Synthesis Anticonvulsant and Cytotoxic Evaluation of Benzimidazole-Quinoline Hybrids Schiff Base Analogs

Both, benzimidazole and quinoline pharmacophores have shown various activities against cancer, tuberculosis, malaria, fungal, viral, and bacterial infections. These moieties are considered highly potent nuclei in medicinal chemistry. Thus, this research work aims to produce a synergistic effect involving both nuclei in a single structure. A Series of literature work resulted in the synthesis of novel derivatives of 2-( 1H -1,3-benzothiazole-2-yl)- N ’ -substituted quinoline-4-carbohydrazide 5a – q using a multiple-step reaction. Characterization of all compounds was successfully done using IR and 1 HNMR, 13 CNMR as well as elemental analysis. Furthermore, screening of all synthesized compounds has been shown a positive response as an anticonvulsant and antidepressant activity in mice. Compounds 5b , 5h , 5l, and 5n have shown potentially active on pentylenetetrazole-induced seizures in mice. The synthesized compounds were also screened on a panel of 60 cell lines by the National Cancer Institute according to standard screening protocol. Ten compounds were selected and evaluated via a single high dose (10 (cid:1) 5 M). In a one-dose assay, compounds 5j and 5o showed the highest activity on leukemia (CCRF-CEM) and renal cancer (A498) with growth inhibition (GI) of 70.91 and 61.15%.


Introduction
Epilepsy is progressively more difficult to treat patients using existing information of antiepileptic drugs (AEDs) and documentation about the clinical treatment increases with time. 1 International League Against Epilepsy (ILAE) defines epilepsy as the occurrence of a minimum of two controlled seizures with 24-h gaps between them or one seizure causing the vulnerability for recurrence of seizure rate as 60% in the following 10 years.Epilepsy is a neurological condition characterized by recurring seizures.According to the World Health Organization (WHO), it is estimated that around 50 million people worldwide have epilepsy.Thus, epilepsy is a global health issue and affects people of all ages, cultures, and income levels.However, it has been reported that 8% of patients with epilepsy in low-and middle-income countries face additional challenges due to limited access to adequate medical care, medications, and resources.The prevalence of epilepsy in India is high, with an estimated 10-12 million people affected by the condition.The incidence of epilepsy is higher in rural areas compared to urban areas.Additionally, studies have shown that the state of Kerala has a higher incidence of epilepsy compared to other states in India. 2 In the last few decades, there has been potential development in the medical treatment of epilepsy involving the identification of novel AEDs and modified dosage forms of existing drugs like first-generation and second-generation drugs.A few examples of first-generation drugs are phenytoin, carbamazepine, phenobarbital, and valproate, and second-generation drugs lamotrigine, vigabatrin, tiagabine, topiramate, gabapentin, and levetiracetam.The goal of AED treatment is to control seizures and minimize their impact on daily life, while minimizing any adverse effects on normal brain function.Despite advancements in AEDs, many patients still suffer from poorly controlled or drug-resistant epilepsy, often due to undiagnosed conditions.Effective management of epilepsy requires a comprehensive approach that includes accurate diagnosis, appropriate medication, and lifestyle modifications as needed.Despite pharmacological treatment of secondgeneration AEDs on resistant epileptic patients in comparison with first-generation AEDs.The problem of either undiagnosed or difficult-to-treat seizures has not changed.The side effects and toxicity of some AEDs can pose challenges in their use and effectiveness.The importance of finding new and better drugs that have increased efficacy, fewer side effects, and are effective against drug-resistant epilepsy.This requires ongoing research and clinical trials to identify and evaluate novel treatment options. 3For purpose to overcome these difficulties, several attempts were made to design a novel drug.Benzimidazole and quinoline, are individually known for their potent pharmacological activities in various conditions like tuberculosis, 4,5 cancer, 6,7 malaria, 8,9 microbial, 10,11 fungal and bacterial infection, 12,13 viral infection, 14,15 inflammation, 16 convulsions, diabetes, hypertension, [17][18][19] and different multi-targeting hybrids inhibitors. 20,21Moreover, Different attempts were made to design and synthesize both pharmacophores in a single structure for exhibit a synergistic effect against the targeting conditions.
Cancer is the biggest cause of mortality in the world, with over 10 million casualties estimated by 2020.The most common cancers globally in 2020 were breast cancer (2.26 million cases), lung cancer (2.21 million cases), colon and rectal cancer (1.93 million cases), prostate cancer (1.41 million cases), non-melanoma skin cancer (1.20 million cases), and stomach cancer (1.20 million cases).Additionally, 1.09 million cases of liver cancer were also reported.These statistics highlight the ongoing need for effective prevention, early detection, and treatment of cancer. 22Around 5.2 million deaths were observed from the most common cancer like lung, colon and rectum, liver, stomach, and breast around the globe (1).Breast cancer is the second most common cause of mortality among various forms of malignant tumors documented so far. 23According to the Indian Council of Medical Research's (ICMR) National Cancer Registry Programme Report 2020, there will be 13.9 lakh cancer cases in India in 2020, and this figure is expected to grow to 15.7 lakh by 2025. 24It is a significant global health challenge despite efforts to develop and improve chemotherapy treatments.Despite some advances, cancer continues to cause significant morbidity and mortality around the world.Addressing the cancer burden requires a multifaceted approach that includes primary prevention, early detection, and effective treatment, as well as research into new and improved treatment options.As a result, there is a tremendous need to discover a new class of compounds that work selectively against cancer cells. 25Regulation of cell proliferation and apoptotic pathways linked to cell death is a well-known method for understanding a wide range of medical diseases, including cancer. 26,27][30][31][32][33] The authors of this research work are exploring the design of a hybrid drug that combines benzimidazole with a 2,4-disubstituted-quinoline hybrid, with the goal of achieving a synergistic effect and overcoming resistance factors in the treatment of seizures and cancer.The aim of this study is to synthesize a new and effective drug that could potentially improve treatment outcomes for these conditions.

Material and methods
The chemical reagents and solvent required were listed and obtained from different industries and companies like Central Drug House, E. Merck India Ltd, and spectrochem, etc., depending on the grade required for the reaction procedures.Reagents and solvents were of LR grade.All synthesized compounds and schemes defining the experimental work performed were drawn and theoretical analytical calculations were completed with the help of chemSketch, MarvinSketch, and ChemDraw Ultra version 14.0 (Waltham MA, United States).Mettler Toledo SF-400C was operated for weighing the content amount.Melting Point, IR, and NMR ( 1 HNMR and 13 CNMR) analysis were obtained using the instrument Lab India Melting Point Apparatus, Perkin Elmer Spectrum 65 FT-IR Spectrometer, and Bruker 300 Hz, respectively.IR and 1 HNMR samples were prepared using KBr pellet and DMSO grade d 6 and CDCl 3 sampling.

Experimental
Synthesis of 1-(1H-benzimidazol-2-yl)ethan-1-ol (1)   o-phenylene diamine (0.17 mol, 18.38 g) and lactic acid (0.17 mol, 15.31 mL) were added in the 200 ml of 4 N HCl using a round bottom flask.The mixture was allowed to reflux for 6-8 h.After completion of the reaction, cooled the solution with ice and neutralized with ammonia solution.The resulting precipitates were filtered and washed with ice-cold water, purified with charcoal, and recrystallized with hot ethanol.

Synthesis of 2-(1H-benzimidazol-2-yl)quinoline-4-carboxylic acid (3)
The solution of 2-acetyl benzimidazole 2 (0.17 mol, 27.22 g) in 150 mL ethanol and isatin (0.17 mol, 25.01 g) was prepared.The solution of potassium hydroxide (0.93 mol, 52.17 g) in 250 mL water was prepared separately.Both solutions were mixed in 250 mL of the round-bottom flask and refluxed for 18 h.Poured the mixture into the ice-cold water.The alcohol was removed under a vacuum and acidified using glacial acetic acid.Filtered and washed the precipitate with cold water. 30 General procedure for the synthesis of 2-(1H-1,3-benzodiazol-2-yl)-N'-substituted quinoline-4-carbohydrazide (5a-q) The equimolar amount of 2-(1H-benzimidazol-2-yl)quinoline-4-carbohydrazide 4 (0.005 mol, 1.51 g) and aromatic aldehyde (0.005 mol) were added in 50 ml ethyl alcohol.The mixture was refluxed for 6-12 h and poured the reaction mixture onto ice with continuous stirring.Filtered off the precipitate and wash it with cold water.The compounds were dried and recrystallized with ethyl alcohol.

Animal selection and maintenance
For the biological evaluation of anticonvulsant activity, adult albino mice (25-30 g) were selected based on their weight and age.These animals were taken from Central Animal House of NIET, Greater Noida.These selected animals were harbor in polypropylene cages under the specified conditions with a temperature of 25 ± 5 C. Animals were given proper 12 h light and dark cycles.Animals were being fed with food and water accordingly.Animal activity testing data included in protocol number IAEC/NIET/2019/01/07 were being conducted and executed as per the guidance of CPCSEA registration number i.e., 10/GO/abc/99/CPCSEA.

Preparation of stock solutions and dose calculation
The standard drugs (phenytoin and fluoxetine) formulated as per Indian Pharmacopeia were used for animal activity.The preparation of a stock solution for each testing compound required quantity of standard drugs (phenytoin & fluoxetine) and tested compound dissolved in vehicle (2% tween 80 þ 0.9% NaCl) solution.Each stock solution was prepared after measuring the weight of albino mice and theoretical calculation of dosage form.After calculating the dose and volume for the standard and each testing compound, stock solutions were prepared accordingly. 34

Acute toxicity studies
The purpose of performing an acute toxicity study was to determine the lethal dose (LD 50 ) of the test compound.Initially, animals were distributed into different groups of three animals, according to the selected dose stated in OECD 423 guidelines.Different dose concentrations of each compound were selected at 50, 100, 300, 500, and 2000 mg/kg, and were administered via the intraperitoneal route.For observation, the mortality rate in each group was recorded along with their gross behavioral activity, and other changes during the first 4 h of observation.After that, they were observed for their mortality. 35

Anticonvulsant activity
This test procedure was executed for calculating the threshold value to produce minimal tonicclonic seizures in the body.This involves the administration of subcutaneous pentylenetetrazole (PTZ) after the intraperitoneal administration of control, standard, and test compounds.Animals were distributed into different groups each having 6 animals.Group I was administered with control (tween 80 2% v/v in 0.9% NaCl solution), standard with standard drug (phenytoin 20 mg/kg) and the other groups will receive test compounds solubilized or suspended in a vehicle.Doses for tested compounds were calculated based on acute toxicity results i.e., 30, 100, and 300 mg/kg were administered via intraperitoneal route prior 0.5 h of the test performance.After 0.5 h of each administration of drugs, PTZ will be administered 75 mg/kg via the subcutaneous route.The animals were initially observed for 0.5 h and then for an additional 4 hr.Each animal was observed for determining the onset of action (clonus and Tonic) and duration of epilepsy along with the symptoms of Straub tail movement, hind-limb tonic extension, and jerky movement.The absence of a single 5-s episode of the hind-limb tonic extension will be taken as end point. 36

Anti-depressant activity
All the test compounds were screened for anti-depressant activity using a forced swimming test using Swiss albino mice.The distribution of mice was similar to the anticonvulsant grouping method.Before treating the animals, each mouse was tested for the Forced Swimming Test for 6 min.Each mouse was placed individually in the chamber (height: 20 cm, diameter: 45 cm) containing water up to the height of 15 cm within normal temperatures ranging 25 ± 2 C. Animals will be distributed into different groups each having six animals.Group I was administered with control (2% tween 80 v/v in 0.9% NaCl) solution, standard with standard drug (fluoxetine 30 mg/kg) and the other groups will receive test compounds solubilized or suspended in vehicle.Doses for tested compounds were calculated based on acute toxicity results i.e., 30, 100, and 300 mg/kg.The compounds to be tested were administered via i.p. route 30 min prior to the test performance.During test performance, animals were kept in the water chamber for 5 min.A test was performed to observe the duration of immobility as passive floating without struggling while using those movements required to keep their head on the surface was observed in mice. 37dditionally, the statistical calculation was accomplished using one-way ANOVA followed by Dunnett's test. 38

Anticancer activity
All the synthesized compounds were submitted online to NCI DTP screening for determining the antiproliferative activity among which the selected compounds were sent to NCI-Development Therapeutic Program, USA, for practical testing of single high dose activity.The antiproliferative activity of the tested compounds was evaluated in single dose concentration (10 mM) against different cell line of leukemia, non-small cell lung cancer, melanoma and colon, CNS, Ovarian, renal, prostate, and breast cancer.The one-dose data were reported as a mean graph of the percent growth of treated cells.The results for one dose assay, and analysis of historical DTP were performed.The growth percent and growth inhibition (GI) percentage were calculated for determining antiproliferative activity.][41]

Chemistry
The synthesis of targeted compounds 5a-p was prepared by the series of reaction described in Scheme 1.The o-phenylene diamine reacted with lactic acid in 4 N hydrochloric acid and then neutralized using ammonia solution to yield a-(2-hydroxyethyl)benzimidazole 1.The compound Scheme 1. synthesis of benzimidazole incorporated 2,4-disubstituted quinoline hybrids.
1 solution in acetic acid (glacial) was refluxed with chromium trioxide solution in acidic medium, followingly extracted with chloroform and then concentrated to give 2-acetyl benzimidazole 2. Separate solutions were prepared of compound 2 in ethyl alcohol and isatin in potassium hydroxide, individually.These solutions were added in RBF and refluxed which subsequently acidified to produce compound 3 (2-(1H-benzimidazol-2-yl)quinoline-4-carboxylic acid).Reactant for the targeted product was then synthesized by the two-step reaction.
Compound 3 was initially treated with dilute sulfuric acid for esterification reaction which on subsequent reaction with hydrazine hydrate yielded a reactant compound 4 (2-(1H-benzimidazol-2-yl)quinoline-4-carbohydrazide).In the last step, compound 4 is treated with different analogs of aldehyde in a sufficient amount of ethyl alcohol to yield the targeted products 5(a-o), 5p, and 5q i.e. (2-(1H-1,3-benzodiazol-2-yl)-N'-substituted-quinoline-4-carbohydrazide).The completion of each reaction was affirmed by TLC spotting in either benzene: acetone [B:A] (9.5: 0.5 OR 9: 1 OR 8: 2) or toluene: ethyl acetate: formic acid [T:E:F] (5: 4: 1 OR 5: 4.5: 0.5), where Rf value (refractive value) of each compound was observed and calculated.Compounds 1 and 5a showed their Rf value near 0.50 while 5f, 5 m, and 5o were identified near 0.90.The identification of synthesized compounds was confirmed and characterized using different analytical instruments like melting point, infrared (IR) spectroscopy, and proton ( 1 H NMR) nuclear magnetic resonance spectroscopy.These solvents were chosen based on the solubility of each compound in solvent.Interpretation of IR spectra defines that the ketonic group can be seen in between 1600 and 1730 cm À1 , while C ¼ N will be in 1500-1610 cm À1 .The NH group in benzimidazole ranges in between 3200 and 3000 cm À1 .The -CH group in substituted aromatic, quinoline, and benzimidazole ranges 2900-3000 cm À1 .The presence of chlorine on substituted quinoline was identified with stretching in peak at 775 cm À1 while the bromo-was identified with full stretch peak at 677 cm À1 .In 1 H NMR spectrum, hydrogen of CH group in benzimidazole and quinoline was identified within d 7.65 and d 7.80, whereas the CH in substituted aromatic group was found within d 7.00 and d 7.70.The hydrogen of NH in the side chain was determined above d 8.00.The methoxy group was identified with the singlet between d 3.79 and d 3.80.

Acute toxicity studies
Selected tested compounds were injected intraperitoneally and observed for the median lethal dose of each tested compound.The dose concentration given was in 50, 100, 300, 500, and 2000 mg/kg of albino mice.Compounds were dissolved in tween 80 (2% v/v) and 0.9% NaCl solution of the vehicle.Each animal was observed for 24 h after the drug administration.On observation, animals with a dose concentration of 2000 mg/kg show full mortality rate.These animals show involuntary movements like unwanted body stiffening and jerky movements.Animals at 500 mg/kg dose concentration, show LD 50 effects with slight spasmodic behavior.At 300 mg/kg dose concentration, it shows mortality in a few drugs which do not define as LD 50 .Whereas, at dose concentrations of 100 mg/kg and 50 mg/kg, animals survived and stays healthy as described in Table 1.

Anticonvulsant activity
The anticonvulsant activity was screened under a pentylenetetrazole-induced model using phenytoin as a standard drug.Mice were injected with the control, standard, and tested compounds intraperitoneally, 30 min prior to the test PTZ injection.On injecting PTZ subcutaneously, animals were observed for anticonvulsant activity.Animals were observed for the onset of tonic and clonic convulsions along with Straub tail and jerky movements.It was observed that most of the synthesized compounds have shown active anticonvulsant activities in mice.The compounds 5b, 5h, 5 l, and 5n show the most potent activity against convulsions at the minimum dose of 30 mg/kg concentration.The mice were stable for 4 h with 100 and 300 mg/kg doses.The compounds 5b, 5e, 5i, and 5 m showed moderate activity within the concentration of 100 mg/kg which was visible as the onset of action within half hour of the PTZ injection.On the same side, compounds 5a, 5d, 5f, 5 g, 5k, 5p, and 5q showed the least anticonvulsant activity in mice while compound 5j does not show any activity (Table 2).These animals had shown the symptoms of tonic-clonic seizures nearly after 3 h with a moderate 100 mg/kg dose.

Antidepressant activity
The forced swim test was used for the antidepressant activity evaluation of the tested compounds.Animals were administered with control (tween80(2%w/v) in 0.9% NaCl solution), standard drug (Fluoxetine 30 mg/kg), and the synthesized compounds before 30 min of the test performance.During the test performance, the animals were observed for the period of immobility.In observation, it was observed that the compounds 5c, 5 g, 5 l, 5 m, 5n, and 5q had shown potent antidepressant activity at 30 mg/kg concentration.While compounds 5a, 5b, 5h, 5k, and 5o showed moderate activity against depressant activity at a concentration of 100 mg/kg in mice.Moreover, the compound 5d, 5e, 5f, 5i, and 5p determines as least activity.In comparison with standard drugs, compounds 5 m and 5n show the most potential efficacy of antidepressant activity (Table 2).

In-vitro anticancer activity
The selected synthesized compounds undergo in vitro anticancer activity in the screening test on all tested cancer cell lines.These compounds were tested on 60 human cancer cell lines derived from nine different panels including breast, colon, CNS, leukemia, melanoma, non-small cell lung, prostate, and renal cancer cell lines, according to National Cancer Institute (NCI US) protocols.The growth percentage of cancer cell lines was determined against the tested compounds as shown in Table 3, where the most sensitive compounds were shown in Table 4.The calculated GI percentage was calculated using the growth percentage data.In a one-dose assay, compounds 5j and 5o showed the highest activity on leukemia (CCRF-CEM) and renal cancer (A498) with a   Anticancer activity of some of the compounds at 10 µM in term of growth percent (GP) Anticancer activity of selected compounds at 10 mM in term of growth percent (GP).

Structure-activity relationship
Based on the findings of the research, the following points can be analyzed regarding the antiepileptic and anticancer activities of the hybrid drug under study: Antiepileptic activity: The results of the study may reveal the effectiveness of the hybrid drug in controlling seizures and improving seizure control in patients with epilepsy.
Anticancer activity: The findings may also show the ability of the hybrid drug to target cancer cells and inhibit their growth, potentially improving treatment outcomes for various forms of cancer.
It is important to note that these are potential outcomes based on the research, and further studies will be needed to confirm the efficacy and safety of the hybrid drug for both antiepileptic and anticancer treatments.The compounds having quinoline carrying benzimidazole moiety have better anticonvulsant (5b, 5h, 5l, and 5n) and anticancer activity (5j and 5o) than those of the compounds having only quinoline nucleus.Groups like chloride, bromides, hydroxyl, and dimethylamino shown good anticonvulsant activity.
The compounds having nitro and hydroxyl group showing good activity that the others.The anticonvulsant activity was improved due to the presence of electron withdrawing groups viz.dimethylamino, 4-chloro, 4-bromo, and (5b, 5h, and 5l) and electron donating groups like hydroxyl substituted phenyl substituted hydrazones.

Conclusion
The objective of this study was to synthesize, characterize, and evaluate the anticonvulsant activity of the Schiff base-bearing benzimidazole incorporating quinoline moieties.Hybrid derivatives were synthesized by the multi-step reaction initiated from o-phenylene diamine.Each compound was validated via their analytical studies, melting point as well as spectral studies involving IR and 1 HNMR spectrum.Furthermore, these synthesized compounds were screened for anticonvulsant activity where, compounds 5c, 5h, 5 l, and 5n have shown the highest potent activity against convulsions in comparison with phenytoin.Although, on antidepressant screening, it has been found that compounds 5c, 5 g, 5 l, 5 m, 5n, and 5q have shown the most potent activity at on 30 mg/kg concentration.The overall result of this research work indicates the potential activity of synthesized compounds against convulsions.

Table 1 .
Acute toxicity studies of standards and synthesized compounds.

Table 2 .
Anticonvulsant and antidepressant activity of the tested synthesized compounds 5a-q.

Table 3 .
The growth percentage of tested compounds on 60 human cancer cell lines.