Benzimidazole Scaffold: Advances In Structural Modifications And Diverse Pharmacological Activities

Benzimidazole constitutes a prominent class of nitrogen-containing heteroaromatic compounds exhibiting extensive biological and pharmacological significance. Structurally, it is characterized by a fused bicyclic system comprising a benzene ring condensed with an imidazole moiety bearing two nitrogen atoms ^[2]. The presence of these heteroatoms imparts unique electronic distribution, hydrogen-bonding capacity, and amphoteric behavior, which collectively contribute to its broad spectrum of biological interactions. Heterocyclic compounds are ubiquitous in nature and play indispensable roles in cellular metabolism and biochemical processes ^[3]. Benzimidazole derivatives demonstrate diverse pharmacokinetic and pharmacodynamic properties, making them valuable scaffolds in medicinal chemistry ^[1]. A wide range of biological activities has been reported, including anti-inflammatory, antimicrobial, antiviral, anticancer, anticonvulsant, antidiabetic, and antioxidant effects ^[2]. Additionally, benzimidazole-based compounds exhibit inhibitory activity against the Zika virus, function as α-glucosidase inhibitors, antitubercular and antiglycation agents, NOX-2 antagonists, antileukemic agents, and possess antihypertensive properties. These multifaceted activities highlight the versatility of the benzimidazole core as a privileged pharmacophore. Several clinically approved drugs incorporate the benzimidazole nucleus, such as Candesartan (antihypertensive), Albendazole (antimicrobial), Bendamustine, Nocodazole, Abemaciclib (antitumor), Enviradene (antiviral), Omeprazole (antiulcer), and Benoxaprofen analogues (anti-inflammatory) ^[4,5]. The successful integration of this scaffold into marketed therapeutics underscores its structural and pharmacological relevance. Furthermore, transition metal complexes of benzimidazole derivatives have been extensively investigated as biologically active molecular models. In several instances, metal coordination enhances biological efficacy compared to the corresponding free ligands ^[6]. Due to its structural resemblance to nucleotide bases, the benzimidazole framework can act as a bioisostere, facilitating interactions with biologically derived polymers and enabling the development of compounds with improved therapeutic profiles and reduced toxicity. The chemical stability and synthetic flexibility of the benzimidazole nucleus allow for extensive structural modification and lead optimization. Consequently, it serves as a robust template for the generation of novel derivatives with enhanced biological potential, contributing significantly to modern drug discovery ^[7].

Figure 1. Chemical structures of benzimidazole scaffolds

BIOLOGICAL ACTIVITY OF BENZIMIDAZOLE-BASED COMPOUNDS

Antimicrobial agents are classified according to their target microorganisms, such as antibacterial and antifungal drugs. Based on their mechanism of action, they are further categorized as microbicidal agents, which eliminate microorganisms, and microbiostatic agents, which inhibit microbial growth. Benzimidazole derivatives exhibit notable antimicrobial activity, primarily attributed to their structural similarity to purine bases ^[3]. This resemblance enables them to interfere with essential microbial processes, including nucleic acid and protein synthesis. In rational drug design, the development of novel antimicrobial agents often involves incorporating structural features of biologically active heterocycles. The fusion of pyrimidine and imidazole rings forms the purine nucleus ^[8], a key structural component of microbial genetic material. Owing to its critical biological role, purine and its bioisosteric analogues, such as benzimidazole, are considered promising scaffolds for the design of new antimicrobial agents with broad-spectrum activity ^[7].

Figure 2. Structural comparison of the benzimidazole and purine heterocyclic

The antibacterial potential of synthesized benzimidazole derivatives has shown promising results. Substituted benzimidazoles prepared from benzene-1,2-diamine and dicarboxylic acids, followed by acylation with benzoyl chloride (NaH), were structurally confirmed by spectral analysis. Among them, compound SB1 exhibited significant activity against Pseudomonas aeruginosa, comparable to ciprofloxacin ^[7]. Similarly, coumarin–benzimidazole hybrids synthesized by Singh L. R. et al. demonstrated potent antibacterial effects. Compound 12 showed strong activity against P. aeruginosa (MIC = 3.12 µg/mL), outperforming ampicillin (25 µg/mL), tetracycline, and kanamycin (450 µg/mL). It also exhibited excellent efficacy against Staphylococcus aureus (MIC = 1.56 µg/mL) and Escherichia coli (MIC = 3.12 µg/mL) ^[9]. Structure–activity relationship studies indicated that antibacterial potency is strongly influenced by halogen substitution, with para-chloro derivatives displaying maximum activity, while meta- and ortho-substituted analogues showed reduced effects.

Kapoor A. et al. synthesized a series of 2-substituted benzimidazole derivatives and evaluated their antimicrobial activity. Among them, compound 5d exhibited the highest potency against Escherichia coli with a MIC of 1.30 µmol/mL. Compounds 5e–5h also demonstrated significant activity (MIC = 1.58–1.88 µmol/mL) against E. coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Aspergillus niger. The observed activity was superior or comparable to standard drugs such as ciprofloxacin (MIC = 2.33 µmol/mL) and fluconazole (MIC = 1.99 µmol/mL). Structure–activity relationship analysis indicated that the introduction of electron-donating substituents on the benzylidene phenyl ring enhances antimicrobial efficacy ^[10].

Yadav et al. reported the synthesis of novel 2-substituted benzimidazole derivatives incorporating a pyridine moiety (4a–4f). The structures were confirmed by elemental analysis, IR, ¹H-NMR, and mass spectroscopy. The synthesized compounds were evaluated in vitro for antibacterial and antifungal activity using the disc diffusion method. At a concentration of 500 µg/mL, the compounds exhibited measurable zones of inhibition against Escherichia coli, Pseudomonas pseudomonas, Bacillus subtilis, and Klebsiella pneumoniae, as well as antifungal activity against Candida albicans and Aspergillus niger. Fluconazole and cefuroxime HCl were used as standard antifungal and antibacterial drugs, respectively ^[11].

Alasmary F.A. et al. synthesized a library of 53 benzimidazole derivatives substituted at the 1-, 2-, and 5-positions and evaluated them against 26 bacterial and 10 fungal strains. Compounds 17 and 18 (2-ethanamine derivatives) exhibited potent antibacterial activity, with MIC values comparable to ciprofloxacin against two MRSA strains. Several 5-halogenated derivatives, particularly 5-bromo analogues, showed strong antifungal activity, with MICs comparable to or better than amphotericin B. Compound 18 was identified as one of the most active antibacterial candidates ^[12]. Gupta S.K. et al. synthesized 2-alkyl and 2-aryl benzimidazoles using acetic, o-chlorobenzoic, benzoic, and cinnamic acids, followed by N-substitution with benzoyl and tosyl chlorides. Compounds 2a and 2d demonstrated significant antibacterial activity against Pseudomonas aeruginosa and antifungal activity against Candida albicans, showing efficacy comparable to standard drugs ^[13]. Tuncbilek M. et al. prepared novel 2-substituted benzimidazole derivatives, where compound 32 exhibited superior antibacterial activity compared to ciprofloxacin ^[7,14]. Shingalapur R.V. et al. synthesized 5-bromostyryl-2-benzimidazoles via condensation of cinnamic acids with 5-bromo-1,2-phenylenediamine in ethylene glycol. Compound 40 showed potent antimicrobial activity against various bacterial and fungal strains, comparable to ciprofloxacin and fluconazole ^[7,15].

Turkey R. H. et al. synthesized novel 5-ethoxy-2-mercaptobenzimidazole derivatives and confirmed their structures by elemental microanalysis, IR, and ^1H-NMR spectroscopy. The antibacterial activity was evaluated by the agar diffusion method at concentrations of 50 and 100 µg/mL against Staphylococcus aureus, Streptococcus agalactiae, Proteus mirabilis, and Pseudomonas aeruginosa. Compounds 6b and 7b exhibited the highest potency, producing zones of inhibition ranging from 21–37 mm, comparable to standard antibiotics cefotaxime and imipenem ^[16].

Alam S.A.M.F. et al. synthesized Schiff base derivatives of 2-phenylbenzimidazole via cyclocondensation of benzene-1,2-diamine with p-aminobenzoic acid in the presence of polyphosphoric acid and xylene, followed by condensation with various aldehydes. The synthesized Schiff bases were evaluated for antibacterial activity using the tube dilution method. Compounds SAM-2 and SAM-10 exhibited superior antibacterial potency compared to the standard drug vancomycin, indicating enhanced efficacy of the azomethine-linked benzimidazole derivatives ^[17].

U. Kankeaw et al. reported a facile synthesis of benzimidazole derivatives via condensation of citronellal with o-phenylenediamine in ethanol. The structures of the obtained products were confirmed by FTIR and 1H-NMR spectral analysis. The antibacterial activity was evaluated using the agar diffusion method, with gentamicin as the reference drug. The synthesized derivatives exhibited measurable zones of inhibition, indicating promising antibacterial potential ^[18].

1. Antifungal Activities

Benzimidazole derivatives are widely recognized as effective antifungal agents due to their potency and favorable safety profiles. Clinically used examples include Carbendazim, Benomyl, and Thiabendazole ^[19]. Their antifungal mechanism is primarily associated with inhibition of ergosterol biosynthesis. Ergosterol is an essential sterol regulating fungal membrane permeability and fluidity ^[20]. A key molecular target is lanosterol 14α-demethylase (CYP51), a cytochrome P450 enzyme. Azole antifungals act as non-competitive inhibitors by coordinating to the heme iron of CYP51, thereby blocking ergosterol formation and exerting fungistatic effects ^[21]. Emir Güzel et al. synthesized benzimidazole–1,2,4-triazole derivatives (6a–l), structurally confirmed by ¹H-NMR, ¹³C-NMR, and HR-MS analysis. The compounds were evaluated in vitro against Candida albicans, C. parapsilosis, C. glabrata, and C. krusei. Compounds 6b, 6i, and 6j demonstrated superior antifungal activity with MIC values of 0.97 µg/mL, particularly against C. glabrata, outperforming standard drugs Voriconazole and Fluconazole ^[22].

The antifungal efficacy of azoles depends on the affinity of the N-1 substituent toward cytochrome P450 and the strength of coordination with the heme iron of lanosterol 14α-demethylase (CYP51). Inhibition of ergosterol biosynthesis disrupts fungal membrane integrity, leading to growth inhibition or cell death ^[23]. Both benzimidazole and triazole rings are recognized as key pharmacophores contributing to potent antifungal activity ^[24]. Can N. Ö. et al. synthesized a series of benzimidazole–triazole derivatives (5a–s) as ergosterol biosynthesis inhibitors. The compounds were evaluated against Candida glabrata (ATCC 90030), C. parapsilosis (ATCC 22019), C. krusei (ATCC 6258), and C. albicans (ATCC 24433). Compounds 5i and 5s demonstrated significant antifungal activity with MIC₅₀ values of 0.78–1.56 µg/mL. Cytotoxicity assessment against NIH/3T3 cells showed IC₅₀ values markedly higher than the MIC₅₀ values, indicating favorable selectivity. LC–MS/MS analysis confirmed that both compounds significantly reduced intracellular ergosterol levels, validating CYP51 inhibition as the probable mechanism of action ^[25].

A series of 12 benzimidazole–thiazole derivatives (3a–l) was synthesized and structurally characterized by 1H-NMR, 13C-NMR, IR, and ESI-MS spectroscopy. The compounds were evaluated for anticandidal activity against Candida krusei, C. glabrata, C. albicans, and C. parapsilosis. In silico ADME analysis predicted favorable pharmacokinetic profiles for all derivatives. Among them, compounds 3c and 3d exhibited the highest antifungal potency against all tested Candida species. Cytotoxicity studies indicated low toxicity, with IC₅₀ values > 500 µg/mL. LC–MS/MS analysis confirmed that compounds 3c and 3d significantly reduced ergosterol levels in C. albicans, supporting inhibition of ergosterol biosynthesis as the probable mechanism of action ^[26].

Khabnadideh S. et al. synthesized novel alkylated benzimidazole and benzotriazole derivatives via N-alkylation with bromoalkanes and by coupling amino thiazole amide derivatives with 2-piperazino-1-ethanol using tetraethylammonium bromide or triethylamine as catalysts. The structures were confirmed by standard spectroscopic techniques. Antifungal activity was evaluated using the CLSI-recommended broth microdilution method. The results indicated that alkylbenzimidazole derivatives exhibited higher antifungal potency than the corresponding alkylbenzotriazoles. Compounds 1a and 2a from the benzimidazole series showed broad-spectrum activity against all tested fungal strains ^[27].

Sadeghian S. et al. synthesized imidazole, benzimidazole, and benzotriazole derivatives (C1–C8) and evaluated their antimicrobial activity using the CLSI broth microdilution method against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Salmonella typhi, and Candida albicans. Among them, imidazole derivative C5 exhibited the highest potency, showing superior activity against S. aureus (MIC = 2–16 µg/mL) compared to ampicillin, and strong anticandidal activity against C. albicans (MIC = 1 µg/mL), comparable to Amphotericin B. MTT cytotoxicity assays indicated low toxicity (< 35%) for all tested compounds ^[28]. Yang L. et al. synthesized benzimidazole derivatives bearing thioether and carbamate functionalities and assessed their antifungal activity using the mycelial growth inhibition method against Cytospora mandshurica, Thanatephorus cucumeris, Botrytis cinerea, Verticillium dahliae, Phytophthora infestans, and Gibberella zeae. Compound E11 showed significant antifungal efficacy at 50 µg/mL, with inhibition rates of 70% against V. dahliae and 75% against P. infestans, outperforming Albendazole ^[29].

Kerimov et al. synthesized novel benzimidazole derivatives and evaluated there in vitro effects on NADPH-dependent lipid peroxidation (LP), ethoxyresorufin O-deethylase (EROD) activity, and antifungal potential. At a concentration of 10⁻³ M, compounds 4c (52%), 4e (58%), and 4h (43%) significantly inhibited lipid peroxidation in male rat liver microsomes. In the EROD assay, compounds 4c (100%), 4h (100%), 5c (98%), and 5h (100%) exhibited stronger inhibitory activity than the reference inhibitor caffeine (85%). Antifungal screening revealed that only compounds 4b and 4h demonstrated notable activity against Candida albicans, whereas other derivatives showed moderate effects ^[30].

Zamani L. et al. synthesized ten 2-substituted benzimidazole derivatives (J1–J10) via condensation of o-phenylenediamine with substituted aldehydes using nano-SnCl₄/SiO₂ as a mild heterogeneous catalyst. The structures were confirmed by spectroscopic analysis. Among the series, compound J2 exhibited the most potent antifungal activity against Cryptococcus neoformans, Candida krusei, C. dubliniensis, Microsporum canis, and C. albicans, with MIC values ranging from 8–64 µg/mL. Cytotoxicity evaluation against MCF-7 and A549 cell lines (MTT assay) revealed that J2 showed promising antitumor activity, J5 and J6 displayed moderate effects, while J7 was essentially non-toxic ^[31].

2. Antiviral Activities

Heterocyclic scaffolds play a crucial role in antiviral drug development, particularly purine-based analogues containing the imidazole ring. Clinically important agents such as Ganciclovir, Valganciclovir, Valacyclovir, Entecavir, and Adefovir dipivoxil derive their antiviral efficacy largely from imidazole- or purine-based structural motifs. In contrast, anti-cytomegalovirus activity in Maribavir (1263W94) and GW275175X is attributed to the benzimidazole nucleus ^[32]. Despite their clinical utility, prolonged antiviral therapy often leads to resistance and systemic toxicity, necessitating the development of novel agents with improved safety and reduced resistance profiles. Benzimidazole nucleosides represent promising candidates due to their structural versatility and distinct mechanisms of action ^[33]. Numerous benzimidazole derivatives have demonstrated antiviral activity against diverse viral pathogens, including human cytomegalovirus (HCMV), human immunodeficiency virus (HIV), and hepatitis B and C viruses, highlighting the therapeutic potential of this heterocyclic scaffold in antiviral drug discovery ^[34].

Vitale et al. synthesized a series of 2-arylbenzimidazole derivatives and evaluated their antiproliferative and antiviral activities using cell-based assays. The compounds were screened against a broad panel of viruses, including members of the Flaviviridae (flaviviruses and pestiviruses), Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae, Reoviridae, as well as DNA viruses from the Herpesviridae and Poxviridae families. Compounds 15, 28, and 29 exhibited moderate antiviral activity against the yellow fever virus, with effective concentrations in the range of 6–27 µM. However, none of the synthesized benzimidazole derivatives demonstrated significant antiviral effects at non-cytotoxic concentrations in resting cell monolayers ^[35].

Tonelli M. et al. synthesized 76 derivatives of 2-phenylbenzimidazole and evaluated their cytotoxic and antiviral profiles against 10 RNA and DNA viruses. The most susceptible viruses were BVDV, CVB-2, HSV-1, Sb-1, and YFV, whereas HIV-1 and VSV were inactive, and RSV, VV, and Reo-1 were minimally affected. Compound 24 (5,6-dichloro-2-(4-nitrophenyl) benzimidazole) exhibited potent and selective activity against vaccinia virus (VV) with an EC₅₀ of 0.1 µM, demonstrating 18- and 110-fold higher potency than reference drugs mycophenolic acid (EC₅₀ = 1.8 µM) and 6-azauridine (EC₅₀ = 11 µM), respectively. Additionally, compounds 50, 51, and 53 showed strong and selective activity against BVDV with EC₅₀ values of 0.8–1.5 µM, comparable to the standard NM 108 (EC₅₀ = 1.7 µM) ^[36].

Francesconi et al. synthesized two series of benzimidazole derivatives bearing 5-(thio) semicarbazone and 5-hydrazone functionalities via modification of 5-acetylbenzimidazole. The compounds were evaluated for antiviral activity against multiple viruses and for cytotoxicity in mammalian cell lines. Derivatives 6, 8, 16, and 17, containing 5-(thio) semicarbazone or 5-hydrazone groups along with a 2-benzyl substituent on the benzimidazole core, exhibited significant inhibitory activity against influenza A virus and human coronavirus. For respiratory syncytial virus (RSV), activity was primarily observed in 5-thiosemicarbazone (25) and 5-hydrazone (22) derivatives possessing a 2-[(benzotriazol-1/2-yl) methyl] benzimidazole framework. These compounds demonstrated potent antiviral efficacy with activity comparable to the reference drug Ribavirin ^[37].

Gudmundsson et al. synthesized amine-substituted N-(1H-benzimidazol-2-ylmethyl)-5,6,7,8-tetrahydro-8-quinolinamines and evaluated their anti-HIV-1 activity. Structural modification of amine substituents significantly influenced antiviral potency, leading to the identification of highly active derivatives exhibiting sub-nanomolar inhibitory activity against HIV-1 ^[38]. Additionally, 2-pyridyl-1H-benzimidazole-4-carboxamide derivatives 59 and 60 demonstrated selective inhibition of Coxsackie virus B3 in VERO cells. Compound 61, a 2-substituted benzimidazole N-carbamate derivative, showed moderate antiviral activity against Coxsackie virus B2 ^{[39, 40]}.

Starčević et al. synthesized C-5 amidino-substituted benzimidazole derivatives bearing heterocyclic moieties at C-2 (pyridine, N-methyl-pyrrole, imidazole) and evaluated their antitumor and antiviral activities against coxsackieviruses, echoviruses, and adenovirus. Among them, 2-(1-methyl-1H-pyrrol-2-yl)-1H-benzimidazole-5-carboxamidine hydrochloride (16) and N-isopropyl-2-pyridin-2-yl-1H-benzimidazole-5-carboxamidine (17) showed significant anti-adenoviral activity, indicating their potential to inhibit adenoviral replication with promising IC₅₀ values in the micromolar range ^[41].

Huo et al. synthesized a series of benzimidazole derivatives and assessed their antiviral potential against the respiratory syncytial virus (RSV). Among the synthesized compounds, a27 exhibited the most potent activity with an EC₅₀ = 9.49 µM, significantly suppressing viral replication and pro-inflammatory cytokine production across multiple RSV strains. In vivo evaluation in a murine model demonstrated reduced body-weight loss and attenuation of lung tissue damage. Structure–activity relationship (SAR) studies indicated that substitution at the C-3 position of the benzimidazole core markedly influenced antiviral potency and cytotoxicity ^[42].

Liu et al. investigated the antiviral mechanism of 7-(4-benzimidazolebutoxy) coumarin (BBC) against Spring viraemia of carp virus (SVCV). Mechanistic studies demonstrated that BBC modulates protein kinase C (PKC) activity, leading to activation of the Nrf2-mediated antiviral pathway. BBC treatment significantly reduced viral replication, enhanced interferon (IFN) response, and upregulated heme oxygenase-1 (HO-1) expression, thereby strengthening host antiviral defense. These findings indicate that BBC exerts antiviral activity through PKC/Nrf2 pathway modulation and immune response enhancement ^[43].

Chen J. et al. designed two series of benzimidazole-based inhibitors targeting Lassa virus (LASV) entry. Lentiviral pseudotypes expressing LASV glycoprotein complex (GPC) were employed to screen viral entry inhibitors, and binding affinities were validated by surface plasmon resonance (SPR). Compounds 7d–z, 7h–z, 13c, 13d, and 13f exhibited potent antiviral activity with IC₅₀ values of 7.58–15.46 nM and selectivity index (SI) > 1251. SPR studies showed strong target binding with Kᴅ < 8.25 × 10⁻⁷ M. Notably, compound 7h-Z demonstrated the highest potency (IC₅₀ = 7.58 nM; SI = 2496), identifying it as a promising lead candidate for further antiviral development ^[44].

Tonelli M. et al. evaluated a library of 86 benzimidazole derivatives for antiviral activity against 10 RNA and DNA viruses. Among them, 52 compounds exhibited measurable antiviral effects, with the most pronounced activity observed against Coxsackievirus B5 (CVB-5), Respiratory syncytial virus (RSV), Bovine viral diarrhea virus (BVDV), and Suid herpesvirus 1 (Sb-1). Most active compounds were based on the 2-benzylbenzimidazole scaffold, while other effective derivatives included 1-phenylbenzimidazole (2), 2-trifluoromethylbenzimidazole (69), dihydropyrido[3,2′:4,5]imidazo[1,2-a][1,4]benzodiazepine-5-one (3), dibenzo[c,e] benzimidazo[1,2-a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazoles (81, 82, 86). These scaffolds demonstrated significant antiviral potential, highlighting benzimidazole-based frameworks as promising lead structures for further optimization against CVB-5, RSV, BVDV, and Sb-1 infections ^[45].

Benzimidazole has emerged as an important scaffold in the development of antidiabetic agents, particularly for managing hyperglycemia, a globally prevalent metabolic disorder. Several benzimidazole-based derivatives have demonstrated significant glucose-lowering potential through enzyme inhibition mechanisms. Rai P. V. et al. synthesized a novel series of benzimidazole–chalcone hybrids via a one-pot nitro reductive cyclization method. The compounds were structurally confirmed by ¹H-NMR, ¹³C-NMR, IR, and mass spectroscopy. Biological evaluation revealed that compound 7l exhibited potent inhibitory activity with IC₅₀ = 22.45 ± 0.36 µg/mL (α-glucosidase) and 20.47 ± 0.60 µg/mL (α-amylase). Molecular docking studies further demonstrate strong binding interactions of 7l within the active sites of both enzymes, supporting its potential as a promising antidiabetic lead molecule ^[2].

Tocco G. et al. synthesized a series of 1,2-disubstituted benzimidazoles via condensation of o-phenylenediamine with substituted aromatic aldehydes. The compounds were evaluated for α-glucosidase and α-amylase inhibitory activity. Notably, compound 8s showed the highest α-glucosidase inhibition with IC₅₀  = 0.39 ± 0.04 µM, followed by 8k (IC₅₀ = 7.4 ± 1.6 µM) and 8r (IC₅₀  = 13.8 ± 2.7 µM). However, all tested derivatives exhibited weak α-amylase inhibitory activity, indicating selective α-glucosidase inhibition and potential utility in postprandial hyperglycemia management ^[46].

Gidado I. et al. synthesized two 2-substituted benzimidazoles using a boric acid–catalyzed protocol, affording high yields with sharp melting points. Structural elucidation was performed by FT-IR, ¹H-NMR, and ¹³C-NMR spectroscopy. Biological evaluation revealed significant antioxidant and α-glucosidase inhibitory activity, with compound BL24 exhibiting the strongest radical scavenging capacity and enzyme inhibition, highlighting its potential as an antidiabetic lead ^[47]. Similarly, Ullah H. et al. synthesized a series of oxadiazole–benzimidazole hybrids (1–16) and characterized them by ¹H-NMR, ¹³C-NMR, and HREI-MS. Among the series, analogue 8 showed the most potent α-glucosidase inhibition, while analogue 2 demonstrated significant α-amylase inhibitory activity. Molecular docking studies confirmed strong binding interactions within the active sites of both enzymes, suggesting that these hybrid scaffolds possess promising dual inhibitory potential for antidiabetic therapy ^[48].

Nikfar P. et al. designed and synthesized a series of quinolone-2-thio-acetamide-propane hydrazide benzimidazole derivatives (12a–o) targeting key carbohydrate-hydrolyzing enzymes involved in type-2 diabetes, namely α-glucosidase and α-amylase. Among the series, compound 12n (a 4-fluorophenylacetamide analogue) exhibited the most potent dual inhibitory activity against both enzymes and showed superior efficacy compared to the standard drug Acarbose. These findings highlight compound 12n as a promising lead candidate for further antidiabetic drug development ^[49].

Khan S. et al. synthesized a series of benzimidazole-derived thiazole-based bis-Schiff bases (1–14) and confirmed their structures by ¹H-NMR, ¹³C-NMR, and HREI-MS analyses. The compounds were evaluated for α-glucosidase and α-amylase inhibitory activity in comparison with the standard drug Acarbose (IC₅₀ = 8.30 ± 0.20 µM and 9.30 ± 0.10 µM, respectively). Among them, compound 8 exhibited superior potency with IC₅₀ = 4.20 ± 0.20 µM (α-glucosidase) and 3.50 ± 0.20 µM (α-amylase), significantly outperforming acarbose. Molecular docking studies revealed favorable protein–ligand interactions within enzyme active sites, while ADME analysis indicated improved pharmacokinetic properties of the active analogues, supporting their potential as promising antidiabetic lead candidates ^[50].

A series of benzimidazole-based thiosemicarbazone derivatives (7–21) was synthesized and structurally characterized by ¹H-NMR, ¹³C-NMR, and HREI-MS spectroscopy. The compounds were evaluated for α-glucosidase and α-amylase inhibitory activity as potential antidiabetic agents. All derivatives exhibited good to excellent enzyme inhibition. Notably, compound 19 showed remarkable potency with IC₅₀ = 1.30 ± 0.20 µM (α-glucosidase) and 1.20 ± 0.20 µM (α-amylase), while compound 20 demonstrated IC₅₀ = 1.60 ± 0.20 µM and 1.10 ± 0.01 µM, respectively. These values were significantly superior to the standard drug Acarbose (IC₅₀ = 11.29 ± 0.07 µM and 11.12 ± 0.15 µM), indicating strong dual inhibitory potential. The results suggest that benzimidazole–thiosemicarbazone hybrids represent promising lead scaffolds for the development of novel antidiabetic therapeutics ^[51].

Azizian H. et al. synthesized a novel series of benzimidazole–Schiff base hybrids (8a–p) and evaluated their α-glucosidase inhibitory activity. Structural characterization was carried out using FT-IR, ¹H-NMR, ¹³C-NMR, and CHNS elemental analysis. The majority of the synthesized compounds exhibited superior α-glucosidase inhibition compared to the standard drug Acarbose. Among them, compounds 8p (thiophene substituent), 8h (phenyl), and 8a (2-fluorophenyl) demonstrated the highest inhibitory potency. In contrast, compound 8g, bearing a 3,4,5-trimethoxyphenyl moiety, was inactive against α-glucosidase. These findings highlight the significant influence of the Schiff base substituent on enzyme inhibition and support further optimization of benzimidazole-based antidiabetic agents ^[52].

4. Antioxidant Activities

Oxidative stress plays a crucial role in the pathogenesis of degenerative disorders such as diabetes, atherosclerosis, ischemic heart disease, cancer, and ageing ^[53]. Consequently, extensive research has focused on identifying potent antioxidant agents ^[54], and benzimidazole derivatives have emerged as promising candidates due to their broad pharmacological potential. Karmaker N. et al. synthesized a series of 2-substituted-5-methylbenzimidazole derivatives (3a–e) by condensing 4-methyl-1,2-phenylenediamine with various p-substituted benzaldehydes (2a–e). Structural characterization was performed using standard spectroscopic techniques. Antioxidant activity was evaluated via the DPPH free radical scavenging assay, where the compounds exhibited significant activity with IC₅₀ values ranging from 1.054 to 19.05 µg/mL, outperforming the reference antioxidant Butylated hydroxytoluene (IC₅₀  = 26.96 µg/mL). These results highlight the strong antioxidant potential of substituted benzimidazole derivatives ^[53].

Ayhan-Kılcıgil G. et al. synthesized a series of novel benzimidazole derivatives (10–24) and evaluated their antioxidant potential using multiple in vitro assays, including microsomal NADPH-dependent lipid peroxidation inhibition, DPPH radical scavenging, and superoxide anion scavenging assays. Among the tested compounds, 12 and 13 exhibited outstanding antioxidant activity. In the DPPH assay, they showed IC₅₀ values of 1.3 × 10⁻⁵ M and 1.2 × 10⁻⁵ M, respectively, which were significantly more potent than the reference antioxidant Butylated hydroxytoluene (IC₅₀ = 2.3 × 10⁻⁴ M). These findings indicate that selected benzimidazole derivatives possess strong free radical scavenging capacity and potential therapeutic relevance against oxidative stress–related disorders ^[54].

Bhandari S. V. et al. synthesized novel benzimidazole–1,3,4-oxadiazole hybrids and evaluated their in vitro antioxidant potential using the DPPH radical scavenging assay. Structural characterization was carried out by ¹H-NMR and FT-IR spectroscopy. Molecular docking studies revealed that compounds 1A, 2A, and 3A exhibited favorable Glide scores of −7.575, −6.932, and −6.911 kcal/mol, respectively, compared to reference antioxidants Propyl gallate (−4.757 kcal/mol) and Ascorbic acid (−4.50 kcal/mol). Biological evaluation showed that compound 2A displayed the highest antioxidant activity with IC₅₀ = 53.00 ± 1.31 µg/mL, though it was less potent than ascorbic acid (IC₅₀ = 11.51 ± 0.31 µg/mL). These findings suggest that benzimidazole–oxadiazole hybrids possess promising antioxidant properties and warrant further structural optimization ^[55].

Odame F. et al. reported a base-catalyzed synthesis of benzimidazoles via condensation of aldehydes with o-phenylenediamine. The synthesized derivatives were characterized by IR, elemental microanalysis, ¹H/¹³C-NMR, and GC–MS techniques. Additionally, single-crystal X-ray analysis was performed for 2-(2-chlorophenyl)-1H-benzimidazole (4) and 2-(1H-benzimidazol-2-yl)-4-nitrophenol (8) to confirm their molecular structures. Antioxidant activity assessed by the DPPH radical scavenging assay showed that 2-(1H-benzimidazol-2-yl) phenol (2), 2-p-tolyl-1H-benzimidazole (3), and 2-(4-methoxyphenyl)-1H-benzimidazole (7) exhibited IC₅₀ values of 1974, 773, and 800 µM, respectively, indicating moderate free radical scavenging potential ^[56].

5. Anticancer Activities

Wang Q. et al. synthesized a series of benzimidazole-containing flavone and chalcone derivatives and evaluated their antiproliferative activity against various tumor cell lines. The benzimidazole–chalcone hybrids displayed stronger growth inhibition compared to benzimidazole–flavone analogues. Among them, compound 19 emerged as a promising lead, demonstrating significant antitumor efficacy in both in vitro and in vivo models ^[57]. In another study, ethyl-2-(4-(piperidine-1-yl) phenyl)-1H-benzo[d]imidazole-5-carboxylate (BZD9L1) showed high selectivity against colorectal cancer (CRC), with IC₅₀ values of 16.82 µM (HCT-116) and 20.11 µM (HT-29) cell lines. Colony formation assays revealed a 96.50% reduction (HCT-116) and 1.33% reduction (HT-29) at 25 µM, compared to the standard drug Fluorouracil (103.5% and 163.7%, respectively). Mechanistic studies indicated that BZD9L1 induces apoptosis through inhibition of SIRT1 and SIRT2 (sirtuins), leading to DNA damage and suppression of tumor cell proliferation ^[58,59].

Nguyen et al. synthesized fourteen transition metal complexes along with five free benzimidazole ligands. Structural characterization was performed using FT-IR, ¹H-NMR, powder XRD, and ESI-MS, while UV–Visible spectroscopy and ICP-OES confirmed metal incorporation in the complexes. Biological evaluation demonstrated that most metal complexes exhibited enhanced anticancer activity compared to their corresponding free ligands. Notably, complexes C1, C3, and C14 showed potent antiproliferative effects with IC₅₀ values of 5.8–10.4 µM, outperforming the reference drug Cisplatin (IC₅₀ = 16.5 ± 1.0 µM for A549 and 19.4 ± 1.7 µM for MDA-MB-231) ^[60]. Similarly, Morais G. R. et al. synthesized fluorinated and hydroxylated alkyl-substituted benzimidazole derivatives and evaluated their antitumor activity. Among them, compound (2) bearing an unsubstituted benzimidazole core with a 2-fluoroethyl chain at the aniline nitrogen displayed the highest activity, showing moderate cytotoxicity against the U87 glioblastoma cell line with IC₅₀ = 45.2 ± 13.0 µM, compared to Doxorubicin (IC₅₀ = 16.6 ± 2.5 µM) ^[61].