Phenol—Benzoxazolone bioisosteres: Synthesis and biological evaluation of tricyclic GluN2B‐selective N‐methyl‐
d‐aspartate receptor antagonists

Tricyclic tetrahydrooxazolo[4,5‐h]‐[3]benzazepin‐9‐ols 22 were designed as phenol bioisosteres of tetrahydro‐3‐benzazepine‐1,7‐diols. Key features of the synthesis are the introduction of the trifluoromethylsulfonyl and allyl protective groups at the heterocyclic N‐atoms. Two methods were developed to convert the triflyl‐protected ketone 16 into tricyclic alcohols 21 bearing various N‐substituents. According to the first method, trifluoromethanesulfinate was removed by K2CO3. Following the selective reduction of the imino moiety of 17 with NaBH(OAc)3 afforded the aminoketone 18, which was reductively alkylated and reduced. According to the second method, both the imine and the ketone of the iminoketone 17 were reduced with NaBH4 to yield the aminoalcohol 20, which was alkylated or reductively alkylated to form tertiary amines 21f–21r. In the last step, the allyl protective group of 21 was removed with RhCl3 and HCl to obtain oxazolones 22. In receptor binding studies using [3H]ifenprodil as radioligand ketone, 22m showed the highest GluN2B affinity (Ki = 88 nM). However, a reduced affinity toward GluN2B subunit‐containing N‐methyl‐ d‐aspartate (NMDA) receptors was observed for oxazolones 22 compared to bioisosteric 3‐benzazepine‐1,7‐diols. High selectivity of 22m for the ifenprodil binding site of GluN2B‐NMDA receptors over the 1‐(1‐phenylcyclohexyl)piperidine binding site and σ2 receptors was observed, but only negligible selectivity over σ1 receptors. In two‐electrode voltage clamp experiments, the 4‐phenylbutyl derivative 22d (Ki = 422 nM) demonstrated 80% inhibition of ion flux at a concentration of 1 µM. The differences in GluN2B affinity and inhibitory activity are explained by docking studies. In conclusion, 22d is regarded as a novel scaffold of highly potent GluN1/GluN2B antagonists.


| INTRODUCTION
In the mammalian brain, (S)-glutamate represents the most important excitatory neurotransmitter. It interacts with metabotropic (G protein-coupled) and ionotropic (ion channel-coupled) receptors.
Three ionotropic glutamate receptor classes are differentiated, which are termed N-methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5methylisoxazol-4-yl)propionate (AMPA), and kainate receptors. All three ionotropic glutamate receptors play a key role in excitatory neurotransmission in the central nervous system. [1][2][3] The heterotetrameric NMDA receptor contributes considerably to processes leading to long-term potentiation (LTP), a special form of synaptic plasticity, which correlates with learning and memory. [4,5] On the other hand, acute and chronic overactivation of the NMDA receptor is involved in the condition of excitotoxicity, a selfamplifying process of neuronal damaging by increased release of the excitatory neurotransmitter (S)-glutamate. Therefore, drugs inhibiting the ion flux of Ca 2+ through the NMDA receptor are of great interest for the treatment of acute neuronal disease states (e.g., stroke, brain injury, epileptic seizure, and migraine) and chronic neurodegenerative processes (e.g., Parkinson's disease, Alzheimer's disease, and multiple sclerosis). [3,[6][7][8] Seven protein variants are known, which are able to form the heterotetrameric NMDA receptor: one GluN1 subunit existing in eight splice variants GluN1a-h, four GluN2 subunits termed GluN2A-D and two GluN3 subunits termed GluN3A and GluN3B. Usually, a functional NMDA receptor consists of two mandatory GluN1 and two additional subunits, that can be different combinations of the GluN2 and GluN3 subunits. GluN3 subunits are found predominantly in the prenatal phase of an individual and are therefore not considered in this project. [9] The activation of the NMDA receptor requires slight depolarization to release the Mg 2+ -block as well as simultaneous binding of two coagonists: (S)-glutamate interacts with its binding site on the GluN2 subunit, and glycine interacts with its binding site on the GluN1 subunit.
Although the GluN1 subunit is expressed ubiquitously throughout the central nervous system, different expression levels of the GluN2 subunits, which are encoded by four different genes, are found in different brain regions. Thus, the features of the GluN2 subunit determine the properties and gating characteristics of different NMDA receptor subtypes. [10][11][12][13] Herein, we focus on NMDA receptors containing the GluN2B subunit (GluN2B-NMDA receptors). The GluN2B subunit is predominantly expressed in the cortex, hippocampus, and striatum of the human brain. [12] Compared to NMDA receptors with the GluN2A subunit, GluN2B-NMDA receptors exert slower channel opening kinetics and thus slower Ca 2+ influx. [14] Negative allosteric modulators selectively targeting GluN2B-NMDA receptors represent promising drug candidates for the treatment of various CNS disorders including neurodegenerative diseases. Compared to nonselective open-channel NMDA receptor antagonists (e.g., phencyclidine, [S]ketamine), selective GluN2B-NMDA receptor antagonists show a better side effect profile. [15,16] β-Aminoalcohol ifenprodil (1) represents the prototypical negative allosteric modulator of GluN2B-NMDA receptors ( Figure 1). It interacts with an allosteric binding site at the interface between the amino-terminal domains of the GluN1 and GluN2B subunit, which is termed the ifenprodil binding site. [16][17][18] In Japan, racemic F I G U R E 1 Design of oxazolo-annulated 3-benzazepines A: The prototypical lead compounds ifenprodil (1) and  (2) were developed into 3-benzazepine WMS-1410 (3) and besonprodil (4). Novel oxazolo-annulated 3-benzazepines A result from merging WMS-1410 (3) with besonprodil (4). *Only one enantiomer of the racemic mixture is depicted for clarity.
unlike-configured ifenprodil is approved as a cerebral vasodilator.
However, the metabolically labile phenol is still present in 3. [35,36] In besonprodil (4), the benzylic secondary alcohol of ifenprodil (1) and  is replaced bioisosterically by a sulfoxide, and the distance between the benzene ring and the piperidine ring is similar to the distance in Ro 25-6981 (2). The most important modification of 4 is the bioisosteric replacement of the phenol present in 1-3 by a benzoxazolone, which is no longer prone to fast glucuronidation [37] ( Figure 1).
Herein, we wish to report on the synthesis of oxazolo-annulated 3-benzazepines of type A. In A, the bioisosteric replacement of the phenol of 1-3 by the benzoxazolone system of 4 and the reorganization of the flexible β-aminoalcohol side chain of 1 in the 3-benzazepine system of 3 are combined. In particular, the Natom (N-3) of the oxazolone system of A should bear a proton allowing the formation of an H-bond similar to the phenolic OH moiety of 1-3.

| Synthesis
Very recently, we have reported the synthesis of oxazolo-annulated 3-benzazepines of type A bearing a p-methoxybenzyl (PMB) moiety at the N-atom of the oxazolone ring (N-3). Unfortunately, it was not possible to remove this N-protective group without destroying the molecule. In particular, the removal of the PMB moiety was tried with H 2 and different catalysts, with (NH 4 ) 2 [Ce(NO 3 ) 6 ], with acid (TFA, HCl), and with α-chloroethyl chloroformate. [38] As the PMB-protective group could not be removed, the synthesis of unsubstituted oxazolo-annulated 3-benzazepines of type A was performed without a protective group at the N-atom (Scheme 1). Thus, Mitsunobu reaction of the alcohol 5 [39] with N-triflylglycine methyl ester (6) [38] led to ester 7, which was hydrolyzed to yield acid 8. P 4 O 10 /CH 3

SO 3 H mediated intramolecular
Friedel-Crafts acylation of acid 8 provided three products: The desired oxazolobenzazepine 9, its regioisomer 10, and the tetrahydroisoquinoline 11. Due to the missing substituent at the oxazolone N-atom, acid 8 can also attack the 4-position leading to the regioisomer 10. The formation of the ring-contracted tetrahydroisoquinoline 11 was unexpected as the analogous PMB-protected acid did not lose CO to form an analogous tetrahydroisoquinoline. [38] Due to separation and purification problems, the yield of 9 did not exceed 31% even after optimization of the cyclization reaction. Elimination of trifluoromethanesulfinate (CF 3 SO 2 − ) with K 2 CO 3 in refluxing acetonitrile [40,41] followed by reduction of cyclic imine 12 with NaBH(OAc) 3 and reductive alkylation with benzaldehyde and NaBH (OAc) 3 [42] provided the benzylamine 13 in 17% yield. In summary, oxazolo-annulated 3-benzazepine 13 without N-3-substituent was available, but in very low yield, which is due to side reactions during the intramolecular Friedel-Crafts acylation and isolation and purification problems.
Thus, the introduction of a protective group at N-3 appeared to be beneficial for the preparation of a large series of oxazoloannulated 3-benzazepines of type A. At first, the introduction of a triisopropylsilyl protective group into ester 7 was performed.
However, the resulting silyl-protected ester could not be saponified without removal of the silyl protective group (see Supporting Information: Scheme S1). Introduction of a methoxyethoxymethyl (MEM) protective group into 7 and subsequent saponification of the ester were successful. However, the intramolecular Friedel-Crafts acylation required acidic conditions, which led to cleavage of the MEM protective group (see Supporting Information: Scheme S2).
Thus, a protective group, which was stable under basic (saponification of the ester) and acidic conditions (intramolecular Friedel-Crafts acylation) had to be selected. We chose the allyl protective group, which should fulfill these requirements, but could be cleaved off at the end of the synthesis by an Rh-catalyzed isomerization and hydrolysis.
The allyl protective group was introduced by reacting the ester 7 with allyl bromide. In the next step, the triflyl protective group had to be removed.
For this purpose, the tricyclic ketone 16 was treated with K 2 CO 3 in acetone to eliminate trifluoromethanesulfinate (CF 3 SO 2 -) [40,41] and form the iminoketone 17. Without isolation, the iminoketone 17 was reduced with NaBH(OAc) 3 [42] to give the aminoketone 18, which was reductively alkylated with benzaldehyde and NaBH(OAc) 3 . [42] For the reaction of iminoketone 17 with NaBH(OAc) 3  To remove the allyl protective group, the allyl derivatives 21 were treated with RhCl 3 , which led to isomerization of the double bond yielding enamines. [43,44]  at the receptor is a measure of the affinity of the test compound. [45] In series 21 with N-allyl moiety, the GlunN2B affinity appears to be rather low. In the series of homologous phenylalkyl substituted derivatives 21a-21f the highest GluN2B affinity was observed for the phenylbutyl derivative 21d (K i = 515 nM). Introduction of an S-atom (21h), an SO 2 moiety (21i), and a 1,3-dioxane ring (21j, 21k) in the side chain or reduction of the conformational flexibility by biphenyl residues (21n-21p) resulted in reduced GluN2B affinity.
Only an O-atom (21g, K i = 400 nM) within the side chain was tolerated by the ifenprodil binding site. Unexpectedly, the piperidinylbenzyl substituted oxazolobenzazepine 21q showed the highest GluN2B affinity (K i = 194 nM) of the N-allyl derivatives 21 (Table 1).  [41,46,47] Therefore, the inhibitory activity of the most promising derivatives 21q (K i = 194 nM), 22d (K i = 422 nM), and 22m (K i = 88 nM) was tested in twoelectrode voltage-clamp (TEVC) experiments using GluN1a/GluN2B expressing oocytes. Addition of 10 µM glycine and 10 µM (S)glutamate induced a characteristic NMDA-specific ion current that was inhibited by the application of 10 µM of 21q, 22d, and 22m. [31,46] To evaluate the activity of the test compounds, the inhibitory activity of ifenprodil (1) was also recorded at concentrations of 1 and 10 µM.
Compounds achieving more than 80% inhibitory activity at 10 µM were tested at a concentration of 1 µM as well. The results are summarized in Table 2.
All tested compounds were able to inhibit the agonist-induced current moderately (21q) or strongly (1, 22d, 22m) at 10 µM concentration ( Table 2). The highest inhibitory activity was obtained by 10 µM of ifenprodil (1), which was not significantly different (p > 0.05) from the inhibitory activity of 10 µM 22d. On the other hand, the activity of 21q and 22m at 10 µM was significantly reduced compared to ifenprodil (1) and 22d (statistical analysis see Table S1).
Since the inhibitory activity of ifenprodil (1) and 22d at 10 µM were close to full inhibition of NMDA receptors, the activity of 1 µM for both test compounds was evaluated. Similar to the observed inhibitory effect at 10 µM, the activity of 1 µM of 22d was not significantly different compared to the respective activity at 1 µM ifenprodil (1). Both compounds were able to inhibit the agonistinduced current by 77% (1) and 80% (22d) suggesting IC 50 values in the nanomolar range. Even at a concentration of 1 µM, the inhibitory activities of 1 and 22d were significantly higher than the inhibitory activity of 21q at 10 µM underlining the high potency of ifenprodil (1) and 22d.

| Interactions with the ifenprodil binding site
The evaluation of the interactions between the ligands and their receptor binding site local in silico docking was performed using the crystal structure of a functional heterotetrameric NMDA receptor (PDB 4PE5). [18] The virtual docking box was placed around the ifenprodil-binding site formed at the interface of the amino-terminal domains of the GluN1 and GluN2B subunits. The subdomains R1 and R2 of the GluN2B subunit form this binding site ( Figure 2). To generate reliable docking results the docking procedure was optimized and tested by redocking of (1R,2S)-1. The lowest RMSD value obtained by redocking of (1R,2S)-1 compared to the crystal structure was 1.4 Å ( Figure 2C).
In previous studies, it was shown that ligands with (R)-configuration at the benzylic position represent the eutomers. [36,46] Therefore, T A B L E 1 Interaction of oxazolobenzazepines 21 and 22 with the ifenprodil and phencyclidine binding sites of the N-methyl-D-aspartate receptor and with both σ receptor subtypes.  The docking poses displayed in Figure 3  Note: Inhibitory activity was evaluated in presence of 10 µM glycine/ 10 µM (S)-glutamate at 1 or 10 µM concentrations of the respective inhibitor. The number of independent oocytes (n) is given for each compound and concentration.
as interactions with a loop formed by GluN1 amino acids S132-L135, are required for high activity of ifenprodil-derived compounds. [ Moreover, bioisosteric replacement of the phenol by a benzoxazolone scaffold in compounds 22d and 22m did not disturb H-bonding with E236, which has also previously been identified as an important interaction partner in the R2 subdomain.
Although a significant difference in activity of 1 and 22d was not observed, the activity of 22m was reduced considerably.
This observation could be explained by the docking studies. While  [18] F I G U R E 3 Docking reveals differences in in silico binding of (1R,2S)-1 (a), (R)-21q (b), (R)-22d (c), (R)-22m (d). Amino acids from GluN1 (violet) are colored in pink, while amino acids from R1 (green) and R2 (blue) are colored in green and blue; H-bonding is indicated by yellow dotted lines, and π,π-interactions are indicated by red lines.
22d and (1R, 2S)-1 can interfere strongly with F114, the tert-butyl moiety of 22m reduces the direct π,π-interactions with this amino acid. We showed in a previous mutagenesis study the necessity of strong π,π-interactions with F114 for high activity. [46] 2.

| Receptor selectivity
In addition to the interaction of the oxazolobenzazepines 21 and 22 with the ifenprodil binding site of the NMDA receptor, their interactions with the phencyclidine (1-(1-phenylcyclohexyl)piperidine, PCP) binding site were investigated using the radioligand [ 3 H]-(+)-MK-801 and pig brain cortex membrane preparations. [48,49] The PCP binding site is located within the ion channel pore of the NMDA receptor. Ligands interacting with the PCP binding site do not differentiate between different NMDA receptor subtypes. [3,14] Therefore, our ligands should not interact with this potential binding site. Indeed, at a concentration of as radioligands. Guinea pig brain preparations (σ 1 ) and rat liver preparations (σ 2 ) were employed as receptor material. [50][51][52] Some of the allyl derivatives 21 showed moderate σ 1 affinity (e.g., 21f, K i = 144 nM), but none of them exhibited a higher σ 1 affinity than 100 nM. The σ 2 affinity of the allyl derivatives 21 was even lower (K i (σ 2 ) > 370 nM). In the series of oxazolobenzazepines 22 without allyl protective group, three compounds 22a, 22f, and 22m achieved K i -values in the low two-digit nanomolar range. All three compounds interacted selectively or preferably with the σ 1 receptor.
Altogether, the piperidinylbenzyl substituted derivative 21q reveals high selectivity for GluN2B-NMDA receptors over the PCP binding site, but no selectivity over both σ receptor subtypes. Although the affinity toward GluN2B-NMDA receptors is only moderate (K i = 422 nM), the phenylbutyl derivative 22d is selective over the PCP binding site and σ 1 receptors, but not over the σ 2 subtype. 22m with a butanoylphenyl substructure showed the highest GluN2B-NMDA receptor affinity (K i = 88 nM) of this series of compounds with high selectivity over the PCP binding site and σ 2 receptors. Unfortunately, 22m interacted with even higher affinity with σ 1 receptors (K i (σ 1 ) = 16 nM).

| CONCLUSION
Due to fast metabolic degradation, in particular conjugation with glucuronic acid, [32,35] the phenol of the potent GluN2B antagonist 3 was replaced bioisosterically by the metabolically more stable benzoxazolone moiety that is also found in the potent GluN2B antagonist besonprodil (4).
Performing the reaction sequence without a protective group at the benzoxazolone moiety using acid 8 for the intramolecular Friedel Crafts acylation, led to the desired tricyclic ketone 9 and finally to the benzylated ketone 13 without a substituent at the oxazolone ring. However, due to side reactions (nonregioselective Friedel Crafts acylation, ring contraction) and purification problems originating from the zwitterionic nature of the intermediates and products (acidity of benzoxazolone, basicity of amine), the yields were rather low. Therefore, a protective group had to be introduced into the benzoxazolone system.
Attempts to conduct the reaction sequence with the MEM or triisopropylsilyl protective group did not result in the tricyclic oxazolobenzazepines due to the low stability of the protective groups. After introduction of the PMB protective group, the desired oxazolobenzazepines could be prepared, but the protective group could not be cleaved off without destroying the compounds. [38] However, the allyl protective group showed the optimal properties surviving all transformations. At the end of the synthesis, it could be removed upon isomerization with Rh 3+ and subsequent hydrolysis of the formed enamines.
To obtain compounds with diverse N-substituents, the triflylprotective group of ketone 16 was removed by β-elimination of trifluoromethanesulfinate. Two main methods were developed to convert the resulting iminoketone 17 into the alcohols

21.
According to the first method, NaBH(OAc) 3 reduced selectively the imino moiety of iminoketone 17 providing aminoketone 18, which was reductively alkylated and finally reduced to give alcohols 21a-21e. Alternatively, the iminoketone 17 was completely reduced with NaBH 4 to form aminoalcohol 20, which was alkylated with alkyl halides or with aldehydes and NaBH(OAc) 3 .
In receptor binding studies, 21q (K i = 194 nM), 22d (K i = 422 nM), and 22m (K i = 88 nM) showed the highest affinity toward the ifenprodil binding site. Since previous studies have shown that high affinity does not always translate into high activity, [41,46,47] TEVC analysis for these compounds was per- Data acquisition: HSM software, manual integration.
Unless otherwise noted, the purity of all test compounds is >95% according to one of these HPLC methods.
The combined organic layers were dried (Na 2 SO 4 ) and the solvent was removed under reduced pressure giving a brown oil as crude product, which was purified by flash column chromatography (Ø     4.2.3 | Preparation of membrane homogenates from pig brain cortex [48,49] Fresh pig brain cortex was homogenized with the potter 4.2.5 | Performance of the GluN2B binding assay [45] The competitive binding assay was performed with the radi- 4.2.6 | Performance of the PCP binding assay [48,49] The assay was performed with the radioligand 4.2.7 | Performance of σ 1 and σ 2 receptor assays [50][51][52] The interaction with σ 1 and σ 2 receptors was performed as described in Meyer et al. [50] , Miyata et al. [51] , and Hasebein et al. [52]

| TEVC measurement
The compound activity was evaluated by TEVC measurements as previously described. [36,46] Xenopus laevis oocytes were purchased from Ecocyte Bioscience (Dortmund) and injected with 0.

| Docking studies
Docking was performed using YASARA 21, implemented AutoDock program, and AMBER15IPQ force field as previously described. [46] Ligands were constructed in YASARA using SMILES codes with defined stereochemistry followed by structure optimization by semiempirical quantum mechanics force field (MOPAC). Generated compound structures were locally docked into the crystal structure of full GluN1b/GluN2B NMDA receptor (PDB ID 4PE5). [18]