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. 2021 Aug 26;6(35):22726–22739. doi: 10.1021/acsomega.1c03056

Substituted Benzimidazole Analogues as Potential α-Amylase Inhibitors and Radical Scavengers

Akinsola Adegboye Akande †,§, Uzma Salar ‡, Khalid Mohammed Khan †,∥,*, Shazia Syed †, Sherifat Adeyinka Aboaba §, Sridevi Chigurupati ⊥, Abdul Wadood #, Muhammad Riaz #, Muhammad Taha ∥, Saurabh Bhatia ∇, Kanwal †, Shahbaz Shamim †, Shahnaz Perveen ○
PMCID: PMC8427641  PMID: 34514244

Abstract

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Benzimidazole scaffolds are known to have a diverse range of biological activities and found to be antidiabetic and antioxidant. In this study, a variety of arylated benzimidazoles 1–31 were synthesized. Except for compounds 1, 6, 7, and 8, all are new derivatives. All compounds were screened for α-amylase inhibitory, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. In vitro screening results revealed that all molecules demonstrated significant α-amylase inhibition with IC50 values of 1.86 ± 0.08 to 3.16 ± 0.31 μM as compared to standard acarbose (IC50 = 1.46 ± 0.26 μM). However, compounds showed significant ABTS and DPPH radical scavenging potentials with IC50 values in the range of 1.37 ± 0.21 to 4.00 ± 0.10 μM for ABTS and 1.36 ± 0.09 to 3.60 ± 0.20 μM for DPPH radical scavenging activities when compared to ascorbic acid with IC50 values of 0.72 ± 0.21 and 0.73 ± 0.05 μM for ABTS and DPPH radical scavenging potentials, respectively. Structure–activity relationship (SAR) was established after critical analysis of varying substitution effects on α-amylase inhibitory and radical scavenging (ABTS and DPPH) potentials. However, molecular docking was also performed to figure out the active participation of different groups of synthetic molecules during binding with the active pocket of the α-amylase enzyme.

Introduction

Diabetes mellitus is associated with a group of metabolic disorders, mainly hyperglycemia, which is characterized by an elevated level of blood glucose, caused by a fail in insulin secretion, its action, or both.1−3 Diabetes is developed through various pathogenic processes including destruction of the pancreatic ß-cells, which consequently leads to deficiency of insulin, resulting in abnormality of insulin action and secretion. Chronic hyperglycemia leads to the dysfunction, damage, and failure of different organs, including eyes, kidneys, nerves, blood vessels, and the heart. The major symptoms of hyperglycemia include weight loss, polyuria, polyphagia, and blurred vision. However, prolonged complications of diabetes include retinopathy, nephropathy, neuropathy, gastrointestinal, genitourinary, cardiovascular symptoms, and sexual dysfunction. Diabetes is categorized as type-I and type-II. Type-I diabetes is triggered by insufficiency of insulin secretion, while type-II diabetes, the most prevalent type of diabetes, is caused due to a combination of insulin resistance and its action.2,4 Type-I and type-II diabetes have similar physiopathological properties but differ in etiology. There are 430 million type-II diabetic patients worldwide, and this number is expected to reach 630 million by 2045.5,6 According to the WHO, deaths from diabetes have increased by 70% globally between the years 2000 and 2019. There has also been an 80% rise in deaths among males. However, in the Eastern Mediterranean, deaths from diabetes have even more than doubled and represent the greatest percentage increase among all WHO regions.7

Diabetes can be treated by the inhibition of α-amylase and α-glucosidase enzymes, involved in carbohydrate digestion. α-Amylase (E.C. 3.2.1.1) is a digestive enzyme (a calcium-ion metalloenzyme), often secreted in the pancreas and salivary glands. It catalyzes the hydrolysis of α-1,4-glycosidic linkages of starch, glycogen, amylose, and amylopectin.8−13 Few pharmacologically active flavonoids, phenolic compounds, and some heterocycles have been identified as α-amylase inhibitors. Since the past few years, our group has been working on the evaluation of various classes of organic compounds for their potential α-amylase inhibitory (α-AMY) activity.14−18

Antioxidants are the chemical entities that trap a free radical or reactive oxygen species (ROS) by contributing an electron to them and changing them into harmless substances. In short, antioxidants are agents that protect the body from oxidative stress by inhibiting the process of oxidation or slowing down the oxidative damage.19−22 Oxidative stress is also a risk factor for several disorders such as diabetes, asthma, inflammation, arthropathies, and Alzheimer’s and Parkinson’s diseases.23−25 Autoxidation and protein glycosylation of glucose are also caused by the excess production of free radicals and other ROS, which lead to oxidative damage to many tissues.15 These reactive oxygen species are commonly associated with diabetic complications.26

Benzimidazole is well known for its promising pharmacological and biological activities such as antidiabetic, antimicrobial, antiviral, antitumor, antidepressant, antihistaminic, antioxidant, antihypertensive, anti-HIV, anticoagulant, antiallergic, and antihelmentic activities.27−35 A range of pharmaceutically important marketed drugs are based on the benzimidazole scaffold (Figure 1). Anthelmintic drugs including albendazole, mebendazole, triclabendazole, etc. possess a benzimidazole nucleus. Many commercial fungicide structures are based on benzimidazole. Furthermore, lansoprazole, omeprazole, rabeprazole, pantoprazole, and tenatoprazole are proton-pump inhibitors (antacids), and all contain a benzimidazole scaffold. Other pharmaceutical drugs such as galeterone, mavatrep, dovitinib, and etonitazene belong to the benzimidazole class. Benzimidazole is a priviliged nucleus that is among the topmost ring systems used in small-molecule drugs listed by the US FDA.36,37

Figure 1.

Figure 1

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Commonly marketed drugs based on the benzimidazole scaffold.

Benzimidazole derivatives are well known as antidiabetic agents after studying different mechanisms such as glycosidase receptor, activated receptor α transcriptional activity, peroxisome proliferator, glucokinase, human glucagon receptor (hGCGR) antagonist, dipeptidyl peptidase IV, aldose reductase enzyme, and stearoyl-CoA desaturase.38 Previously, we have reported benzimidazole-based derivatives as potent α-glucosidase and α-amylase inhibitors (Figure 2);1,39,40 however, there is still need to further explore this class to identify potential molecules for future advanced research. In the current study, we synthesized new arylated benzimidazoles by incorporating furan, pyrene, anthracene, and benzyloxy moieties to identify new α-amylase inhibitors having the ability to scavenge 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals (Figure 1). Furthermore, our intention was to synthesize small molecules that were easy to synthesize, stable enough, and somehow follow Lipinski’s rule of five.

Figure 2.

Figure 2

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Already identified α-glucosidase and α-amylase inhibitors based on the benzimidazole nucleus and currently synthesized molecules as new α-amylase inhibitors, DPPH, and ABTS radical scavengers.

Results and Discussion

Chemistry

A variety of substituted o-phenylene diamines were reacted with aromatic and pseudoaromatic aldehydes to afford the diversely substituted benzimidazoles (Table 1). All reactions were carried out in N,N-dimethylformamide (DMF) in the presence of sodium metabisulfite (Na2S2O5) as a catalyst (Scheme 1). o-Phenylene diamine condensed with aldehydes under oxidative conditions, in which Na2S2O5 acts as a reducing agent. Na2S2O5 first reacts with aldehyde by donating electrons to form a metabisulfite adduct of aldehyde. Then, o-phenylene diamine readily condenses with this reactive adduct to form 2-substituted benzimidazole. Use of metabisulfite has several advantages, including short reaction time, simple workup, easy purification of products, and mild reaction conditions.41,42 Structures of synthetic compounds were identified by spectroscopic techniques such as UV, IR, electron ionization mass spectroscopy (EI-MS), high-resolution electron impact mass spectrometry (HREI-MS), 1H NMR, and 13C NMR. Structures of compounds 1 and 6–843−45 are already known, while the rest are new.

Table 1. In Vitro α-Amylase Inhibitory and Antioxidant (ABTS and DPPH) Activities of Arylated Benzimidazoles 1–31.

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a

Standard error mean (SEM).

b

Acarbose (standard inhibitor for α-amylase enzyme).

c

Ascorbic acid (standard for ABTS and DPPH radical scavenging activities).

Scheme 1. Synthesis of Arylated Benzimidazoles 1–31.

Scheme 1

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α-Amylase Inhibitory and Antioxidant (ABTS and DPPH) Activities In Vitro

Synthetic compounds 1–31 were subjected to in vitro α-amylase inhibitory and antioxidant (ABTS and DPPH) activities. All compounds demonstrated α-amylase inhibitory activity in the range of IC50 = 1.86 ± 0.08 to 3.16 ± 0.31 μM and ABTS and DPPH radical scavenging potentials in the ranges of IC50 =1.37 ± 0.21 to 4.00 ± 0.10 and 1.36 ± 0.09–3.60 ± 0.20 μM, respectively. Results were compared to the standard inhibitors acarbose with IC50 = 1.46 ± 0.26 μM for α-amylase inhibition and ascorbic acid with IC50 = 0.72 ± 0.21 and 0.73 ± 0.05 μM for ABTS and DPPH radical scavenging activities,2,19,39 respectively. Derivatives 3, 4, and 9 with IC50 values of 1.91 ± 0.02, 1.89 ± 0.25, and 1.86 ± 0.08 μM against the α-amylase enzyme; 1.40 ± 0.08, 1.38 ± 0.05, and 1.37 ± 0.21 μM for ABTS; and 1.40 ± 0.30, 1.39 ± 0.12, and 1.36 ± 0.09 μM for DPPH, respectively, were identified to be significantly active, while the rest of the analogues showed moderate α-amylase inhibition and antioxidant (ABTS and DPPH) potential (Table 1).

Structure–Activity Relationship (SAR) and In Silico Studies

The structure–activity relationship (SAR) was established by inspecting the influence of varying groups (R1 and R2) on α-amylase inhibitory (α-AMY) and ABTS and DPPH radical scavenging activities (Table 1). However, an in silico study was conducted on all compounds 1–31. The binding energies of the derived ligands (compounds) and protein (α-amylase) were calculated and are given in Table 2.

Table 2. Binding Energy Details of Compounds 1–31 in the Active Site of the Enzyme.

compounds binding energy (kcal/mol) compounds binding energy (kcal/mol)
1 –5.23 17 –5.90
2 –5.44 18 –7.61
3 –5.49 19 –5.74
4 –5.90 20 –7.92
5 –6.27 21 –5.30
6 –5.49 22 –6.54
7 –5.10 23 –7.62
8 –5.84 24 –6.50
9 –5.07 25 –6.94
10 –5.45 26 –6.09
11 –7.04 27 –6.46
12 –6.32 28 –6.05
13 –6.62 29 –5.39
14 –7.22 30 –6.62
15 –6.19 31 –5.97
16 –5.79    
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Compounds 1–10 having a furan and a methylated furan ring (R4) established different interactions with the important residues, including Tyr151, Leu162, Arg195, Asp197, Ala198, Lys200, Glu233, and Ile235, of the active site of the target enzyme, shown by in silico inhibition studies. The brief interaction detail is given in Table S1 (Supporting Information). Among compounds 1–10, methylated furan containing derivative 9 with 5-fluoro substitution was identified as the most active α-amylase inhibitor (IC50 = 1.86 ± 0.08 μM) and ABTS (IC50 = 1.37 ± 0.21 μM) and DPPH (IC50 = 1.36 ± 0.09 μM) radical scavenger. Its good activity might be due to participation of both methyl and fluoro groups. This analogue also showed two important interactions with the residues Lys200 and Tyr151, revealed by in silico studies (Figure 3). Besides, the bond energy of compound 9 (−5.07) also shows it as a strong inhibitor than other structurally close analogues. Participation of the methyl group on furan can be assessed by comparing the activities of compound 9 with its nonmethylated derivative 3, showing slightly decreased α-amylase inhibitory (IC50 = 1.91 ± 0.02 μM), ABTS (IC50 = 1.40 ± 0.08 μM), and DPPH (IC50 = 1.40 ± 0.30 μM) scavenging activities. An in silico study showed three interactions made by compound 3 with active site residues including ASP197, GLU233, and ALA198 (Table S1). The bond energy of compound 3 revealed somehow less in silico inhibition than its analogue, i.e., compound 9. As far as antioxidant activity is concerned, the absence of methyl on furan also makes its comparatively less susceptible to scavenging and stabilizing electron-deficient DPPH and ABTS radicals.

Figure 3.

Figure 3

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Compound 9, the methylfuran substituted analogue, showing two important interactions with the residues Lys200 and Tyr151. Overlay conformation of compound 9 (IC50 of 1.86 μM for α-amylase inhibition) and acarbose, the reference ligand, in the binding pocket. Blue, native cocrystallized acarbose; green, docked compound 9.

Comparison of 5-chloro containing methylated and nonmethylated derivatives 10 (α-AMY IC50 = 2.10 ± 0.09; ABTS IC50 = 1.46 ± 0.03; DPPH IC50 = 1.46 ± 0.10 μM) and 4 (α-AMY IC50 = 1.89 ± 0.25; ABTS IC50 = 1.38 ± 0.05; DPPH IC50 = 1.39 ± 0.12 μM) revealed that the nonmethylated compound 4 was more active than the methylated one 10. It means that the methyl group does not participate in the presence of the chloro group. Similarly, the bond energy of compound 10 revealed rather low in silico inhibition compared to its analogue 4. Further, comparison of compounds 10 and 4 with 9 and 3 revealed that the fluoro group is more important for the activities than the chloro group. The fluoro group, being more electronegative and small in size, can interact more strongly than the chloro group, which is comparatively less electronegative and bigger in size. In addition, fluoro substitution can scavenge radicals more effectively than chloro substitution. IC50 values of derivatives 9 and 3 can be compared with compounds 1 (α-AMY IC50 = 2.08 ± 0.19; ABTS IC50 = 1.42 ± 0.10; DPPH IC50 = 1.40 ± 0.09 μM) and 7 (α-AMY IC50 = 2.07 ± 0.09; ABTS IC50 = 1.42 ± 0.04; DPPH IC50 = 1.43 ± 0.08 μM) lacking fluoro groups at position-5. This activity comparison showed a decreased α-amylase inhibitory and ABTS and DPPH radical scavenging activities, which further confirms the active participation of the fluoro group in the inhibitory and scavenging potentials.

Likewise, 5,6-dimethyl substituted derivatives 2 and 8 [(α-AMY IC50 = 2.11 ± 0.10; 2.14 ± 0.10 μM), (ABTS IC50 = 1.52 ± 0.10; 1.55 ± 0.20 μM), (DPPH IC50 = 1.50 ± 0.09; 1.54 ± 0.07 μM)] showed close activities, which reveals that the methyl present at the furan ring does not really affect the inhibitory and scavenging properties of both compounds. In addition, the smaller inhibitory and radical scavenging potentials of compounds 2 and 8 compared to compounds 3, 4, 9, and 10 might be due to the steric hindrance created by methyl groups while binding with the active site of the enzyme, as well as the smaller susceptibility of the electron-donating methyl itself to scavenge free radicals than electronegative fluoro and chloro groups. Furthermore, activities of compound 1 compared with compound 5 (α-AMY IC50 = 2.12 ± 1.33; ABTS IC50 = 1.59 ± 0.20; DPPH IC50 = 1.59 ± 0.10 μM) bearing 5-nitro substitution and 6 (α-AMY IC50 = 2.13 ± 0.14 μM; ABTS IC50 = 1.62 ± 0.06; DPPH IC50 = 1.61 ± 0.07 μM) having a phenyl ring as R3. Both compounds showed almost similar α-amylase inhibitory and radical scavenging activities, which showed that these groups are not actively participating in inhibiting the enzyme and scavenging the reactive species. An in silico study revealed that as a whole electron-rich structural motifs like halogen, NH, and π-electron system of aromatic rings in compounds 1–10 were observed to be very responsible for the good interaction mode as well as the active nature of the molecules. All of these electron-rich groups were observed to report almost similar/close charisma regarding interactions with active site residues. In addition, electron-rich moieties also showed good radical scavenging potential.

Compounds 11–18 had pyrene and anthracene rings at position-2 (R4). Anthracene-bearing compounds 15–18 showed slightly decreased activities than pyrene-containing molecules 11–14. The moderate α-amylase inhibitory activities revealed by compounds 11–18 might be due to bulky fused ring structures, which may create steric hindrance during binding with the active pocket of the enzyme. Nonetheless, a molecular docking study reveals that the pyrene and anthracene group substitutions in this group of compounds showed a comparatively similar behavior in the binding pocket of the enzyme regarding interactions (Table S1). Compound 15, an anthracene bearing analogue of compound 11, formed a π–H bond like compound 11, making with the active site residues Ile235. Thus, the pyrene and anthracene group substitutions in these two analogues offered almost the same behavior regarding interactions. Compound 16, an anthracene substituted analogue of compound 12, showed binding interactions exactly similar to those of compound 15. Similarly, compound 18, the anthracene substituted analogue of compound 14, presented three interactions with the residues Lys200 and Ile235. Lys200 presented two H-acceptor bonds with the nitro group, and Ile235 also formed a H-acceptor interaction with the same nitro group of the compound.

Compounds 19–24 have a methoxy and benzyloxy substituted phenyl ring at position-2 (R4). It is interesting to note that compounds 19–23 having a para-methoxy and a meta-benzyloxyphenyl ring as R4 showed almost similar α-amylase inhibitory as well as ABTS and DPPH radical scavenging activities. However, reverting the positions to meta-methoxy and para-benzyloxy as in compound 24 (α-AMY IC50 = 2.16 ± 0.02; ABTS IC50 = 1.66 ± 0.09; DPPH IC50 = 1.66 ± 0.08 μM) showed slightly better activities. An in silico inhibition study showed that this compound showed four interactions with Asp197, Glu233, and Leu162, which means that the positions of methoxy and benzyloxy are ideal in compound 24, to interact strongly with the active pocket of the enzyme. Nevertheless, the moderate α-amylase inhibitory activities revealed by compounds 19–23 might be due to unfavorable positions of substitutions and the extra carbon load on R3.

Compounds 25–31, having a benzyloxy substituted phenyl ring as R4, showed α-AMY IC50 = 2.15 ± 0.06 to 2.47 ± 0.11 μM; ABTS IC50 = 1.64 ± 0.40 to 1.85 ± 0.09 μM; and DPPH IC50 = 1.56 ± 0.07 to 2.36 ± 0.17 μM activities. It is interesting to note that compounds 25–27 having an ortho-benzyloxy substitution and compound 28 with meta-benzyloxy showed similar α-amylase inhibitory and ABTS scavenging activities but different DPPH radical scavenging potentials. Among compounds 25–29, analogues 26 and 28 bearing electronegative fluoro groups showed enhanced DPPH radical scavenging potential and confirms the active participation of fluoro in stabilization of the DPPH radical. An in silico inhibition study revealed that compound 27 showed three interactions with ASP197, LYS200, and TRP59 and compound 28 presented two interactions with GLU233 and LEU165 in its predicted binding mode, while all other members showed one interaction each (Table S1). Compounds 30 (α-AMY IC50 = 2.16 ± 0.07 μM; ABTS IC50 = 1.66 ± 0.12 μM; DPPH IC50 = 1.58 ± 0.10 μM) and 31 (α-AMY IC50 = 2.15 ± 0.06 μM; ABTS IC50 = 1.64 ± 0.40 μM; DPPH IC50 = 1.62 ± 0.02 μM) with para-benzyloxy showed similar activities (Table 1). Nevertheless, the in silico inhibition study displayed some unexpected binding modes in cases of compounds 30 and 31 as compared to their biological activities. Both of these compounds showed one π–H bond each with the active site residues Leu162 and Ile235.

Conclusions

Arylated and heteroarylated benzimidazoles 1–31 were synthesized and screened for their in vitro α-amylase inhibitory as well as ABTS and DPPH radical scavenging activities. All compounds were identified to have significant to moderate α-amylase inhibitory as well as ABTS and DPPH radical scavenging potentials as compared to standards acarbose and ascorbic acid, respectively. Limited SAR revealed that better α-amylase inhibitory potential was perceived on the order of 2-furanyl/2-methylated furanyl > 2-benzyloxyphenyl > 2-pyrenyl ∼ 2-anthracenyl substituted benzimidazole. Compounds bearing halogens, especially the fluoro group, showed good radical scavenging potential. The molecular docking study recognized various structural features involved in binding with the active site of the enzyme. This study has identified some potential molecules including 3, 4, and 9, which can be used for further future research to obtain better and potent α-amylase inhibitors and ABTS and DPPH radical scavengers.

Experimental Section

Materials and Methods

Analytical-grade reagents and solvents were purchased from Sigma-Aldrich and used as received. Thin-layer chromatography (TLC) was performed on precoated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). TLC chromatograms were visualized under ultraviolet light at 254 and 366 nm. Mass spectra were recorded by electron impact (EI) on MAT 312 and MAT 113D mass spectrometers. The 1H and 13C NMR spectra were recorded on Avance Bruker AM spectrometers, operating at 300 and 400 MHz instrument. The chemical shift values are presented in ppm (δ), relative to tetramethylsilane (TMS) as an internal standard, and the coupling constants (J) are in Hz. Multiplicities are reported as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of triplets (dt), quartet (q), or multiplet (m). Melting points of the compounds were determined on Stuart SMP10 melting point apparatus. IR spectra (KBr discs) were run on an FTS 3000 MX, Bio-RAD Merlin (Excalibur Model) spectrophotometer.

General Procedure for the Synthesis of 2-Aryl and Heteroaryl Benzimidazoles

Aryl or heteroaryl benzaldehyde derivative (0.5 mmol), o-phenylene diamine derivative (0.5 mmol), and a pinch of sodium metabisulfite (Na2S2O5) were mixed in N,N-dimethylformamide (10 mL) into a round-bottomed flask (100 mL). After mixing, the reaction mixture was refluxed for 4 h with continuous stirring. Reaction completion was monitored by periodic thin-layer chromatography (TLC). Precipitates appeared after pouring the reaction mixture onto crushed ice (100 mL). Precipitates were filtered, washed with distilled water, and recrystallized from ethanol. Product purity was again checked by TLC analysis. Structures for all compounds were identified by various spectroscopic techniques.

2-(Furan-2′-yl)-1H-benzo[d]imidazole (1)

Brown solid; yield: 69%; mp 283–285 °C [lit. 285–287 °C (Temirak et al.43)]; Rf: 0.43 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.88 (s, 1H, −NH), 7.93 (d, 1H, J5′,4′ = 1.2 Hz, H-5′), 7.60 (br s, 1H, H-7), 7.47 (br s, 1H, H-4), 7.18–7.17 (m, 3H, H-5, H-6, H-3′), 6.72–6.71 (m, 1H, H-4′); 13C NMR (100 MHz, DMSO-d6): δC 145.5 (C-2′), 144.5 (C-5′), 143.5 (C-8, C-9), 122.4 (C-6), 121.8 (C-5), 118.7 (C-2), 112.2 (C-7, C-4), 110.3 (C-4′, C-3′); IR (KBr, cm–1): ≈3400 (N–H), 1621 (C=N), 1521, 1490, 1414 (C=C), 1315 (C–N), 1227, 1012 (C–O); UV–vis: λmax 306 nm; EI-MS: m/z (rel. abund. %), 184 [M+] (100), 156 (21), 129 (9), 102 (5), 92 (8), 64 (4), 52 (3); HREI-MS: m/z calcd for C11H8N2O [M+] 184.0637; found 184.0639.

2-(Furan-2′-yl)-5,6-dimethyl-1H-benzo[d]imidazole (2)

Brown solid; yield: 72%; mp 166–168 °C; Rf: 0.41 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.60 (s, 1H, −NH), 7.88 (d, 1H, J5′,4′ = 0.8 Hz, H-5′), 7.36 (s, 1H, H-7), 7.23 (s, 1H, H-4), 7.10 (d, 1H, J3′,4′ = 3.2 Hz, H-3′), 6.69–6.68 (m, 1H, H-4′), 2.30 (s, 3H, 5-CH3), 2.28 (s, 3H, 6-CH3); IR (KBr, cm–1): ≈3400 (N–H), 3120, 2926, 2856 (C–H), 1643 (C=N), 1524 (C=C), 1448 (C–H), 1312 (C–N), 1233, 1013 (C–O); UV–vis: λmax 312 nm; EI-MS: m/z (rel. abund. %), 212 [M+] (100), 197 (73), 183 (38), 169 (26), 106 (15), 91 (50), 81 (54), 65 (38); HREI-MS: m/z calcd for C13H12N2O [M+] 212.0950; found 212.0948.

5-Fluoro-2-(furan-2′-yl)-1H-benzo[d]imidazole (3)

Brown solid; yield: 88%; mp 194–198 °C; Rf: 0.47 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.97 (s, 1H, H-5′), 7.57–7.54 (m, 1H, H-4), 7.38 (dd, 1H, J7,6 = 9.2 Hz, J7,5F = 2.0 Hz, H-7), 7.24 (d, J3′,4′ = 3.2 Hz, H-3′), 7.11 (dt, 1H, J6,7/6,5F = 10.0 Hz, J6,4 = 2.4 Hz, H-6), 6.75–6.73 (m, 1H, H-4′); 13C NMR (100 MHz, DMSO-d6): δC 159.8 (C-5, C-2′), 157.5 (C-9, C-8, C-2), 145.2 (C-5′), 144.7 (C-7), 112.2 (C-4, C-3′), 110.6 (C-6, C-4′); IR (KBr, cm–1): ≈3400 (N–H), 1639 (C=N), 1523, 1449, 1414 (C=C), 1312 (C–N), 1230 (C–O), 1142 (C–F); UV–vis: λmax 309 nm; EI-MS: m/z (rel. abund. %), 202 [M+] (100), 174 (71), 147 (62), 121 (30), 108 (39), 81 (18); HREI-MS: m/z calcd for C11H7FN2O [M+] 202.0542; found 202.0539.

5-Chloro-2-(furan-2′-yl)-1H-benzo[d]imidazole (4)

Black solid; yield: 91%; mp 109–111 °C; Rf: 0.49 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.97 (d, 1H, J5′,4′ = 1.2 Hz, H-5′), 7.60 (s, 1H, H-4), 7.57 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.24–7.21 (m, 2H, H-6, H-3′), 6.75–6.73 (m, 1H, H-4′); IR (KBr, cm–1): ≈3400 (N–H), 3118 (C–H), 1636 (C=N), 1519 (N–H), 1408 (C=C), 1304 (C–N), 1231, 1018 (C–O), 1063 (C–Cl); UV–vis: λmax 311 nm; EI-MS: m/z (rel. abund. %), 218 [M+] (100), 220 [M+ + 2] (33), 190 (26), 155 (65), 124 (18), 109 (8), 63 (27); HREI-MS: m/z calcd for C11H7ClN2O [M+] 218.0247; found 218.0241.

5-Nitro-2-(furan-2′-yl)-1H-benzo[d]imidazole (5)

Brown solid; yield: 69%; mp 199–201 °C; Rf: 0.37 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 8.42 (s, 1H, H-5′), 8.13 (dd, 1H, J6,7 = 8.8 Hz, J6,4 = 2.0 Hz, H-6), 8.04 (s, 1H, H-4), 7.73 (d, 1H, J7,6 = 8.8 Hz, H-7), 7.35 (d, 1H, J3′,4′ = 3.2 Hz, H-3′), 6.79–6.78 (m, 1H, H-4′); IR (KBr, cm–1): 3374 (N–H), 3121 (C–H), 1633 (C=N), 1515 (N=O), 1471 (C=C), 1340 (N=O), 1235, 1067 (C–O); UV–vis: λmax 278 nm; EI-MS: m/z (rel. abund. %), 229 [M+] (100), 199 (46), 183 (62), 156 (60), 128 (28), 101 (26), 90 (29), 81 (54), 78 (66), 63 (57); HREI-MS: m/z calcd for C11H7N3O3 [M+] 229.0487; found 229.0484.

1-Phenyl-2-(furan-2′-yl)-1H-benzo[d]imidazole (6)

Dark-brown solid; yield: 80%; mp 121–123 °C; Rf: 0.73 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.77 (d, 1H, J5′,4′ = 0.8 Hz, H-5′), 7.75 (d, 1H, J7,6 = 8.0 Hz, H-7), 7.66–7.62 (m, 3H, H-14, H-13, H-12), 7.52–7.50 (m, 2H, H-15, H-11), 7.31 (dt, 1H, J6,7/6,5 = 7.2 Hz, J6,4 = 1.2 Hz, H-6), 7.26 (dt, 1H, J5,4/5,6 = 8.4 Hz, J5,7 = 1.2 Hz, H-5), 7.06 (d, 1H, J4,5 = 7.6 Hz, H-4), 6.55–6.54 (m, 1H, H-4′), 6.30 (d, 1H, J3′,4′ = 3.2 Hz, H-3′); IR (KBr, cm–1): 1632, 1499 (C=C), 1316 (C–N), 1258, 1008 (C–O); UV–vis: λmax 308 nm; EI-MS: m/z (rel. abund. %), 260 [M+] (100), 243 (52), 231 (59), 206 (78), 77 (63), 69 (63), 51 (79); HREI-MS: m/z calcd for C17H12N2O [M+] 260.0950; found 260.0959.

2-(5′-Methylfuran-2′-yl)-1H-benzo[d]imidazole (7)

Brown solid; yield: 76%; mp 274–276 °C [lit. 275-277 °C (Temirak et al.43)]; Rf: 0.46 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.53–7.50 (m, 2H, H-4, H-7), 7.18–7.16 (m, 2H, H-5, H-6), 7.08 (d, 1H, J3′,4′ = 3.2 Hz, H-3′), 6.34 (d, 1H, J4′,3′ = 2.4 Hz, H-4′), 2.40 (s, 3H, 5′-CH3); 13C NMR (75 MHz, DMSO-d6): δC 153.7 (C-5′, C-2′), 143.7 (C-9), 143.6 (C-8), 122.0 (C-6, C-5), 114.8 (C-2), 111.6 (C-7, C-4), 108.5 (C-4′, C-3′), 13.4 (C-6′); IR (KBr, cm–1): 3447 (N–H), 3054, 2953, 2805 (C–H), 1632 (C=N), 1570 (C=C), 1506 (N–H), 1423 (C–H), 1334 (C–N), 1275, 1020 (C–O); UV–vis: λmax 311 nm; EI-MS: m/z (rel. abund. %), 198 [M+] (100), 183 (34), 169 (41), 155 (21), 90 (22), 63 (32); HREI-MS: m/z calcd for C12H10N2O [M+] 198.0793; found 198.0800.

5,6-Dimethyl-2-(5′-methylfuran-2′-yl)-1H-benzo[d]imidazole (8)

Dark-brown solid; yield: 62%; mp 225–227 °C; Rf: 0.43 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.31 (s, 2H, H-7, H-4), 7.09 (d, 1H, J3′,4′ = 2.8 Hz, H-3′), 6.35 (d, 1H, J4′,3′ = 2.4 Hz, H-4′), 2.40 (s, 3H, 5′-CH3), 2.30 (s, 6H, 6-CH3, 5-CH3); IR (KBr, cm–1): 3407 (N–H), 3028, 2917, 2851 (C–H), 1644 (C=N), 1570 (C=C), 1511 (N–H), 1439 (C–H), 1310 (C–N), 1207, 1018 (C–O); UV–vis: λmax 312 nm; EI-MS: m/z (rel. abund. %), 226 [M+] (100), 211 (64), 197 (20), 183 (34), 169 (18), 113 (27), 105 (916), 91 (38), 69 (49); HREI-MS: m/z calcd for C14H14N2O [M+] 226.1106; found 226.1091.

5-Fluoro-2-(5′-methylfuran-2′-yl)-1H-benzo[d]imidazole (9)

Brown solid; yield: 69%; mp 148–151 °C; Rf: 0.50 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.55–7.52 (m, 1H, H-4), 7.36 (dd, 1H, J7,6 = 9.2 Hz, J7,5F = 2.0 Hz, H-7), 7.15 (d, 1H, J3′,4′ = 3.2 Hz, H-3′), 7.10 (dt, 1H, J6,7/6,5F = 10.0 Hz, J6,4 = 2.4 Hz, H-6), 6.37 (d, 1H, J4′,3′ = 2.4 Hz, H-4′), 2.41 (s, 3H, 5′-CH3); 13C NMR (125 MHz, DMSO-d6): δC 159.7 (C-5), 157.8 (C-5′), 154.5 (C-2′), 144.5 (C-9, C-8, C-2), 112.8 (C-7), 110.6 (C-4), 110.4 (C-6), 108.8 (C-4′, C-3′), 13.4 (C-6′); IR (KBr, cm–1): 3380 (N–H), 3111, 2924, 2850 (C–H), 1639 (C=N), 1570 (C=C), 1511 (N–H), 1424 (C–H), 1338 (C–N), 1215, 1022 (C–O), 1145 (C–F); UV–vis: λmax 301 nm; EI-MS: m/z (rel. abund. %), 216 [M+] (100), 201 (32), 187 (50), 173 (24), 147 (16), 108 (18), 91 (8), 69 (37); HREI-MS: m/z calcd for C12H9FN2O [M+] 216.0699; found 216.0704.

5-Chloro-2-(5′-methylfuran-2′-yl)-1H-benzo[d]imidazole (10)

Black solid; yield: 78%; mp 163–165 °C; Rf: 0.51 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.55 (s, 1H, H-4), 7.52 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.20 (dd, 1H, J6,7 = 8.8 Hz, J6,4 = 2.0 Hz, H-6), 7.11 (d, 1H, J3′,4′ = 3.6 Hz, H-3′), 6.35 (d, 1H, J4′,3′ = 2.4 Hz, H-4′), 2.40 (s, 3H, 5′-CH3); IR (KBr, cm–1): ≈3400 (N–H), 3007, 2918, 2834 (C–H), 1630 (C=N), 1569, 1418 (C=C), 1305 (C–N), 1212, 1019 (C–O), 1059 (C–Cl); UV–vis: λmax 311 nm; EI-MS: m/z (rel. abund. %), 232 [M+] (100), 234 [M+ + 2] (48), 217 (25), 203 (23), 189 (17), 116 (8), 95 (11); HREI-MS: m/z calcd for C12H9ClN2O [M+] 232.0403; found 232.0383.

5,6-Dimethyl-2-(pyren-1′-yl)-1H-benzo[d]imidazole (11)

Brownish-yellow solid; yield: 75%; mp 301–304 °C; Rf: 0.67 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.83 (s, 1H, −NH), 9.52 (d, 1H, J3′,2′ = 9.2 Hz, H-3′), 8.52 (d, 1H, J6′,7′ = 8.0 Hz, H-6′), 8.44 (d, 1H, J2′,3′ = 8.0 Hz, H-2′), 8.37–8.31 (m, 3H, H-8′, H-5′, H-4′), 8.29 (d, 1H, J10′,9′ = 8.8 Hz, H-10′), 8.26 (d, 1H, J9′,10′ = 9.2 Hz, H-9′), 8.14 (t, 1H, J7′,8′ = 7.6 Hz, H-7′), 7.60 (s, 1H, H-7), 7.38 (s, 1H, H-4), 2.37 (s, 6H, 6-CH3, 5-CH3); IR (KBr, cm–1): 3406 (N–H), 3042, 2923, 2853 (C–H), 1626 (C=N), 1587, 1431 (C=C), 1453 (C–H), 1315 (C–N); UV–vis: λmax 233 nm; EI-MS: m/z (rel. abund. %), 346 [M+] (94), 330 (38), 316 (6), 227 (24), 200 (11), 172 (11), 165 (11), 91 (10), 65 (11); HREI-MS: m/z calcd for C25H18N2 [M+] 346.1500; found 346.1470.

5-Fluoro-2-(pyren-1′-yl)-1H-benzo[d]imidazole (12)

Brownish-yellow solid; yield: 98%; mp 272–274 °C; Rf: 0.69 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 13.24 (br s, 1H, −NH), 9.45 (d, 1H, J3′,2′ = 9.6 Hz, H-3′), 8.54 (d, 1H, J6′,7′ = 8.0 Hz, H-6′), 8.47 (d, 1H, J2′,3′ = 8.0 Hz, H-2′), 8.39–8.33 (m, 3H, H-8′, H-5′, H-4′), 8.32 (d, 1H, J10′,9′ = 8.8 Hz, H-10′), 8.28 (d, 1H, J9′,10′ = 8.8 Hz, H-9′), 8.16 (t, 1H, J7′,8′/7′,6′ = 7.6 Hz, H-7′), 7.73 (br s, 1H, H-7), 7.53 (br s, 1H, H-4), 7.18 (dt, 1H, J6,7/6,5F = 9.6 Hz, J6,4 = 2.4 Hz, H-6); IR (KBr, cm–1): 3542 (N–H), 3042 (C–H), 1676 (C=N), 1627, 1597, 1425 (C=C), 1329 (C–N), 1137 (C–F); UV–vis: λmax 233 nm; EI-MS: m/z (rel. abund. %), 336 [M+] (100), 227 (12), 200 (19), 168 (19), 149 (6), 119 (6), 83 (71), 69 (35), 55 (36); HREI-MS: m/z calcd for C23H13FN2 [M+] 336.1077; found 336.1063.

5-Chloro-2-(pyren-1′-yl)-1H-benzo[d]imidazole (13)

Brownish-yellow solid; yield: 72%; mp 267–270 °C; Rf: 0.71 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 13.30 (br s, 1H, −NH), 9.43 (d, 1H, J3′,2′ = 9.2 Hz, H-3′), 8.54 (d, 1H, J6′,7′ = 8.0 Hz, H-6′), 8.47 (d, 1H, J2′,3′ = 8.0 Hz, H-2′), 8.39–8.33 (m, 3H, H-8′, H-5′, H-4′), 8.32 (d, 1H, J10′,9′ = 8.8 Hz, H-10′), 8.28 (d, 1H, J9′,10′ = 9.2 Hz, H-9′), 8.16 (t, 1H, J7′,8′/7′,6′ = 7.6 Hz, H-7′), 7.78 (br d, 2H, H-7, H-4), 7.32 (dd, 1H, J6,7 = 8.4 Hz, J6,4 = 1.6 Hz, H-6); IR (KBr, cm–1): 3554 (N–H), 3042 (C–H), 1672 (C=N), 1594, 1425 (C=C), 1325 (C–N), 1056 (C–Cl); UV–vis: λmax 210 nm; EI-MS: m/z (rel. abund. %), 352 [M+] (100), 354 [M+ + 2] (33), 316 (60), 227 (15), 200 (13), 176 (5), 158 (6), 83 (74), 69 (33), 55 (30); HREI-MS: m/z calcd for C23H13ClN2 [M+] 352.0767; found 352.0766.

5-Nitro-2-(pyren-1′-yl)-1H-benzo[d]imidazole (14)

Orange solid; yield: 77%; mp 310–313 °C; Rf: 0.60 (ethyl acetate/hexane, 1:1); 1H NMR (600 MHz, DMSO-d6): δH 13.81 (br s, 1H, −NH), 9.43 (d, 1H, J3′,2′ = 9.2 Hz, H-3′), 8.63 (br s, 1H, H-4), 8.59 (d, 1H, J6′,7′ = 8.0 Hz, H-6′), 8.51 (d, 1H, J2′,3′ = 8.0 Hz, H-2′), 8.42–8.37 (m, 3H, H-8′, H-5′, H-4′), 8.35 (d, 1H, J10′,9′ = 8.9 Hz, H-10′), 8.30 (d, 1H, J9′,10′ = 8.9 Hz, H-9′), 8.22 (d, 1H, J6,7 = 8.8 Hz, H-6), 8.17 (t, 1H, J7′,8′/7′,6′ = 7.6 Hz, H-7′), 7.89 (br d, 1H, H-7); IR (KBr, cm–1): 3567 (N–H), 3102 (C–H), 1626 (C=N), 1594, 1542 (C=C), 1516, 1333 (N=O); UV–vis: λmax 237 nm; EI-MS: m/z (rel. abund. %), 363 [M+] (100), 333 (37, loss of NO), 316 (69, loss of HNO2), 227 (69, loss of fragment A), 200 (41, loss of fragment B), 158 (41), 90 (20, loss of fragment C), 63 (45); HREI-MS: m/z calcd for C23H13N3O2 [M+] 363.1008; found 363.1001.

5,6-Dimethyl-2-(anthracen-9′-yl)-1H-benzo[d]imidazole (15)

Yellow solid; yield: 85%; mp 335–336 °C; Rf: 0.68 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.79 (br s, 1H, −NH), 8.82 (s, 1H, H-10′), 8.20 (d, 2H, J5′,6′ = J4′.3′ = 8.4 Hz, H-5′, H-4′), 7.68 (d, 2H, J8′,7′ = J1′,2′ = 8.8 Hz, H-8′, H-1′), 7.58 (t, 2H, J7′,6′/7′,8′ = J2′,3′/2′,1′ = 8.0 Hz, H-7′, H-2′), 7.51 (t, 2H, J6′,5′/6′,7′ = J3′,2′/3′,4′ = 8.0 Hz, H-6′, H-3′), 7.46 (s, 2H, H-7, H-4); IR (KBr, cm–1): 3373 (N–H), 3045, 2913, 2858 (C–H), 1670 (C=N), 1625, 1578, 1400 (C=C), 1530 (N–H), 1319 (C–N); UV–vis: λmax 248 nm; EI-MS: m/z (rel. abund. %), 322 [M+] (57), 321 (100), 311 (39), 306 (16), 236 (11), 153 (8), 44 (5); HREI-MS: m/z calcd for C23H18N2 [M+] 322.1470; found 332.1452.

5-Fluoro-2-(anthracen-9′-yl)-1H-benzo[d]imidazole (16)

Brown solid; yield: 87%; mp 301–303 °C; Rf: 0.73 (ethyl acetate/hexane, 1:1); 1H NMR (500 MHz, DMSO-d6): δH 13.17 (br s, 1H, −NH), 8.85 (s, 1H, H-10′), 8.21 (d, 2H, J5′,6′ = J4′.3′ = 8.5 Hz, H-5′, H-4′), 7.70 (br s, 1H, H-4), 7.68 (d, 2H, J8′,7′ = J1′,2′ = 8.5 Hz, H-8′, H-1′), 7.59 (t, 2H, J7′,6′/7′,8′ = J2′,3′/2′,1′ = 7.0 Hz, H-7′, H-2′), 7.53–7.50 (m, 3H, H-7, H-6′, H-3′), 7.19 (dt, 1H, J6,7/6,5F = 10.0 Hz, J6,4 = 2.5 Hz, H-6); IR (KBr, cm–1): 3388 (N–H), 3050 (C–H), 1626 (C=N), 1598, 1446 (C=C), 1530 (N–H), 1334 (C–N), 1130 (C–F); UV–vis: λmax 248 nm; EI-MS: m/z (rel. abund. %), 312 [M+] (70), 311 (100), 284 (2), 203 (2), 176 (2), 156 (16); HREI-MS: m/z calcd for C21H13N2F [M+] 312.1063; found 312.1065.

5-Chloro-2-(anthracen-9′-yl)-1H-benzo[d]imidazole (17)

Brown solid; yield: 74%; mp 330–332 °C; Rf: 0.77 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 13.2 (br s, 1H, −NH), 8.85 (s, 1H, H-10′), 8.22 (d, 2H, J5′,6′ = J4′.3′ = 8.4 Hz, H-5′, H-4′), 7.87–7.80 (br d, 1H, H-4), 7.67 (d, 3H, J8′,7′ = J1′,2′ = J7,6 = 8.8 Hz, H-8′, H-1′, H-7), 7.59 (t, 2H, J7′,6′/7′,8′ = J2′,3′/2′,1′ = 7.6 Hz, H-7′, H-2′), 7.53 (t, 2H, J6′,5′/6′,7′ = J3′,1′/3′,2′ = 7.6 Hz, H-6′, H-3′), 7.34 (d, 1H, J6,7 = 8.4 Hz, H-6); IR (KBr, cm–1): 3387 (N–H), 3050 (C–H), 1664 (C=N), 1619, 1444, 1401 (C=C), 1527 (N–H), 1326 (C–N), 1058 (C–Cl); UV–vis: λmax 278, 249 nm; EI-MS: m/z (rel. abund. %), 328 [M+] (58), 327 (100), 292 (27), 203 (3), 164 (7), 146 (9), 63 (2); HREI-MS: m/z calcd for C21H13N2Cl [M+] 328.0767; found 328.0748.

5-Nitro-2-(anthracen-9′-yl)-1H-benzo[d]imidazole (18)

Yellow solid; yield: 82%; mp 295–297 °C; Rf: 0.73 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 13.75 (br s, 1H, −NH), 8.90 (s, 1H, H-10′), 8.73 (br s, 1H, H-6), 8.24 (d, 3H, J5′,6′ = J4′,3′ = J7,6 = 8.4 Hz, H-5′, H-4′, H-7), 7.82 (br s, 1H, H-4), 7.69 (d, 2H, J8′,7′ = J1′,2′ = 8.4 Hz, H-8′, H-1′), 7.61 (t, 2H, J7′,6′/7′,8′ = J2′,3′/2′,1′ = 6.8 Hz, H-7′, H-2′), 7.55 (t, 2H, J6′,5′/6′,7′ = J3′,2′/3′,4′ = 6.8 Hz, H-6′, H-3′); IR (KBr, cm–1): 3377 (N–H), 3052 (C–H), 1623 (C=N), 1595, 1446 (C=C), 1520, 1338 (N=O); UV–vis: λmax 248 nm; EI-MS: m/z (rel. abund. %), 339 [M+] (84), 338 (100), 308 (39), 292 (80), 206 (68), 178 (64), 147 (15), 88 (9), 44 (25); HREI-MS: m/z calcd for C21H13N3O2 [M+] 339.1008; found 339.1006.

2-(3′-(Benzyloxy)-4′-methoxyphenyl)-1H-benzo[d]imidazole (19)

White solid; yield: 89%; mp 116–119 °C; Rf: 0.44 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.89 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.78 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 1.6 Hz, H-6′), 7.60–7.58 (m, 2H, H-7, H-4), 7.51 (d, 2H, J12′,11′= J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.43 (t, 2H, J11′,10′/11′,12′ = J9′,10′/9′,8′ = 7.6 Hz, H-11′, H-9′), 7.36 (t, 1H, J10′,9′/10′,11′ = 7.6 Hz, H-10′), 7.23–7.21 (m, 2H, H-6, H-5), 7.20 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 5.19 (s, 2H, −OCH2−), 3.85 (s, 3H, 4′-OCH3); 13C NMR (75 MHz, DMSO-d6): δC 151.0 (C-2), 150.9 (C-4′, C-3′), 148.0 (C-7′), 136.7 (C-9, C-8), 128.4 (C-11′, C-9′′), 127.9 (C-10′), 127.9 (C-12′, C-8′), 122.4 (C-6, C-5), 121.4 (C-1′), 120.1 (C-6′), 114.5 (C-7, C-4), 112.2 (C-2′), 111.5 (C-5′), 70.0 (C-13′), 55.7 (C-14′); IR (KBr, cm–1): 3419 (N–H), 3063, 2927 (C–H), 1601, 1505 (C=C), 1450 (C–H), 1325 (C–N), 1265, 1018 (C–O); UV–vis: λmax 311 nm; EI-MS: m/z (rel. abund. %), 330 [M+] (52), 301 (6), 239 (87), 211 (12), 91 (100); HREI-MS: m/z calcd for C21H18N2O2 [M+] 330.1368; found 330.1350.

5,6-Dimethyl-2-(3′-(benzyloxy)-4′-methoxyphenyl)-1H-benzo[d]imidazole (20)

White solid; yield: 98%; mp 106–109 °C; Rf: 0.45 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.85 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.73 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 1.2 Hz, H-6′), 7.51 (d, 2H, J12′,11′= J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.43 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.2 Hz, H-11′, H-9′), 7.36 (m, 3H, H-10′, H-7, H-4), 7.16 (d, 1H, J5′,6′ = 8.8 Hz, H-5′), 5.18 (s, 2H, −OCH2−), 3.84 (s, 3H, 4′-OCH3), 2.31 (s, 6H, 6-CH3, 5-CH3); IR (KBr, cm–1): 3416 (N–H), 3160, 2925 (C–H), 1606, 1504 (C=C), 1455 (C–H), 1315 (C–N), 1263, 1019 (C–O); UV–vis: λmax 316 nm; EI-MS: m/z (rel. abund. %), 358 [M+] (77), 329 (8), 267 (100), 253 (14), 239 (28), 91 (58); HREI-MS: m/z calcd for C23H22N2O2 [M+] 358.1681; found 358.1690.

5-Fluoro-2-(3′-(benzyloxy)-4′-methoxyphenyl)-1H-benzo[d]imidazole (21)

Brown solid; yield: 91%; mp 176–178 °C; Rf: 0.51 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.87 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.77 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 1.6 Hz, H-6′), 7.60–7.56 (m, 1H, H-4), 7.51 (d, 2H, J12′,11′= J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.43 (t, 3H, J11′,12′/11′,10′ = J9′,8′/9′,10′ = J6,5F/6,7 = 7.2 Hz, H-11′, H-9′, H-6), 7.36 (t, 1H, J10′,11′ = J10′,9′ = 7.2 Hz, H-10′), 7.20 (d, 1H, J5′,6′ = 8.8 Hz, H-5′), 7.10 (dd, 1H, J7,6 = 10.0 Hz, J7,5F = 2.0 Hz, H-7), 5.18 (s, 2H, −OCH2−), 3.85 (s, 3H, 4′-OCH3); 13C NMR (75 MHz, DMSO-d6): δC 160.3 (C-5), 157.1 (C-2), 152.2 (C-3′), 151.2 (C-4′), 148.0 (C-7′), 136.7 (C-9, C-8), 128.3 (C-11′, C-9′′), 127.8 (C-10′), 127.8 (C-12′, C-8′), 121.0 (C-1′), 120.2 (C-7, C-6′), 112.3 (C-2′), 111.7 (C-5′), 110.6 (C-4), 110.2 (C-6), 70.1 (C-13′), 55.7 (C-14′); IR (KBr, cm–1): 3418 (N–H), ≈3050, 2924, 2853 (C–H), 1631 (C=N), 1600, 1508 (C=C), 1447 (C–H), 1329 (C–N), 1267, 1025 (C–O), 1145 (C–F); UV–vis: λmax 313 nm; EI-MS: m/z (rel. abund. %), 348 [M+] (46), 257 (77), 227 (9), 186 (6), 91 (100), 65 (6), 18 (13); HREI-MS: m/z calcd for C21H17FN2O2 [M+] 348.1274; found 348.1286.

5-Chloro-2-(3′-(benzyloxy)-4′-methoxyphenyl)-1H-benzo[d]imidazole (22)

Brown solid; yield: 94%; mp 112–114 °C; Rf: 0.55 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.87 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.77 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 1.6 Hz, H-6′), 7.62 (s, 1H, H-4), 7.59 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.51 (d, 2H, J12′,11′= J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.43 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.2 Hz, H-11′, H-9′), 7.36 (t, 1H, J10′,9′/10′,11′ = 7.2 Hz, H-10′), 7.24 (dd, 1H, J6,7 = 8.4 Hz, J6,4 = 1.6 Hz, H-6), 7.19 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 5.18 (s, 2H, −OCH2−), 3.85 (s, 3H, 4′-OCH3); 13C NMR (75 MHz, DMSO-d6): δC 152.5 (C-2), 151.2 (C-4′, C-3′), 148.0 (C-7′), 136.7 (C-9, C-8), 128.4 (C-11′, C-9′′), 127.9 (C-10′), 127.9 (C-12′, C-8′), 126.5 (C-5), 122.4 (C-6), 121.2 (C-1′), 120.2 (C-6′), 112.2 (C-7, C-4), 111.6 (C-5′, C-2′), 70.0 (C-13′), 55.7 (C-14′); IR (KBr, cm–1): 3418 (N–H), 3067, 2929 (C–H), 1603, 1502 (C=C), 1452 (C–H), 1322 (C–N), 1267, 1019 (C–O), 1058 (C–Cl); UV–vis: λmax 316 nm; EI-MS: m/z (rel. abund. %), 364 [M+] (81), 366 [M+ + 2] (48), 335 (11), 273 (91), 259 (11), 245 (24), 91 (100), 65 (18), 18 (7); HREI-MS: m/z calcd for C21H17ClN2O2 [M+] 364.0979; found 364.0983.

5-Nitro-2-(3′-(benzyloxy)-4′-methoxyphenyl)-1H-benzo[d]imidazole (23)

Orange solid; yield: 91%; mp 110–113 °C; Rf: 0.45 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 8.42 (s, 1H, H-4), 8.12 (dd, 1H, J6,7 = 8.8 Hz, J6,4 = 2.0 Hz, H-6), 7.92 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.83 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 2.0 Hz, H-6′), 7.74 (d, 1H, J7,6 = 8.8 Hz, H-7), 7.51 (d, 2H, J12′,11′ = J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.43 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.2 Hz, H-11′, H-9′), 7.36 (t, 1H, J10′,9′/10′,11′ = 7.2 Hz, H-10′), 7.22 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 5.20 (s, 2H, −OCH2−), 3.86 (s, 3H, 4′-OCH3); 13C NMR (75 MHz, DMSO-d6): δC 155.8 (C-2), 151.6 (C-4′, C-3′), 148.0 (C-7′), 142.5 (C-5), 136.7 (C-9, C-8), 128.3 (C-11′, C-9′′), 127.9 (C-10′), 127.8 (C-12′, C-8′), 121.2 (C-1′), 120.6 (C-6′), 117.7 (C-7, C-6), 112.2 (C-5′, C-2′), 111.9 (C-4), 70.1 (C-13′), 55.7 (C-14′); IR (KBr, cm–1): 3325 (N–H), 3072, 2931 (C–H), 1600 (C=C), 1504, 1338 (N=O), 1449 (C–H), 1268, 1019 (C–O); UV–vis: λmax 218 nm; EI-MS: m/z (rel. abund. %), 375 [M+] (61), 345 (13), 284 (87), 254 (9), 227 (9), 210 (8) 91 (100), 65 (10), 28 (30), 18 (18); HREI-MS: m/z calcd for C21H17N3O4 [M+] 375.1219; found 375.1233.

5-Chloro-2-(4′-(benzyloxy)-3′-methoxyphenyl)-1H-benzo[d]imidazole (24)

Dark-brown solid; yield: 94%; mp 116–119 °C; Rf: 0.42 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.77 (d, J2′,6′ = 1.8 Hz, H-2′), 7.73 (dd, 1H, J6′,5′ = 8.4 Hz, J6′,2′ = 2.1 Hz, H-6′), 7.64 (d, 1H, J4,6 = 1.8 Hz, H-4), 7.60 (d, 1H, J7,6 = 8.7 Hz, H-7), 7.48–7.34 (m, 5H, H-12′, H-11′, H-10′, H-9′, H-8′), 7.26 (m, 2H, H-5′, H-6), 5.18 (s, 2H, −OCH2−), 3.89 (s, 3H, 3′-OCH3); IR (KBr, cm–1): ≈3400 (N–H), 3066, 2931 (C–H), 1600, 1504 (C=C), 1458 (C–H), 1331 (C–N), 1269 (C–O), 1061 (C–Cl); UV–vis: λmax 316 nm; EI-MS: m/z (rel. abund. %), 364 [M+] (31), 366 [M+ + 2] (11), 258 (10), 245 (23), 202 (8), 91 (100), 65 (24), 51 (10); HREI-MS: m/z calcd for C21H17ClN2O2 [M+] 364.0979; found 364.0977.

5,6-Dimethyl-2-(2′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (25)

Brown solid; yield: 75%; mp 130–132 °C; Rf: 0.63 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.23 (s, 1H, −NH), 8.21 (dd, 1H, J6′,5′ = 7.6 Hz, J6′,4′ = 1.2 Hz, H-6′), 7.48 (d, 2H, J12′,11′ = J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.39 (s, 2H, H-7, H-4), 7.36 (t, 3H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = J4′,3′/4′,5′ = 7.2 Hz, H-11′, H-9′, H-4′), 7.29–7.17 (m, 2H, H-10′, H-3′), 7.08 (t, 1H, J5′,6′ = 7.6 Hz, H-5′), 5.48 (s, 2H, −OCH2−), 2.32 (s, 6H, 6-CH3, 5-CH3); IR (KBr, cm–1): 3306 (N–H), 3029, 2923, 2860 (C–H), 1581 (C=C), 1528 (N–H), 1451 (C–H), 1379 (C–N), 1217, 1014 (C–O); UV–vis: λmax 316 nm; EI-MS: m/z (rel. abund. %), 328 [M+] (100), 311 (42), 251 (22), 237 (72), 222 (54), 209 (29), 91 (93), 65 (14), 44 (16); HREI-MS: m/z calcd for C22H20N2O [M+] 328.1576; found 328.1579.

5-Fluoro-2-(2′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (26)

Brown solid; yield: 82%; mp 125–127 °C; Rf: 0.68 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 12.40 (br s, 1H, −NH), 8.24 (dd, 1H, J6′,5′ = 8.0 Hz, H-6′), 7.64–7.61 (m, 1H, H-4), 7.48 (d, 2H, J12′,11′ = J8′,9′ = 7.6 Hz, H-12′, H-8′), 7.41 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.38–7.32 (m, 3H, H-11′, H-9′, H-4′), 7.28 (t, 1H, J10′,9′/10′,11′ = 7.2 Hz, H-10′), 7.20 (d, 1H, J3′,4′ = 8.4 Hz, H-3′), 7.09 (t, 2H, J6,7/6,5F = J5′,4′/5′,6′ = 7.2 Hz, H-6, H-5′), 5.51 (s, 2H, −OCH2−); IR (KBr, cm–1): 3413 (N–H), 3062, 2925, 2876 (C–H), 1629 (C=N), 1593 (C=C), 1528 (N–H), 1463 (C–H), 1390 (C–N), 1234, 1007 (C–O), 1131 (C–F); UV–vis: λmax 313 nm; EI-MS: m/z (rel. abund. %), 318 [M+] (78), 301 (35), 227 (11), 212 (28), 199 (16), 77 (5), 65 (13); HREI-MS: m/z calcd for C20H15FN2O [M+] 318.1168; found 318.1158.

5-Chloro-2-(2′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (27)

Dark-brown solid; yield: 76%; mp 127–129 °C; Rf: 0.69 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 8.25 (dd, 1H, J6′,5′ = 8.0 Hz, J6′,4′ = 1.6 Hz, H-6′), 7.67 (d, 1H, J4,6 = 1.6 Hz, H-4), 7.65 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.48 (d, 2H, J12′,11′ = J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.41 (dt, 1H, J4′,3′/4′,5′= 8.8 Hz, J4′,6′ = 1.6 Hz, H-4′), 7.36 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.2 Hz, H-11′, H-9′), 7.28–7.22 (m, 2H, H-10′, H-6′), 7.21 (d, 1H, J3′,4′ = 8.4 Hz, H-3′), 7.09 (t, 1H, J5′,6′/5′,4′ = 7.6 Hz, H-5′), 5.51 (s, 2H, −OCH2−); IR (KBr, cm–1): 3409 (N–H), 3032, 2923, 2868 (C–H), 1594 (C=C), 1525 (N–H), 1463 (C–H), 1386 (C–N), 1237, 1006 (C–O), 1048 (C–Cl); UV–vis: λmax 214 nm; EI-MS: m/z (rel. abund. %), 333 [M+] (46), 335 [M+ + 2] (23), 317 (13), 228 (9), 197 (7), 91 (100), 65 (7), 44 (7); HREI-MS: m/z calcd for C20H15ClN2O [M+] 334.0873; found 334.0872.

5-Fluoro-2-(3′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (28)

Brown solid; yield: 88%; mp 201–204 °C; Rf: 0.65 (ethyl acetate/hexane, 1:1); 1H NMR (500 MHz, DMSO-d6): δH 13.05 (br d, 1H −NH), 7.82 (s, 1H, H-2′), 7.75 (d, 1H, J6′,5′ = 7.5 Hz, H-6′), 7.58 (br s, 1H, H-4), 7.50 (d, 2H, J12′,11′ = J8′,9′ = 7.5 Hz, H-12′, H-8′), 7.48 (t, 1H, J5′,4′/5′,6′ = 8.0 Hz, H-5′), 7.42 (m, 3H, H-11′, H-9′, H-7), 7.35 (t, 1H, J10′,9′/10′,11′ = 7.5 Hz, H-10′), 7.15 (dd, 1H, J4′5′ = 8.0 Hz, J4′6′ = 2.0 Hz, H-4′), 7.08 (dt, 1H, J6,5F/6,7 = 10.0 Hz, J6,4 = 2.5 Hz, H-6), 5.20 (s, 2H, −OCH2−); IR (KBr, cm–1): 3449 (N–H), 3061, 2922 (C–H), 1597 (C=C), 1537 (N–H), 1451 (C–H), 1357 (C–N), 1229 (C–O), 1139 (C–F); UV–vis: λmax 299 nm; EI-MS: m/z (rel. abund. %), 318 [M+] (70), 228 (4), 91 (100), 65 (6), 28 (4), 18 (37); HREI-MS: m/z calcd for C20H15FN2O [M+] 318.1168; found 318.1185.

5-Chloro-2-(3′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (29)

Dark-brown solid; yield: 87%; mp 102–104 °C; Rf: 0.66 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 7.83 (s, 1H, H-2′), 7.76 (d, 1H, J6′,5′ = 7.6 Hz, H-6′), 7.66 (s, 1H, H-4), 7.62 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.50 (m, 3H, H-12′, H-8′, H-5′), 7.42 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.6 Hz, H-11′, H-9′), 7.36 (t, 1H, J10′,9′/10′,11′ = 7.6 Hz, H-10′), 7.26 (dd, 1H, J6,7 = 8.4 Hz, J6,4 = 1.6 Hz, H-6), 7.18 (dd, 1H, J4′,5′ = 8.0 Hz, J4′,2′ = 1.6 Hz, H-4′), 5.21 (s, 2H, −OCH2−); IR (KBr, cm–1): 3418 (N–H), 3063, 2921, 2866 (C–H), 1658 (C=N) 1591, 1484 (C=C), 1453 (C–H), 1224, 1024 (C–O); UV–vis: λmax 222 nm; EI-MS: m/z (rel. abund. %), 334 [M+] (67), 336 [M+ + 2] (26), 244 (3), 215 (6), 91 (100), 65 (12), 18 (17); HREI-MS: m/z calcd for C20H15ClN2O [M+] 334.0873; found 334.0895.

5,6-Dimethyl-2-(4′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (30)

White solid; yield: 95%; mp 251–253 °C; Rf: 0.46 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 8.08 (d, 2H, J6′,5′ = J2′,3′ = 8.8 Hz, H-6′, H-2′), 7.48 (d, 2H, J12′,11′ = J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.42 (t, 2H, J11′,10′/11′,12′ = J9′,8′/9′,10′ = 7.2 Hz, H-11′, H-9′), 7.37 (m, 3H, H-10′, H-7, H-4), 7.22 (d, 2H, J5′,6′ = J3′,2′ = 8.8 Hz, H-5′, H-3′), 5.20 (s, 2H, −OCH2−), 2.32 (s, 6H, 6-CH3, 5-CH3); IR (KBr, cm–1): 3424 (N–H), 3036, 2923, 2859 (C–H), 1610, 1502 (C=C), 1456 (C–H), 1304 (C–N), 1257 (C–O); UV–vis: λmax 311 nm; EI-MS: m/z (rel. abund. %), 328 [M+] (66), 237 (100), 209 (15), 197 (9), 91 (62), 65 (5), 44 (3); HREI-MS: m/z calcd for C22H20N2O [M+] 328.1576; found 328.1563.

5-Fluoro-2-(4′-(benzyloxy)phenyl)-1H-benzo[d]imidazole (31)

Brown solid; yield: 94%; mp 223–226 °C; Rf: 0.60 (ethyl acetate/hexane, 1:1); 1H NMR (400 MHz, DMSO-d6): δH 8.09 (d, 2H, J6′,5′ = J2′,3′ = 8.8 Hz, H-6′, H-2′), 7.57–7.54 (m, 1H, H-4), 7.48 (d, 2H, J12′,11′ = J8′,9′ = 7.2 Hz, H-12′, H-8′), 7.42–7.32 (m, 4H, H-11′, H-10′, H-9′, H-7), 7.21 (d, 2H, J5′,6′ = J3′,2′ = 8.8 Hz, H-5′, H-3′), 7.08 (dt, 1H, J6,7/6,5F = 9.6 Hz, J6,4 = 2.0 Hz, H-6), 5.19 (s, 2H, −OCH2−); IR (KBr, cm–1): 3419 (N–H), 3063, 2928, 2877 (C–H), 1609, 1500 (C=C), 1442 (C–H), 1300 (C–N), 1251 (C–O), 1141 (C–F); UV–vis: λmax 299 nm; EI-MS: m/z (rel. abund. %), 318 [M+] (30), 227 (8), 197 (9), 91 (100), 65 (6), 44 (5); HREI-MS: m/z calcd for C20H15FN2O [M+] 318.1168; found 318.1161.

α-Amylase Inhibition Assay

The α-amylase inhibitory activity was determined as recently reported protocol by our group.39 A volume of 500 μL of the test sample (100, 200, 400, 800, 1000 μg/mL) and 500 μL of α-amylase solution (0.5 mg/mL) in 0.2 mM phosphate buffer (pH 6.9) was incubated at 25 °C for 10 min. After preincubation, 500 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube and incubated at 25 °C for 10 min. The reaction was arrested with 1 mL of dinitrosalicylic acid color reagent. The tubes were then incubated in boiling water for 5 min and cooled to room temperature. The solutions were diluted after adding 10 mL of distilled water, and the absorbance was measured at 540 nm. Acarbose was used as the standard, and the assay was carried out in triplicate. The percent inhibition was calculated as follows:

graphic file with name ao1c03056_m001.jpg

where Acontrol and Asample are the absorbances of the control and sample, respectively. The value of IC50, the concentration required to inhibit the α-amylase activity by 50%, was calculated by nonlinear regression of a plot of concentration (y-axis) versus percent inhibition (x-axis) using Graph Pad Prism Software (version 5).

Molecular Docking

A molecular docking study was performed to dock the synthetic 2-furanyl, -pyrenyl, -anthracenyl, and -benzyloxyphenyl substituted benzimidazoles on the active site of the α-amylase enzyme using Molecular Operating Environment (MOE) software. The crystal structure of α-amylase was retrieved from Protein Data Bank (PDB ID: 3BAJ). The retrieved protein structure was then subjected to three-dimensional (3D) protonation and energy minimization up to 0.05 Gradient using an MMFF 94s forcefield implemented in MOE.46

The 3D structures of all synthetic derivatives were built using the Molecular Builder Module program implemented in MOE and were energy-minimized. Molecular docking carried out using the triangular matching docking method allowed 10 different conformations for each compound to be generated. To obtain structures of minimum energy, the ligands were allowed to be flexible during docking. The ligands were ranked by the scores from the GBVI/WSA binding free energy calculation. The GBVI/WSA is a scoring function that estimates the free energy of binding of the ligand from a given pose. For all scoring functions, lower scores indicate more favorable poses. The unit for all scoring functions is kcal/mol. At the end of docking, the predicted ligand–protein complexes were analyzed for molecular interactions and their 3D images were taken using LigPlot implemented in MOE.

DPPH Free Radical Scavenging Assay

The ability of the sample to scavenge 2-diphenyl-1-picrylhydrazyl (DPPH) free radicals was evaluated by a standard method.2 The sample solutions were prepared in absolute alcohol, ranging from 0.01 to 1 mg/mL. A total of 500 μL of the sample was added with 500 μL of 2 μmol DPPH solution. After 20 min of incubation, the samples were placed in the dark at room temperature, and the absorbance was taken at 517 nm. A total of 500 μL of the prepared DDPH solution and 500 μL of absolute alcohol were used as the control. A similar procedure was repeated for ascorbic acid as the standard. The percent inhibition of the radical scavenging activity was calculated as follows:

graphic file with name ao1c03056_m002.jpg

ABTS Free Radical Cation Scavenging Assay

The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) free radical cation (ABTS+•) scavenging ability of the compounds was determined by a standard method.2 A total of 7 mM ABTS was dissolved in distilled water, and 2.45 mM potassium persulfate was added. The solution was kept in the dark for 12–16 h at room temperature. The sample solutions were prepared in absolute alcohol ranging from 0.01 to 1 mg/mL. The samples were added with ABTS solution and incubated for 30 min. The absorbance was taken at 734 nm, and the procedure was repeated for ascorbic acid as the standard. The percent inhibition of the radical scavenging activity was calculated as follows:

graphic file with name ao1c03056_m003.jpg

Acknowledgments

A.A.A. is thankful to The World Academy of Sciences, Trieste, Italy, for awarding the prestigious Fellowship for the year 2015–2016 under the TWAS fellowship program. The authors are thankful to the Pakistan Academy of Sciences for providing financial support via Project No. (5-9/PAS/440).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03056.

  • Interaction details of compounds 1–31 (PDF)

  • Scans of EI-MS and 1H NMR spectra (PDF)

Author Present Address

School of Health Science, University of Petroleum and Energy Studies, Dehradun, Uttarakhand 248007, India

The authors declare no competing financial interest.

Supplementary Material

ao1c03056_si_001.pdf (112.5KB, pdf)
ao1c03056_si_002.pdf (14.9MB, pdf)

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