Hydroxymethyl bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists: Design, synthesis and pharmacological evaluation

Accepted Manuscript Hydroxymethyl bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists: Design, synthesis and pharmacological evaluation Louisa Temme, Bastian Frehland, Dirk Schepmann, Dina Robaa, Wolfgang Sippl, Bernhard Wünsch PII:

S0223-5234(17)31085-1

DOI:

10.1016/j.ejmech.2017.12.054

Reference:

EJMECH 10032

To appear in:

European Journal of Medicinal Chemistry

Received Date: 24 October 2017 Revised Date:

14 December 2017

Accepted Date: 15 December 2017

Please cite this article as: L. Temme, B. Frehland, D. Schepmann, D. Robaa, W. Sippl, B. Wünsch, Hydroxymethyl bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists: Design, synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Hydroxymethyl bioisosteres of phenolic GluN2B-selective NMDA receptor antagonists: Design, synthesis and pharmacological evaluation

Louisa Temme,[a,b] Bastian Frehland,[a] Dirk Schepmann,[a] Dina Robaa,[c] Wolfgang

[a]

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Sippl,[c] Bernhard Wünsch*[a,b]

L. Temme, Dr. D. Schepmann, Prof. Dr. Wünsch, Institut für Pharmazeutische und

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Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149

[email protected] [b]

Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische WilhelmsUniversität Münster, Germany

[c]

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Münster, Germany Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail:

Dr. Dina Robaa, Prof. Dr. W. Sippl, Institut für Pharmazie der Martin-Luther-

Abstract

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(Saale), Germany

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Universität Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120 Halle

Antagonists addressing selectively NMDA receptors containing the GluN2B subunit

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are of particular interest for the treatment of various neurological disorders including neurodegenerative

diseases.

With

the

aim

to

bioisosterically

replace

the

metabolically labile phenol of 7-amino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-ols, several analogs were docked into the ifenprodil binding site leading to the hydroxymethyl derivatives 4 as promising candidates. They display the same binding pose as Ro 25-6981 and the same H-bond interactions with Gln110 and Glu236 within the GluN2B subunit. The phenylalkyl moieties occupy the hydrophobic pocket formed predominantly by Pro78 (GluN2B), Phe114 (GluN2B), and Tyr109 (GluN1b).

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ACCEPTED MANUSCRIPT Starting from o-phthalaldehyde, the hydroxymethyl derivatives 4 were prepared in a 7-step synthesis with a haloform reaction of trichloroacetophenone 7 as key step. In receptor binding studies, the phenylpropyl derivative 4a shows promising GluN2B affinity (Ki = 101 nM) and high selectivity over the PCP binding site and both σ

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receptor subtypes. 4a was able to inhibit the glutamate/glycine induced cytotoxicity at mouse fibroblasts with an IC50 value of 5.2 µM. It is assumed that the hydroxymethyl moiety of 4a stabilizes the closed channel conformation by an H-bond with Glu236 as

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does the phenolic OH moiety of 3, Ro 25-6981 and ifenprodil.

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Keywords: docking studies; NMDA receptor; GluN2B antagonists; ifenprodil binding site; affinity; selectivity; cytoprotective activity; structure-affinity relationships; structure-activity relationships

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1. Introduction

The N-Methyl-D-aspartate (NMDA) receptor consisting of two GluN1 and two GluN2B subunits is involved in the development of various neurodegenerative diseases, like

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Alzheimer´s disease, Huntington´s disease, Parkinson´s disease, cerebral ischemia

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and traumatic brain injury, mood disorders, neuropsychiatric systemic lupus erythematosus, cognitive impairments and pain.[1-5] As example, the amyloid-β protein is suggested to play an important role in the development of Alzheimer’s disease. The soluble oligomeric forms of amyloid-β are believed to disturb synaptic function.[6] Amyloid-β induces a deficit in glutamate uptake by excitatory amino acid transporters (EAATs). The resulting increase in glutamate in the synaptic cleft probably activates extrasynaptic GluN2B subunit containing NMDA receptors which affects long-term potentiation (LTP) involved in memory and learning.[4] GluN2B-

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ACCEPTED MANUSCRIPT selective antagonists prevent this amyloid-β induced impairment of LTP [7-9] and amyloid-β induced synaptic loss.[4.7]

The binding mode of GluN2B antagonists was analyzed with the highly potent

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GluN2B selective NMDA receptor antagonist Ro 25-6981 (1). (Figure 1) Its interaction with the ifenprodil binding pocket, which is termed after the prototypical GluN2B antagonist ifenprodil, was studied in detail on the molecular level.[10]

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Binding of Ro 25-6981 (1) influences the amino terminal domain (ATD) and the ligand binding domain (LBD) located extracellularly, as well as the transmembrane domain

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(TMD) forming the ion channel. After binding of an allosteric inhibitor to the ATD, the coupling between the ATD and the LBD is strengthened and interactions within the LBD are stabilized. As a result, the agonists (S)-glutamate and glycine bound in the LBD cannot cause the required rotation by around 13.5° anymore and the movement

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of the LBD-TMD linker leading to ion channel opening is inhibited. Thus, binding of an allosteric inhibitor at the ATD, although far away from the TMD, stabilizes the

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receptor in an “agonist-bound desensitized state” with a closed ion channel pore.[10]

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ACCEPTED MANUSCRIPT Figure 1. Design of GluN2B selective ligands 4 with benzo[7]annulene scaffold by conformational restriction and bioisosteric replacement of the phenolic OH moiety by a hydroxymethyl moiety.

Based on these findings, it is of great interest to determine the structural

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requirements for functionally active, GluN2B selective ligands. Therefore, we investigated compounds obtained by a conformational restriction approach on Ro 256981 (1). (Figure 1) The flexible three-carbon spacer of 1 was connected to the

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phenol, which led formally to benzo[7]annulen-7-amines.[11] As an example, the cis-

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configured phenylpropylamine 2 was prepared and proved the concept by showing high GluN2B affinity (Ki = 16 nM). Removal of the benzylic OH moiety of 1 and 2 resulted in the benzo[7]annulen-2-ols 3 with still high GluN2B affinity indicating that the benzylic OH moiety is not essential for high GluN2B affinity. Moreover, the phenols

3

displayed

inhibition

of

cytotoxicity

(cytoprotection)

in

a

(S)-

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glutamate/glycine induced cytotoxicity assay using mouse fibroblasts.[12]

In order to get rid of the phenolic OH moiety in 2-position,[13,14] which is prone to

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fast biotransformation (e.g. glucuronidation),[15,16] several analogs of 3 with different

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substituents in 2-position were prepared. Compounds with a NO2, Cl and OCH3 group in 2-position showed very high GluN2B affinity. Unfortunately, these derivatives could not inhibit the opening of the ion channel. It was concluded that benzo[7]annulenamines of type 3 with NO2, Cl or OCH3 moiety in 2-position fit nicely into the ifenprodil binding pocket, but did not stabilize the inactive conformation of the ion channel. Obviously, the phenolic OH moiety of 3a and 3b is essential for the stabilization of the heterotetrameric NMDA receptor in the inactive conformation resulting in the inhibition of ion channel opening.[12]

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ACCEPTED MANUSCRIPT Therefore, the idea came up to replace the OH moiety in 2-position of 3 by another H-bond donor group, i.e. the hydroxymethyl moiety (4). The lower conversion of aliphatic alcohols in comparison to phenols is described for five out of eight human UDP-glucuronosyltransferases.[17] Moreover, only one out of seven human cytosolic

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sulfotransferases converts a benzylic alcohol while phenolic compounds are xenobiotic substrates of the remaining six enzymes.[18] In this report, at first docking studies were performed to predict the possible interactions of the hydroxymethyl

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derivatives 4 with GluN2B subunit containing NMDA receptors. Subsequently, the

evaluated.

2. Results and Discussion

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hydroxymethyl derivatives 4 were synthesized and their pharmacological activity was

2.1. Docking of hydroxymethyl derivatives 4 into the ifenprodil binding site

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In order to analyze the ligand-protein interactions, a set of hydroxymethyl substituted benzo[7]annulen-7-amines was docked into the ifenprodil binding site of GluN2B subunit containing NMDA receptors. For this purpose, the crystal structure of the

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GluN1b/GluN2B NMDA receptor (PDB ID 3QEM) was used. 3QEM represents a dimer of the ATDs of a GluN1b and GluN2B subunit co-crystallized with the

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antagonist Ro 25-6981.[19] The docking process was performed using the docking software Glide® (Schrödinger®) as described in the Methods section.

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Figure 2: Predicted binding modes of the hydroxymethyl derivatives 4a (Figure 2A) and 4b (Figure 2B) and the phenol 3b (Figure 2C). Binding mode of the co-

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crystallized ligand Ro 25-6981 (Figure 2D). Docking was performed with Glide® and images were generated with Pymol®. The GluN1b subunit (white) is orientated in the bottom and the GluN2B (pale cyan) subunit at the top. Blue-dashed lines indicate H-

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bond interactions while green-dashed lines display CH-π interactions. Docked ligands are shown as yellow sticks, co-crystallized Ro 25-6981 as purple sticks and

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the water molecule as red sphere. Oxygen and nitrogen atoms are colored in red and blue, respectively.

Compounds 4a and 4b with a phenylpropyl- or phenylbutylamino moiety at 7-position were of particular interest, because they nicely fulfill the basic requirements for binding at the ifenprodil binding site of GluN2B subunit containing NMDA receptors. (Figures 2A and 2B) The docking results showed that the hydroxymethyl compounds 4a and 4b adopt the same orientation in the binding pocket as the co-crystallized

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ACCEPTED MANUSCRIPT potent GluN2B antagonist Ro 25-6981. (Figure 2D) As the co-crystallized ligand Ro 25-6981, both hydroxymethyl derivatives 4a and 4b form H-bonds between their protonated (positively charged) amino groups and the carbonyl moiety in the side chain of Gln110 (GluN2B). The phenylpropyl group of 4a and the phenylbutyl group

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of 4b occupy a hydrophobic pocket formed predominantly by Pro78 (GluN2B), Phe114 (GluN2B), and Tyr109 (GluN1b). This is in good accordance to the previously reported docking results of benzo[7]annulen-7-amines.[12,18] Compounds

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4a and 4b do not only meet these basic requirements, but the hydroxymethyl group also displays H-bond interactions with the carboxy group of Glu236 (GluN2B) and the

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conserved water molecule (H2O410). Interactions of this conserved water molecule with the phenolic OH moiety also contributes to the stabilization of Ro 25-6981 and ifenprodil

(PDB

ID

3QEL)

in

their

binding

sites.[12]

Docking

of

the

benzo[7]annulenamine 3b with phenol substructure (Figure 2C) [12] into the

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ifenprodil binding site resulted in the same binding pose and the same interactions with Glu236 and Gln110 as obtained for the hydroxymethyl derivatives.

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Since the phenolic benzo[7]annulenamines 3a and 3b with a phenylpropyl and phenylbutyl side chain displayed high GluN2B affinity [12], these N-substituents were

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kept unchanged in the analogous hydroxymethyl derivatives 4a and 4b. Although the replacement of the hydroxy moiety in 3 by the hydroxymethyl moiety in 4 should result in a shift of the bound molecule to the hydrophobic pocket, the polar interactions between the central protonated amino group of 4 and the amide of Gln110 were not affected in the obtained docking results. The observed polar interactions of 4 with Gln110 and Glu236 together with the occupation of the hydrophobic pocket by the phenylalkyl side chains stimulated the synthesis and pharmacological evaluation of hydroxymethyl derivatives 4.

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2.2. Synthesis The synthesis of hydroxymethyl derivatives 4 started with ketone 5, which was obtained

in

three

reaction

steps

from

and

dimethyl

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oxoglutarate.[21]

o-phthalaldehyde

Scheme 1: Synthesis of hydroxymethyl substituted benzo[7]annulen-7-amines 4a and

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4b. Reagents and reaction conditions: (a) CH3COCl, AlCl3, CH2Cl2, 0 °C, 42 %. (b) Cl3CCOCl. AlCl3. CH2Cl2, 0 °C, 17 %. (c) 2 M NaOH, 1,4-dioxane, rt, 93 %. (d) 1. RNH2, CH3OH, Na2SO4; 2. NaBH4, rt, 93 % (9a), 61 % (9b). (e) LiAlH4, THF, 0 °C to

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rt, 18 % (4a), 39 % (4b).

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Initial attempts to introduce the hydroxy- or halomethyl moiety into ketone 5 by hydroxymethylation with paraformaldehyde,[22] chloromethylation according to Blanc or alkylation with CH2Br2[23] failed to give any substitution product. Next, ketone 5 was reacted with acetyl chloride and AlCl3 in a Friedel Crafts acylation [24,25] to yield the acetophenone 6 in 42 % yield. However, various attempts to transform the acetophenone 6 into the sodium salt of carboxylic acid 8 by a haloform reaction (Br2/NaOH,

I2/KI/NaOH,

KI/NaOCl,

NaOCl/NaOH/Na2CO3)

[26-28]

led

to

decomposition of the starting material, probably due to similar reactivity of the methyl and methylene moieties in α-position of the ketones. (Scheme 1)

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In order to circumvent the halogenation of the methyl moiety of 6 the Friedel-Crafts acylation of ketone 5 was performed with trichloroacetyl chloride [29] providing the

carboxylate 8 in 93 % yield upon removal of CHCl3.

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trichloroacetophenone 7. Reaction of ketone 7 with NaOH [30] led to the sodium

In previous studies it was shown that phenylpropyl- and phenylbutylamino groups in

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7-position led to high GluN2B affinity.[12] Therefore, ketone 8 was reacted with 3phenylpropyl- and 4-phenylbutylamine to form imines, which were reduced

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subsequently with NaBH4 [31] to afford the amino acids 9a and 9b. Finally, LiAlH4 reduction [32] provided the hydroxymethyl substituted benzo[7]annulenamines 4a and 4b in 18 % and 39 % yield, respectively.

2.3.1. GluN2B affinity

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2.3. Pharmacological evaluation

The GluN2B affinity of the phenols 3 and the hydroxymethyl derivatives 4 was

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determined in a radioligand binding assay.[33] L(tk-) cells stably transfected with a vector containing the genetic information of GluN1a and GluN2B subunits expressed

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the NMDA receptor upon incubation with dexamethasone. The compounds were tested in a competition assay using membrane preparations of these cells and [3H]labeled ifenprodil.[33]

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ACCEPTED MANUSCRIPT Table 1. Receptor affinity of hydroxymethyl derivatives 4 compared with the analogous phenols 3 and reference compounds. NH R

n

R

n

GluN2B

PCP

3a [12]

OH

3

28 ± 5

0%

3b [12]

OH

4

21 ± 5

25 %

4a

CH2OH

3

101 ± 21

4b

CH2OH

4

186 ± 64

dexoxadrol haloperidol

123

32 ± 13

211

96 ± 24

565

592

0%

41 ± 6

271

-

125 ± 24

98 ± 34

13 ± 2.0

-

-

-

-

32 ± 7.4

-

-

-

-

6.3 ± 1.6

78 ± 2.3

10 ± 0.7

Eliprodil

σ2

0%

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Ifenprodil

σ1

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compd.

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Ki ± SEM [nM] (n = 3)a

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di-o89 ± 29 57 ± 18 tolylguanidine a The Ki values of potent compounds were recorded three times (n = 3). For lowaffinity or very low-affinity compounds the competition curves were recorded only once (single value) or the inhibition (in %) of the radioligand binding at a test

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compound concentration of 1 µM is given.

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In a previous study the GluN2B affinity of the phenols 3a and 3b was determined resulting in Ki values of 28 nM and 21 nM, respectively.[12] The GluN2B affinities of the homologous hydroxymethyl derivatives 4a and 4b are 4-fold and 9-fold reduced compared to the GluN2B affinity of the phenols. Although the GluN2B affinity of the hydroxymethyl derivatives 4a and 4b is decreased, they still show moderate GluN2B affinity (Ki = 101 nM, Ki = 186 nM).

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ACCEPTED MANUSCRIPT The CO2Na moiety of 9a and 9b also represents an interesting bioisostere of the phenolic OH moiety of 3a and 3b, which is comparable with the hydroxymethyl moiety of 4a and 4b and the 2-NO2 moiety of analogous potent ligands.[20]

and Dulbecco Modified Earl’s Medium.

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Unfortunately, 9a and 9b could not be tested due to their low solubility both in DMSO

2.3.2. Selectivity against related binding sites and receptors

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For further characterization of the hydroxymethyl derivatives, the affinity towards the phencyclidine (PCP) binding site and σ1 and σ2 receptors was determined.[34-38]

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The PCP binding site in the TMD of the NMDA receptor is of particular interest, because it should be excluded as a possible binding site for novel NMDA receptor antagonists. Both hydroxymethyl derivatives 4a and 4b did not show any affinity to the PCP binding site in a competition assay with the radioligand [3H](+)-MK-801.

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(Table 1) Thus, both compounds show high selectivity for the ifenprodil binding site of GluN2B receptors over the PCP binding site, which is in good accordance with the

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high selectivity of phenols 3a and 3b.

Due to the moderate affinity of ifenprodil towards σ1 and σ2 receptors, the affinity of

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the hydroxymethyl derivatives 4a and 4b towards these receptors was also investigated in radioligand binding assays.[36-38] The phenylpropyl derivative 4a displays 5-6-fold lower affinity towards σ1 and σ2 receptors, indicating moderate selectivity over these receptors. (Table 1) For the homolog 4b increased σ1 and σ2 affinity was observed leading to the same affinity towards both ifenprodil binding site and σ2 receptor. Importantly, the recorded Ki-value for the interaction of 4b with σ1 receptors is approximately 4-fold lower than the Ki-value indicating GluN2B affinity resulting in reversed selectivity.

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With respect to selectivity, the phenylpropyl derivative 4a represents the most promising GluN2B ligand, as its affinities towards the PCP binding site of the NMDA receptor and both σ receptor subtypes are considerably lower than the affinities of

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the corresponding phenols 3a,b and the phenylbutyl homolog 4b. Altogether, the hydroxymethyl group is able to maintain GluN2B affinity, but increases the selectivity

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over both σ receptor subtypes or at least keeps it constant.

2.3.3. Functional activity

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The functional activity of the GluN2B ligands 4a and 4b and the reference compound Ro 25-6981 was determined in a lactate dehydrogenase (LDH) based cytotoxicity assay and the results were compared with the previously recorded activity of the phenols 3a and 3b.[12] In this cytotoxicity assay, the cell death of mouse fibroblast

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L(tk-) cells overexpressing NMDA receptors containing only GluN1a and GluN2B subunits upon treatment with (S)-glutamate and glycine is determined. As a correlate for cell death the amount of released LDH is measured. Cytoprotective compounds

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and glycine.

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decrease the amount of LDH in the supernatant after stimulation with (S)-glutamate

As described previously, phenols 3a and 3b showed low cytoprotective effects with IC50-values of 25 µM and 3.0 µM, respectively. (Table 2) [12] The homologous hydroxymethyl derivatives 4 behaved differently: Whereas the phenylpropyl derivative 4a revealed an IC50-value of 5.2 µM, the phenylbutyl derivative 4b could not protect the cells against the toxic effects of (S)-glutamate and glycine even at the highest concentration of 100 µM. (Table 2)

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ACCEPTED MANUSCRIPT Table 2. Cytoprotective effects of benzo[7]annulen-7-amines 3 and 4 and reference compounds ifenprodil and Ro 25-6981 correlated with their GluN2B affinity.

R

n

Ki ± SEM (nM) (n = 3)

cytoprotective activity IC50 ± SEM (µM) (n = 3)

3a [12]

OH

3

28 ± 5

25 ± 8.0

3b [12]

OH

4

21 ± 5

4a

CH2OH

3

101 ± 21

4b

CH2OH

4

186 ± 64

ifenprodil

-

-

10 ± 0.7

Ro 25-6981

-

-

173 ± 26

3.0 ± 0.2

5.2 ± 3.0

>100 µM

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0.59 ± 0.2

0.018 ± 0.002

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compd.

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GluN2B affinity

The dose-dependent cytoprotective activity of 4a is displayed in Figure 3. The cytoprotective effects of the hydroxymethyl derivatives 4 correlate with their GluN2B affinity as the phenylpropyl derivative 4a possesses higher GluN2B affinity than the

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phenylbutyl derivative 4b. In case of the phenols 3 the cytoprotective effect also

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correlates with the GluN2B affinity.

Figure 3: Cytoprotective effect of hydroxymethyl derivative 4a (curve resulting from one experiment is shown).

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The IC50 values of the hydroxymethyl derivative 4a and phenol 3b are comparable which can be explained by similar interactions within the binding pocket. (see Figure 2) However, the moderate to high potency of ifenprodil and Ro 25-6981 (1) was not

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achieved. (Table 2) These observations extend the previously described structural requirements for active compounds.[12] A phenolic OH moiety in 2-position contributes considerably to the cytoprotective activity.[12] Herein it was shown that a

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hydroxymethyl moiety in 2-position is also able to preserve the cytoprotective activity

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of benzo[7]annulene based GluN2B ligands such as 4a.

3. Conclusion

In order to obtain metabolically more stable negative allosteric modulators of GluN2B subunit containing NMDA receptors, replacement of the phenolic hydroxy group of 3

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by the hydroxymethyl moiety was envisaged. Both the phenolic hydroxy and the hydroxymethyl moiety can react as H-bond donor. However, the hydroxymethyl moiety is less acidic than the phenolic hydroxy moiety and, moreover, is less prone to

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glucuronidation and sulfatation during biotransformation in the liver.

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In docking studies, the hydroxymethyl derivatives 4 adopt the same binding pose as the phenols 3 and Ro 25-6981. Furthermore, the same crucial interactions with the ifenprodil binding site were found: H-bond between protonated (positively charged) amino moiety and the carbonyl moiety of Gln110 (GluN2B), H-bond between hydroxymethyl moiety and Glu236 (GluN2B) and between hydroxymethyl moiety and a conserved water molecule, hydrophobic interactions between the phenylalkyl substituent and Phe114 (GluN2B), Pro78 (GluN2B) and Tyr109 (GluN1b).

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ACCEPTED MANUSCRIPT The key steps in the synthesis of 4 were the Friedel-Crafts acylation of ketone 5 with trichloroacetyl chloride and subsequent cleavage of the trichloromethyl ketone 7 with NaOH to provide the carboxylate 8. Reductive amination of the ketone 8 led to secondary amines 9, which were reduced with LiAlH4 to obtain the hydroxymethyl

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derivatives 4.

In receptor binding studies the hydroxymethyl derivatives 4a and 4b show promising

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GluN2B affinity of 101 nM and 183 nM. The phenylpropylamine 4a exhibits high selectivity over the PCP binding site of the NMDA receptor and moderate selectivity

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over σ1 and σ2 receptors. Its cytoprotective potential is approximately 10-fold lower than that of ifenprodil. It is concluded that the H-bond between Glu236 and the hydroxymethyl moiety of 4a is responsible for stabilization of the closed receptor

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conformation.

Variation of the arylalkyl side chain length led to opposite effects of the phenols 3 and hydroxymethyl derivatives 4. Whereas the phenol 3a with phenylpropyl side chain

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shows lower GluN2B affinity and cytoprotective activity than the phenylbutyl homolog

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3b, the phenylpropyl derivative 4a with hydroxymethyl moiety represents the more potent GluN2B antagonist. A reason for the different behavior of the phenylpropyl and phenylbutyl derivatives could be the additional methylene moiety between the benzene ring and the OH moiety of the hydroxymethyl derivatives 4, which shifts the whole molecule towards the hydrophobic pocket. (see Figure 2) Due to this shift the smaller phenylpropyl substituent fits better into the hydrophobic pocket.

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ACCEPTED MANUSCRIPT 4. Experimental Part 4.1. Chemistry, General Methods Oxygen and moisture sensitive reactions were carried out under nitrogen, dried with silica gel with moisture indicator (orange gel, VWR, Darmstadt, Germany) and in dry

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glassware (Schlenk flask or Schlenk tube). Temperatures were controlled with dry ice/acetone (-78 °C), ice/water (0 °C), Cryostat (Julabo TC100E-F, Seelbach, Germany), magnetic stirrer MR 3001 K (Heidolph, Schwalbach, Germany) or RCT CL

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(IKA, Staufen, Germany), together with temperature controller EKT HeiCon (Heidolph) or VT-5 (VWR) and PEG or silicone bath. All solvents were of analytical or

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technical grade quality. Demineralized water was used. CH2Cl2 was distilled from CaH2; THF was distilled from sodium/benzophenone; MeOH was distilled from magnesium methanolate. Thin layer chromatography (tlc): tlc silica gel 60 F254 on aluminum sheets (VWR). Flash chromatography (fc): Silica gel 60, 40–63 µm (VWR);

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parentheses include: diameter of the column (d), length of the stationary phase (l), fraction size (V) and eluent. Automated flash chromatography: IsoleraTM Spektra One (Biotage®); parentheses include: cartridge size, flow rate, eluent, fractions size was

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always 20 mL. Melting point: Melting point system MP50 (Mettler Toledo, Gießen, Germany), open capillary, uncorrected. MS: MicroTOFQII mass spectrometer (Bruker

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Daltonics, Bremen, Germany); deviations of the found exact masses from the calculated exact masses were 5 mDa or less; the data were analyzed with DataAnalysis® (Bruker Daltonics). NMR: NMR spectra were recorded in deuterated solvents on Agilent DD2 400 MHz and 600 MHz spectrometers (Agilent, Santa Clara CA, USA); chemical shifts (δ) are reported in parts per million (ppm) against the reference substance tetramethylsilane and calculated using the solvent residual peak of the undeuterated solvent; coupling constants are given with 0.5 Hz resolution; assignment of 1H and

13

C NMR signals was supported by 2-D NMR techniques

17

ACCEPTED MANUSCRIPT where necessary.IR: FT/IR IR Affinity®-1 spectrometer (Shimadzu, Düsseldorf, Germany) using ATR technique.

4.2. HPLC method for the determination of the purity

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Pump: LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface: DIONEX UltiMate 3000, data acquisition: Chromeleon 7 (equipment and software from Thermo Fisher Scientific, Lauenstadt, Germany);

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column: LiChrospher® 60 RP-select B (5 µm), LiChroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A:

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demineralized water with 0.05 % (V/V) trifluoroacetic acid, B: CH3CN with 0.05 % (V/V) trifluoroacetic acid; gradient elution (% A): 0 - 4 min: 90 %; 4 - 29 min: gradient from 90 % to 0 %; 29 - 31 min: 0 %; 31 - 31.5 min: gradient from 0 % to 90 %; 31.5 -

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40 min: 90 %.

4.3. Synthetic procedures

4.3.1. 2-Acetyl-5,6,8,9-tetrahydrobenzo[7]annulen-7-one (6)

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Under N2, a suspension of AlCl3 (approx. 6 g) in dry CH2Cl2 (20 mL) was cooled down to 0 °C and acetyl chloride (2.3 mL, 30 mmol, 4 eq.) was added dropwise. Then, a

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solution of ketone 5 (1.19 g, 7.4 mmol, 1 eq.) in dry CH2Cl2 (5 mL) was added at 0 °C. After stirring for 1.5 h at 0 °C, the reaction mixture was poured into a mixture of CH2Cl2 (10 mL) and ice (10 mL). The aqueous layer was separated and extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were washed with water (2 x 20 mL) and brine (20 mL), dried (Na2SO4) and concentrated under reduced pressure. The crude product was purified by fc (d = 3 cm, l = 15 cm, V = 20 mL, cyclohexane/ethyl acetate 80:20). Yellow oil, yield 632 mg (42 %), C13H14O2 (202.3 g/mol). Rf = 0.42 (cyclohexane/ethyl acetate 67:33). FT-IR (neat): ν̃ (cm-

18

ACCEPTED MANUSCRIPT 1

) = 2955 (C-Haliph.); 1678 (C=O), 1605, 1570 (C=Carom.), 883, 833 (Ar-Hout

of plane).

1

H NMR (400 MHz, CDCl3): δ (ppm) = 2.60 (s, 3H, C(O)CH3), 2.61 – 2.66 (m, 4H, 6-

CH2, 8-CH2), 2.94 – 3.01 (m, 4H, 5-CH2, 9-CH2), 7.32 (d, J = 7.8 Hz, 1H, 4-H), 7.80 (dd, J = 7.7/1.9 Hz, 1H, 3-H), 7.83 (d, J = 1.5 Hz, 1H, 1-H).

13

C NMR (151 MHz,

RI PT

CDCl3): δ (ppm) = 26.8 (1C, C(O)CH3), 30.6 (1C, C-9), 30.7 (1C, C-5), 44.3 (1C, C-6 or C-8), 44.5 (1C, C-8 or C-6), 127.6 (1C, C-3), 129.1 (1C, C-1), 129.6 (1C, C-4), 136.3 (1C, C-2), 141.1 (1C, C-9a), 146.3 (1C, C-4a), 197.9 (1C, C(O)CH3), 210.5

M AN U

[M+H]+). Purity (HPLC): 98.6 % (tR = 16.7 min).

SC

(1C, C-7). Exact mass (APCI): m/z = 203.1069 (calcd. 203.1067 for C13H15O2

4.3.2. 2-(Trichloroacetyl)-5,6,8,9-tetrahydrobenzo[7]annulen-7-one (7) Under N2 atmosphere, a suspension of AlCl3 (approx. 4 g) in dry CH2Cl2 (15 mL) was cooled down to -10 °C. Successively trichloroacetyl chloride (1.4 mL, 13 mmol, 2 eq.)

TE D

and ketone 5 (1.03 g, 6.5 mmol, 1 eq.) dissolved in dry CH2Cl2 (5 mL) were added dropwise. The reaction mixture was stirred for 62 h at 0 °C. After pouring into a mixture of CH2Cl2 (25 mL) and ice (25 mL), the aqueous layer was extracted with

EP

CH2Cl2 (3 x 20 mL). The combined organic layers were washed with water (2 x 20 mL) and brine (20 mL), dried (Na2SO4) and concentrated in vacuo. The crude

AC C

product was purified by automated flash chromatography (cartridge: SNAP 50 g, flow rate 50 mL/min, cyclohexane/ethyl acetate 95:5). Yellow solid, mp 96 °C, yield 331 mg (17 %), C13H11Cl3O2 (305.6 g/mol). Rf = 0.59 (cyclohexane/ethyl acetate 67:33). FT-IR (neat): ν̃ (cm-1) = 2951 (C-Haliph.), 1701 (C=O), 1605, 1566 (C=Carom.), 894, 833 (Ar-Hout of plane). 1H NMR (600 MHz, CDCl3): δ (ppm) = 2.64 – 2.68 (m, 4H, 6CH2, 8-CH2), 2.98 – 3.02 (m, 4H, 5-CH2, 9-CH2), 7.35 (d, J = 8.0 Hz, 1H, 4-H), 8.11 (d, J = 1.9 Hz, 1H, 1-H), 8.15 (dd, J = 8.0/2.0 Hz, 1H, 3-H).

13

C NMR (151 MHz,

CDCl3): δ (ppm) = 30.7 (1C, C-9), 30.8 (1C, C-5), 44.0 (1C, C-6 or C-8), 44.4 (1C, C-

19

ACCEPTED MANUSCRIPT 8 or C-6), 128.1 (1C, C-2), 129.4 (1C, C-4), 130.7 (1C, C-1), 132.4 (1C, C-3), 141.2 (1C, C-9a), 147.8 (1C, C-4a), 181.0 (1C, COCCl3), 210.0 (1C, C-7). Exact mass (APCI): m/z = 304.9892 (calcd.

304.9897

for

[M+H]+).

C13H12Cl3O2

Purity

RI PT

(HPLC): 94.5 % (tR = 22.3 min, strong fronting).

4.3.3. Sodium 7-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-2-carboxylate (8) NaOH

(2 M,

590 µL,

1.2 mmol,

1.1 eq.)

was

added

to

a

solution

of

SC

trichloroacetophenone 7 (331 mg, 1.1 mmol, 1 eq.) in 1,4-dioxane (1 mL). The reaction mixture was stirred for 6 min at rt. The resulting suspension was filtered

M AN U

under reduced pressure and the residue was washed with 1,4-dioxane (3 x 1 mL). The filtrate was filtered again without reduced pressure and washing. The combined solid residues were dried (Na2SO4), concentrated in vacuo and the residue was used without further purification. Colorless solid, mp 287 °C, yield 215 mg (88 %),

TE D

C12H11O3Na (226.2 g/mol). Rf = 0.57 (cyclohexane/ethyl acetate 67:33). FT-IR (neat): ν̃ (cm-1) = 2954 (C-Haliph.), 1690 (C=O), 1589 (C=Carom.), 1547 (CO2-asym), 1369, 1346 (CO2-sym), 856, 795 (Ar-Hout of plane). 1H NMR (600 MHz, DMSO-D6): δ (ppm) = 2.47 –

EP

2.53 (m, 4H, 6-CH2, 8-CH2), 2.84 – 2.90 (m, 4H, 5-CH2, 9-CH2), 7.14 (d, J = 7.7 Hz, 1H, 4-H), 7.68 (dd, J = 7.6/1.6 Hz, 1H, 3-H), 7.74 (d, J = 1.6 Hz, 1H, 1-H).

13

C NMR

AC C

(151 MHz, DMSO-D6): δ (ppm) = 29.3 (1C, C-5), 29.7 (1C, C-9), 44.0 (1C, C-6 or C8), 44.2 (1C, C-8 or C-6), 127.5 (1C, C-3), 128.0 (1C, C-4), 129.9 (1C, C-1), 138.8 (1C, C-2), 139.2 (1C, C-9a), 141.1 (1C, C-4a), 169.5 (1C, CO2Na), 210.5 (1C, C-7). Exact mass (APCI): m/z = 205.0887 (calcd. 205.0859 for C12H13O3 [M-Na+2H]+). Purity (HPLC): 99.7 % (tR = 14.5 min).

20

ACCEPTED MANUSCRIPT 4.3.4.

Sodium

7-[(3-phenylpropyl)amino]-6,7,8,9-tetrahydro-5H-

benzo[7]annulene-2-carboxylate (9a) Under N2 atmosphere, a mixture of sodium carboxylate 8 (31 mg, 0.14 mmol, 1 eq.) and 3-phenylpropan-1-amine (37 µL, 0.26 mmol, 1.8 eq.) in dry CH3OH (4 mL) was

RI PT

stirred for 18 h at rt. After addition of NaBH4 (29 mg, 0.76 mmol, 5.4 eq.), stirring was continued for 5 h. Then, NaOH (1 M, 7 mL) was added and the organic solvent was evaporated under reduced pressure. Addition of CH2Cl2 (10 mL) resulted in a

SC

colorless solid, which was slowly filtered off, washed with water and ethyl acetate and dried in vacuo. The resulting product was used without further purification. The Rf

M AN U

value could not be determined due to zwitterionic character of 9a. Colorless solid, mp 142 °C, yield 44 mg (93 %), C21H24NO2Na (345.4 g/mol). FT-IR (neat): ν̃ (cm1

) = 2931, 2855 (C-Haliph.), 1566 (CO2-asym), 1497 (C=Carom.), 1412 (CO2-sym), 783, 748,

698 (Ar-Hout of plane). 1H NMR (400 MHz, CD3CO2D): δ (ppm) = 1.45 – 1.62 (m, 2H, 6-

TE D

H, 8-H), 2.01 – 2.07 (m, 2H, PhCH2CH2CH2), 2.36 – 2.48 (m, 2H, 6-H, 8-H), 2.65 – 2.75 (m, 2H, PhCH2CH2CH2), 2.80 – 2.92 (m, 2H, 5-H, 9-H), 2.91 – 3.03 (m, 2H, 5-H, 9-H), 3.11 – 3.21 (m, 2H, PhCH2CH2CH2), 3.49 – 3.62 (m, 1H, 7-H), 7.16 – 7.23 (m,

EP

3H, 2-Hph, 4-Hph, 6-Hph), 7.24 – 7.35 (m, 3H, C-4, 3-Hph, 5-Hph), 7.83 – 7.91 (m, 2H, 1H, 3-H). A signal for the NH proton is not seen in the spectrum.

13

C NMR (151 MHz,

AC C

CD3CO2D): δ (ppm) = 28.6 (1C, PhCH2CH2CH2), 30.9 (1C, C-6 or C-8), 31.0 (1C, C-8 or C-6), 31.9 (1C, C-9), 32.2 (1C, C-5), 33.3 (1C, PhCH2CH2CH2), 45.5 (1C, PhCH2CH2CH2), 62.3 (1C, C-7), 127.3 (1C, C-4ph), 128.9 (1C, C-2), 129.2 (2C, C-2ph, C-6ph), 129.5 (2C, C-3ph, C-5ph), 129.7 (1C, C-3), 130.4 (1C, C-4), 131.5 (1C, C-1), 141.4 (1C, C-1ph), 142.6 (1C, C-9a), 148.6 (1C, C-4a), 172.2 (1C, CO2Na). Exact mass (APCI): m/z = 324.1972 (calcd. 324.1958 for C21H26NO2 [M-Na+H]+). Purity (HPLC): 90.2 % (tR = 16.9 min).

21

ACCEPTED MANUSCRIPT

4.3.5.

Sodium

7-[(4-phenylbutyl)amino]-6,7,8,9-tetrahydro-5H-

benzo[7]annulene-2-carboxylate (9b) Under N2 atmosphere, 4-phenylbutan-1-amine (220 µL, 1.4 mmol, 1.7 eq.) and

RI PT

Na2SO4 (approx. 2 g) were successively added to a solution of sodium carboxylate 8 (186 mg, 0.82 mmol, 1 eq.) in dry CH3OH (4 mL). After stirring for 24 h at rt, NaBH4 (137 mg, 3.6 mmol, 4.4 eq.) was added and stirring was continued for 20 h. Then,

SC

NaOH (1 M, 5 mL) was added dropwise and the mixture was concentrated in vacuo. Upon CH2Cl2 addition to the aqueous layer, a colorless solid precipitated, which was

M AN U

slowly filtered off, washed with ethyl acetate and water and dried under reduced pressure. The product was used without further purification. The Rf value could not be determined due to zwitterionic character of 9b. Colorless solid, mp 213 °C, yield 181 mg (61 %), C22H26NO2Na (359.4 g/mol). FT-IR (neat): ν̃ (cm-1) = 3318 (NH),

TE D

2943 (C-Haliph.), 1593 (CO2-asym), 1555, 1493 (C=Carom.), 1373 (CO2-sym), 783, 748, 698 (Ar-Hout of plane). 1H NMR (600 MHz, CD3CO2D): δ (ppm) = 1.47 – 1.59 (m, 2H, 6H, 8-H), 1.64 – 1.77 (m, 4H, PhCH2CH2CH2CH2), 2.38 – 2.47 (m, 2H, 6-H, 8-H), 2.63

EP

(t, J = 7.1 Hz, 2H, PhCH2CH2CH2CH2), 2.82 – 2.91 (m, 2H, 5-H, 9-H), 2.92 – 3.01 (m, 2H, 5-H, 9-H), 3.12 – 3.19 (m, 2H, PhCH2CH2CH2CH2), 3.54 (tt, J = 11.7/3.4 Hz, 1H,

AC C

7-H), 7.14 – 7.18 (m, 3H, 2-Hph, 4-Hph, 6-Hph), 7.24 – 7.29 (m, 3H, 4-H, 3-Hph, 5-Hph), 7.85 – 7.88 (m, 2H, 1-H, 3-H). A signal for the NH proton is not seen in the spectrum. 13

C NMR (151 MHz, CD3CO2D): δ (ppm) = 26.6 (1C, PhCH2CH2CH2CH2), 29.0 (1C,

PhCH2CH2CH2CH2), 30.9 (1C, C-6 or C-8), 31.0 (1C, C-8 or C-6), 31.9 (1C, C-9), 32.2 (1C, C-5), 35.9 (1C, PhCH2CH2CH2CH2), 45.8 (1C, PhCH2CH2CH2CH2), 62.2 (1C, C-7), 126.9 (1C, C-4ph), 128.9 (1C, C-2), 129.27 (2C, C-2ph, C-6ph), 129.33 (2C, C-3ph, C-5ph), 129.7 (1C, C-3), 130.3 (1C, C-4), 131.5 (1C, C-1), 142.60 (1C, C-9a), 142.63

(1C,

C-1ph),

148.6

(1C,

C-4a),

172.1

(1C,

CO2Na).

22

ACCEPTED MANUSCRIPT Exact mass (APCI): m/z = 338.2083 (calcd. 338.2115 for C22H28NO2 [M-Na+2H]+). Purity (HPLC): 96.7 % (tR = 17.8 min).

4.3.6.

{7-[(3-Phenylpropyl)amino]-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-

RI PT

yl}methanol (4a)

A suspension of LiAlH4 (22 mg, 0.57 mmol, 2.5 eq.) in THF (2 mL) was added dropwise to a suspension of sodium carboxylate 9a (80 mg, 0.23 mmol, 1 eq.) in THF

SC

(2 mL) at 0 °C. After stirring for 29 h at rt, the reaction mixture was carefully treated with water (200 µL), NaOH (1 M, 200 µL) and water (200 µL). Then, the mixture was

M AN U

dried (Na2SO4) and filtered through Celite®. The filter cake was washed with THF and the filtrate was concentrated in vacuo. The crude product was purified by fc (d = 1.5 cm,

l = 17 cm,

V = 5 mL,

cyclohexane/CH2Cl2

50:50,

20:80,

0:100,

CH2Cl2/CH3OH 99:1, 95:5; 1 %, N N-dimethylethylamine was added to each solvent

TE D

mixture). The product (161 mg) was further purified by dissolving in CH2Cl2, filtration through cotton and removal of the solvent in vacuo. Yellow solid, mp 114 °C, yield 13 mg (18 %), C21H27NO (309.5 g/mol). Rf = 0.35 (CH2Cl2/CH3OH 90:10). FT-

EP

IR (neat): ν̃ (cm-1) = 3264 (OH, NH), 3024 (C-Harom.), 2924, 2851 (C-Haliph.), 1605, 1497 (C=Carom.), 872, 814, 748, 698 (Ar-Hout of plane). 1H NMR (400 MHz, CD3OD): δ

AC C

(ppm) = 1.11 – 1.25 (m, 2H, 6-H, 8-H), 1.84 (quint, J = 7.6 Hz, 2H, PhCH2CH2CH2), 2.07 – 2.17 (m, 2H, 6-H, 8-H), 2.62 – 2.72 (m, 4H, PhCH2CH2CH2), 2.72 – 2.81 (m, 5H, 5-CH2, 7-H, 9-CH2), 4.52 (s, 2H, CH2OH), 7.07 (m, 3H, 1-H, 3-H, 4-H), 7.11 – 7.31 (m, 5H, Ph-H). Signals for the NH and OH protons are not seen in the spectrum. 13

C NMR (101 MHz, CD3OD): δ (ppm) = 32.3 (1C, PhCH2CH2CH2), 32.9 (1C, C-5),

33.3 (1C, C-9), 34.7 (1C, PhCH2CH2CH2), 34.8 (1C, C-6 or C-8), 34.9 (1C, C-8 or C6), 47.3 (1C, PhCH2CH2CH2), 62.9 (1C, C-7), 65.1 (1C, CH2OH), 126.1 (1C, C-3), 126.9 (1C, C-4ph), 128.8 (1C, C-1), 129.398 (2C, Cph), 129.404 (2C, Cph), 129.9 (1C,

23

ACCEPTED MANUSCRIPT C-4), 140.6 (1C, C-2), 142.6 (1C, C-4a), 143.2 (1C, C-1ph), 143.5 (1C, C-9a). Exact mass (APCI): m/z = 310.2180 (calcd. 310.2165 for C21H28NO [M+H]+). Purity (HPLC): 90.9 % (tR = 16.3 min).

{7-[(4-Phenylbutyl)amino]-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-

RI PT

4.3.7.

yl}methanol (4b)

Under N2 atmosphere, sodium carboxylate 9b (101 mg, 0.28 mmol, 1 eq.) was

SC

suspended in THF (2.5 mL). Then, a suspension of LiAlH4 (28 mg, 0.75 mmol, 2.7 eq.) in THF (2.5 mL) was added dropwise at 0 °C. The reaction mixture was

M AN U

stirred for 19 h at rt, and afterwards carefully treated with water (200 µL), NaOH (1 M, 200 µL) and water (200 µL). The mixture was dried (Na2SO4), filtered, the residue was washed with THF and the filtrate was concentrated in vacuo. The crude product was purified by fc (d = 2 cm, l = 15 cm, V = 10 mL cyclohexane/CH2Cl2 50:50,

TE D

CH2Cl2/CH3CH2OH 99:1, 90:10, 1 % N,N-dimethylethylamine was added to each solvent mixture). The product was further purified by dissolving in CH2Cl2, filtration through cotton and removal of the solvent in vacuo. Yellow oil, yield 34 mg (39 %), (323.5 g/mol).

Rf = 0.29

EP

C22H29NO

(cyclohexane

/

ethyl

acetate

/

N,N-

dimethylethylamine 22:67:11). FT-IR (neat): ν̃ (cm-1) = 3287 (OH, NH), 2928, 2851

AC C

(C-Haliph.), 1585, 1497 (C=Carom.), 891, 826, 748, 698 (Ar-Hout

1

of plane).

H NMR (400

MHz, CD3OD): δ (ppm) = 1.11 – 1.25 (m, 2H, 6-H, 8-H), 1.49 – 1.58 (m, 2H, PhCH2CH2CH2CH2), 1.62 – 1.72 (m, 2H, PhCH2CH2CH2CH2), 2.07 – 2.18 (m, 2H, 6H, 8-H), 2.61 – 2.68 (m, 4H, PhCH2CH2CH2CH2), 2.73 – 2.79 (m, 5H, 5-CH2, 7-H, 9CH2), 4.52 (s, 2H, CH2OH), 7.03 – 7.10 (m, 3H, 1-H, 3-H, 4-H), 7.10 – 7.29 (m, 5H, Ph-H). Signals for NH and OH protons are not seen in the spectrum. MHz,

CD3OD):

δ

(ppm)

=

30.2

(1C,

13

PhCH2CH2CH2CH2),

C NMR (151 30.4

(1C,

PhCH2CH2CH2CH2), 33.0 (1C, C-5), 33.3 (1C, C-9), 34.9 (1C, C-6 or C-8), 35.0 (1C,

24

ACCEPTED MANUSCRIPT C-8 or C-6), 36.7 (1C, PhCH2CH2CH2CH2), 47.6 (1C, PhCH2CH2CH2CH2), 63.0 (1C, C-7), 65.1 (1C, CH2OH), 126.0 (1C, C-3), 126.7 (1C, C-4ph), 128.8 (1C, C-1), 129.3 (2C, C-2ph, C-6ph), 129.4 (2C, C-3ph, C-5ph), 129.8 (1C, C-4), 140.5 (1C, C-2), 142.7 (1C,

C-4a),

143.59

(1C,

C-9a),

143.60

(1C,

C-1ph).

Exact

mass

RI PT

(APCI): m/z = 324.2319 (calcd. 324.2322 for C22H30NO [M+H]+). Purity (HPLC): 93.1 % (tR = 17.5 min).

SC

4.4. Receptor binding studies 4.4.1. Materials

M AN U

Guinea pig brains and rat livers were commercially available (Harlan-Winkelmann, Borchen, Germany). Pig brains were a donation of the local slaughterhouse (Coesfeld, Germany). The recombinant L(tk-) cells stably expressing the GluN2B receptor were obtained from Prof. Dr. Dieter Steinhilber (Frankfurt, Germany).

TE D

Homogenizers: Elvehjem Potter (B. Braun Biotech International, Melsungen, Germany) and Soniprep® 150, MSE, London, UK). Centrifuges: Cooling centrifuge model Rotina® 35R (Hettich, Tuttlingen, Germany) and High-speed cooling centrifuge

EP

model Sorvall® RC-5C plus (Thermo Fisher Scientific, Langenselbold, Germany). Multiplates: standard 96 well multiplates (Diagonal, Muenster, Germany). Shaker:

AC C

self-made device with adjustable temperature and tumbling speed (scientific workshop of the institute). Harvester: MicroBeta® FilterMate 96 Harvester. Filter: Printed Filtermat Typ A and B. Scintillator: Meltilex® (Typ A or B) solid state scintillator. Scintillation analyzer: MicroBeta® Trilux (all Perkin Elmer LAS, RodgauJügesheim, Germany).

4.4.2. Cell culture and preparation of membrane homogenates from GluN2B cells

25

ACCEPTED MANUSCRIPT Mouse L(tk-) cells stably transfected with the dexamethasone-inducible eukaryotic expression vectors pMSG GluN1a, pMSG GluN2B (1:5 ratio) were grown in Modified Earl’s Medium (MEM) containing 10 % of standardized FCS (Biochrom AG, Berlin, Germany). The expression of the NMDA receptor at the cell surface was induced

RI PT

after the cell density of the adherent growing cells had reached approximately 90 % of confluency. For the induction, the original growth medium was replaced by growth medium containing 4 µM dexamethasone and 4 µM ketamine (final concentration).

SC

After 24 h, the cells were rinsed with phosphate buffered saline solution (PBS, Biochrom AG, Berlin, Germany), harvested by mechanical detachment and pelleted

M AN U

(10 min, 5,000 x g).

For the binding assay, the cell pellet was resuspended in PBS solution and the number of cells was determined using a Scepter® cell counter (MERCK Millipore,

TE D

Darmstadt, Germany). Subsequently, the cells were lysed by sonication (4 °C, 6 x 10 s cycles with breaks of 10 s). The resulting cell fragments were centrifuged with a high performance cool centrifuge (23,500 x g, 4 °C). The supernatant was

EP

discarded and the pellet was resuspended in a defined volume of PBS yielding cell fragments of approximately 500,000 cells/mL. The suspension of membrane

AC C

homogenates was sonicated again (4 °C, 2 x 10 s cycles with a break of 10 s) and stored at -80 °C.

4.4.3. Preparation of membrane homogenates from pig brain cortex Fresh pig brain cortex was homogenized with the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 31,000 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of

26

ACCEPTED MANUSCRIPT TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA, pH 7.5) and centrifuged again at 31,000 x g (20 min, 4 °C). The final pellet was resuspended in 5-6 volumes of buffer

RI PT

and frozen (-80 °C) in 1.5 mL portions containing about 0.8 mg protein/mL.

4.4.4. Preparation of membrane homogenates from guinea pig brain

5 guinea pig brains were homogenized with the potter (500-800 rpm, 10 up and down

SC

strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at

M AN U

23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500 x g (20 min, 4 °C). This procedure was repeated twice. The final pellet was resuspended in 5-6 volumes of

TE D

buffer and frozen (-80 °C) in 1.5 mL portions containing about 1.5 mg protein/mL.

4.4.5. Preparation of membrane homogenates from rat liver Two rat livers were cut into small pieces and homogenized with the potter (500-

EP

800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was

AC C

separated and centrifuged at 31,000 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 8.0) and incubated at rt for 30 min. After the incubation, the suspension was centrifuged again at 31,000 x g for 20 min at 4 °C. The final pellet was resuspended in 5-6 volumes of buffer and stored at -80 °C in 1.5 mL portions containing about 2 mg protein/mL.

4.4.6. General procedures for the binding assays

27

ACCEPTED MANUSCRIPT The test compound solutions were prepared by dissolving approximately 10 µmol (usually 2-4 mg) of test compound in DMSO so that a 10 mM stock solution was obtained. To obtain the required test solutions for the assay, the DMSO stock solution was diluted with the respective assay buffer. The filtermats were presoaked

RI PT

in 0.5 % aqueous polyethylenimine solution for 2 h at rt before use. All binding experiments were carried out in duplicates in the 96 well multiplates. The concentrations given are the final concentration in the assay. Generally, the assays

SC

were performed by addition of 50 µL of the respective assay buffer, 50 µL of test compound solution in various concentrations (10-5, 10-6, 10-7, 10-8, 10-9 and 10mol/L), 50 µL of the corresponding radioligand solution and 50 µL of the respective

M AN U

10

receptor preparation into each well of the multiplate (total volume 200 µL). The receptor preparation was always added last. During the incubation, the multiplates were shaken at a speed of 500-600 rpm at the specified temperature. Unless

TE D

otherwise noted, the assays were terminated after 120 min by rapid filtration using the harvester. During the filtration, each well was washed five times with 300 µL of water. Subsequently, the filtermats were dried at 95 °C. The solid scintillator was

EP

melted on the dried filtermats at a temperature of 95 °C for 5 min. After solidifying of the scintillator at rt, the trapped radioactivity in the filtermats was measured with the

AC C

scintillation analyzer. Each position on the filtermat corresponding to one well of the multiplate was measured for 5 min with the [3H]-counting protocol. The overall counting efficiency was 20 %. The IC50 values were calculated with the program GraphPad Prism® 3.0 (GraphPad Software, San Diego, CA, USA) by non-linear regression analysis. Subsequently, the IC50 values were transformed into Ki values using the equation of Cheng and Prusoff.[39] The Ki values are given as mean value ± SEM from three independent experiments.

28

ACCEPTED MANUSCRIPT 4.4.7. Affinity for the GluN2B binding site of the NMDA receptor The competitive binding assay was performed with the radioligand [3H]ifenprodil (60 Ci/mmol; BIOTREND, Cologne, Germany). The thawed cell membrane preparation from the transfected L(tk-) cells (about 20 µg protein) was incubated with

RI PT

various concentrations of test compounds, 5 nM [3H]-ifenprodil, and TRIS/EDTAbuffer (5 mM TRIS/1 mM EDTA, pH 7.5) at 37 °C. The non-specific binding was

SC

determined with 10 µM unlabeled ifenprodil. The Kd value of ifenprodil is 7.6 nM.[40]

4.4.8. Affinity for the PCP binding site of the NMDA receptor

M AN U

The assay was performed with the radioligand [3H]-(+)-MK-801 (22.0 Ci/mmol; Perkin Elmer).[34,35] The thawed membrane preparation of pig brain cortex (about 100 µg of the protein) was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-MK-801, and TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA, pH 7.5) at rt. The

TE D

non-specific binding was determined with 10 µM unlabeled (+)-MK-801. The Kd value of (+)-MK-801 is 2.26 nM. [41]

assay[36-38]

was

performed

with

the

radioligand

[3H]-(+)-pentazocine

AC C

The

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4.4.9. Affinity for the σ1 receptor

(22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation of guinea pig brain cortex (about 100 µg of the protein) was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-pentazocine, and TRIS buffer (50 mM, pH 7.4) at 37 °C. The non-specific binding was determined with 10 µM unlabeled (+)-pentazocine. The Kd value of (+)-pentazocine is 2.9 nM.[42]

4.4.10. Affinity for the σ2 receptor

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ACCEPTED MANUSCRIPT The assay[36-38] was performed with the radioligand [3H]di-o-tolylguanidine (specific activity 50 Ci/mmol; ARC, St. Louis, MO, USA). The thawed rat liver membrane preparation (about 100 µg protein) was incubated with various concentrations of the test compound, 3 nM [3H]di-o-tolylguanidine and buffer containing (+)-pentazocine

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(500 nM (+)-pentazocine in TRIS buffer (50 mM TRIS, pH 8.0)) at rt. The non-specific binding was determined with 10 µM non-labeled di-o-tolylguanidine. The Kd value of

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di-o-tolylguanidine is 17.9 nM.[43]

4.5. Cytoprotection

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Mouse L(tk-) cells stably transfected with the dexamethasone-inducible eukaryotic expression vectors pMSG NR1-1a, pMSG NR2B (1:5 ratio) were grown and harvested as described in chapter 4.4.2. The assay was based on ref.[44,45].

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After careful removal of the medium the plate was blocked twice with 200 µL DMEM containing 1 % BSA and rinsed once with 200 µL DMEM. Then the cells in the inner wells (B2-G11) were incubated with 50 µL DMEM and 50 µL of the respective test

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compound in 5 different concentrations (e.g. 4 x 10-5, 10-6, 10-7, 10-8, 10-9 mol/L) and 100 µL DMEM was added to the remaining outer wells. Each concentration of the

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test compound was incubated at least in triplicates for 30 min at 37 °C. Afterwards 100 µL of a glutamate/glycine solution (each 20 µM) were added and the cells were incubated for 6 h at 37 °C.

Subsequently, 50 µL of the supernatant were transferred into a 96-well plate and incubated with 50 µL of the LDH-assay mixture (1 U/mL diaphorase, 1 % Na lactate, 0.1 % NAD+, 0.08 % BSA, 0.4 % iodonitrotetrazolium chloride in 75 mM PBS) at 37 °C for 45 min. The UV absorption was measured at λ = 485 nm in a plate reader

30

ACCEPTED MANUSCRIPT (TECAN, Crailsheim, Germany). Total LDH activity was determined with buffer instead of ligand solution and a solution of (S)-ketamine with a concentration of 4 x 10-5 mol/L served as positive control.

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4.6 Docking studies

The crystal structure of the NMDA-GluN1/GluN2B dimer in complex with Ro 25-6981 (PDB ID: 3QEM) was taken from the Protein Data Bank (PDB; www.rcsb.org). Since

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no water molecules were resolved in this X-ray structure, the conserved water molecule was taken from the resolved X-ray structure of the NMDA-GluN1/GluN2B

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dimer in complex with ifenprodil (PDB ID: 3QEL; H2O410). Renumbering of the amino acid sequence in 3QEM was also performed, since the annotated numbers were not conform to the reported primary sequence of the protein. The protein structure was subsequently prepared

using Schrödinger’s

Protein

Preparation Wizard.[46]

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Hydrogen atoms were added, protonation states were assigned, and a restrained minimization was performed.

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MOE modeling software 2012.10 [47] was used to generate the molecular structures of all compounds at the physiological pH. The ligands were subsequently prepared

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for docking using the LigPrep tool [48] as implemented in Schrödinger’s software, where all possible tautomeric forms as well as stereoisomers were generated and energy minimized using the OPLS force field.

The receptor grid preparation for the docking procedure was carried out by assigning the co-crystallized ligand as the centroid of the grid box while keeping the conserved water molecule (H2O410) in the binding pocket. The prepared ligand structures were docked into the Ro 25-6981 binding pocket using the program Glide (Schrödinger,

31

ACCEPTED MANUSCRIPT LLC, New York, NY, USA, version 9.8) [49] in the Standard Precision mode. A total of 20 poses per ligand conformer were included in the post-docking minimization step and a maximum of two docking poses were output for each ligand conformer. Using

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into the NMDA-GluN1/GluN2B binding pocket.

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this setup, the co-crystallized inhibitors could be correctly docked (RMSD below 1 Å)

Acknowledgements

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We thank Benedicte Doerner and Christina Jordan for their assistance during laboratory work. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), which is gratefully acknowledged. Moreover, we are grateful to Cells-inMotion (CiM) Cluster of Excellence for funding a lab visit at the University of Halle-

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Wittenberg to learn molecular modelling.

Supporting Information

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The Supporting Information contains purity data of prepared compounds, protein determination of biological material, experimental details of docking studies, and

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NMR spectra.

Conflict of interest

The authors declare no conflict of interest.

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ACCEPTED MANUSCRIPT

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Graphical Abstract

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Prof. Dr. B. Wünsch

|Institut für Pharmazeutische und Medizinische Chemie | Corrensstraße 48 | 48149 Münster

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Corrensstraße 48 48149 Münster

Bearbeiterin

Laura Prause Tel. +49 251 83-33310 Fax +49 251 83-32144

Research Highlights

Datum

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[email protected]

> Bioisosteric replacement of phenolic OH moiety by a CH2OH moiety > Crucial interactions with the GluN2B subunit, such as H-bonds with

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Gln110 and Glu236, are retained.

> Benzo[7]annulen-7-amines with 2-OH and 2-CH2OH moieties have similar GluN2B affinity and selectivity.

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> The cytoprotective potential of hydroxymethyl derivatives confirms the importance of the H-bond donor stabilizing the closed channel

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conformation.

18.12.2017