Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-d -aspartate receptor (NMDAR) containing GluN2B subunit

Accepted Manuscript Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-d-aspartate receptor (NMDAR) containing GluN2B subunit Maria Rosa Buemi, Laura De Luca, Stefania Ferro, Emilio Russo, Giovambattista De Sarro, Rosaria Gitto PII: DOI: Reference:

S0968-0896(16)30101-8 http://dx.doi.org/10.1016/j.bmc.2016.02.021 BMC 12823

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

12 November 2015 21 January 2016 13 February 2016

Please cite this article as: Buemi, M.R., Luca, L.D., Ferro, S., Russo, E., Sarro, G.D., Gitto, R., Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-d-aspartate receptor (NMDAR) containing GluN2B subunit, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc. 2016.02.021

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Bioorganic & Medicinal Chemistry

Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-D-aspartate receptor (NMDAR) containing GluN2B subunit Maria Rosa Buemi,a Laura De Luca,a Stefania Ferro,a Emilio Russo,b Giovambattista De Sarro,b Rosaria Gittoa, a

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali (CHIBIOFARAM) Università degli Studi di Messina, Viale Annunziata, I-98168 Messina, Italy. b Dipartimento di Medicina Sperimentale e Clinica, Università Magna Graecia, Viale Europa Località Germaneto, I-88100 Catanzaro, Italy.

A RT I C L E I N F O

A BS T RA C T

Article history: Received Received in revised form Accepted Available online

Negative allosteric modulators (NAMs) of GluN2B-containing NMDARs provide pharmacological tools for the treatment of chronic neurodegenerative diseases. Novel NAMs have been designed on the basis of computational studies focused on the “hit compound” 3. This series of indoles has been tested in competition assay. Compounds 16 and 17 were the most active ligands (IC 50 values of 83nM and 71nM, respectively) and they showed a potency close to that of reference compounds ifenprodil (1, IC50 = 47 nM) and 3 (IC50 = 25 nM). Furthermore, docking studies have been performed for active ligand 16 and the results were in a good agreement with biological data .

Keywords: Glutamate GluN2B/NMDA synthesis indoles molecular docking

1. Introduction Glutamate (Glu) is the main excitatory neurotransmitter in the mammalian central nervous system (CNS). Glu activates and binds to membrane receptors and specific transporters. The glutamate-gated ion channel receptors (named iGluRs) are ligand-gated ion channels composed of four subunits organized around a central ion channel. iGluRs can be subdivided into three families classified on the basis of their selective exogenous ligands: AMPA receptors (AMPARs), kainate receptors (KARs) and NMDA receptors (NMDARs).[1] Among them NMDARs play a crucial role for neuronal development, coincidence detection for long-term potentiation (LTP), synaptic plasticity, learning, memory and cell survival. NMDARs display highly calcium permeability, voltage-dependence by magnesium block, slow deactivation kinetics. These features can be different among NMDARs which are heterotetrameric assembly being composed of two GluN1 subunits and two GluN2 subunits (more rarely GluN3 subunits) encoded by different genes. The “diheteromeric” GluN2 assembly combines two of the four different GluN2A, GluN2B, GluN2C and GluN2D subunit. The specific combination of subunits controls gating and ligandbinding properties. [2, 3] Actually, GluN1 subunit is ubiquitously expressed in the CNS at all stages of development; in the case of GluN2B subunits, the

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expression varies on the basis of neuronal region as well as brain development. The binding of two agonists is necessary for the activation of NMDARs containing GluN1 and GluN2: glutamate binds the GluN2 subunit and glycine binds the GluN1 subunit. From a structural point of view, the NMDAR subunits contain specific modular domains including the amino-terminal domain (ATD), ligand-binding domain (LBD), transmembrane domain (TMD), and carboxy-terminal domain (CTD). Thus there are different sites capable to bind small extracellular ligands acting as agonists, antagonists, co-agonists, competitive agonists, channel blockers and allosteric modulators. Among them allosteric modulators are considered the most promising compounds both as pharmacological tools and prototypes for the identification of neurotherapeutics.[4] The allosteric modulators might offer the advantage that they can act through interaction to some specific residues that are poor conserved in iGluRs. Thus the allosteric modulators could induce subunit selectivity and reduced side-effects. We focused our interest on NMDARs as potential drug targets for the treatment of neurodegenerative diseases associated to hyper-activation or hypo-activation of glutamatergic transmission. In particular, NMDARs are implicated in epilepsy, Alzheimer, chronic and neuropathic pain, schizophrenia, depression and mental retardation, as well as neuronal loss following stroke [5] [6].

 Rosaria Gitto Tel.: +39 0906766413; fax: +390906766402; e-mail: [email protected]

The antihypertensive agent ifenprodil (1) [7] might be considered the prototype of the phenylethanolamines displaying subunit selective allosteric inhibition of NMDARs containing GluN2B subunit. The allosteric modulation of NMDARs happens at the level ATDs of dimeric interface of GluN1/GluN2B subunits. X-ray structures of the hetero-tetrameric GluN1/GluN2B receptor in complex with 1 have provided insights into the binding sites and mechanism of action of negative allosteric modulators (NAMs) of GluN2B-containing NMDARs. The co-crystal structures highlighted that 1 and its analogue Ro 25-6981 (2) exhibit interactions into the heterodimeric GluN1/GluN2B interface that can be summarized as follow: (a) hydrophobic interactions between benzylpiperidine group and a cluster of aliphatic and aromatic residues from GluN1 and GluN2B; (b) a crucial H-bonding contact between hydroxyphenyl moiety and the key residue Glu236 from GluN2B; (c) interaction between the nitrogen atom of piperidine nucleus and carbonyl Gln110 from GluN2B. [8]

N N

N HO

HO

Me

Me

O

HN

OH 1, ifenprodil

OH 2, Ro 25-6981

OH 3

Figure 1. GluN2B subunit-containing NMDA receptor ligands.

We have previously disclosed a series of 3-substituted-indoles as a novel class of NAMs that bind GluN2B-containing NMDARs [9-14]. Notably, the 3-(4-benzylpiperidin-1-yl)-1-(5hydroxy-1H-indol-3-yl)ethan-1-one (3) proved to be one of the most potent ligands of this class of NAMs. Compound 3 yielded of IC50 = 25 nM in competition assay with [3H]ifenprodil and produced reversible blockade of NMDA-induced current in electrophysiological experiments. Moreover, 3 was able to protect DBA/2 mice against audiogenic seizures.[15] Thus, compound 3 has been identified as a new lead structure for the development of neuroprotective agents.[15, 16] On the basis of our previous findings we decided to synthesize new indole derivatives designed in order to optimize the interaction within GluN1/Glu2NB interface of NMDARs. Given that the 5-hydroxyindole fragment emerged as a structural requirement for promoting high affinity for this class of NAMs of NMDARs [16], we chose to introduce several structural modification at level of other portions of “hit compound” 3: (a) we modified the linker between the indole ring and benzylpiperidine fragment; (b) we changed the bridge connecting phenyl ring to piperidine fragment; (c) we introduced new substituents on aromatic ring of benzylpiperidine portion. Thereby we present in vitro and in silico studies on this series of new 3-substituted-indole derivatives structurally related to the prototype 3. 2. Results and discussion At beginning of our rational design we analyzed the interactions of “hit compound” 3 within the so-called ifenprodil pocket at GluN1/GluN2B interface retrieved from RCSB Protein DataBank (PDB code: 3QEL). [8, 16] As displayed in Figure 2 compound 3 establishes hydrogen bond interactions and hydrophobic contacts within the heterodimer composed of

GluN1/GluN2B subunits. In more details the compound 3 engages profitable contacts both with specific residues from GluN1 (Tyr109, Lys131 and Leu135) and GluN2B (Gln110, Ile111, Phe114, Phe176, Pro177, Glu236) subunits.

Figure 2. Panel A: Docking pose of compound 3 at the GluN1-GluN2B subunit interface (PDB code 3QEL). [6] Crucial residues are drawn in stick and coloured in cyan (GluN2B) and in green (GluN1). Hydrogen bonds are shown as dashed yellow lines. Panel B: Structural modification carried out on “hit compound” 3.

Our earlier studies on a library of indole derivatives provided us the understanding that the presence of a 5-hydroxyl substituent combines an optimal binding mode with the best pharmacological profile.[16, 17] This evidence prompted us to synthesize and test a new series of indole derivatives retaining the 5-hydroxy-indole fragment for which we hypothesize a crucial interaction in the bottom region of GluN1/GluN2B interface with the key residues Glu236, Phe176 and Pro177 from GluN2B subunit (see Figure 2) and Lys131 from GluN1. As shown in Figure 2 (Panel B), to define the structural requirements for an optimal binding to the middle region of so-called ifenprodil site, we changed the ethanone fragment linking the indole ring with benzylpiperidine moiety of “hit compound 3”. Furthermore we introduced a different bridge between the phenyl and piperidine nucleus. New compounds have been also designed to probe the leading role of hydrophobic interactions at the pocket hosting the benzyl moiety in the upper area. Thus, we added an alkyl/alkoxy-substituent as well as a fluorine atom on the phenyl moiety of benzylpiperidine fragment. Therefore we studied how the enhancement of the degree of occupation can be beneficial to improve binding affinity to GluN2B containing NMDARs. Synthesis of the designed compounds has been accomplished as highlighted in Schemes 1-2. Specifically, in Scheme 1 it is depicted the synthetic procedure to obtain derivatives 3-[2-(4benzylpiperidin-1-yl)ethyl]1H-indoles 8 and 9 which were prepared through a multistep synthetic route. Accordingly, the synthesis was started from the appropriate commercially available (5-methoxy-1H-indol-3-yl)acetic acid (4) which was transformed into its methyl ester 6 by treatment with SOCl2 in CH3OH [18] that upon reduction with lithium borohydride yielded the corresponding alcohol 7. To obtain 5-methoxyindole derivative 8, the 4-benzylpiperidine was readily coupled with the not isolated methane-sulfonate ester prepared by activation of alcohol 7. Finally, by reaction with boron tribromide the cleavage of methoxy group was successfully carried-out to give desired compound 9. In Scheme 1 it was also reported the synthesis of 1[4-(4-benzyl)piperidin-1-yl]-2-(5-hydroxy-1H-indol-3yl)ethanone derivatives 10-11, which were prepared following a

standard amide coupling reaction already reported by us [15] for analog derivatives. To access the target compounds 15-19 we carried out a synthetic procedure through an adaptation of our previously reported procedures [14, 16]. Firstly, starting material 5methoxyindole 12 has been acylated by Vilsmeier Haack reaction. Then, methoxy group of the intermediate 2-chloro-1-(5methoxy-1H-indol-3-yl)ethanone 13 was converted into the corresponding hydroxyl analogue 14. In turn, this intermediate 14 has been coupled with 4-phenoxypiperidine to give the desired compound 15. Similarly, intermediate 14 furnished desired compounds 16-19 by treatment with suitable 4-benzylpiperidine derivatives. Scheme 1 OH

R1 N H

ii

N H

N H

R1 = OMe

X

v

iii O N

R1

R

N H

N

R1 2

N H

10 R2 = H 11 R2= F

8, R1 = OMe 9, R1 = OH

iv

Reagents and conditions: i) SOCl2, CH3OH, r. t., 2 h; ii) (a) Et2O, 0°C, LiBH4, CH3OH, r. t., 5 h; (b) DCM, TEA, CH3SO2Cl, r.t., 45’; iii) 4benzylpiperidine, K2CO3, CH3COCH3, Δ 50°C, 24h; iv) BBr3 (1.0 M in DCM), r.t., 10h; v) piperidine derivative, HBTU, TEA, DMF, r.t., 2h.

Scheme 2 O MeO

i

12

ii

Cl

HO

N H

N H

13

14 iii O

15 R = H, X = O 16 R = 4-Me, X = CH2 17 R = 4-Et, X = CH2 18 R = 4-OMe, X = CH2 19 R = 3,4-(Me)2, X = CH2

Y

3b

COCH2

CH2

H

75%

25.0

9

CH2CH2

CH2

H

42%

N.D.

10

CH2CO

CH2

H

5%

N.D.

11

CH2CO

CH2

F

19%

N.D.

9%

N.D.

15

COCH2

O

H

16

COCH2

CH2

4-Me

66%

83.0

17

COCH2

CH2

4-Et

75%

71.0

18

COCH2

CH2

4-MeO

16%

1,280

CH2

3,4-(Me)2

<5%

3,330

-

47.0

19

COCH2

1

Displacement of [ H]ifenprodil. Three concentration (1 M, 0.1 M, 0.001 M, in duplicate) of test compounds were used in displacement assay. bData from reference [11]. ND= not determined. a

3

O Cl

MeO

N H

R

N

N H

7

6

4, R 1 = OMe 5, R1 = OH R1 = OH

OH

R1

OMe

R1

i

HO

Table 1. GluN2B/NMDA binding affinities of indole derivatives and reference compound ifenprodil (1). % IC50 inhibition X Y R (nM) a @ 0.1μM

O

O

been screened for their ability to prevent audiogenic seizures in DBA/2 mice, which has been considered an excellent animal model for generalized epilepsy and for screening new anticonvulsant drugs [15]. Unexpectedly, 16 and 17 were not able to exert in vivo effects at the highest tested doses (100 mol/kg) when compared with prototype 3 (ED50 value of 11.5 mol/kg in clonic phase) [15]. Finally, the other synthesized indole derivatives 9-11, 15 and 18-19 were inefficacious to displace [3H]ifenprodil at fixed dose of inhibitor (0.1 μM).

N

HO

R N H

X 15-19

Reagents and conditions: i) ClCH2CON(CH3)2, POCl3, r. t., 2.5 h; ii) BBr3 (1.0 M in DCM), r. t., 10 h; iii) 4-phenoxypiperidine or 4-benzylpiperidine derivatives, K2CO3, DMF, MW: 10 min 50°C, 200W.

All designed compounds were preliminary evaluated for their ability to interact with the GluN2B subunit and the results have been compared with previously results reported for the active indole 3 and ifenprodil (1) as reference compounds [16]. Data presented in the Table 1 show that a 0.1μM concentration of two of the eight examined indoles produced greater than 50% displacement of [3H]ifenprodil suggesting nanomolar affinity (IC50) toward the target receptor [19]. For the most active compounds 16 and 17 we measured IC50 values of 83 and 71 nM, respectively (see Table 1). Although they were able to bind GluN2B/NMDA receptors they display lower affinity than parent compound 3 and prototype ifenprodil (1). Given that the NAMs of GluN2B-containing NMDARs generally show anticonvulsant effects in animal models of epilepsy, the ligands 16 and 17 have

By analyzing data displayed in Table 1, we recovered some information about structure-affinity relationships (SARs) for this small series of compounds. The loss of affinity measured for ligands 9-11 suggested that there were detrimental structural modifications introduced into the middle area (highlighted as “X”) of the “hit compound” 3. Specifically, for the amide analogues 10-11 the loss of basicity of the piperidine might impair the H-bond interaction between the piperidine nitrogen atom and crucial residue Gln110 (see Figure 2). The replacement of methylene bridge of compound 3 with an oxygen atom furnished the analog 15 showing very low potency (9% of inhibition @ 0.1μM). By varying the phenyl tail of benzylpiperidine fragment we identified active ligands for which the substituent was a small alkyl group (Me or Et). On the contrary, the introduction of a methoxyl group (i.e. compound 18) or a couple of methyl groups (i.e. compound 19) significantly decreases up 130-fold the ability to bind GluN2B (IC50 at micromolar concentration). In a good agreement with previous findings [9], the introduction of a fluorine atom improves receptor affinity. In order to rationalize these SARs we docked one the most active compound (16, IC50 value of 83 nM) into the so-called ifenprodil pocket at GluN1/GluN2B interface retrieved from RCSB Protein DataBank (PDB code: 3QEL) [8]. As drawn in Figure 3, the best binding pose of compound 16 has been compared with the orientation of “hit compound” 3 (cfr supra). The superimposition of compound 3 (coloured in orange) and 16 (coloured in magenta) reveals that these two active ligands share a very similar network of contacts within GluN1/GluN2B

interface of NMDARs. Such that the compound 16 engages a crucial “three-point interaction” through three H-bonding contacts (dashed yellow lines) with residues Gln110 and Glu236 from GluN2B and Lys131 from GluN2B. We can hypothesize that this “three-point interaction” forces the ligand to adopt a specific binding pose. Moreover, the 4-methylphenyl moiety of compound 16 well maps the hydrophobic pocket lined by aromatic residues Phe114 and Tyr109 as well as hydrophobic residue Ile111.

We assume that the low active ligand 19 might adopt an alternative orientation for which the “middle area” lose the Hbond interaction between the piperidine nitrogen atom and crucial residue Gln110. Therefore, the ligand 19 appears lacking for a key driving force for binding interaction. The key role of Hbond interaction between the piperidine nitrogen atom and Gln110 is coherent with the measured poor affinity of amides 1011. 3. Conclusion In conclusion, using the 3-(4-benzylpiperidin-1-yl)-1-(5hydroxy-1H-indol-3-yl)ethan-1-one (3) as the lead structure, we designed and synthesized a novel series of indole derivatives by modification of two portions on its scaffold. The most active ligands 16 and 17 showed affinity comparable to that of reference compounds ifenprodil (1) and “hit molecule” 3. For other compounds the introduced modifications led to an immense drop of binding affinity. Overall, it has been collected novel information about the SARs, which control the recognition process within GluN1/GluN2B subunit interface.

4. Experimental section Figure 3. Superimposition of “hit compound” 3 with 16 at the GluN1/GluN2B subunit interface (PDB code 3QEL). Crucial residues are drawn in stick and coloured in cyan (GluN2B) and in green (GluN1). Hydrogen bonds are shown as dashed yellow lines.

As shown in Figure 3 the arylmethylene-piperidine tails of the two ligands 3 and 16 are almost superimposable and they could form  stacking interaction with residue Phe114 and/or hydrophobic interactions within pocket. So there is a good agreement between biological data and the results of our docking studies for these NMDA ligands into the so-called ifenprodil pocket. This would be possible that the 4-methyl or 4-ethyl substituent might fill the hydrophobic pocket thus justifying the IC50 values of 83 nM and 71 nM for the active ligands 16 and 17, respectively. In contrast the bulky 4-methoxysubstituent or 3,4dimethyl groups could clash with residues lining the hydrophobic pocket. Thus we hypothesize that a detrimental steric hindrance occurs for analogs 18 and 19 thus furnishing a plausible explanation for the loss of affinity. This hypothesis can be supported by analyzing the docking pose of compound 19 at the GluN1/GluN2B subunit interface (see Figure 4).

4.1. Chemistry All starting materials and reagents commercially available (Sigma-Aldrich Milan, Italy and Alfa Aesar, Karlsruhe, Germany) were used without further purification. Microwaveassisted reactions were carried out in a CEM focused Microwave Synthesis System, working at the power necessary for refluxing under atmospheric conditions. Melting points were determined on a BUCHI Melting Point B-545 apparatus and are uncorrected. Elemental analyses (C, H, N) were carried out on a C. Erba Model 1106 Elemental Analyzer and the results were within ± 0.4% of the theoretical values. Merck silica gel 60 F254 plates were used for TLC; column chromatography was performed on Merck silica gel 60 (230-400 mesh) and Flash Chromatography (FC) on Biotage SP1 EXP. Rf values were determined on TLC plates using a mixture of DCM/MeOH (90:10) as eluent. 1H NMR spectra were measured with a Varian Gemini-300 spectrometer in CDCl3 with TMS as internal standard or in DMSO-d6. Coupling constants (J) are reported in hertz and chemical shifts are expressed in δ (ppm). All exchangeable protons were confirmed by addition of deuterium oxide (D2O).

4. 1. 1. Sy nthe s is of m e thy l (5 -m e thox y -1H -indol -3 y l)ac e tate (6) A solution of 5-methoxy-3-indoleacetic acid (4) (205 mg, 1 mmol) in dry MeOH (500 mL) was cooled to 0 °C and SOCl2 (5 mmol) was added slowly. After stirring for 2 h at room temperature, the mixture was concentrated under reduced pressure, the residue taken up in EtOAc (10 mL) and subsequently washed with saturated aqueous NH4Cl (10 mL), saturated aqueous NaHCO3 (3 x 15 mL) and brine (3 x 10 mL). After drying over Na2SO4, the mixture was concentrated to dryness to give the pure methyl ester. Experimental data are in accordance with literature.[20] Yield 60%; mp 73-75 °C. Figure 4. Superimposition of compound 19 at the GluN1/GluN2B subunit interface (PDB code 3QEL). Crucial residues are drawn in stick and coloured in cyan (GluN2B) and in green (GluN1). Hydrogen bonds are shown as dashed yellow lines.

4. 1. 2. Sy nthe s is of 2-(5 -m e thox y -1 H -indol -3 y l)e thanol (7) The 2-(5-methoxy-1H-indol-3-yl)ethanol (7) was synthesized following a procedure reported in literature [20] with slight modification. In particular a reaction mixture containing the

intermediate 6 (419 mg, 2.2 mmol) in Et2O (16 mL) and dry MeOH (0.1 mL) was treated with LiBH4 (97 mg, 4.4 mmol) at 0 °C and under nitrogen atmosphere. The reaction mixture was allowed to attain to room temperature and stirred at that for 5 h. The solution obtained was diluted with water (15 mL) and extracted with Et2O (3 x 15 mL). The organic solution was washed with H2O (15 mL), dried, filtered and the solvent was concentrated in vacuo. The crude alcohol was purified by flash chromatography (cyclohexane/EtOAc 70:30) and crystallized from methanol. Spectral data are in agreement with data reported in literature. [20]

for 2 h at room temperature. The reaction mixture was then quenched with H2O (10 mL) and extracted with EtOAc (3 x 10 mL). The organic phases were dried with Na 2SO4 and concentrated in vacuo. The crude compound was purified by flash chromatography (cyclohexane/EtOAc 40:60) and crystallized by treatment with Et2O to give the desired final products 10-11.

4 . 1 . 3 . Sy nthe s is of 3-[ 2-(4 -be nz y lpipe r idin -1 y l)e thy l] -5 -m e thox y -1 H -indole (8) A solution of the alcohol 7 (516 mg, 2.7 mmol), and TEA (0.5 mL) in DCM (5 mL) was cooled to 0 °C, and methanesulfonyl chloride (271 L, 3.5 mmol) dissolved in DCM (1 mL) was added dropwise. After the mixture was stirred 45 min at room temperature and then H2O (15 mL) and DCM (10 mL) were added. The organic phase was separated, dried, and concentrated in vacuo. The methanesulfonate ester (748 mg, 2.8 mmol) obtained, was used without further purification. To a solution of ester in acetone (10 mL), the 4-benzylpiperidine (492 L, 2.8 mmol) and K2CO3 (540 mg, 3.9 mmol) were added and the solution was refluxed for 24h. Then, the solvent was evaporated, and the residue was dissolved in H2O (15 mL) and Et2O (10 mL). The organic phase was washed with H2O (3 x 5 mL), saturated aqueous NaHCO3 (3 x 5 mL) and dried with Na2SO4. The final compound 3-[2-(4-benzylpiperidin-1-yl)ethyl]-5-methoxy-1Hindole (8) was purified by flash chromatography (DCM/CH3OH 90:10) and crystallized from a mixture of Et2O and CH3OH.

1-[4-(4-Fluorobenzyl)piperidin-1-yl]-2-(5-hydroxy-1H-indol3-yl)ethanone (11):

Yield 40%; mp 110-112 °C; 1H NMR (CDCl3) 1.30-3.21 (m, 15H), 3.91 (s, 3H, OCH3), 6.90 (dd, 1H, J= 8.8, J= 2.4, ArH, H-6), 7.00 (s, 1H, ArH), 7.12 (s, 1H, H-2), 7.20 (s, 1H, ArH), 7.22-7.39 (m, 5H, ArH), 8.61 (bs, 1H, NH). Anal.(C23H28N2O): C 79.27, H 8.10, N 8.04. Found: C 79.17, H 8.20, N 7.99. 4 . 1 . 4 . Sy nthe s is of 3-[ 2-(4 -be nz y lpipe r idin -1 y l)e thy l] -5 -hy dr ox y -1H -indole (9 ) The 3-[2-(4-benzylpiperidin-1-yl)ethyl]-5-hydroxy-1H-indole (9) was prepared following a synthetic procedure fully described by us earlier [16]. In particular 3-[2-(4-benzylpiperidin-1yl)ethyl]-5-methoxy-1H-indole (348 mg, 1 mmol) was dissolved in methylene chloride (DCM) (5 mL), treated with BBr3 (1 M in DCM) (6 mmol, 6 mL) under nitrogen atmosphere and stirred overnight. After completion of reaction, MeOH (7 mL) was carefully added at 0C and the solvent removed under reduced pressure. The residue was dissolved in EtOAc (10 mL) and washed with H2O (3 x 10 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo. The desired compound was obtained by crystallization with Et2O. Yield 40%; mp 205-207 °C; Rf = 0.17; 1H NMR (DMSO-d6) 1.06-2.77 (m, 15H), 6.53-6.58 (m, 2H, ArH), 6.66 (s, 1H, ArH), 7.12 (s, 1H, H-2), 7.14-7.28 (m, 5H, ArH), 8.91 (bs, 1H, OH), 9.99 (bs, 1H, NH). Anal.(C22H26N2O): C 79.01, H 7.84, N 8.38. Found: C 79.17, H 7.90, N 8.48. 4 . 1 . 5 . G e ne r al proc e dur e f or the sy nthe s is of de r iv ativ es 1-[ 4-(4 -f luor obe nz y l)pipe r idin -1-y l] -2 (1 H -indol -3 -y l)e thanone s (10 -11) A mixture of (5-hydroxy-1H-indol-3-yl)acetic acid (5) (0.5 mmol) with N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1yl)uronium hexafluorophosphate (HBTU) (190 mg, 0.5 mmol) in DMF (2 mL) was stirred for 30 min at room temperature. Successively, the appropriate benzylpiperidine (1 mmol) and TEA (70 L, 0.5 mmol) were added and the mixture was stirred

Spectral data of compounds 1-(4-benzylpiperidin-1-yl)-2-(5hydroxy-1H-indol-3-yl)ethanone (10) are in accordance with literature. [15]

Yield 96%; mp 190-191 C; Rf = 0.30 1H NMR (DMSO-d6) 0.89-4.38 (m, 11H), 3.63 (s, 2H, CH2CO), 6.58 (d, 1H, ArH),6.88 (s, 1H, ArH), 7.07-7.18 (m, 6H, ArH), 8.57 (s, 1H, OH), 10.54 (bs, 1H, NH). Anal. Calcd for C22H23FN2O2: C, 72.11; H, 6.33; N, 7.64. Found: C, 72.22; H, 6.23; N, 7.75. The 2-chloro-1-(5-methoxy-1H-indol-3-yl)ethanone (13), and 2-chloro-1-(5-hydroxy-1H-indol-3-yl)ethanone (14) were synthesized following a previously reported procedure and spectral data are in accordance with literature. [14, 16] 4. 1. 6. G e ne r al proc e dur e f or the sy nthe s is of c om pounds 15 -19 To a solution of 2-chloro-1-(5-hydroxy-1H-indol-3yl)ethanone (1 mmol) (14) in DMF dry (5 mL) the proper benzylpiperidines (1 mmol) or 4-phenoxypiperidine (1.5 mmol) and K2CO3 (0.5 mmol) were added. The resulting mixture was subjected to microwave irradiation (100 °C, 200 W) for 15 min and then was treated with a saturated NaHCO3 (10 mL) aqueous solution and extracted with EtOAc (3 x 10 mL). The organic layer was dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash chromatography (DCM/CH3OH 90:10) and crystallized from Et2O and EtOH to give the desired compounds. 1-(5-Hydroxy-1H-indol-3-yl)-2-(4-phenoxypiperidin-1yl)ethanone (15) Yield 30%; mp 199-200 °C; Rf = 0.44; 1H NMR (DMSO-d6) 1.61-2.81 (m, 9H), 3.54 (s, 2H, CH2N), 6.66 (dd, 1H, J= 8.8, J= 2.4, ArH, H-6), 6.88-7.27 (m, 6H, ArH),7.58 (d, 1H, J= 2.3, ArH, H-4), 8.37 (s, 1H, H-2), 8.95 (bs, 1H, OH), 11.63 (bs, 1H, NH). Anal.(C21H22N2O3): C 71.98, H 6.33, N 7.99. Found: C 71.86, H 6.44, N 7.89. 1-(5-Hydroxy-1H-indol-3-yl)-2-(4-(4-methylbenzyl)piperidin1-yl)ethanone (16) Yield 30%; mp 215-216 °C; Rf = 0.23; 1H NMR (DMSO-d6) 1.09-2.90 (m, 11H), 2.25 (s, 3H, CH3), 3.47 (s, 2H, CH2N), 6.69 (dd, 1H, J= 8.5, J= 2.1, ArH, H-6), 7.02-7.06 (m, 4H, ArH), 7.24 (d, 1H, J= 8.5, ArH, H-7), 7.57 (d, 1H, J= 2.1, ArH, H-4), 8.35 (d, 1H, J= 3.2, H-2), 8.98 (s, 1H, OH), 11.63 (bs, 1H, NH). Anal.(C23H26N2O2): C 76.21, H 7.23, N 7.73. Found: C 76.31, H 7.33, N 7.63. 2-(4-(4-Ethylbenzyl)piperidin-1-yl)-1-(5-hydroxy-1H-indol-3yl)ethanone (17) Yield 50%; mp 199-200 °C; Rf = 0.28; 1H NMR (DMSO-d6) 1.06-2.90 (m, 16H), 3.47 (s, 2H, CH2N), 6.67 (dd, 1H, J= 8.8, J= 3.0, ArH, H-6), 7.03-7.11 (m, 4H, ArH), 7.24 (d, 1H, J= 8.8, ArH, H-7), 7.56 (d, 1H, J= 3.0, ArH, H-4), 8.34 (d, 1H, J= 2.9, H-2), 8.96 (s, 1H, OH), 11.62 (bs, 1H, NH). Anal.(C24H28N2O2): C 76.56, H 7.50, N 7.44. Found: C 76.46, H 7.55, N 7.53.

1-(5-Hydroxy-1H-indol-3-yl)-2-(4-(4 methoxybenzyl)piperidin-1yl)ethanone (18) Yield 60%; mp 195-196 °C; Rf = 0.24; 1H NMR (DMSO-d6) 1.05-2.88 (m, 11H), 3.50 (s, 2H, CH2N), 3.71 (s, 1H, OCH3), 6.68 (dd, 1H, J= 9.1, J= 2.7, ArH, H-6), 6.81-7.08 (m, 4H, ArH), 7.24 (d, 1H, J= 9.1, ArH, H-7), 7.57 (d, 1H, J= 2.7, ArH, H-4), 8.34 (d, 1H, J= 3.2, H-2), 8.96 (s, 1H, OH), 11.64 (bs, 1H, NH). Anal.(C23H26N2O3): C 72.99, H 6.92, N 7.40. Found: C 72.85, H 6.82, N 7.49. 2-(4-(3,4-Dimethylbenzyl)piperidin-1-yl)-1-(5-hydroxy-1Hindol-3-yl)ethanone (19) Yield 35%; mp 198-200 °C; Rf = 0.26; 1H NMR (DMSO-d6) 1.17-2.89 (m, 11H), 2.15 (s, 3H, CH3), 2.17 (s, 3H, CH3), 3.45 (s, 2H, CH2N), 6.67 (dd, 1H, J= 8.5, J= 2.6, ArH, H-6), 6.83-7.02 (m, 4H, ArH), 7.24 (d, 1H, J= 8.5, ArH, H-7), 7.57 (d, 1H, J= 2.6, ArH, H-4), 8.35 (s, 1H, H-2), 8.95 (s, 1H, OH), 11.61 (bs, 1H, NH). Anal.(C24H28N2O2): C 76.56, H 7.50, N 7.44. Found: C 76.48, H 7.59, N 7.49. 4.2. Receptor binding studies The radioligand binding assays against NMDA receptor containing GluN2B-subunit were carried out using [3H]ifenprodil (Custom Screen by Eurofin Panlab, LCC, USA) [19, 21]. Cerebral cortices of male Wistar derived rats weighing 175 ± 25 g are used to prepare glutamate NMDA receptors in Tris-HCl buffer pH 7.4. A 5 mg aliquot is incubated with 2 nM [3H]Ifenprodil (plus 5 M GBR-12909 to block non-polyamine sensitive sites) for 120 minutes at 4°C. Non-specific binding is estimated in the presence of 10 M ifenprodil (1). Membranes are filtered and washed, the filters are then counted to determine [3H]Ifenprodil specifically bound. Three concentration (10 M, 0.1 M, 0.001 M, in duplicate) of test compounds were used in displacement assay. 4.3. Anticonvulsant activity in DBA/2 mice All experiments were performed in DBA/2 mice which are genetically susceptible to sound-induced seizures [22]. DBA/2 mice (8-12 g; 22-25-days-old) were purchased from Harlan Italy (Corezzano, Italy). Groups of 10 mice of either sex were exposed to auditory stimulation 30 min following administration of either the vehicle or each dose of the drugs studied. The compound was administered intraperitoneally (ip) (0.1 mL/10 g of mouse body weight ) as a freshly-prepared solution in 50% dimethylsulfoxide (DMSO) and 50% sterile saline (0.9% NaCl). Individual mice were placed under a hemispheric perspex dome (diameter 58 cm), and 60 s were allowed for habituation and assessment of locomotor activity. Auditory stimulation (12-16 kHz, 109 dB) was applied for 60 s or until tonic extension occurred, and induced a sequential seizure response in control DBA/2 mice, consisting of an early wild running phase, followed by generalized myoclonus and tonic flexion and extension sometimes followed by respiratory arrest. The control and drugtreated mice were scored for latency to and incidence of the different phases of the seizures. The experimental protocol and all the procedures involving animals and their care were conducted in conformity with the institutional guidelines and the European Council Directive of laws and policies. Statistical comparisons between groups of control and drug-treated animals were made using Fisher's exact probability test (incidence of the seizure phases). The ED50 values of each phase of audiogenic seizures was determined for each dose of the compound administered, and dose-response curves were fitted using a computer program by Litchfield and Wilcoxon's method [23].

4.4. Automated Molecular Docking Experiments The crystal structure of amino terminal domains of the NMDA receptor subunit GluN1 and GluN2B in complex with ifenprodil (1) was retrieved from the RCSB Protein Data Bank (entry code 3QEL) [8]. The ligand and water molecules were discarded and the hydrogen atoms were added to protein by Discovery Studio 2.5[24]. The ifenprodil (1) structure was extracted from X-ray complex, and the other structures of the ligands were constructed using Discovery Studio 2.5[24]. The conformational behaviour of simulated compounds was investigated by a MonteCarlo procedure (as implemented in the VEGA suite of programs which generated 1000 conformers by randomly rotating the rotors) [25]. All geometries obtained were stored and optimized to avoid high-energy rotamers. The 1000 conformers were clustered by similarity to discard redundancies; in this analysis, two geometries were considered non-redundant when they differed by more than 60° in at least one torsion angle. For each derivative, the lowest energy structure was then submitted to docking simulations The ligands minimized in this way were docked in their corresponding proteins by means of Gold Suite 5.0.1. The region of interest used by Gold was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side chain of residue Leu135 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore [26] was chosen as a fitness function and the standard default settings were used in all the calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom RMSD, were clustered together. The best ranked GOLDcalculated conformation was used for analysis and representation. Minimization process. The GluN2B/ligand complex obtained by docking studies was minimized using 1000 iterations of SD and 1000 interaction of Polak-Ribiere Conjugate Gradient. Minimization of complexes was performed using OPLS-2005 [27] force field and im plicit GB/SA water model [28] as solvation treatment using Macromodel.

Acknowledgments. Financial support for this research by Fondo di Ateneo per la Ricerca (PRA grant number ORME09SPNC - Università di Messina).

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Graphical Abstract

Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-Daspartate receptor (NMDAR) containing GluN2B subunit

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M.R. Buemi, L. De Luca, S.Ferro, E. Russo, G De Sarro, R Gitto*

O N

HO

Dept Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali - Università degli Studi di Messina. Dept Medicina Sperimentale e Clinica, Università Magna Graecia Catanzaro, Italy

N H IC50 = 71 nM (GluN2B/NMDA)

Et