Negative allosteric modulators of the GluN2B NMDA receptor with phenylethylamine structure embedded in ring-expanded and ring-contracted scaffolds

European Journal of Medicinal Chemistry 190 (2020) 112138

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Negative allosteric modulators of the GluN2B NMDA receptor with phenylethylamine structure embedded in ring-expanded and ringcontracted scaffolds Louisa Temme a, b, Elena Bechthold a, Julian A. Schreiber a, b, c, Sandeep Gawaskar a, b, Dirk Schepmann a, Dina Robaa d, Wolfgang Sippl d, Guiscard Seebohm c, Bernhard Wünsch a, b, * €t Münster, Corrensstraße 48, D-48149, Münster, Germany Institut für Pharmazeutische und Medizinische Chemie der Universita €lische Wilhelms-Universita €t, Münster, Germany Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), Westfa c €t Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120, Halle (Saale), Germany Institut für Pharmazie der Martin-Luther-Universita d Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Münster, Robert-Koch-Str. 45, D-48149, Münster, Germany a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 December 2019 Received in revised form 20 January 2020 Accepted 9 February 2020 Available online 10 February 2020

A set of GluN2B NMDA receptor antagonists with conformationally restricted phenylethylamine substructure was prepared and pharmacologically evaluated. The phenylethylamine substructure was embedded in ring expanded 3-benzazocines 4 as well as ring-contracted tetralinamines 6 and indanamines 7. The ligands 4, 6 and 7 were synthesized by reductive alkylation of secondary amine 11, reductive amination of ketones 12 and 16 and nucleophilic substitution of nosylates 14 and 17. The moderate GluN2B affinity of 3-benzazocine 4d (Ki ¼ 32 nM) translated into moderate cytoprotective activity (IC50 ¼ 890 nM) and moderate ion channel inhibition (60% at 10 mM) in two-electrode voltage clamp experiments with GluN1a/GluN2B expressing oocytes. Although some of the tetralinamines 6 and indanamines 7 showed very high GluN2B affinity (e.g. Ki (7f) ¼ 3.2 nM), they could not inhibit glutamate/ glycine inducted cytotoxicity. The low cytoprotective activity of 3-benzazocines 4, tetralinamines 6 and indanamines 7 was attributed to the missing OH moiety at the benzene ring and/or in benzylic position. Docking studies showed that the novel GluN2B ligands adopt similar binding poses as Ro 25e6981 with the central H-bond interaction between the protonated amino moiety of the ligands and the carbamoyl moiety of Gln110. However, due to the lack of a second H-bond forming group, the ligands can adopt two binding poses within the ifenprodil binding pocket. © 2020 Elsevier Masson SAS. All rights reserved.

Keywords: NMDA receptor GluN2B antagonists Ifenprodil binding site GluN2B affinity Selectivity Cytoprotective activity Electrophysiology TEVC Structure-affinity relationships Structure-activity relationships Docking studies Ligand-receptor interactions

1. Introduction The N-methyl-D-aspartate (NMDA) receptor belongs to the class of ionotropic receptors activated by the excitatory amino acid neurotransmitter (S)-glutamate. It is associated with neurodegenerative disorders including Parkinson’s, Alzheimer’s and Huntington’s disease and cerebral ischemia. Therefore, the NMDA receptor represents a promising target for the development of novel drugs

* Corresponding author. Institut für Pharmazeutische und Medizinische Chemie €t Münster, Corrensstraße 48, D-48149, Münster, Germany. der Universita E-mail address: [email protected] (B. Wünsch). https://doi.org/10.1016/j.ejmech.2020.112138 0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

for the therapy of these diseases. However, the strong blockade of the NMDA receptor associated ion channel by open-channel blockers, such as phencyclidine (PCP) and (þ)-MK-801 leads to strong psychotomimetic side effects (e.g. hallucinations, psychosis, amnesia). On the other hand, open-channel blockers with reduced affinity towards the PCP binding site of the NMDA receptor are in clinical use as anti-Parkinson drugs (amantadine, budipine), antiAlzheimer drugs (memantine) and as dissociative anesthetics ((S)-ketamine) [1e6]. During the last decades, subtype selective NMDA receptor antagonists interacting selectively with NMDA receptors containing the GluN2B subunit emerged as attractive drug candidates. NMDA receptors with GluN2B subunit (shortly GluN2B receptors) present

2

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

an additional binding site at the interface between the GluN1 and GluN2B subunits, which is addressed by ifenprodil (1) as prototypical negative allosteric modulator (NAM). Therefore, this binding site is referred to as ifenprodil binding site [7e10]. (Fig. 1). Interestingly, a H-bond interaction between the benzylic OH moiety of ifenprodil-like compounds and either the carbonyl Oatom of Ser132 (GluN1) in case of ifenprodil [12] and the terminal carbamoyl moiety of Gln110 (GluN2B) [12,13] or the backbone NH group of Leu135 (GluN1) in case of Ro 25-698113 was found. According to X-ray crystal structures of the GluN2B receptor together with ifenprodil (1), the phenolic OH moiety interacts with a conserved water molecule and Glu236 (GluN2B subunit). The benzylic OH moiety and the protonated amino moiety form H-bonds with the carbonyl O-atom of Ser132 (GluN1) [12] and the amide of Gln110 (GluN2B) [12,13]. Ifenprodil is further stabilized in the allosteric binding site by lipophilic interactions formed by the benzylpiperidine part, the methyl moiety and the phenol [12,13]. In a previous study, ifenprodil (1) was reorganized into 2,3,4,5tetrahydro-1H-3-benzazepine-1,7-diols bearing various side chains at the N-atom. 3-Benzazepine WMS-1410 (2) with the phenylbutyl side chain at the N-atom imitating the benzylpiperidine substructure of ifenprodil showed high GluN2B affinity (Ki ¼ 84 nM), high cytoprotective activity (IC50 ¼ 18.4 nM) and high activity in two-electrode voltage clamp experiments (IC50 ¼ 116 nM) [11,14]. The impact of the OH moieties in 1- and 7position of WMS-1410 (2) was analyzed by their stepwise removal. The 3-benzazepine without phenolic OH moiety showed a Ki value of 73 nM15 and even the 3-benzazepine 3d (R ¼ 4-phenylbutyl) without both OH moieties displayed a GluN2B affinity of 230 nM [15,16]. Obviously, the contribution of the phenolic and benzylic OH moieties to the interaction with the ifenprodil binding site is limited. Therefore, it was planned to synthesize and pharmacologically evaluate further analogs of 3-benzazepines 3 without OH moieties. Herein, we wish to report the synthesis and pharmacological evaluation of 3-benzazocines of type 4 and tetralinamines and indanamines of type 6 and 7, respectively (Fig. 2). Compared to 3benzazepines 3, 3-benzazocines 4 have a larger (8-membered) Nheterocycle. In contrast, the tetralinamines 6 and indanamines 7 represent ring-contracted analogs of benzo [7]annulen-7-amines 5, which also show high GluN2B affinity [17,18]. The common feature of the ring-expanded and ring-contracted analogs 4, 6, and 7 is a phenylethylamine substructure. The phenylethylamine substructure is a crucial structural element of typical GluN2B selective NAMs such as ifenprodil (1), WMS-1410 (2), eliprodil and traxoprodil (Fig. 1).

Fig. 2. Design of novel GluN2B selective ligands without benzylic and phenolic OH moieties. In the hexahydro-3-benzazocines 4 the N-heterocycle is expanded and in the tetralines 6 and indanes 7 the carbocycle is contracted compared to the lead compounds 3 and 5, respectively.

2. Synthesis The synthesis of 3-benzazocines 4 started with a Schmidt rearrangement of ketone 8 (Scheme 1). Unexpectedly, treatment of ketone 8 with NaN3 and H2SO4/HOAc led exclusively to tetrazole 10, the desired lactam 9 could not be detected. Formation of tetrazole 10 is due to trapping of intermediate nitrilium ion by N 3 in a 1,3dipolar cycloaddition. However, using Cl3CCO2H instead of H2SO4 at 65  C provided the lactam 9 in 84% yield without formation of tetrazole 10 [19,20]. Reduction of lactam 9 with LiAlH4 afforded the secondary amine 11, which was reductively alkylated with 3phenylpropionaldehyde or 4-phenylbutyraldehyde and NaBH(OAc)3 to give the tertiary amines 4c and 4d (Scheme 1). Reductive amination of b-tetralone (12) with homologous phenylalkylamines and NaBH(OAc)3 led to the homologous phenylalkylamines 6a-d in 80e95% yield. In addition to phenylalkylamino groups at the tetraline ring system, 4-phenyl- and 4benzylpiperidino groups should be introduced, since these structural elements are found in the GluN2B NAMs ifenprodil, traxoprodil and Ro 25e6981. Since reductive amination of ketone 12 with 4-phenyl- and 4-benzylpiperidine did not lead to amines 6e and 6f, a nucleophilic substitution was envisaged. After reduction of b-tetralone 12 with NaBH4 and activation of the resulting alcohol 13 with 4-nitrobenzenesulfonyl chloride (nosyl chloride), SN2 substitution with 4-phenyl- and 4-benzylpiperidine provided the desired amines 6e and 6f in moderate yields of 33% and 39%, respectively (Scheme 2). Indanamines 7a and 7b were obtained upon PCC oxidation of commercially available indanol 15 and subsequent reductive amination of the resulting indanone 16 with benzylamine or 2phenylethan-1-amine and NaBH(OAc)3. Alternatively, indanol 15 was reacted with nosyl chloride to afford the nosylate 17, which underwent nucleophilic substitution with 3-phenylpropan-1amine and 4-phenylbutan-1-amine to yield the homologous phenylalkylamines 7c and 7d. The piperidine derivatives 7e and 7f were also obtained by SN2 substitution of the nosylate 17 employing 4-phenyl- and 4-benzylpiperidine, respectively. In case of the indanyl nosylate 17 nucleophilic substitution with primary and secondary amines led to moderate to high yields of the substitution products (Scheme 3).

3. Pharmacological evaluation

Fig. 1. Ifenprodil (1), WMS-1410 (2) [11], eliprodil and traxoprodil - prototypical negative allosteric modulators of GluN2B subunit containing NMDA receptors.

Affinity towards GluN2B subunit containing NMDA receptors and related receptors. In the GluN2B assay, test compounds were competing with the radioligand [3H]ifenprodil for the ifenprodil binding site of NMDA receptors. Standardized membrane fragments from L (tk-)cells

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

3

Scheme 1. Synthesis of hexahydro-3-benzazocines 4. Reagents and reaction conditions: (a) NaN3, H2SO4, HOAc, rt, 9: 0%; 10: 41%. (b) NaN3, Cl3CCO2H, 65  C, 9: 84%; 10: 0%. (c) LiAlH4, THF, 75  C, 16 h then rt, 5 d, 40%. (d) Ph(CH2)2CH]O or Ph(CH2)3CH]O, NaBH(OAc)3, CH2Cl2, Na2SO4, rt, 14 h, 4c: 24%; 4d: 57%.

Scheme 2. Synthesis of tetrahydronaphthalenamines 6. Reagents and reaction conditions: (a) Ph(CH2)nNH2, CH2Cl2, NaBH(OAc)3, rt, 14 h, 6a: 80%; 6b: 95%, 6c: 84%; 6d: 87%. (b) NaBH4, CH3OH, 0  C, 2 h, 98%. (c) nosyl chloride, CH2Cl2, NEt3, DMAP, rt, 16 h, 64%. (d) 4-phenylpiperidine or 4-benzylpiperidine, CH3CN, 75  C, 48 h, 6e: 33%; 6f: 39%.

Scheme 3. Synthesis of indanamines 7. Reagents and reaction conditions: (a) PCC, CH2Cl2, rt, 4 h, 68%. (b) Ph(CH2)nNH2, CH2Cl2, NaBH(OAc)3, rt, 14 h, 7a: 50%; 5b: 35%. (c) nosyl chloride, CH2Cl2, NEt3, rt, 14 h, 70%. (d) Ph(CH2)nNH2, 4-phenylpiperidine or 4-benzylpiperidine, CH3CN, 60  C, 16e20 h, 7c: 59%; 7d: 45%, 7e: 59%; 7f: 68%.

stably expressing recombinant human GluN1a and GluN2B subunits of the NMDA receptor were employed as receptor material. Unspecific binding was determined with an excess of ifenprodil (100 nM) [21].

In addition to the GluN2B affinity, the interaction with s1 and s2 receptors was recorded in receptor binding studies [22e24]. In general, we always include the s affinity in our GluN2B projects and vice versa, since the lead compound ifenprodil shows high affinity

4

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

Table 1 Affinity towards the ifenprodil binding site of GluN2B subunit containing NMDA receptors and s receptors.

compd.

11,14

2 3d15,16 4c 4d 5c17 or 18 5d17 or 18 6a 6b 6c 6d 6e 6f 7b 7c 7d 7e 7f ifenprodil eliprodil haloperidol di-o-tolylguanidine

n

e 4 3 4 3 4 1 2 3 4 e e 2 3 4 e e

Ki ± SEM [nM] (n ¼ 3)

m

e e e e e e e e e e 0 1 e e e 0 1

GluN2B

s1

s2

84 ± 18 230 36 ± 3 32 ± 1 16 ± 4 17 ± 2 4000 237 163 ± 51 266 8.4 ± 3.4 8.0 ± 4.0 455 117 ± 15 66 ± 8 4.5 ± 1.5 3.2 ± 0.8 10 ± 0.7 13 ± 2.0 e

190 10 ± 8 17 ± 3 22 ± 8 150 215 285 51 ± 14 59 ± 16 46 ± 10 1.9 ± 0.6 6.0 ± 1.4 168 58 ± 13 31 ± 7 44 ± 9 51 ± 9 125 ± 24 e 6.3 ± 1.6

>10 mM 35 ± 13 178 50 ± 1 27 ± 12 55 ± 7 65 ± 15 70 ± 4 29 ± 9 50 ± 19 2.9 ± 0.6 2.1 ± 0.3 694 38 ± 9 150 25 ± 7 28 ± 2 98 ± 34 e 78 ± 2.3

e

89 ± 29

57 ± 18

The affinity of compounds with Ki values > 170 nM was recorded only once (n ¼ 1). Due to low purity, compound 7a was not evaluated pharmacologically. 3c, 3d: see Fig. 2, R ¼ (CH2)3Ph (3c), R ¼ (CH2)4Ph (3d). 5c, 5d: see Fig. 2, NR2 ¼ NH(CH2)3Ph (5c); NR2 ¼ NH(CH2)4Ph (5d).

towards both s receptor subtypes (see Table 1). Moreover, the pharmacophores for potent s1 ligands [25] and negative allosteric modulators at the ifenprodil binding site [26] are very similar. The cross reactivity at s receptors and GluN2B-NMDA receptors has been shown in several projects [27e29]. The GluN2B, s1 and s2 affinities of the 3-benzazocines 4, tetralinamines 6 and indanamines 7 together with the GluN2B affinity of some lead and reference compounds are summarized in Table 1. Expansion of the 3-benzazepine ring (3d) to a 3-benzazocine ring led to almost 10-fold increased GluN2B affinity of 4c and 4d. Moreover, the affinity towards both s1 and s2 receptors is reduced resulting in an increased selectivity over these receptors. In particular, the low s2 affinity renders the phenylpropyl derivative 4c an interesting GluN2B ligand with 5-fold selectivity over the s2 receptor. Up to a concentration of 10 mM both 3-benzazocines 4c and 4d did not interact with the PCP binding site [30,31] of the NMDA receptor. Phenylpropyl- or phenylbutylamino substituents seem to be favorable for high GluN2B affinity of ring-contracted tetralinamines 6 and indanamines 7. However, neither the tetralinamines 6c,d nor the indanamines 7c,d reach the GluN2B affinity of the benzo[7] annulenamines 5c,d. Introduction of the 4-phenyl- or 4benzylpiperidino moiety led to very potent GluN2B ligands with very low Ki values (e.g. 6e: Ki ¼ 8.4 nM; 7f: Ki ¼ 3.2 nM). Unfortunately, the s1 and s2 receptor affinities of all tetralinamines 6a-f are higher than their GluN2B affinity, indicating preference for these receptors instead for the ifenprodil binding site of the NMDA receptor. For the indanamines 7c and 7d with a phenylalkyl side chain a similar result was obtained. However, the 4-phenylpiperidine 7e and the 4-benzylpiperidine 7f show a considerable selectivity for GluN2B receptors over both s receptor subtypes.

3.1. Functional activity The cytoprotective activity of GluN2B ligands with various ring sizes was determined in an assay recording the released amount of lactate dehydrogenase (LDH) after stimulation of cells with (S)glutamate and glycine. The same mouse fibroblast L (tk-) cells expressing only GluN1a and GluN2B subunits as described in the receptor binding assay were employed in this assay. The amount of released LDH correlates with cell damage and/or death. A reduced release of LDH upon treatment with the GluN2B ligands is regarded as cytoprotective effect [32]. In order to get information about the influence of the ring size on the cytoprotective activity, compounds with phenylpropyl moiety (c-series) were investigated systematically. In case of 3benzazocines 4 with the basic amino moiety located within the

Table 2 Cytoprotective effects of GluN2B ligands with various ring sizes and reference compound ifenprodil correlated with their GluN2B affinity. compd.

4c 4d 5c 6c 7c 7f Ifenprodil a

n

3 4 3 3 3 e

GluN2B affinity

cytoprotective activity

Ki ± SEM (nM) (n ¼ 3)

IC50 ± SEM (mM) (n ¼ 3)

36 ± 3 32 ± 1 16 ± 4 163 ± 51 117 ± 15 3.2 ± 0.8 10 ± 0.7

7.8 ± 6.3 0.89 ± 0.38 29%[a] 30%[a] 31[b] 7% 0.59 ± 0.2

values in % indicate the cytoprotective effect at a test compound concentration of 10 mM. due to low activity, the IC50 value was recorded only once.

b

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

Fig. 3. Inhibition (in %) of ion currents evoked by 10 mM glycine and 10 mM (S)glutamate in presence of 1 mM and 10 mM 4d and 10 mM ifenprodil at GluN1a/GluN2B expressing oocytes (n ¼ 5 per concentration). Significance of mean differences was evaluated by one-way-ANOVA and posthoc mean comparison Tukey test (*** indicate p < 0.001).

eight-membered ring, the 4-phenylbutyl derivative 4d was also investigated, because the exocyclic 4-phenylbutyl moiety of 4d correlates nicely with the exocyclic 3-phenylpropylamino moiety of benzo[7]annulene 5c, tetraline 6c and indane 7c. Data in Table 2 clearly show that the eight-membered 3benzazocines 4c and 4d display considerable protection against cell damaging agents (S)-glutamate and glycine. Particularly high activity was found for the 4-phenylbutyl derivative 4d with an IC50value of 890 nM. Shortening of the side chain (4c) and contraction of the eight-membered ring to a smaller seven-, six- or fivemembered ring led to reduced or loss of cytoprotective activity. For the most protective compound 4d the inhibition of GluN2B receptors was evaluated directly in two-electrode voltage clamp (TEVC) measurements with GluN1a/GluN2B expressing oocytes [33] and compared to the activity of ifenprodil. The results are shown in Fig. 3. The results of TEVC measurements clearly confirm 4d as GluN2B receptor inhibitor. Increasing the concentration from 1 mM to 10 mM of 4d led to a significantly increased inhibition of the ion current from 8% (n ¼ 5) to 60% (n ¼ 5). However, 10 mM ifenprodil resulted in significantly higher ion current inhibition (95%, n ¼ 5) than 10 mM 4d. In general, the cytoprotective activity of the test compounds in Table 2 correlates nicely with their GluN2B affinity in receptor binding studies. However, the 3-benzazepine 5c and the indanamine 7f represent exceptions of this correlation, as 5c and 7f display high GluN2B affinity (Ki ¼ 16 nM and 3.2 nM), but negligible cytoprotective activity. Recently it was observed that affinity and activity of 3-benzazepines are also not correlating completely indicating a mechanism of inhibition, which is not only based on the binding of the compounds [33]. 4. Docking studies The docking studies were carried out using the X-ray crystal structure of the GluN1b/GluN2B dimer co-crystallized with the prototypical negative allosteric modulator Ro 25e6981 (PDB-ID 3QEM) [13]. Three important H-bonds are responsible for the orientation of Ro 25e6981 in the ifenprodil binding pocket: Hbonds between (1) protonated piperidine N-atom and the O-atom of the carbonyl moiety of Gln110 (GluN2B), (2) phenolic OH group and the carboxylate group of Glu236 (GluN2B) and a conserved

5

water molecule, and (3) benzylic OH moiety and the backbone Natom of Leu135 (GluN1b). The benzyl moiety interacts with a hydrophobic pocket formed by the residues of Ile111 (GluN2B), Phe114 (GluN2B) and Tyr109 (GluN1b) via hydrophobic and aromatic interactions. In addition to the H-bond interactions, the phenol of Ro 25e6981 undergoes hydrophobic and aromatic interactions with Phe176 (GluN2B) and Arg115 (GluN1b). (Fig. 4F). In order to prove the reliability of the docking results, the cocrystallized ligand Ro 25e6981 was removed from its binding site and the binding pose after re-docking of Ro 25e6981 into the binding pocket was compared with the original pose in the crystal structure. The RMSD value was lower than 1 Å indicating a reliable docking procedure. All docking studies were performed with the €dinger®) [34e36]. docking software Glide® version 2014 (Schro In contrast to Ro 25e6981, the benzazocine derivatives 4c and 4d as well as the indanamines 7e and 7f contain only one polar functional group, which is able to form H-bonds. Thus, the docking studies show a conserved central H-bond interaction between the protonated amino moiety of the ligands and the amide-carbonyl group of Gln110. However, the H-bond donating phenol and benzylic alcohol of Ro 25e6981 are missing in the new ligands, which hampers their clear orientation in the binding pocket. Thus, all ligands can adopt two poses in the binding pocket with the phenylalkyl moiety embedded either in the right hydrophobic binding pocket with Phe114 or alternatively in the left hydrophobic binding pocket with Phe176 and Arg115. This is illustrated for the phenylpropyl derivative 4c in Fig. 4A and B as example. The hydrophobic pocket on the right hand side (Ile111, Phe114, Tyr109) can accommodate either the terminal phenyl moiety of the phenylalkyl substituent (Fig. 4A and C) or the annulated benzene ring of the ligands (Fig. 4B, D, 4E). In particular, the piperidinylindanes 7e and 7f show an opposite orientation, with the indane ring located in the right hydrophobic pocket (Fig. 4D and E). 5. Conclusion Herein, novel GluN2B receptor antagonists with a conformationally constrained 2-phenylethylamine substructure are reported. The phenylethylamine structure is incorporated in larger (3-benzazocine ring, compounds 4) or smaller rings (tetralin-2amines (6), indan-2-amines (7)). An expanded eight-membered 3-benzazocine ring increased considerably the GluN2B affinity (e.g. Ki (4d) ¼ 32 nM. 3Benzazocine 4d was identified as the most active antagonist of this series of compounds showing moderate cytoprotective activity (IC50 (4d) ¼ 890 nM) and moderate ion current inhibition (60% at 10 mM) in TEVC measurements. Ring-contraction of the [7]annulene ring of benzo [7]annulenamines 5 resulted in reduced GluN2B affinity of tetralinamines 6 and indanamines 7. Phenylpropyl- and phenylbutylamino substituents appear to be favorable for GluN2B affinity, but 4-phenyland 4-benzylpiperidino groups led to high-affinity GluN2B ligands (e.g. Ki (7f) ¼ 3.2 nM). Despite their high GluN2B affinity, the tetralinamine 6c and the indanamines 7c and 7f do not show cytoprotective activity. It was hypothesized that at least one hydroxy moiety either at the benzene ring (phenol) or in benzyl position has to be present in order to induce a conformational change and thus a closure of the NMDA receptor associated ion channel. Moreover, a recent study showed, that both hydroxy moieties contribute to the inhibitory activity in an additive manner [33]. The novel GluN2B ligands 4, 6 and 7 contain only one functional group for the establishment of H-bonds. This protonated amino moiety is located in the middle of the molecule and undergoes a Hbond interaction with the carbonyl moiety of Gln110 in the ifenprodil binding site. However, due to rather similar structural

6

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

Fig. 4. Binding modes of the 3-benzazocine derivative 4c (A and B) and 4d (C), and indanamines 7e (D) and 7f (E) compared with the binding mode of Ro 25e6981 (F) cocrystallized in the ifenprodil binding pocket of the NMDA receptor (PDB-ID 3QEM). For the phenylpropyl derivative 4c two poses with the phenylpropyl moiety located either in the right (A) or left (B) hydrophobic binding pocket are shown.

elements on the central amino moiety two possible orientations in the binding site were found. The hydrophobic binding pocket formed by Ile111, Phe114 and Tyr109 can accommodate either the terminal phenyl moiety of the phenylalkyl side chain or the benzene ring of the annulated ring system.

6. Experimental part 6.1. 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 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 (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 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, 40e63 mm (VWR); parentheses include: diameter of the column (d), length of the stationary phase (l), fraction size (V) and eluent. Automated flash chromatography: Isolera™ Spektra One (Biotage®); parentheses include: cartridge size, flow rate, eluent, fractions size was always 20 mL. Melting point: Melting point system MP50 (Mettler Toledo, Gieben, Germany), open capillary, uncorrected. MS: MicroTOFQII mass spectrometer (Bruker 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 (d) 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 13C NMR signals was supported by 2-D NMR techniques where necessary. IR: FT/IR Affinity®-1 spectrometer (Shimadzu, Düsseldorf, Germany) using ATR technique.

6.2. HPLC methods for the determination of the purity In method 1 equipment 1 and in method 2 equipment 2 were used. All other parameters are the same for both methods. Equipment 1: Pump: L-7100, degasser: L-7614, autosampler: L7200, UV detector: L-7400, interface: D-7000, data transfer: D-line, data acquisition: HSM-Software (all from LaChrom, Merck Hitachi). Equipment 2: Pump: LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface: DIONEX UltiMate 3000, data acquisition: Chromeleon 7 (Thermo Fisher Scientific, Lauenstadt, Germany). Column: LiChrospher® 60 RP-select B (5 mm), LiChroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 mL; detection at l ¼ 210 nm; solvents: A: demineralized water with 0.05% (V/V) trifluoroacetic acid, B: CH3CN with 0.05% (V/V) trifluoroacetic acid; gradient elution (A %): 0e4 min: 90%; 4e29 min: gradient from 90% to 0%; 29e31 min: 0%; 31e31.5 min: gradient from 0% to 90%; 31.5e40 min: 90%. The purity of all compounds was determined by one of these methods.

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

6.3. Synthetic procedures 6.3.1. 3-(3-Phenylpropyl)-1,2,3,4,5,6-hexahydro-3-benzazocine (4c) Under N2 atmosphere, 3-phenylpropanal (57 mL, 0.43 mmol, 1.1 eq.) and Na2SO4 (approx. 400 mg) were added to a solution of secondary amine 11 (69 mg, 0.39 mmol, 1 eq.) in CH2Cl2 (3 mL). The solution was stirred for 5 h at rt. NaBH(OAc)3 (126 mg, 0.59 mmol, 1.5 eq.) was added and the reaction mixture was stirred for 14 h at rt. After treatment with saturated aqueous solution of NaHCO3 (10 mL) and water (10 mL), the organic layer was separated and the aqueous phase was washed with CH2Cl2 (3  30 mL). The combined organic layers were dried (Na2SO4), filtered and the solvent was removed in vacuo. The crude product was purified by fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, gradient elution: cyclohexane/ethyl acetate 99:1 / 95:5 / 90:10 þ 1% N,N-dimethylethylamine / 80:20 þ 1% N,N-dimethylethylamine). Yellow oil, yield 27 mg (24%). C20H25N (279.4 g/mol). TLC: Rf ¼ 0.67 (cyclohexane/ethyl acetate/N,Ndimethylethylamine 82:9:9). FT-IR: ~n [cm1] ¼ 3059, 3021 (CHarom.); 2924, 2855 (C-Haliph.); 1601, 1493 (C¼Carom.); 756, 698 (ArHout of plane). 1H NMR (400 MHz, CDCl3): d [ppm] ¼ 1.60e1.77 (m, 4H, 5-CH2, PhCH2CH2CH2), 2.32e2.41 (m, 2H, 4-CH2), 2.46e2.57 (m, 4H, PhCH2CH2CH2), 2.71e2.79 (m, 2H, 2-CH2), 2.79e2.92 (m, 4H, 1CH2, 6-CH2), 7.06e7.21 (m, 7H, AreH, 1-Hphenyl, 3-Hphenyl, 5-Hphenyl), 7.26 (m, 2H, 2-Hphenyl, 6-Hphenyl). 13C NMR (101 MHz, CDCl3): d [ppm] ¼ 29.9 (1C, PhCH2CH2CH2), 31.6 (1C, C-6), 32.7 (1C, C-5), 33.3 (1C, PhCH2CH2CH2), 35.6 (1C, C-1), 52.8 (1C, C-4), 58.2 (1C, PhCH2CH2CH2), 59.2 (1C, C-2), 125.7 (1C, C-4phenyl), 126.3 (1C, C-8 or C-9), 126.6 (1C, C-9 or C-8), 128.3 (2C, C-2phenyl, C-6phenyl), 128.6 (2C, C-3phenyl, C-5phenyl), 129.0 (1C, C-7), 129.2 (1C, C-10), 140.8 (1C, C-6a or C-10a), 141.2 (1C, C-10a or C-6a), 142.6 (1C, C-1phenyl). Exact mass (APCI): m/z ¼ 280.2046 (calcd. 280.2060 for C20H26N [MþH]þ). Purity (HPLC, method 2): 98.1% (tR ¼ 18.76 min). 6.3.2. 3-(4-Phenylbutyl)-1,2,3,4,5,6-hexahydro-3-benzazocine (4d) Under N2 atmosphere, 4-phenylbutanal (93 mL, 0.63 mmol, 1.1 eq.) was added to a mixture of secondary amine 11 (100 mg, 0.57 mmol, 1 eq.) and Na2SO4 (approx. 800 mg) in dry CH2Cl2 (4.5 mL). After stirring for 3 h at rt, NaBH(OAc)3 (184 mg, 0.87 mmol, 1.5 eq.) was added and the reaction mixture was stirred for additional 14 h at rt. Sequentially a saturated aqueous solution of NaHCO3 (10 mL) and water (10 mL) were added. The organic layer was separated and the water layer was washed with CH2Cl2 (3  30 mL). The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude product was purified by fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, gradient elution: cyclohexane/ ethyl acetate 99:1 / 90:10 / 80:20 / 50:50, 1% N,N-dimethylethylamine was added to every solvent mixture). The product (121 mg) was further purified by a second fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, cyclohexane/ethyl acetate 99:1 þ 1% N,N-dimethylethylamine) and a third fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, cyclohexane/ethyl acetate 99:1). Yellow oil, yield 95 mg (57%). C21H27N (293.5 g/mol). TLC: Rf ¼ 0.20 (cyclohexane/ethyl acetate 90:10). FTIR: ~n [cm1] ¼ 3283 (NeH); 2955, 2916, 2847 (C-Haliph.); 1605, 1493 (C¼Carom.); 752, 729, 698 (Ar-Hout of plane). 1H NMR (600 MHz, CD3OD): d [ppm] ¼ 1.40e1.47 (m, 2H, PhCH2CH2CH2CH2), 1.49e1.55 (m, 2H, PhCH2CH2CH2CH2), 1.59e1.64 (m, 2H, 5-CH2), 2.27e2.31 (m, 2H, 4-CH2), 2.48e2.52 (m, 2H, PhCH2CH2CH2CH2), 2.56 (t, J ¼ 7.5 Hz, 2H, PhCH2CH2CH2CH2), 2.71e2.75 (m, 2H, 2-CH2), 2.77e2.81 (m, 2H, 6-CH2), 2.81e2.85 (m, 2H, 1-CH2), 7.05e7.15 (m, 7H, AreH, 1Hphenyl, 3-Hphenyl, 5-Hphenyl), 7.21e7.25 (m, 2H, 2-Hphenyl, 6Hphenyl). 13C NMR (151 MHz, CD3OD): d [ppm] ¼ 27.9 (1C, PhCH2CH2CH2CH2), 30.2 (1C, PhCH2CH2CH2CH2), 32.0 (1C, C-6), 33.3 (1C, C-5), 35.7 (1C, C-1), 36.7 (1C, PhCH2CH2CH2CH2), 53.5 (1C, C-4), 59.9 (1C, PhCH2CH2CH2CH2), 60.5 (1C, C-2), 126.7 (1C, C4phenyl), 127.4 (1C, C-8 or C-9), 127.7 (1C, C-9 or C-8), 129.2 (2C, C-

7

2phenyl, C-6phenyl), 129.4 (2C, C-3phenyl, C-5phenyl), 129.8 (1C, C-7), 130.1 (1C, C-10), 141.68 (1C, C-6a), 141.71 (1C, C-10a), 143.7 (1C, C1phenyl). Exact mass (APCI): m/z ¼ 294.2241 (calcd. 294.2216 for C21H28N [MþH]þ). Purity (HPLC, method 2): 99.0% (tR ¼ 19.77 min). 6.3.3. N-(3-phenylpropyl)-1,2,3,4-tetrahydronaphthalen-2-amine (6c) A solution of b-tetralone (12, 30 mL, 33.18 mg, 0.23 mmol, 1 eq.), 3-phenylpropan-1-amine (66 mL, 62.5 mg, 0.46 mmol, 2 eq.) and NaBH(OAc)3 (98.9 mg, 0.47 mmol, 2 eq.) in CH2Cl2 (4 mL) was vigorously stirred for 14 h at rt. A saturated solution of NaHCO3 (6 mL) was added and the reaction mixture was extracted with CH2Cl2 (3  5 mL). The CH2Cl2 layer was dried (Na2SO4), filtered and the solvent was evaporated in vacuo. The crude product was purified by fc (d ¼ 3 cm, l ¼ 12 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.24) to obtain a yellow oil, yield 50.8 mg (84%). C19H23N (265.4 g/mol). FT-IR (neat): n (cm1) ¼ 2920 (CeH), 740 and 698 (1,2-disubst. arom.). 1H NMR (600 MHz, CD3OD): d (ppm) ¼ 1.51 (dtd, J ¼ 12.6/10.9/5.8 Hz, 1H, 3-H), 1.85 (tt, J ¼ 9.1/6.8 Hz, 2H, PhCH2CH2CH2), 2.01e2.08 (m, 1H, 3-H), 2.53 (dd, J ¼ 15.9/10.0 Hz, 1H, 1-H), 2.63e2.72 (m, 4H, PhCH2CH2CH2), 2.73e2.88 (m, 3H, 2-H, 4-H), 2.93e3.00 (m, 1H, 1-H), 6.96e7.08 (m, 4H, AreH), 7.13e7.17 (m, 1H, Ph-H), 7.18e7.21 (m, 2H, Ph-H), 7.23e7.27 (m, 2H, Ph-H). A signal for the NH proton is not observed. 13C NMR (151 MHz, CD3OD): d (ppm) ¼ 29.2 (1C, C-4), 30.1 (1C, C-3), 32.4 (1C, PhCH2CH2CH2), 34.7 (1C, PhCH2CH2CH2), 36.8 (1C, C-1), 47.3 (1C, PhCH2CH2CH2), 55.0 (1C, C-2), 126.7 (1C, Carom), 126.9 (1C, CPh), 126.9 (1C, Carom), 129.4 (4C, CPh), 129.6 (1C, Carom), 130.2 (1C, Carom), 136.0 (1C, Carom ), 137.1 (1C, Carom ), 143.2 (1C, CPh q q q ). Exact Mass (APCI): m/z ¼ 266.1933 (calcd. 266.1903 for C19H24N [M þ Hþ]). Purity (HPLC, method 2): 96.3% (tR ¼ 19.51 min). 6.3.4. 4-Phenyl-1-(1,2,3,4-tetrahydronaphthalen-2-yl)piperidine (6e) A solution of nosylate 14 (50.5 mg, 0.15 mmol, 1 eq.) and 4phenylpiperidine (116.7 mg, 0.72 mmol, 4.8 eq.) in CH3CN (5 mL) was stirred vigorously for 48 h at 75  C. The reaction mixture was concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm, l ¼ 16 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.24) to obtain a yellow solid, mp 104  C, yield 14.6 mg (33%). C21H25N (291.4 g/mol). FT-IR (neat): n (cm1) ¼ 2931 (CeH), 744 and 698 1 (1,2-disubst. arom.). H NMR (600 MHz, CD3OD): d (ppm) ¼ 1.64e1.72 (m, 1H, 3-H), 1.78e1.87 (m, 2H, 3-CH2pip, 5CH2pip), 1.87e1.92 (m, 2H, 3-CH2pip, 5-CH2pip), 2.18e2.25 (m, 1H, 3-H), 2.45e2.55 (m, 2H, 2-CH2pip, 6-CH2pip), 2.59 (tt, J ¼ 12.1/4.1 Hz, 1H, 4-CHpip), 2.79e2.90 (m, 3H, 2-H, 4-H), 2.90e2.96 (m, 1H, 1-H), 2.99e3.05 (m, 1H, 1-H), 3.14e3.24 (m, 2H, 2-CH2pip, 6-CH2pip), 7.01e7.12 (m, 4H, AreH), 7.15e7.20 (m, 1H, Ph-H), 7.21e7.32 (m, 4H, Ph-H). 13C NMR (151 MHz, CD3OD): d (ppm) ¼ 26.9 (1C, C-4), 30.4 (1C, C-3), 32.4 (1C, C-1), 34.4 (1C, C-3pip), 34.4 (1C, C-5pip), 43.9 (1C, C-4pip), 50.7 (1C, C-2pip), 51.2 (1C, C-6pip), 62.4 (1C, C-2), 126.8 (1C, Carom), 126.9 (1C, Carom), 127.2 (1C, CPh), 127.8 (2C, CPh), 129.4 (1C, Carom), 129.5 (2C, CPh), 130.4 (1C, Carom), 136.6 (1C, Carom ), 137.2 (1C, q Carom ), 147.4 (1C, CPh q q ). Exact Mass (APCI): m/z ¼ 292.2096 (calcd. 292.2060 for C21H26N [M þ Hþ]). Purity (HPLC, method 2): 93.2% (tR ¼ 18.93 min). 6.3.5. 4-Benzyl-1-(1,2,3,4-tetrahydronaphthalen-2-yl)piperidine (6f) A solution of nosylate 14 (50.5 mg, 0.15 mmol, 1 eq.) and 4benzylpiperidine (126.7 mg, 0.72 mmol, 4.8 eq.) in CH3CN (6 mL) was stirred vigorously for 48 h at 75  C. The reaction mixture was concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm, l ¼ 14 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.22) to obtain a yellow oil, yield 18.2 mg (39%). C22H27N (305.5 g/mol). FT-

8

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

IR (neat): n (cm1) ¼ 2916 (CeH), 740 and 698 (1,2-disubst. arom.). 1 H NMR (600 MHz, CD3OD): d (ppm) ¼ 1.30e1.38 (m, 2H, 3-H, 4Hpip), 1.57e1.66 (m, 2H, 3-CH2pip, 5-CH2pip), 1.67e1.73 (m, 2H, 3CH2pip, 5-CH2pip), 2.14 (ddt, J ¼ 12.6/5.5/2.8 Hz, 1H, 3-H), 2.27e2.36 (m, 2H, 2-CH2pip, 6-CH2pip), 2.56 (d, J ¼ 7.2 Hz, 2H, PhCH2), 2.72e2.83 (m, 3H, 2-H, 4-H), 2.85e2.92 (m, 1H, 1-H), 2.93e2.98 (m, 1H, 1-H), 3.01e3.10 (m, 2H, 2-CH2pip, 6-CH2pip), 7.02e7.07 (m, 4H, AreH), 7.14e7.18 (m, 3H, Ph-H), 7.23e7.28 (m, 2H, Ph-H). 13C NMR (151 MHz, CD3OD): d (ppm) ¼ 26.8 (1C, C-4), 30.4 (1C, C-3), 32.3 (1C, C-1), 32.9 (2C, C-3pip, C-5pip), 39.3 (1C, C-4pip), 44.0 (1C, PhCH2), 50.3 (1C, C-2pip), 50.7 (1C, C-6pip), 62.4 (1C, C-2), 126.8 (1C, Carom), 126.9 (1C, Carom), 126.9 (1C, CPh), 129.2 (2C, CPh), 129.4 (1C, Carom), 130.1 (2C, CPh), 130.3 (1C, Carom), 136.5 (1C, Carom ), q 137.2 (1C, Carom ), 141.7 (1C, CPh q q ). Exact Mass (APCI): m/z ¼ 306.2227 (calcd. 306.2216 for C22H28N [M þ Hþ]). Purity (HPLC, method 2): 87.2% (tR ¼ 19.88 min).

was concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm, l ¼ 11 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.22) to obtain a colorless solid, mp 102  C, yield 26.6 mg (68%). C21H25N (291.4 g/mol). FT-IR (neat): n (cm1) ¼ 2924 (CeH), 732 and 698 (1,2-disubst. arom.). 1H NMR (300 MHz, CD3OD): d (ppm) ¼ 1.26e1.42 (m, 2H, 3-CH2pip, 5-CH2pip), 1.53e1.64 (m, 1H, 4-CHpip), 1.64e1.74 (m, 2H, 3-CH2pip, 5-CH2pip), 1.98e2.13 (m, 2H, 2CH2pip, 6-CH2pip), 2.56 (d, J ¼ 6.8 Hz, 2H, PhCH2), 2.77e2.92 (m, 2H, 2-CH2pip, 6-CH2pip), 3.01e3.17 (m, 5H, 1-H, 2-H, 3-H), 7.06e7.31 (m, 9H, AreH, Ph-H). 13C NMR (101 MHz, CDCl3): d (ppm) ¼ 32.1 (2C, C3pip, C-5pip), 37.3 (2C, C-1, C-3), 37.9 (1C, C-4pip), 43.3 (1C, PhCH2), 52.2 (2C, C-2pip, C-6pip), 67.3 (1C, C-2), 124.5 (2C, Carom), 125.9 (1C, CPh), 126.5 (2C, Carom), 128.3 (2C, CPh), 129.2 (2C, CPh), 140.7 (1C, CPh q ), 141.7 (2C, Carom ). Exact Mass (APCI): m/z ¼ 292.2061 (calcd. q 292.2060 for C21H26N [M þ Hþ]). Purity (HPLC, method 2): 98.3% (tR ¼ 18.15 min).

6.3.6. N-(4-phenylbutyl)indan-2-amine (7d) A solution of nosylate 17 (57.1 mg, 0.18 mmol, 1 eq.) and 4phenylbutan-1-amine (142 mL, 134.3 mg, 0.9 mmol, 5 eq.) in CH3CN (4 mL) was stirred vigorously for 16 h at 60  C. The reaction mixture was concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm, l ¼ 11 cm, v ¼ 10 mL, CH2Cl2:CH3OH 99:1 þ 1% NH3, Rf ¼ 0.17) to obtain an orange oil, yield 21.3 mg (45%). C19H23N (265.4 g/mol). FT-IR (neat): n (cm1) ¼ 2927 (CeH), 740 and 698 1 (1,2-disubst. arom.). H NMR (400 MHz, CD3OD): d (ppm) ¼ 1.52e1.62 (m, 2H, PhCH2CH2CH2CH2), 1.63e1.73 (m, 2H, PhCH2CH2CH2CH2), 2.60e2.69 (m, 4H, PhCH2CH2CH2CH2), 2.75 (dd, J ¼ 15.6/7.2 Hz, 2H, 1-H, 3-H), 3.14 (dd, J ¼ 15.6/7.3 Hz, 2H, 1-H, 3-H), 3.56 (quint, J ¼ 7.2 Hz, 1H, 2-H), 7.06e7.12 (m, 2H, AreH), 7.12e7.21 (m, 5H, AreH), 7.21e7.28 (m, 2H, AreH). A signal for the NH proton is not observed. 13C NMR (101 MHz, CD3OD): d (ppm) ¼ 30.2 (1C, PhCH2CH2CH2CH2), 30.5 (1C, PhCH2CH2CH2CH2), 36.8 (1C, PhCH2CH2CH2CH2), 40.2 (2C, C-1, C-3), 60.7 (1C, C-2), 125.5 (2C, Carom), 126.7 (1C, CPh), 127.5 (2C, Carom), 129.3 (2C, CPh), 129.4 (2C, CPh), 142.6 (2C, Carom ), 143.6 (1C, CPh q q ). The NHCH2 signal cannot be assigned due to interference with the methanol signal. However, the presence of the signal is confirmed by 2D-HSQC NMR spectroscopy. Exact Mass (APCI): m/z ¼ 266.1903 (calcd. 266.1903 for C19H24N [M þ Hþ]). Purity (HPLC, method 2): 90.0% (tR ¼ 19.17 min).

6.4. Receptor binding studies

6.3.7. 1-(Indan-2-yl)-4-phenylpiperidine (7e) A solution of nosylate 17 (55.0 mg, 0.17 mmol, 1 eq.) and 4phenylpiperidine (135.4 mg, 0.84 mmol, 4.9 eq.) in CH3CN (3 mL) was stirred vigorously for 20 h at 60  C. The reaction mixture was concentrated in vacuo and the residue was purified by fc (d ¼ 1 cm, l ¼ 12 cm, v ¼ 10 mL, cyclohexane:EtOAc 7:3 þ 1% NH3, Rf ¼ 0.28) to obtain a colorless solid, mp 119  C, yield 28.2 mg (59%), C20H23N (277.4 g/mol). FT-IR (neat): n (cm1) ¼ 2927 (CeH), 740 and 698 1 (1,2-disubst. arom.). H NMR (400 MHz, CD3OD): d (ppm) ¼ 1.76e1.95 (m, 4H, 3-CH2pip, 5-CH2pip), 2.20e2.32 (m, 2H, 2-CH2pip, 6-CH2pip), 2.54e2.65 (m, 1H, 4-CHpip), 2.86e2.98 (m, 2H, 2. CH2pip, 6-CH2pip), 3.10e3.26 (m, 5H, 1-H, 2-H, 3-H), 7.06e7.37 (m, 9H, AreH, Ph-H). 13C NMR (101 MHz, CD3OD): d (ppm) ¼ 34.1 (2C, C-3pip, C-5pip), 38.1 (2C, C-1, C-3), 43.5 (1C, C-4pip), 53.7 (2C, C-2pip, C-6pip), 68.5 (1C, C-2), 125.4 (2C, Carom), 127.3 (1C, CPh), 127.7 (2C, Carom), 127.8 (2C, CPh), 129.5 (2C, CPh), 142.3 (2C, Carom ), 147.3 (1C, q CPh q ). Exact Mass (APCI): m/z ¼ 278.1927 (calcd. 278.1903 for C20H24N [M þ Hþ]). Purity (HPLC, method 2): 98.3% (tR ¼ 17.07 min). 6.3.8. 4-Benzyl-1-(indan-2-yl)piperidine (7f) A solution of nosylate 17 (42.6 mg, 0.13 mmol, 1 eq.) and 4benzylpiperidine (115 mL, 114.7 mg, 0.65 mmol, 5 eq.) in CH3CN (3 mL) was stirred vigorously for 20 h at 60  C. The reaction mixture

Performance of receptor binding studies is described in the Supporting Information and in Refs. [21e24] and references [30,31]. 6.5. Cytoprotection Mouse L (tk-) cells stably transfected with the dexamethasoneinducible 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 reference [32]. After careful removal of the medium the plate was blocked twice with 200 mL DMEM containing 1% BSA and rinsed once with 200 mL DMEM. Then the cells in the inner wells (B2-G11) were incubated with 50 mL DMEM and 50 mL of the respective test compound in 5 different concentrations (e.g. 4  105, 106, 107, 108, 109 mol/L) and 100 mL DMEM was added to the remaining outer wells. Each concentration of the test compound was incubated at least in triplicates for 30 min at 37  C. Afterwards 100 mL of a glutamate/ glycine solution (each 20 mM) were added and the cells were incubated for 6 h at 37  C. Subsequently, 50 mL of the supernatant were transferred into a 96-well plate and incubated with 50 mL of the LDH-assay mixture (1 U/mL diaphorase, 1% Na lactate, 0.1% NADþ, 0.08% BSA, 0.4% iodonitrotetrazolium chloride in 75 mM PBS) at 37  C for 45 min. The UV absorption was measured at l ¼ 485 nm in a plate reader (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  105 mol/L served as positive control. 6.6. Molecular biology and two-electrode voltage clamp (TEVC) Two-electrode voltage clamp experiments were carried out as previously described [14,33]. Xenopus laevis oocytes were purchased from EcoCyte Bioscience (Dortmund, Germany) and injected with 0.8 ng GluN1a and 0.8 ng GluN2B cRNA. cRNAs were generated by in vitro transcription with the T7 mMessage mMachine kit (Life Technologies, Darmstadt, Germany) from linearized cDNA templates (wildtype rat). Injected oocytes were stored for 5 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. Barth’s solution was supplemented with 80 mg/L theophylline, 63 mg/L benzylpenicillin, 40 mg/L streptomycin and 100 mg/L gentamycin. Ifenprodil (þ)-tartrate was purchased from Sigma Aldrich. Compound 4d and ifenprodil were used as 10 mM solutions in DMSO, which were diluted with agonist solution (Ba2þ-Ringer supplemented with 10 mM glycine and 10 mM (S)-glutamate) to obtain the desired concentration of compound and constant concentration of

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

agonists. All test solutions were adjusted to equal DMSO concentrations of 0.1%. Ion current inhibition of compounds was tested by two-electrode voltage clamp (TEVC) in Xenopus laevis oocytes at room temperature. For measurements, oocytes were constantly superfused with Ba2þ Ringer (pH 7.4) containing (mmol/L): 10 HEPES, 90 NaCl, 1 KCl, 1.5 BaCl2. Measurements were performed at a holding potential of 70 mV and recording pipettes (0.5e1.5 MU) were backfilled with 3 M KCl. Each compound concentration was tested at five different oocytes.

6.7. Data analysis and statistics Data recording and data analysis was carried out like previously described [14,33]. Electrophysiological data were recorded with GePulse (Dr. Michael Pusch, Genova, Italy; http://users.ge.ibf.cnr.it/ pusch/) and analyzed with Ana (Dr. Michael Pusch, Genova, Italy; http://users.ge.ibf.cnr.it/pusch/) and OriginPro 2019 (OriginLab Corporation, Northampton, MA, USA). Inhibitory activity was calculated as previously described by the following equation:

Ic  Ih inhibtion ¼ 1  Ia  Ih Ih represents the holding current of the oocyte without agonists; Ia is defined as the steady-state current after activation by 10 mM glycine and 10 mM (S)-glutamate; Ic is the resulting steady-state current in presence of agonists and compound. Significance of mean data differences was tested by one-way ANOVA and Tukey post-hoc mean comparison test and indicated by *** for p-values < 0,001, ** for p-values < 0,01, * for p-values < 0,05 and ns for pvalues > 0,05.

6.8. Docking studies The crystal structure of the NMDA-GluN1/GluN2B dimer in complex with Ro 25e6981 (PDB ID: 3QEM) was taken from the Protein Data Bank (PDB; www.rcsb.org). Since 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 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 €dinger’s Protein Preparawas subsequently prepared using Schro tion Wizard [34]. Hydrogen atoms were added, protonation states were assigned, and a restrained minimization was performed. MOE modelling software 2012.1035 was used to generate the molecular structures of all compounds at the physiological pH. The ligands were subsequently prepared for docking using the LigPrep €dinger’s software, where all tool [36] as implemented in Schro 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 25e6981 binding pocket using the program Glide €dinger, LLC, New York, NY, USA, version 9.8) [49] in the (Schro 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 this setup, the co-crystallized inhibitors could be correctly docked (RMSD below 1 Å) into the NMDA-GluN1/GluN2B binding pocket.

9

Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We thank Mark Worrmann for his assistance during laboratory work. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), which is gratefully acknowledged. Moreover, we are grateful to Cells-in-Motion (CiM) Cluster of Excellence for funding a lab visit at the University of Halle-Wittenberg to learn molecular modelling. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2020.112138. References [1] H. Stark, S. Graßmann, U. Struktur Reichert, Funktion und potentielle therapeutische Bedeutung von NMDA-Rezeptoren: Teil 1, Pharm, Unserer Zeit 29 (2000) 159e166. [2] S. Davies, D.B. Ramsden, Huntington’s disease, J. Clin. Pathol.: Mol. Pathol. 54 (2001) 409e413. [3] S.E. Lakhan, M. Caro, N. Hadzimichalis, NMDA receptor activity in neuropsychiatric disorders, Front. Psychiatr. 4 (2013) 52. [4] P. Paoletti, C. Bellone, Q. Zhou, NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease, Nat. Rev. Neurosci. 14 (6) (2013) 383e400. [5] Q. Zhou, M. Sheng, NMDA receptors in nervous system diseases, Neuropharmacology 74 (2013) 69e75. €uner-Osborne, J. Egebjerg, E.Ø. Nielsen, U. Madsen, P. Krogsgaard[6] H. Bra Larsen, Ligands for glutamate receptors: Design and therapeutic prospects, J. Med. Chem. 43 (2000) 2609e2645. [7] F.S. Menniti, M.J. Pagnozzi, P. Butler, B.L. Chenard, S.S. Jaw-Tsai, W. Frost White, CP-101,606, an NR2B subunit selective NMDA receptor antagonist, inhibits NMDA and injury induced c-fos expression and cortical spreading depression in rodents, Neuropharmacology 39 (2000) 1147e1155. [8] R.A. Al-Hallaq, T.P. Conrads, T.D. Veenstra, R.J. Wenthold, NMDA Diheteromeric receptor populations and associated proteins in rat Hippocampus, J. Neurosci. 27 (2007) 8334e8343. [9] H. Chaffey, P.L. Chazot, NMDA receptor subtypes: structure, function and therapeutics, Curr. Anaesth. Crit. Care 19 (4) (2008) 183e201. [10] I. Borza, G. Domany, NR2B selective NMDA antagonists: the evolution of the ifenprodil-type pharmacophore, CTMC 6 (7) (2006) 687e695. [11] B. Tewes, B. Frehland, D. Schepmann, K.-U. Schmidtke, T. Winckler, B. Wünsch, Conformationally constrained NR2B selective NMDA receptor antagonists derived from ifenprodil: synthesis and biological evaluation of tetrahydro-3benzazepine-1,7-diols, Bioorg. Med. Chem. 18 (2010) 8005e8015, https:// doi.org/10.1016/j.bmc.2010.09.026. [12] D. Stroebel, D.L. Buhl, J.D. Knafels, P.K. Chanda, M. Green, S. Sciabola, L. Mony, P. Paoletti, J. Pandit, A novel binding mode reveals two Distinct classes of NMDA receptor GluN2B-selective antagonists, Mol. Pharmacol. 89 (5) (2016) 541e551. [13] E. Karakas, N. Simorowski, H. Furukawa, Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors, Nature 475 (2011) 249e253. €rgel, M. Szermerski, J.A. Schreiber, L. Temme, N. Strutz-Seebohm, [14] F. Bo K. Lehmkuhl, D. Schepmann, S.M. Ametamey, G. Seebohm, T.J. Schmidt, B. Wünsch, Synthesis and pharmacological evaluation of enantiomerically pure GluN2B selective NMDA receptor antagonists, ChemMedChem 13 (2018) 1580e1587. [15] S. Dey, D. Schepmann, B. Wünsch, Role of the phenolic OH moiety of GluN2Bselective NMDA antagonists with 3-benzazepine scaffold, Bioorg. Med. Chem. Lett 26 (2016) 889e893. [16] S. Dey, L. Temme, J.A. Schreiber, D. Schepmann, B. Frehland, K. Lehmkuhl, N. Strutz-Seebohm, G. Seebohm, B. Wünsch, Deconstruction e reconstruction approach to analyze the essential structural elements of tetrahydro-3benzazepine-based antagonists of GluN2B subunit containing NMDA receptors, Eur. J. Med. Chem. 138 (2017) 552e564. [17] S. Gawaskar, D. Schepmann, A. Bonifazi, B. Synthesis Wünsch, GluN2B affinity and selectivity of benzo[7]annulen-7-amines, Bioorg. Med. Chem. 22 (2014) 6638e6646. [18] S. Gawaskar, L. Temme, J.A. Schreiber, D. Schepmann, A. Bonifazi, D. Robaa, W. Sippl, N. Strutz-Seebohm, G. Seebohm, B. Wünsch, Design, synthesis, pharmacological evaluation and docking studies of GluN2B-selective NMDA receptor antagonists with a benzo[7]annulen-7-amine scaffold, ChemMedChem 12 (2017) 1212e1222.

10

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138

 ska, B. Musielak, P. Serda, M. Owin  ska, B. Rys, Conformation of [19] A. Witosin eight-membered benzoannulated lactams by combined NMR and DFT studies, J. Org. Chem. 77 (2012) 9784e9794. [20] L.W. Deady, N.H. Pirzada, R.D. Topsom, Synthesis of tetrahydro-2- and 3benzazepines, and of hexahydro-3-benzazocine, J. Chem. Soc., Perkin Trans. 1 (1973) 782e783. [21] D. Schepmann, B. Frehland, K. Lehmkuhl, B. Tewes, B. Wunsch, Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors, J. Pharmaceut. Biomed. Anal. 53 (2010) 603e608. € hlich, D. Schepmann, B. Wünsch, [22] P. Hasebein, B. Frehland, K. Lehmkuhl, R. Fro Synthesis and pharmacological evaluation of like- and unlike-configured tetrahydro-2-benzazepines with the a-substituted benzyl moiety in the 5position, Org. Biomol. Chem. 12 (29) (2014) 5407e5426. [23] C. Meyer, B. Neue, D. Schepmann, S. Yanagisawa, J. Yamaguchi, E.U. Würthwein, K. Itami, B. Wünsch, Improvement of s1 receptor affinity by late-stage C-H-bond arylation of spirocyclic lactones, Bioorg. Med. Chem. 21 (7) (2013) 1844e1856. [24] K. Miyata, D. Schepmann, B. Wünsch, Synthesis and s receptor affinity of regioisomeric spirocyclic furopyridines, Eur. J. Med. Chem. 83 (2014) 709e716. €hlich, D. Schepmann, B. Wünsch, Design, synthesis and phar[25] E. Rack, R. Fro macological evaluation of spirocyclic s1 receptor ligands with exocyclic amino moiety (increased distance 1), Bioorg. Med. Chem. 19 (2011) 3141e3151. [26] P.B. Burger, H. Yuan, E. Karakas, M. Geballe, H. Furukawa, D.C. Liotta, P. Snyder, S.F. Traynelis, Mapping the binding of GluN2B-selective N-methyl-D-aspartate receptor negative allosteric modulators, Mol. Pharmacol. 82 (2012) 344e359. [27] S. Thum, D. Schepmann, R.F. Reinoso, I. Alvarez, S.M. Ametamey, B. Wünsch, Synthesis and pharmacological evaluation of fluorinated benzo[7]annulen-7amines as GluN2B-selective NMDA receptor antagonists, J. Label. Compd.

Radiopharm. 69 (2019) 354e379. [28] S. Thum, D. Schepmann, E. Ayet, M. Pujol, F.R. Nieto, S.M. Ametamey, B. Wünsch, Tetrahydro-3-benzazepines with fluorinated side chains as NMDA and s1 receptor antagonists: synthesis, receptor affinity, selectivity and antiallodynic activity, Eur. J. Med. Chem. 177 (2019) 47e62. [29] S. Baumeister, D. Schepmann, B. Wünsch, Thiophene bioisosteres of GluN2B selective NMDA receptor antagonists: synthesis and pharmacological evaluation of [7]annuleno[b]thiophen-6-amines, Bioorg. Med. Chem. 15 (2) (2020) 115245, https://doi.org/10.1016/j.bmc.2019.115245, 28. €hler, E.-U. Würthwein, B. Wünsch, Synthesis [30] A. Banerjee, D. Schepmann, J. Ko and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists, Bioorg. Med. Chem. 18 (22) (2010) 7855e7867. € hler, K. Bergander, J. Fabian, D. Schepmann, B. Wünsch, Enantiomerically [31] J. Ko pure 1,3-dioxanes as highly selective NMDA and s₁ receptor ligands, J. Med. Chem. 55 (20) (2012) 8953e8957. [32] L. Temme, D. Schepmann, J.A. Schreiber, B. Frehland, B. Wünsch, Comparative pharmacological study of common NMDA receptor open channel blockers regarding their affinity and functional activity towards GluN2A and GluN2B NMDA receptors, ChemMedChem 13 (2018) 446e452. [33] J.A. Schreiber, D. Schepmann, B. Frehland, S. Thum, M. Datunashvili, T. Budde, M. Hollmann, N. Strutz-Seebohm, B. Wünsch, G. Seebohm, A common mechanism allows selective targeting of GluN2B subunit-containing Nmethyl-D-aspartate receptors, Commun. Biol. 2 (2019) 420, https://doi.org/ 10.1038/s42003-019-0645-6. €dinger Release 2014-2, LigPrep, Schro €dinger, LLC, New York, NY, 2014. [34] Schro [35] Molecular Operating Environment (MOE), 2012.10 H3A 2R7, Chemical Computing Group Inc., Montreal, QC, Canada, 2012, p. 1010. Sherbooke St. West, Suite #910. € dinger, LLC, New [36] Small-Molecule Drug Discovery Suite 2014-2: Glide, Schro York, NY, 2014 version 9.8.