Synthesis, Molecular Docking, and Bioactivity Study of Novel Hybrid Benzimidazole Urea Derivatives: A Promising α-Amylase and α-Glucosidase Inhibitor Candidate with Antioxidant Activity

A novel series of benzimidazole ureas 3a–h were elaborated using 2-(1H-benzoimidazol-2-yl) aniline 1 and the appropriate isocyanates 2a–h. The antioxidant and possible antidiabetic activities of the target benzimidazole-ureas 3a–h were evaluated. Almost all compounds 3a–h displayed strong to moderate antioxidant activities. When tested using the three antioxidant techniques, TAC, FRAP, and MCA, compounds 3b and 3c exhibited marked activity. The most active antioxidant compound in this family was compound 3g, which had excellent activity using four different methods: TAC, FRAP, DPPH-SA, and MCA. In vitro antidiabetic assays against α-amylase and α-glucosidase enzymes revealed that the majority of the compounds tested had good to moderate activity. The most favorable results were obtained with compounds 3c, 3e, and 3g, and analysis revealed that compounds 3c (IC50 = 18.65 ± 0.23 μM), 3e (IC50 = 20.7 ± 0.06 μM), and 3g (IC50 = 22.33 ± 0.12 μM) had good α-amylase inhibitory potential comparable to standard acarbose (IC50 = 14.21 ± 0.06 μM). Furthermore, the inhibitory effect of 3c (IC50 = 17.47 ± 0.03 μM), 3e (IC50 = 21.97 ± 0.19 μM), and 3g (IC50 = 23.01 ± 0.12 μM) on α-glucosidase was also comparable to acarbose (IC50 = 15.41 ± 0.32 μM). According to in silico molecular docking studies, compounds 3a–h had considerable affinity for the active sites of human lysosomal acid α-glucosidase (HLAG) and pancreatic α-amylase (HPA), indicating that the majority of the examined compounds had potential anti-hyperglycemic action.


Introduction
Diabetes mellitus (DM) is a broad and complex endocrine disease, which is a major global health issue. It is a disease characterized by marked multiple organ dysfunction and failure caused by hyperglycemia resulting from insulin deficiency or dysregulation of the insulin action pathway [1]. insulin secretion and insulin resistance, as well as environmental factors such as obesity, overeating, a lack of exercise, stress, and aging. However, type 1 diabetes is the result of an autoimmune reaction to proteins of the islets cells of the pancreas [2]. Since 2019, an estimated 463 million people (8.8% of the adult population) have been diagnosed with diabetes; type II diabetes represents about 85% to 95% of all diabetes cases in developed countries, with an even higher percentage in developing nations [3,4].
α-Amylase is a calcium-based metalloenzyme that degrades saccharides into maltose by cleaving α (1-4) glycosidic bonds. It is abundant in human pancreatic juice and saliva, as well as in the saliva of some other mammals [5]. The small intestine contains some αglucosidases, such as the hexamer-based hydrolyze enzyme, which converts polysaccharides and disaccharides into glucose by processing the non-reducing terminal 1,4-linked α-glucose residues. In diabetes, the inhibition of these two enzymes becomes very important as it reduces the digestion rate of carbohydrates, resulting in less glucose absorption in the bloodstream [6]. Three drugs are commercially available for diabetes: naturally derived glycosidase inhibitor molecules (acarbose, miglitol, and voglibose), as well as many other types of drugs [7] (Figure 1).
There appears to be a strong direct correlation between antioxidant and anti-diabetic effects; this relationship may be attributed to the significant role of oxidative stress in hyperglycemia and overall diabetic complications [8]. The evaluated levels of blood glucose are combined with the overproduction of free radicals "reactive oxygen species" [8]. In addition, part of the immunogenic damage to beta cells of the pancreas is mediated by ROS [9]. Therefore, natural and synthetic antioxidant compounds, e.g., phenolics and flavonoids, are promising candidates for the protection, treatment, and management of diabetic complications [10,11].  There appears to be a strong direct correlation between antioxidant and anti-diabetic effects; this relationship may be attributed to the significant role of oxidative stress in hyperglycemia and overall diabetic complications [8]. The evaluated levels of blood glucose are combined with the overproduction of free radicals "reactive oxygen species" [8].
In addition, part of the immunogenic damage to beta cells of the pancreas is mediated by ROS [9]. Therefore, natural and synthetic antioxidant compounds, e.g., phenolics and flavonoids, are promising candidates for the protection, treatment, and management of diabetic complications [10,11].
Furthermore, free radical scavengers and chelators have been reported for their potential role in diabetes management, such as reducing the vascular relaxation and neurovascular impairments associated with hyperalgesia, improving endoneurial blood flow, and overall reducing the production of OH-and the initiation of lipid peroxidation associated with vascular reactivity impairment and nerve function in diabetes [12]. Benzimidazole species have an excellent structural range that covers large biological and therapeutic application fields. The research describing the importance of structures containing benzimidazole cores has been extensively developed and possesses a wide range of bioactive activities, including antifungal [13], antitubercular [14,15], antiulcer and antimicrobial [16], antileishmanial [17], antiglycation and antioxidant [18], antimycobacterial [19], anti-HIV [20], antitumor [21], antiproliferative [22], anti-inflammatory [23,24], antiviral [25], and antihelminthic activities [26]. Several candidates of this class were tested for anti-tumor properties and showed activities against several tumor cell lines, including breast cancer, human cells, and human lung cancer cells [27][28][29][30][31][32][33].
Following the previous scientific report, we elaborated herein a new target structure, containing both benzimidazole and diarylurea moieties in one component. Our purpose was to combine their properties, which represent a new route to improve the effectiveness of bioactive molecules ( Figure 2).
There is a critical need to find better drugs to treat type I and type II diabetes. The discovery of new α-glucosidase and α-amylase inhibitor agents is an interesting method of obtaining such drugs. To contribute to the advancement of the design and the discovery of new bioactive species [66][67][68][69][70][71], we combined the two patterns, benzimidazole and urea, in the same molecule, allowing for potential antidiabetic agents, and having good anti-oxidant activity.
To estimate the interaction with α-amylase and α-glucosidase enzymes, an in silico molecular docking study of benzimidazole-urea was performed. There is a critical need to find better drugs to treat type I and type II diabetes. The discovery of new α-glucosidase and α-amylase inhibitor agents is an interesting method of obtaining such drugs. To contribute to the advancement of the design and the discovery of new bioactive species [66][67][68][69][70][71], we combined the two patterns, benzimidazole and urea, in the same molecule, allowing for potential antidiabetic agents, and having good antioxidant activity.
A simple route to novel hybrid benzimidazole urea compounds 3a-h was utilized from the reaction of 2-(1H-benzimizadol-2-yl) aniline 1 and appropriate isocyanates substrates 2a-h. The evaluation of antioxidant, α-amylase, and α-glucosidase activities was assessed. To estimate the interaction with α-amylase and α-glucosidase enzymes, an in silico molecular docking study of benzimidazole-urea was performed.

Chemistry
The synthesis of target benzimidazole-ureas 3a-f was realized from commercially available 2-(1H-benzoimidazol-2-yl)aniline 1 and six different isocyanates 2a-f. In a first attempt, the reaction of 2-(1H-benzoimidazol-2-yl)aniline 1 and naphthylisocyanate 2b in anhydrous acetone produced the expected benzimidazole-urea 3b with a moderate yield of 60% (Scheme 1). To enhance the performance of this synthetic method, different reaction conditions were studied. The principal reaction parameters, including the solvent and number of equivalent reagents, were varied using the same substrates (1 and 2b). Among the investigated solvents: tetrahydrofuran, acetone, dichloromethane, and mixtures of dichloromethane/acetone, this latter mixture, in the respective proportions of 80/20, gave the best results with total substrate conversion and a 90% reaction yield.
As a result of optimizing the equivalent number of the substrate 2b suitable for the reaction process, the substrate was fully converted with 1.5 equivalents of the isocyanate 2b.
1,4-phenelenediisocyanate 2g and 1,4-bis(isocyanatomethyl)cyclohexane 2h reacted with two equivalents of substrate 1 to afford the corresponding products 3g and 3h in good yields, 93% and 85%, respectively (Scheme 2). To enhance the performance of this synthetic method, different reaction conditions were studied. The principal reaction parameters, including the solvent and number of equivalent reagents, were varied using the same substrates (1 and 2b). Among the investigated solvents: tetrahydrofuran, acetone, dichloromethane, and mixtures of dichloromethane/acetone, this latter mixture, in the respective proportions of 80/20, gave the best results with total substrate conversion and a 90% reaction yield.
As a result of optimizing the equivalent number of the substrate 2b suitable for the reaction process, the substrate was fully converted with 1.5 equivalents of the isocyanate 2b. 1,4-phenelenediisocyanate 2g and 1,4-bis(isocyanatomethyl)cyclohexane 2h reacted with two equivalents of substrate 1 to afford the corresponding products 3g and 3h in good yields, 93% and 85%, respectively (Scheme 2).
were studied. The principal reaction parameters, including the solvent and number of equivalent reagents, were varied using the same substrates (1 and 2b). Among the investigated solvents: tetrahydrofuran, acetone, dichloromethane, and mixtures of dichloromethane/acetone, this latter mixture, in the respective proportions of 80/20, gave the best results with total substrate conversion and a 90% reaction yield.
As a result of optimizing the equivalent number of the substrate 2b suitable for the reaction process, the substrate was fully converted with 1.5 equivalents of the isocyanate 2b.
The reactions progressed easily, offering good yields of the proposed substrates 3ah, and the results are collected in Table 1. It is worth noting that the reaction yields of the aryl substrates were higher than those of the alkyl and cycloalkyl ones. The low reaction yields as well as the rates of the reactions observed with alkyl and cycloalkyl isocyanate Scheme 2. Synthesis of compounds 3g and 3h from 2-(1H-benzoimidazol-2-yl)aniline 1.
The reactions progressed easily, offering good yields of the proposed substrates 3a-h, and the results are collected in Table 1. It is worth noting that the reaction yields of the aryl substrates were higher than those of the alkyl and cycloalkyl ones. The low reaction yields as well as the rates of the reactions observed with alkyl and cycloalkyl isocyanate substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity.
The structure of novel prepared benzimidazole urea was determined by spectroscopic data from 1 H and 13 C NMR, FT-IR, and HRMS. The IR spectra of compound 3a showed the absorption at 3249 cm −1 related to the NH group of the benzimidazole ring. The spectra also displayed stretching at 1672, 1586, and 1536 cm −1 assigned to (C=O), (C=N), and (C=C), respectively.
The 1 H NMR spectra of compound 3a showed a band at 13.08 ppm attributed to the NH group of benzimidazole. The amino proton of the urea group resonated at 12.02 ppm. Aromatic protons resonated in the range of 9.69-7.01 ppm. The compound 3a was identified with 13 C NMR. The spectra exhibited a singlet at 153.2 ppm corresponding to the carbon atom of the urea carbonyl group. In addition, the carbon of the benzimidazole ring appeared at 151.45 ppm. The high-resolution mass spectra of compound 3a revealed the presence of a molecular peak with m/z: 328.  substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. substrates may be due principally to the low reactivity of such radicals, which exert an inductive effect that decreases the isocyanate carbon electrophilicity. The structure of novel prepared benzimidazole urea was determined by spectroscopic data from 1 H and 13 C NMR, FT-IR, and HRMS. The IR spectra of compound 3a showed the absorption at 3249 cm −1 related to the NH group of the benzimidazole ring. The spectra also displayed stretching at 1672, 1586, and 1536 cm −1 assigned to (C=O), (C=N), and (C=C), respectively.
The 1 H NMR spectra of compound 3a showed a band at 13.08 ppm attributed to the NH group of benzimidazole. The amino proton of the urea group resonated at 12.02 ppm. Aromatic protons resonated in the range of 9.69-7.01 ppm. The compound 3a was identified with 13 C NMR. The spectra exhibited a singlet at 153.2 ppm corresponding to the carbon atom of the urea carbonyl group. In addition, the carbon of the benzimidazole ring appeared at 151.45 ppm. The high-resolution mass spectra of compound 3a revealed the presence of a molecular peak with m/z: 328.12280 attributed to [M] + compatible with the formula for C20H16N4O (see Supplementary Materials).

Antioxidant Activity
Four in vitro assays were used to determine the antioxidant activity of the compounds 3a-h. The methods used provide information about the compounds' reducing capacity (TAC and FRAP methods), scavenging capability (DPPH-SA), and metal chelating activity (MCA). Table 2 gives the antioxidant levels for the compounds, measured in a comparable manner using three different in vitro mechanisms, namely, reducing, scavenging, and metal chelating activities. The structure of novel prepared benzimidazole urea was determined by spectroscopic data from 1 H and 13 C NMR, FT-IR, and HRMS. The IR spectra of compound 3a showed the absorption at 3249 cm −1 related to the NH group of the benzimidazole ring. The spectra also displayed stretching at 1672, 1586, and 1536 cm −1 assigned to (C=O), (C=N), and (C=C), respectively.
The 1 H NMR spectra of compound 3a showed a band at 13.08 ppm attributed to the NH group of benzimidazole. The amino proton of the urea group resonated at 12.02 ppm. Aromatic protons resonated in the range of 9.69-7.01 ppm. The compound 3a was identified with 13 C NMR. The spectra exhibited a singlet at 153.2 ppm corresponding to the carbon atom of the urea carbonyl group. In addition, the carbon of the benzimidazole ring appeared at 151.45 ppm. The high-resolution mass spectra of compound 3a revealed the presence of a molecular peak with m/z: 328.12280 attributed to [M] + compatible with the formula for C20H16N4O (see Supplementary Materials).

Antioxidant Activity
Four in vitro assays were used to determine the antioxidant activity of the compounds 3a-h. The methods used provide information about the compounds' reducing capacity (TAC and FRAP methods), scavenging capability (DPPH-SA), and metal chelating activity (MCA). Table 2 gives the antioxidant levels for the compounds, measured in a comparable manner using three different in vitro mechanisms, namely, reducing, scavenging, and metal chelating activities. The structure of novel prepared benzimidazole urea was determined by spectroscopic data from 1 H and 13 C NMR, FT-IR, and HRMS. The IR spectra of compound 3a showed the absorption at 3249 cm −1 related to the NH group of the benzimidazole ring. The spectra also displayed stretching at 1672, 1586, and 1536 cm −1 assigned to (C=O), (C=N), and (C=C), respectively.
The 1 H NMR spectra of compound 3a showed a band at 13.08 ppm attributed to the NH group of benzimidazole. The amino proton of the urea group resonated at 12.02 ppm. Aromatic protons resonated in the range of 9.69-7.01 ppm. The compound 3a was identified with 13 C NMR. The spectra exhibited a singlet at 153.2 ppm corresponding to the carbon atom of the urea carbonyl group. In addition, the carbon of the benzimidazole ring appeared at 151.45 ppm. The high-resolution mass spectra of compound 3a revealed the presence of a molecular peak with m/z: 328.12280 attributed to [M] + compatible with the formula for C20H16N4O (see Supplementary Materials).

Antioxidant Activity
Four in vitro assays were used to determine the antioxidant activity of the compounds 3a-h. The methods used provide information about the compounds' reducing capacity (TAC and FRAP methods), scavenging capability (DPPH-SA), and metal chelating activity (MCA). Table 2 gives the antioxidant levels for the compounds, measured in a comparable manner using three different in vitro mechanisms, namely, reducing, scavenging, and metal chelating activities. The structure of novel prepared benzimidazole urea was determined by spectroscopic data from 1 H and 13 C NMR, FT-IR, and HRMS. The IR spectra of compound 3a showed the absorption at 3249 cm −1 related to the NH group of the benzimidazole ring. The spectra also displayed stretching at 1672, 1586, and 1536 cm −1 assigned to (C=O), (C=N), and (C=C), respectively.
The 1 H NMR spectra of compound 3a showed a band at 13.08 ppm attributed to the NH group of benzimidazole. The amino proton of the urea group resonated at 12.02 ppm. Aromatic protons resonated in the range of 9.69-7.01 ppm. The compound 3a was identified with 13 C NMR. The spectra exhibited a singlet at 153.2 ppm corresponding to the carbon atom of the urea carbonyl group. In addition, the carbon of the benzimidazole ring appeared at 151.45 ppm. The high-resolution mass spectra of compound 3a revealed the presence of a molecular peak with m/z: 328.12280 attributed to [M] + compatible with the formula for C20H16N4O (see Supplementary Materials).

Antioxidant Activity
Four in vitro assays were used to determine the antioxidant activity of the compounds 3a-h. The methods used provide information about the compounds' reducing capacity (TAC and FRAP methods), scavenging capability (DPPH-SA), and metal chelating activity (MCA). Table 2 gives the antioxidant levels for the compounds, measured in a comparable manner using three different in vitro mechanisms, namely, reducing, scavenging, and metal chelating activities. Four in vitro assays were used to determine the antioxidant activity of the compounds 3a-h. The methods used provide information about the compounds' reducing capacity (TAC and FRAP methods), scavenging capability (DPPH-SA), and metal chelating activity (MCA). Table 2 gives the antioxidant levels for the compounds, measured in a comparable manner using three different in vitro mechanisms, namely, reducing, scavenging, and metal chelating activities. In the series of benzimidazole ureas 3a-h, compound 3g demonstrated the highest reducing power for molybdenum (VI) with 10.06 ± 1.51 mM trolox equivalents in the total antioxidant capacity (TAC). The results of reducing activity also revealed that some com- pounds reduced molybdenum (VI) ions selectively in the TAC. For instance, compounds 3b, 3c, and 3f showed significant molybdenum (VI) reducing activity compared to other compounds at levels of 5.16 ± 0.69, 4.43 ± 0.53, and 8.50 ± 0.87 mM trolox equivalents.
Regarding Table 2, the results of the ferric reducing antioxidant power (FRAP) of compounds 3a-h indicated higher activities of derivatives 3a and 3c with 9.25 ± 0.20 and 8.65 ± 0.29 mM trolox equivalents, respectively. The most potent compound 3g yielded the best results, with 16.12 ± 0.29 mM trolox equivalents.
The results of DPPH-SA of hybrid benzimidazole urea revealed the potency of derivatives 3d and 3g as free radical scavengers. Compound 3d presented good activity with 2.89 ± 1.07 mM trolox equivalents. Compound 3g also gave an excellent result with 3.86 ± 0.04 mM trolox equivalents. The other compounds exerted moderate DPPH-SA activity at values ranging from 1.70 to 0.96 mM trolox equivalents.
The metal chelating activity (MCA) of compounds 3a-h manifested potential activity for the compound 3h at the MCA level of 2.95 ± 0.21 mM EDTA equivalents due to the presence of two urea groups. In addition, compounds 3a-c exhibited moderate MCA in the range of 1.36-1.62 mM EDTA equivalents.
The overall antioxidant activities of compounds 3a-h confirmed that the majority manifested excellent to moderate activities. Benzimidazole-urea 3b and 3c displayed good activity with the three-antioxidant methods TAC, FRAP, and MCA. Targeted compound 3g represented the most derivatives in this family, manifesting excellent activity in three methods: TAC, FRAP, and DPPH-SA. It is worth noting that compound 3g also had significant activity for the TAC method, with 10.06 ± 1.51 mM trolox equivalents. Compound 3g also gave higher activity with 16.12 ± 0.29 mM trolox equivalents for the FRAP method. Concerning the DPPH-SA method, compound 3g also gave the best results with 3.86 ± 0.04 mM trolox equivalents.

α-Amylase and α-Glucosidase Inhibition Activity
All the synthesized novel hybrid benzimidazole urea compounds 3a-h were subjected for the evaluation of their possible α-amylase and α-glucosidase inhibitory activities. It is worth noting that the majority of the analogs were found to have good to moderately active IC 50 values ranging from 18.65 ± 0.23 to 28.33 ± 0.02 µM for α-amylase inhibition and with the IC 50 values ranging from 17.47 ± 0.03 to 29.01 ± 0.12 µM for α-glucosidase inhibition except compounds 3a, 3b, and 3h. The inhibitory activities of α-amylase and α-glucosidase, according to Table 3, revealed that the tested compounds in the current study demonstrate higher activities than those reported in previous studies [47,72]. Table 3. α-amylase and α-glucosidase inhibition activity of benzimidazole-urea 3a-h.
Indeed, the whole molecule played an important role in the inhibitory potential; however, limited SAR was rationalized by emphasizing the effects of varying structural features such as different substitutions on nitrogen of the amide side chain of urea, on the inhibitory potential in terms of IC50 values.
Derivative 3c having a methoxy phenyl group displayed a significant α-amylase and α-glucosidase inhibitory activity. Substrate 3e with an isopropyl group and 3g substituted with a di-phenyl urea group on nitrogen also exhibited good inhibitory potential compared to standard acarbose.
In summary, in the series of aromatic substituents, compounds containing phenyl group 3a and naphthyl group 3b exhibited a weak activity for inhibition of both α-glucosidase and α-amylase. The excellent results were shown with target analog 3c bearing 4-methoxyphenyl, showing remarkable inhibitory action against both α-amylase and α-glucosidase. The inclusion of a methoxy substituent in the phenyl group at the para-position significantly improved activity, which might be attributed to the favorable interaction for the inhibition activity of both α-amylase and α-glucosidase (Figure 4). interaction for the inhibition activity of both α-amylase and α-glucosidase (Figure 4).
In the series of alkane and cycloalkane, it is clear that compounds 3d and 3f exhibited higher potential activity in comparison with aromatic compounds 3a and 3b. This observation is explained based on the donor effect of cyclohexyl and butyl groups when compared to phenyl or naphthyl groups. The same result was observed with compound 3e bearing an isopropyl group manifesting a good inhibition of enzyme α-amylase and αglucosidase. The above observation was interpreted by the sufficient donor effect of the isopropyl group, which exerted a favorable interaction and demonstrated the highest activity. Compound 3g also indicated a strong inhibition of α-amylase and α-glucosidase compared with 3d and 3f. The presence of two phenylbenzimidazoles in the structure of compound 3g allowed favorable interactions involving a good inhibition activity of αamylase and α-glucosidase (Figure 4).

In Silico Docking Study
The objective of the in-silico study was to clarify and rationalize the type of interactions of the new benzimidazole urea 3a-h in the target catalytic site. Molecular docking is a useful tool in the design of structure-based drugs. The optimized structures of the most potent benzimidazole urea 3c, 3e, and 3g used in the docking study are presented in Figure 5. We notice that the most stable structures for 3c and 3e had a cis structure with intramolecular interactions. The most stable structure of 3g had almost a Ci symmetry also with intramolecular interactions. In the series of alkane and cycloalkane, it is clear that compounds 3d and 3f exhibited higher potential activity in comparison with aromatic compounds 3a and 3b. This observation is explained based on the donor effect of cyclohexyl and butyl groups when compared to phenyl or naphthyl groups. The same result was observed with compound 3e bearing an isopropyl group manifesting a good inhibition of enzyme α-amylase and α-glucosidase. The above observation was interpreted by the sufficient donor effect of the isopropyl group, which exerted a favorable interaction and demonstrated the highest activity. Compound 3g also indicated a strong inhibition of α-amylase and α-glucosidase compared with 3d and 3f. The presence of two phenylbenzimidazoles in the structure of compound 3g allowed favorable interactions involving a good inhibition activity of α-amylase and α-glucosidase ( Figure 4).

In Silico Docking Study
The objective of the in-silico study was to clarify and rationalize the type of interactions of the new benzimidazole urea 3a-h in the target catalytic site. Molecular docking is a useful tool in the design of structure-based drugs. The optimized structures of the most potent benzimidazole urea 3c, 3e, and 3g used in the docking study are presented in Figure 5. We notice that the most stable structures for 3c and 3e had a cis structure with intramolecular interactions. The most stable structure of 3g had almost a Ci symmetry also with intramolecular interactions.
Our study was interested in explaining the interactions of the most effective analogs molecules 3c, 3e, and 3g manifesting appropriate interactions (2D) and (3D) with the different amino acids illustrated in Figures 6 and 7. Due to their high inhibition activity amongst molecules, the structures 3c, 3e, and 3g were chosen for docking studies with 5E0F. Table 4  Benzimidazole urea derivatives showed good values of binding energies with αamylase (HPA) human pancreatic and also the human lysosomal acid-α-glucosidase (HLAG) ( Table 4). Our study was interested in explaining the interactions of the most effective analogs molecules 3c, 3e, and 3g manifesting appropriate interactions (2D) and (3D) with the different amino acids illustrated in Figures 6 and 7. Due to their high inhibition activity amongst molecules, the structures 3c, 3e, and 3g were chosen for docking studies with 5E0F. Table 4 reveals that compounds 3c, 3e, and 3g with potent inhibitory activities occupied suitable positions at the binding center of HPA, with energies of −8.8, −8.0, and −11.2 kcal.mol −1 , respectively.

Conclusions
A prominent series of benzimidazole-urea analogs 3a-h was designed and synthesized from 2-(1H-benzoimidazol-2-yl) aniline 1 and the appropriate isocyanates 2a-h. The antioxidant and antidiabetic activities of the target benzimidazole-ureas 3a-h were evaluated. When measured using four different methods, the antioxidant data revealed that the most desired benzimidazole-urea exhibited high activity. The results indicated that among the series, compounds 3a and 3b displayed good inhibition with three methods: FRAP, MCA, and DPPH-SA. The most potent antioxidant compound was 3g, which demonstrated strong activity in all four methods. Furthermore, the ability of benzimidazole-urea scaffolds to inhibit α-glycosidase and α-amylase was investigated. According to our predictions, the majority of tested compounds exhibited good to moderate activities. The results indicated that compounds 3e and 3g exhibited good inhibition for both enzymes, α-amylase, and α-glucosidase. Compound 3c bearing the 4-methoxy moiety on the phenyl group at the para-position was found to be a more potent inhibitor of α-amylase, and α-glucosidase similar to the standard drug acarbose. The study confirms that compounds 3c, 3e, and 3g have strong in vitro antioxidant properties, as well as α-glucosidase and α-amylase inhibition activities comparable to those of acarbose, suggesting that they could be used as anti-diabetic medications.

General Details
Without any further purification, all commercially available reagents were used. Unless otherwise specified, all reactions were carried out in a dry nitrogen-protected atmosphere using oven-dried glassware. FT-IR spectra were recorded on an Agilent (Agilent Technologies, Inc. Headquarters, address: 5301 Stevens Creek Blvdv, Santa Clara, CA 95051, USA) apparatus type Cary 600 in the range of 400-4000 cm −1 . A Bruker (Bruker Daltonics Inc., Bremen, Germany) NMR spectrometer Avance III HD 400 MHz was used to analyze 1 H and 13 C NMR spectra at 400 and 100 MHz, respectively. All spectra were recorded and referenced to TMS using DMSO d 6 as the solvent. According to an internal standard of residual DMSO d 6 at 2.50 ppm, chemical shifts in 1 H NMR spectra were mentioned in parts per million (ppm) on the scale. Spin multiplicities are denoted by the letters s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Chemical shifts in 13 C NMR spectra were also indicated in ppm from the peak position of DMSO d 6 (39.52 ppm).
The values of the coupling constant J are given in hertz (Hz). High-resolution mass spectra were recorded on an Agilent 6545 LC/Q-TOF MS (Agilent Technologies, Inc. Headquarters, address: 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA). Using the Stuart SMP30 apparatus (New Jersey 07740, États-Unis), the melting points in open capillary tubes were determined. For analytical TLC, Silica Gel 60 F254 plates (Sigma 40-60 m) were employed, and the chromatogram was developed using a UV lamp 254 nm. DMSO-d 6 (99.9 atom % D; CAS no. 2206-27-1) were purchased from Sigma-Aldrich and used without further purification.

Chemistry: Benzimidazole-Ureas 3a-h Synthesis: General Procedure
The reaction was carried out under a nitrogen atmosphere. In a 100 mL round bottom flask, 0.52 g (2.5 mmol) of 2-(1H-benzoimidazol-2-yl) aniline was placed in 20 mL of dichloromethane and 5 mL of acetone, and the appropriate isocyanate (3.75 mmol) was added dropwise in 20 mL of dichloromethane and 5 mL of acetone. The reaction was stirred under reflux. TLC and diethyl ether/hexane (50/50) as the eluent were used to monitor the reaction progress (see Table 3). After filtering the reaction mixture, the raw solid was purified through recrystallization from ethanol (see Supplementary Materials). An amount of 2 mL of freshly prepared 0.6 M solution of sulfuric acid, a solution of ammonium molybdate (4 mM), and a buffer solution of sodium phosphate (28 mM) were placed in small glass test tubes and thoroughly mixed with the compound solution (200 µL in DMSO with a concentration of 1 mM) [76]. An amount of 2 mL of molybdate reagent was mixed with 200 µL of distilled water to make a blank test. The cylinders were warmed to 85 • C for 1.5 h before cooling to ambient temperature. At 695 nm, the produced blue color was assessed with a UV spectrophotometer, with the blank reading having to serve as the auto-zero point. The TAC results were calculated using the Trolox standard calibration curve.

DPPH Scavenging Activity (DPPH-SA)
The ability of the compounds to scavenge free radicals was tested against DPPH-stable free radicals. The degree of reduction in the DPPH violet color is directly proportional to the scavenging efficacy of the compounds. According to the method of Aroua et al. [52], 1 mL of the DMSO solutions of the tested compounds (1 mM final concentration) was thoroughly mixed in test tubes with the DPPH solution prepared at the concentration of 300 µM in methanol. After 30 min at room temperature, the reduction in DPPH-violet color was measured at 517 nm. The DPPH-SA of the compounds was calculated using the DPPH-Trolox calibration curve.
Iron Chelating Activity Assay (MCA) Using the method of Zengin et al. [78] with modification, the ability of compounds to chelate iron compared to the EDTA was estimated. To inchoate the color, a compound solution in DMSO (2 with a final concentration of 1 mM) plus ferrous chloride (25 µL, 2 mM, FeCl 2 ) was added to 100 µL of ferrozine. At 562 nm, the absorbance of the mixture was measured in comparison to a blank (prepared similarly to a test without ferrozine). The chelating activity of the extract was calculated in equivalents to the EDTA standard calibration curve.

α-Amylase Inhibition Assay
The α-amylase inhibition activity was measured using the previously reported methodologies [52]. An amount of 500 µL of test sample (10-1000 M) and 500 µL of substrate α-amylase (source: Aspergillus oryzae) solution (0.5 mg/mL) in 0.2 mM phosphate buffer (pH 6.9) were incubated for 10 min at 25 • C. Following pre-incubation, 500 µL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added and incubated for 10 min at 25 • C. An amount of 1 mL of dinitro salicylic acid color reagent was used to stop the reaction. The tubes were then placed in boiling water for 5 min before being cooled to room temperature. After diluting the solutions with 10 mL of distilled water, the absorbance at 540 nm was measured. The standard medication was acarbose [79]. The percentage of enzyme inhibition was calculated using the following equation: % of Enzyme Inhibition = (Absorbance of control -Absorbance of compound) × 100 Absorbance of control (1) The IC 50 values, concentrations necessary to reduce α-amylase activity by 50%, were measured and used Graph Pad Prism Software and a non-linear correlation graph plotting percentage inhibition (x axis) versus concentrations (y axis) (Version 5).

α-Glucosidase Inhibition Assay
The inhibition of α-glucosidase activity was measured using a modified version of a previously published method [80]. In 100 mL of phosphate buffer (pH 6.8) containing 200 mg of bovine serum albumin, one milligram of α-glucosidase (Saccharomyces cerevisiae, Sigma-Aldrich, St. Louis, MO, USA) was dissolved (Merck, Darmstadt, Germany). The reaction mixture was made up of 10 µL of sample at various concentrations (10-1000 M), 490 µL of phosphate buffer pH 6.8, and 250 µL of 5 mM p-nitrophenyl-D-glucopyranoside (Sigma-Aldrich, Buchs, Switzerland). After a 5 min preincubation at 37 • C, 250 µL of α-glucosidase (0.15 unit/mL) was added and incubated at 37 • C for 15 min. The reaction was stopped by adding 2000 µL Na 2 CO 3 200 mM. α-glucosidase activity was measured using a spectrophotometer UV-Vis (Shimadzu 265, Kyoto, Japan) at 400 nm by measuring the amount of p-nitrophenol released from p-NPG. As a positive control for α-glucosidase inhibitor, acarbose was used [81]. The IC 50 value was defined as the concentration of the extract required to inhibit 50% of α-glucosidase activity under the assay conditions. Equation (1) was used to calculate the percentage of inhibition.

Statistical Analysis
The IC 50 values, concentrations necessary to inhibit the α-amylase and α-glucosidase activities by 50%, were determined using Graph Pad Prism Software (Version 5) and a nonlinear regression graph plotting percentage inhibition (x axis) versus concentrations (y axis).

Molecular Docking
The new benzimidazole-urea 3a-h and their interactions with HPA and HLAG were studied by molecular docking in order to clarify the appropriate position for the ligands in the catalytic site of the proteins. We imported HPA (PDB code: 5E0F) and HLAG (PDB code: 5NN8) from the Protein Data Bank. Molecules of water and co-crystallized ligand from the receptors were removed. AutoDockTools1.5.2 (ADT) was used to add Gasteiger charges and polar hydrogens to the files, which were then converted to PDBQT format [82,83]. A docking grid was chosen by ADT. The grid box in HPA was centered at x = −7.946 Å, y = 10.438 Å, and z = −21.863 Å using a grid of x = 80, y = 72, and z = 66 points. The grid box for HLAG was centered at x = −12.175 Å, y = −35.415 Å, and z = 88.753 Å using a grid of x = 74, y = 70, and z = 90 points. A spacing of 0.375 Å was chosen.
The optimization of the compounds was performed using a conjugate gradient AMMP implemented in VEGA ZZ software [84]. The file format was converted from PDB to PDBQT using ADT. The docking calculations were performed by AutoDock Vina software [85] with an exhaustiveness parameter of 32. ADT allowed the analysis of the conformations obtained from the docking calculations. The ligand-receptor interaction types were determined using Discovery Studio Visualizer [86].