NaHCO3, H2O, 1 min, 76%; (d) R-SO2Cl, Na2B4O7, H2O, rt, 3e16 h, R is defined in Table 1; (e) PhCONHNH2, COMU®, DIPEA, THF, rt, 16 h; (f) 1. SOCl2, 75 ꢀC, 1 h; 2. tert-Butyl carbazate,
pyridine, CH2Cl2, reflux, 2 h; (g) 1. TFA, CH2Cl2, rt, 1 h; 2. Et3N, MeOH, rt, 5 min; (h) PhCOCl, pyridine, CH2Cl2, reflux, 2 h.
COMU® ¼ (1-Cyano-1-ethoxycarbonylmethylenaminoxy)-dimethylamino-morpholino-carbenium hexafluorophosphate.
formed sulfonamides 5e7 were insoluble in the aqueous system
and precipitated resulting in high reaction rates and simple puri-
fication by filtration and recrystallization.
The reference compound TCN-201 (2) was synthesized via Path
B. It was prepared by reaction of 3-chloro-4-fluorophenylsulfonyl
chloride with amine 8 in Na2B4O7 buffer and subsequent coupling
of carboxylic acid 12h [27] with benzoylhydrazine and COMU®.
Since Path B requires only two reaction steps, the preparation of
large amounts of reference compound 2 via Path B was more effi-
cient than the synthesis via Path A.
The synthesis of sulfonamides 5k, 5w, 6c, and 7a via Path B
required the carboxylic acids 12b, 12d, 12f, and 12g with appro-
priate sulfonyl moieties (Scheme 2).
For the synthesis of the 3-iodophenylsulfonamide 5k the car-
boxylic acid 12a was prepared by reaction of 3-
nitrobenzenesulfonyl chloride with amine 8. Subsequent esterifi-
cation with methanol and SOCl2 provided the ester 15a, which was
reduced by Zn to give the primary amine 15b. Diazotation and
subsequent reaction with KI afforded the 3-iodo derivative 15c,
which was hydrolyzed with NaOH to yield the carboxylic acid 12b.
The 4-aminophenylsulfonamide 5w was obtained from acet-
amide 12c [25]. Treatment of 12c with boiling NaOH led to hy-
drolysis of the acetamide and the amine 12d [26] was isolated in
94% yield.
A Sonogashira reaction [22] of 4-iodophenylsulfonamide 12e
with
phenylethyne
and
PdI2
led
to
the
phenyl-
ethynylbenzenesulfonamide 12f in 60% yield.
The preparation of the methanesulfonamide 7a was performed
starting with the carboxylic acid 12g, which was synthesized in a
similar manner as reported in literature [23]. Thus, amine 8 was
transformed into methyl ester 15d, which reacted with mesyl
chloride to form the sulfonamide 15e. Hydrolysis with NaOH gave
the desired carboxylic acid 12g.
Finally, the carboxylic acids 12b, 12d, 12f, and 12g were coupled
with benzoylhydrazine in the presence of the coupling agent
COMU® to provide the desired sulfonamides 5k, 5w, 6c, and 7a.

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
127
O O
S
O O
S
N
X
O2
N
N
b)
H
H
OR
OCH3
+Cl-
O
O
NH3
=
=
=
12a: OR OH
X
X
15b:
15c:
a)
c)
=
15a: OR OCH3
I
O O
S
I
N
d)
H
OH
O
12b
O O
O O
S
N
H
S
N
H
O
e)
OH
OH
H
3C
N
H2N
H
O
O
12c
12d
O O
S
O O
S
N
H
N
f)
OH
H
OH
O
I
O
12e
12f
O O
S
H3N
Cl
=
15e: OR OCH3
H
N
3C
g)
h)
8
H
i)
OCH3
OR
=
OR OH
12g:
O
O
15d
Scheme 2. Reagents and reaction conditions: (a) SOCl2, MeOH, rt, 16 h, 79%; (b) 1. Zn, AcOH, MeOH, 0ꢀC-rt, 16 h; 2. 2 M HCl/ether, 43%; (c) 1. NaNO2, HCl, H2O, 0 ꢀC, 30 min; 2. KI,
H2O, reflux, 10 min, 32%; (d) NaOH, dioxane, H2O, reflux, 30 min. 99%; (e) 20% NaOH reflux, 4 h, 94%; (f) PhC≡CH, PdI2, TBAA, DMF, rt, 24 h, 60%; (g) SOCl2, MeOH, rt, 16 h, 97%; (h)
MsCl, DIPEA, CH2Cl2, rt, 16 h, 96%; (i) NaOH, dioxane, H2O, rt, 2 h, 89%.
The sulfonamide 5a without further substituents in the scaffold
should be included into the study as basis for the structure-activity-
relationship discussions. 5a as well as 5j, 5m, and 6e were already
described in a patent [24], but the synthesis differed from our
synthesis reported herein. For the synthesis of 5a, Path C (Scheme
1) was followed. At first amine 8 was reacted with benzenesulfonyl
chloride to give the carboxylic acid 12i [28]. After conversion of the
acid 12i into the acid chloride, reaction with Boc-protected hydra-
zine led to the hydrazide 13. Removal of the Boc group provided the
monoacylhydrazine 14, which was transformed into the diacylhy-
drazine 5a upon reaction with benzoyl chloride.
subunits were used for screening and dose response curves. In the
assay 10 M (S)-glutamate and 10 M glycine were added for
m
m
channel opening and the effect of 500 nM test compound on the
resulting current was recorded and compared to the effect of lead
compound 2. Each concentration level of the different compounds
was tested in at least five independent oocytes (n ¼ 5) except for 5r
(n ¼ 3) and the lead compound 2 (n ¼ 30). The screening results are
shown in Fig. 3 and Table 2.
The derivatives 5c, 5k, 5f, and 5i with only one halogen atom in
3-position of the phenylsulfonyl moiety show high activity at
GluN2A containing NMDARs. Even the least active 3-fluoro deriv-
ative 5c reveals a significantly higher normalized inhibition than
the unsubstituted derivative 5a (p < 0.001). The type of halogen in
3-position has a major impact on GluN2A inhibition. Whereas the
3-chloro-, 3-bromo- and 3-iodobenzenesulfonamides 5f, 5i and 5k
The structures of all final test compounds are summarized in
Table 1 together with the obtained yields and the Path (Path A, B or
C) of synthesis. The exact procedures for the synthesis of all com-
pounds are given in the Supporting Information.
show
high
but
similar
channel
blockade,
the
3-
fluorobenzenesulfonamide 5c was significantly less active.
(p < 0.001). Compared to the lead compound 2, the 3-fluoro de-
rivative 5c is less active, whereas the 3-chloro, 3-bromo and 3-iodo
derivatives 5f, 5i and 5k show higher ion channel blockade. The
most potent 3-bromobenzenesulfonamide 5i displays a significant
increased inhibition of GluN2A containing NMDARs (p < 0.001). In
3. Pharmacological activity
The channel blocking activity of the synthesized test com-
pounds was investigated electrophysiologically in two electrode
voltage clamp (TEVC) measurements. For this purpose, Xenopus
laevis oocytes expressing NMDARs with GluN1a and GluN2A

128
S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
Table 1
Summary of all prepared sulfonamides 5e7 including yields and path of synthesis.
Substituted phenyl derivatives 2 and 5
Cpd.
R
Yield [%]
78
Path
B
Cpd.
R
Yield [%]
73
Path
A
2
5m
5a
5b
5c
54
74
62
C
A
A
5n
5o
5p
94
90
78
A
A
A
5d
5e
80
61
A
A
5q
5r
87
87
A
A
5f
63
77
26
67
A
A
A
A
5s
5t
52
77
75
49
A
A
A
A
5g
5h
5i
5u
5v
5j
93
93
A
B
5w
5x
58
53
B
5k
A

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
129
Table 1 (continued )
Substituted phenyl derivatives 2 and 5
Cpd.
R
Yield [%]
60
Path
A
Cpd.
R
Yield [%]
74
Path
5l
5y
A
Aryl residues with an extended
p
system and heterocycles 6
Cpd.
6a
R
Yield [%]
Path
Cpd.
6e
R
Yield [%]
Path
30
A
80
A
6b
44
A
6f
70
A
6c
75
68
B
6g
6h
64
81
A
A
6d
A
Alkylated derivatives 7
Cpd.
7a
R
Yield [%]
Path
Cpd.
7c
R
Yield [%]
Path
71
B
17
A
7b
8
A
7d
21
A
particular
the
high
GluN2A
activity
of
the
3-
Halogenation in 2-position of the benzenesulfonamide moiety
(5b, 5e, 5h) causes a complete loss of activity. A NO2 group in 2-
position (5m) led to a channel inhibition, which is comparable
with the activity of the unsubstituted derivative 5a. Nevertheless,
5m is still less active than the lead compound 2.
chlorobenzenesulfonamide 5f indicates that the fluoro atom in 4-
position of the lead compound 2 is not essential for high GluN2A
inhibition.
Replacement of the halogen atom in 3-position by a methyl (5p),
nitro (5n) or cyano moiety (5t) led to decreased ion channel inhi-
bition compared to the 3-fluoro derivative 5c and the lead com-
pound 2 (p < 0.001). The 3-cyano derivative 5t was significantly less
active than the unsubstituted compound 5a. At a concentration of
500 nM the 3-methoxybenzenesulfonamide 5r did not inhibit
GluN1/GluN2A receptors. These results confirm the particular
properties of halogen atoms in 3-position. It can be speculated that
the halogen atom, in particular the bromo substituent, forms a
halogen bond with the receptor increasing the overall ligand ac-
tivity. The precise task of the halogen atom in 3-position can be
determined only after identification of the exact binding site of
these ligands at GlunN1/GluN2A channels.
Introduction of substituents in 4-position of 2 did not result in
highly active GluN2A antagonists. All compounds of this type (5o,
5l, 5d, 5g, 5x, 5j, 5w) display a decreased activity compared to lead
compound 2. Whilst 4-amino-, 4-bromo-, 4-carboxy- and 4-
chlorobenzenesulfonamides 5w, 5j, 5x, 5g show comparable
normalized inhibition as 5a, 4-fluoro-, 4-iodo- and 4-
nitrobenzesulfonamides 5d, 5l and 5o were almost inactive at a
concentration of 500 nM.
Replacement of the phenyl ring of the benzenesulfonamide 5a
by an electron deficient 3-pyridiyl ring (6g) led to a considerable
decrease of channel inhibition. However, introduction of an elec-
tron rich 2-thienyl ring (6h) instead of the phenyl ring of 5a
increased the antagonistic activity. Obviously, a ring with high
electron density (e.g. thiophene) favors the antagonistic activity,
whereas electron deficient heterocycles (e.g. pyridine) reduce it.
Based on the screening results, dose-response curves were
recorded for the lead compound 2 and the most promising 3-bromo
derivative 5i under the conditions of the screening. In Fig. 4, the
dose response curves of 2 and 5i are compared. The left-shift of the
dose-response curve for 5i compared to the curve for 2 confirms its
higher antagonistic activity. Analysis of the curves resulted in IC50
values of 512 nM ( 64 nM (SEM)) for the lead compound 2 and
204 nM ( 28 nM (SEM)) for the 3-bromobenzenesulfonamide 5i.
Altogether the 3-bromo derivative 5i is 2.5-fold more potent than
the lead compound 2 in GluN1/GluN2A inhibition.
In literature, an IC50 value of 109 nM is reported for the lead
compound 2 [12,14]. The higher IC50 value (512 nM) in our assay is
supposedly due to different cells (Xenopus laevis oocytes instead of

130
S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
5e
5r
***
***
5o
5b
5h
5l
***
***
***
***
5d
5t
***
***
***
***
6g
5n
5g
5p
5x
5j
***
***
***
***
***
5a
5w
5m
6h
5c
2
***
***
***
ns
5k
5f
ns
ns
5i
***
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
Inorm
Fig. 3. Normalized Inhibition Inorm of the test compounds (light grey). Inhibition of ion flux at a test compound concentration of 500 nM. The inhibition was normalized relative to
the inhibition of 500 nM of 2 (black, n ¼ 30); n ¼ 3e5, SEM, One-Way ANOVA, post hoc mean comparison Tukey Test compared to 2, ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.
Unsubstituted derivative 5a for comparison shown in grey.
Table 2
2
5i
Normalized Inhibition Inorm of compounds based on screening data by two-electrode
voltage clamp (TEVC). Inhibition at a test compound concentration of 500 nM was
normalized relative to the inhibition of 500 nM 2 from the same experiment. n
100
reflects the number of independent oocytes.
90
80
70
60
50
Compd.
R
n
Normalized Inhibition Inorm
SEM
40
30
20
10
0
2
3-Cl-4-F-phenyl
Phenyl
2-F-phenyl
3-F-phenyl
4-F-phenyl
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
2-Br-phenyl
3-Br-phenyl
4-Br-phenyl
3-I-phenyl
30
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
3
5
5
5
5
5
1.0
0.022
0.029
0.067
0.079
0.069
0.024
0.065
0.024
0.026
0.034
0.040
0.086
0.052
0.065
0.014
0.045
0.012
0.046
0.008
0.050
0.038
0.011
0.027
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.351
0.019
0.818
0.103
ꢁ0.092
1.214
0.279
0.045
1.273
0.315
1.193
0.051
0.397
0.161
0.002
0.280
ꢁ0.062
0.151
0.379
0.283
0.151
0.529
0,001
0,01
0,1
1
10
5j
5k
5l
Concentration (μM)
4-I-phenyl
Fig. 4. Dose response curves of 5i (grey) and 2 (black). Compounds were measured in a
concentration range from 1 nM to 10 M. Each concentration of each compound was
measured in 5 different oocytes (n ¼ 5).
5m
5n
5o
5p
5r
5t
5w
5x
6g
6h
2-NO2-phenyl
3-NO2-phenyl
4-NO2-phenyl
3-CH3-phenyl
3-OCH3-phenyl
3-CN-phenyl
4-NH2-phenyl
4-COOH-phenyl
Pyridin-3-yl
Thien-2-yl
m
containing NMDARs was investigated electrophysiologically. Xen-
opus laevis oocytes were injected with cRNA for GluN1a and
GluN2B subunits. Due to limited solubility, 2 and 5a were tested at a
concentration of 10 mM. The inhibition of the GluN2B channel by
both sulfonamides 2 and 5a at this very high concentration is
comparable to the effect of the solvent DMSO (negative control)./
Fig. 5) Obviously, both compounds do not affect NMDARs with
GluN2B subunits indicating high selectivity for GluN2A over
GluN2B subunit containing NMDARs.
HEK cells) and/or higher glycine concentrations in our protocol.
In addition to the effect of the sulfonamides 2 and 5i on GluN2A
subunit containing NMDARs, the effect on GluN2B subunit

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
131
coupling constants are given with 0.5 Hz resolution; the assign-
ments of 13C and 1H NMR signals were supported by 2D NMR
techniques. The signals of the 13C and 1H spectra were assigned
according to the following scheme.
100
90
80
70
60
50
40
30
20
10
0
ns
ns
Aromatic signals, which are part of the 4-(aminomethyl)ben-
zoate moiety, were marked with B. Signals, which are part of the
benzoylhydrazide moiety, were marked with C. Aromatic signals,
which are part of the arylsulfonyl moiety, were marked with A. The
purity of all compounds was determined by HPLC analysis. HPLC
(method ACN): Merck Hitachi Equipment; UV detector: L-7400;
autosampler: L-7200; pump: L-7100; degasser: L-7614; column:
5i
2
0,1 % DMSO
Ifenprodil
Compound
Fig. 5. Inhibition of GluN2B containing NMDARs at a test compound concentration of
10
m
M (n ¼ 5, DMSO as negative control, ifenprodil as positive control. One-Way
LiChrospher® 60 RP-select B (5
tridge; flow rate: 1.0 mL/min; injection volume: 5.0
m
m); LiCroCART® 250-4 mm car-
L; detection at
ANOVA, post hoc mean comparison Tukey Test, ns p > 0.05).
m
l
¼ 210 nm; solvent A: demineralized H2O with 0.05% (v/v) tri-
4. Conclusion
fluoroacetic acid; solvent B: acetonitrile with 0.05% (v/v) tri-
fluoroacetic acid: gradient elution (% A): 0e4 min: 90.0%;
4e29 min: gradient from 90% to 0%; 29e31 min: 0%; 31e31.5 min:
gradient from 0% to 90.0%; 31.5e40 min: 90%. According to HPLC
analysis the purity of all test compounds is >95%, if not mentioned
otherwise.
The sulfonamide 2 (TCN-201) represents the most interesting
NMDAR antagonist selectively inhibiting GluN1/GluN2A receptor
function. Herein the benzenesulfonamide part of 2 was diversely
modified including different substituents at the phenyl ring and
replacement of the phenyl ring by other aryl, hetaryl, alkyl and
alkenyl residues. Introduction of substituents in 2- and 4-position
led to almost complete elimination or considerable reduction of
GluN1/GluN2A inhibition. Compounds with a halogen atom in 3-
position of the phenyl ring show promising GluN2A antagonistic
activity. Compared to the lead compound 2, the 3-fluoro derivative
5c shows slightly reduced GluN2A inhibition, whereas the 3-chloro
and 3-iodo derivatives 5f, 5k reveal similar GluN2A activity and the
3-bromo derivative 5i is even more potent than 2. The superior
activity of 5i is reflected by the left shifted dose-response curve
resulting in an IC50 value of 204 nM. Thus, 5i is 2.5-fold more potent
than 2 (IC50 ¼ 512 nM). Both sulfonamides 2 and 5i did not block
GluN1/GluN2B containing NMDARs indicating high selectivity for
GluN2A subunit containing NMDARs. These first promising results
stimulate further modifications of our new lead compound 5i to
increase the GluN2A antagonistic activity.
5.2. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl}-3-chloro-4-
fluorobenzenesulfonamide (2) [12]
12h (1.03 g, 3.00 mmol), benzoylhydrazine (409 mg, 3.00 mmol)
and COMU® (1413 mg, 3.29 mmol) were suspended in THF (10 mL).
After addition of DIPEA (1.05 mL, 6.03 mmol), the resulting yellow
solution was stirred at rt overnight. The solvent was removed under
reduced pressure and the resulting orange oil was dissolved in 2 M
NaOH. The solution was extracted twice with CH2Cl2 to remove
byproducts. Afterwards, the aqueous layer was acidified with conc.
HCl to adjust pH 1 and the formed precipitate was filtered, washed
with H2O and dissolved in THF. After removal of the solvent under
reduced pressure, the formed solid was recrystallized from MeOH/
H2O to yield 2. Colorless solid, mp 222 ꢀC, yield 1.08 g (78%).
C
21H17ClFN3O4S (461.9). Rf
¼
0.80 (ethyl acetate, detection:
[ppm] ¼ 4.14 (d,
254 nm). 1H NMR (400 MHz, DMSO-D6):
d
5. Experimental part
J ¼ 3.8 Hz, 2H, NHCH2), 7.34e7.41 (m, 2H, 2-HB, 6-HB), 7.50e7.55 (m,
2H, 3-HC, 5-HC), 7.57e7.66 (m, 2H, 5-HA, 4-HC), 7.81 (ddd, J ¼ 8.7/
4.6/2.4 Hz, 1H, 6-HA), 7.83e7.87 (m, 2H, 3-HB, 5-HB), 7.89e7.97 (m,
3H, 2-HA, 2-HC, 6-HC), 8.44 (t, J ¼ 5.2 Hz, 1H, SO2NHCH2), 10.46 (s,
1H, ArBCONHNH), 10.49 (s, 1H, NHNHCOArC). HPLC: tR ¼ 18.7 min,
purity 98.6%.
5.1. Chemistry, general
Moisture sensitive reactions were conducted under dry nitro-
gen. THF was dried with sodium/benzophenone and was freshly
distilled before use. Thin layer chromatography: Silica gel 60 F254
plates (Merck). Flash chromatography (fc): Silica gel 60, 40e43
m
m
5.3. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl}
(Macherey-Nagel); parentheses include: diameter and length of the
column, eluent, Rf value. In order to obtain high yields and due to
the poor solubility some compounds were adsorbed on silica gel by
addition of silica gel to a solution of the compound in an appro-
priate solvent, removal of the solvent in vacuo and giving the
mixture on top of the column. Melting point: Melting point system
MP50 (Mettler Toledo), uncorrected. NMR spectra were recorded
on Agilent DD2 spectrometers (1H NMR: 600 MHz, 400 MHz; 13C
benzenesulfonamide (5a) [24]
14 (100 mg, 0.33 mmol) was dissolved in a mixture of CH2Cl2
(8 mL) and pyridine (2 mL) and a solution of benzoyl chloride
(100 mL, 0.87 mmol) in CH2Cl2 (2 mL) was added dropwise to the
solution. The resulting mixture was heated to reflux for 2 h. After
cooling down to rt, 2 M NaOH (10 mL) was added and the organic
layer was extracted twice with 2 M NaOH (10 mL). The combined
aqueous layers were acidified by addition of conc. HCl to adjust pH
NMR: 151 MHz, 100 MHz);
d in ppm related to tetramethylsilane;

132
S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
1. The formed precipitate was filtered and purified by flash column
2H, 2-HB, 6-HB), 7.47 (t, J ¼ 6.2 Hz, 1H, NHCH2), 7.50e7.56 (m, 2H, 3-
HC, 5-HC), 7.58e7.63 (m, 1H, 4-HC), 7.88 (d, J ¼ 8.1 Hz, 2H, 3-HB, 5-
HB), 7.91e7.95 (m, 2H, 2-HC, 6-HC), 10.45 (s, 1H, ArBCONHNH), 10.48
(s, 1H, NHNHCOArC). HPLC: tR ¼ 17.6 min, purity 98.6%.
chromatography (ethyl acetate þ 1% NH3
25%, Ø ¼ 2 cm,
(aq)
h ¼ 20 cm, V ¼ 10 mL, Rf ¼ 0.58) to give 5a. Colorless solid, mp
195 ꢀC, yield 73 mg (54%). C21H19N3O4S (409.5). Rf ¼ 0.58 (ethyl
acetate þ 1% NH3 (aq) 25%, detection: 254 nm). 1H NMR (400 MHz,
DMSO-D6):
d
[ppm] ¼ 4.08 (s, 2H, NHCH2), 7.36e7.40 (m, 2H, 2-HB,
6-HB), 7.50e7.56 (m, 2H, 3-HC, 5-HC), 7.56e7.67 (m, 4H, 3-HA, 4-HA,
5-HA, 4-HC), 7.81e7.87 (m, 4H, 2-HA, 6-HA, 3-HB, 5-HB), 7.91e7.95
5.7. 4-(Aminomethyl)-N'-benzoylbenzohydrazide (11)
(m, 2H, 2-HC, 6-HC), 8.27 (br s, 1H, SO2NHCH2), 10.48 (s, 2H, ArB
CONHNHCOArC). HPLC: tR ¼ 15.1 min, purity 98.1%.
-
10 (3.70 g, 10.0 mmol) was dissolved in CH2Cl2 (25 mL) and
trifluoroacetic acid (15 mL) by stirring the mixture at rt for 1 h. After
complete conversion monitored by TLC, the solvent was removed
under reduced pressure to yield the trifluoroacetate salt of 11 as
pale yellow solid (3.80 g, 9.91 mmol, 99%). To obtain the free amine
this solid was dissolved in 2 M NaOH. The solution was acidified
with conc. HCl to pH 1 and neutralized with saturated NaHCO3
solution. The precipitate was filtered, washed with H2O and dried
to yield the free amine 11. Pale yellow solid, mp 187 ꢀC, yield 2.05 g
(76%). C15H15N3O2 (269.3). Rf ¼ 0.03 (ethyl acetate, detection:
5.4. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl}-3-
bromobenzenesulfonamide (5i)
11 (135 mg, 0.50 mmol) and Na2B4O7 (1.00 g, 4.97 mmol) were
suspended in H2O (30 mL) under ultra-sonication. 3-
Bromobenzenesulfonyl chloride (70
mL, 0.49 mmol) was added
and the reaction mixture was treated by ultra-sonication for 5 min,
inducing precipitation of a colorless solid. After stirring at rt over-
night, the suspension was treated with conc. HCl to adjust pH 1. The
solid was filtered and washed with H2O and CH2Cl2 to remove
byproducts and was dissolved in THF. Removal of the solvent under
reduced pressure gave 5i. Colorless solid, mp 203 ꢀC, yield 164 mg
(69%). C21H18BrN3O4S (488.4). Rf ¼ 0.81 (ethyl acetate, detection:
254 nm, Dragendorff reagent). 1H NMR (600 MHz, DMSO-D6):
d
[ppm] ¼ 3.78 (s, 2H, H2NCH2), 7.45 (d, J ¼ 7.9 Hz, 2H, 3-HB, 5-HB),
7.51 (t, J ¼ 7.6 Hz, 2H, 3-HC, 5-HC), 7.57 (t, J ¼ 7.4 Hz, 1H, 4-HC), 7.87
(d, J ¼ 7.9 Hz, 2H, 2-HB, 6-HB), 7.93 (d, J ¼ 7.4 Hz, 2H, 2-HC, 6-HC).
Signals for the protons of the NH2 and NH-NH group are not
observed in the spectrum. HPLC: tR ¼ 6.6 min, purity 98.1%.
254 nm). 1H NMR (400 MHz, DMSO-D6):
d
[ppm] ¼ 4.13 (d,
J ¼ 4.4 Hz, 2H, NHCH2), 7.38 (d, J ¼ 8.3 Hz, 2H, 2-HB, 6-HB), 7.50e7.57
(m, 3H, 5-HA, 3-HC, 5-HC), 7.57e7.63 (m,1H, 4-HC), 7.80 (ddd, J ¼ 7.8/
1.7/1.1 Hz, 1H, 6-HA), 7.82e7.87 (m, 3H, 4-HA, 3-HB, 5-HB), 7.90e7.95
(m, 3H, 2-HA, 2-HC, 6-HC), 8.42 (t, J ¼ 5.5 Hz, 1H, SO2NHCH2), 10.46
(s, 1H, ArBCONHNH), 10.48 (s, 1H, NHNHCOArC). HPLC:
tR ¼ 18.6 min, purity 99.2%.
5.8. 4-(3-Chloro-4-fluorophenylsulfonamidomethyl)benzoic acid
(12h) [27]
4-(Aminomethyl)benzoic acid (3.03 g, 20.0 mmol) and Na2B4O7
(13.0 g, 64.6 mmol) were suspended in H2O (1 L) under ultra-
sonication. 3-Chloro-4-fluorobenzenesulfonyl chloride (2.95 mL,
20.7 mmol) was added and the reaction mixture was treated with
ultra-sonication for 5 min. After vigorously stirring at rt for 4 h,
conc. HCl was added slowly to adjust pH 1 and the formed pre-
cipitate was filtered, washed with H2O and dissolved in EtOAc.
Removal of the solvent under reduced pressure gave 12h. Colorless
solid, mp 247 ꢀC, yield 6.75 g (98%). C14H12ClFNO4S (343.8).
5.5. 4-{[(tert-Butoxycarbonyl)amino]methyl}benzoic acid (9) [19]
4-(Aminomethyl)benzoic acid (5.0 g, 33.1 mmol) was dissolved
in a mixture of dioxane (60 mL), H2O (30 mL) and 1 M NaOH
(40 mL). The resulting solution was cooled to 0 ꢀC in an ice bath and
di-tert-butyl dicarbonate (7.46 g, 34.2 mmol) was added. The re-
action mixture was stirred for 1 h at 0 ꢀC and a colorless precipitate
was formed. The mixture was concentrated to 40 mL under reduced
pressure and 1 M NaHSO4 solution was added to adjust pH 2. The
resulting suspension was extracted with EtOAc (ca. 100 mL), the
organic layer was washed with brine and dried (Na2SO4). The sol-
vent was removed under reduced pressure to yield 9. Colorless
solid, mp 169 ꢀC, yield 7.63 g (92%). C13H17NO4 (251.1). Rf ¼ 0.18
(ethyl acetate:cyclohexane ¼ 1:1, detection: 254 nm). 1H NMR
Rf ¼ 0.88 (ethyl acetate þ 1% formic acid, detection: 254 nm). 1
H
NMR (400 MHz, DMSO-D6):
d
[ppm] ¼ 4.13 (d, J ¼ 6.3 Hz, 2H,
NHCH2), 7.30e7.35 (m, 2H, 3-HB, 5-HB), 7.59 (t, J ¼ 8.9 Hz, 1H, 5-HA),
7.78 (ddd, J ¼ 8.7/4.5/2.3 Hz, 1H, 6-HA), 7.80e7.84 (m, 2H, 2-HB, 6-
HB), 7.85 (dd, J ¼ 6.9/2.3 Hz, 2H, 2-HA), 8.54 (t, J ¼ 6.3 Hz, 1H,
SO2NHCH2), 12.89 (br s, 1H, CO2H). HPLC: tR ¼ 17.3 min, purity
98.9%.
(400 MHz, DMSO-D6):
d
[ppm] ¼ 1.39 (s, 9H, (H3C)3C), 4.19 (d,
J ¼ 6.2 Hz, 2H, NHCH2), 7.34 (d, J ¼ 8.0 Hz, 2H, 3-HB, 5-HB), 7.46 (t,
J ¼ 6.1 Hz, 1H, NHCH2), 7.89 (d, J ¼ 8.1 Hz, 2H, 2-HB, 6-HB), 12.84 (s,
1H, CO2H). HPLC: tR ¼ 17.8 min, purity 99.9%.
5.9. 4-(Phenylsulfonamidomethyl)benzoic acid (12i) [28]
4-(Aminomethyl)benzoic acid (3.02 g, 20.0 mmol) and Na2B4O7
(13.0 g, 64.6 mmol) were suspended in H2O (1 L) under ultra-
sonication. Benzenesulfonyl chloride (2.56 mL, 20.0 mmol) was
added and the reaction mixture was treated with ultra-sonication
for 5 min. After vigorously stirring at rt for 4 h, conc. HCl was
added slowly to adjust pH 1 and the formed precipitate was filtered,
washed with H2O and dissolved in THF. Removal of the solvent
under reduced pressure gave 12i. Colorless solid, mp 231 ꢀC, yield
5.33 g (91%). C14H13NO4S (291.3). Rf ¼ 0.48 (ethyl acetate/cyclo-
hexane 1:1þ1% acetic acid, detection: 254 nm). 1H NMR (400 MHz,
5.6. tert-Butyl N-[4-(2-benzoylhydrazine-1-ylcarbonyl)benzyl]
carbamate (10)
9 (3.52 g, 14.0 mmol), benzoylhydrazine (1.91 g, 14.0 mmol) and
COMU® (6.6 g, 15.4 mmol) were dissolved in THF (100 mL). After
addition of DIPEA (5.0 mL, 28.7 mmol), the resulting yellow solu-
tion was stirred at rt overnight. The solvent was removed under
reduced pressure and the resulting solid was recrystallized from
EtOAc. The obtained solid was filtered, washed with EtOAc and
dissolved in THF. The solvent was removed under reduced pressure
to yield 10. Colorless solid, mp 166 ꢀC, yield 4.65 g (90%).
DMSO-D6):
d
[ppm] ¼ 4.06 (d, J ¼ 6.4 Hz, 2H, NHCH2), 7.35 (d,
J ¼ 8.2 Hz, 2H, 3-HB, 5-HB), 7.57 (t, J ¼ 7.5 Hz, 2H, 3-HA, 5-HA),
7.60e7.64 (m, 1H, 4-HA), 7.80 (d, J ¼ 7.3 Hz, 2H, 2-HA, 6-HA), 7.84 (d,
J ¼ 8.1 Hz, 2H, 2-HB, 6-HB), 8.26 (t, J ¼ 6.4 Hz, 1H, SO2NHCH2), 12.81
(br s, 1H, CO2H). HPLC: tR ¼ 15.1 min, purity 98.2%.
C
20H23N3O4 (369.4). Rf ¼ 0.20 (ethyl acetate:cyclohexane ¼ 1:1,
detection: 254 nm). 1H NMR (400 MHz, DMSO-D6):
[ppm] ¼ 1.41
(s, 9H, (H3C)3C), 4.20 (d, J ¼ 6.2 Hz, 2H, NHCH2), 7.37 (d, J ¼ 8.2 Hz,
d

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
133
5.10. tert-Butyl 2-[4-(phenylsulfonamidomethyl)benzoyl]
hydrazine-1-carboxylate (13)
oocytes were constantly perfused with Ba2þ-Ringer containing
(mmol/L): 10 HEPES, 90 NaCl, 1 KCl, 1.5 BaCl2. pH was adjusted with
1 M NaOH to 7.4. Agonist solution contained 10 mM glycine and
A mixture of carboxylic acid 12i (5.00 g, 17.2 mmol) and SOCl2
(12 mL) was heated to 75 ꢀC for 1 h. Then excess of SOCl2 was
removed under reduced pressure. The resulting solid was sus-
pended in CH2Cl2 (50 mL) and a mixture of tert-butyl carbazate
(2.27 g, 17.2 mmol) in CH2Cl2 (100 mL) and pyridine (15 mL) was
added to the suspension. The reaction mixture was heated to reflux
for 2 h. Afterwards, 1 M HCl (200 mL) was added and the biphasic
system was treated with ultra-sonication for 5 min. The formed
precipitate was filtered off, washed with H2O and dissolved in THF.
Removal of the solvent under reduced pressure gave 13. Colorless
solid, mp 159 ꢀC, yield 6.91 g (99%). C19H23N3O5S (405.5). Rf ¼ 0.75
(ethyl acetate/CH2Cl2 1:1, detection: 254 nm). 1H NMR (600 MHz,
10 mM glutamate was freshly prepared from 100 mM stock solu-
tions in Ba2þ-Ringer for each measurement. Solutions of com-
pounds 5e6 and 2 were prepared from 10 mM DMSO stock
solutions by dilution with agonist solution to final concentrations of
1 nMe10 mM. For the screening of activity, compounds 5e6 and 2
were diluted to final concentrations of 500 nM. Recordings were
performed at a holding potential of ꢁ70 mV. Recording pipettes
were backfilled with 3 M KCl and had resistances in a range of
0.5e1.5 MU.
6.3. Data analysis
DMSO-D6):
d
[ppm] ¼ 1.43 (s, 9H, (H3C)3C), 4.06 (d, J ¼ 6.3 Hz, 2H,
Data were analyzed with Ana (Dr. Michael Pusch, Genova, Italy)
and OriginPro 2015 (OriginLab Corporation, Northampton, USA).
Inhibition was calculated by following equation:
NHCH2), 7.34 (d, J ¼ 7.9 Hz, 2H, 3-HB, 5-HB), 7.51e7.61 (m, 2H, 3-HA,
5-HA), 7.61e7.66 (m, 1H, 4-HA), 7.76 (d, J ¼ 7.9 Hz, 2H, 2-HB, 6-HB),
7.79e7.84 (m, 2H, 2-HA, 6-HA), 8.24 (t, J ¼ 6.3 Hz, 1H, SO2NHCH2),
8.88 (s, 1H, NHNHCO2), 10.14 (d, J ¼ 1.5 Hz, 1H, ArBCONHNH). HPLC:
tR ¼ 16.3 min, purity 99.7%.
Ic ꢁ Ih
Inhibtion ¼ 1 ꢁ
Ia ꢁ Ih
Ih is the holding current before adding agonist solution; Ia is the
current after adding of agonist solution; Ic is the resulting current in
presence of compound solution. To compare the activity of com-
pounds, the average inhibition of the compound was normalized to
the average inhibition of 2 in the same experimental measurement.
Inorm is determined as normalized Inhibition.
5.11. N-[(4-Hydrazinocarbonyl)benzyl]benzenesulfonamide (14)
A mixture of 13 (2.47 g, 6.09 mmol), CH2Cl2 (10 mL) and tri-
fluoroacetic acid (10 mL) was stirred at rt for 1 h. After complete
conversion monitored by TLC, the solvent was removed under
reduced pressure to yield the trifluoroacetate salt of 14 as colorless
solid. To obtain the free hydrazine this solid was dissolved in MeOH
and Et3N (6 mL) was added. The solvent was removed under
reduced pressure and the resulting solid was recrystallized from
MeOH/H2O to yield 14. Colorless solid, mp 148 ꢀC, yield 1.64 g (88%).
Inhibition500 nM compound
Inorm
¼
Inhibition500 nM TCNꢁ201
Dose response curves of 2 and 5i were fitted to logistic sigmoid
C
14H15N3O3S (305.4). Rf ¼ 0.34 (ethyl acetate, detection: 254 nm).
equitation:
1H NMR (400 MHz, DMSO-D6):
d
[ppm] ¼ 4.03 (s, 2H, NHCH2), 4.48
A1 ꢁ A2
(s, 2H, ArBCONHNH2), 7.29 (d, J ¼ 7.9 Hz, 2H, 2-HB, 6-HB), 7.54e7.66
(m, 3H, 3-HA, 4-HA, 5-HA), 7.73 (d, J ¼ 8.2 Hz, 2H, 3-HB, 5-HB),
7.78e7.83 (m, 2H, 2-HA, 6-HA), 8.23 (s, 1H, SO2NHCH2), 9.72 (s, 1H,
ArBCONHNH2). HPLC: tR ¼ 11.5 min, purity 98.2%.
y ¼
p þ A2
ꢀ ꢁ
x
1 þ
x0
A1 and A2 are defined as the minimal and maximal inhibition of
compound and were determined as A1 ¼ 0% and A2 ¼ 100%; p is
defined as slope of the curve; x0 is the concentration of half-
maximal inhibition and x is the tested concentration.
Significance of Inorm was tested for all screenings by One-way-
ANOVA and post hoc mean comparison Tukey Test.
6. Electrophysiological evaluation
6.1. Molecular biology
Defoliculated oocytes stage V were commercially obtained from
EcoCyte Bioscience (Castrop-Rauxel, Germany). They were coin-
jected with 0.8 ng GluN1a cRNA and 0.8 ng GluN2A cRNA using
nanoliter injector 2000 (WPI, Berlin, Germany). For selectivity
testing 0.8 ng of GluN2A cRNA was replaced by 0.8 ng GluN2B cRNA.
cRNA was generated by in vitro transcription with Ambion T7
mMessage mMachine kit (Life Technologies, Darmstadt, Germany)
from linearized cDNA templates (wildtype rat, subloned into
pSGEM vector, linearized with restriction enzyme PacI). Injected
oocytes were stored for 3e6 d at 18 ꢀC in Bath's solution containing
(mmol/L): 88 NaCl, 1 KCl, 0.4 CaCl2, 0.33 Ca(NO3)2, 0.6 MgSO4, 5
Tris-HCl, 2.4 NaHCO3 and supplemented with 80 mg/L theophyl-
line, 63 mg/L penicillin, 40 mg/L streptomycin and 100 mg/L
gentamycin.
Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinschaft which is gratefully acknowledged. Moreover,
we are grateful to Cells-in-Motion (CiM) Cluster of Excellence for
supporting this project.
Abbreviations
AMPA
2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic
acid
APCI
CNS
atmospheric pressure chemical ionization
central nervous system
6.2. Two electrode voltage clamp (TEVC)
COMU® (1-Cyano-1-ethoxycarbonylmethylenaminoxy)-
dimethylamino-morpholino-carbenium
hexafluorophosphate
Functional activity was tested by two electrode voltage clamp
(TEVC) at room temperature in Xenopus laevis oocytes using a Turbo
Tec 10CX amplifier (NPI electronic, Tamm, Germany), NI USB 6221
DA/AD Interface (National Instruments, Austin, USA) and GePulse
Software (Dr. Michael Pusch, Genova, Italy). For measurements
DIPEA
HRMS
NAM
N,N-diisopropylethylamine
high resolution mass spectroscopy
negative allosteric modulator
NMDA N-methyl-d-aspartate

134
S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134
modulation of NMDA receptor activity and the advent of negative and positive
PAM
SEM
positive allosteric modulator
standard error of the mean
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.02.018.
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