Pharmacological evaluation of newly synthesized benzimidazole derivative for anti-Alzheimer potential

Abstract Backgound: Alzheimer disease (AD) is a disastrous disease characterized by accretion of amyloid-beta plaques, neurofibrillary tangles inducing oxidative stress, loss of neuronal functions and continuous progression of cognitive impairment leading to severe dementia. Material and Methods: The newly synthesized benzimidazole derivative 4-chloro-3-(2-phenyl-1H-benzimidazole-1-sulfonyl) benzoic acid (CB) was evaluated for its anti-Alzheimer activity using in silico, in vivo, in vitro and molecular techniques (ELISA, WB & IHC). Results: In-silico studies revealed that CB has atomic contact energy values of −3.9 to −8.9 kcal/mol against selected targets. In vitro assay showed that CB caused acetylcholinesterase (AChE) and diphenyl-1-picrylhydrazyl inhibition. In-vivo findings revealed improvement in dementia as observed in the morris water maze test and Ymaze test. Amyloid-beta disaggregation, increased level of anti-oxidants, decreased expressions of inflammatory markers and enhanced cellular architecture were found in the cortex and hippocampus of treated rats in the histopathological examination, immunohistochemistry analysis, enzyme-linked immunosorbent assay and western blot analysis. Conclusions: This study revealed that CB possess different binding affinities with the Alzheimer-related targets and it possess anti-Alzheimer activity, mediated via AChE and amyloid-beta inhibition, anti-oxidant and anti-inflammatory pathways.


Introduction
Neurodegenerative diseases are a threat to humanity which means it's a considerable challenge to medical field because these diseases are irremediable as they are life-threatening, characterized by irreversible loss of specific neurons [ 1 ]. it is becoming an issue in developed countries with large proportion of old age people [ 2 ]. it is the most ordinary cause of dementia that worsens over time and is associated with a decrease in the ability of thinking, decline in memory, judgment and difficulty in performing daily life tasks normally [ 3 ].aD is the most common neurodegenerative disease distinguished by extracellular aggregates of amyloid-beta (aβ) plaques and intracellular neurofibrillary tangles that are made by hyperphosphorylated proteins which ultimately cause synaptic dysfunction, vascular disorders, oxidative stress and neuroinflammation [ 4 ]. the major pathological hallmark of aD is an alteration in amyloid precursor protein that create small fragments of peptide commonly soluble amyloid precursor protein that control and inhibit the function of other proteins too.apart from this, the amyloid-beta peptide is also a key molecule in the pathogenesis of aD that leads to neurofibrillary tangles [ 5 ]. the forebrain and cholinergic neurons degenerate due to abnormally low levels of acetylcholine and tau protein phosphorylation, disrupting the neuronal pathways and neurotransmitters involved in the process [ 6 ]. these events led to the significant increase in the NlR family pyrin domain containing 3 (NlRP3) inflammasomes that generate pro-inflammatory cytokines interleukin-1 beta (il-1β), interleukin-18 (il-18), cyclooxygenase-2 (cOX-2), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), Reactive oxygen species (ROs) and tumor necrosis factor-alpha (tNF-α) that reduces the memory potential in neurodegenerative disorders such as aD, Parkinson's disease and ischemic brain injury [ 7 ].
scopolamine is a muscarinic receptor blocker that disturbs the cholinergic activity, increasing amyloid-beta deposition and oxidative stress that leads to different physiological, cellular and neurobehavioral deficiency.as a result, the use of scopolamine in research related to aD has been proven to be significant.the relationship between aD and scopolamine was explored in this study by focusing on its effects on cholinergic function and the amyloid cascade [ 8 ].Various FDa-approved drugs are available in the market for aD, i.e. donepezil, glunatamine, rivastigmine, however their use is limited because of efficacy mainly and side effects [ 9 , 10 ].
the N-containing heterocyclic compound benzimidazole derivative has well known biological activity because of its structural similarity with naturally occurring nucleotides.it gained importance in medicinal chemistry due to its activity with different enzymes and receptors.anti-ulcer and anthelmintic drugs contain benzimidazole and its derivatives which made them an important pharmacological agent.apart from that benzimidazole derivatives possess antimicrobial, antiviral, anticancer, anti-inflammatory, analgesic and other pharmacological activities [ 11 ].

Animals
Male adult sprague-Dawely rats weighing 250 ± 20 g (age: 10-12 weeks) were gleaned from the local breeding facility of Riphah institute of Pharmaceutical sciences, Riphah international University islamabad and experimentation was conducted according to guidelines of Research and ethics committee of RiPs (Ref.No. Rec/ RiPs/2021/22). the animals were divided into five groups each having six animals (n = 5), provided them controlled conditions, i.e. light and dark cycle of 12/12 h, 44-55% relative humidity, and 25 ± 1 °c temperature.throughout the experiment, animals were facilitated with water and food ad libitum [ 12 ].after completion of behavioral studies, animals were sacrificed according to the standard protocols.Brain tissues were extracted and stored at − 80° c temperature that was further processed by tissue homogenization to collect the supernatant for further biochemical analysis of each group.

Synthesis
cB was prepared by two-step procedure following scheme 1 .in the first step 4-chlorobenzoic was treated with sulfonic acid and substituted sulfonyl chloride was obtained.in the next step already prepared 2-phenyl benzimidazole was condensed with prepared sulfonyl chloride in equimolar ratios.the reaction mixture was stirred continuously for 30 to 50 min at very low temperature −5°C.White precipitate (solid) of 4-chloro-3-(2-phenyl-1h-benzimidazole-1-sulfonyl) benzoic acid were obtained, which were filtered and recrystallized in a suitable solvent to yield the respective compound.tlc was used to determine the completion of the reaction and further ethyl acetate was used to extract the reaction.to obtain the crude final compound, anhydrous magnesium sulphate was used to dry the separated organic layer, which was further filtered and solvent was removed by using rotary evaporator.silica gel was used to ensure the purity of final yield.

Computational study
For preliminary studies and perception of their binding affinities against various targets involved in aD pathogenesis, in-silico analysis of cB and donepezil was performed.target proteins were downloaded from online protein database (RcsB PD) whereas 3D crystallographic structure of tested compounds were drawn on chem draw that are saved as Mol files.PDB files of compound and ligands minimized by swiss PDB viewer, before docking.Proteins and compounds were prepared by using MGlt tools software [ 13 ]. the molecular docking studies of the tested compound were performed by using auto Dock Vina software a digital docking software that will further interpret results in the form of binding energies.target proteins selected were: amyloid beta (aβ), Nuclear factor-erythroid factor-2 (Nrf2), nuclear factor kappa-B cells (NF-ĸB), NlR family pyrin domain containing 3 (NlRP3), tissue necrosis factor-alpha (tNF-α), ionized calcium-binding adaptor molecule (iba-1), interleukin-10 (il-10), cyclooxygenase-2 (cOX-2), and interleukin-18 (il-18).Once docking complex formed then the best pose was generated of the compound for all the targets [10].each complex was evaluated in a 2D pattern to examine the maximum binding interactions formed between ligands and amino acid residues including: arginine (aRG), asparagine (asN), aspartic acid (asP), cysteine (cYs), glutamic acid (GlU), glycine (GlY), histidine (his), leucine (leU), lysine (lYs), methionine (Met), phenylalanine (Phe), serine (seR), threonine (thR), tyrosine (tYR) and valine (Val).

DPPH free radical scavenging assay
to evaluate the free radical scavenging potential of cB in vitro antioxidant activity was performed using 1, 1-diphenyl, 2-picrylhydrazyl (DPPh) free radicals.Different concentrations of tested compound (1.25, 2.5, 5, 10, 20 µg/ml) were taken up to 0.1 ml added to 0.004% methanol solution of DPPh and kept this mixture in dark for 30 min [ 14 ].through UV spectrophotometer absorbance was calculated at 517 nm.ascorbic acid was used as a standard.the scavenging activity was calculated based on the following equation: Further maximal inhibitory concentration (ic 50 ) was determined.Which is the concentration (μg/ml) of sample required for 50% scavenging of DPPh, calculated from the graph plotted for scavenging percentage against the sample concentration.

Acetylcholinestrase enzyme (AChE) inhibitory assay
ellman's method with acetlthiocholine iodide as a substrate was used to perform ache assay of compound.Reaction mixture contained 50 mM concentration of Na 2 hPO 4 buffer (ph 7.7), 0.5 mM test compound of different dilutions (1.25, 2.5, 5, 10, 20 µg/ml) in each well further DtNB, ellman's reagent 5, 5-dithio-bis-(2-nitrobenzoic acid) 1 Mm is added.the enzyme hydrolyses the substrate acetlthiocholine which leads to the formation of thiocholine that reacts with DtNB to produce a 5-thio-2-nitrobenzoate, yellow color complex.a microplate reader was used to assay yellow colored complex at a wavelength of 412 nm.Readings were taken for 10 min with 2 min intervals for confirmation of reaction occurred linearly [ 15 ].ic 50 value calculated by linear regression analysis between inhibition percentages versus the sample concentration by using the excel Program.

Morris water maze test
Rats were placed in a large circular tub (120 cm diameter × 50 cm height) with fixed platform that was 1 cm under the water surface (± 25 °c) to a depth of 15.5 cm, and the maze was divided into four equal quadrants.animals were trained on different days in order to find out the visible fixed platform that is present in any one of the quadrants that allow it to escape the water through different cues.two learning trials were conducted on the basis of visible platform and on the third trial rat is allowed to find the invisible cue with latency time of maximum 60 s to determine escape latency which is the time it takes to reach the platform, whereas water was made opaque through white ink.Rat's performance can be influenced by drug which is administered to it in each trial.Rats had used various strategies to find the hidden platform through visual cues, using distal cues as a point of reference and through remembering the movements needed to reach the hidden platform [ 16 ].the number of crossing of rats in the target quadrant was observed using video recording by utilizing a digital recording camera.

Y-maze test
the Y-maze test was performed in a Y-shaped apparatus consisting of 3 arms, each arm 12 cm wide, 35 cm in height and 40 cm in length.Food reward was placed in one of the arm of Y maze while rat was placed in the center of arm and allowed to move freely to find the food to consume it.Now again rat was placed in the Y-maze without food reward if it chooses the previous arm to go it chooses correctly.With this method different trials were performed to calculate the number of uninterrupted continuous entries to the correct arm and was visually recorded [ 17 ]. the alternation behavior % was calculated through the following formula: successive three sessions entries into correct arm successively

Anti-oxidant profile
Pre homogenized samples of cortex and hippocampus in 0.1-0.2ml were mixed with the PBs followed by the addition of 2-nitro benzoic acid solution, reduced glutathione (Gsh) was measured when it reacted with 0.6 mM DtNB in 0.2 M sodium phosphate (ph 8.0) in which 0.2 M phosphate buffer is added for the final volume up to 3 ml and absorbance values were calculated at 412 nm by using a microplate reader and the values were expressed as µmoles/mg of proteins [ 18 ].likewise, the levels of glutathione s-transferase (Gst) were assayed by using the reported protocol with slight modifications.the same amount of Gst with 1-chloro-2, 4-di-nitro benzene was diluted with the 0.1 M PBs as a result cDNB conjugate formed, whose absorbance was measured at 340 nm. the values were expressed as µmoles/mg of proteins.catalase activity was measured according to the method of aebi.catalase assay mixture consist of 0.05 M PBs (ph 7.0), 1 ml freshly prepared 30 nmol/l h 2 O 2 and 0.05 ml of supernatant sample of a total volume of 3 ml.the rate of decomposition of h 2 O 2 was measured in the presence of catalase.spectrophotometrically the absorbance was recorded at 340 nm and values were expressed as µmol/h 2 O 2 [ 19 ]. thiobarbituric acid reactive substances (tBaRs) assay was utilized to quantify the malonaldehyde level that is a marker resultant of an end product of lipid per oxidation (lPO) that is directly involved in cell damage.the mixture is formed by supernatant solution with the addition of 200 µl of 100 mM ascorbic acid, 580 µl of 0.1 Mm PBs and lastly 20 µl of ferric chloride followed by the incubation for 20 min in a water bath, and finally centrifuged for 10 min.then microplate reader was utilized to measure absorbance at 532 nm and values were shown in tBaRs nmoles/min/mg of proteins [19].

Hematoxylin and eosin (H&E) staining
tissue slides were de-paraffinized by using absolute xylene and then rehydrated with 100% to 70% gradient ethyl alcohol dilutions.sections were washed with distilled water followed by incubation in hematoxylin for not less than 10 min.similarly treated with the eosin solution for 5-10 min.slides were then cleaned, dried and lastly dehydrated with 70%, 95% and 100% alcohol washed with xylene and covered them with coverslips [12].Microscopic images were taken using a light microscope (Olympus, Japan) and analyzed with image J. total 5 images were taken of each group in order to examine for neuron morphology, neuron cell size, vacuolation and neuron cell shape.

Immunohistochemistry (IHC)
after deparaffinization of slides sections were made permeable to the three different xylene concentrations for 5-10 min.then treated with different concentrations of alcohol (100%, 90%, 80% and 70%) for rehydration in a graded fashion manner followed by washing of slides with the distilled water for the removal of alcohol traces.then these slides were exposed to protein kinase for antigen retrieval.then washed with 0.1 M PBs and peroxidase activity was inhibited by immersing slides in 3% h 2 O 2 for 10 min.Now sections were immersed in primary rat antibodies (anti-aβ, anti NF-ĸB and anti-tNFα) followed by immersion of sections in the PBs overnight at 4 °c. the next morning sections were immersed in biotinylated secondary antibodies for 2 h in a humidified chamber.the reaction product was treated with 3, 3′-diaminobenzidine-tetrahydrochloride (DaB) for staining of slides.Further slides were processed with dehydration in graded ethanol, followed by fixation in xylene and then cover with coverslips [ 20 ]. a light microscope (Olympus, Japan) was used for capturing 3 microscopic images for each group at 40X magnification.expression of aβ, NF-ĸB and tNF-α measured by analysis through image J and calculated as relative integrity density.

Enzyme linked immunosorbent assay (ELISA)
elisa of tNF-α, NF-ĸB, NlRP3, cOX-2 & il-18 were performed according to the procedure mentioned on the kit to measure the respective marker's expression.this assay was carried out in two main steps. in the first step brain tissue measuring 50 mg was homogenized and centrifuged at 4000 rpm for 30 min and finally supernatant was collected.the bicinchoninic acid (Bca) method using a 96 well plate was utilized to calculate the protein concentration in each group.the absorbance value was calculated by a microplate reader.total protein content values were measured in pictograms per milliliter (pg/ml) whereas all steps were performed in a triplicate pattern [ 21 ].

Western blot
10% (w/v) hippocampus homogenates were taken for centrifugation at 20,000 × g for 20 min at 4 °c which was processed by transferring of supernatant to clean tubes.the concentration of protein was measured by Bca method [ 22 ].Protein homogenates were separated on a sodium dodecyl sulfate and 12% polyacrylamide gel for the purpose of electrophoresis and further transferred to a 0.2 µm polyvinylidene fluoride membrane.the membrane was then incubated with the primary antibody NF-ĸB and tNF-α overnight at 4 °c then blocked for 1 h at room temperature with 5% bovine serum albumin.Followed by treatment with 0.1% tween after being washed three times with tris-buffered saline.then reacted with 1:1000 dilution of secondary antibody like anti-rabbit, anti-goat for two hours at room temperature.Quantification of protein bands was evaluated by densitometry with image J software [ 23 ].

Statistical analysis
the data was presented as mean ± standard error of mean (seM).statistical parameters applied one-way analysis of variance (aNOVa) followed by a post hoc tukey's multiple comparison test using Graph pad Prism 8 software.Morphological data were analyzed using image J software (Nih, Usa). the symbol # indicates a significant difference relative to saline and * shows significant difference relative to scopolamine group.p < 0.05 considered statistically significant.

In-silico analysis
cB and donepezil against amyloid-beta showed atomic contact energy (ace) value of −5.7 and −6.9 kcal/mol respectively.cB and donepezil binding against Nrf2 exhibit ace value of −8.8 and −9.9 kcal/mol respectively.cB and donepezil against NF-ĸB have ace value of −7.7 and −7.8 kcal/mol respectively.cB and donepezil against NlRP3 possess ace value −8.9 and −8.8 kcal/mol.cB and donepezil against tNF-α have ace value of −8.7 and −8.2 kcal/mol respectively.cB and donepezil against iba-1 have ace value of −7.9 and −1.8 kcal/mol binding energy.cB and donepezil against il-10 present ace value of −6.9 and −7.1 kcal/ mol.cB and donepezil against cOX-2 have ace value of −8.6 and −8.8 kcal/mol.cB and donepezil against il-18 displayed ace value of −3.9 and −6.9 kcal/mol binding energy.the best-docked poses of drug-target complex, along with binding energy values, hydrogen bonding and π-π bonding and residues involved in hydrogen bonding and π-π bonding presented in table 1 .2D interaction of cB and donepezil with targets ( Figures s1-s9 ).

Histopathological examination
in the saline group (10 ml/kg) group, no significant pathological alteration has been observed in the brain tissues of the cortex and hippocampus region.in the scopolamine (5 mg/kg) disease group depicts zealous results compared to saline control group including an abnormal change in cell size and shape abnormality in the morphology of neurons associated with pyknosis, cytoplasmic eosinophilia and nuclear basophilia in cortex and hippocampus.cB (10 mg/kg) and donepezil (5 mg/kg) treated group showed significant mitigation and great degree of cellular integrity against the scopolamine-induced aD group ( Figure 8 ).

IHC analysis
in the saline (10 ml/kg) group, there was no change observed in the regulation of aβ, NF-ĸB and tNF-α.the scopolamine (5 mg/kg) group, showed marked elevation in the expression of aβ, NF-ĸB and tNF-α, in the cortex and hippocampus tissues. in cB (5 mg/ kg), cB (10 mg/kg) and donepezil (5 mg/kg) treated groups reduced the expression of aβ, NF-ĸB and tNF-α shown to be down-regulated ( Figures 9-11 ).benzimidazole derivative (cB) for the attenuation of aD. the compound was characterized by spectroscopic techniques including 1 hNMR and 13 cNMR.computational studies were carried out through computer-assisted technique to obtain preliminary information about the affinity of cB and donepezil to their respective protein targets through which we interpret ace values and hydrogen bonding [7 ]. the present study revealed that cB has binding energy between −3.9 to −8.9 kcal/mol against selected targets.the following is the order in which cB binds with target proteins: NlRP3 > Nrf2 > tNF-α > cOX-2 > iba-1 > Nf-ĸB > il-10 > aβ > il-18.Order of binding affinity of d o n e p e z i l w i t h t a r g e t p r o t e i n s : Nrf2 > NlRP3 > cOX-2 > tNF-α > Nf-ĸB > il-10 > aβ, il-18 > iba-1.
acetylcholinestrase enzyme inhibition activity was conducted as acetylcholine is a major neurotransmitter that regulates cognitive function and refers to learning, reasoning, problem-solving, and decision making capabilities.ache inhibitors elevate the endogenous levels of acetylcholine in the brain as a result it enhanced cholinergic transmission which led to better communication between nerve cells.Results depict that cB had concentration-dependent ache inhibition potential.Moreover, free radical scavenging property of cB was observed, by comparing different concentrations of cB against standard ascorbic acid in order to find the capacity to scavenge superoxide ions formation and interaction with stable free radical DPPh compound [ 24 ].Results showed that cB exhibited a higher anti-oxidant potential.
scopolamine is a nonselective muscarinic receptor antagonist that affects learning capabilities and memory by inhibiting the cholinergic signaling pathway and decreasing levels of brain acetylcholine.according to conducted study, scopolamine caused deposition of aβ, synaptic dysfunction oxidative stress and increased the ache activity that enhanced the neurodegeneration in the cortex and hippocampus region of the brain [ 25 ].scopolamine is a prominent pharmacological paradigm for producing cognitive impairment that is observed in the behavioral tests by evaluating learning and memory ability in the Morris water maze and Y-maze test [ 26 ]. in Morris water maze test escape latency help us to assess hippocampus dependent spatial learning aptitude [ 27 ].escape latency and total number of the entries in target arm indicate general locomotor results while spontaneous alteration behavior indicates short-term memory [ 28 ]. in water maze test escape latency time was recorded during the training trials which showed that scopolamine group took more time to reach the platform cue compared to the treated one.the second observable dimension in morris water maze test was average number of the rat crossings in specific quadrant.Rats in the treated group showed more number of platform crossings as compared to disease group.according to the findings of morris water maze test, cB demonstrate neuroprotective effects against scopolamine-induced memory deficits.in Y-maze test, results showed that the treated group of rats have more number of entries as compared to disease group.Whereas higher percentage of alteration behavior represents cognitive performance.Both behavioral tests illustrate the success of aD model establishment as the scopolamine group shows memory impairment, while the treated groups shows improvement in cognitive impairment.
Previous studies suggest that oxidative stress is one of the earliest events in pathogenesis of memory impairment.the neurotoxic production of aβ and tau protein mediates neurotoxicity that instigate the oxidative stress in which increase level of free radicals produced hydrogen peroxide followed by the reduction process that produced reactive oxygen species, causing severe cell damage which leads to aD [ 29 ]. in this study, oxidative damage was identified by observing low contents of Gsh, Gst, catalase and high content of the lPO in scopolamine group as compared to cB and donepezil treated groups where elevation in Gsh, Gst, catalase and reduction of lPO were noticed in cortex and hippocampus region.
histopathological results demonstrate that scopolamine group exhibited severe pathological changes characterized by abnormal cell architecture and inflammatory cell infiltration.cB, donepezil treated group attenuated these abnormalities.Furthermore ihc, elisa and western blot were performed to validate the expression of aβ, tNF-α, NF-ĸB, cOX-2, il-18 and NlRP3.the hyper expression was observed in cortex and hippocampus of scopolamine group in rats.Normally there is a balance between pro and anti-inflammatory markers [ 30 ]. aβ, produced by the cleavage of aPP, forms aggregates that activate microglia by signaling through toll-like receptors, which produced the reactive oxygen species and inflammatory mediators such as cytokines by activating transcription factor NF-ĸB [ 31 ].tNF-α is a key pro-inflammatory cytokine in aD.aβ activates the transcription factor NF-ĸB directly that stimulates tNF-α production.cOX-2 is also highly expressed in neurons, and its expression correlates with the presence of aβ deposits and tau tangles.Overexpression of cOX-2 in neurons contributed to neuronal cell death in an animal model of aD by promoting the formation of aβ plaques and by production of reactive oxygen species, resulting in   exacerbated cognitive impairments.Glycogen synthase kinase and cyclin-dependent kinase are involved in the hyperphosphorylation of tau protein, and il-18 increase their expression.similarly inflammasomes, i.e.NlRP3 activation enhances tau hyper phosphorylation that drives the pathology of aD [ 32 ].Up regulation of these markers was observed in hippocampus and cortex region of scopolamine while, treatment with cB and donepezil attenuate the expression significantly.
considering every experimental procedure, the present data supported our hypothesis that cB has possible anti-alzheimer effect by inhibiting amyloid-beta aggregation, neurofibrillary tangles formation and exhibit anti-inflammatory and anti-oxidant effect by suppressing NF-ĸB and tNF-α which decreased expression of pro-inflammatory cytokines.

Conclusions
the present study revealed that newly synthesized cB possess binding energy values of −3.9 to −8.9 kcal/mol against the selected targets.cB exhibits anti-alzheimer's effect, mediated via ache and aβ inhibition, anti-oxidant and anti-inflammatory pathways, demonstrating its therapeutic potential in aD management.