Copper-Catalyzed Synthesis , Bio-Evaluation , and in Silico Studies of 2-Aryl-N-alkylbenzimidazoles as Neuroprotective Agents

2-aryl-N-alkylbenzimidazole derivatives synthesized by CuI/PPh3 promoted direct coupling of N-alkylbenzimidazoles with aryl bromides. In vitro neurotoxicities of 20 compounds were evaluated, and the neuroprotective abilities of low-neurotoxic compounds (3b, 3g, 3h, 3i, 3j, 3k, 3o, 3q, 3s and 3t) were investigated against toxicity induced by 1-methyl-4-phenylpyridinium ion (MPP+) in SH-SY5Y neuronal cells. In silico studies revealed that compound 3g could have molecule docking with the following proteins: the bone morphogenetic protein receptor type 1B (BMPR1B), human cytochrome P450 1B1(CYP1B1), Metabotropic glutamate receptor 7 (GRM7), histone deacetylase 6 (HDAC6), 5-hydroxytryptamine receptor 5A (HTR5A), human topoisomerase II beta (TOP2B). A molecular docking simulation of model compound 3g and model protein CYP1B1 has been shown.


Introduction
Benzimidazole derivatives are known to display a wide range of biological activities [1].Among them, the neuroprotective activity is significant in the treatment of neurodegenerative diseases including ischemia [2], epilepsy [3], and Alzheimer's and Parkinson's disease [4].For example, two clinically-promising anti-Parkinson's disease agents, Istradefylline and Preladenant, are both benzimidazole derivatives [5].In particular, 2-arylbenzimidazole derivatives were evaluated as inhibitors of c-Jun N-terminal kinase 3 (JNK3) in the treatment of neurodegenerative symptoms [6,7].In addition, a novel cholinesterase (ChE) inhibitor with 2-arylbenzoimidazoles skeleton was helpful in reversing the memory impairments associated with Alzheimer's disease [8].2-arylbenzimidazole derivatives also inhibited the increase of inducible nitric oxide synthase (iNOS) when peripheral nerve injury occurred (Figure 1) [9].Consequently, there is a great demand for the generation a library of novel 2-arylbenzimidazoles for the evaluation of their neuroprotective activity.

Copper-Catalyzed Synthesis of 2-Aryl-N-Alkylbenzimidazoles
We investigated the best reaction conditions of the Cu-catalyzed direct arylation of 1-methylbenzimidazole (1a) with 1-bromo-4-tert-butylbenzene (2d) (Table 1).Initially, the screening of various bases was carried out under the CuI/ PPh 3 (10 mol %/30 mol %) catalytic system at 140 • C in DMF for 24 h.There was small quantity of 2-[4-(1,1-Dimethylethyl)phenyl]-1-methyl-1H-benzimidazole (3d) when KOH, NaOH and t-BuOK (Table 1, entry 1-3) was used as base.In contrast, a mild base such as K 2 CO 3 , Na 2 CO 3 or KH 2 PO 4 (Table 1, entry 4-6) promoted the arylation reaction dramatically.Next, we examined the effect of temperatures ranging from 120 • C to 150 • C (Table 1, entries 4, 7-8).When the temperature decreased to 120 • C, the yield dropped to 45%.After the temperature screening, the selection of a high boiling-point solvent drew our attention.DMF and xylene positively affected this arylation reaction.We also studied the reasons why, as the temperature changed from 140 • C to 130 • C (Table 1, entries 9 and 10), the solvent became xylene.The yield increased as the temperature rose to 140 • C. The reaction-time difference of group 1 (Table 1, entries 11 and 8) and group 2 (Table 1, entries 9 and 12) revealed that increasing time benefits the yield.Therefore, optimized conditions (Table 1, entry 12) were obtained by following the scope of aryl bromides.In a study of 2-arylations of 1-methyl-benzimidazole (Table 2), compared with optimized conditions using 40 h, the majority of products could be produced in the same yield with a shorter reaction time (24 h). the synthesis of diverse 2-arylbenzimidazoles.The classical cross-coupling of benzimidazoles with aryl electrophiles catalyzed by a noble metal such as Palladium has yielded various 2arylbenzimidazoles [17][18][19][20][21][22].However, the high cost limits the scale-up development of Pd-catalyzed cross coupling reaction.Fortunately, cheap copper is an efficient counterpart of palladium in the direct arylation of benzoheterocyclic compounds [23][24][25][26][27][28].Previously, we have reported that Cu/PPh3 directly catalyzed the 2-arylation of benzoxazoles with economical aryl bromides [29], and the same catalysis system has been used in our further research.In this work, a series of 2-aryl-Nalkylbenzimidazole derivatives were prepared via copper-catalyzed direct arylation of Nalkylbenzimidazoles with aryl bromides, and their in vitro neuroprotective activities were evaluated.

Copper-Catalyzed Synthesis of 2-Aryl-N-Alkylbenzimidazoles
We investigated the best reaction conditions of the Cu-catalyzed direct arylation of 1-methylbenzimidazole (1a) with 1-bromo-4-tert-butylbenzene (2d) (Table 1).Initially, the screening of various bases was carried out under the CuI/ PPh3 (10 mol %/30 mol %) catalytic system at 140 °C in DMF for 24 h.There was small quantity of 2-[4-(1,1-Dimethylethyl)phenyl]-1-methyl-1H-benzimidazole (3d) when KOH, NaOH and t-BuOK (Table 1, entry 1-3) was used as base.In contrast, a mild base such as K2CO3, Na2CO3 or KH2PO4 (Table 1, entry 4-6) promoted the arylation reaction dramatically.Next, we examined the effect of temperatures ranging from 120 °C to 150 °C (Table 1, entries 4, 7-8).When the temperature decreased to 120 °C, the yield dropped to 45%.After the temperature screening, the selection of a high boiling-point solvent drew our attention.DMF and xylene positively affected this arylation reaction.We also studied the reasons why, as the temperature changed from 140 °C to 130 °C (Table 1, entries 9 and 10), the solvent became xylene.The yield increased as the temperature rose to 140 °C.The reaction-time difference of group 1 (Table 1, entries 11 and 8) and group 2 (Table 1, entries 9 and 12) revealed that increasing time benefits the yield.Therefore, optimized conditions (Table 1, entry 12) were obtained by following the scope of aryl bromides.In a study of 2-arylations of 1-methyl-benzimidazole (Table 2), compared with optimized conditions using 40 h, the majority of products could be produced in the same yield with a shorter reaction time (24 h). a The reactions performed using 1.0 equiv.of1a, 1.2 equiv. of 2d and 2.0 equiv. of base on a 0.1 mmol scale.b This is NMR assay yield.c Isolated yield.
a 1b was recovered from 50% to 60% yield.b 1b was recovered in 78% yield.c 1c was recovered from 55% yield.d 1c was recovered in 62% yield.
Catalysts 2018, 8, x FOR PEER REVIEW 4 of 14 1H-benzimidazole (3r), was produced at a yield rate of 35%.Although yields of heterocyclic structures (Table 3. 3k and 3m, Table 4. 3r, 3s and 3t) were moderate, the economical Cu-catalyzed protocol helped to meet the demands of the activity tests.
a 1b was recovered from 50% to 60% yield.b 1b was recovered in 78% yield.c 1c was recovered from 55% yield.d 1c was recovered in 62% yield.
a 1b was recovered from 50% to 60% yield.b 1b was recovered in 78% yield.c 1c was recovered from 55% yield.d 1c was recovered in 62% yield.

Neuroprotective Performances of 2-Aryl-N-Alkylbenzimidazoles against MPP +
The ability to prevent SH-SY5Y cells from MPP + -induced damage is shown in Table 4 and Figure 1.Unfortunately, our results indicated that the protective capcities of compounds (3b, 3g, 3h, 3i, 3j, 3k, 3o, 3q, 3s, 3t) under 10 and 50 µM were low.We chose these compounds for the aforementioned experiment based on the fact that they did not show appreciable neurotoxicities on SH-SY5Y cells under 50 µM.(The inhibition rates of 20 compounds are illustrated in the supplementary materials).

Neuroprotective Performances of 2-Aryl-N-Alkylbenzimidazoles against MPP +
The ability to prevent SH-SY5Y cells from MPP + -induced damage is shown in Table 4 and Figure 1.Unfortunately, our results indicated that the protective capcities of compounds (3b, 3g, 3h, 3i, 3j, 3k, 3o, 3q, 3s, 3t) under 10 and 50 μM were low.We chose these compounds for the aforementioned experiment based on the fact that they did not show appreciable neurotoxicities on SH-SY5Y cells under 50 μM.(The inhibition rates of 20 compounds are illustrated in the supplementary materials)

The Computer Anaysis of Neuroprotective Targets Metablising 3g and Docking Analysis
In order to gain some insights into the neuroprotective potential of 2-aryl-Nalkylbenzimidazoles, we prepared proteins with neuroprotective functions and performed molecular docking studies to identify the neuroprotective enzymes that play a key role in metablising 2-aryl-Nalkylbenzimidazoles.Compound 3g was listed as a model to select enzymes.

The Preparation of Neuroprotective Targets
The preparation began by searching for protein targets using a similarity ensemble approach (SEA) [30].Only proteins from human and mouse samples were saved for subsequent analyses.Gene ontology (GO) annotation and cluster analysis were used as a filter for proteins with neuroprotective functions [31].Then, 19 targets were selected for further discussion of potential targets of compound 3g.There were 14 proteins whose crystal structures were available in the Protein Data Bank (PDB) at the time of the work: APP, BMPR1B, CXCL8, CYP1B1, GLRA1, GRIK2, GRM7, HDAC6, NR4A2, PRKCG, RELA, TLR8, TOP2B, TSHR.As no crystallographic structures were available for the other five targets, CCKAR, FPR2, GABRB3, HTR5A, NPBWR1, a homology modeling methodology was used to build 3D structures of these enzymes.The appropriate template structures corresponded to proteins with PDB accession codes 5ZBH, 3ODU, 4COF, 4IAR, 4N6H, respectively [32][33][34][35][36].

The Computer Anaysis of Neuroprotective Targets Metablising 3g and Docking Analysis
In order to gain some insights into the neuroprotective potential of 2-aryl-N-alkylbenzimidazoles, we prepared proteins with neuroprotective functions and performed molecular docking studies to identify the neuroprotective enzymes that play a key role in metablising 2-aryl-N-alkylbenzimidazoles.Compound 3g was listed as a model to select enzymes.

The Preparation of Neuroprotective Targets
The preparation began by searching for protein targets using a similarity ensemble approach (SEA) [30].Only proteins from human and mouse samples were saved for subsequent analyses.Gene ontology (GO) annotation and cluster analysis were used as a filter for proteins with neuroprotective functions [31].Then, 19 targets were selected for further discussion of potential targets of compound 3g.There were 14 proteins whose crystal structures were available in the Protein Data Bank (PDB) at the time of the work: APP, BMPR1B, CXCL8, CYP1B1, GLRA1, GRIK2, GRM7, HDAC6, NR4A2, PRKCG, RELA, TLR8, TOP2B, TSHR.As no crystallographic structures were available for the other five targets, CCKAR, FPR2, GABRB3, HTR5A, NPBWR1, a homology modeling methodology was used to build 3D structures of these enzymes.The appropriate template structures corresponded to proteins with PDB accession codes 5ZBH, 3ODU, 4COF, 4IAR, 4N6H, respectively [32][33][34][35][36].

The Molecular Docking Simulation of 3g and Neuroprotective Targets
After the preparation of 19 neuroprotective targets, molecular docking simulations between compound 3g and neuroprotective targets were performed by use of Discovery Studio.The CHARMm force field was applied to the molecules, and CDOCKER module was used in a molecular docking simulation [37].The final binding results were determined by -CDOCKING ENERAGE, which means that six proteins (BMPR1B, CYP1B1, GRM7, HDAC6, HTR5A, and TOP2B) whose -CDOCKING ENERAGE is above zero are much more likely to interact with compound 3g (Figure 2a).The score of -CDOCKING ENERAGE cannot illustrate actual levels of interactions between compound 3g and the six targets.During the preliminary docking study, the major 3g-binding sites on cytochromes P450 1B1 (CYP1B1) were analyzed through comparison with the docking study of interaction between polycyclic aromatic compounds and CYP1B1.Polycyclic aromatic compounds and 2-aryl-N-alkylbenzimidazoles were found to bind to the same site (Phe231) [38].There is no discussion about the binding mode between 2-aryl-N-alkylbenzimidazoles and CYP1B1 in the litterature.As such, a molecular docking study between protein CYP1B1 and 3g was listed as a model to obtain more insight into the binding sites (Figure 2).The binding site was mainly delimitated by Phe 231 through π-π interaction.The 2-naphthalenyl group also interacted with Ala330 and Ala133 residues through CH-π interaction.In addition, the naphthalene moiety in compound 3g could form a hydrogen bond with the Gly329 residue of protein CYP1B1.For the benzimidazole moiety of 3g, Val395, Ile399 and Cys470 residues were implicated in Pi bonds.The van der Waals interactions are also shown in Figure 2d.These interactions contributed to the binding affinity of 2-naphthalenyl-alkylbenzimidazole, compound 3g, and model protein CYP1B1.(Figure 2b-d) The polycyclic aromatic rings at 2-position of N-alkylbenzimidazoles seems to be important for ligands to dock into CYP1B1 through π-π interactions.After the preparation of 19 neuroprotective targets, molecular docking simulations between compound 3g and neuroprotective targets were performed by use of Discovery Studio.The CHARMm force field was applied to the molecules, and CDOCKER module was used in a molecular docking simulation [37].The final binding results were determined by -CDOCKING ENERAGE, which means that six proteins (BMPR1B, CYP1B1, GRM7, HDAC6, HTR5A, and TOP2B) whose -CDOCKING ENERAGE is above zero are much more likely to interact with compound 3g (Figure 2a).The score of -CDOCKING ENERAGE cannot illustrate actual levels of interactions between compound 3g and the six targets.During the preliminary docking study, the major 3g-binding sites on cytochromes P450 1B1 (CYP1B1) were analyzed through comparison with the docking study of interaction between polycyclic aromatic compounds and CYP1B1.Polycyclic aromatic compounds and 2-aryl-N-alkylbenzimidazoles were found to bind to the same site (Phe231) [38].There is no discussion about the binding mode between 2-aryl-N-alkylbenzimidazoles and CYP1B1 in the litterature.As such, a molecular docking study between protein CYP1B1 and 3g was listed as a model to obtain more insight into the binding sites (Figure 2).The binding site was mainly delimitated by Phe 231 through π-π interaction.The 2-naphthalenyl group also interacted with Ala330 and Ala133 residues through CH-π interaction.In addition, the naphthalene moiety in compound 3g could form a hydrogen bond with the Gly329 residue of protein CYP1B1.For the benzimidazole moiety of 3g, Val395, Ile399 and Cys470 residues were implicated in Pi bonds.The van der Waals interactions are also shown in Figure 2d.These interactions contributed to the binding affinity of 2-naphthalenylalkylbenzimidazole, compound 3g, and model protein CYP1B1.(Figure 2b-d) The polycyclic aromatic rings at 2-position of N-alkylbenzimidazoles seems to be important for ligands to dock into CYP1B1 through π-π interactions.

General Methods
All reactions were carried out under a nitrogen atmosphere with ovendried glassware.Unless otherwise stated, reagents were commercially available and used as purchased.The tetrahydrofuran (THF) was steamed through sodium to remove the aqueous part when using as a solvent, and N,N-Dimethylformamide (DMF) and xylene were dried through activated 4 Å Molecular Sieves under argon.TLC was performed with silica gel plates (GF 254) with detection under UV light at 254 nm.The NMR spectra were recorded by a Brüker 400 MHz Fourier-transform NMR spectrometer (Swiss Brüker, Chengdu, China).Chemical shifts (δ) are reported in units of parts per million (ppm) downfield from tetramethylsilane (TMS).

A Typical Procedure for the Cu-Catalyzed Arylation of Benzoimidazoles
A Schlenk 15 mL flask equipped with a magnetic bar was loaded with the benzoimidazoles with substituents at the nitrogen position (0.2 mmol) and K 2 CO 3 (69.1 mg, 2.5 equiv), while CuI (10 mol %) and PPh 3 (30 mol %) were prepared in another flask.Then, the vessels were purged by a sequence of seven vacuum-argon refill-evacuation cycles.A solution of catalytic Cu/PPh 3 system was prepared through the addition of 1 mL dry DMF (or xylene) with a syringe.Preparation of the stock solution of CuI and PPh 3 was stirred for 2 h at 45 • C under argon.The catalytic solution and aryl bromide (0.3 mmol, 1.5 equiv) were added to the reaction mixture by syringe, but the solid aryl bromides were added to the reaction at the beginning.The reaction mixture was stirred for 20-44 h at 140 or 150 • C, and quenched with two drops of H 2 O.The crude products were filtered over Na 2 SO 4 and silica to remove salts, and rinsed with additional ethyl acetate.Finally, the solution was concentrated in vacuo.The arylated products were obtained by flash chromatography.

Determination of Protective Rate by MTT Assay
Cells were cultured in 96-well plates with a density of 5 × 10 3 edia for one day, and then pre-treated at 37 • C for 4 h with compounds 3b, 3g, 3h, 3i, 3j, 3k, 3o, 3q, 3s and 3t (10 and 50 µM, n = 3).The cells were treated with 1mm MPP + in the continued presence of low-neurotoxicity compounds for 24 h, and then assessed for cell viability.Cell viability was determined by a mitochondrial enzymedependent reaction of MTT assay.In brief, 10 µL of MTT 5 mg/mL was added directly to each culture media with SH-SY5Y cells.Following a two-hour incubation at 37 • C, the supernatants were removed, and then 150 µL solubilizing agent was added to dissolve the purple formazan crystals formed by metabolism of the yellow MTT tetrazolium salt.The plate was then incubated for 1 h or overnight, and the absorbance was measured using a microplate reader (Wellscan MK3, Labsystems Dragon) at a wavelength of 570 nm.Cell death following MPP + treatment was shown by the inhibition rate of untreated cells.Relying on the structural similarity, possible targets related to 3g were predicted by the similarity ensemble approach (SEA) (http://sea.bkslab.org/)[30].After the screening of targets, only proteins from human and mouse were continuously analyzed through gene ontology (GO) annotation and cluster analysis.According to the results from GO annotation and cluster analysis, the following six results related to neuroprotective function (inhibitory postsynaptic potential, cellular response to hydrogen peroxide, chemical synaptic transmission, inflammatory response, adult locomotory behavior, and axonogenesis) were obtained, and 19 targets were selected [31].Among them, the 3D crystal structures of 14 targets were retrieved from the Protein Data Bank (PDB); their names and PDB codes were APP (PDB ID: 3KTM), BMPR1B (PDB ID: 3MDY), CXCL8 (PDB ID: 5D14), CYP1B1 (PDB ID: 3PM0), GLRA1 (PDB ID: 3JAF), GRIK2 (PDB ID: 5CMM), GRM7 (PDB ID: 3MP4), HDAC6 (PDB ID: 6CE6), NR4A2 (PDB ID: 1OVL), PRKCG (PDB ID: 2UZP), RELA (PDB ID: 3GUT), TLR8 (PDB ID: 3W3J), TOP2B (PDB ID: 4G0W), TSHR (PDB ID: 3G04).The 3D structures of another 5 proteins, CCKAR, FPR2, GABRB3, HTR5A, NPBWR1, that were absent from this study were constructed by homology modeling using SWISS-MODEL.This protein structure homology-modeling server is one of the most accurate computational methods (https://swissmodel.expasy.org/workspace/)[33][34][35][36][37].The template structures for CCKAR, FPR2, GABRB3, HTR5A, and NPBWR1 were downloaded from PDB for the homology methodology; their template codes are 5ZBH, 3ODU, 4COF, 4IAR, 4N6H, respectively (The results of SEA, GO annotation, cluster analysis, homology modeling and the summarizing scheme of in silico studies are shown in the Supplementary Materials).

A Procedure for Molecular Docking Simulation
The Accelrys Discovery Studio docking program (version 3.5, Accelrys Inc., Bedminster Township, NJ, USA) was adopted here [32].For the module, CDOCKER is a molecular dynamics (MD) simulated-annealing-based algorithm, and ligand conformations are generated using a hightemperature molecular dynamics methodology and energy minimized into the protein active site using simulated annealing molecular dynamics [45].Although the docking accuracy decreases when ligands with eight or more rotatable bonds, low-flexible 2-aryl-N-alkylbenzimidazoles could have appropriate docking accuracy rates [37,46].In this program, the preparations of protein structures included adding hydrogen atoms, removing water molecules, and assigning a CHARMm forcefield.After clicking the CDOCKER module, the input receptors were set up as the corresponding targets, and the input ligand was set up as compound 3g.Top hits were selected as 10, and the other parameters were set as default.

Conclusions
We demonstrated a copper-catalyzed direct arylation of N-alkylbenzimidazoles with diverse aryl bromides to furnish the corresponding 2-aryl-N-alkylbenzimidazoles.All these compounds were evaluated for their in vitro neurotoxicities, but low-neurotoxic compounds (3b, 3g, 3h, 3i, 3j, 3k, 3o, 3q, 3s and 3t) were found to exert a low neuroprotective effect.This preliminary study provided basic research about 2-aryl-N-alkylbenzimidazoles based on neurodegenerative diseases.Six targets, BMPR1B, CYP1B1, GRM7, HDAC6, HTR5A, and TOP2B, could be potential goals for the study of 2-aryl-N-alkylbenzimidazoles as neuroprotective agents.The dicyclic aromatic rings at the 2-position of N-alkylbenzimidazoles could be important structure moieties for the interaction with CYP1B1, according to our atomic-level study of the interaction between 3g and CYP1B1.This work generated a series of compounds for the 2-arylbenzimidazoles library using a direct protocol, and provided a basic evaluation of 2-aryl-N-alkylbenzimidazoles as neuroprotective agents.Six potential targets and suggestions about the structure of 2-aryl-N-alkyl benzimidazoles as a ligand for CYP1B1 were provided in terms of atomic level.

Figure 2 .
Figure 2. Molecular docking simulations.(a) CDOCKER ENERGY of potential targets; (b) Molecular docking of compound 3g into CYP1B1 protein; (c) Three-dimensional representation of the complex (CYP1B1 protein and 3g compound); (d) Two-dimensional interactions diagram of the complex.
Microsoft ® Excel 2010 was used to analyze data.The neuroprotective effects by the compounds were expressed as the protective rate, and it was calculated according to the following formulas: Inhibition rate = (mean OD control − mean OD drug reated )/(mean OD control − mean OD vacuity ) × 100% Cell viability = 1 − Inhibition rate Protective rate = (Cell viability drug reated − Cell viability MPP + )/Cell viability MPP + × 100% And Cell viability MPP + = 46.35%3.5.A Procedure for the Preparation of Neuroprotective Targets

Table 4 .
The protective rate of compounds on MPP + -induced SH-SY5Y cells.

Table 4 .
The protective rate of compounds on MPP + -induced SH-SY5Y cells.