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

European Journal of Medicinal Chemistry 129 (2017) 124e134

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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 b c

Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, D-48149 Münster, Germany Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Muenster, D-48149 Muenster, Germany 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: Received 14 December 2016 Received in revised form 20 January 2017 Accepted 7 February 2017 Available online 14 February 2017

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-[(2benzoylhydrazino)carbonyl]benzyl}-3-bromobenzenesulfonamide) has 2.5-fold higher antagonistic activity than the lead compound 2 and represents our new lead compound. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: NMDA receptor GluN2A selective antagonists TCN-201 Synthesis Electrophysiology Two electrode voltage clamp Structure activity relationships Antagonistic activity

1. Introduction N-Methyl-D-aspartate receptors (NMDARs) are glutamate and 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: [email protected] (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.

Both endogenous agonists glycine and (S)-glutamate are required to activate the NMDA receptor [1,2]. With these three subtype families diheteromeric receptors, containing GluN1 and one type of GluN2 subunits can be formed. Additionally, triheteromeric receptors, 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 influence 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 developmental 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 containing 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 implicates a possible involvement of GluN2A subunit containing

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substitution pattern of 2. Since 2 was the result of a highthroughput screening [12e14]. systematic structure activity relationships 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 responsible 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).

NMDARs in the pathophysiology of these diseases [6]. Different studies demonstrated the involvement of GluN2A subunit hypofunction in the pathophysiology of chronic schizophrenia [7,8]. Moreover, GluN2A subunit containing NMDARs are also correlated to other neurological diseases like cerebral ischemia, seizure disorder, 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 selective 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 compounds 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]. 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 noncompetitive 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 modulators (PAMs) binding at the same or overlapping binding site as the NAM 2. One of the most active compounds with sufficient selectivity for GluN2A over other GluN2 subunits was the thiazolopyrimidine 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. 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

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

Br H N H 3C

PO3H2 H N

O

N H

O

Cl

H

H F3C

O

N S

N

O

O N H

TCN-201 (2)

CF3 N N

F F

O O S N H H3C

CH3

N H N

N O

Cl

GNE-0723 (3)

H N

F

NVP-AAM077 (1) NC

O O S N H

MPX-007 (4) Fig. 1. GluN2A selective NMDAR ligands.

S

N

126

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134

2

O O S N H

H N

X O

O O S N Alkyl H

O

H N

N H

O

O N H

7

5 O O S N Ar H

H N O

O N H

6 Fig. 2. Design of novel GluN2A selective NMDAR antagonists by systematic variation of lead compound 2.

Path A

H2N

BOC

a)

OH O

8

BOC

N H

OH

b)

N H O

O

9

H N

O N H

10

d)

c) O O

O O S N H

Path B S N R e) H

R

H N

OH

O

O

12

O

d)

N H

O O S R N H

H N

13

O

O N H

11 h)

g) N H

H N O

5-7

f) Path C

H2 N

BOC

O O S R N H

H N

14

NH2

O

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 purification 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 efficient 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 appropriate sulfonyl moieties (Scheme 2). For the synthesis of the 3-iodophenylsulfonamide 5k the carboxylic acid 12a was prepared by reaction of 3nitrobenzenesulfonyl chloride with amine 8. Subsequent esterification 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 acetamide 12c [25]. Treatment of 12c with boiling NaOH led to hydrolysis 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 phenylethynylbenzenesulfonamide 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

O O S N H

O 2N

O O S N H

X

b) OR

c)

15a: OR = OCH3 O O S N H

I

d)

O O S N H

O

15c: X = I

O

O O S N H

e) OH

N H

15b: X = NH3+Cl-

OH

12b

H3C

OCH3 O

O

12a: OR = OH

a)

O O S N H

OH

I

12d

f)

12f

H3N OCH3

Cl

15d

OH O

O

g)

O

O O S N H

12e

8

OH

H2N

O

12c

127

h)

O

O O S H 3C N H

15e: OR = OCH3 OR

12g: OR = OH

i)

O

Scheme 2. Reagents and reaction conditions: (a) SOCl2, MeOH, rt, 16 h, 79%; (b) 1. Zn, AcOH, MeOH, 0 C-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-activityrelationship 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 hydrazine led to the hydrazide 13. Removal of the Boc group provided the monoacylhydrazine 14, which was transformed into the diacylhydrazine 5a upon reaction with benzoyl chloride. 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 compounds are given in the Supporting Information.

3. Pharmacological activity The channel blocking activity of the synthesized test compounds was investigated electrophysiologically in two electrode voltage clamp (TEVC) measurements. For this purpose, Xenopus laevis oocytes expressing NMDARs with GluN1a and GluN2A

subunits were used for screening and dose response curves. In the assay 10 mM (S)-glutamate and 10 mM glycine were added for 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 derivative 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 show high but similar channel blockade, the 3fluorobenzenesulfonamide 5c was significantly less active. (p < 0.001). Compared to the lead compound 2, the 3-fluoro derivative 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

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 [%]

Path

Cpd.

2

78

B

5a

54

5b

R

Yield [%]

Path

5m

73

A

C

5n

94

A

74

A

5o

90

A

5c

62

A

5p

78

A

5d

80

A

5q

87

A

5e

61

A

5r

87

A

5f

63

A

5s

52

A

5g

77

A

5t

77

A

5h

26

A

5u

75

A

5i

67

A

5v

49

A

5j

93

A

5w

58

B

5k

93

B

5x

53

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 [%]

Path

Cpd.

60

A

5y

Yield [%]

Path

74

A

Yield [%]

Path

Cpd.

Yield [%]

Path

6a

30

A

6e

80

A

6b

44

A

6f

70

A

6c

75

B

6g

64

A

6d

68

A

6h

81

A

Yield [%]

Path

Cpd.

Yield [%]

Path

7a

71

B

7c

17

A

7b

8

A

7d

21

A

5l

R

Aryl residues with an extended p system and heterocycles 6 Cpd.

R

R

Alkylated derivatives 7 Cpd.

R

particular the high GluN2A activity of the 3chlorobenzenesulfonamide 5f indicates that the fluoro atom in 4position 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 inhibition compared to the 3-fluoro derivative 5c and the lead compound 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 activity. 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 4chlorobenzenesulfonamides 5w, 5j, 5x, 5g show comparable normalized inhibition as 5a, 4-fluoro-, 4-iodo- and 4nitrobenzesulfonamides 5d, 5l and 5o were almost inactive at a concentration of 500 nM.

R

Halogenation in 2-position of the benzenesulfonamide moiety (5b, 5e, 5h) causes a complete loss of activity. A NO2 group in 2position (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. 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 electron 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

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134

Compound

130

5e 5r 5o 5b 5h 5l 5d 5t 6g 5n 5g 5p 5x 5j 5a 5w 5m 6h 5c 2 5k 5f 5i

*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ns ns ns ***

-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.

2 5i

Table 2 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 reflects the number of independent oocytes.

100 90 80

Compd.

R

n

Normalized Inhibition Inorm

SEM

2 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5r 5t 5w 5x 6g 6h

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 4-I-phenyl 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

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 0.351 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

0.022 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

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

Inhibition (%)

70 60 50 40 30 20 10 0 0,001

0,01

0,1

1

10

Concentration (μM) Fig. 4. Dose response curves of 5i (grey) and 2 (black). Compounds were measured in a concentration range from 1 nM to 10 mM. Each concentration of each compound was measured in 5 different oocytes (n ¼ 5).

containing NMDARs was investigated electrophysiologically. Xenopus 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.

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134

131

coupling constants are given with 0.5 Hz resolution; the assignments 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

Inhibition (%)

70 60 50

ns

40

ns

30 20 10 0 5i

2

0,1 % DMSO

Ifenprodil

Compound Fig. 5. Inhibition of GluN2B containing NMDARs at a test compound concentration of 10 mM (n ¼ 5, DMSO as negative control, ifenprodil as positive control. One-Way ANOVA, post hoc mean comparison Tukey Test, ns p > 0.05).

4. Conclusion 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 3position 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. Experimental part 5.1. Chemistry, general Moisture sensitive reactions were conducted under dry nitrogen. 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 mm (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 appropriate 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 NMR: 151 MHz, 100 MHz); d in ppm related to tetramethylsilane;

Aromatic signals, which are part of the 4-(aminomethyl)benzoate 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: LiChrospher® 60 RP-select B (5 mm); LiCroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 mL; detection at l ¼ 210 nm; solvent A: demineralized H2O with 0.05% (v/v) trifluoroacetic acid; solvent B: acetonitrile with 0.05% (v/v) trifluoroacetic 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. 5.2. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl}-3-chloro-4fluorobenzenesulfonamide (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%). C21H17ClFN3O4S (461.9). Rf ¼ 0.80 (ethyl acetate, detection: 254 nm). 1H NMR (400 MHz, DMSO-D6): d [ppm] ¼ 4.14 (d, 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%. 5.3. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl} 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

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1. The formed precipitate was filtered and purified by flash column chromatography (ethyl acetate þ 1% NH3 (aq) 25%, Ø ¼ 2 cm, 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 (m, 2H, 2-HC, 6-HC), 8.27 (br s, 1H, SO2NHCH2), 10.48 (s, 2H, ArBCONHNHCOArC). HPLC: tR ¼ 15.1 min, purity 98.1%. 5.4. N-{4-[(2-Benzoylhydrazino)carbonyl]benzyl}-3bromobenzenesulfonamide (5i) 11 (135 mg, 0.50 mmol) and Na2B4O7 (1.00 g, 4.97 mmol) were suspended in H2O (30 mL) under ultra-sonication. 3Bromobenzenesulfonyl 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 overnight, 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). 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%. 5.5. 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 reaction 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 solvent 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 (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%. 5.6. 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 solution 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%). C20H23N3O4 (369.4). Rf ¼ 0.20 (ethyl acetate:cyclohexane ¼ 1:1, detection: 254 nm). 1H NMR (400 MHz, DMSO-D6): d [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,

2H, 2-HB, 6-HB), 7.47 (t, J ¼ 6.2 Hz, 1H, NHCH2), 7.50e7.56 (m, 2H, 3HC, 5-HC), 7.58e7.63 (m, 1H, 4-HC), 7.88 (d, J ¼ 8.1 Hz, 2H, 3-HB, 5HB), 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%.

5.7. 4-(Aminomethyl)-N'-benzoylbenzohydrazide (11) 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: 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%.

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 ultrasonication. 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 precipitate 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). Rf ¼ 0.88 (ethyl acetate þ 1% formic acid, detection: 254 nm). 1H 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, 6HB), 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%.

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 ultrasonication. 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/cyclohexane 1:1þ 1% acetic acid, detection: 254 nm). 1H NMR (400 MHz, 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%.

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134

5.10. tert-Butyl 2-[4-(phenylsulfonamidomethyl)benzoyl] hydrazine-1-carboxylate (13) 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 suspended 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, DMSO-D6): d [ppm] ¼ 1.43 (s, 9H, (H3C)3C), 4.06 (d, J ¼ 6.3 Hz, 2H, 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%. 5.11. N-[(4-Hydrazinocarbonyl)benzyl]benzenesulfonamide (14) A mixture of 13 (2.47 g, 6.09 mmol), CH2Cl2 (10 mL) and trifluoroacetic 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%). C14H15N3O3S (305.4). Rf ¼ 0.34 (ethyl acetate, detection: 254 nm). 1 H NMR (400 MHz, DMSO-D6): d [ppm] ¼ 4.03 (s, 2H, NHCH2), 4.48 (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%. 6. Electrophysiological evaluation 6.1. Molecular biology Defoliculated oocytes stage V were commercially obtained from EcoCyte Bioscience (Castrop-Rauxel, Germany). They were coinjected 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 theophylline, 63 mg/L penicillin, 40 mg/L streptomycin and 100 mg/L gentamycin.

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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 10 mM glutamate was freshly prepared from 100 mM stock solutions in Ba2þ-Ringer for each measurement. Solutions of compounds 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 Data were analyzed with Ana (Dr. Michael Pusch, Genova, Italy) and OriginPro 2015 (OriginLab Corporation, Northampton, USA). Inhibition was calculated by following equation:

Inhibtion ¼ 1 

Ic  Ih 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 compounds, 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.

Inorm ¼

Inhibition500 nM compound Inhibition500 nM TCN201

Dose response curves of 2 and 5i were fitted to logistic sigmoid equitation:

y¼

A1  A2  p þ A2 1 þ xx0

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 halfmaximal inhibition and x is the tested concentration. Significance of Inorm was tested for all screenings by One-wayANOVA and post hoc mean comparison Tukey Test. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft which is gratefully acknowledged. Moreover, we are grateful to Cells-in-Motion (CiM) Cluster of Excellence for supporting this project. Abbreviations AMPA

6.2. Two electrode voltage clamp (TEVC)

APCI CNS COMU®

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 NMDA

2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid atmospheric pressure chemical ionization central nervous system (1-Cyano-1-ethoxycarbonylmethylenaminoxy)dimethylamino-morpholino-carbenium hexafluorophosphate N,N-diisopropylethylamine high resolution mass spectroscopy negative allosteric modulator N-methyl-d-aspartate

134

PAM SEM

S.L. Müller et al. / European Journal of Medicinal Chemistry 129 (2017) 124e134

positive allosteric modulator standard error of the mean

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Appendix A. Supplementary data

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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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[24] [25] [26]

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