Benzimidazole derivatives as potent α-amylase inhibitors: synthesis, characterization and in vitro α-amylase inhibition

ABSTRACT

Aim

This study aims to evaluate the α-amylase inhibitory potential of newly synthesized benzimidazole derivatives, assessing their viability as prospective antidiabetic agents.

Materials & methods

A series of 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-phenylacetamide derivatives (7a–7j) were synthesized via an efficient synthetic route. The structural elucidation of these compounds was accomplished using advanced spectroscopic techniques, including mass spectrometry, FT-IR, ¹H & ¹³C NMR, and elemental analysis. The α-amylase inhibitory activity of the synthesized compounds was evaluated in vitro, with IC₅₀ values determined to quantify their efficacy. To gain insights into the molecular interactions, molecular docking studies were conducted, followed by extensive molecular dynamics (MD) simulations.

Result & discussion

All synthesized derivatives exhibited varying degrees of α-amylase inhibitory activity, with IC₅₀ values ranging from 1.10 ± 0.05 to 12.50 ± 0.30 μM. Notably, compounds 7b, 7c, and 7i demonstrated superior inhibitory effects, with IC₅₀ values of 1.20 ± 0.05, 1.40 ± 0.10, and 1.10 ± 0.05 μM, respectively, surpassing the standard drug acarbose (IC₅₀ = 1.70 ± 0.10 μM).

Conclusion

The synthesized benzimidazole derivatives, notably compounds 7b, 7c, and 7i, demonstrated potent α-amylase inhibitory activity, surpassing the standard drug acarbose. These findings highlight their potential as lead compounds for developing novel antidiabetic agents.

GRAPHICAL ABSTRACT

1. Introduction

Diabetes mellitus is a chronic, non-communicable disorder marked by impaired carbohydrate, protein, and lipid metabolism, leading to hyperglycemia [1]. It primarily results from inadequate insulin secretion. Previously, it caused significant mortality, affecting around 387 million people [2], and is projected to impact 366 million by 2030 [3,4]. Complications include cardiovascular diseases, neuropathy, and retinopathy [5,6]. The enzymes α-amylase and α-glucosidase contribute to this condition by breaking down carbohydrates into glucose, raising blood sugar levels. α-amylase converts polysaccharides into starch and maltose, while α-glucosidase converts disaccharides into glucose [7]. Common treatments include sulfonylureas, biguanides, and α-glucosidase inhibitors like acarbose, miglitol, and voglibose. However, side effects such as nausea, dizziness, and hypoglycemia limit their use [8]. Acarbose is preferred due to its dual enzyme inhibition [9].

Nitrogen-based heterocycles, particularly benzimidazoles, have gained attention for their resemblance to bioactive natural molecules and their potential as chemotherapeutic agents with diverse clinical applications [10]. As a bioactive scaffold, the benzimidazole nucleus is involved in various biological functions, including its role in vitamin B12, highlighting its physiological significance [11]. It is a crucial pharmacophore in drug design, showing a wide range of biological activities [12]. In modern drug discovery, benzimidazole derivatives are of great interest due to their broad therapeutic potential. These include anticancer [13,14], antiulcer [15,16], antifungal [17,18], antihypertensive [19,20], antiviral [21,22], antiparasitic [23,24], antidiabetic [25–27], analgesic [28,29], and antimalarial activities [30,31]. Additionally, they show promise as anticonvulsants [32,33], anti-inflammatory agents [34,35], anticoagulants [36,37], and in treating neurodegenerative disorders [38]. Figure S1 presents marketed drugs featuring the benzimidazole nucleus. Their clinical approval and therapeutic success underscore the practical relevance of benzimidazole-based compounds in modern medicine.

In our design of benzimidazole derivatives, we were inspired by existing α-amylase inhibitors featuring benzimidazole based structures. Champa et al [39] synthesized benzimidazole derivatives 1a and 1b exhibiting IC₅₀ values of 64.74–65.14 µg/mL for α-amylase, comparable to acarbose. Rai PV et al [40] reported chalcone-based benzimidazole 2 with superior activity with IC₅₀: 20.47 ± 0.60 µg/mL for α-amylase. Khan et al [41] reported benzimidazole derivative 3 with superior activity with IC₅₀: 3.50 ± 0.20 µM for α-amylase. In another work, Khan et al [42] introduced thiadiazole-benzimidazole derivative 4 with remarkable potency, achieving IC₅₀ values as low as 1.10 µM for α-amylase, surpassing acarbose (Figure 1).

Figure 1.

Reported benzimidazole derivatives with α-amylase inhibitory potential.

The present investigation centers on synthesizing and thoroughly characterizing 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-phenylacetamide derivatives using o-Phenylenediamine and Isonipecotic acid. The synthesized compound underwent lead discovery and optimization processes to explore its potential biological activities. The outcomes of this research significantly advance our understanding of the compound’s attributes and highlight its promising potential for future development in biological applications.

2. Materials and methods

2.1. Chemistry

The reagents used for all synthetic steps were procured commercially from Sigma-Aldrich, Spectrochem, Loba Chemie, and Merck and employed as received, without any additional purification. The progress of each reaction was tracked by thin-layer chromatography on aluminum sheets precoated with silica gel 60 F₂₅₄. Infrared spectra were recorded on a Shimadzu FTIR-8400 spectrometer using the KBr pellet technique. Mass spectral data were acquired with a Shimadzu GCMS-QP2010 SE system via direct injection probe analysis. Proton (¹H) NMR spectra were collected on a Bruker Avance III 400 MHz instrument, employing tetramethylsilane (TMS) as the internal chemical shift reference. Similarly, carbon-13 nuclear magnetic resonance (¹³C NMR) spectra were acquired on the same instrument, utilizing TMS as the internal reference.

2.1.1. Synthesis of 2-(piperidin-4-yl)-1H-benzo[d]imidazole 3

A reaction mixture, consisting of piperidine-4-carboxylic acid(0.01 mol), o-phenylenediamine(0.01 mol), and toluene(10 mL), was blended with ρ-TsOH(1 equiv) serving as a catalyst. The resulting mixture underwent reflux for a duration of 2–3 h. Following the cooling phase, the mixture was subjected to filtration and subsequent drying, culminating in the production of the compound 3.

2.1.2. Synthesis of 2-chloro-N-phenylacetamide derivatives (6a-6j)

Various substituted amines (1 equiv) dissolved in acetone underwent the gradual addition of chloroacetylchloride (1 equiv). The resultant mixture was stirred for 2–3 h at rt. Following this, the reaction mixture was carefully poured onto crushed ice, leading to the formation of a solid intermediate product. The solid was separated through filtration, washed with water, and subsequently dried. The ensuing intermediate product was then utilized in the subsequent step of the synthesis without undergoing any additional purification. We followed the same synthetic parameters and procedures described by Kangad et al [43].

2.1.3. Synthesis of substituted 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-phenylacetamide (7a-7j)

2-chloro-N-phenylacetamide derivatives (6a-6j) (1 equiv) was introduced into a 100 mL round-bottom flask containing Compound 3 (1 equiv) and acetonitrile (2 volumes) as the solvent. Subsequently, K₂CO₃ (1.2 equiv) was added as a base. The reaction mixture was stirred at room temperature for 10–15 minutes, followed by refluxing for 5–6 h. The progress of the reaction was monitored using Thin Layer Chromatography (TLC). Upon completion of the reaction, the mixture was cooled and filtered to obtain a solid product. The solid product was washed with cooled acetonitrile solvent, followed by a water wash, resulting in the formation of the desired compound.

2.1.4. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(2-nitrophenyl)acetamide (7a)

Pale yellow; Yield: 91%; Chemical Formula: C₂₀H₂₁N₅O₃; mp 224 °C; FT-IR (KBr, cm⁻¹): 3627 (−NH), 3050 (=CH, str), 2941 (-CH, str), 1687 (C=O, amide), 1537 and 1348 (NO₂, str), 1454 (−CH Ben.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H, benzimidazole), 10.24 (s, 1H, amide), 8.71 (s, 1H, Ar. ring), 8.05–8.03 (d, J = 8.4, 1H, Ar. ring), 7.94–7.92 (d, J = 8.4, 1H, Ar. ring), 7.63–7.61 (d, J = 8.0, 1H, Ar. ring), 7.53–7.52 (d, J = 6.4, 1H, Ar. ring), 7.41–7.40 (d, J = 7.6, 1H, Ar. ring), 7.14–7.08 (m, 2H, Ar. ring), 3.22 (s, 2H, methylene), 2.99 (q, 2H, piperidine), 2.88–2.81 (m, 1H, piperidine), 2.38–2.32 (q, 2H, piperidine), 2.02–1.93 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.47, 157.81, 147.89, 143.04, 139.80, 134.21, 130.04, 125.55, 121.42, 120.76, 118.24, 117.88, 113.64, 110.75, 62.09, 52.98, 39.50, 35.33, 30.18; MS (m/z) 380.27 (M⁺); micro analysis: Calc. C, 63.31; H, 5.58; N, 18.46; Found. C, 63.16; H, 5.42; N, 18.21.

2.1.5. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(3-nitrophenyl)acetamide (7b)

Pale yellow; Yield: 93%; Chemical Formula: C₂₀H₂₁N₅O₃; mp 228 °C; FT-IR (KBr, cm⁻¹): 3277 (−NH), 2938 (=CH, str), 2815 (-CH, str), 1698 (C=O, amide), 1580 and 1347 (NO₂, str), 1432 (−CH Ben.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.20 (s, 1 H, benzimidazole), 11.53 (s, 1 H, amide), 8.64–8.62 (d, J = 8.4, 1H, Ar. ring), 8.21–8.19 (d, J = 8.4, 1H, Ar. ring), 7.80–7.76 (t, 1H, Ar. ring), 7.57–7.55 (d, J = 6.0, 1H, Ar. ring), 7.43–7.41 (d, J = 7.2, 1H, Ar. ring), 7.33–7.31 (d, J = 8.8, 1H, Ar. ring), 7.15–7.09 (m, 2H, Ar. ring), 3.24 (s, 2H, methylene), 3.02 (q, 2H, piperidine), 2.92–2.86 (m, 1H, piperidine), 2.44 (q, 2H, piperidine), 2.11–1.97 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.01, 157.58, 143.06, 137.08, 135.65, 134.23, 133.56, 125.68, 123.62, 121.73, 120.77, 118.30, 110.78, 62.04, 53.17, 39.50, 35.00, 30.27; MS (m/z) 380.27 (M⁺); micro analysis: Calc. C, 63.31; H, 5.58; N, 18.46; found. C, 63.16; H, 5.42; N, 18.21.

2.1.6. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(4-nitrophenyl)acetamide (7c)

Off white, Yield: 89%; Chemical Formula: C₂₀H₂₁N₅O₃; mp 218 °C; FT-IR (KBr, cm⁻¹): 3638 (−NH), 3052 (=CH, str), 2946 (-CH, str), 1710 (C=O, amide), 1536 and 1349 (NO₂, str), 1456 (−CH Ben.); ¹H NMR (400 MHz, DMSO-d₆) δ 12.17 (s, 1H, benzimidazole), 10.36 (s, 1H, amide), 8.24–8.22 (d, J = 9.2, 2H, Ar. ring), 7.94–7.92 (d, J = 9.2, 2H, Ar. ring), 7.54–7.52 (d, J = 6.8, 1H, Ar. ring), 7.41–7.40 (d, J = 6.4, 1H, Ar. ring), 7.14–7.08 (m, 2H, Ar. ring), 3.25 (s, 2H, methylene), 2.98 (q, 2H, piperidine), 2.88–2.82 (m, 1H, piperidine), 2.38–2.33 (q, 2H, piperidine), 2.04–1.91 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.66, 157.81, 144.85, 142.27, 124.84, 120.91, 119.09, 62.10, 52.94, 39.50, 35.35, 30.24; MS (m/z) 380.27 (M⁺); micro analysis: Calc. C, 63.31; H, 5.58; N, 18.46; found. C, 63.16; H, 5.42; N, 18.21.

2.1.7. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(o-tolyl)acetamide (7d)

Off white, Yield: 84% Chemical Formula: C₂₁H₂₄N₄O, mp 180 °C, FT-IR (KBr, cm⁻¹): 3292 (−NH), 3051 (=CH, str), 2934 (-CH, str), 1665 (C=O, amide), 1451 (−CH Ben.), ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H, benzimidazole), 9.62 (s, 1H, amide), 7.53–7.51 (d, J = 8.4, 3H, Ar. ring), 7.418–7.407 (d, J = 4.4, 1H, Ar. ring), 7.13–7.09 (m, 4H, Ar. ring), 3.13 (s, 2H, methylene), 2.97 (q, 2H, piperidine), 2.88–2.81 (m, 1H, piperidine), 2.35–2.30 (q, 2H, piperidine), 2.25 (s, 3H, methyl), 2.03–1.91 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.18, 157.81, 136.06, 132.27, 129.03, 119.37, 62.04, 53.00, 39.50, 35.37, 30.31, 20.43; MS (m/z) 349.29 (M⁺), micro analysis: Calc. C, 72.39; H, 6.94; N, 16.08; found. C, 72.14; H, 6.71; N, 15.98.

2.1.8. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide (7e)

Off white, Yield: 87% Chemical Formula: C₂₁H₂₄N₄O, mp 174 °C, FT-IR (KBr, cm⁻¹): 3286 (−NH), 2942 (=CH, str), 2823 (-CH, str), 1656 (C=O, amide), 1454 (−CH Ben.), ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H, benzimidazole), 9.62 (s, 1H, amide), 7.54–7.52 (d, J = 6.8, 1H, Ar. ring), 7.46–7.40 (m, 3H, Ar. ring), 7.20–7.18 (d, J = 7.6, 1H, Ar. ring), 7.12–7.08 (m, 2H, Ar. ring), 6.88–6.87 (d, J = 6.8, 1H, Ar. ring), 3.24 (s, 2H, methylene), 2.97 (q, 2H, piperidine), 2.88–2.82 (m, 1H, piperidine), 2.37 (q, 2H, piperidine), 2.26 (s, 3H, methyl), 2.00–1.94 (m, 4 H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.41, 157.86, 138.47, 137.92, 128.53, 124.10, 121.39, 119.88, 116.54, 62.09, 53.02, 39.50, 35.40, 30.37, 21.13; MS (m/z) 349.29 (M⁺), micro analysis: Calc. C, 72.39; H, 6.94; N, 16.08; found. C, 72.14; H, 6.71; N, 15.98.

2.1.9. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(p-tolyl)acetamide (7f)

Off white, Yield: 86% Chemical Formula: C₂₁H₂₄N₄O, mp 172 °C, FT-IR (KBr, cm⁻¹): 3664 (−NH), 3288 (=CH, str), 2819 (-CH, str), 1681 (C=O, amide), 1454 (−CH Ben.),¹H NMR (400 MHz, DMSO-d₆) δ 12.19 (s, 1 H, benzimidazole), 9.42 (s, 1H, amide), 7.85–7.83 (d, J = 9.6, 1H, Ar. ring), 7.55–7.53 (d, J = 9.2, 1H, Ar. ring), 7.42–7.41 (d, J = 6.0, 1H, Ar. ring), 7.23–7.03 (m, 5H, Ar. ring), 3.17 (s, 2H, methylene), 3.03 (q, 2H, piperidine), 2.91–2.85 (m, 1H, piperidine), 2.42 (q, 2H, piperidine), 2.26 (s, 3H, methyl), 2.10–1.90 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.09, 157.66, 136.05, 130.25, 128.47, 126.27, 124.22, 121.73, 61.90, 53.15, 39.49, 35.19, 30.62, 17.40; MS (m/z) 349.29 (M⁺), micro analysis: Calc. C, 72.39; H, 6.94; N, 16.08; found. C, 72.14; H, 6.71; N, 15.98.

2.1.10. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(4-chlorophenyl)acetamide (7 g)

Off white, Yield: 94%, Chemical Formula: C₂₀H₂₁ClN₄O, mp 194 °C, FT-IR (KBr, cm⁻¹): 3101 (−NH), 2952 (=CH, str), 2831 (-CH, str), 1587 (C=O, amide), 1455 (−CH Ben.), 759 (C-Cl, str), ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H, benzimidazole), 9.87 (s, 1H, amide), 7.71–7.70 (d, J = 2.0, 1H, Ar. ring), 7.68–7.67 (d, J = 2.8, 1H, Ar. ring), 7.52–7.51 (d, J = 1.2, 1H, Ar. ring), 7.41–7.35 (m, 3H, Ar. ring), 7.14–7.09 (m, 2H, Ar. ring), 3.16 (s, 2H, methylene), 2.96 (q, 2H, piperidine), 2.85–2.81 (m, 1H, piperidine), 2.35 (q, 2H, piperidine), 2.03–1.94 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.71, 157.81, 137.58, 128.52, 126.90, 121.00, 62.05, 52.98, 39.50, 35.35, 30.25; MS (m/z) 369.2 (M⁺), micro analysis: Calc. C, 65.12; H, 5.74; N, 15.19, found. C, 64.76; H, 5.62; N, 15.02.

2.1.11. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(4-bromophenyl)acetamide (7 h)

Off white, Yield: 90%, Chemical Formula: C₂₀H₂₁BrN₄O, mp 246 °C, FT-IR (KBr, cm⁻¹): 3291 (−NH), 2934 (=CH, str), 2769 (-CH, str), 1676 (C=O, amide), 1455 (−CH Ben.), 668 (C-Br, str), ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H, benzimidazole), 9.87 (s, 1H, amide), 7.67–7.65 (d, J = 5.2, 1H, Ar. ring), 7.64–7.63 (d, J = 2.0, 1H, Ar. ring), 7.51–7.48 (m, 4H, Ar. ring), 7.14–7.09 (t, 2H, Ar. ring), 3.16 (s, 2H, methylene), 2.97 (q, 2H, piperidine), 2.87–2.81 (m, 1H, piperidine), 2.35 (q, 2H, piperidine), 2.04–1.94 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ168.74, 157.81, 138.00, 131.44, 121.38, 120.77, 118.22, 114.93, 110.75, 62.09, 52.98, 39.50, 35.36, 30.25; MS (m/z) 413.21 (M⁺), micro analysis: Calc. C, 58.12; H, 5.12; N, 13.56; found. C, 57.80; H, 4.97; N, 13.36.

2.1.12. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(4-fluorophenyl)acetamide (7i)

Off white, Yield: 84%, Chemical Formula: C₂₀H₂₁FN₄O, M.P. mp 188 °C, FT-IR (KBr, cm⁻¹): 3665 (−NH), 2934 (=CH, str), 2770 (-CH, str), 1666 (C=O, amide), 1453 (−CH Ben.), 1212 (C-F, str), ¹H NMR (400 MHz, DMSO-d₆) δ 12.18 (s, 1H, benzimidazole), 9.78 (s, 1H, amide), 7.70–7.69 (d, J = 4.4, 1H, Ar. ring), 7.67–7.65 (d, J = 4.8, 1H, Ar. ring), 7.54–7.52 (d, J = 6.8, 1H, Ar. ring), 7.42–7.41 (d, J = 6.4, 1H, Ar. ring), 7.14–7.08 (m, 4H, Ar. ring), 3.15 (s, 2H, methylene), 2.97 (q, 2H, piperidine), 2.86–2.81 (m, 1H, piperidine), 2.35 (q, 2H, piperidine), 1.99–1.87 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.43, 159.25, 157.82, 156.86, 143.03, 135.00, 134.22, 121.29, 120.76, 118.24, 115.28, 110.75, 62.05, 53.00, 39.50, 35.37, 30.64, 30.26; MS (m/z) 353.29 (M⁺), micro analysis: Calc C, 68.16; H, 6.01; N, 15.90; found. C, 68.03; H, 5.89; N, 5.76.

2.1.13. 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-(2-chloro-3-methylphenyl)acetamide (7j)

Off white, Yield: 82% Chemical Formula: C₂₁H₂₃ClN₄O, mp 234 °C, FT-IR (KBr, cm⁻¹): 3589 (−NH), 2922 (=CH, str), 2850 (-CH, str), 1624 (C=O, amide), 1447 (−CH Ben.), 782 (C-Cl, str), ¹H NMR (400 MHz, DMSO-d₆) δ 12.17 (s, 1H, benzimidazole), 9.59 (s, 1H, amide), 7.68–7.66 (d, J = 7.6, 1H, Ar. ring), 7.54–7.52 (d, J = 6.8, 1H, Ar. ring), 7.42–7.40 (d, J = 8.0, 1H, Ar. ring), 7.32–7.31 (d, J = 3.2, 1H, Ar. ring), 7.26–7.08 (m, 3H, Ar. ring), 3.18 (s, 2H, methylene), 3.03 (q, 2H, piperidine), 2.88–2.86 (m, 1H, piperidine), 2.41–2.35 (q, 2H, piperidine), 2.28 (s, 3H, methyl), 2.07–1.91 (m, 4H, piperidine); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.51, 157.68, 137.63, 133.55, 127.99, 127.05, 125.27, 122.07, 61.81, 53.12, 39.50, 35.21, 30.48, 14.51; MS (m/z) 383.20 (M⁺), micro analysis: Calc. C, 65.88; H, 6.05; N, 14.63, found. C, 65.52; H, 5.93; N, 14.38.

2.2. In silico studies/computational studies

Computational analyses were performed on a Linux-based workstation equipped with a 16-core (hyper-threaded) processor and an NVIDIA Titan V GPU. Ligand structures were generated using OpenBabel (version 3.1.1) [44] and molecular docking studies were conducted with AutoDock Vina (version 1.2.5) [45,46]. Physicochemical properties, absorption, distribution, metabolism, and excretion (ADME) parameters, along with toxicity predictions, were evaluated using the pkCSM web server [47] and DataWarrior [48] software. Binding interactions between the most promising ligands and the active site residues were examined in detail using the Protein – Ligand Interaction Profiler (PLIP) web tool [49,50]. Molecular dynamics simulations of the synthesized compounds were carried out using the MD simulations software Desmond/Maestro non-commercial version 2023.4 from DE Shaw Research.

2.2.1. Docking studies

The X-ray crystal structure of porcine α-amylase (PDB ID: 1OSE) was obtained from the Protein Data Bank (rcsb.org) [51–53] for docking studies, with a resolution of less than 2.0 Å. Protein preparation was performed using UCSF Chimera (v. 1.17.3) [54], where all solvent molecules and ions were removed, highest occupancy conformations retained, and incomplete side chains were modeled based on the Dunbrack 2010 rotamer library [55]. Hydrogens were added to satisfy valencies, and Kollman charges were assigned to the protein. A docking grid measuring 40 Å in each dimension (x, y, z) was centered on the co-crystallized ligand acarbose, with a grid spacing of 1.0 Å, using AutoDock Vina. The exhaustiveness parameter for exploring binding orientations within the grid was set to 512. Benzimidazole derivatives were prepared for docking via OpenBabel (v. 3.1.1), generating three-dimensional conformations, adding hydrogens at physiological pH (7.4) with appropriate ionization states, and assigning EEM partial charges [56]. The best docking poses of the synthesized compounds in complex with α-amylase were analyzed for ligand – binding site interactions using the Protein – Ligand Interaction Profiler (PLIP).

2.2.2. Molecular dynamics simulations

The synthesized compounds in their optimal docked conformations complexed with α-amylase were subjected to molecular dynamics simulations. The inhibitor-bound α-amylase complexes were placed in an orthorhombic simulation box containing TIP3P water molecules, buffered with a 10 Å margin along the x, y, and z axes. The orientation of the complexes within the box was optimized by minimizing the simulation box volume to reduce computational expense. All simulation energies were calculated using the OPLS force field. Sodium and chloride ions were added to neutralize the system’s net charge and to maintain a physiological salt concentration of 0.15 M. The solvated enzyme-inhibitor complexes underwent an initial relaxation using the built-in protocol for a brief equilibration period before the main 100 ns production run. Simulations were performed with Desmond/Maestro non-commercial version 2023.4, saving trajectory and energy data every 100 ps. The simulations were conducted under the NPT ensemble at 300 K and 1 bar, with temperature and pressure controlled via coupling to a Langevin thermostat [57] and barostat [58], using relaxation times of 1.0 ps and 2.0 ps respectively, and isotropic coupling for the barostat. Subsequent trajectory analyses evaluated protein backbone and ligand atomic fluctuations using metrics such as RMSD and RMSF.

2.3. Biological screening

The activity of α-amylase inhibition was determined using a slightly modified assay based on the method by Kwon et al. All the synthesized compounds (7a-7j) were thoroughly dissolved in dimethyl sulfoxide (DMSO). A mixture containing 40 μL of the sample solution and 40 μL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) with α-amylase solution (0.5 mg/mL) was incubated at 25 ℃ for 10 minutes. Subsequently, 40 μL of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to each tube at 5-second intervals. The reaction mixtures were incubated again at 25 ℃ for an additional 10 minutes. To terminate the reaction, 100 μL of dinitrosalicylic acid color reagent was added. The test tubes were then placed in a boiling water bath for 5 minutes and subsequently cooled to room temperature. The reaction mixture was diluted with 900 μL of distilled water, and the absorbance was measured at 540 nm. The percentage of inhibition was calculated using the following formula:

$$\mathrm{\%}\mathit{\hspace{0.17em}}\mathit{Inhibition}\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\left[\left(A_c-\mathit{\hspace{0.17em}}{A}_s\right)/A_c\right]\mathit{\hspace{0.17em}}\times \mathit{\hspace{0.17em}}100\mathit{\hspace{0.17em}}\mathrm{\%}$$

Where: A_c = absorbance of the control

A_s = absorbance of the sample

3. Result & discussion

3.1. Chemistry

In this research, we synthesized a series of new 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-phenylacetamide derivatives (7a-7j) (Table S1) from 2-chloro-N-phenylacetamide derivatives (6a-6j) and 2-(piperidin-4-yl)-1H-benzo[d]imidazole 3 using acetonitrile as a solvent and K₂CO₃ as a base through the process discussed above (Figure 2).

Figure 2.

Synthetic pathway for the synthesis of piperidine-benzimidazole derivatives (7a-7j).

All the synthesized compounds were characterized by advanced characterization techniques such as Mass spectrometry, FT-IR, and ¹H & ¹³C NMR spectroscopy. e.g., Mass spectrum of representative compound, Compound 7a shows molecular ion peak [M+H]⁺ at 380.24 m/z. Further, FT-IR analysis revealed several significant spectral features indicative of specific functional groups within the molecule under investigation. A broad peak observed at 3627 cm⁻¹ corresponded to the N-H stretching vibration, thereby confirming the presence of a secondary amine group. Additionally, distinct signals at 3050 cm⁻¹ and 2941 cm⁻¹ were consistent with the stretching vibrations associated with alkene and alkane-like C-H bonds, respectively. The presence of an amide group was substantiated by a carbonyl stretching band observed at 1687 cm⁻¹ in the spectrum. Furthermore, signals observed at 1537 & 1348 cm⁻¹ provided evidence for the presence of a Nitro group within the molecule. Lastly, a distinct signal at 1454 cm⁻¹ represents C-H bending vibration, within the molecular structure. Further, Different types of protons present in the molecule were distinguished through ¹H NMR spectroscopic analysis. In the ¹H NMR spectrum of compound 7a, a singlet corresponding to a single proton was observed at 12.16 δppm, indicative of the N-H proton of benzimidazole ring. Additionally, a singlet at 10.24 δppm was attributed to the proton of N-H of amide functional group. Protons associated with the aromatic ring resonated within the range of 8.71–7.08 δppm. Furthermore, a singlet at 3.22 δppm was attributed to the methylene group. Aliphatic protons of piperidine ring resonated within the range of 2.99–1.93 δppm. Further, Different types of carbons present in the molecule were distinguished through ¹³C NMR spectroscopic analysis. In the ¹³C NMR spectrum of compound 7a, a signal at 169.47 δppm was observed indicative of carbonyl carbon of amide functional group. Carbons associated with the aromatic ring resonated within the range of 157.81–110.75 δppm. Furthermore, a signal at 62.09 δppm was attributed to the methylene group adjacent to amide functional group. Aliphatic protons of piperidine ring resonated within the range of 52.98–30.18 δppm.

3.2. In silico studies/computational studies

3.2.1. Molecular docking studies

Molecular docking is a pivotal tool in structure-based drug design, enabling the prediction of interactions between small molecules and target proteins. This computational approach is both cost-effective and efficient, providing valuable insights into ligand-receptor binding affinities. In this study, benzimidazole derivatives (7a–7j) were docked into the active site of α-amylase, exhibiting binding energies ranging from −9.64 to −8.87 kcal/mol as shown in Table 1. These values are comparable to the standard inhibitor acarbose, which demonstrated a binding energy of −10.38 kcal/mol. However, small score differences may not reflect biological significance. Docking provides an initial affinity estimate, but factors like protein flexibility and solvent effects can impact activity. The in vitro α -amylase inhibition data support the potential of these compounds as effective inhibitors.

Table 1.

The predicted binding of synthesized compounds in terms of docking scores (kcal/mol).

Compound No.	Docking Score (kcal/mol)	Compound No.	Docking Score (kcal/mol)
7a	−9.50	7 g	−8.89
7b	−9.64	7 h	−8.89
7c	−9.12	7i	−8.87
7d	−9.30	7j	−9.53
7e	−9.51	Acarbose	−10.38
7f	−9.30

The interactions of compounds with the binding site amino acid residues were analyzed using the web-based server − PLIP (Protein – Ligand Interaction Profiler) for representative compounds 7a and 7b as shown in Figure 3 and Table 2 as compared to acarbose. The binding site interactions analysis of the inhibitors with porcine α-amylase revealed that the compounds exhibit hydrophobic interactions with Trp58, Tyr62, Gln63, Tyr151, Val163, Leu165, Lys200, and Ile235 and hydrogen bond with His201. Furthermore compounds also formed π-cation interactions with His201 and salt bridges with Asp197, Glu233, and Asp300. Few compounds formed π-stacking with Trp59, and His201.

Figure 3.

The interactions of compounds 7a & 7b with the binding site amino acid residues.

Table 2.

Binding site interactions analysis of synthesized compounds (represented by 7a and 7b) along with acarbose for the target enzyme porcine α-amylase.

Compound Nos.	Hydrophobic Interactions	Hydrogen Bond Interactions	Others
7a 7c, 7d, 7f, 7 g, 7 h, 7i	Trp58, Tyr62, Gln63, Tyr151, Val163, Leu165, Lys200, Ile235	His201	π-Cation Interactions: His201 Salt Bridges: Asp197, Glu233, Asp300
7b, 7e	Trp58, Trp59, Tyr62, Val163, Leu165, Lys200, Ile235	–	π-Stacking: Trp59, His201 Salt Bridges: Asp197, Asp300
7j	Trp59, Tyr62, Tyr151, Val163, Leu165, Lys200, Ile235	–	π-Stacking: Trp59, His201 Salt Bridges: Asp300
acarbose	Trp59	Gln63, His101, Ser105, Gly106, Arg195, His201, Glu233, His299, His305	–

3.2.2. Molecular dynamics simulations

While molecular docking predicts potential binding modes of inhibitors within target sites, the stability and dynamics of these interactions require validation through extended molecular dynamics (MD) simulations. In this study, the binding poses of synthesized compounds 7a and 7b with porcine α-amylase were subjected to 100 ns MD simulations using the Desmond/Maestro non-commercial version 2023.4. Structural stability was assessed by analyzing root mean square deviation (RMSD) and root mean square fluctuation (RMSF) over 1000 simulation frames, as detailed in Table 3.

Table 3.

Maxima, minima and averages of the RMSD and RMSF computed over MD simulation trajectories for compounds 7a and 7b in complex with the target enzyme porcine α-amylase.

Compound ID	Parameters		7a	7b
RMSD (Å)	Protein Backbone	Maximum	1.87	2.22
		Minimum	0.93	0.87
		Average	1.42	1.71
	Ligand	Maximum	7.425	6.506
		Minimum	0.790	1.129
		Average	4.649	2.912
RMSF (Å)	Protein Backbone	Maximum	3.69	4.13
		Minimum	0.35	0.39
		Average	0.74	0.83
	Ligand	Maximum	3.62	2.66
		Minimum	0.63	0.72
		Average	1.69	1.50

Root Mean Square Deviation (RMSD) quantifies the average displacement of atoms between corresponding frames in a molecular dynamics trajectory relative to a reference structure. It is computed using the formula:

$$\mathit{RMS}{D}_x=\sqrt{\frac{1}{N}\sum _{\mathit{i}=1}^N\left(r_i^{\prime }\left(t_x\right)\right)-r_i\left(t_{\mathit{ref}}\right){)}^2}$$

Where N represents the number of atoms included in the selection; t_ref denotes the reference time, commonly taken as the first frame, which is assigned as time t = 0; and r’ corresponds to the position of the chosen atoms in frame ×following alignment to the reference frame, with frame ×recorded at time t_x. This process is conducted for each frame throughout the simulation trajectory.

The atomic deviations observed during the molecular dynamics (MD) simulations for compounds 7a and 7b in complex with porcine α-amylase were evaluated using Root Mean Square Deviation (RMSD). The RMSD values for the protein backbone, calculated over 1000 trajectory frames, are presented in Figure 4. Similarly, the atomic deviations of the bound ligands are depicted in the same figure. The porcine α-amylase backbone exhibited atomic fluctuations ranging from 0.93 to 2.22 Å, with an average of 1.57 Å across the compounds. In contrast, the ligand atoms displayed fluctuations between 0.79 and 7.43 Å, averaging 3.78 Å. Notably, compound 7b demonstrated an RMSD consistently below 3.0 Å, indicating a stable binding conformation throughout the simulation.

Figure 4.

The atomic fluctuations shown by the compounds 7a and 7b.

Root Mean Square Fluctuation (RMSF) is useful for characterizing the local changes along the protein chain and is calculated per residue using the formula:

$$\mathit{RMS}{F}_i=\sqrt{\frac{1}{T}\sum _{\mathit{t}=1}^T⟨\left(r_1^{\prime }\left(\mathit{t}\right)\right)-r_i\left(t_{\mathit{ref}}\right){)}^2⟩}$$

Where T represents the trajectory duration over which the RMSF is determined; t_ref denotes the reference time; r_i indicates the position of residue i; r’ refers to the coordinates of atoms within residue i after alignment to the reference structure. The angle brackets signify that the mean squared displacement is averaged over all atoms within the residue.

Root Mean Square Fluctuation (RMSF) analysis elucidates the structural integrity of the protein and the flexibility of amino acid residues during molecular dynamics simulations. The RMSF values for porcine α-amylase in complex with compounds 7a and 7b were calculated over 1000 trajectory frames obtained during a 100 ns MD simulation, as depicted in Figure 4. Amino acid residues located in the terminal regions and loop regions exhibited higher fluctuations, whereas residues situated in the core and those interacting with the ligands demonstrated lower RMSF values. The RMSF calculated for porcine α-amylase amino was arranged between 0.35–4.13 Å. The atomic fluctuations shown by the compounds 7a and 7b is depicted in Figure 4, was observed between 0.63–2.66 Å.

During the simulations the interactions of the compounds 7a and 7b with the binding site amino acids of the porcine α-amylase were observed in docking studies were retained during the simulations. The interactions are depicted in Figure 5.

Figure 5.

The interactions of the compound 7a & 7b with the binding site amino acids of the porcine α-amylase.

4. Drug likeness and ADMET study

The pharmacokinetic properties of the synthesized molecules were evaluated using the pkCSM web server. Various molecular attributes pertinent to drug-likeness were analyzed, including molecular weight (MW) ≤ 500, the number of hydrogen bond donors (≤5) and acceptors (≤10), as well as the partition coefficient (LogP) ≤ 5. These parameters were assessed to predict the drug-like behavior of the compounds. As shown in Table 4, all the compounds well corroborated with the drug likeness properties without any violation.

Table 4.

The calculated drug-likeness properties of the synthesized compounds (7a-7j).

Compound	Molecular weight	LogP	Rotatable Bonds	H-Bond Acceptors	H-Bond Donors	Surface Area	Lipinski Violation
7a	379.42	3.2892	5	5	2	161.272	No
7b	379.42	3.2892	5	5	2	161.272	No
7c	379.42	3.2892	5	5	2	161.272	No
7d	348.45	3.68942	4	3	2	152.984	No
7e	348.45	3.68942	4	3	2	152.984	No
7f	348.45	3.68942	4	3	2	152.984	No
7 g	368.86	4.0344	4	3	2	156.922	No
7 h	413.319	4.1435	4	3	2	160.486	No
7i	352.413	3.5201	4	3	2	150.784	No
7j	382.895	4.34282	4	3	2	163.287	No
Acarbose	645.608	−8.7219	12	19	14	250.281	Yes

The role of cytochrome P450 (CYP) enzymes in the biotransformation and elimination of drugs is well-documented. Inhibition of CYP enzymes often leads to impaired drug elimination, resulting in drug-induced toxicity. Therefore, in silico studies of the most active compounds with CYP enzymes can provide insights into their potential drug transformation and elimination pathways.

In this study, we investigated the inhibition of cytochrome P450 by compounds 7a-7j using in silico methods. The findings indicated that all the synthesized compounds exhibited no significant potential to inhibit CYP2D6 and CYP2C9 enzymes while only some compounds exhibited potential to inhibit CYP2C19 and CYP3A4 as shown in Table S2. These results suggest that compounds with minimal inhibitory effects on CYP isoforms are likely to possess favorable metabolism and elimination profiles, reducing the risk of drug-induced toxicity.

The toxicity profiles of all synthesized compounds (7a-7j) were evaluated using the pkCSM web server. This tool predicts toxicity profiles against multiple targets and parameters, including hERG I/II inhibition, oral rat acute and chronic toxicity (LD50 and LOAEL), hepatotoxicity, skin sensitization, AMES toxicity (Salmonella/microsome mutagenicity), and minnow toxicity.

As shown in Table S3, the synthesized compounds exhibited no toxicity in terms of skin sensitization. Furthermore, compounds 7a-7j demonstrated similar oral rat acute toxicity to standard drugs, with LD50 values ranging from 2.501 to 2.619. The predicted oral rat chronic toxicity (LOAEL) for these compounds ranged from 1.858 to 2.617. Additionally, many of the synthesized compounds were identified as non-inhibitors of hERG-I, indicating a non-cardiotoxic profile, although they did inhibit hERG-II. Despite the prediction of hepatotoxicity, other toxicity parameters such as AMES, minnow toxicity, and skin sensitization were found to be favorable for this class of compounds.

4.1. Biological screening

All the synthesized compounds then underwent for their in-vitro α-amylase inhibitory activity by taking acarbose as a standard drug. The results showed that all the synthesized compounds showed variable degree of inhibition with IC₅₀ values ranging from 1.10 ± 0.05 to 12.50 ± 0.30 as shown in Table 5. More concisely, Compounds 7b, 7c & 7i showed excellent potential with IC₅₀ values of 1.20 ± 0.05, 1.40 ± 0.10, and 1.10 ± 0.05 μM respectively, when compared to standard drug acarbose (IC₅₀ = 1.70 ± 0.10 μM). All other compounds exhibited good to moderated potential.

Table 5.

α-amylase inhibitory potential of newly synthesized benzimidazole derivatives (7a-7j).

Compounds	−R	IC50, μM	Compounds	−R	IC50, μM
7a	2-NO2	2.70 ± 0.20	7 g	4-Cl	3.20 ± 0.10
7b	3-NO2	1.20 ± 0.05	7 h	4-Br	12.50 ± 0.30
7c	4-NO2	1.40 ± 0.10	7i	4-F	1.10 ± 0.05
7d	2-CH3	4.90 ± 0.20	7j	3-CH3-2-Cl	5.70 ± 0.20
7e	3-CH3	7.90 ± 0.20	Acarbose	–	1.70 ± 0.10
7f	4-CH3	3.10 ± 0.10

4.1.1. Structure-activity relationship

A comparative evaluation of IC₅₀ values and docking scores revealed that the type and position of substituents significantly influence α-amylase inhibitory activity.

Within the nitro-substituted series, the compound bearing 3-NO₂ group exhibited the highest α-amylase inhibitory potency, with an IC₅₀ value of 1.20 ± 0.05 µM, surpassing that of the reference standard acarbose (IC₅₀ = 1.70 ± 0.10 µM). This compound also showed a strong binding affinity, as reflected by a docking score of –9.64 kcal/mol. The compounds having 2-NO₂ and 3-NO₂ groups demonstrated slightly diminished activity (IC₅₀ = 2.70 ± 0.20 and 1.40 ± 0.10 µM, respectively), consistent with somewhat lower docking scores (–9.50 and –9.14 kcal/mol, respectively). These observations suggest that a nitro group at the meta position is particularly favorable for enhancing both binding affinity and inhibitory efficacy.

Methyl-substituted analogues displayed a general reduction in inhibitory activity. The 3-CH₃ derivative showed the weakest activity in the series (IC₅₀ = 7.90 ± 0.20 µM), despite a relatively strong docking score (–9.51 kcal/mol). This discrepancy may be attributed to steric hindrance or unfavorable binding orientation. The 2-CH₃ and 4-CH₃ analogues exhibited moderate inhibitory effects (IC₅₀ = 4.90 ± 0.20 and 3.10 ± 0.10 µM, respectively) and similar docking scores (–9.30 kcal/mol), indicating that the position of the methyl group plays a key role in influencing biological activity.

Halogenated derivatives substituted at the para position yielded varied results. The 4-F derivative showed excellent activity (IC₅₀ = 1.10 ± 0.05 µM) despite a comparatively lower docking score (–8.87 kcal/mol), suggesting the involvement of favorable electrostatic or hydrogen bonding interactions within the enzyme’s active site. The 4-Cl compound demonstrated moderate activity (IC₅₀ = 3.20 ± 0.10 µM, docking score: –8.89 kcal/mol), whereas the 4-Br analogue was the least active in this group (IC₅₀ = 12.50 ± 0.30 µM), possibly likely due to its larger size hindering effective binding. 3-CH₃-2-Cl compound displayed moderate activity (IC₅₀ = 5.70 ± 0.20 µM) and a favorable docking score (–9.53 kcal/mol). While each substituent may support binding individually, their combination could introduce steric interference.

In summary, electron-withdrawing groups like 3-NO₂ and 4-F enhance α-amylase inhibition, highlighting the importance of both electronic properties and substituent positioning in optimizing enzyme binding.

5. Conclusion

In this research, we synthesized a series of new 2-(4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)-N-phenylacetamide derivatives (7a-7j) from 2-chloro-N-phenylacetamide derivatives (6a-6 u) and 2-(piperidin-4-yl)-1H-benzo[d]imidazole (3). Structures of all the synthesized compounds were confirmed using advance spectroscopic techniques such as Mass spectrometry, FT-IR spectroscopy, and ¹H & ¹³C NMR spectroscopy. In silico study of synthesized compounds showed that all the ligands snugly fit into the binding site of α-amylase. Further, the synthesized compounds were evaluated for their in vitro α-amylase inhibitory activity and in the result we found variable degree of inhibition with IC₅₀ values ranging from 1.10 ± 0.05 to 12.50 ± 0.30 μM. More precisely, Compounds 7b, 7c & 7i showed excellent potential with IC₅₀ values of 1.20 ± 0.05, 1.40 ± 0.10, and 1.10 ± 0.05 μM respectively, when compared to standard drug acarbose (IC₅₀ = 1.70 ± 0.10 μM). So, we can conclude those molecules for further research. The predicted drug-likeness and toxicity properties of the synthesized compounds indicate that they are nontoxic and potentially safer agents.

Funding Statement

This paper was not funded.

Article highlights

• Efficient synthesis and spectral characterization of a library of benzimidazole derivatives

• In silico molecular docking study showed strong α-amylase inhibition

• Compound with Flouro substitution showed highest α-amylase inhibitory potential

• Benzimidazole derivatives as promising antidiabetic agents

Authorship contributions

Shantaben K. Kangad: Conceptualization, Methodology, Investigation, Analysis of all Data, Writing – original draft. V. N. Patolia: Project administration, Conceptualization, Writing – review & approval of original draft. Sachin M. Sitapara, Chintan Somaiya: Resources, Formal analysis. Deepika Maliwal, Raghuvir R.S. Pissurlenker : Molecular Docking studies and the subsequent analysis There-off

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties

Writing assistance

No writing assistance was utilized in the production of this manuscript

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2527585