Introduction

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It is a significant global health concern affecting millions of individuals worldwide and is associated with various complications, including cardiovascular disease, nephropathy, neuropathy, and retinopathy1,2. Diabetes mellitus encompasses different types, such as type 1 diabetes, type 2 diabetes, gestational diabetes, and other specific forms. Type 1 diabetes is an autoimmune disease that destroys pancreatic β-cells, leading to an absolute insulin deficiency. In contrast, type 2 diabetes, the most prevalent form, arises from a combination of insulin resistance and impaired insulin secretion. Gestational diabetes occurs during pregnancy and typically resolves after childbirth3. The pathophysiology of type 2 diabetes involves the development of insulin resistance, wherein target cells exhibit reduced responsiveness to insulin. To compensate for this resistance, the pancreas increases insulin secretion, resulting in elevated insulin levels in the blood. Over time, excessive insulin production can lead to β-cell exhaustion and a decline in insulin production4,5.

α-Glucosidase (EC 3.2.1.20) is a hydrolase enzyme found in the intestinal cells at the brush border surface6. α-Glucosidase plays a crucial role in the small intestine's final steps of carbohydrate digestion. It breaks down the α-1,4 glycosidic bonds present in complex carbohydrates, such as disaccharides and polysaccharides, releasing absorbable monosaccharides, including glucose. This enzymatic activity regulates postprandial blood glucose levels. Inhibition of α-glucosidase effectively slows down the digestion of complex carbohydrates, leading to reduced glucose absorption and a blunted glycemic response after meals7. α-Glucosidase inhibitors like acarbose and miglitol are widely used as oral anti-diabetic medications to manage type 2 diabetes. However, the currently approved α-glucosidase inhibitors have notable side effects such as abdominal distention, bloating, and diarrhea8. Consequently, scientists have been actively pursuing discovering and developing new compounds that can offer enhanced therapeutic benefits while minimizing adverse effects. They aim to identify and synthesize novel inhibitors that improve treatment outcomes and reduce the risk of undesirable reactions6,9.

Coumarin, a naturally occurring compound, and its derivatives have demonstrated various pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties10,11,12,13,14. These characteristics make it an attractive pharmacophore for designing and developing new compounds with potential therapeutic applications15. In the case of α-glucosidase inhibition, coumarin derivatives A–C (Fig. 1) demonstrated α-glucosidase inhibitory activities16,17,18. As can be seen in Fig. 1, coumarincarbohydrazone derivative A with hydroxy substituent on 2-position of pendant phenyl group was 35.8-fold more potent than standard inhibitor acarbose. Moreover, in α-glucosidase inhibitor B coumarin by a carboxylate linker attached to styrene (Fig. 1). It is worthy to note that coumarin derivative C was 252-fold more potent than acarbose (Fig. 1). Furthermore, the potency of anti- α-glucosidase activity of coumarin derivatives was also confirmed in other studies19,20,21.

Figure 1
figure 1

Rationale for the design of coumarin linked to 2-phenylbenzimidazole derivatives as the new α-glucosidase inhibitors.

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Benzoimidazole is a nitrogen containing heterocycle that has gained significant attention in medicinal chemistry due to its diverse biological activities and therapeutic potential22. It exhibits various pharmacological properties, including antimicrobial, anticancer, anti-inflammatory, and antioxidant activities23,24,25,26. Also, the benzoimidazole is a valuable structural motif in design of α-glucosidase inhibitors27,28,29. For example, 2-phenylbenzimidazole derivative D exhibited excellent inhibitory activity yeast α-glucosidase (IC50 = 8.40 μM comparing with acarbose IC50 = 774.5 μM)30. Furthermore, 2-phenylbenzimidazole derivatives E and F showed high anti-α-glucosidase activities in comparison to standard inhibitor31,32.

In view of the mentioned scaffolds, herein, by connection of coumarin to 2-phenylbenzimidazole by carboxylate linker, a novel series of α-glucosidase inhibitors was designed. Next, all designed compounds were examined against yeast α-glucosidase. Finally, in silico assessments were done to get insight into the structure–activity relationships (SARs) and binding interaction modes of the title compounds.

Results and discussion

Chemistry

Synthesis of the target compounds 5a–m was schematically described in Scheme 1. Briefly, 3,4-aminobenzoic acid 1 was added to different aldehydes 2 in DMF, and the mixture was stirred at 110 °C for 1 h to synthesize 2-phenylbenzimidazole-5-carboxylic acids 3. Next, intermediate 3 was reacted with 7-hydroxy-2H-chromen-2-one 4 in the presence of N,N-diisopropylethylamine (DIPEA), and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in DMF to synthesize final products 5a–m. FTIR, 1H-NMR, and 13C-NMR characterized all synthesized compounds.

Scheme 1
scheme 1

Synthesis of coumarin linked to 2-phenylbenzimidazole derivatives 5a–m.

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In vitro α-glucosidase inhibition and SAR discussion

The potency of the target compounds 5a–m was evaluated in vitro against α-glucosidase, compared with acarbose used as a positive control. The IC50 values obtained for each compound are listed in Table 1. Notably, compound 5k, which contains a 2-hydroxyphenyl moiety, exhibited promising activity with an IC50 value of 10.8 ± 0.1 µM, making it the most potent inhibitor compared to acarbose.

Table 1 α-Glucosidase inhibition assay of the target compounds 5a–m.
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In the present study, compound 5a, an un-substituted derivative (R=H), demonstrated an IC50 value of 50.0 ± 0.8 µM, which was approximately 15-fold better than acarbose.

Different moieties were substituted at the R position to explore SAR and optimization of the inhibitory activity. Initially, halogens were introduced at the para position of the phenyl ring, and the following potency order was observed: 4-Br (5d, IC50 = 22.9 ± 0.2 µM) > 4-Cl (5c, IC50 = 26.9 ± 1.1 µM) > 4-F (5b, IC50 = 30.6 ± 0.4 µM).

Substituting chlorine at the ortho position (5e; IC50 = 34.7 ± 0.7 µM) or bromine at the meta position (5f.; IC50 = 34.7 ± 0.7 µM) resulted in reduced activity compared to their para-substituted counterparts. However, despite the decrease in activity, these derivatives still exhibited better inhibition compared to un-substituted derivative 5a.

Furthermore, it was observed that the introduction of second fluorine atom on 4-fluorophenyl ring of compound 5b and the second chlorine atom on 2-chlorophenyl ring of derivative 5e, as in compounds 5g and 5h, respectively, reduced anti-α-glucosidase activity.

SAR study also demonstrated that electron-withdrawing groups at the para position of the phenyl ring are more effective than electron-donating groups (halogen derivatives 5b–d vs. methyl and methoxy derivatives 5i–j). In contrast, 2-hydroxy derivative 5k was significantly more active than 2-chlorophenyl derivative 5e.

Moreover, SAR study of compounds with two deference substitutions, involving both electron-donating and electron-withdrawing groups, was performed. The lowest potency was observed in 2-CH3-3-NO2 derivative 5l, with an IC50 value of 119.5 ± 0.1 µM. Replacing of 3-NO2 group of compound 5l with 3-Cl substituent, as in compound 5m, improved the potency.

In addition to the listed of IC50 values in Table 1, for example, two dose response curves for two compounds 5c and 5d were showed in Fig. 2.

Figure 2
figure 2

Dose response curves of compounds 5c and 5d.

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Enzyme kinetic studies

According to Fig. 3a, the Lineweaver–Burk plot showed that the Michaelis–Menten constant (Km) gradually increased and maximum velocity (Vmax) remained unchanged with increasing inhibitor concentration indicating a competitive inhibition. The results show 5k binds to the active site on the enzyme and compete with the substrate for binding to the active site. Furthermore, the plot of the Km versus different concentrations of inhibitor gave an estimate of the inhibition constant (Ki) of 10.4 µM (Fig. 3b).

Figure 3
figure 3

Kinetics of α-glucosidase inhibition by 5k (inhibitor): (a) the Lineweaver–Burk plot in the absence and presence of different concentrations of 5k; (b) the secondary plot between Km and various concentrations of 5k.

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Molecular docking

In the enzyme assay section, it was reported that the assay was conducted utilizing the enzyme Saccharomyces cerevisiae α-glucosidase (EC. 3. 2. 1. 20). However, due to the unavailability of the 3D crystallographic structure of this enzyme in the corresponding databases, a new structure was developed using homology modeling33. After that, based on obtained mode of representative compound (the most potent compound 5k) in the kinetic study (competitive mode), docking study of the target compounds was performed in the α-glucosidase active site. Superimposed structure of the standard inhibitor (acarbose, pink) and the most potent compound 5k (orange) is showed in Fig. 4a. as can be seen in Fig. 4b, acarbose as positive control established hydrogen bonds with residues Thr307, Thr301, Asn241, Glu304, Ser308, Phe157, and Pro309, Arg312, and Gln322, non-classical hydrogen bonds with residues Val305, His239, Arg312, and Glu304, a hydrophobic interaction with His279, and two unfavorable interactions with residues Thr307 and Arg312 in the α‐glucosidase active site.

Figure 4
figure 4

(a) Acarbose (pink) and compound 5k (orange) in the α-glucosidase active site and (b) interaction mode of acarbose.

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For this study, we considered the compounds 5c–e, 5k, and 5l–m because they were either potent or had interesting points in term of SARs. Interaction modes of compounds 5c–e, 5k, and 5l–m were showed in Figs. 5 and 6 and binding energies of these compounds and acarbose were listed in Table 2.

Figure 5
figure 5

Interaction modes of compounds 5c–e and 5k in the α-glucosidase active site.

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Figure 6
figure 6

Interaction modes of compounds 5m and 5l in the α-glucosidase active site.

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Table 2 Binding energies of the selected compounds 5c–e, 5k, and 5l-m.
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The comparison of binding energies of the compounds 5c–e, 5k, and 5l–m with acarbose revealed that these new compounds can be bind to the active site easier than the standard inhibitor (Table 2). These results were confirmed by the obtained results of in vitro assessment.

Interaction mode of the most potent compound 5k showed that this compound established three hydrogen bonds with residues Thr307, Thr301 (via 2-hydroxy group), and Glu304 (Fig. 5). This compound also formed a π-anion interaction with Glu304 and a π-cation interaction with His239. Moreover, hydrophobic interactions between compound 5k and residues Glu304, His239, Val305, Pro309, and Arg312 were observed. In vitro study demonstrated that compound 5k with 2-hydroxy substituent was twofold more potent thah the second potent compound 5d with 4-bromo substituent (Table 1). The comparison of binding energies of these compounds showed that compound 5k had lower binding energy in comparison to compound 5d. Compound 5d formed four classical hydrogen bonds with residues Asn241, Asn412, Ser308, and His239 and a non-classical hydrogen bond with His279 (Fig. 5). Glu304 established a π-anion interaction with compound 5d. Furthermore, the latter compound created hydrophobic interactions with residues Arg312 and Pro309. Replacement of 4-bromo substituent of compound 5d with 4-chloro substituent, in case of compound 5c, led to a negligible decrease in inhibitory activity and a negligible increase in binding energy (Table 1 and 2). As can be seen in Fig. 5, 4-chloro derivative 5c formed three classical hydrogen bonds (with Asn241, His239, and Ser308) and a non-classical hydrogen bond (with Arg312) with the active site of α-glucosidase. This compound established π-anion interactions with Glu304 and hydrophobic interactions with Arg312, Pro309, and Val305. The comparison of IC50 values of 4-chloro derivative 5c and 2-chloro derivatives 5e demonstrated that translocation of chlorine atom on the 2-phenyl ring had a moderate effect on anti-α-glucosidase activity, and 4-chloro was preferred (Table 1). The obtained binding energies of compounds 5c and 5e were in agreement with in vitro study (Table 2). The comparison of binding modes of the latter compounds showed that 4-chloro derivative established three classical hydrogen bonds and a non-classical hydrogen bond while 2-chloro derivative formed two classical hydrogen bonds with Gly306 and Arg312 and two non-classical hydrogen bonds with His239 and Pro309. According to the mode of interaction of acarbose (Fig. 4b), it seems that Gly306 is not very important in the occurrence of the effect. π-Anion interactions were similar in both of compounds 5c and 5e. Moreover, hydrophobic interactions also were similar in these compound only compound 5e interacted with Phe311 while compound 5c interacted Arg312.

As can be seen Table 1, replacement of 3-chloro substituent of compound 5m with 3-nitro substituent, as in case of compound 5l, the inhibitory activity diminished to three folds. This finding with obtained binding energies for these compounds was confirmed. Binding mods of compound 5m and 5l showed that compound 5m formed interactions with nine amino acids Val305, Gly306, Thr307, Glu304, Pro309, His279, Arg312, Phe158, and Phe157 and compound 5l established interactions with eight amino acids His239, Arg312, Phe311, Ser308, Pro309, Glu304, Val305, and Val316 (Fig. 6).

Conclusion

Following our expertise in the rational design of α-glucosidase inhibitors, a series of coumarin-benzimidazole derivatives were designed and synthesized. The chemical structures of these derivatives were thoroughly characterized using various analytical techniques, including 1H-NMR, 13C-NMR, and FTIR analysis. Our results revealed that all the synthesized compounds exhibited significant anti-α-glucosidase activity, as demonstrated by their IC50 values ranging from 10.8 ± 0.1 μM to 119.5 ± 0.1 μM, in comparison to the reference compound acarbose (IC50 = 750.0 µM). These findings highlight the efficacy of the designed backbone in targeting α-glucosidase. Among the tested compounds, compound 5k emerged as the most potent inhibitor with an IC50 value of 10.8 ± 0.1 μM. Further kinetic experiments revealed that compound 5k exhibited a competitive inhibition pattern. To gain deeper insights into the interaction mechanism of these derivatives at the α-glucosidase active site, we performed molecular docking study. By this study, the observed SARs were rationalized and possible binding modes were detected.

Experimental

General synthesis of 2-aryl-1H-benzo[d]imidazole-5-carboxylic acids 3

A mixture of 3,4-aminobenzoic acid 1 (1 mmol) and aldehydes 2 (1 mmol) in DMF (10 mL) was stirred at 110 °C for 1 h. Then, water was added to the reaction mixture and observed participates were separated by filtration to give pure 2-aryl-1H-benzo[d]imidazole-5-carboxylic acids 3.

General synthesis of the target compounds 5a–m

A mixture of 2-aryl-1H-benzo[d]imidazole-5-carboxylic acids 3 (1 mmol), 7-hydroxy-2H-chromen-2-one 4 (1 mmol), DIPEA (1.1 mmol), and TBTU (1 mmol) in DMF (15 mL) was stirred at room temperature for 24 h. Then, cold water was added to the reaction mixture and formed participates were separated by filtration to give pure target compounds 5a–m.

2-Oxo-2H-chromen-7-yl 2-phenyl-1H-benzo[d]imidazole-5-carboxylate (5a)

Brown solid; yield: 65%; MP = 180–183 °C; IR (KBr, vmax) 3320(NH), 3040 (C–H aromatic), 1680(C = O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H, NHImidazole), 8.34 (s, 1H, HAr), 8.00 (d, J = 9.5 Hz, 1H, HAr), 7.79 (d, J = 7.6 Hz, 1H, HAr), 7.65 (d, J = 8.6 Hz, 1H, HAr), 7.59 (d, J = 8.0 Hz, 2H, HAr), 7.45 (d, J = 7.6 Hz, 1H, HAr), 7.34 (t, J = 7.8 Hz, 2H, HAr), 7.20 (s, 1H, HAr), 7.09 (t, J = 7.4 Hz, 1H, HAr), 7.05 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 164.64, 161.62, 160.76, 155.79, 152.95, 147.45, 144.79, 142.25, 138.87, 136.74, 130.00, 129.41, 127.19, 124.26, 123.43, 119.66, 118.79, 115.08, 113.40, 113.14, 113.05, 102.01.

2-Oxo-2H-chromen-7-yl 2-(4-fluorophenyl)-1H-benzo[d]imidazole-5-carboxylate (5b)

Cream solid; yield:81%; MP = 195–197 °C; IR (KBr, vmax) 3401 (NH), 3065(CH aromatic), 1670 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H, NHImidazole), 8.33 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.80 (d, , J = 7.9 Hz, 1H, HAr), 7.66 (d, J = 8.6 Hz, 1H, HAr), 7.63–7.57 (m, 2H, HAr), 7.46 (d, , J = 8.1 Hz, 1H, HAr), 7.22–7.15 (m, 3H, HAr), 7.05 (dd, J = 8.7, 2.4 Hz, 1H, HAr), 6.32 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 164.60, 161.62, 160.76, 159.90, 157.51, 155.80, 152.91, 147.17, 144.80, 142.22, 138.74, 135.27, 135.25, 130.01, 127.15, 125.19, 123.24, 121.52, 121.44, 119.16, 117.70, 116.13, 115.91, 113.41, 113.15, 113.05, 102.01.

2-Oxo-2H-chromen-7-yl 2-(4-chlorophenyl)-1H-benzo[d]imidazole-5-carboxylate (5c)

Cream solid; yield:68%; MP = 180–182 °C; IR (KBr, vmax) 3235 (NH), 3030 (CH Aromatic), 1684 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 8.88 (s, 1H, NHImidazole), 8.27 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.86 (d, J = 7.2 Hz, 1H, HAr), 7.65 (d, J = 8.6 Hz, 1H, HAr), 7.52 (d, J = 7.2 Hz, 1H, HAr), 7.37–7.29 (m, 2H, HAr), 7.24–7.13 (m, 3H, HAr), 7.04 (dd, J = 8.7, 2.4 Hz, 1H, HAr), 6.32 (d, J = 9.4 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.92, 162.95, 161.62, 160.76, 160.54, 155.79, 152.13, 144.80, 142.12, 139.68, 135.42 (d, J = 3.0 Hz), 130.00, 129.92, 129.84, 127.04, 125.90, 123.05, 119.14, 115.68, 115.47, 113.40, 113.14, 113.04, 101.99.

2-Oxo-2H-chromen-7-yl 2-(4-bromophenyl)-1H-benzo[d]imidazole-5-carboxylate (5d)

Brown solid; yield: 65%; MP = 179–181 °C; IR (KBr, vmax) 3336(NH), 3030 (C–H Aromatic), 1651(C=O) cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H, NHImidazole), 8.33 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.84 (d, J = 8.1 Hz, 1H, HAr), 7.66 (d, J = 8.7 Hz, 1H, HAr), 7.61–7.50 (m, 4H, HAr), 7.36 (d, J = 7.9 Hz, 1H, HAr), 7.20 (s, 1H, HAr), 7.05 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.32 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 164.88, 161.62, 160.76, 155.79, 152.93, 147.62, 144.80, 142.23, 138.24, 132.25, 130.01, 127.16, 125.07, 123.08, 121.60, 115.89, 113.41, 113.15, 113.05, 102.01.

2-Oxo-2H-chromen-7-yl 2-(2-chlorophenyl)-1H-benzo[d]imidazole-5-carboxylate (5e)

Cream solid; yield: 68%; MP = 189–191 °C; IR (KBr, vmax) 3221 (NH), 3025 (CH aromatic), 1679 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H, NH Imidazole), 8.34 (s, 1H, HAr), 7.99 (d, J = 9.5 Hz, 1H, HAr), 7.84 (d, J = 7.5 Hz, 1H, HAr), 7.75 (d, J = 7.5 Hz, 1H, HAr), 7.64 (d, J = 8.6 Hz, 1H, HAr), 7.53 (d, J = 7.5 Hz, 1H, HAr), 7.43 (d, J = 7.5 Hz, 1H, HAr), 7.34 (t, J = 7.4 Hz, 1H, HAr), 7.23 (d, J = 7.4 Hz, 1H, HAr), 7.19 (s, 1H, HAr), 7.04 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.36, 161.60, 160.76, 155.79, 152.86, 146.56, 144.76, 142.26, 138.61, 134.61, 125.32, 123.25, 119.57, 115.34, 113.39, 113.13, 113.04, 101.99.

2-Oxo-2H-chromen-7-yl 2-(3-bromophenyl)-1H-benzo[d]imidazole-5-carboxylate (5f)

Cream solid; yield: 66%; MP = 172–174 °C; IR (KBr, vmax) 3243 (NH), 3050 (CH aromatic), 1679 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H, NHImidazole), 8.33 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.93 (s, 1H, HAr), 7.80 (d, J = 7.8 Hz, 1H, HAr), 7.66 (d, J = 8.7 Hz, 1H, HAr), 7.54 (d, J = 7.5 Hz, 1H, HAr), 7.49 (d, J = 7.3 Hz, 2H, HAr), 7.37–7.26 (m, 2H, HAr), 7.20 (s, 1H, HAr), 7.05 (dd, J = 8.7, 2.4 Hz, 1H, HAr), 6.32 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.11, 161.62, 160.76, 155.80, 152.74, 148.19, 144.79, 142.24, 140.41, 137.08, 131.45, 130.00, 127.16, 126.91, 125.48, 123.81, 122.13, 122.05, 118.48, 115.58, 113.40, 113.15, 113.05, 102.00.

2-Oxo-2H-chromen-7-yl 2-(2,4-difluorophenyl)-1H-benzo[d]imidazole-5-carboxylate (5g)

Brown solid; yield: 65%; MP = 201–203 °C; IR (KBr, vmax) 3343(NH), 3045 (C–H aromatic), 1654 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H, NHImidazole), 8.33 (s, 1H, HAr), 7.99 (d, J = 9.5 Hz, 1H, HAr), 7.92–7.82 (m, 1H, HAr), 7.78 (d, J = 7.8 Hz, 1H, HAr), 7.64 (d, J = 8.6 Hz, 1H, HAr), 7.49 (d, J = 7.9 Hz, 1H, HAr), 7.44–7.31 (m, 1H, HAr), 7.18 (s, 1H, HAr), 7.08 (t, J = 8.7 Hz, 1H, HAr), 7.03 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.32, 161.60, 160.76, 160.40, 160.29, 157.98, 157.86, 155.78, 155.60, 155.47, 153.13, 153.00, 146.81, 144.75, 142.24, 139.57, 129.97, 127.17, 125.77, 125.74, 125.67, 125.64, 122.52, 122.49, 122.40, 122.37, 113.38, 113.13, 113.04, 111.89, 111.85, 111.67, 111.64, 105.03, 104.79, 104.76, 104.53, 101.99.

2-Oxo-2H-chromen-7-yl 2-(2,3-dichlorophenyl)-1H-benzo[d]imidazole-5-carboxylate (5h)

Cream solid; yield: 71%; MP = 182–184 °C; IR (KBr, vmax) 3364 (NH), 3035(CH aromatic) 1661 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H, NHImidazole), 8.34 (s, 1H, HAr), 7.99 (d, J = 9.5 Hz, 1H, HAr), 7.84 (d, J = 7.9 Hz, 1H, HAr), 7.73 (d, J = 8.0 Hz, 1H, HAr), 7.64 (d, J = 8.6 Hz, 1H, HAr), 7.49 (d, J = 8.0 Hz, 1H, HAr), 7.37 (t, J = 8.1 Hz, 1H, HAr), 7.27 (d, J = 8.0 Hz, 1H, HAr), 7.18 (s, 1H, HAr), 7.03 (d, J = 8.2 Hz, 1H, HAr), 6.30 (d, J = 9.4 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.54, 161.59, 160.75, 155.78, 152.12, 146.53, 144.76, 138.22, 136.59, 132.48, 129.98, 128.61, 127.56, 127.28, 125.44, 124.93, 122.06, 113.39, 113.14, 113.04, 101.99.

2-Oxo-2H-chromen-7-yl 2-(p-tolyl)-1H-benzo[d]imidazole-5-carboxylate (5i)

Cream solid; yield: 71%; MP = 193–195 °C; IR (KBr, vmax) 3213 (NH), 3020 (CH aromatic), 2965 (CH aliphatic), 1671 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H, NH Imidazole), 8.33 (s, 1H, HAr), 8.00 (d, J = 9.5 Hz, 1H, HAr), 7.83 (d, J = 7.3 Hz, 1H, HAr), 7.82 (d, J = 7.4 Hz, 1H, HAr), 7.65 (d, J = 8.6 Hz, 1H, HAr), 7.47 (d, J = 8.4 Hz, 2H, HAr), 7.33 (d, J = 7.1 Hz, 1H, HAr), 7.19 (s, 1H, HAr), 7.14 (d, J = 8.2 Hz, 2H, HAr), 7.04 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr)2.25(s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 164.37, 161.62, 160.76, 155.79, 147.43, 144.78, 142.20, 139.37, 136.36, 133.23, 129.99, 129.77, 127.15, 119.67, 116.32, 113.39, 113.14, 113.04, 102.00, 20.92.

2-Oxo-2H-chromen-7-yl 2-(4-methoxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (5j)

Cream solid; yield: 68%;MP = 169–171 °C; IR (KBr, vmax) 3355 (NH), 3070(CH aromatic), 2965(CH aliphatic), 1670 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H, NHImidazole), 8.32 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.88–7.80 (d, J = 7.4 Hz, 1H, HAr), 7.66 (d, J = 8.6 Hz, 1H, HAr), 7.55–7.45 (m, 2H, HAr), 7.39–7.30 (d, J = 7.3 Hz, 1H, HAr), 7.23–7.18 (m, 1H, HAr), 7.05 (dd, J = 8.7, 2.4 Hz, 1H, HAr), 6.91 (d, J = 9.0 Hz, 2H, HAr), 6.32 (d, J = 9.5 Hz, 1H, HAr), 3.73 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6) δ 164.09, 161.63, 160.76, 155.99, 155.80, 153.19, 145.93, 144.80, 142.18, 131.96, 130.01, 127.14, 125.15, 123.60, 121.21, 119.56, 114.49, 113.41, 113.15, 113.05, 102.01, 55.63.

2-Oxo-2H-chromen-7-yl 2-(2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (5k)

Brown solid;Yield:70%;MP = 187–189 °C; IR (KBr, vmax) 3320(NH), 3030 (C–H aromatic), 1650 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H, OH), 8.88 (s, 1H, NHImidazole), 8.28 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.85 (d, J = 7.8 Hz, 1H, HAr), 7.65 (d, J = 8.6 Hz, 1H, HAr), 7.53 (d, J = 7.6 Hz, 1H, HAr), 7.38–7.32 (m, 2H, HAr), 7.32–7.24 (m, 2H, HAr), 7.20 (d, J = 2.4 Hz, 1H, HAr), 7.04 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ 165.88, 161.63, 160.76, 158.15, 155.79, 152.87, 147.25, 144.80, 142.11, 139.17, 130.00, 128.85, 127.87, 127.49, 127.05, 125.06, 123.65, 119.66, 115.78, 113.40, 113.14, 113.04, 101.99.

2-Oxo-2H-chromen-7-yl 2-(2-methyl-3-nitrophenyl)-1H-benzo[d]imidazole-5-carboxylate (5l)

Cream solid; yield: 71%;MP = 194–196 °C; IR (KBr, vmax) 3375 (NH), 3045(CH aromatic), 2960(CH aliphatic), 1665 (C=O) cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H, NHImidazole), 8.35 (s, 1H, HAr), 8.00 (d, J = 9.5 Hz, 1H, HAr), 7.84 (d, J = 7.4 Hz, 1H, HAr), 7.76 (d, J = 8.1 Hz, 1H, HAr), 7.71 (d, J = 8.0 Hz, 1H, HAr), 7.64 (d, J = 8.6 Hz, 1H, HAr), 7.55 (d, J = 7.4 Hz, 1H, HAr), 7.45 (t, J = 8.1 Hz, 1H, HAr), 7.19 (s, 1H, HAr), 7.04 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.31 (d, J = 9.5 Hz, 1H, HAr), 2.30 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.49, 161.60, 160.75, 155.78, 152.54, 151.36, 144.77, 142.32, 138.76, 137.75, 130.29, 129.99, 127.29, 127.18, 126.99, 121.69, 113.40, 113.14, 113.04, 101.99, 14.27.

2-Oxo-2H-chromen-7-yl 2-(3-chloro-2-methylphenyl)-1H-benzo[d]imidazole-5-carboxylate (5m)

Brown solid; yield: 74%; MP = 182–184 °C; IR (KBr, vmax) 3326(NH), 3045 (C–H aromatic), 2990 (CH aliphatic), 1679 (C=O) cm−1;1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H, NHImidazole), 8.33 (s, 1H, HAr), 8.01 (d, J = 9.5 Hz, 1H, HAr), 7.86 (d, J = 7.8 Hz, 1H, HAr), 7.65 (d, J = 8.7 Hz, 1H, HAr), 7.50 (d, J = 7.9 Hz, 1H, HAr), 7.37 (d, J = 7.9 Hz, 1H, HAr), 7.32 (d, J = 7.9 Hz, 1H, HAr), 7.23 (d, J = 8.0 Hz, 1H, HAr), 7.20 (s, 1H, HAr), 7.04 (dd, J = 8.6, 2.4 Hz, 1H, HAr), 6.32 (d, J = 9.5 Hz, 1H, HAr), 2.26 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.15, 161.61, 160.76, 155.80, 152.98, 147.74, 144.80, 142.21, 139.82, 137.47, 134.34, 130.86, 130.01, 127.46, 127.14, 126.92, 124.80, 122.29, 119.04, 115.78, 113.42, 113.15, 113.05, 102.00, 15.57.

In vitro α-glucosidase inhibition assay

α-Glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/mg) and the substrate, p-nitrophenyl-β-d-glucopyranoside (p-NPG) were purchased from Sigma-Aldrich, and the assay was performed exactly according to our previous report34,35.

Enzyme kinetic studies

The mode of inhibition of the most active compound 5k identified with the lowest IC50 was investigated against α-glucosidase activity with different concentrations of p-nitrophenyl α-d-glucopyranoside (1–16 mM) as substrate in the absence and presence of sample 5k at different concentrations (0, 2.7, 5.4, and 10.8 µM)36,37. A Lineweaver–Burk plot was generated to identify the type of inhibition and Km value was determined from the plot between the reciprocal of the substrate concentration (1/[S]) and reciprocal of enzyme rate (1/V) over various inhibitor concentrations. Experimental Ki value was constructed by secondary plots of the inhibitor concentration [I] versus Km.

Docking study

Docking study of the selected compounds 5c–e, 5k, and 5l–m were performed exactly according to our previous reported work33 (Supplementary Information).