Synthesis, antioxidant activity, molecular docking and ADME studies of novel pyrrole-benzimidazole derivatives

Several 5-(alkylsulfonyl)-1-substituted-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole derivatives were synthesized and their antioxidant activities were investigated using lipid peroxidation (LPO) and 7-ethoxyresorufin O-deethylase (EROD) assays. Docking analysis with Human NAD[P]H-Quinone oxidoreductase 1 (NQO1) was also performed to gather thorough information about these compounds that have antioxidant activities. Moreover, their molecular descriptors and ADME properties were calculated using the SwissADME online program. As a result, most of our compounds possessed better affinity and created ample interactions with NQO1. The most potent compound 5j had LP inhibition value of 3.73 nmol/mg/min. Other compounds exhibited moderate activity on LP levels comparing to standard butylated hydroxy toluene (BHT). However, the inhibitory effect on EROD activity was not significant.

. In our previous papers, we applied the strategy of using particular benzimidazole conjugates as antioxidant agents [1,17,18].In light of data obtained from our previous studies, we still explore the potential new benzimidazole derivatives with strong antioxidant activities.For instance, we have synthesized and characterized some new 6-fluoro-5-substituted-benzimidazole compounds bearing an indole ring at the second position of the benzimidazole ring and then evaluated their antioxidant capacities in vitro.At the 10 -3 M concentrations, almost all the synthesized compounds exhibited remarkable superoxide anion formation inhibitory effects compared to that of superoxide dismutase [19].In this study, we have aimed to conjugate pyrrole fragments instead of an indole ring to the benzimidazole from the second position to evaluate their antioxidant activities and structure-activity relationships.Herein, some new 5-(alkylsulfonyl)-1substituted-2-(1H-pyrrol-2-yl)-1H-benzo [d]imidazole derivatives (5a-n) were designed, synthesized, characterized, and were analyzed in silico and in vitro.Their free radical scavenging properties were then probed in vitro by employing lipid peroxidation (LPO) and 7-ethoxyresorufin O-deethylase (EROD) assays to verify their potential antioxidant activities and structure-activity relationship.

General procedure for synthesis of 3a-n
To the solution of 4-(alkylsulfonyl)-1-chloro-2-nitrobenzene (2a-c) (5 mmol) in ethanol (5 mL), amine derivative (15 mmol) was added and heated under reflux, until the starting material was consumed (determined by TLC, 8-48 h).Upon cooling the mixture, water was added.The resultant yellow residue was crystallized from ethanol or purified by cc by using a mixture of hexane and ethyl acetate in varying concentrations as eluent [20].

General procedure for synthesis of 4a-n
Compounds 3a-n (3.5 mmol) in EtOH (75 mL) reduced by hydrogenation using 40 psi of H 2 and 10% Pd/C (40 mg) until cessation of H 2 uptake to obtain the catalyst before filtering off on a bed of celite and washing with EtOH; and concentrating the filtrate in vacuo [22].The crude amine was used without purification [23].

In vitro antioxidant activity 2.2.1. Treatment of animals
Albino male Wistar rats with 200-225 g were used in the present study.They were individually housed in standard cages with free access to tap water and standard rat chow ad libitum and maintained at room temperature of 22-25 °C, a 12 h light-dark cycle, and 60% relative humidity.Rats were deprived of feed for 24 h before decapitation under anaesthesia.The liver tissues were dissected quickly, rinsed thoroughly with deionized water, weighed, and then immediately stored at −80 °C to minimize any potential changes before processing.All housing and experimental procedures were approved by Ankara University Animal Ethics Committee.

Isolation of rat liver microsomes
Rat liver tissues were homogenized in cold 1.15% KCI (w/v) with a homogenizer on ice at 250 x g.The homogenates were first centrifuged at 11,000 x g for 25 min at 4 °C and the resulting supernatants were collected and further ultracentrifuged at 108,000 x g for 60 min at 4 °C.The latter pellets were suspended with 20% glycerol and maintained at −80 °C until further analysis.Total microsomal protein concentrations were quantified by the protocol of Lowry et al. [25], using bovine serum albumin as a standard.

Lipid peroxidation (LPO) assay
Lipid peroxidation (LPO) level was determined based on the protocols reported by Wills [26,27], and Bishayee and Balasubramanian [28], with some modifications described previously [29].The assay employs the measurement of thiobarbituric acid reactive substances (TBARS) that yields a pink color and can be measured spectrophotometrically at 532 nm, resulting from the reaction between lipid peroxidation products, mainly malondialdehyde (MDA), and thiobarbituric acid (TBA) under acidic conditions and high temperature.The final reaction mixture (1 mL) in the test tube consists of 0.2 mg microsomal protein, 10 -3 M test compound, 62.5 mM potassium phosphate buffer (pH: 7.4), 90 mM KCl, 0.2 mM Fe 2+ , in which cofactor (NADPH -generating system containing of 2.5 mM MgCI 2 , 0.25 mM NADP + , 2.5 mM glucose-6-phosphate, 14.2 mM potassium phosphate buffer (pH 7.8), and 1.0 U glucose-6-phosphate dehydrogenase) was added to initiate the reaction.Next, the mixture was incubated at 37 °C in a shaking water bath.After 30 min of incubation, the reaction was terminated by the addition of 500 µL of 25 % (w/v) trichloroacetic acid (TCA) and the mixture was then centrifuged at 7000 x g for 20 min to remove denatured proteins.One milliliter of the supernatant was mixed with 0.5 mL of TBA and then heated for 20 min in a boiling water bath.The absorbance of TBARS was measured at 532 nm against the blank, which contains all reagents without microsomal proteins.The results were then expressed as nmol TBARS/mg of protein.In this protocol, while dimethyl sulfoxide (DMSO), in which the synthesized compounds were dissolved, was employed as the control, butylated hydroxyl toluene (BHT) was used as a standard.

7-Ethoxyresorufin O-deethylase (EROD) assay
7-Ethoxyresorufin O-deethylase (EROD) activity was assayed by the protocol described by Burke et al. [30].EROD activity is measured by following the CYP1A1-mediated deethylation of the substrate 7-ethoxyresorufin to form the product resorufin that can be monitored fluorometrically [30].The final reaction mixture (1 mL) in the test tube consists of 0.2 mg microsomal protein, 10 -3 M test compound, 1.0 mM 7-ethoxyresorufin as a substrate, 12 mM albumin, 100 mM Tris -HCl buffer (pH 7.8), in which cofactor (NADPH -generating system consisting of 0.25 mM NADP + , 2.5 mM MgCI 2 , 2.5 mM glucose-6-phosphate, 14.2 mM potassium phosphate buffer (pH 7.8), and 1.0 U glucose-6-phosphate dehydrogenase) was added to initiate the reaction.Next, the mixture was incubated at 37 °C for 5 min in a shaking water bath.After incubation, the reaction was terminated by the addition of 3 mL ice cold methanol and the mixture was then centrifuged at 7 000 x g for 20 min to remove denatured proteins.Following the centrifugation, the fluorescence intensity of the supernatant (3 mL) was read at the excitation/emission wavelengths of 538 nm/587 nm.While caffeine was employed as a standard, the control used in this protocol was dimethyl sulfoxide (DMSO) in which the synthesized compounds were dissolved.

Docking method and ADME property calculation
Human NAD[P]H-Quinone oxidoreductase 1 (PDB ID : 1dxo, resolution: 2.5 Å) file was obtained from the RCSB Protein Data Bank website [31].AutoDockTools v.1.5.6 was used for deleting water molecules and defining the grid box [32].Following this process, polar hydrogens and Gasteiger charges were added, and the grid was also prepared using the same software.Assigned grid's center were X = -2.718,Y = 16.674,Z = 5.139 and dimensions were X = 40, Y = 40, Z = 40.Spacing was defined as 0.375 Å.The 2D structures of the compounds were drawn on ChemDraw Ultra 12.0, minimized with MMFF94 and UFF force fields (number of steps: 5000 with steepest descent algorithm and convergence value of 10e-7) and then these files were converted to pdb files using Avogadro software [33].Subsequently, Gasteiger charges and torsion were added to ligand files with AutoDockTools.Prepared ligands were docked with AutoDock Vina [34].Finally, 3-D docked poses and interaction diagrams of the ligands were generated and interpreted using Discovery Studio Visualizer Ligand interaction module [35].
The evaluation of physicochemical properties and the prediction of ADME parameters were determined with SwissADME online tools [36].Besides, using BOILED-Egg representation brain penetration and gastrointestinal absorption of synthesized compounds were assessed [37].With the aid of the abovementioned calculations, we aimed to gather medicinal chemistry information about our compound set and to suggest further solutions for the improvement of these properties.

Validation of the docking method
This molecular docking method was validated for proving the reliability of the results.For this purpose, DockRMSD online program was used.This program can calculate RMSD value between two docked poses of a ligand and can render the result as text output [38].For the validation of the docking method, co-ligand duroquinone was extracted from the protein file (pdb id:1dxo) then after adding the polar hydrogens and Gasteiger charges, docked again with the same protein.After this, docked and crystallographic poses of duroquinone were converted to convenient format (.pdbqt to .mol2) using Open Babel GUI [39].In the end, these two files were submitted as input to DockRMSD online program.

Biological activity
The in vitro effects of these novel pyrrole-benzimidazole derivatives on rat liver microsomal NADPH-dependent lipid peroxidation (LPO) levels and ethoxyresorufin O-deethylase (EROD) activity were monitored [9].When compared to that observed for standard BHT, all synthesized compounds had moderate LPO inhibitory activity, particularly compounds 5b, 5d, and 5i-m displayed high inhibitory activity on LPO with the inhibition rates of 77%-65%.However, none of these compounds had a marked inhibitory effect on EROD activity.
According to the comparison of substituents and activity results which are given in Table 1, we observe that when R 1 substituent is ethyl, the activity becomes elevated dramatically.Also, by using our R 1 ethyl-substituted compounds as a starting point, we can determine that nucleophilic groups such as benzyl at R 2 also would enhance the activity.Benzyl group needs to maintain electron density, therefore we encounter the decreasing values for compounds that are substituted with deactivating halogens: 5k, 5l, 5m, and 5n.If we examine 5b, 5d, and 5e closely, R 1 is methyl and R 2 is ethyl.For 5d (R 2 = butyl) and 5e (R 2 = cyclohexyl), the lipid peroxidation inhibition slightly decreases.Moreover, the poor activity of compound 5n can be explained by having propyl at R 1 and an electronically sparse aromatic ring at R 2 .5m also has this aromatic portion at R 2 but has ethyl instead of propyl at R 1 .This should possibly explain the relatively poor activity of 5n.According to the LPO assay; compounds 5b, 5d, 5i, 5j, 5k, 5l, 5m were elected as candidates.These compounds were compared to standard compound BHT.Among them, the most potent compound was 5j.

Molecular docking and ADME studies
Initially, necessary positioning and interactions have been understood with the aid of a reference study, which mandates that the bound acceptor's aromatic moieties should become stacked with FAD and maintain certain interactions.Moreover, ligand should offer several interactions with aromatic residues such as Trp105, Phe106, Phe178, Tyr126, and Tyr128 and polar residue His161 [6].Regarding Figure 2, compound 5j docked into the binding site while maintaining hydrophobic interactions with Tyr128 through its benzyl substituent and benzimidazole moiety also constitutes Pi-alkyl interaction with Pro68.We can determine that the sulfonyl group acts as an acceptor in H-bond interactions with existing coenzyme FAD and Gly193.
The reliability of this method was proved using the DockRMSD distance calculation program.Out of 1,048,576 possible mappings, an optimal mapping was chosen by the same program.RMSD value for this mapping was calculated as 1.270 Å.For a reliable docking method, this value should be below 2 Å [40].Fortunately, the RMSD value for these two duroquinone ligands was below this upper limit, therefore our method was proved to be rather versatile and reliable in conducting docking analysis with 1dxo.The H-bond donor/acceptor count represents the atoms that are capable of interacting with the polar residues in the domains or cavities in the protein besides having an impact on P-glycoprotein transport, permeability, and many other parameters [36,41].Number of rotatable bonds increases the overall flexibility of the compounds and give them the ability to interact with various enzymes and plasma proteins which have pharmacokinetic functions [36].Using the Lipinski's rule of five (RO5) we can predict the oral activity of small molecules.This rule includes the following thresholds: the molecular mass should be below 500 daltons, calculated logP (clogP) must be less than 5.5, hydrogen bond donors should be less than or equal to 5 and hydrogen bond acceptors should be less than or equal to 10. Ligands that fail to conform to at least one of these parameters are flagged as orally undesirable [36,42,43].Muegge's filter consists of the limits: 200 ≤ molecular weight ≤ 600, −2 ≤ XLOGP ≤ 5, total polar surface area ≤ 150, the number of rings ≤ 7, the number of carbon > 4, the number of heteroatoms > 1, the number of rotatable bonds ≤ 15, the hydrogen bond acceptors ≤ 10, and the hydrogen bond donors ≤ 5 [44].Leadlikeness is on the other hand, is a rule-based method that includes 250 ≤ molecular weight ≤ 350, XLOGP ≤ 3.5, number of rotatable bonds ≤ 7 [36,45].
Moreover, according to Table 2, compounds in our pyrrole-benzimidazole set have exhibited much better docking scores, interactions, and overall drug-likeness.Amongst these derivatives, docking scores for most effective LP inhibitors were found to be relatively lower than BHT and also other compounds.Additionally, the compounds 5g, 5h, 5l, 5m, and 5n offered the lowest energy values and 5b, 5d, and 5i passed all of the medicinal chemistry friendliness filters.Since the binding region consists of hydrophobic residues, H-bond acceptor/donor count has lost its relevance.One must note that all these parameters may affect the binding of drug-like compounds to proteins although a certain correlation is yet to be created.We have the knowledge that these ADME properties mainly have an impact on the bioavailability of substances.
The BOILED-Egg representation is a useful tool for the interpretation of permeation to the central nervous system (CNS) and gastrointestinal reabsorption.As result, all of the synthesized derivatives were found to be passively absorbable via the gastrointestinal tract.This finding increases the overall bioavailability of our compounds.Hereby we can determine that BHT can effectively pass through the blood-brain barrier (BBB) thus may cause mild CNS adverse effects.Besides, we can witness similar but milder hazards for the compounds except 5l, 5m, and 5n.Among these, 5n was found to have a much higher WLOGP (Figure 3).This may prevent the permeation of the compound through BBB thus having fewer adverse effects on CNS.These compounds have good absorption through the gastrointestinal tract while possessing a lower chance of permeating through BBB.Fortunately, they also exhibit high antioxidant activity.Overall, these attributes render these three compounds safe and versatile lead-like compounds.

Discussion
In this study, novel pyrrole-benzimidazole derivatives were designed, synthesized, and characterized and their antioxidant activities were then analyzed through lipid peroxidation and EROD activity.These derivatives exhibited moderate activities relative to BHT.Among all of them, compound 5j emerged as a potential LPO inhibitor while having no considerable inhibitory effect against EROD activity.This characteristic may increase the chance of it being a potential agent against  lipid peroxidation.SAR studies suggested that when R 1 becomes ethyl and R 2 is benzyl, the activity greatly enhances.Though when this benzyl group is substituted with the halogen group, activity is decreased.Results of our studies suggest that pyrrole-benzimidazole derivatives have expressed much higher affinity values and a higher number of interactions in docking analysis, compared to that of standard BHT.Among them, compound 5j which ensures certain interactions with the enzyme has shown promise as potential lipid peroxidation (LPO) inhibitor.This compound has offered hydrophobic interactions with Tyr128, a steric interaction with Pro68, and H-bond interactions with FAD and Gly193.Pharmacokinetic parameters were also calculated to discuss whether our compounds lack certain requirements for their pharmaceutical development or need certain improvements in this area.Among our derivatives, 5b and 5d have passed all the filters although other derivatives failed these requirements.These approaches have helped  our team to both discover a novel potent antioxidant agent and to gain a better understanding of certain drug-protein interactions which play a role in this particular activity.

Figure 2 .
Figure 2. Binding pose (A) and ligand interaction diagram (B) of the most potent compound.

Table 1 .
The inhibition values of synthesized pyrrole-benzimidazole derivatives 5a-n, standards BHT and Caffeine, and control DMSO against EROD and LPO.

Table 2 .
AutoDock Vina docking scores and SwissADME results of pyrrole-benzimidazole derivatives and BHT.