iNOS inhibitors: Benzimidazole-coumarin derivatives to combat inflammation European Journal of Chemistry 13 (3) (2022) 307-318 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2022 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.13.3.307-318.2282 European Journal of Chemistry View Journal Online View Article Online iNOS inhibitors: Benzimidazole-coumarin derivatives to combat inflammation Richa Minhas and Yogita Bansal * Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147002 India. * Corresponding author at: Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala - 147002 India. e-mail: yogita_pharma@pbi.ac.in (Y. Bansal). 10.5155/eurjchem.13.3.307-318.2282 Received: 22 April 2022 Received in revised form: 16 June 2022 Accepted: 19 June 2022 Published online: 30 September 2022 Printed: 30 September 2022 Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory processes via accelerating the production of nitric oxide (NO). The efforts to develop small molecules as selective inhibitors of iNOS are being reported across the globe. The current study explores varied benzimidazole-coumarin derivatives as anti-iNOS agents. Literature survey suggests 2-aminobenzimidazole, coumarin nucleus, and 4-atom linker as important structural components for iNOS inhibition. Target compounds were designed and synthesized by coupling 2-aminobenzimidazole with (un)substituted coumarin through different linkers. These were docked in iNOS (1QW4) and nNOS (1QW6) targets to ascertain their iNOS selectivity, and evaluated for NO and iNOS inhibitory activities in vitro. The most active inhibitors were subsequently evaluated for acute toxicity and anti-inflammatory activity using carrageenan-induced rat paw edema model in vivo. All compounds possessed moderate to good NO and iNOS inhibitory activities. Compounds 14a, 14b, 14d, and 14e were the most potent inhibitors in vitro. These were found to significantly reduce the inflammation. Compounds 14d and 14e have been identified as the most potent iNOS inhibitors to combat inflammation. These derivatives may serve as potential compounds as such against iNOS, or as leads for the development of novel anti-iNOS agents. Docking Coumarin Carrageenan Inflammation Benzimidazole Biological activity Cite this: Eur. J. Chem. 2022, 13(3), 307-318 Journal website: www.eurjchem.com 1. Introduction Any physical injury, alteration in the normal immune response or in general any pathological condition triggers a series of biochemical events that involve the release of several pro-inflammatory mediators. These mediators induce inflame- mation, which is actually a protective mechanism to get rid of the pathological condition(s). However, prolonged or untreated inflammation causes associated tissue damage and/or modula- tion of normal immune system, culminating in several complex and multifactorial diseases. Alzheimer’s disease, Parkinson’s disease, rheumatoid arthritis and osteoarthritis, ulcerative colitis, and all cancers are such diseases that are mainly charac- terized by inflammation. Although non-steroidal anti-inflam- matory drugs (NSAIDs) are the first line of drugs for the treatment of inflammation in general, but these have proven inefficient in completely manipulating the inflammatory pathological indications in these multifactorial diseases. Moreover, their prolonged usage is associated with several undesirable effects, and the most common of these include hepatotoxicity and gastric ulcers [1,2]. Some drugs of biological origin such as etanercept and infliximab that act at different inflammatory targets have offered novel treatments for such diseases. However, their use is limited because of their high cost and poor bioavailability [3]. These limitations leave the door wide open for continued efforts toward the discovery of small molecules to counter inflammatory mechanisms. There are several molecular targets identified from the inflammatory cascades that could be antagonized to block the output of these cascades. These include cyclooxygenases (COXs), lipooxyge- nases (LOXs), tumor necrosis factor (TNF), and nitric oxide synthases (NOSs). NOS catalyze the formation of nitric oxide that plays a pivotal role in the inflammatory progression. It exists in three isoforms, i.e., nNOS, iNOS and eNOS. Of these, iNOS were identified as an important inflammatory target in the past few years [4]. Several molecules have been developed and reported as iNOS inhibitors by different research groups across the globe. Some of these molecules entered the clinical trials but none reach the market because of their toxicity, lack of selecti- vity, and/or poor pharmacokinetics [5]. Thus, the discovery of selective and druggable compounds capable of inhibiting iNOS is still a research area needing more exploration. An extensive and critical analysis of the literature on iNOS inhibitors by our research group has revealed that a compound having two appropriately substituted aryl or heteroaryl moieties that are connected to each other linked through nitrogen, sulfur or oxygen containing 3-5 atoms long linker has a probability of being a potent and selective inhibitor of iNOS [6]. In the current study, 2-aminobenzimidazole is selected as one heteronucleus because it is a wonderful mimic of guanidine moiety of arginine (the natural substrate of NOS). Coumarin is selected as the other heteronucleus because it is a well-reported free radical scavenger [7,8] (NO is a free radical). Therefore, 4-substituted coumarin is linked to a 5-(un)substituted-2-aminobenzimi- ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.13.3.307-318.2282 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.13.3.307-318.2282 mailto:yogita_pharma@pbi.ac.in http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.13.3.307-318.2282&domain=pdf&date_stamp=2022-09-30 308 Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 N N H NH R' (R' = H, CH3, Cl, NO2, OCH3)O Pharmacophore for iNOS inhibition 14a-e 15a-e 16a-e 17a-e Molecular mimic of guanidine of arginine Linker O O Pharmacophore for NO scavenging R R O O O NH NH N R' O O NH O NH N O R' R (R = H, 7-OCH3) Figure 1. Designing of target compounds. dazole through an alkoxy and amide linker to design target compounds 14a-e, 15a-e, 16a-e and 17a-e (Figure 1). To assess the binding efficiency and selectivity, all synthesized compounds are docked into iNOS and nNOS taken as target proteins. The compounds are found to possess good docking scores towards iNOS. Results of docking analysis are supported by in vitro pharmacological evaluations (NO determination and iNOS enzyme assay). The compounds showing good potential in vitro are evaluated through an in vivo carrageenan-induced rat paw edema model using L-NAME and AG, respectively, as reference nonselective and selective iNOS inhibitors. Previously, our research group synthesized 3- substituted coumarin derivatives as anti-iNOS agents [9]. To check the effect of orientation of -OH group of coumarin on biological activity, the current study was designed. 2. Experimental 2.1. Instrumentation Synthetic grade chemicals, reagents and solvents as procured from commercial sources including Loba Chem (India), SD-Fine Ltd. (India), Avra Synthesis Pvt. Ltd. (India), and Sigma Aldrich (India) were used for the synthesis of inter- mediates and target compounds. The solvents were dried, wherever required, by the standard methods. Pre-coated aluminum plates (Merck, Germany) were used for monitoring the progress of chemical reactions as well as to ascertain the purity of compounds. Melting points of all intermediates and target compounds were determined in open capillary tubes, and were uncorrected. IR spectrometer (Bruker Optik) was used for the analysis of IR spectra. 1HNMR and 13CNMR spectra were recorded in CDCl3 and DMSO-d6 solvents on a Bruker Avance II, 400 MHz spectrophotometer using tetramethylsilane (TMS) as an internal standard. Mass spectral analyses were performed using a Waters Q-TOF mass spectrometer. Docking study was performed with Schrodinger 2016-1 LLC (NY-USA) controlled with Maestro 10.5 software [10]. For the enzyme assay, an iNOS kit (ELISA) was procured from Krishgen Biosystems (Mumbai). Wistar rats of either sex weighing 190- 200 g were used for in vivo anti-inflammatory activity evalua- tion of target compounds. The animals were taken care of out as per the guidelines issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, India). The animals were housed in cages at ambient temperature 22-27°C with 12 h light/dark cycle in the animal house of Punjabi University, Patiala. They had free access to a standard laboratory diet (Ashirwad Industries, Chandigarh, India) and water ad libitum. Both feed and water were withdrawn 12 h prior to the experiment. The compounds and reference drugs were administered through intraperitoneal route as suspension in 0.5% sodium carboxymethylcellulose solution (SCMC). 2.2. Chemistry For the synthesis of the designed compounds, 5-(un)subs- tituted-2-aminobenzimidazole intermediate (13a-e) was coupled with (un)substituted coumarin-based intermediates (7-8 and 11-12) by using the methods already reported in literature by our research group [9]. Intermediate 3 was procured commercially and used as such. Other intermediates were synthesized by the methods reported in literature with slight modifications [11] (Figure 2). 2.2.1. Synthesis of 1-(2,4-dihydroxyphenyl)ethan-1-one (2) Compound 2 was synthesized by using the Nencki’s reaction which involves acetylation with acetic acid in the presence of ZnCl2 [10] with some modifications. Briefly, the fused and powdered ZnCl2 (0.24 mol) was added in glacial acetic acid, and the mixture was heated up to boiling. Dried resorcinol (0.2 mol) was added to the mixture with stirring at 140 °C. The mixture was heated until it turns blood red in color. The temperature was maintained at 150 °C for 20 min. The solution was acidified with dilute HCl (1:1), and cooled. The separated product was filtered, washed with dil. HCl (1:3), and re- crystallized from hot water containing a little amount of hydrochloric acid as brownish small needles (Figure 2). 1-(2,4-Dihydroxyphenyl)ethan-1-one (2): Color: Orange. Yield: 78%. M.p.: 144-145 °C. 2.2.2 Synthesis of 2-hydroxy-4-methoxyacetophenone (4) A solution of intermediate 2 (0.099 mol) in dry acetone was refluxed with dimethylsulphate (0.116 mol) and anhydrous potassium carbonate for 4 h under anhydrous conditions. The inorganic salts were filtered and washed with hot acetone. The filtrate and the washings were combined, and the solvent was recovered under reduced pressure. The left-over oily residue was macerated with ice-cold water, and extracted with solvent ether. The ethereal layer was washed with water, potassium carbonate solution (5%, w/v), and finally extracted with aq. sodium hydroxide (5%, w/v). The clear alkaline solution was acidified with dilute HCl and cooled at 8 °C. The separated product was filtered, washed with water, and re-crystallized from alcohol as colorless long needles (Figure 2). 2-Hydroxy-4-methoxyacetophenone (4): Color: Orange. Yield: 75%. M.p.: 53-54 °C. Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 309 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 Figure 2. Synthetic scheme employed for the synthesis of target compounds. 2.2.3. Synthesis of 4-hydroxycoumarin (5) and 4-hydroxy-7- methoxycoumarin (6) Intermediate 3 was procured commercially and used as such. A solution of compounds 3 (0.0147 mol) or 4 (0.006 mol) in dry diethyl carbonate (10-15 mL) was added to a suspension of sodium methoxide (0.0185 mol) in diethyl carbonate (10 mL). The reaction mixture was heated on a steam bath under anhydrous conditions for 5 h. It was cooled, diluted with water (25 mL) and extracted with solvent ether (2×40 mL) to remove the residual diethyl carbonate. The aqueous layer was acidified with hydrochloric acid. The separated product was filtered, washed with water, and re-crystallized from ethanol as white powder (Figure 2). 4-Hydroxycoumarin (5): Color: White powder. Yield: 80%. M.p.: 210-211 °C. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.31 (s, 1H, OH), 7.82 (d, 1H, H-8, J = 1.54, Ar-H), 7.59 (t, 1H, H-6, J = 7.82 Hz, Ar-H), 7.30 (m, 2H, H-7, H-5, Ar-H), 5.61 (s, 1H, H-3, Ar- H). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 166.1 (C-4), 163.1 (C- 2), 152.5 (C-8a'), 128.3 (C-7), 125.4 (C-6), 123.2 (C-5), 116.4 (C- 4a', C-8), 91.1 (C-3). 4-Hydroxy-7-methoxycoumarin (6): Color: White powder. Yield: 37%. M.p.: 252-253 °C. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.28 (s, 1H, OH), 7.70 (d, 1H, H-5, J = 6.1 Hz, Ar-H), 6.88 (m, 2H, H-6, H-8, Ar-H), 5.45 (s, 1H, H-3, Ar-H), 3.85 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 165.8 (C-4), 162.4 (C-2), 152.3 (C-8a'), 160.3 (C-7), 124.3 (C-5), 111.0 (C-6), 108.5 (C-4a'), 100.5 (C-8), 88.7 (C-3), 55.8 (OCH3). 2.2.4. Synthesis of 4-(3-bromopropoxy) coumarin (7) and 4-(3-bromopropoxy)-7methoxycoumarin (8) Anhydrous potassium carbonate (1.2 mmol) was added to a solution of compounds 5 or 6 (0.99 mmol) in 40 mL of acetone or DMF, respectively. Following this, dibromopropane (1 mmol) was added, and the mixture was stirred under reflux until the reaction is complete (4-5 h). Solvent was partly recovered under vacuum, and the concentrated reaction mixture is transferred into water to get the white colored powder. This product was used for the next step of synthesis without any further purification (Figure 2). 4-(3-Bromopropoxy)coumarin (7): Color: White powder. Yield: 52%. M.p.: 90-92 °C. Rf: 0.64 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.79 (d, 1H, H- 8, J = 7.92 Hz, Ar-H), 7.56 (t, 1H, H-6, J = 7.82 Hz, 1.58 Hz, Ar-H), 7.29 (m, 2H, H-7, H-5, Ar-H), 5.73 (s, 1H, H-3, Ar-H), 4.30 (t, 2H, H-4a, J = 5.84 Hz), 3.63 (t, 2H, H-4c, J = 6.32 Hz), 2.46 (q, 2H, H- 4b, J= 6.08 Hz). 13C NMR (100 MHz, CDCl3, δ, ppm): 168.56 (C- 4), 162.31 (C-2), 152.50 (C-8a'), 128.2 (C-7), 123.2 (C-5), 125.2 (C-6), 116.17 (C-4a', C-8), 88.5 (C-3), 66.73 (C-4a), 34.17 (C-4c), 30.7 (C-4b). MS (m/z): 283.98 [M+H]+. 4-(3-Bromopropoxy)-7-methoxycoumarin (8): Color: Off white powder. Yield: 35%. M.p.: 105-106 °C. Rf: 0.45 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.74 (d, 1H, H-5,J = 8.84), 6.92 (m, 2H, H-6, H-8), 5.79 (s, 1H, H-3), 4.42 (t, 2H, H-4a, J = 5.9 Hz), 3.85 (s, 3H, OCH3), 2.38 (t, 2H, H-4c, J = 6 Hz), 1.94 (m, 2H, H-4b). 13C NMR (100 MHz, 310 Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 DMSO-d6, δ, ppm): 168.54 (C-4), 162.25 (C-2), 150.12 (C-8a'), 159.5 (C-7), 111.0 (C-6), 123.6 (C-5), 108.5 (C-4a'), 100.3 (C-8), 88.9 (C-3), 65.9 (C-4a), 55.8 (OCH3), 34.17 (C-4c), 30.5 (C-4b). MS (m/z): 313.97 [M+H]+. 2.2.5. Synthesis of ethyl-2-((2-oxo-2H-chromen-4-yl)oxy) acetate (9) and ethyl 2-((7-methoxy-2-oxo-2H-chromen-4- yl)oxy)acetate (10) A mixture of anhydrous potassium carbonate (5.5 mmol) and a solution of compounds 5 or 6 (5 mmol) in DMF was stirred at room temperature for 30-45 minutes. Thereafter, ethyl chloroacetate (5.2 mmol) was added drop-wise with stirring. After the addition was complete, temperature of the mixture was raised to 90 °C, and stirred for another 2-3 h. After the reaction was complete, the temperature of reaction mixture was brought to ambient conditions. Addition of water to the reaction mixture afforded the product as cream-colored precipitates, which are filtered, washed with water, and dried for further use (Figure 2). Ethyl-2-((2-oxo-2H-chromen-4-yl)oxy)acetate (9): Color: Off white powder. Yield: 85%. M.p.: 110-111 °C. Rf: 0.55 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.91 (d, 1H, H-5, J = 7.9 Hz), 7.57 (t, 1H, H-7, J = 7.8 Hz), 7.29 (m, 2H, H-6, H-8), 5.58 (s, 1H, H-3), 4.77 (s, 2H, H-4a), 4.31 (q, 2H, CH2), 1.33 (t, 3H, CH3, J = 7.14 Hz). 13C NMR (100 MHz, CDCl3, δ, ppm): 169.7 (C=O, C-4), 162.4 (C-2), 152.12 (C-8a'), 128.3 (C-7), 125.4 (C-6), 123.3 (C-5), 116.2 (C-4a', C-8), 88.5 (C- 3), 62.5 (C-4a), 60.9 (CH2), 15.2 (CH3). MS (m/z): 249.07 [M+H]+. Ethyl 2-((7-methoxy-2-oxo-2H-chromen-4-yl)oxy)acetate (10): Color: White powder. Yield: 20%. M.p.: 129-130 °C. Rf: 0.68 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.79 (d, 1H, H-5, J = 6.8 Hz), 6.85 (d, 1H, H-6, J = 7.0 Hz), 6.80 (s, 1H, H-8), 5.44 (s, 1H, H-3), 4.74 (s, 2H, 4a), 4.31 (q, 2H, CH2), 3.87 (s, 3H, OCH3), 1.32 (t, 3H, CH3, J = 5.8 Hz). 13C NMR (100 MHz, CDCl3, δ, ppm): 169.6 (C=O, C-4), 162.3 (C-2), 160.2 (C-7), 152.10 (C-8a'), 123.6 (C-5), 111.0 (C-6), 108.5 (C-4a'), 100.7 (C-8), 87.3 (C-3), 62.5 (C-4a), 60.0 (CH2), 14.1 (CH3). MS (m/z): 279.08 [M+H]+. 2.2.6. 2-((2-Oxo-2H-chromen-4-yl)oxy)acetic acid (11) and 2-((7-methoxy-2-oxo-2H-chromen-4-yl)oxy)acetic acid (12) Compounds 9 or 10 (0.5 mol) was refluxed with aqueous NaOH (40 g/160 mL) in a round bottom flask for 15-20 min to hydrolyze the ester reactant. An equal volume of water was added to the reaction mixture; the dilute mixture was cooled to room temperature, and poured with vigorous stirring into conc. HCl (125 mL). Cooling of the acidic solution to room tempe- rature afforded the crude product that was filtered, washed with water, and re-crystallized from 1% HCl (Figure 2). 2-((2-Oxo-2H-chromen-4-yl)oxy)acetic acid (11): Color: White powder. Yield: 36%. M.p.:155-156 °C. Rf: 0.21 in Chloro- form: Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 13.29 (brs, 1H, OH), 7.87 (m, 2H, H-5, H-8), 7.67 (t, 1H, H-7, J = 7.8 Hz), 7.52 (t, 1H, H-6, J = 7.75 Hz), 5.88 (s, 1H, H-3), 4.99 (s, 2H, H-4a). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 173.9 (C=O), 169.8 (C-4), 162.5 (C-2), 152.15 (C-8a'), 128.5 (C-7), 125.6 (C- 6), 123.3 (C-5), 116.2 (C-4a', C-8), 87.5 (C-3), 64.9 (C-4a). MS (m/z): 221.04 [M+H]+. 2-((7-Methoxy-2-oxo-2H-chromen-4-yl)oxy)acetic acid (12): Color: White powder. Yield: 10%. M.p.: 167-168 °C. Rf: 0.35 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 13.20 (brs, 1H, OH), 7.80 (d, 1H, H-5, J = 7.0 Hz), 6.84 (d, 1H, H-6, J = 6.8 Hz), 6.78 (s, 1H, H-8), 5.39 (s, 1H, H-3), 4.72 (s, 2H, CH2), 3.87 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 173.8 (C=O), 169.7 (C-4), 162.4 (C-2), 160.1 (C-7), 152.12 (C-8a'), 122.8 (C-5), 111.2 (C-6), 108.2 (C-4a'), 101.2 (C-8), 87.3 (C-3), 64.7 (C-4a), 55.8 (OCH3). MS (m/z): 251.05 [M+H]+. 2.2.7. Synthesis of 2-aminobenzimidazole intermediates (13a-e) These were prepared by using a method already optimized in the laboratory. An aqueous solution of methanol (50%, v/v) was divided into two parts. To the first part CNBr (0.03 mol), and to the second part substituted diaminophenylenes (0.02 mol) were added. The solutions were poured into 100 mL RBF followed by stirring at 30-35 °C for 24-48 h. Thereafter methanol was distilled in vacuum; the solution was cooled to the room temperature and made alkaline by adding aq. ammo- nia solution. The precipitates were separated by filtration and re-crystallized from aq. ethanol (Figure 2). 2-Aminobenzimidazole (13a): Color: Brown crystal. Yield: 71%. M.p.: 229-230 °C. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.11 (m, 2H, H-4 and H-7), 6.85 (m, 2H, H-6, H-5), 6.07 (s, 2H, NH2), 5.57 (s, H, NH). 2-Amino-5-methylbenzimidazole (13b): Color: Light brown powder. Yield: 75%. M.p.: 198-199 °C. 1H NMR (400 MHz, CDCl3, δ, ppm): 7.01 (d, 1H, H-7, J = 7.8 Hz), 6.95 (s, 1H, H-4), 6.71 (d, 1H, H-6, J = 7.6 Hz), 5.90 (s, 2H, NH2), 2.33 (s, 3H, CH3). 2-Amino-5-nitrobenzimidazole (13c): Color: Yellow powder. Yield: 83%. M.p.: 133-134 °C. 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.96 (s, 1H, H-4), 7.85 (dd, 1H, H-6, J = 8.6 Hz, 1.4Hz), 7.18 (d, 1H, H-7, J = 8.2 Hz), 6.92 (s, 3H, 1-NH, NH2). 2-Amino-5-chlorobenzimidazole (13d): Color: Yellow brown powder. Yield: 48%. M.p.: 169-171°C. 1H NMR (400 MHz, CDCl3, δ, ppm): 6.14 (d, 1H, H-4, J = 2 Hz), 6.10 (d, 1H, H-7, J = 8.12 Hz), 5.87 (dd, 1H, H-6, J = 8.24 Hz, 2.04 Hz), 5.31 (s, 2H, NH2). 2-Amino-5-methoxybenzimidazole (13e): Color: Light brown powder. Yield: 42%. M.p.: 170-171 °C. 1H NMR (400 MHz, CDCl3, δ, ppm): 7.33 (d, 1H, H-7, J = 8 Hz), 6.93 (s, 1H, H-4), 6.79 (d, 1H, H-6, J = 7.6 Hz), 6.51 (s, 2H, NH2), 3.83 (s, 3H, OCH3). 2.2.8. Synthesis of target molecules (14a-e and 15a-e) Several 3-substituted compounds were synthesized by our research group [9] to produce the target molecules. A suspen- sion of dried potassium carbonate (2.5 mmol) and a solution of compounds 7 or 8 (0.5 mmol) in DMF (10 mL) was treated with 5-(un)substituted-2-aminobenzimidazole 13a-e (1 mmol). During the 24 h reaction, the mixture was stirred at 60 °C. A vacuum was used to recover the solvent, and then cold water was poured over the concentrated mixture. With chloroform and methanol as mobile phases, the precipitates were further purified by column chromatography (silica gel (60-120 mesh) column) (Figure 2). 4-(3-(1H-Benzimidazol-2yl)aminopropoxy)coumarin (14a): Color: Brown. Yield: 28%. M.p.: 230-232 °C. Rf: 0.39 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.75 (d, 1H, H-8, J = 7.52 Hz), 7.59 (t, 1H, H-6, J = 7.2 Hz), 7.30 (m, 2H, H-7, H-5), 7.12 (m, 2H, H-4', H-7'), 6.90 (m, 2H, H- 5', H-6'), 6.24 (s, 1H, NH), 5.72 (s, 1H, H-3), 4.43 (t, 2H, H-4a, J = 5.91 Hz), 4.24 (t, 2H, H-4c, J = 6.32 Hz), 2.32 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.64 (C-4), 161.29 (C-2), 154.30 (C-8a'), 152.45 (C-2'), 133.77 (C-3a'), 132.00 (C-7a'), 123.50 (C-5'), 122.80 (C-7), 122.50 (C-6), 120.07 (C-6'), 118.03 (C-5), 116.15 (C-7'), 116.02 (C-8), 115.00 (C-4a'), 114.57 (C-4'), 106.75 (C-3), 90.08 (C-4a), 66.56 (C-4c), 27.30 (C-4b). FT-IR (KBr, ν, cm-1): 3478 (NH str), 3130 (Ar C-H str), 2815 (Al C- Hstr), 1710 (C=O lactone str), 1625 (C=N str), 1600 (C=C str), 1055 (C-O str). HRMS (ESI+, m/z) calcd. for C19H17N3O3 [M+H]+ 336.1303, found 336.1302 [M+H]+. 4-(3-(5-Methyl-1H-benzimidazol-2yl)aminopropoxy) couma rin (14b): Color: Reddish brown. Yield: 25%. M.p.: 160-162 °C. Rf: 0.27 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.74 (d, 1H, H-8, J = 7.50 Hz), 7.56 (t, 1H, H- 6, J = 7.21 Hz), 7.20 (m, 2H, H-7, H-5), 7.01 (d, 1H,H-7', J = 7.81), Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 311 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 6.85 (s, 1H, H-4'), 6.70 (m, 2H, H-6'), 6.19 (s, 1H, NH), 5.38 (s, 1H, H-3), 4.20 (t, 2H, H-4a, J = 5.90 Hz), 3.24 (t, 2H, H-4c, J = 6.23 Hz), 2.33 (s, 3H, CH3), 2.16 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.32 (C-4), 161.00 (C-2), 154.15 (C-8a'), 152.17 (C-2'), 133.50 (C-3a'), 131.89 (C-7a'), 123.28 (C-5'), 122.53 (C-7), 122.29 (C-6), 119.99 (C-6'), 118.00 (C-5), 116.02 (C-7'), 115.92 (C-8), 114.97 (C-4a'), 114.50 (C-4'), 106.68 (C-3), 90.00 (C-4a), 66.37 (C-4c), 27.26 (C-4b),21.3 (CH3). FT-IR (KBr, ν, cm-1): 3320 (NH str), 3133 (Ar C-H str), 2815 (Al C-Hstr), 1715 (C=O lactone str), 1610 (C=N str), 1605 (C=C str), 1050 (C- O str). HRMS (ESI+, m/z) calcd. for C20H19N3O3 [M+H]+ 350.1460, found 350.4159 [M+H]+. 4-(3-(5-Nitro-1H-benzimidazol-2yl)aminopropoxy)couma rin (14c): Color: Yellow brown. Yield: 22%. M.p.: 239-240 °C. Rf: 0.5 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.20 (s, 1H, H-4'), 8.02 (m, 1H,H-6'), 7.76 (m, 1H, H-6), 7.75 (m, 1H, H-8), 7.36 (m, 2H, H-7, H-5), 7.26 (m, 1H, H-7'), 6.69 (s, 1H, H-3), 5.98 (s, 1H, NH), 5.21 (m, 4H, H-4a, 4c), 2.19 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.77 (C-4), 161.58 (C-2), 152.71 (C-8a'), 152.00 (C-2'), 133.66 (C-3a'), 131.97 (C-7a'), 123.65 (C-5'), 122.78 (C-7), 122.55 (C-6), 120.14 (C-6'), 118.87 (C-5), 116.10 (C-7'), 115.98 (C-8), 115.52 (C-4a'), 114.96 (C-4'), 106.79 (C-3), 90.28 (C-4a), 65.84 (C-4c), 27.25 (C- 4b). FT-IR (KBr, ν, cm-1): 3485 (NH str), 3130 (Ar C-H str), 2818 (Al C-H str), 1710 (C=O lactone str), 1620 (C=N str), 1610 (C=C str), 1055 (C-O str). HRMS (ESI+, m/z) calcd. for C19H16N4O5 [M+H]+ 381.1154, found 381.1153 [M+H]+. 4-(3-(5-Chloro-1H-benzimidazol-2yl)aminopropoxy) couma rin (14d): Color: Grey. Yield: 32%. M.p.: 220-222 °C. Rf: 0.4 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.78 (d, 1H, H-8, J = 7.9 Hz), 7.66 (m, 1H, H-6), 7.35 (m, 2H, H-7, H-5), 7.17 (m, 1H, H-4'), 7.07 (m, 1H, H-7'), 6.84 (m, 1H, H-6'), 6.66 (s, 1H, H-3), 6.61 (s, 1H, NH), 4.21 (m, 4H, H-4a, 4c), 2.21 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.84 (C-4), 161.57 (C-2), 154.62 (C-8a'), 152.72 (C-2'), 134.00 (C-3a'), 132.32 (C-7a'), 123.76 (C-5'), 123.06 (C-7), 122.84 (C- 6), 120.27 (C-6'), 118.05 (C-5), 116.17 (C-7'), 116.07 (C-8), 115.12 (C-4a'), 114.79 (C-4'), 106.99 (C-3), 90.28 (C-4a), 66.73 (C-4c), 27.39 (C-4b). FT-IR (KBr, ν, cm-1): 3487 (NH str), 3135 (Ar C-H str), 2820 (Al C-Hstr), 1715 (C=O lactone str), 1620 (C=N str) 1615 (C=C str), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C19H16ClN3O3 [M+H]+ 370.0958, found 370.0983 [M+H]+ and 372.0941 [(M+2)+H]+. 4-(3-(5-Methoxy-1H-benzimidazol-2-yl)amino propoxy) cou marin (14e): Color: Dark brown powder. Yield: 27%. M.p.: 253- 254 °C. Rf: 0.32 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.75 (d, 1H, H-8, J = 7.50 Hz), 7.57 (t, 1H, H-6, J = 7.21 Hz), 7.20 (m, 2H, H-7, H-5), 7.01 (d, 1H, H-7', J = 7.81), 6.85 (s, 1H, H-4'), 6.69 (m, 2H, H-6'), 6.19 (s, 1H, NH), 5.38 (s, 1H, H-3), 4.20 (t, 2H, H-4a, J = 5.90 Hz), 3.83 (s, 3H, OCH3), 2.24 (t, 2H, H-4c, J = 6.23 Hz), 2.16 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.03 (C-4), 160.89 (C-2), 154.08 (C-8a'), 152.07 (C-2'), 133.37 (C-3a'), 131.70 (C-7a'), 123.14 (C-5'), 122.37 (C-7), 122.12 (C-6), 119.87 (C-6'), 117.99 (C-5), 116.00 (C-7'), 115.87 (C-8), 114.92 (C-4a'), 114.42 (C-4'), 106.62 (C-3), 89.97 (C-4a), 66.35 (C-4c), 56.3 (OCH3), 27.22 (C- 4b). FT-IR (KBr, ν, cm-1): 3325 (NH str), 3130 (Ar C-H str), 2810 (Al C-Hstr), 1705 (C=O lactone str), 1610 (C=N str), 1605 (C=C str), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C20H19N3O4 [M+H]+ 366.1376, found 366.1375 [M+H]+. 4-(3-(1H-Benzimidazol-2-ylamino)propoxy)-7-methoxy cou marin (15a): Color: Light green. Yield: 35%. M.p.: 210-211 °C. Rf: 0.39 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.73 (d, 1H, H-5, J = 7.50 Hz), 7.10 (m, 2H, H- 4', H-7'), 6.87 (m, 2H, H-6, H-8), 6.85 (m, 2H, H-6', H-5'), 6.24 (s, 1H, NH), 5.79 (s, 1H, H-3), 4.41 (t, 2H, H-4a, J = 5.9 Hz), 3.83 (s, 3H, OCH3), 2.19 (t, 2H, H-4c, J = 6 Hz), 2.13 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.29 (C-4), 161.00 (C-2), 153.80 (C-8a'), 152.07 (C-2'), 133.57 (C-3a'), 131.98 (C-7a'), 123.42 (C-5'), 122.59 (C-7), 122.27 (C-6), 120.00 (C-6'), 117.95 (C-5), 116.02 (C-7'), 115.84 (C-8), 114.98 (C-4a'), 114.50 (C-4'), 106.59 (C-3), 90.00 (C-4a), 66.34 (C-4c), 55.4 (OCH3), 27.22 (C- 4b). FT-IR (KBr, ν, cm-1): 3460 (NH str), 3125 (Ar C-H str), 2810 (Al C-Hstr), 1690 (C=O lactone str), 1615 (C=N str). 1605 (C=C str), 1045 (C-O str). HRMS (ESI+, m/z) calcd. for C20H19N3O4 [M+H]+ 366.1409, found 366.1408 [M+H]+. 7-Methoxy-4-(3-((5-methyl-1H-benzimidazol-2-yl) amino) propoxy)coumarin (15b): Color: Brown powder. Yield: 40%. M.p.: 180-181 °C. Rf: 0.24 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.74 (d, 1H, H-5, J = 7.50 Hz), 7.01 (d, 1H, H-7', J = 7.8 Hz), 6.89 (m, 2H, H-6, H-8), 6.82 (s, 1H, H-4'), 6.71 (d, 1H, H-6', J = 7.6 Hz), 6.19 (s, 1H, NH), 5.73 (s, 1H, H-3), 4.40 (t, 2H, H-4a, J = 5.9 Hz), 3.83 (s, 3H, OCH3), 2.30 (s, 3H, CH3), 2.18 (t, 2H, H-4c, J = 6 Hz), 2.12 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.32 (C-4), 161.00 (C-2), 154.15 (C- 8a'), 152.17 (C-2'), 133.50 (C-3a'), 131.89 (C-7a'), 123.28 (C-5'), 122.53 (C-7), 122.29 (C-6), 119.99 (C-6'), 118.00 (C-5), 116.02 (C-7'), 115.92 (C-8), 114.97 (C-4a'), 114.50 (C-4'), 106.68 (C-3), 90.00 (C-4a), 66.37 (C-4c), 55.7 (OCH3), 27.26 (C-4b),21.3 (CH3). FT-IR (KBr, ν, cm-1): 3310 (NH str), 3125 (Ar C-H str), 2810 (Al C-Hstr), 1695 (C=O lactone str), 1615 (C=N str), 1610 (C=C str), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C21H21N3O4 [M+H]+ 380.1566, found 380.1565 [M+H]+. 7-Methoxy-4-(3-((5-nitro-1H-benzimidazol-2-yl) amino) pro poxy)coumarin (15c): Color: Yellow brown. Yield: 38%. M.p.: 243-244 °C. Rf: 0.45 in Chloroform:Methanol (8:2).1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.20 (s, 1H, H-4'), 7.84 (dd, 1H, H-6', J = 8.6 Hz, 1.4 Hz), 7.71 (d, 1H, H-5, J = 7.50 Hz), 7.18 (d, 1H, H-7', J = 8.2 Hz), 6.89 (m, 2H, H-6, H-8), 6.70 (s, 1H, NH), 5.73 (s, 1H, H- 3), 4.40 (t, 2H, H-4a, J = 5.9 Hz), 3.89 (s, 3H, OCH3), 2.16 (t, 2H, H-4c, J = 6 Hz), 2.09 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO- d6, δ, ppm): 164.70 (C-4), 161.51 (C-2), 152.67 (C-8a'), 151.96 (C-2'), 133.59 (C-3a'), 131.90 (C-7a'), 123.58 (C-5'), 122.71 (C- 7), 122.49 (C-6), 120.07 (C-6'), 118.80 (C-5), 116.02 (C-7'), 115.91 (C-8), 115.45 (C-4a'), 114.89 (C-4'), 106.72 (C-3), 90.21 (C-4a), 65.76 (C-4c), 55.7 (OCH3), 27.22 (C-4b). FT-IR (KBr, ν, cm-1): 3470 (NH str), 3125 (Ar C-H str), 2815 (Al C-Hstr), 1700 (C=O lactone str), 1620 (C=N str), 1605 (C=C str), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C20H18N4O6 [M+H]+ 411.1260, found 411.1259 [M+H]+. 4-(3-((5-Chloro-1H-benzimidazol-2-yl)amino) propoxy)-7- methoxycoumarin (15d): Color: Light brown. Yield: 45%. M.p.: 272-273 °C. Rf: 0.4 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.34 (s, 1H, H-4'), 7.83 (dd, 1H, H-6', J = 8.6 Hz, 1.4Hz), 7.71 (d, 1H, H-5,J = 7.50 Hz), 7.18 (d, 1H, H-7', J = 8.2 Hz), 6.89 (m, 2H, H-6, H-8), 6.70 (s, 1H, NH), 5.70 (s, 1H, H- 3), 4.40 (t, 2H, H-4a, J = 5.9 Hz), 3.89 (s, 3H, OCH3), 2.17 (t, 2H, H-4c, J = 5.8 Hz), 2.10 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO- d6, δ, ppm): 164.80 (C-4), 161.50 (C-2), 154.60 (C-8a'), 152.66 (C-2'), 134.00 (C-3a'), 132.30 (C-7a'), 123.69 (C-5'), 123.00 (C- 7), 122.78 (C-6), 120.19 (C-6'), 118.00 (C-5), 116.09 (C-7'), 116.01 (C-8), 115.06 (C-4a'), 114.70 (C-4'), 106.91 (C-3), 90.21 (C-4a), 66.67 (C-4c), 55.7 (OCH3),27.32 (C-4b). FT-IR (KBr, ν, cm-1): 3482 (NH str), 3130 (Ar C-H str), 2815 (Al C-Hstr), 1710 (C=O lactone str), 1620 (C=N str), 1610 (C=C str), 1050 (C-O str). HRMS (ESI+, m/z) C20H18ClN3O4 [M+H]+ 400.0956, found 400.980 [M+H]+ and 402.0937 [(M+2)+H]+. 7-Methoxy-4-(3-((5-methoxy-1H-benzimidazol-2-yl) amino) propoxy)coumarin (15e): Color: Light brown. Yield: 39%. M.p.: 225-226 °C. Rf: 0.35 in Chloroform:Methanol (8:2). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 7.69 (d, 1H, H-5, J = 7.6 Hz), 7.01 (d, 1H, H-7', J = 7.8 Hz), 6.90 (s, 1H, H-4'), 6.89 (m, 2H, H-6, H-8), 6.73 (d, 1H, H-6', J = 7.6 Hz), 6.19 (s, 1H, NH), 5.73 (s, 1H, H-3), 4.40 (t, 2H, H-4a, J = 5.9 Hz), 3.83 (s, 6H, OCH3), 2.24 (t, 2H, H-4c, J = 6 Hz), 2.13 (m, 2H, H-4b). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 164.00 (C-4), 160.78 (C-2), 154.01 (C-8a'), 152.01 (C-2'), 133.29 (C-3a'), 131.61 (C-7a'), 123.10 (C-5'), 122.31 (C-7), 122.07 (C- 6), 119.79 (C-6'), 117.92 (C-5), 116.01 (C-7'), 115.85 (C-8), 312 Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 114.88 (C-4a'), 114.37 (C-4'), 106.56 (C-3), 89.91 (C-4a), 66.27 (C-4c), 56.27 (OCH3), 27.23 (C-4b). FT-IR (KBr, ν, cm-1): 3300 (NH str), 3125 (Ar C-H str), 2810 (Al C-H str), 1690 (C=O lactone str), 1610 (C=N str), 1605 (C=C str), 1047 (C-O str). HRMS (ESI+, m/z) calcd. for C21H21N3O5[M+H]+ 396.1515, found 396.1514 [M+H]+. 2.2.9. Synthesis of target molecules (16a-e and 17a-e) Previously, our research team reported a method for preparing these compounds [9]. A solution of compounds 11 or 12 (0.25 mol) in THF (10 mL) was added to an excess of SOCl2 (2-5 mL) and allowed to heat at 40 °C for 30 min. In the reaction mixture, 5-substituted-2-aminobenzimidazole solutions (13a- e, 0.25 mol) were poured slowly over 1 hour. During the reaction, the mixture was stirred at 40-50 °C for 12-24 hours. By using a rotary vacuum evaporator, the solvent was recove- red after the reaction. In order to get the target compounds in pure form, the crude solid was fractionated by column chromatography using a gradient solvent system (Petroleum ether:ethyl acetate) (Figure 2). N-(1H-Benzimidazol-2-yl)-2-((2-oxo-2H-chromen-4-yl) oxy) acetamide (16a): Color: Yellow brown. Yield: 25%. M.p.: 205- 206 °C. Rf: 0.55 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.60 (s, 1H, NH), 7.91 (d, 1H, H- 5, J = 6.4 Hz), 7.57 (t, 1H, H-7, J = 6.2 Hz), 7.27 (m, 2H, H-6, H-8), 6.96 (m, 2H, H-5', H-6'), 6.89 (m, 2H, H-4', H-7'), 5.60 (s, 1H, H- 3), 4.80 (s, 2H, H-4a). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.3 (C-4b, C-4), 162.3 (C-2), 152.5 (C-8a'), 146.7 (C-2'), 136.4 (C-3a', C-7a'), 128.3 (C-7), 125.4 (C-6), 123.3 (C-5, C-5', C-6'), 116.2 (C-4a', C-8), 115.2 (C-4', C-7'), 87.5 (C-3), 64.6 (C-4a). FT- IR (KBr, ν, cm-1): 3360 (NH str), 3100 (Ar C-H str), 2900 (Al C-H str), 1720 (C=O, lactone str), 1705 (C=O), 1680 (NH bending), 1056 (C-O str). HRMS (ESI+, m/z) calcd. for C18H13N3O4 [M+H]+ 336.0940, found 336.0938 [M+H]+. N-(5-Methyl-1H-benzimidazol-2-yl)-2-( (2-oxo-2H-chromen- 4-yl)oxy)acetamide (16b): Color: Brown. Yield: 21%. M.p.: 165- 166 °C. Rf: 0.60 inPetroleum ether: Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.59 (s, 1H, NH), 7.91 (d, 1H, H- 5, J = 6.4 Hz), 7.57 (t, 1H, H-7, J = 6.2 Hz), 7.26 (m, 2H, H-6, H-8), 7.01 (d, 1H, H-7', J = 7.8 Hz), 6.98 (s, 1H, H-4'), 6.75 (d, 1H, H-6', J = 7.6 Hz), 5.60 (s, 1H, H-3), 4.82 (s, 2H, H-4a), 3.54 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 168.2 (C-4b, C-4), 162.0 (C-2), 152.3 (C-8a'), 146.2 (C-2'), 136.4 (C-3a'), 133.5 (C-7a'), 132.7 (C-5'), 128.28 (C-7), 125.4 (C-6), 123.0 (C-5), 120.8 (C-6'), 116.19 (C-4a', C-8), 113.8 (C-4', C-7'), 87.3 (C-3), 64.3 (C-4a), 21.1 (CH3). FT-IR (KBr, ν, cm-1): 3355 (NH str), 3095 (Ar C-H str), 2890 (Al C-H str), 1715 (C=O, lactone str), 1705 (C=O), 1678 (NH bending), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C19H15N3O4 [M+H]+ 350.1096, found 350.1095 [M+H]+. N-(5-Nitro-1H-benzimidazol-2-yl)-2-( (2-oxo-2H-chromen-4- yl)oxy)acetamide (16c): Color: Brown. Yield: 16%. M.p.: 245- 246 °C. Rf: 0.47 in Petroleum ether:Ethyl acetate (5:5).1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.60 (s, 1H, NH), 7.95 (s, 1H, H- 4'), 7.91 (d, 1H, H-5, J = 5.8 Hz), 7.85 (dd, 1H, H-6', J = 8.6 Hz, 1.4Hz), 7.58 (t, 1H, H-7, J = 6.0 Hz), 7.30 (m, 2H, H-6, H-8), 7.17 (d, 1H, H-7', J = 8.2 Hz), 5.60 (s, 1H, H-3), 4.79 (s, 2H, H-4a). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.6 (C-4b, C-4), 162.4 (C- 2), 152.57 (C-8a'), 146.8 (C-2'), 145.5 (C-3a'), 144.2 (C-5'), 142.7 (C-7a'), 128.33 (C-7), 125.46 (C-6), 123.5 (C-5), 122.0 (C- 6'), 116.23 (C-4a', C-8, C-7'), 114.4 (C-4'), 87.8 (C-3), 66.2 (C-4a). FT-IR (KBr, ν, cm-1): 3355 (NH str), 3105 (Ar C-H str), 2905 (Al C-H str), 1720 (C=O, lactone str), 1680 (NH bending), 1050 (C- O str). HRMS (ESI+, m/z) calcd. for C18H12N4O6 [M+H]+ 381.0790, found 381.0788 [M+H]+. N-(5-Chloro-1H-benzimidazol-2-yl)-2-( (2-oxo-2H-chromen- 4-yl)oxy)acetamide (16d): Color: Light green. Yield: 27%. M.p.: 233-234 °C. Rf: 0.50 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 12.59 (s, 1H, NH), 7.97 (d, 1H, H-5, J = 7.8 Hz), 7.71 (t, 1H, H-7, J = 7.8 Hz), 7.58 (m, 2H, H- 6', H-7'), 7.41 (m, 2H, H-6, H-8), 7.32 (d, 1H, H-4', J = 1.74 Hz), 6.02 (s, 1H, H-3), 5.30 (s, 2H, H-4a). 13C NMR (100 MHz, DMSO- d6, δ, ppm): 169.0 (C-4b, C-4), 161.9 (C-2), 152.52 (C-8a'), 146.5 (C-2'), 139.7 (C-7a'), 135.3 (C-3a'), 133.8 (C-5'), 128.3 (C-7), 125.4 (C-6), 123.1 (C-5), 121.6 (C-6'), 116.2 (C-4a', C-8, C-7'), 114.7 (C-4'), 87.5 (C-3), 65.2 (C-4a). FT-IR (KBr, ν, cm-1): 3360 (NH str), 3105 (Ar C-H str), 2900 (Al C-H str), 1715 (C=O, lactone str), 1675 (NH bending), 1052 (C-O str). HRMS (ESI+, m/z) calcd. for C18H12ClN3O4 [M+H]+370.0568, found 370.0598 [M+H]+ and 372.0556 [(M+2)+H]+. N-(5-Methoxy-1H-benzimidazol-2-yl)-2-((2-oxo-2H-chromen -4-yl)oxy)acetamide (16e): Color: Light brown. Yield: 12%. M.p.: 178-179 °C. Rf: 0.58 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 11.69 (s, 1H, NH), 7.89 (d, 1H, H-5, J = 6.2 Hz), 7.54 (t, 1H, H-7, J = 6.0 Hz), 7.23 (m, 2H, H- 6, H-8), 6.97 (d, 1H, H-7', J = 7.2 Hz), 6.90 (s, 1H, H-4'), 6.65 (d, 1H, H-6', J = 7.6 Hz), 5.58 (s, 1H, H-3), 4.76 (s, 2H, H-4a), 3.87 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 168.7 (C-4b, C-4), 162.0 (C-2), 155.7 (C-5'), 152.49 (C-8a'), 146.0 (C-2'), 135.0 (C-3a'), 128.9 (C-7a', C-7), 125.40 (C-6), 123.3 (C-5), 116.19 (C-8, C-7'), 113.7 (C-6'), 108.0 (C-4a'), 105.8 (C-4'), 87.3 (C-3), 64.3 (C-4a), 55.3 (OCH3). FT-IR (KBr, ν, cm-1): 3357 (NH str), 3090 (Ar C-H str), 2892 (Al C-H str), 1718 (C=O, lactone str), 1678 (NH bending), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C19H15N3O5 [M+H]+ 366.1045, found 366.1044 [M+H]+. N-(1H-Benzimidazol-2-yl-)-2-((7-methoxy-2-oxo-2H chromen -4-yl)oxy)acetamide (17a): Color: Green. Yield: 22%. M.p.: 233- 234 °C. Rf: 0.57 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.57 (s, 1H, NH), 7.78 (d, 1H, H-5, J = 6.8 Hz), 6.91 (m, 2H, H-5', H-6'), 6.82 (m, 2H, H-4', H-7'), 6.77 (d, 1H, H-6, J = 7.0 Hz), 6.74 (s, 1H, H-8), 5.35 (s, 1H, H-3), 4.71 (s, 2H, H-4b), 3.87 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.3 (C-4b, C-4), 162.4 (C-2), 159.2 (C-7), 154.50 (C- 8a'), 146.7 (C-2'), 136.6 (C-3a', C-7a'), 123.6 (C-5, C-5', C-6'), 115.8 (C-4', C-7'), 111.0 (C-6), 108.9 (C-4a'), 101 (C-8), 87.5 (C- 3), 65.9 (C-4a), 55.8 (OCH3). FT-IR (KBr, ν, cm-1): 3353 (NH str), 2955 (Ar C-H str), 2895 (Al C-H str), 1715 (C=O lactone str), 1678 (NH bending), 1052 (C-O str). HRMS (ESI+, m/z) calcd. for C19H15N3O5 [M+H]+ 366.1045, found 366.1041 [M+H]+. 2-((7-Methoxy-2-oxo-2H-chromen-4-yl)oxy)-N-(5-methylben zimidazol-2-yl)acetamide (17b): Color: Red brown. Yield: 20%. M.p.: 203-204 °C.Rf: 0.62 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.56 (s, 1H, NH), 7.79 (d, 1H, H-5, J = 6.4 Hz), 7.01 (d, 1H, H-7', J = 7.6 Hz), 6.95 (s, 1H, H- 4'), 6.79 (d, 2H, H-6, J = 6.8 Hz), 6.77 (s, 1H, H-8), 6.73 (d, 1H, H- 6', J = 7.8 Hz), 5.40 (s, 1H, H-3), 4.70 (s, 2H, H-4b), 3.87 (s, 3H, OCH3), 3.54 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 168.0 (C-4b, C-4), 162.1 (C-2), 159.8 (C-7), 153.42 (C-8a'), 146.2 (C-2'), 138.4 (C-3a'), 133.4 (C-7a'), 131.4 (C-5'), 125.3 (C-6'), 123.2 (C-5), 114.8 (C-4', C-7'), 110.3 (C-6), 108.3 (C-4a'), 99.7 (C-8), 87.3 (C-3), 64.3 (C-4a), 55.7 (OCH3), 21.3 (CH3). FT-IR (KBr, ν, cm-1): 3355 (NH str), 2952 (Ar C-H str), 2895 (Al C-H str), 1710 (C=O lactone str), 1700 (C=O), 1675 (NH bending), 1048 (C-O str). HRMS (ESI+, m/z) calcd. for C20H17N3O5 [M+H]+ 380.1202, found 380.1201 [M+H]+. 2-((7-Methoxy-2-oxo-2H-chromen-4-yl)oxy)-N-(5-nitrobenzi midazol-2-yl)acetamide (17c): Color: Yellow brown. Yield: 25%. M.p.: 246-247 °C. Rf: 0.49 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.57 (s, 1H, NH), 7.97 (s, 1H, H-4'), 7.92 (d, 1H, H-5, J = 5.8 Hz), 7.85 (dd, 1H, H-6', J = 8.0 Hz, 1.2 Hz), 7.79 (d, 1H, H-7', J = 7.8 Hz), 7.52 (m, 2H, H-6, H-8), 5.40 (s, 1H, H-3), 4.78 (s, 2H, H-4a), 3.89 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 169.3 (C-4b, C-4), 162.4 (C-2), 160.2 (C-7), 152.5 (C-8a'), 146.7 (C-2'), 145.7 (C-3a'), 144.1 (C- 5'), 142.7 (C-7a'), 123.5 (C-5), 120.6 (C-6'), 113.9 (C-4'), 111.3 (C-6, C-7'), 108.3 (C-4a'), 100.5 (C-8), 87.5 (C-3), 65.9 (C-4a), 55.7 (OCH3). FT-IR (KBr, ν, cm-1): 3360 (NH str), 2950 (Ar C-H str), 2890 (Al C-H str), 1710 (C=O lactone str), 1680 (NH Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 313 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 bending), 1050 (C-O str). HRMS (ESI+, m/z) calcd. forC19H14N4O7 [M+H]+ 411.0896, found 411.0895 [M+H]+. N-(5-Chlorobenzimidazol-2-yl-)-2-((7-methoxy-2-oxo-2H-ch romen-4-yl)oxy)acetamide (17d): Color: Brown. Yield: 20%. M.p.: 267-268 °C. Rf: 0.51in Pet ether: Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.58 (s, 1H, NH), 7.95 (s, 1H, H-4'), 7.85 (d, 1H, H-7', J = 7.8 Hz), 7.75 (dd, 1H, H-6', J = 7.8 Hz, 1.2 Hz), 7.69 (d, 1H, H-5, J = 6.2 Hz), 7.52 (m, 2H, H-6, H-8), 5.39 (s, 1H, H-3), 4.75 (s, 2H, H-4a), 3.87 (s, 3H, OCH3). 13CNMR (100 MHz, DMSO-d6, δ, ppm): 169.1 (C-4b, C-4), 161.4 (C-2), 160.1 (C- 7), 152.51 (C-8a'), 145.7 (C-2'), 140.3 (C-3a'), 134.7 (C-7a', C- 5'), 124.1 (C-6'), 123.6 (C-5), 116.4 (C-7'), 113.8 (C-4'), 111.2 (C- 6), 108.3 (C-4a'), 100.5 (C-8), 87.5 (C-3), 65.0 (C-4a), 55.8 (OCH3). FT-IR (KBr, ν, cm-1): 3358 (NH str), 2955 (Ar C-H str), 2895 (Al C-H str), 1715 (C=O lactone str), 1678 (NH bending), 1048 (C-O str). HRMS (ESI+, m/z)calcd. forC19H14ClN3O5 [M+H]+ 400.0562, found 400.0590 [M+H]+ and 402.0939 [(M+2)+H]+. N-(5-Methoxybenzimidazol-2-yl-)-2-( (7-methoxy-2-oxo-2H- chromen-4-yl)oxy)acetamide (17e): Color: Brown. Yield: 18%. M.p.: 289-290 °C. Rf: 0.60 in Petroleum ether:Ethyl acetate (5:5). 1H NMR (400 MHz, DMSO-d6, δ, ppm): 8.55 (s, 1H, NH), 7.69 (d, 1H, H-5, J = 6.2 Hz), 7.52 (m, 2H, H-6, H-8), 7.29 (d, 1H, H-7', J = 8.0 Hz), 6.90 (s, 1H, H-4'), 6.75 (dd, 1H, H-6', J = 7.6 Hz, 1.2 Hz), 5.39 (s, 1H, H-3), 4.75 (s, 2H, H-4a), 3.82 (s, 6H, OCH3). 13C NMR (100 MHz, DMSO-d6, δ, ppm): 168.3 (C-4b, C-4), 162.0 (C-2), 159.1 (C-7, C-5'), 152.23 (C-8a'), 146.1 (C-2'), 135.3 (C-3a'), 128.9 (C-7a'), 123.6 (C-5), 116.19 (C-7'), 111.2 (C-6, C-6'), 100.8 (C-4', C-8, C-4a'), 87.0 (C-3), 64.2 (C-4a), 55.8 (OCH3). FT-IR (KBr, ν, cm-1): 3353 (NH str), 2945 (Ar C-H str), 2885 (Al C-H str), 1715 (C=O lactone str), 1675 (NH bending), 1050 (C-O str). HRMS (ESI+, m/z) calcd. for C20H17N3O6 [M+H]+ 396.1151, found 396.1150 [M+H]+. 2.3. Docking analysis 2.3.1. Computer and software Specifications of the workstation used for molecular model- ling studies are Intel® CoreTMi7-4558V@280GHz, 2801 MHz, 2 cores, SMBIOS Version 2.7 (Samsung-A740). Schrodinger 2016- 1 LLC (NY-USA) controlled with Maestro 10.5 software [10] was employed for docking studies. Chemical structures of the designed molecules were drawn in ChemBioDraw Ultra-12.0 [12]. Windows 10 and Centos-6.6 operating systems were used for all computational analyses. 2.3.2. Molecular docking and ADME studies All compounds were sketched and cleaned in Maestro using the molecular modeling workspace followed by energy mini- mization in LIGPREP program of Schrodinger software [13] using OPLS_2005 force field [14] at pH = 7.4. Structures of both the protein (1QW4 and 1QW6) with co-crystalline ligand 3AR (N-propyl-L-arginine) were downloaded from the protein data bank [15]. For the docking analysis, protein optimization was done for both proteins that include the addition of hydrogen atoms, deletion of water molecules, completion of bond orders, assignment of hydrogen bonds, and complex minimization using 3AR. Extra precision (XP) docking mode of ‘glide’ was used for docking. The results were analysed on the basis of glide docking. QIKPROP program [16] of Maestro was used for calcu- lating the theoretical absorption, distribution, metabolism, and excretion (ADME) properties of target compounds. 2.4. In vitro evaluations 2.4.1. Nitrite determination assay Splenocytes suspension (100 µL), Roswell Park Memorial Institute (RPMI) media (700 µL) and lipopolysaccharide (LPS, 100 µL) were transferred into each of the test tubes marked as control, test, standard L-NG-nitro arginine methyl ester (L- NAME), and standard aminoguanidine (AG). A 100 µL aliquot of RPMI media, test compound (10 µM concentration), L-NAME and AG was transferred into the respective test tubes. All tubes were incubated at 37 °C for 2 h. Thereafter, the contents in each tube were mixed well by gentle shaking, and a solution of arginine (20 µL; 0.034 g arginine in 10 mL normal saline) was added to each tube. The tubes were incubated in a CO2 chamber for 24 h. Finally, Griess reagent was transferred in each test tube, the contents are mixed well, and the tubes are kept in the dark for 10 min. Each tube was shaken gently to mix the content, and the absorbance (OD) is read at 540 nm taking Griess reagent as blank [9,17,18]. NO inhibitory activity of the test and standard compounds was calculated using the Equation (1): % Inhibition of NO = [(Control OD− Test OD) ÷ Control OD] × 100 (1) 2.4.2. iNOS assay Inducible nitric oxide synthase (iNOS) inhibitory activity of standard as well as test compounds was evaluated through a rat iNOS enzyme-linked immunosorbent assay (ELISA) kit using a 96-well microtiter plate by the method as provided by the kit manufacturer. 2.5. In vivo evaluations 2.5.1. Acute toxicity study Acute toxicity studies were performed for test compounds 14a, 14b, 14d, and 14e by on rats using acute toxic class limit test dose in accordance with guidelines 425 of Organization for Economic Co-operation and Development (OECD 2001) [19]. The control group animals were administered 0.5% sodium carboxymethyl cellulose (SCMC) i.p., whereas the test group animals received the test compounds (300 mg/kg body weight, i.p.). The toxicological effects were assessed in terms of mortality and behavioral changes that occur during 48 h after the administration of compounds. 2.5.2. Anti-inflammatory activity Wistar rats of either sex weighing 200-250 g were used for the evaluation of anti-inflammatory activity of the test and standard compounds using carrageenan-induced rat paw edema model as reported by Salvemini et al. [20]. The rats were randomly divided into control, test-treated, AG-treated, and L- NAME-treated groups, with five rats in each group. SCMC (0.5%, used as vehicle for test and standard compounds) was administered to control group. The four test-treated groups were administered compounds 14a, b, d and e. The AG- and L- NAME-treated groups were administered AG and L-NAME, respectively. Dose of each test and standard compound was 30 mg/kg i.p. After 1h of administration of test or standard compound, carrageenan 1.0% w/v suspension in normal saline was injected in right paw of each rat in each group. The paw volume was measured just before the injection of carrageenan as well as at hourly intervals for 10 h in each animal in all groups with a plethysmometer. Anti-inflammatory activity of each compound was determined from a plot obtained by using reduction in the paw volume as ordinate and time as abscissa with the help of Graphpad prism software [21]. 3. Results and discussion 3.1. Chemistry Target compounds were synthesized by using the methods optimized earlier in our laboratory as well as those reported in the literature [9,11,22]. The structural characterization of all 314 Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 Table 1. Docking scores of the target compounds and the amino acids in iNOS (1QW4), and nNOS (1QW6) involved in interactions with compounds. Compound Docking scores 1QW4/1QW6 Amino acid residues interacting with 1QW4 Amino acid residues interacting with 1QW6 14a -6.37/-3.30 Heme900, Glu371, Tyr367, Ash376, Arg382 Heme900, Arg481, Glu592 14b -6.38/-2.66 Heme900, Glu371, Tyr367, Ash376, Arg382, Arg260 Heme900, Arg603, Glu592 14c -4.41/-3.04 Heme900, Ash376, Tyr367 Asn569, Tyr706, Gln478, Tyr588 14d -6.46/-2.53 Heme900, Glu371, Tyr367, Ash376, Arg382, Arg260 Heme900, Asn569, Gln478, Tyr588 14e -6.15/-1.39 Heme900, Glu371, Arg260, Ash376, Arg382 Heme900, Glu592 15a -4.82/-3.31 Heme900, Glu371, Arg260, Asn348 Heme900, Arg603, Ash597 15b -4.94/-4.00 Heme900, Glu371, Asn115 Heme900, Glu592, Arg603 15c -4.49/-4.14 Heme900, Gln486, Ash376, Tyr367 Tyr588, Ash597, Tyr706 15d -4.76/-4.51 Heme900, Tyr485, Ash376, Tyr367, Glu486 Heme900, Tyr706, Tyr588, Ash597 15e -5.35/-4.32 Heme900, Tyr485, Ash376, Tyr367 Heme900, Glu592, Arg481 16a -5.00/-4.32 Heme900, Glu371, Tyr367, Ash376, Arg382, Arg260 Tyr588, Ash597 16b -4.64/-3.38 Tyr367, Ash376, Arg382 Gln478, Tyr588, Ash597 16c -4.70/-4.63 Heme900, Glu371, Tyr367, Ash376 Heme900, Arg596, Gln478, Ash597 16d -4.85/-4.11 Heme900, Glu371, Tyr367, Ash376, Arg260 Tyr588, Ash597 16e -5.22/-0.92 Heme900, Glu371, Tyr367, Ash376, Arg260 Heme900, Glu592, Arg481, Arg603, Gln478 17a -4.97/-4.09 Heme900, Glu371, Tyr367 Tyr588, Ash597 17b -4.25/-4.28 Heme900, Glu371, Asn348 Tyr588, Ash597, Gln478 17c -4.10/-2.07 Heme900, Tyr485, Glu488, Ash376, Tyr367 Tyr706, Tyr588, Ash597 17d -3.94/-2.66 Tyr367, Ash376, Arg260, Asn348 Heme900, Arg596, Ash597, Tyr588 17e -0.71/-4.42 Tyr367, Tyr485 Tyr588, Ash597, Arg596 AG -4.80/-5.01 Heme900, Glu371, Trp366 Heme900, Glu592 NAME -5.59/-4.65 Heme900, Glu371, Trp366, Ash376, Tyr367 Heme900, Glu592, Trp587, Tyr588, Ash597 3AR -6.86/-5.65 Heme900, Glu371, Ash376, Arg260 Heme900, Glu592, Gln478, Tyr588, Ash597 compounds was carried out using IR, NMR, and mass spectrometry. In 1H NMR spectra of intermediates 5 and 6, all protons of the coumarin ring were detected in the range of δ 5- 8 ppm, the OH protons appeared as singlets at δ 12.31 (5) and 12.28 ppm (6), and the OCH3 protons were detected as a 3- proton singlet at δ 3.85 ppm (6). Intermediates 7 and 8 were synthesized by o-alkylation of compounds 5 and 6, respectively in the presence of a base. Initial experiments used cesium carbonate as a basic catalyst [22], but the yields were poor. A second base, K2CO3, and solvents (Acetonitrile, acetone, and DMF) were used to optimize the reaction [23]. At the end, DMF as a solvent and potassium carbonate as a catalyst produced good yields. In 1H NMR analyses, aromatic protons were observed in a range of δ 7.79-5.73 ppm, while aliphatic protons were observed in a range of δ 4.30-2.46 ppm. Protons in compound 8 were slightly upfield than those in compound 7. This is the result of the presence of an electron-releasing group (-OCH3) on the 7th position of the coumarin nucleus. Both inter- mediates were also analysed by mass spectrometry. Through proton NMR spectroscopy, aromatic protons were observed in the range of δ 7.91-5.58 and 7.79-5.44 ppm, respectively, in intermediates 9 and 10. The aliphatic protons of compounds 9 and 10 appear in the range of δ 4.31-1.32 ppm. A peak of the OH group was observed in intermediates 11 and 12 (δ 13.29 and 13.20 ppm, respectively). Benzimidazole intermediates (13a-e) were synthesized by reacting different 4-(un)substituted phenylenediamines with CNBr in the presence of aq. methanol. The aromatic protons of all these intermediates were noted at the δ values as reported in literature [24]. The chemical shifts were observed upfield or downfield according to the electronic nature of the functional group present at 5th position of benzimidazole nucleus in compounds 13a-e. To synthesize the target compounds 14a-e and 15a-e intermediates 7 or 8, and 13a-e were coupled in the presence of K2CO3. IR, NMR (1H NMR and 13C NMR), and HRMS spectral analyses were performed. IR spectra showed a signal of NH in the range of 3300-3478 cm-1 in all the target compounds. Appearance of a single NH signal confirmed the coupling of compounds 13a-e intermediates with coumarin intermediates (7 and 8). 1H NMR spectra of the target compounds showed a singlet of one proton of NH in the range of δ 5.98-7.00 ppm for the target compounds 14a-e and 15a-e. Observed 13C NMR signals of these target compounds were in the predicted ranges. HRMS analysis showed that the mass of all target compounds did not deviate by more than 0.005 Da. In order to synthesize compounds 16a-e and 17a-e (series with amide linkage), intermediates 11/12 were used. In situ conversion of these intermediates into the corresponding acid, chloride was performed by dissolving them in THF and adding an excess of SOCl2. Thereafter, a solution of compounds 13a-e in THF was added to the reaction mixture containing the acid chloride to form the corresponding amidic compound. In IR spectra of these compounds, amide stretch is observed above 3350 cm-1 for all the target compounds. Protons and carbons of the compounds were detected at δ values almost similar to the δ values of corresponding compounds 14a-e and 15a-e. Masses of these compounds were also found to deviate by not more than 0.005 Da during HRMS analysis. These spectral data confir- med that all target compounds are synthesized as designed. 3.2. Docking analysis The docking scores of all the target compounds and the amino acid residues with which they are interacting in the active sites of both iNOS (1QW4) and neuronal nitric oxide synthase (nNOS) (1QW6) are summarized in Table 1. In these studies, it was discovered that all designed compounds might act as selective anti-iNOS agents. According to literature reports on selective iNOS inhibitors, ligand H- bonding with Glu371 or Glu592 determines the ligand’s selec- tivity toward iNOS or nNOS [25]. Interactions with synthesized compounds (docking score > -6 kcal/mol, Figure 3) indicate that their 2-aminobenzimidazole moiety occupies a pocket lined by Phe363, Asn364, Gly365, Trp366, Tyr367, and Met368, and interacts with Glu371 via a hydrogen bond and Heme900 via a co-ordinate bond. 3AR exhibited a docking score of -6.868 and -5.652 kcal/mol for 1QW4 and 1QW6, respectively, with RMSD (Root mean square deviation) < 1 Å. This observation suggested that both the target proteins are validated and suitable for predic- ting the binding affinity of test compounds. All compounds, except compounds 17d and 17e, exhibited docking scores equivalent to or greater than that of AG and L-NAME for 1QW4. Conversely, docking scores with 1QW6 were significantly lower than that of AG and L-NAME. Coumarin nucleus in all test compounds occupies the other pocket(s) in binding site and affects the binding pattern, which may be responsible for the lower docking scores of all test compounds vis-a-vis 3AR. Nevertheless, the coumarin nucleus incurs hydrophobicity to the compounds, which is conducive to better oral absorption. This statement is in agreement with ADME properties of the compounds predicted by the QIKPROP module (Table 2). In addition to this, free-radical scavenging activity of coumarin nucleus is predicted to enhance thera- peutic potential of the compounds [7,26]. Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 315 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 Table 2. ADME properties * of 3AR, AG, NAME, and target compounds 14a-e, 15a-e,16a-e and 17a-e. Compound Mol wt Donor, HB Acceptor, HB QP, Po/w QP, Logs QPP, Caco QPlog, BB QPPMDCK % Oral Abs. 14a 335.36 2 5.25 3.26 -4.88 550.49 -1.164 259.49 95.12 14b 349.38 2 5.25 3.60 -5.55 549.22 -1.225 258.85 100.00 14c 380.35 2 6.25 2.60 -5.14 68.78 -2.311 27.40 75.04 14d 369.80 2 5.25 3.76 -5.65 573.69 -1.010 667.01 100.00 14e 365.38 2 6.00 3.41 -5.28 549.60 -1.287 259.04 96.34 15a 365.38 2 6.00 3.36 -5.11 550.05 -1.258 259.27 96.91 15b 379.41 2 6.00 3.70 -5.79 548.85 -1.318 258.66 100.00 15c 410.38 2 7.00 2.73 -5.45 68.72 -2.448 27.38 75.78 15d 399.83 2 6.00 3.85 -5.72 641.06 -1.021 752.09 100.00 15e 395.41 2 6.75 3.50 -5.42 584.68 -1.336 276.96 96.81 16a 335.31 0 4.25 2.78 -3.67 183.76 -1.366 79.26 84.63 16b 349.34 0 4.25 3.45 -4.89 184.64 -1.502 79.68 86.07 16c 380.31 1 7.25 1.70 -4.45 31.49 -2.450 11.77 63.72 16d 369.76 0 4.25 3.64 -5.08 184.42 -1.323 196.22 87.06 16e 365.34 0 5.00 2.83 -3.80 183.69 -1.476 79.23 84.43 17a 365.34 0 5.00 2.78 -3.56 185.17 -1.414 79.92 84.80 17b 379.37 0 5.00 3.13 -4.33 183.35 -1.518 79.07 86.58 17c 410.34 1 8.00 1.77 -4.63 31.46 -2.571 11.76 51.34 17d 399.79 0 5.00 3.64 -5.04 183.76 -1.405 187.05 87.63 17e 395.37 0 5.75 2.88 -3.89 183.71 -1.573 79.24 85.40 3AR 201.26 4 4.00 1.29 -2.41 37.37 -1.80 18.02 62.67 AG 74.08 6 3.00 -2.64 1.92 16.56 -0.87 6.50 20.30 NAME 233.22 5 7.50 -1.92 -0.35 2.53 -2.40 0.85 22.90 * MW (Molecular weight in Da); Donor HB (hydrogen bond that can be donated, 0.0 to 6.0); Acceptor HB (hydrogen bond that can be accepted, 2.0 to 20.0); QPLogPo/w (Predicted octanol/water partition coefficient, –2.0 to 6.5); QPLogS (Predicted aqueous solubility, log S, S in mol/dm3, -6.5 to -0.5); QPPCaco (Predicted apparent Caco-2 cell permeability, nm/sec, <25 poor and >500 good); QPLogBB (Predicted brain/blood partition coefficient,–3.0 to –1.2); QPPMDCK (Predicted apparent Madin-Darby canine kidney (MDCK) cell permeability, nm/sec, <25 poor and >500 great); %HOA (Predicted human oral absorption on 0 to 100% scale). (a) (b) (c) (d) Figure 3. Interactions of compounds 14a (a), 14b (b), 14d (c) and 14e (d) with iNOS. 3.3. In vitro evaluations 3.3.1. Nitrite assay All test compounds significantly inhibited the LPS-induced NO production. Compounds 14a, b, d, and e possessed docking score almost equivalent to 3AR (Table 3), and also showed NO inhibition in the range of 70.33-78.62%. Among all the test compounds, compounds14d (78.62% inhibition) and 14e (73.74% inhibition) are maximally active. It is even more active than L-NAME (68.27%), and almost equipotent to AG (81.41%). 316 Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 Table 3. NO and iNOS inhibition of target compounds *. Compound % Inhibition of NO radical (10 µM conc.) % Inhibition of iNOS (10 µM conc.) 14a 70.33±0.63a 67.23±0.34a,b 14b 71.17±0.96a,b 63.10±3.32a,b 14c 52.30±0.86a,b 40.42±0.40a,b 14d 78.62±0.91a,b 77.19±1.99a,b 14e 73.74±0.74a,b 73.97±0.24a,b 15a 62.58±0.69a,b 44.10±1.16a,b 15b 63.65±0.97a,b 44.06±0.19a,b 15c 55.25±0.86a,b 38.94±0.41a,b 15d 61.41±0.73a,b 43.97±0.24a,b 15e 67.26±0.81a 59.97±1.09a,b 16a 67.48±0.72a 54.11±2.15a,b 16b 57.61±0.89a,b 48.69±1.18a,b 16c 60.28±0.92a 41.52±0.31a,b 16d 61.49±0.90a,b 47.28±0.43a,b 16e 65.75±0.68a 61.47±0.43a,b 17a 63.62±0.99a,b 58.93±0.37a,b 17b 55.20±0.99a,b 50.56±0.37a,b 17c 50.79±0.97a,b 42.04±0.45a,b 17d 50.98±0.95a,b 41.21±0.37a,b 17e 31.83±0.96a,b 28.49±0.47a,b AG 81.41±0.93b 91.81±0.28b NAME 40.17±0.84a 83.26±0.62a * One-way ANOVA (Analysis of variance) followed by Tukey test were used for statistical analysis of the data. a,b Values are statistically different from AG and NAME, respectively, at p<0.05. Figure 4. Paw volume is expressed as mean±S.E.M. from five rats and analysed by two-way ANOVA followed by Bonferroni’s test. 3.3.2. iNOS assay All test compounds were found to inhibit iNOS by 28.49 to 77.19%. However, the compounds are less active than both AG and NAME (91.82 and 87.58%, respectively) (Table 3). Compounds 14a, b, d, and e possessed the maximum docking score, and inhibited the NO production maximally, and also exhibited good iNOS inhibition. The maximum iNOS inhibitory activity was exhibited by compound 14d (77.19% inhibition). These findings of biological activity evaluation are in concordance with the docking analysis of the compounds, indicating that 1QW4 can be used as a dependable target for the prediction of both NO production and iNOS inhibitory activities of new compounds. 3.4. In vivo evaluation Based on the research studies reported in literature, in vivo activities of AG, NAME as well as test compounds 14a, b, d and e were evaluated at a dose of 30 mg/kg. 3.4.1. Acute toxicity evaluation The maximum inhibitory activity against NO production as well as iNOS was exhibited by compounds 14a, 14b, 14d and 14e. Hence, these were selected for evaluating in vivo activity. None of the selected test compounds showed any toxic effect in any animal at a dose level of 300 mg/kg. No mortality, no weight loss, and no abnormal behaviour were seen in any treated animal during 48 h of the study. 3.4.2. Anti-inflammatory activity Inflammation response is usually divided into two phases, i.e., early and delayed phases. The early phase inflammation can be attenuated by a non-selective iNOS inhibitor, and the delayed phase inflammation is characterized by an increased expres- sion of iNOS. Therefore, a compound capable of inhibiting iNOS selectively would intervene in the delayed phase of inflame- mation [20]. All tested compounds were found to reduce the paw volume to significant levels and proved themselves as good anti-inflammatory agents. AG being a selective iNOS inhibitor starts inhibiting the inflammation in the late stage of inflamma- tion whereas L-NAME which is a non-selective iNOS inhibitor starts suppressing the inflammation from an early-stage (up to 12.16% inhibition in initial 4 h). All tested compounds showed marked reduction in paw volume after 4 h of carrageenan administration along with some reduction in the early phase also (Figure 4). Results of in vivo activity indicated that although these compounds are more potent inhibitors of iNOS than the reference drugs, but these may act as anti-inflammatory agents via acting on some other mechanisms also since they are showing inhibition in the early phase. Amongst all the test Minhas and Bansal / European Journal of Chemistry 13 (3) (2022) 307-318 317 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.3.307-318.2282 compounds, compounds 14d and 14e were found to be the most potent rat paw edema inhibitors, and act as potential anti- inflammatory agents. 4. Conclusion Compounds 14a-e, 15a-e, 16a-e, and 17a-e were synthe- sized by coupling of 5-(un)substituted-2-aminobenzimidazole with 7-(un)substituted coumarin through an alkoxy and amide linker (at 4th position of the coumarin nucleus). Docking analysis indicated that majority of the synthesized compounds are selective inhibitors of iNOS. The test compounds exhibited in vitro NO production and iNOS inhibitory activities complying with the findings of docking analysis. All compounds are found to be non-toxic. Compounds 14d and 14e were identified as the most potent inhibitors of NO production and iNOS enzyme. The reason for this may be their affinity for the iNOS enzyme, which explains their maximum docking score as well. The in vivo anti- inflammatory activity of the tested compounds indicated that all compounds possessed marked reduction in the late phase of inflammation along with some reduction in the initial phase also. In carrageenan-induced rat paw edema model compounds 14d and e were found to be the potent anti-inflammatory agents. These findings support the hypothesis that the benzimi- dazole-coumarin hybrids can be potential drug candidates for multifactorial inflammatory disorders. Using structural activity relationships of these compounds, we have observed that the most potent iNOS inhibitors are found in the series in which the coumarin nucleus is unsubstituted. Additionally, five atoms separate two heteronuclear moieties. There is no relationship between the biological activity of the synthesized compounds and the electronic nature of the descriptor on the 5th position of the benzimidazole nucleus. Acknowledgements Ms. Richa Minhas would like to acknowledge Department of Science and Technology, Ministry of Science and Technology, India for providing financial support to carry out this research work under the INSPIRE fellowship program (IF170459). Authors are also thankful to Dr. Raj Kumar, Professor, Central University of Punjab, Bathinda (India), for providing his expert guidance on in silico studies. Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. This study was approved (Reference No. 107/GO/ReBi/S/99/CPCSEA/2019-7) by the institutional animal ethics committee of Punjabi University, Patiala (India). Human and animal rights: No human subjects were used in this study. All the animal procedures were in accordance with the CPCSEA guidelines. Sample availability: Samples of the compounds are available from the authors. CRediT authorship contribution statement Conceptualization: Richa Minhas, Yogita Bansal; Methodology: Richa Minhas, Yogita Bansal; Software: Raj Kumar; Validation: Richa Minhas; Formal Analysis: Richa Minhas; Investigation: Yogita Bansal; Resources: Punjabi University, Patiala, Department of Science and Technology (DST); Data Curation: Richa Minhas, Yogita Bansal; Writing - Original Draft: Richa Minhas; Writing - Review and Editing: Richa Minhas, Dr. Yogita Bansal; Visualization: Richa Minhas, Yogita Bansal; Funding acquisition: Richa Minhas; Supervision: Yogita Bansal; Project Administration: Yogita Bansal. Funding Department of Science & Technology, Ministry of Science and Technology, India http://dx.doi.org/10.13039/501100001409 ORCID and Email Richa Minhas richaminhas14@gmail.com https://orcid.org/0000-0001-9083-5295 Yogita Bansal yogitabansal101@gmail.com yogita_pharma@pbi.ac.in https://orcid.org/0000-0001-8894-066X References [1]. Kahlenberg, J. M.; Fox, D. A. Advances in the medical treatment of rheumatoid arthritis. Hand Clin. 2011, 27, 11–20. [2]. Handa, R.; Rao, U. R. K.; Lewis, J. F. M.; Rambhad, G.; Shiff, S.; Ghia, C. J. Literature review of rheumatoid arthritis in India. Int. J. Rheum. Dis. 2016, 19, 440–451. [3]. Upadhyay, K.; Bavishi, A.; Thakrar, S.; Radadiya, A.; Vala, H.; Parekh, S.; Bhavsar, D.; Savant, M.; Parmar, M.; Adlakha, P.; Shah, A. Synthesis and biological evaluation of 4-styrylcoumarin derivatives as inhibitors of TNF-α and IL-6 with anti-tubercular activity. Bioorg. Med. Chem. Lett. 2011, 21, 2547–2549. [4]. Abdelazeem, A. H.; Khan, S. I.; White, S. 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The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Instrumentation 2.2. Chemistry 2.2.1. Synthesis of 1-(2,4-dihydroxyphenyl)ethan-1-one (2) 2.2.2 Synthesis of 2-hydroxy-4-methoxyacetophenone (4) 2.2.3. Synthesis of 4-hydroxycoumarin (5) and 4-hydroxy-7-methoxycoumarin (6) 2.2.4. Synthesis of 4-(3-bromopropoxy) coumarin (7) and 4-(3-bromopropoxy)-7methoxycoumarin (8) 2.2.5. Synthesis of ethyl-2-((2-oxo-2H-chromen-4-yl)oxy) acetate (9) and ethyl 2-((7-methoxy-2-oxo-2H-chromen-4-yl)oxy)acetate (10) 2.2.6. 2-((2-Oxo-2H-chromen-4-yl)oxy)acetic acid (11) and 2-((7-methoxy-2-oxo-2H-chromen-4-yl)oxy)acetic acid (12) 2.2.7. Synthesis of 2-aminobenzimidazole intermediates (13a-e) 2.2.8. Synthesis of target molecules (14a-e and 15a-e) 2.2.9. Synthesis of target molecules (16a-e and 17a-e) 2.3. Docking analysis 2.3.1. Computer and software 2.3.2. Molecular docking and ADME studies 2.4. In vitro evaluations 2.4.1. Nitrite determination assay 2.4.2. iNOS assay 2.5. In vivo evaluations 2.5.1. Acute toxicity study 2.5.2. Anti-inflammatory activity 3. Results and discussion 3.1. Chemistry 3.2. Docking analysis 3.3. In vitro evaluations 3.3.1. Nitrite assay 3.3.2. iNOS assay 3.4. In vivo evaluation 3.4.1. Acute toxicity evaluation 3.4.2. Anti-inflammatory activity 4. Conclusion Disclosure statement CRediT authorship contribution statement Funding ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField111: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: PrintField211: