Novel iodoquinazolinones bearing sulfonamide moiety as potential antioxidants and neuroprotectors

In a search for new antioxidants, a set of new iodoquinazolinone derivatives bearing benzenesulfonamide moiety and variable acetamide pharmacophores 5–17 were designed and synthesized. The structures of the synthesized compounds were confirmed based on spectral data. Compounds 5–17 were screened using in vitro assay for their antioxidant potential and acetylcholinesterase (AChE) inhibitory activity. The 2-(6-iodo-4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydroquinazolin-2-ylthio)-N-(pyrazin-2-yl) acetamide 14 was the most active scaffold with potent AChE inhibitory activity. Compound 14 showed relative safety with a median lethal dose of 300 mg/kg (LD50 = 300 mg/kg), in an acute toxicity study. The possible antioxidant and neuroprotective activities of 14 were evaluated in irradiated mice. Compound 14 possessed in vivo AChE inhibitory activity and was able to modify the brain neurotransmitters. It was able to cause mitigation of gamma radiation-induced oxidative stress verified by the decline in Myeloperoxidase (MPO) and increase of glutathione (GSH) levels. Also, 14 restored the alterations in behavioral tests. Molecular docking of 14 was performed inside MPO and AChE active sites and showed the same binding interactions as that of the co-crystallized ligands considering the binding possibilities and energy scores. These findings would support that 14 could be considered a promising antioxidant with a neuromodulatory effect.


Biological evaluation
In vitro evaluation Acetylcholine esterase (AChE) inhibitory activity.The cholinesterase enzymes' inhibitory activity was evaluated using modified Osman et al. 45 method.Briefly, samples were initially dissolved in DMSO to achieve a concentration of 10 mg/mL.Then 0.1 mL of the sample was diluted to 1 mL methanol to reach the concentration of 1 mg/mL (concentration 1).From concentration (1), 0.1 mL was further diluted to 1 mL to reach the concentration of 0.1 mg/mL (concentration 2).Concentration 1 (1 mg/mL) will become 0.1 mg/mL inside the plate and concentration 2 will become 0.01 mg/mL due to the addition of the reagents of the assay that dilute the sample ten folds.Samples that achieved inhibition above 50% were further analyzed to determine their IC 50 values.Donepezil and Physostigmine were used as a reference standard, 10 μL of the indicator solution (0.4 mM in buffer (1): 100 mM tris buffer PH = 7.5 was transferred to a 96-well plate followed by 20 μL of enzyme solution (acetylcholine esterase 0.02 U/mL final concentration in buffer (2): 50 mM tris buffer PH = 7.5 containing 0.1% bovine serum albumin).Then 20 μL of the sample/standard solution was added followed by 140 μL of buffer (1).The mixture was allowed to stand for 15 min at room temperature.Afterward, 10 μL of the substrate (0.4 mM acetylcholine iodide buffer (1) was added immediately to all wells.The plate was incubated in a dark chamber for 20 min at room temperature.At the end of the incubation period, the color was measured at 412 nm.Data are represented as means ± SD.The results were recorded using a microplate reader FluoStar Omega.Each test was conducted in triplicate.The Absorbances of the test samples were corrected by subtracting the absorbance of their respective blank (methanol/ethyl acetate in 50 mmol/L Tris-HCl, (pH 8).
The percentage inhibition was calculated using the equation: Inhibition (%) = 1 -(A sample/A control) × 100 where A sample is the absorbance of the sample extracts and A control is the absorbance of the blank.Extract concentration providing 50% inhibition (IC 50 ) was obtained by plotting the percentage inhibition against extract concentration.
Where, 1 mL of the test compounds or ascorbic acid at concentrations (12.5, 25, 50 and 100 mg/mL) were mixed with 1 ml of the DPPH solution.The mixtures were incubated for 30 min at room temperature in dark.The absorbance was read using a JENWAY 6315 spectrophotometer (Keison Products, Chelmsford, England) at 517 nm against blank.
It was carried out in triplicate, and average values were used to determine the inhibitory percentage of DPPH according to the following equation: percent inhibition = [(B − A)/B]*100, where A is the absorbance of the compound or standard and B is the absorbance of blank.IC 50 (µM) of each compound was calculated according to the nonlinear regression dose response-inhibition curve using Prism 5.03 (GraphPad, San Diego, CA, USA) and expressed as mean ± standard error (S.E).

In vivo evaluation
Animals.Swiss albino male mice (20-25 g) were obtained from the breeding unit of the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt.Animals were retained in polypropylene cages under well-preserved laboratory conditions (25 ± 5 °C, 40-60% humidity), with alternating cycles of 12 h light/ dark.They were fed a standard mouse pellet diet, had water ad libitum, and were housed for acclimatization to the lab environment for 1 week before the experimental study.Mice were treated gently; squeezing, pressure and tough handling were avoided, this was in accordance with ARRIVE guidelines.Irradiation processes.Mice were exposed to gamma radiation as a single dose of 5 Gy using a Canadian Gamma Cell-40 biological irradiator ( 137 Cs) located at the NCRRT, Cairo, Egypt.The dose rate was 0.622 rad/s.Acute toxicity study.Acute toxicity of the most active acetylcholinesterase inhibitor was evaluated through the determination of median lethal dose (LD 50 ) which was determined according to Chinedu et al. 47 .
Experimental design.Twenty-one mice were blindly allocated into three groups (n = 7).The first group (control) was injected i.p. with 10% DMSO, daily, for 7 days.The second one (irradiated) was treated as a control, and after 1 h from the last DMSO injection, the mice were exposed to 5 Gy gamma radiation 48 .A third group (Compound 14 + irradiation) received 30 mg/kg/day i.p. (1/10 LD 50 ) of compound 14, daily for 7 days.Then on the last day, after 1 h of injection, animals were irradiated at a dose of 5 Gy.After 24 h. of the last injection, behavioral tests were carried out.Immediately after behavioral test, mice were anesthetized using urethane (1.2 mg/kg i.p.) 49 and sacrificed by cervical dislocation then brain tissues were dissected out, rinsed with ice-cold saline, and dried on a filter paper.Then it was homogenized in ice-cold 0.1 M phosphate buffer saline (pH = 7.4) and stored at -80 °C for subsequent biochemical analysis.
Behavioral tests.All behavioral tests have been carried out under blind conditions to avoid any bias in evaluating the animal's behavior.Mice were allowed to a pre-test session a day before scarification for habituation.
Open field test (OFT).This evaluates locomotors and exploratory activities in mice where, the apparatus is transparent walls and a white floor 30 × 30 × 15 cm, divided into 16 squares of equal area.Each mouse was placed in the center of the open field test apparatus and allowed to roam freely for 5 min and latency time (ie, time taken to start moving) was detected as well as locomotion frequency as ambulation (ie, number of squares crossed), rearing frequency (ie, number of times the mice stand on its hind legs or with its forearm against the wall or in the free air) 50 .
Forced swimming test.Mice were independently forced to swim in an open cylindrical container (22 cm diameter, 40 cm height) that contained water (23-25 °C).Each mouse was observed for 5 min to record the immobility time 51 .
Biochemical parameters investigated in brain tissue homogenate.Brain homogenates were used for measuring AChE using Mouse Acetylcholinesterase (AChE) ELISA Kit content using the Mouse Acetylcholinesterase ELISA Kit from MyBioSource Cat.No.MBS260553 (San Diego, CA., USA) according to the manufacturer's instructions.The generation of norepinephrine (NE) in brain tissues was measured using an Eliza kit (MBS2600834) from MyBioSource (San Diego, CA, USA) according to the manufacturer's instructions.
The antioxidant activity of compound 14 was measured by estimation of MPO activity using an ELISA Kit from MyBioSource Cat.No.: CSB-E08723m (Houston, TX., USA) as well as the level of GSH was evaluated using a colorimetric kit according to the method of E. Beutler 52 .Statistical analysis.Prism 5.0 (GraphPad, San Diego, CA, USA) was used for analyzing the data, and it was expressed as means ± standard error.Comparisons between groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test.IC 50 values were calculated by the nonlinear regression dose response-inhibition curve.

Molecular docking
The Molecular Operating Environment (MOE, 10.2008) software was employed to conduct molecular docking study 53,54 .The X-ray structure of the native ligand 2-((3,5-bis(trifluoromethyl)benzyl)amino)-N-hydroxy-4-oxo-1,4-dihydropyrimidine-5-carboxamide complexed to human MPO (PDB 4C1M) 55 and the co-crystallized ligand (1R)-1,6-dimethyl-1,2-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione (Dihydrotanshinone I) complexed to AChE (PDB: 4M0E) 56 were obtained from the protein data bank and utilized as the target receptors.Water molecules were removed from the protein and protonation using the 3D Protonate protocol was performed to get the receptors prepared for the docking simulation.Using the Triangle Matcher placement and London dG scoring, the co-crystallized ligands were employed to determine the binding location and score.The MMFF94X force field was used to minimize energy with an RMSD grade of 0.01 kcal/mol/Å, taking partial charges into account.The native ligands were re-docked into the active sites of both enzymes to validate the docking technique.The validation showed that the docking approach is appropriate because it accurately recreated the docked pose of the co-crystallized ligands and their binding mode in the active site.

Chemistry
The synthesis of the quinazolinone derivatives 5-17, featuring a biologically active benzenesulfonamide moiety is illustrated in Scheme 1.The starting material 4-(6-iodo-2-mercapto-4-oxoquinazolin-3(4H)-yl) www.nature.com/scientificreports/benzenesulfonamide 4 44 was synthesized in a quantitative yield by cyclo-condensing 4-isothiocyanatobenzenes ulfonamide 2 57 with 2-amino-5-iodobenzoic acid 3 in refluxing ethanol containing a few drops of triethylamine (TEA).The corresponding iodoquinazolinone derivatives 5-17 were obtained by reaction of 4 with 2-chloro-N-arylacetamide derivatives in dry acetone containing anhydrous K 2 CO 3 (1.38 g) (Fig. 2).Based on elemental analysis, IR, 1 H-NMR and 13 C-NMR, the structures of all the synthesized compounds were established.IR spectra of 5-17 revealed an additional acetamide group showing NH and CO bands at their designated regions.Three singlet signals were detected in 1 H-NMR spectra of 5-17, one signal in the range of 3.62-4.17ppm referring to the SCH 2 , the second at 7.35-8.71ppm attributed to the NH proton and the third at 7.39-8.09ppm for SO 2 NH 2 protons, and aromatic protons displayed in the range of 6.13-8.34ppm. 13C-NMR of 5-17 displayed three characteristic signals peculiar to the SCH 2 and 2CO carbons.www.nature.com/scientificreports/

Biological activity
In vitro screening AChE inhibition activity.Acetylcholinesterase (AChE) is an enzyme that plays a crucial role in cholinergic transmission by catalyzing the rapid hydrolysis of the neurotransmitter acetylcholine 21,58 .The application of acetylcholinesterase inhibitors leads to various effects, which can alleviate the symptoms of Alzheimer's disease 59  Therefore, compound 14 was further chosen as the most active compound to be tested for its antioxidant activity and in vivo evaluations as an antioxidant and neuroprotector.Recent studies confirmed that as Alzheimer's (AD) progresses, the activity of AChE decreases 62 .Thus, the new cholinesterase inhibitor 14 could act as a valuable therapeutic agent as a neuroprotector, especially in AD.
Free radical scavenging activity (DPPH) assay.In the present study, the DPPH assay was used for evaluating the free radical scavenging activity of all the newly synthesized compounds by measuring their ability to quench free radicals (Table 2).Compound 14 was able to reduce the stable radical DPPH to the yellow-colored diphenyl picryl hydrazine.Thus, compound 14 displayed significant concentration-dependent inhibition of DPPH activity, with IC 50 95.54µM.compared to vitamin C (IC 50 = 112.78µM).These results indicate that compound 14 is considered a potent antioxidant as it could act as an electron or hydrogen donator to scavenge DPPH ⋅ radicals.These results indicate that 14 has strong scavenging power.This complies with the previous studies that confirmed the free radical scavenging activity of quinazolinones bearing Sulfonamide derivatives 42,63 .www.nature.com/scientificreports/Heterocyclic compounds are well known for their capability to bind free radicals 64 .Furthermore, sulfonamides are known for their redox properties 65 , the 2-pyrazinyl derivative 14 has excellent antioxidant properties.Pyrazine derivatives have the potential to defend cells from oxidative harm caused by ionizing radiation through the transfer of electrons and hydrogen.The aminopyrazine derivatives are inhibitors of lipid peroxidation and good scavengers of peroxynitrite 66 .The mechanism by which compound 14 exerts its antioxidant activity is likely through its ability to donate the hydrogens of the NH 2 group of benzenesulfonamide, thereby interacting with the divalent nitrogen atom of the DPPH and forming hydrazine DPPH-H 67 .These findings suggest that compound 14 has the potential to provide radioprotective effects.

In vivo evaluation
Lethal dose fifty (LD 50 ).Lethal dose fifty (LD 50 ) was determined for the most promising compound 14 to estimate its acute toxicity in albino male mice.The LD 50 value was found to be 300 mg/Kg (i.p.) bodyweight.Consequently, one-tenth of this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of 14.This was following our previous studies 42,63,68 .
Behavior study.Open field test (OFT) As shown in Fig. 3A, latency time was significantly elevated (4.8 folds) in the irradiation group in comparison with the control.Mice treated with 14 followed by irradiation exhibited a significant decrease in latency time by 72.4% compared with mice that received radiation alone.Furthermore, www.nature.com/scientificreports/irradiation intensely decreased locomotor activity of mice expressed as ambulation (by 33%) as compared with control mice.However, compound 14 abolished the effect of radiation on ambulation (by 31%) (Fig. 3B).This complied with the reported studies 69,70 .Whereas, data revealed a decrease in rearing in radiation group relative to control (by 25.7%) (Fig. 3C).Remarkably, administration of 14 before irradiation normalized all open field parameters.
Forced swimming test As shown in Fig. 4, irradiation significantly increased immobility time in the forced swimming test (FST) 7 folds, as compared to the control group.The prior administration of 14 significantly decreased the mean immobility time in mice as compared to animals that received radiation only (by 33%) 71 .
Biochemical parameters investigated in brain tissue homogenate.A significant increase in MPO levels by 33.5% as well as a decrease in the levels of GSH by 18%, was observed in irradiated mice brains when compared to the non-irradiated group.Treatment of irradiated mice with 14 led to a decrease in MPO by 39.6% and an increase in GSH levels by 17% respectively, as compared to irradiated mice (Fig. 5A,B).
As shown in Fig. 6A, irradiation significantly increased AChE by 24%, as compared to the control group.The prior administration of 14 significantly decreased AChE by 21% as compared to the animals that received www.nature.com/scientificreports/radiation only.Besides, irradiation intensely decreased Norepinephrine (NE) by 22% as compared with control mice.However, compound 14 abolished the effect of radiation on ambulation by 25% (Fig. 6B).
It is well-known that radiation generates excessive amounts of reactive oxygen species (ROS) leading to oxidative stress which is responsible for tissue damage following irradiation 72,73 .Oxidative stress (OS) plays a critical role in the pathophysiology of several brain-related disorders, including neurodegenerative diseases and ischemic stroke, which are the major causes of dementia 74,75 .The present study revealed amplified MPO activity in the brain samples of irradiated mice, which indicates the induction of inflammatory response 76 .Besides, the reduction in brain antioxidant status in mice is evidenced by depleted GSH levels, mostly due to its consumption by ROS 77 .GSH is a free radical scavenger that exerts its antioxidant function by reaction with superoxide radicals, hydrogen peroxide (H 2 O 2 ), peroxide free radicals (OH˙), and singlet oxygen that are considered important inhibitors of free radical arbitrated lipid peroxidation 78,79 since membranes within the brain are known to be rich in peroxidizable fatty acids, thus they undergo peroxidation under oxidative damages [80][81][82] .It is well-considered that inflammation underlies a wide range of pathological processes and is closely linked to oxidative stress via generation of ROS that can prolong and augment inflammatory cascades and prompt tissue damage 83 .Inflammation is involved in the pathogenesis of radiation-induced brain injury 84,85 .www.nature.com/scientificreports/In this study, the iodoquinazolinone derivative 14 revealed beneficial effects in relieving oxidative damage, as compound 14 administration boosted GSH levels as compared to irradiated group, which could be attributed to its high scavenging power as potent antioxidant findings.As proved in current study findings (in vitro DPPH assay) appears to support this idea.This coincided with other previous studies that reported the antioxidant ability of quinazolinones derivatives to improve the antioxidant status in irradiation-induced hepatotoxicity 63,68 .Previous studies reported that iodoquinazolinones ameliorated oxidative/antioxidant parameters after liver injury in rats 42 .In the current study, the observed significant upsurge in AChE activity and depletion of NA level in irradiated animals is an indication of neuro-damages.Previous investigators accounted for comparable data 29,86,87 .
Acetylcholinesterase (AChE) plays a key role in cholinergic transmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine which is an essential neurotransmitter in the regulation of motor function and locomotion 88,89 .The use of AChE inhibitors elicits several responses, which mediate the symptoms of Alzheimer's disease 39 .The in vitro study indicates that 14 is the most active AChE inhibitor in this series.Likewise, the administration of 14 restored AChE activity to basal levels, which could be one of the mechanisms involved in mitigating neurobehavioral dysfunction.Furthermore, the observed decline in MPO and AChE activity after treatment with 14 indicates amelioration of inflammation and consequently, improving cholinergic neurotransmission and restoring locomotor functions as that confirmed through behavioral evaluation findings in the current study.These results are in concordance with [90][91][92] .
Thus, current research has shed light on the effects of iodoquinazolinone-bearing sulfonamide derivative 14 as a promising antioxidant and neuroprotector.These neuroprotective effects should be kept in mind for further research to evaluate the possible therapeutic use for Alzheimer's treatment.

Docking on myeloperoxidase
To explore the binding mode of the designed iodoquinazolinone derivatives, we obtained and analyzed the docked structures of the synthesized compounds within the catalytic site of MPO and compared it with that co-crystallized ligand 2-(7-methoxy-4-methylquinazolin-2-yl)guanidine (Fig. 7A,B).Soubhye et al. 93 reported that the 2-(7-methoxy-4-methylquinazolin-2-yl)guanidine was found to be a mechanism-based MPO inhibitor of high inhibitory potency.It achieved the best binding mode with a remarkable docking score of − 16.20 kcal/mol.The binding mode of the aforementioned guanido compound, with the receptor, revealed the interaction between the amino group of guanidine (Hydrogen bond donor HBD) attached to Thr100 and Glu102 through a hydrogen bond.While the methoxy group (Hydrogen bond acceptor HBA) of the fused benzene ring attached to Gln91.
Compound 14 binds to the active site of MPO in the same manner as that of the native ligand, through the amino group of sulfonamide (have the same role as guanido group) binds to Thr100 and Glu102 as hydrogen bond donar, the benzene of sulfonamide group with Arg333 through a cation-pi interaction.Also, the nitrogen of pyrazine ring acts as HBA group and is attached to Arg424 93 (Fig. 8).Table 3 revealed the binding interactions and energy scores of the co-crystallized ligand and compound 14 inside the active site of MPO.

Docking on acetylcholinesterase
The hypothetical binding modes of the most active compound 14 were calculated using MOE docking in the active site of AChE-dihydrotanshinone I complex.Based on the structural similarity between 14 and acetylcholinesterase inhibitors, a crystal structure of the inactive conformation of AChE (PDB: 4M0E) was chosen.This crystal structure was also selected because of the similar structural motifs in the tested compound  and the structure of the co-crystallized ligand, (1R)-1,6-dimethyl-1,2-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione (Dihydrotanshinone I).First, the docking algorithm was validated for its ability to reproduce the co-crystal binding mode of the reference compound.The reference compound was extracted from the complex and re-docked in the active site using the same parameters that will be used for the tested compound.The calculated binding modes of 14 revealed very interesting results as shown in Fig. 9A,B, the oxygen atom of sulfonamide moiety binds to the Arg296 by one hydrogen bonding.The benzene ring attached to sulfonamide moiety gives a hydrophobic interaction with Trp286.On the other hand, another hydrophobic binding interaction occurred between Tyr341 and pyrazine ring.The nitrogen atom of the pyrazine ring is attached through a hydrogen bond to Tyr124. Figure 9A,B demonstrated the binding mode of dihydrotanshinone and its overlay with the re-docked lead compound.Table 1 revealed the energy scores and binding interactions of the native ligand and 14 inside the active site of AChE.

Conclusion
In summary, a hybridization strategy was adopted using the iodoquinazolinone scaffold and benzenesulfonamide moiety to produce were evaluated in irradiated mice.Before irradiation, compound 14 was guarded against the changes in all the measured parameters.Also, it was able to cause mitigation of gamma radiation-induced oxidative stress verified by the decline in MPO and increase in GSH levels.It was able to modify the brain neurotransmitters (NA).Finally, molecular docking of 14 inside the active sites of MPO and AChE proved the same binding interactions as that of the co-crystallized ligands confirming its possible inhibitory effect against both receptors.These outcomes hold great promise for the use of compound 14 as a safe antioxidant agent and its inhibition of cholinesterase is of interest concerning neurodegenerative disorders such as Alzheimer's disease.Future preclinical investigations would be carried out to confirm the specific and exact mechanism of action.
The experimental protocol received ethical approval from the Animal Care Committee of the National Centre for Radiation Research and Technology (NCRRT), Cairo, Egypt (Permit Number: 34 A/22), which complies with the guidelines of the US National Institutes of Health for the appropriate care and use of laboratory animals (NIH Publication No.85-23, revised 2011).

Figure 3 .
Figure 3.Effect of compound 14 on open field test: latency time (A), ambulation (B) and rearing (C).Each value represents mean ± SE.Statistical analysis was carried out by one way ANOVA followed by Bonferroni's multiple comparison test.@ Significantly different from control group, # significantly different from combined group (Radiation + compound 14).P < 0.05 (n = 7).

Figure 4 .Figure 5 .
Figure 4. Effect of compound 14 on immobility time in a forced swimming test.Each value represents mean ± SE.Statistical analysis was carried out by one way ANOVA followed by Bonferroni's multiple comparison test.@ Significantly different from control group, # significantly different from combined group (Radiation + compound 14).P < 0.05 (n = 7).

Figure 8 .
Figure 8. (A) 2D view of 14 and (B) 3D surface view of 14 inside the active site of MPO (4C1M).

Figure 9 .
Figure 9. (A) 2D view and 3D surface view of docking validation and binding mode of the lead compound (yellow) in AChE pocket 4M0E.(B) 2D view and 3D surface view of 14 inside AChE pocket 4M0E.

Table 1 .
AChE inhibition activity assay for compounds 5-17 in comparison to Donepezil and Physostigmine.IC 50 values were calculated using non-linear regression analysis.a Each value indicates the mean ± S.E (n = 3).

Table 2 .
Free radical scavenging activity using DPPH assay for all the newly synthesized compounds in comparison to ascorbic acid.IC 50 values were calculated using non-linear regression analysis.

Table 3 .
Docking scores of the co-crystallized ligand and compound 14 inside the active site of 4C1M and 4M0E enzymes.