A Comprehensive Account on Recent Progress in Pharmacological Activities of Benzimidazole Derivatives

Nowadays, nitrogenous heterocyclic molecules have attracted a great deal of interest among medicinal chemists. Among these potential heterocyclic drugs, benzimidazole scaffolds are considerably prevalent. Due to their isostructural pharmacophore of naturally occurring active biomolecules, benzimidazole derivatives have significant importance as chemotherapeutic agents in diverse clinical conditions. Researchers have synthesized plenty of benzimidazole derivatives in the last decades, amidst a large share of these compounds exerted excellent bioactivity against many ailments with outstanding bioavailability, safety, and stability profiles. In this comprehensive review, we have summarized the bioactivity of the benzimidazole derivatives reported in recent literature (2012–2021) with their available structure-activity relationship. Compounds bearing benzimidazole nucleus possess broad-spectrum pharmacological properties ranging from common antibacterial effects to the world’s most virulent diseases. Several promising therapeutic candidates are undergoing human trials, and some of these are going to be approved for clinical use. However, notable challenges, such as drug resistance, costly and tedious synthetic methods, little structural information of receptors, lack of advanced software, and so on, are still viable to be overcome for further research.


INTRODUCTION
Benzimidazole, alternatively known as 1H-benzimidazole and 1,3-benzodiazole, consists of benzene ring fused with a five-membered imidazole ring, and is an important heterocyclic pharmacophore. Benzimidazole is regarded as a "privileged structure" in heterocyclic chemistry due to its association with a wide range of biological activities (Barot et al., 2013;Alaqeel, 2017).
Back in 1940s, benzimidazole was speculated to act similarly as purines to provide biological responses and the first investigation on biological activity of benzimidazole nucleus was reported in 1944 (Woolley, 1944). Interest among the researchers about the synthetic procedure of benzimidazole and its derivatives escalated when Brink et al. (Brink and Folkers, 1949;Emerson et al., 1950) found that 5,6-dimethylbenzimidaozle was a degradation product of vitamin B 12 and some of its derivatives also possessed vitamin B 12 like activity. These early reports led researchers to the exploration of benzimidazole nucleus for numerous activities. Through the course of many years of research, benzimidazole has emerged as an important heterocyclic system because of its existence in diverse biologically active compounds, such as antiparasitics, antimicrobials, antivirals, antifungals, anticonvulsants, antihypertensives, antihistaminics, analgesics, antiinflammatory agents, anticancers, anticoagulants and proton pump inhibitors (Figure 1) (Fei and Zhou, 2013;Wang et al., 2015). As a result of changing substituents around the core structure, many drugs of a wide variety of therapeutic lines have been developed such as albendazole, mebendazole, thiabendazole as antihelmintics; enviradine as antiviral; carbendazim as fungicidal; omeprazole, lansoprazole, pantoprazole as proton pump inhibitors; candesartan cilexitil and telmisartan as antihypertensives, and astemizole as antihistaminic agent (Figure 2) (Bansal and Silakari, 2012;Alaqeel, 2017). The high therapeutic potential of benzimidazole related drugs has inspired the medicinal chemists to carry out the synthesis of several novel chemotherapeutic agents containing benzimidazole moiety (Morais et al., 2017).
Numerous researches have been accomplished in the past couple of years which produced very intriguing results concerning the chemistry, structure-activity relationship and biological activities of different benzimidazole based compounds. The diverse biological activities displayed by compounds bearing benzimidazole moiety have prompted researchers all around the globe to design and synthesize various benzimidazole analogues. A number of recently published patents on the benzimidazole moiety are listed in Table 1. Several review articles have been published emphasizing on the contribution of benzimidazole nucleus in particular biological activity, e.g. anticancer, analgesic, antiinflammatory, antimicrobial, antiviral, antitubercular, antiulcer, antihypertensive and antidiabetic property (Bansal and Silakari, 2012; Barot et al., 2013;Keri et al., 2015;Wang et al., 2015;Akhtar et al., 2017). To the best of our knowledge, there is no review article available in the literature which has focused on the most updated information of the diverse biological and therapeutic applications of benzimidazole derivatives. The present review gives a comprehensive account of all biological aspects of benzimidazole derivatives and has included information from the recent studies reported up to 2021. Apart from literature Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762807 2 study, this review also provides a thoughtful insight into the latest ongoing research on benzimidazole derivatives in a variety of therapeutic fields.

BIOLOGICAL ACTIVITIES
The wide variety of benzimidazole derivatives synthesized during the last few years and their diverse biological applications are discussed in the following sections.

Antimicrobial Activity
Antimicrobial and Antifungal Activity The antimicrobial potential of benzimidazole moiety has been explored notably since late 1990s and early 2000s (Özkay et al., 2011). Considering the huge dimension of research conducted on antimicrobial property of benzimidazole derivatives after 2012, the following section focuses on the up-to-date information on antibacterial and antifungal activities, while antiviral, antiulcer, antiprotozoal and antitubercular properties are discussed in separate sections. Different benzimidazole based compounds with antibacterial and antifungal activities are shown in Figure 3. Kathrotiya and Patel (Kathrotiya and Patel, 2013) synthesized a series of indole-based pyrido [1,2-a] benzimidazole derivatives (1)(2)(3)(4) and evaluated in vitro antimicrobial activity against some Gram-positive and Gram-negative bacteria and fungi using broth microdilution minimum inhibitory concentration (MIC) method. Compounds 1,3 and 4 (MIC 50,62.5 and 12.5 μg/ ml, respectively) displayed prominent antibacterial activity against S. typhi compared to standards ampicillin, chloramphenicol and ciprofloxacin (MIC 100, 50 and 25 μg/ ml, respectively). Compounds 2 and 3 exhibited notable antifungal activity against C. albicans (MIC 250 μg/ml) in comparison with standard griseofulvin (MIC 500 μg/ml). The derivatives with 4-methoxy (1) and cyano (4) group at 2position of indole nucleus were found to possess excellent inhibitory activity against most of the tested organisms. Birajdar et al. (Birajdar et al., 2013) synthesized some amino alcohol derivatives of 2-methyl benzimidazole (5)(6)(7)(8) by epoxide ring opening of 2-methyl benzimidazole with different substituted cyclic amines.  demonstrated moderate to good activity against Gram-positive (S. aureus) and Gram-negative (E. coli) pathogens in comparison with reference drugs ciprofloxacin and norfloxacin.
A series of benzimidazole derivatives were synthesized and evaluated by Padalkar et al. (Padalkar et al., 2016) (32-34) and Chandrika et al. (Chandrika et al., 2016) (35-37), where the compounds 32-34 showed prominent antibacterial activity against S. aureus strain and compounds 35-37 were found to be the most potent antifungal agents against azole-resistant fungal strain C. albicans ATCC 64124 (strain B). Another study reported that among the total 22 synthesized novel 2substituted fluorinated benzimidazoles, compounds 38-40 showed antimicrobial activity. In contrast, compound 40 containing a trifluoromethyl substituent showed the highest antifungal activity against the fungus C. albicans (Shintre et al., 2017). Similarly, El-Gohary and Shaaban (El-Gohary and Shaaban, 2017) synthesized a series of benzimidazole derivatives (41-43), where compounds 41 and 43 showed notable activity against S. aureus, and compound 42 was found to be the most effective against B. cereus. Compound 41 displayed the highest antifungal potential against C. albicans, whereas 43 exhibited prominent activity against A. fumigatus.
Singh et al. (Singh LR. et al., 2017) prepared a library of coumarin-benzimidazole hybrids and screened them for antimicrobial activity. Compound 44 was found to be the promising broad-spectrum antibacterial agent against P. aeruginosa, S. aureus, B. subtilis and P. vulgaris. A series of N-(substitutedbenzylidene)-4-(1-((dimethylamino) methyl)-1H-benzimidazol-2-yl)thiazol-2-amine derivatives were investigated for antimicrobial activity using agar streak dilution test. Compound 45 appeared to be the most potent among the series. Notably, the presence of an electron-withdrawing group might have contributed to the improved antimicrobial property of the compound (Prasad and Sundararajan, 2017).
Recently, Yadav et al. (Yadav et al., 2018) synthesized 2substituted benzimidazole derivatives (46-47), where compound 46 emerged as the most potent antibacterial agent against both Gram-positive and Gram-negative bacteria compared to standard cefadroxil. All derivatives exhibited better antifungal activity than the standard fluconazole, and compound 47 showed maximum activity against A. niger (MIC 0.018 mM). Liu et al. (Liu et al., 2018) designed a series of novel aminopyrimidinyl benzimidazoles as potential antimicrobial agents. Among them, compound 48 showed effective growth inhibition of MRSA, E. coli, and fungus A. flavus, compared to standard drugs chloromycin, norfloxacin, and fluconazole.

Antiviral Activity
The antiviral properties of benzimidazole derivatives have been tested against different viral strains; human immunodeficiency virus (HIV), hepatitis B and C virus (HBV and HCV), enteroviruses, respiratory syncytial virus (RSV), human cytomegalovirus (HCMV), bovine viral diarrhea virus (BVDV) and herpes simplex virus-1 (HSV-1) are some to mention (Abu-Bakr et al., 2012). This section focuses on the recent studies involving varied antiviral properties of different benzimidazole derivatives, and their structures are shown in Figure 4.

Benzimidazole Against HIV
A number of substituted benzimidazole derivatives were synthesized as reverse transcriptase inhibitors (RTIs) against HIV-1 replication, among them compounds 97-98 showed notable antiviral activity against laboratory-adapted strains HIV-1 IIIB and HIV-1 Ada5 (EC 50 15.4-40 µM) and primary isolates of HIV-1 UG07O and HIV-1 VB59 strains (EC 50 5.28-31.86 µM) . Besides, Ferro et al. (Ferro et al., 2017) synthesized two series of N 1 -aryl-benzimidazol-2one derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs) against HIV-1, where the compounds 99-100 were more potent than the standard drug nevirapine (IC 50 : 1.3 and 0.79 vs. 1.55 µM). The sulfone derivatives 101-102, synthesized by the same research group were also found to be potent HIV-1 NNRTIs with IC 50 values of 47 and 50 nM, respectively. The substitution at C-4 position of the arylacetamide portion of the compounds might have contributed for their notable activity against HIV-1 IIIB strain in cell-based assays . Finally, Srivastava et al. (Srivastava et al., 2020) has recently reported a promising anti-HIV benzimidazole derivative 103 with a low IC 50 value of 0.386 × 10 -5 μM.

Benzimidazole Against Hepatitis B and C Viruses (HBV and HCV)
The hepatitis B surface antigen (HBsAg), an HBV surface protein is an important mediator of HBV life cycle (Wang et al., 2016). Xu et al. (Xu et al., 2014) carried out a highthroughput screening (HTS), and concluded that the compound 104 inhibited the secretion of HBsAg and HBV virions indicated by EC 50 values of 1.5 and 0.6 μM, respectively, along with half cytotoxicity concentration (CC 50 ) value of 24.5 μM.

Benzimidazole Against Bovine Viral Diarrhea Virus (BVDV), Rotavirus and Arenaviruses
Among a library of 5-acetyl-2-arylbenzimidazoles analogues, compound 120 appeared to be the most effective antiviral agent against Bovine Viral Diarrhea virus (BVDV, EC 50 1.11 mM) due to the presence of 2,4dimethoxy group in the phenyl moiety (Vitale et al., 2012). Shaker et al. (Shaker et al., 2015) synthesized a series of 5nitro-1H-benzimidazole derivatives (121-124) bearing substitution of heterocyclic rings at position 1. Compounds 121 and 122 exhibited equal potency as standard doxorubicin against A-549, HCT-116, MCF-7 and human liver carcinoma HepG2 cell lines. Besides, compounds 122-124 showed great potential to be used as potent antiviral agents due to their inhibitory effect against rotavirus Wa strain. Finally, compound 125, identified by Dai and co-workers, was found to be potent antiviral agent against Lassa virus envelope glycoprotein (LASV GP) pseudotypes with EC 50 value of 1.1 nM (Dai et al., 2013).

Anti-inflammatory and Analgesic Activity
Benzimidazole based compounds are of great importance as antiinflammatory and analgesic agents because of their property to inhibit cyclooxygenases (COXs), enzymes involved in biosynthesis of important inflammatory mediators called prostaglandins . Apart from the cyclooxygenases (COX), the benzimidazole derivatives interact with transient receptor potential vanilloid-1, cannabinoid receptors, bradykinin receptors, specific cytokines, and 5lipoxygenase (5-LOX) activating protein. Thus, the compounds derived from benzimidazole moiety show the anti-inflammatory property (Veerasamy et al., 2021). Different benzimidazole derivatives with analgesic and anti-inflammatory properties are shown in Figure 5.
Mariappan et al. (Mariappan et al., 2015) synthesized a set of 2-substituted benzimidazole derivatives and reported that compounds 126-128 were found to be the most promising agents among the series displaying significant (p < 0.01) analgesic and anti-inflammatory effect at a dose level of 100 mg/kg p.o. Li et al.  assessed the synthesized two series of 2-(piperidin-4-yl)-1H-benzo [d] imidazole derivatives for anti-inflammatory activity and found that the compound 129 exhibited the most potent inhibitory activity on nitric oxide and TNF-α production (IC 50 0.86 and 1.87 µM, respectively). Interestingly, the compound 129 also prevented 33.30 and 50.60% ear oedema in xylene-treated mice at doses of 4 and 12 mg/kg, respectively, compared to standard ibuprofen (26.77 and 39.34% inhibition at 4 and 12 mg/kg dose levels, respectively). Besides, compounds 130-132 showed the potentiality as gastroprotective lead compounds which can be developed into orally active analgesic and anti-inflammatory agents (Gaba and Mohan, 2015). Compounds 133-134, synthesized by Kumar et al. displayed significant analgesic and anti-inflammatory properties, (Kumar et al., 2015) and compound 135 showed better inhibition of acetic acid induced writhing at 20 mg/kg showed around 55% inhibition of inflammation compared to standard diclofenac (65.4% inhibition). Furthermore, Bukhari et al. (Bukhari et al., 2016) reported about anti-inflammatory activity of a series of benzimidazole derivatives where compound 146 was found to be a potent inhibitor of 5-LOX, COX, TNF-α, IL-6 and cytokines among the series. Figure 6 depicts a clear diagram for understanding the association of structural modifications with bioactivities of benzimidazole derivatives against inflammation.

Antiulcer Activity
Many benzimidazole derivatives are known to possess potent antiulcer activity and H + /K + -ATPase inhibitory properties. During recent times, several new synthetic benzimidazolebased compounds were developed which exhibited similar or better antiulcerogenic potentials compared to the established market preparations. The benzimidazole derivatives with antiulcer activity are shown in Figure 7.
A series of pyrimidylthiomethyl benzimidazoles (e.g. 174) and pyrimidylsulfinylmethyl benzimidazole derivatives (e.g. 175) were developed and screened for antiulcer activity. Compounds 174-175 significantly reduced gastric acid secretion, free acidity and gastric ulcers in the pylorus-ligated rats at 10 and 30 mg/kg doses, where the sulfinyl derivative (175) was found to be more effective than thio derivative (174) (Bariwal et al., 2008). In another study, compounds 176-177 exhibited most prominent antiulcer activity against pylorus ligationinduced, aspirin induced, and ethanol induced ulcer in rat model at a dose level of 10 and 20 mg/kg compared to omeprazole . Besides, Reddy et al. (Reddy et al., 2011) prepared a series of 2-substituted mercaptobenzimidazole derivatives and reported that compounds 178-180 produced notable antiulcer potentiality at a dose level of 10 mg/kg comparable to omeprazole. Furthermore, compound 181 prevented H + /K + -ATPase enzymatic activity with an IC 50 value of 1.6 × 10 -5 M and compound 182 displayed prominent effects on inhibition of gastric lesions and gastric acid secretion in a dose dependant manner (0.3-30 mg/kg) (Tanaka et al., 2011;Yan et al., 2011). Moreover, some benzimidazole-piperazine conjugated analogues were assessed for their in vivo antiulcer property.

Antioxidant Activity
Several benzimidazole derivatives have been explored through years for their capacity to act as antioxidants. Different benzimidazole derivatives with antioxidant activity are shown in Figure 8. Two series of benzimidazole compounds made through coupling of coumarin derivatives with benzimidazole nucleus either directly or through amide linkage at 2-position were evaluated for antioxidant property (Arora et al., 2014).   Among a library of N-substituted pyrazole-containing benzimidazoles, compounds 202-204 attributed prominent antioxidant activity in both DPPH and hydrogen peroxide assay supposed to the presence of benzyl substituent on imidazole nitrogen (Bellam et al., 2017). Besides, Anastassova et al. (Anastassova et al., 2018a) evaluated antioxidant property of compounds 205-206 using tert-butyl hydroperoxide (tert-BOOH) induced oxidative stress on rat hepatocytes, and reported that both compounds showed significant effect comparable to standard quercetin. Similarly, compounds 207-212 displayed notable cytoprotective effect on rat hepatocytes (Anastassova et al., 2018b).

Anticancer Activity
Among the anticancer drugs discovered in the recent years, different benzimidazole derivatives occupy an important place. The current review accounts the anticancer activity of benzimidazoles reported after 2013. The benzimidazole derivatives with anticancer activity are shown in Figure 10.
A series of substituted benzimidazole derivatives were evaluated for in vitro anticancer activity in human lung adenocarcinoma A549 cell line at normoxic and hypoxic conditions. Compound 230 was found to be the most cytotoxic agent with hypoxia/normoxia cytotoxic coefficient of 4.75, compared to standard tirapazamine (5.59) (Błaszczak-Świątkiewicz et al., 2014). The benzimidazole-thiazole derivatives 231-232 showed notable anticancer effect against human liver carcinoma cell line (HepG2: IC 50 0.518 and 0.578 mM) and pheochromocytoma of the rat adrenal medulla cell line (PC12: IC 50 0.309 and 0.298 mM) (Nofal et al., 2014). Compounds 233-234 with dual inhibition of Aurora A kinase and kinesin spindle protein were found to be the most prominent antitumor agents against various tested cell lines in comparison with standard drug CK0106023 (Abd El-All et al., 2015). Kalalbandi and Seetharamappa synthesized a series of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives among which compound 235 displayed notable antiproliferative activity against nine tumor subpanels, indicated by its selectivity ratios within the range of 0.79-1.53 and 0.47 to 1.69 at the GI 50 (growth inhibitory 50%) and TGI level, respectively (Kalalbandi and Seetharamappa, 2015). Similarly, compounds 236-238 with GI 50 values of 9.79, 2.58 and 3.81 µM, respectively exhibited broad spectrum antitumor activities, while the compound 236 was found to be the most potent DHFR inhibitor with IC 50 value of 1.05 µM (Singla et al., 2015).

Antitubercular Activity
Compounds containing heterocyclic moieties, such as pyrrole, imidazole and benzimidazole have been reported to demonstrate excellent antitubercular properties . Benzimidazole scaffold has been on target of the scientists for producing novel antitubercular agents. Different benzimidazole derivatives with antitubercular property are shown in Figure 12. Warekar et al. (Warekar et al., 2016) synthesized a series of 4-(4-nitro-phenyl)-2-phenyl-1,4-dihydro-benzo [4,5]imidazo [1,2a]pyrimidine-3-carboxylic acid ethyl ester derivatives and reported that compounds 320-321 appeared to be the most promising antitubercular agents among the series with minimum inhibitory concentration (MIC) value of 25 μg/ml against Mycobacterium tuberculosis H37Rv strain. Compound 322 also displayed good activity against same strain under aerobic conditions, as indicated by its IC 90 and MIC values (77 µg/ml and >100 μM, respectively) (Mantu et al., 2016). In a different study (Anguru et al., 2017), compounds 323-328 exhibited good activity against M. tuberculosis H37Rv strain while compound 325 emerged as the most promising agent with MIC value of 16 μg/ml.
A number of substituted fluorobenzimidazole derivatives (329-330) were synthesized and evaluated for in vitro antimycobacterial property against pathogenic M. tuberculosis H37Rv strain (ATCC 27294) using MABA method. Compounds 329-330 exhibited notable antitubercular activity against H37Rv strain and their activity was contributed by the incorporation of methylenedioxyphenyl moiety at 2-and 6-position of benzimidazole ring (Nandha et al., 2017). Harika et al. (Harika et al., 2017) synthesized a series of 2-substituted benzimidazole derivatives (331-333) using condensation of ophenylenediamine with different aliphatic, aromatic, fatty acids, and amino acids, and depicted remarkable antitubercular property against M. tuberculosis H37Rv strain compared to reference drugs pyrazinamide, streptomycin and ciprofloxacin. In addition, Yeong et al. (Yeong et al., 2017) designed two series of benzimidazole derivatives among which compound 334 having trifluoromethyl group displayed antimycobacterial effect against both M. tuberculosis H37Rv strain and the drug-resistant-tuberculosis strain. Compounds 335-336, synthesized by Prasad and Sundararajan, exhibited notable antitubercular activity against M. tuberculosis H37Rv strain with MIC value of 3.9 µg/ml compared to the standard isoniazid (Prasad and Sundararajan, 2017). Recently, Ashok et al. (Ashok et al., 2018) synthesized a series of indole-benzimidazole-based 1,2,3-triazole hybrids (337-339) by conventional and microwave-assisted methods and evaluated for in vitro antitubercular activity against M. tuberculosis H37Rv strain. The derivatives 337-339 showed prominent antitubercular activity with MIC values in the range of 3.125-6.25 μg/ml. Compound 338 was the most potent among all (MIC 3.125 μg/ml) which was likely due to the presence of nitro group at ortho position of phenyl ring. Compound 340 displayed notable activity (MIC 0.05 μg/ml) and emerged as a promising antitubercular agent among the reported series (Shaikh et al., 2018). Compound 341 showing 67.56, 53.45, and 47.56% inhibition against mycobacterial enzymes isocitrate lyase, pantothenate synthetase and chorismate mutase, respectively appeared to be the most potent antitubercular agent among the series (Yadav et al., 2018). Very recently, compound 342 exerted excellent potency against M. tuberculosis H37Rv with IC 50 value of 0.78 mg/ml compared to standard ethambutol (IC 50 1.56 mg/ml) (Sirim et al., 2020). Gobis et al. (Gobis et al., 2015) have prepared a series of compounds containing novel 2-(2-phenalkyl)-1H-benzo [d]imidazole where the compound bearing methyl groups at the benzimidazole system and phenethyl substituent at the C-2 position with electronegative chlorine atom at the phenyl ring exhibited prominent tuberculostatic property against Mycobacterium tuberculosis strains with MIC values ranging from 0.8 to 1.6 μg/ml (Figure 13).

Antiprotozoal Activity
The exploration of benzimidazole nucleus to discover new structural features required for the optimization of novel antiprotozoal agents is of utmost importance. Benzimidazole derivatives with antileishmanial, antimalarial, and antiprotozoal activities against different species are shown in Figure 14.

Benzimidazole Against Malaria
Among the four Plasmodium species responsible for human malaria, P. falciparum has already started to show resistance to available antimalarial drugs, thus causing the urgency to develop drugs with novel drug targets and new mechanism of action (Singh et al., 2012). In search for compounds with comparable activity to chloroquine, Camacho et al. (Camacho et al., 2011) developed a series of benzimidazole-5carbohydrazide derivatives and reported that the compounds 351-352 showed prominent in vivo antimalarial potential against rodent P. berghei and appeared to be as effective as chloroquine. Based on structureactivity relationship studies and pharmacokinetics optimization, compound 353 was found to display noticeable efficacy in the humanized P. falciparum mouse model of malaria (Pf/SCID model) with ED 90 value of 28.6 mg/kg. The attachment of the neutral hydrophobic group at position 6 of the benzimidazole moiety enhanced this excellent anti-malarial property (Hameed et al., 2014). Singh et al. (Singh K. et al., 2017) reported a series of pyrido [1,2-a]benzimidazole (PBI) antimalarial agents having in vitro anti-plasmodial activity with IC 50 values of 0.02-0.95 μM against Pf NF54 strain, and 0.02-1.07 μM against multidrugresistant Pf K1 strain of P. falciparum. Among which, compounds 354-355 exhibited most prominent in vivo efficacy in mouse P. berghei model due to the presence of chlorine group at C-7, C-8 and C-9 position of benzimidazole moiety. In another study, compounds 356-358 displayed excellent activity with IC 50 values of 0.69, 1.60 and 1.61 μM, respectively against chloroquine-sensitive 3D7 strain compared to standard chloroquine (IC 50 1.53 µM) . Recently, compounds 359-360 were synthesized as highly potent antimalarial agents having IC 50 values of 0.098 and 0.062 μM, respectively against NF54 strain of P. falciparum (Mueller et al., 2020).

Benzimidazole Against Different Protozoa
Torres-Gómez et al. (Torres-Gómez et al., 2008) synthesized a library of benzimidazole-pentamidine hybrids and evaluated them for antiprotozoal activity against T. vaginalis, E. histolytica, G. lamblia, L. Mexicana and P. berghei. Compound 361 was found to be the most potent from the series, showing 3and 9-times more activity than standards metronidazole and pentamidine, respectively. Alp et al. (Alp et al., 2009)   Compounds 372 and 373 showed notable effect against T. vaginalis and G. intestinalis, respectively in comparison with standards albendazole and metronidazole, and compound 374 emerged as a broad-spectrum antiprotozoal agent with activity against all three tested protozoans. Farahat et al. (Farahat et al., 2018) synthesized a series of benzimidazole bichalcophene diamidine derivatives. Compound 375 showed prominent antiparasitic activity towards mice model infected with T. brucei rhodesiense at a dose of 4 × 5 mg/kg i.p. and was found to be more potent than pentamidine, the usual drug of choice to treat African sleeping sickness. Similarly, compounds 376-378 exerted superior efficacy against T. brucei in the treatment of human African trypanosomiasis with IC 50 (mean ± SEM) values of 0.47 ± 0.02, 3.67 ± 0.30, and 0.71 ± 0.22, respectively compared to standard nifurtimox (IC 50 2.0 ± 0.2). The presence of diethylaminoethyl group substantially augmented the antitrypanosomal property of the compounds (376-378). Unsubstituted or methyl substituted aromatic rings or inclusion of an imidazole ring at C-5 also potentiated the activity (Figure 15) (Popov et al., 2020).

Antihypertensive Activity
A number of marketed antihypertensive drugs comprise benzimidazole moiety, Candesartan cilexetil and Telmisartan are two major examples (Figure 2). Categorically they are the antagonists of angiotensin II receptor playing important role in managing hypertension (Keri et al., 2015). In recent years, a number of scientists have conducted research to prepare benzimidazole based novel antihypertensive agents which provided similar or even better efficacy than the conventional types of antihypertensive drugs. Different benzimidazole derivatives with antihypertensive activity are shown in Figure 16. Sharma et al. (Sharma et al., 2010) synthesized a series of substituted benzimidazole derivatives and evaluated for their property as angiotensin II receptor antagonists or sartans using invasive (direct) method in Wister rats. Compounds 379 and 380 appeared to be the most prominent antihypertensive agents from the series compared to standard losartan. Kusumoto et al. (Kusumoto et al., 2011) reported about a novel, long-lasting and potent AT 1 blocker azilsartan medoxomil and its active metabolite azilsartan (381) and investigated its pharmacological profile in rat and dog models. Oral administration of 0.1-1 mg/kg azilsartan medoxomil in spontaneously hypertensive rats (SHRs) and renal hypertensive dogs demonstrated better effect in reduction of blood pressure at all doses compared to standard drug olmesartan medoxomil. Abou-Seri et al. (Abou-Seri et al., 2011) synthesized a series of 2alkoxy-4-aryl-6-(1H-benzimidazol-2-yl)-3-pyridinecarbonitrile derivatives. All compounds in the series displayed significant vasodilation properties. Compounds 382-385 showed most prominent activity (IC 50 0.145, 0.202, 0.210, and 0.214 mM, respectively) compared to standard prazosin hydrochloride (IC 50 0.487 mM). Datani et al. (Datani et al., 2012) synthesized a series of twenty novel 5-nitro benzimidazole analogues and screened them for ex vivo vasorelaxant property in rat aorta rings pre-contracted with phenylephrine. Compounds 386-390 exhibited prominent vasorelaxant activity with EC 50 value less than 30 μM. Among a new set of 5-nitro benzimidazole derivatives, compound 391 emerged as the most active agent against AT 1 with IC 50 value of 1.03 ± 0.26 nM (Zhu et al., 2014). The presence of butyl chain on 2-position of benzimidazole moiety (391) helped the derivative to interact and bind tightly with lipophilic pocket of the receptor. Several benzimidazole derivatives containing indazole moiety were synthesized by Lamotte et al. (Lamotte et al., 2014) among which compound 392 displayed potent AT 1 receptor antagonism as indicated by IC 50 value (0.006 mM).
Two series of nitric oxide (NO) releasing benzimidazole derivatives were synthesized by coupling benzimidazole biphenyl skeleton with nitro ester and furoxan NO-donor moieties, where compounds 393-394 were reported to possess comparable activity to positive control losartan . Hao et al. (Hao et al., 2015) designed and synthesized a series of 4′-[(benzimidazol-1-yl)methyl]biphenyl-2sulphonamide derivatives and reported that compound 395 was found to be the most potent AT 1 and Endothelin ET A receptor antagonist with IC 50 values 28 and 10 nM, respectively. Upon the identification of a new series of benzimidazole oxazolidinediones as mineralocorticoid receptor (MR) antagonists by high-throughput screening, compound 396 showed similar efficacy as standard drug spironolactone at a dose of 100 mg/kg (p.o.) in rat natriuresis model (Yang et al., 2015). Bao et al. (Bao et al., 2017) synthesized a series of benzimidazole derivatives which reduced blood pressure in dose-dependent manner in spontaneously hypertensive rats. Among the series, compound 397 exhibited long-lasting efficiency in decreasing blood pressure, with a maximal lowered response of 35.82 ± 6.20 mmHg at 5 mg/kg and 55.98 ± 4.74 mmHg at 10 mg/kg. The compound also showed potent affinity towards AT 1 receptor compared to standard telmisartan with IC 50 value of 1.13 ± 1.68 nM. In a separate study, Khan et al. (Khan et al., 2018) synthesized a series of 2phenyl substituted benzimidazoles and assessed antihypertensive activity of these derivatives by using tail cuff method and confirmed excellent antihypertensive property of compound 398 in spontaneously hypertensive rats compared to FIGURE 20 | Benzimidazole derivatives as neuro-protective agents.

Antidiabetic Activity
Several benzimidazole based compounds have displayed promising antidiabetic activity by acting as targets of varied stages of carbohydrate metabolism and some of them have been marketed for the treatment of type 2 diabetes. The structure of benzimidazole derivatives with antidiabetic activity reported within recent years are shown in Figure 17.
Several 2-(pyridine-2-yl)-1H-benzimidazole derivatives were synthesized by Ishikawa et al. (Ishikawa et al., 2009) among which compound 402 emerged as the most potent and metabolically stable glucokinase activator. Besides, the compound proved its oral glucose lowering efficacy in rat oral glucose tolerance test (OGTT) model. A new series of 4-thiazolidinones and 1,3,4-oxadiazoles bearing 2-mercapto benzimidazole moiety were prepared by Shingalapur et al. (Shingalapur et al., 2010) among which compounds 403-406 produced notable results in OGTT model. All the potent derivatives contained hydroxyl group which might have contributed for their antidiabetic property. Some 2-benzoyl benzimidazole derivatives were assessed for antidiabetic and lipid-lowering effects (Ushiroda et al., 2011). Compound 407 appeared to be an effective peroxisome proliferator-activated receptor (PPARγ) partial agonist. Shaikh et al. (Shaikh et al., 2012) reported one-pot synthesis of carbonyl-amide linkage based benzimidazole derivatives (408-413) via Passerini reaction and demonstrated their antidiabetic potential using rat OGTT model compared to the standard drug glibenclamide.
Diacylglycerol O-acyltransferase-1 (DGAT-1) is an enzyme which catalyzes the formation of triglycerides from diacylglycerol and Acyl-CoA and thereby plays an important role in intestinal fat absorption. DGAT-1 has emerged as an important therapeutic target for the management of different metabolic disorders, such as obesity and diabetes (Birch et al., 2010). Kwak et al. (Kwak et al., 2013) prepared some benzimidazole derivatives as inhibitors of DGAT-1 which contained a phenylcyclohexyl acetic acid group in benzimidazole moiety. Among the derivatives, compound 414 displayed potent in vivo antidiabetic potential in a 4-week study with diet-induced obesity (DIO) mouse model. The α-glucosidase inhibitors are an important class of antidiabetic agents because of their property to reduce the postprandial glucose level in type-2 diabetic patients (Özil et al., 2016). Mobinikhaledi et al. (Mobinikhaledi et al., 2015) synthesized several new benzimidazole derivatives from amino acids in the presence of phosphorus oxychloride (POCl 3 ). Compounds 415 and 416, upon evaluation of yeast and rat intestinal α-glucosidases inhibitory effect produced most notable results. The IC 50 values for compound 416 against yeast and rat intestinal α-glucosidases were reported as 9.1 and 36.7 μM, respectively and thus it appeared to be the most potent benzimidazole among the series. Özil and coworkers (Özil et al., 2016) prepared a series of bisbenzimidazole derivatives by the reaction of o-phenylenediamine and 4-nitro-o-phenylenediamine with oxalic acid using both conventional and microwave techniques. Compounds 417-420 demonstrated prominent α-glucosidase inhibition with IC 50 values of 0.54 ± 0.01, 0.44 ± 0.04, 1.24 ± 0.05 and 0.49 ± 0.01 µM, respectively compared to standard acarbose (IC 50 13.34 ± 1.26 µM). Some novel 1,3,4-thiadiazole substituted 2-methyl benzimidazole derivatives were synthesized by conventional methods among which compound 421 exhibited significant in vitro antidiabetic property (Nair et al., 2016). A series of hybrid benzimidazole-thiourea derivatives were synthesized by Zawawi et al. (Zawawi et al., 2017) and evaluated for α-glucosidases inhibitory potential. Compounds 422-423 displayed significant inhibitory properties with IC 50 values of 50.57 ± 0.81 and 35.83 ± 0.66 µM, respectively. Besides, Singh et al. (Singh et al., 2018) synthesized a library of N-substitutedbenzimidazolyl linked para substituted benzylidene derivatives. Compound 424 bearing 2,4-thiazolidinedione group at 4position of phenyl ring exhibited pronounced in vitro α-amylase and α-glucosidase inhibitory properties (IC 50 0.54 ± 0.01 µM) and appeared as a promising antidiabetic agent from the series.

Anticoagulant Activity
Different substituted benzimidazole derivatives have been explored for several years for their anticoagulant activity and potential use in clinical practice. Benzimidazole derivatives acting as anticoagulants are shown in Figure 18.
Thrombin (fIIa) is a multifunctional serine protease responsible for the proteolytic cleavage of fibrinogen. Inhibition of thrombin is a crucial mechanism for inhibition of coagulation. Benzimidazole moiety serves as a suitable template for placing a wide range of substituents required for interaction with thrombin (Hauel et al., 2002). Hauel et al. (Hauel et al., 2002) designed and synthesized a series of new benzimidazole derivatives bearing structural similarity to α-NAPAP (N-alpha-(2-naphthylsulfonylglycyl)-4amidinophenylalanine piperidide), a benzamidine-based powerful inhibitor of thrombin, trypsin and other serine proteases. With the addition of ethyl ester and hexyloxycarbonyl carbamide hydrophobic side chains, improved pharmacokinetic profile was obtained leading to the invention of orally absorbed prodrug, 437 (Dabigatran etexilate). The prodrug 437 reached clinical trials and its active form 438 (Dabigatran) was discovered with excellent thrombin inhibitory potency and tolerability. Recently, Ren et al. ) designed a series of benzimidazole derivatives and tested them for thrombin inhibitory effects. Compound 439 with IC 50 value of 3.11 ± 0.21 nM appeared to be a potent thrombin inhibitor exhibiting better activity than the standard argatroban (IC 50 9.88 ± 2.26 nM). A series of 1,2,5-trisubstituted benzimidazole fluorinated derivatives (440-442) were also evaluated for in vitro inhibitory activity against thrombin . Compounds 440-442 with IC 50 values of 2.26 ± 0.38, 1.54 ± 0.09 and 3.35 ± 0.87 nmol/L, respectively showed improved result compared to argatroban (IC 50 9.88 ± 2.26 nmol/L), thus showing their potential as thrombin inhibitors. A library of benzimidazole derived 1,3,4-oxadiazole derivatives (443-445) were assessed for ex vivo anticoagulant activity by determining the effect of compounds in increasing prothrombin time (PT) and activated partial thromboplastin time (aPTT) (Vishwanathan and Gurupadayya, 2015). Compounds 443-445 displayed significant increase in PT (32 ± 0.7, 36 ± 0.5 and 41 ± 0.4 s, respectively) compared to standard drug acenocoumarol (48 ± 0.5 s). The compounds, however, caused a slight increase in aPTT in comparison with the reference drug, unfractionated heparin (500 IU/kg).
Factor IXa (fIXa), an important coagulation factor, is a useful target for developing potent and selective antithrombotic agents. A research involving pharmacophore modelling of a new series of benzimidazole analogues presented the chemical features necessary for designing fIXa inhibitors and showed that benzimidazole derivatives have the potential to be developed into effective antithrombotic agents (Kumbhar et al., 2017). Compound 446 was found to be the most active compound from the series, indicated by fIXa binding affinity (K i ) value of 0.016 µM.
Recently, Zhang et al. (Zhang et al., 2020) designed and synthesized ten novel dabigatran derivatives (447-456) with high docking score. The study uncovered that all the compounds showed more than 50% in vitro thrombin inhibitory property at 1 mg/ml concentration, where the IC 50 values of compounds 447, 450 and 456 were 1.92, 2.17 and 1.54 nM, respectively, comparable to the IC 50 value of positive control dabigatran (1.20 nM). The derivatives 425 and 428, previously reported in this review for antidiabetic property, exerted anticoagulant activity by augmenting the clotting duration. However, only compound 425 exhibited excellent inhibition (93.4%) of epinephrine-induced platelet aggregation (Bharadwaj et al., 2018).

Anticonvulsant Activity
Epilepsy is one of the most prevalent and serious neurological disorders, and recurrent seizures or convulsions are its characteristic syndrome. Around one-third of patients in the world show poor response to currently available antiepileptic drugs (Keri et al., 2015). In search of novel clinically effective anticonvulsant medications, benzimidazole nucleus has recently been explored by scientists with promising results. The benzimidazole derivatives with anticonvulsant property are shown in Figure 19.
A library of 2-mercaptobenzimidazole derivatives were evaluated for anticonvulsant activity using maximal electroshock seizure (MES) model. The synthesized compounds displayed anticonvulsant property at a dose of 20 mg/kg (i.p.) compared to standard phenytoin and compounds 457-458 appeared to be the most potent of all (Anandarajagopal et al., 2010). A series of 4-thiazolidinones and 1,3,4-oxadiazoles bearing 2-mercaptobenzimidazole moiety were assessed for in vivo anticonvulsant activity (Shingalapur et al., 2010). Compounds 459-462 emerged as the most promising anticonvulsants in MES model.
Siddiqui and co-workers described the synthesis of several imidazol-2-yl) methyl}-3-arylthioureas which have been mentioned earlier for their analgesic potential. The research group in a previous study reported about the anticonvulsant and cytotoxic effects of the same series of compounds. Compounds 463-465 were found to possess potent anticonvulsant property in comparison with standard drug phenytoin (Siddiqui and Alam, 2010). A series of nitro-benzimidazole derivatives were synthesized by Jain et al. and screened for anticonvulsant activity using MES and subcutaneous pentylenetetrazole (scPTZ) models. Compound 466 displayed the most promising result in inhibiting convulsion induced in mice by both methods (Jain et al., 2010).  hydrazine]-N-phenyl-1H-benzo[d] imidazole-1-carbothioamides (467-471) from the reaction of 2-mercaptobenzimidazole with hydrazine hydrate, substituted acetophenones and phenylisothiocyanate. Compounds 467-471 were found to be active compounds in MES and scPTZ models, and devoid of neurotoxicity (Bhrigu et al., 2012).
A series of benzimidazole substituted semicarbazones were synthesized and tested for anticonvulsant activity using MES model. Compound 472 at a dose of 50 mg/kg (i.p.) appeared to be the most potent among the derivatives (Rajak, 2015). In another study, some oxadiazole bearing benzimidazoles and several derivatives of 2-[2-(phenoxymethyl)-1H-benzimidazol-1-yl]-N0-[(Z)-phenylmethylidene] acetohydrazide (473-474) were evaluated employing MES and scPTZ methods. Compounds 473-474 were found to be effective anticonvulsant agents (Shaharyar et al., 2016). A new series of hybrid benzimidazole containing pyridazinones were developed to assess anticonvulsant property. Compound 475 emerged as an effective and safe anticonvulsant in both MES and scPTZ models. The compound also exhibited notable increase in GABA level (1.7-fold) compared to control which was attributed to its good binding property with the GABA A receptor (Partap et al., 2017). Recently, Sahoo et al. (Sahoo et al., 2019)

Benzimidazole as Neuro-protective Agent
Neurological disorders comprise the disorders of central and peripheral nervous system. Alzheimer disease (AD), dementia, stroke, Parkinson's disease, multiple sclerosis, brain tumor etc. are the most common diseases affecting a large number of people. AD is a chronic neurodegenerative disorder and the most common form of dementia manifested by loss of memory, language, cognitive functions, behavior and emotion. The factors which mostly contribute in the development of AD include the levels of acetylcholine (ACh) and deposits of neurotoxic amyloid-β-peptide (Aβ) . A number of substituted benzimidazole derivatives have been developed for the management and treatment of neurological diseases. The benzimidazole derivatives explored recently as neuro-protective agents are shown in Figure 20. Khan et al. synthesized a series of 6-nitrobenzimidazole derivatives and screened their phosphodiesterase inhibitory property. Among them, compounds 481 (IC 50 1.5 ± 0.043 µM) and 482 (IC 50 2.4 ± 0.049 µM) were found to possess better activity than the standard EDTA (IC 50 274 ± 0.007 µM). The presence of 2,3-dihydroxyphenyl (481) and 4hydroxyphenyl (482) moiety might have contributed for their notable property (Khan et al., 2012). Hu et al. designed and synthesized a series of keto-benzimidazole derivatives (483-484) as potent and selective inhibitors of Phosphodiesterase 10A (PDE10A). Compound 484 showed 55% receptor occupancy (RO) of PDE10A at 30 mg/kg p.o. in rat brain. Further research led to the identification of compound 484, with improvements in in vivo efficacy (57% RO in rats at 10 mg/kg p.o.), rat clearance and oral bioavailability . Hamaguchi et al. (Hamaguchi et al., 2013) prepared a series of benzimidazole derivatives as PDE10A inhibitors with reduced CYP1A2 inhibition among which the compound 485 appeared to be the most prominent.
Tamura and co-workers synthesized a series of benzimidazole derivatives for neuropeptide Y (NPY) receptor antagonistic activity which is an important pharmacological target for the treatment of different neurodegenerative disorders. Compound 486 appeared to be the most promising agent (Tamura et al., 2012a). In another study by the same research group (Tamura et al., 2012b), compound 487 was reported to show high NPY Y5 receptor binding affinity along with good absorption, distribution, metabolism and elimination (ADME) profile resulting in notable in vivo efficacy as NPY Y5 receptor antagonist.
Kim et al. evaluated a series of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as inhibitors of c-Jun N-terminal kinases (JNK3), a group of degenerative signal transducers and potential target of neurodegenerative diseases, e.g. Alzheimer's and Parkinson's diseases. Majority of the compounds exhibited high affinity (K d 10 µM-46 nM) to JNK3 when investigated through SPR, JNK3 kinase assay and cell-viability of human neuroblastoma cells. The most potent compound 488 showed notable cell protective effect (IC 50 1.09 µM) against toxicity induced by anisomycin .
Human Presequence Protease (hPreP) is a mitochondrial metalloprotease capable of degrading amyloid-β peptide and increasing its proteolysis in human neuronal cells. Identification of potential agonists of hPreP is of great importance for Alzheimer's drug design.  showed marked enhancement of hPreP-mediated proteolysis of Aβ, pF1β and fluorogenic-substrate V and thus presented great potential in the treatment of Alzheimer's disease (Vangavaragu et al., 2014).
Among the therapeutic agents used for the treatment of traumatic brain injury (TBI), positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGluR5) are important. He et al. (He et al., 2015) designed and synthesized a series of acyl-2-aminobenzimidazole derivatives based on the chemical structure of a well-known mGluR5 PAM called 3,3'difluorobenzaldazine (DFB). The compounds were tested for binding affinity to transmembrane domain of mGluR5 using nitric oxide (NO) production assay, among which compound 491 (IC 50 6.4 µM) was found to be around 20 times more potent than DFB (IC 50 136 µM) . Zhu (Zhu et al., 2013). In another study, the compounds (495-496) showed selective inhibition on BChE and appeared to be the most potent BuChE inhibitors (IC 50 5.18 and 5.22 µM, respectively) (Ozadali-Sari et al., 2017). Besides, compound 497 was found to be the most potent AChE inhibitor (IC 50 0.93 ± 0.04 µM) with marked selectivity ratio (13.68) (Alpan et al., 2017). Similarly, compound 498 carrying a methyl group at 5position of benzimidazole ring and 2-fluorobenzyl group connected to 1,2,3-triazole system was found to be the most potent inhibitor of AChE displaying 84% inhibition at 100 μM concentration (Faraji et al., 2017). The nitrophenyl piperazine substituted derivative 499 showed 57.25 and 77.92% inhibition of Rho-associated protein kinase II (ROCK II) enzyme at a concentration of 0.5 and 1 mM, respectively and exerted prominent IOP lowering effect (51.56%) compared to the standard fasudil (Abbhi et al., 2017).
Recently, compounds 500-501 were reported as promising AChE and BuChE inhibitors (IC 50 0.14 and 0.22 μM, respectively), highly neuroprotective against hydrogen peroxide mediated toxicity, metal chelators, and free radical scavengers in the treatment of Alzheimer's disease (Sarıkaya et al., 2018). Similarly, compounds 502-503 demonstrated duel effects as anti-Alzheimer agents by beta amyloid cleavage enzyme-1 (BACE1) inhibition and neuroprotection (Gurjar et al., 2020). In a 6-hydroxydopamine (6-OHDA)-induced oxidative stress in vitro model assay, compound 504 exerted neuroprotective action compared to melatonin, and reduced superoxide anion radical and hypochlorite level in a luminol-dependent chemiluminescent assay (Anastassova et al., 2020). In another study, compound 505 displayed significant antineuroinflammatory property (IC 50 5.07 mM to prevent nitric oxide generation) and 65.7% hBACE1 inhibition. Besides, the compound (505) increased glutathione (GSH) level, reduced ROS production, and subsequently opened a door for further development to be established as a promising treatment option against Alzheimer's disease (Fang et al., 2019). Finally, compound 506 exhibited promising neuroprotective role on SH-SY5Y cells by preserving the synaptosomal viability and minimizing GST level compared to the standards melatonin and rasagiline (Anastassova et al., 2021).

Miscellaneous Activities
Several other classes of benzimidazole derivatives have been prepared by different scientists during the last few years. Some of these novel benzimidazole based compounds are shown in Figure 21.
A series of 2-pyridylbenzimidazole derivatives were designed and synthesized as Cannabinoid 1 (CB1) receptor agonists amongst which compound 509 appeared to be the most potent with a binding affinity value (K iCB1 ) of 0.53 nM (Mella-Raipán et al., 2013). The same research group synthesized a series of N-acyl-2,5-dimethoxyphenyl-1H-benzimidazole derivatives and assessed them for human CB1 receptor binding affinity using competitive binding assays (Espinosa-Bustos et al., 2015). Compound 510, carrying 3-nitrophenyl group in the aroyl region of benzimidazole moiety was found to be the most promising agent (K i 1.2 nM). More recently, they synthesized a new series of benzimidazole derivatives which displayed more selectivity towards human Cannabinoid 2 (CB2) receptor than that towards CB1 receptor (Romero-Parra et al., 2016). Compound 511 demonstrated the best binding affinity (K i 0.08 µM), high selectivity index (K i CB1/K i CB2 >125) and low toxicity.
Mochizuki and co-workers (Mochizuki et al., 2016) assessed several benzimidazole analogues for in vivo corticotropin releasing factor type 1 (CRF1) receptor antagonistic property. Compound 514 with an electron withdrawing cyano group at the 4-position of benzimidazole nucleus emerged as the most promising among the series. In continuation with their research to develop CRF1 receptor antagonists, compound 515 was reported as a potent CRF1 binding inhibitor (IC 50 4.1 nM) and in vitro CRF1 antagonist (IC 50 44 nM) (Mochizuki et al., 2017). More recently they designed a series of 1,2,3,4tetrahydropyrimido [1,2-a]benzimidazole analogues as novel CRF1 receptor antagonists. Compound 516 displayed very prominent CRF1 receptor binding activity (IC 50 58 nM) and oral bioavailability (F 68% in rat model) indicating its potential in developing clinically effective CRF1 receptor antagonist in the future (Kojima et al., 2018).
A library of small molecules were tested for their property as human gonadotropin releasing hormone (GnRH) receptor antagonists. The derivative 517 (K i 13.8 nM) exhibited highest affinity towards human GnRH receptor and emerged as the most prominent GnRH receptor antagonist (Fjellaksel et al., 2017). Furthermore, a recent study reported several benzimidazole analogs (518-521) as intraocular pressure (IOP) reducer in the treatment of dexamethasone-induced ocular hypertension. Among these, compound 521 exerted the best anti-glaucoma action by the maximum IOP reduction of 22.32% from baseline following single drop administration (0.1%) (Marcus et al., 2019). Maltsev et al. (Maltsev et al., 2020) evaluated a series of Diazepino [1,2-a]benzimidazole derivatives for their possible psychotropic properties. Compound 522 at a dose of 2.34 mg/kg showed prominent anxiolytic, antidepressant and anticonvulsant activities in both rat and mice models. Vasil'ev et al. (Vasil'ev et al., 2017) investigated a series of imidazo [1,2-a] benzimidazole derivatives using corazole-induced seizure model. The compounds 523-526 displayed notable anticonvulsive activity in comparison with standard valproic acid. The prominent activity is attributed to the presence of a 4-fluoro substituent in the 2-position and a dialkylaminoalkyl or cycloalkylaminoalkyl substituent in the 9-position of the fused ring.
Change et al. (Chang et al., 2017) investigated the antiplatelet activity of some novel saccharide-based benzimidazole derivatives. Compound 527 exhibited concentration-dependent inhibitory property against thrombin (0.01 U/ml) and collagen (1 µg/ml)-induced human platelet aggregation in an in vitro model. Its inhibitory effect might be attributed to the presence of 1-imidazolyl moiety at one end carrying a long chain of three sugar moieties. A series of benzimidazole derivatives bearing a sterically hindered phenolic group in their structures were evaluated for in vitro antiplatelet activity using the Adenosine diphosphate (ADP)-induced platelet aggregation model of rabbit's plasma . Compound 528 showed notable antiplatelet activity by exceeding the standard acetylsalicylic acid by 21.8 times. In the in vivo study of inhibition of intravascular platelet aggregation, the same compound displayed 1.5 times superior activity than Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762807 acetylsalicylic acid and slightly inferior activity than another standard clopidogrel. Idris and his co-workers (Idris et al., 2019) reported that benzimidazole derivatives 529 and 530 decreased repeated morphine administration-induced thermal hyperalgesia and tactile allodynia as well as the expression of spinal Tumor Necrosis Factor-alpha (TNF-α) in Balb-c mice. The compounds displayed PPARγ agonist activity, indicating their potential in the management of morphine-induced paradoxical pain. Akhtar et al. (Akhtar et al., 2021) evaluated the role of benzimidazole derivative 531 in managing nalbuphine-induced tolerance in cisplatininduced neuropathic pain in mice. Cisplatin, a platinum-based anticancer medication, is often coupled with Nalbuphine, an opioid analgesic used for treating acute and chronic pain. The compound 531 attenuated the tolerance to the analgesic activity of nalbuphine developed due to its long-term use, as well as TNF-α expression in the spinal cord of mice.
Moreover, Raka et al. (Raka et al., 2021) reported a series of substituted benzimidazole derivatives and found a notable inhibitory property of compound 532 against the acetylcholinesterase enzyme with an IC 50 value of 29.64 μg/ml compared to the standard drug donepezil (IC 50 9.54 μg/ml).
Among the heterocyclic compounds, benzimidazole derivatives have been playing the most pulsing and eye-catching pioneer role in synthetic pharmaceutical and agrochemical sectors for the last couple of decades (Pardeshi et al., 2021). As having the similarity of the benzimidazole nucleus with many naturally occurring nucleotides and its presence in several natural compounds, the benzimidazole derivatives can easily interact with various biomacromolecules or target proteins. Thus, the compounds derived from the benzimidazole ring system exert broadspectrum efficacy against various human disorders like cancer, hypertension, diabetes, bacterial or viral infection, inflammation, gastritis, neurodegenerative disorders, and so on .
As stated in the comprehensive review, there are diverse classes of benzimidazole derivatives synthesized by several groups, which showed prominent bioactivities even sometimes better than the existing drugs. Although several structure-activity relationship studies were accomplished by several groups in a particular area of bioactivities, it is challenging to draw any conclusion on the structural features needed for a specific activity. In many cases, further studies on specific benzimidazole derivatives leading to the development of potential drug candidates were not continued. The synthesis of a new library of benzimidazole structures is expanding day by day, with a unique spectrum of bioactivity being reported regularly. Despite having extensive biological and diagnostic applications with the lustrous futuristic potentiality of benzimidazole scaffolds, barriers and challenges are still viable in the clinical development process. Drug resistance is the major drawback of the antiviral, antifungal, antibacterial, antimalarial, anti-cancer, anti-hepatitis, and anti-HIV agents . Another notable concern is that several benzimidazole derivatives such as PPIs are showing in vitro and in vivo drugdrug interactions with several classes of therapeutics such as anticancer (Bezabeh et al., 2012) and antidiabetics (Hossain et al., 2020;Hossain et al., 2021).
Benzimidazole derivatives containing pyrrolidine side-chain exhibited a beacon of hope in malignancy treatment where several available drugs (for example, sorafenib) have become resistant due to long-term exposure against several cancers, such as hepatocellular carcinoma (Suk et al., 2019). It is a crucial fact that benzimidazole derivatives have been synthesized and screened against many disease conditions; among them, very few enter into clinical trials. For example, selumetinib (NCT01933932) and galeterone (NCT04098081) are two promising agents undergoing several clinical trials to be established as effective cancer agents. Besides, several ongoing clinical trials (For instance, NCT01611974) of various drugs (maribavir) bearing benzimidazole moiety showed promising effects against resistant cytomegalovirus (Maertens et al., 2019;Papanicolaou et al., 2019). Dexlansoprazole completed the phase IV trial (NCT01093755) and showed extensive effect against esophageal inflammation (Sharma et al., 2020). However, these reported trials are still very negligible compared to the preclinical status of the ample amount of benzimidazole drug candidates.
It is crystal clear that an exciting number of benzimidazole pharmacophore derivatives have become potential drug candidates to treat several disease conditions in ongoing clinical trials. However, this promising moiety is copious to be further investigated for designing a number of different target molecules, which might reveal some encouraging outcomes in medicinal research. It can easily be assumed at the current stage that several new strategies might be designed and developed for multi-target agents with the highest affinity, Frontiers in Pharmacology | www.frontiersin.org November 2021 | Volume 12 | Article 762807 specificity, and enhanced bioactivities. In the future, the benzimidazole scaffolds might be fused with another heterocyclic ring system to be explored several novel chemically stable and potent pharmacological agents against several rare or critical diseases or with better pharmacokinetic and pharmacodynamic profiles. This might be an outrageous and worldwide revolutionary achievement in the next century. Toxicity, drug resistance, and poor bioavailability are the significant concerns during drug development or the underlying reasons behind the compounds be untouched in the human trial. Structural modifications of the existing synthesized molecules might be an excellent technique to be brought about improved or novel activities with a better safety profile. Besides, the prodrug strategy can also be applied to ameliorate the bioavailability of the present derivatives. Furthermore, combinations of the benzimidazole derivatives with other existing drugs might be explored for finding synergistic effects or exciting pharmacological activities against multidrug-resistant microorganisms.
Moreover, several benzimidazole-containing clinically used medications, e.g., omeprazole, lansoprazole, pantoprazole, and rabeprazole, are actually in the form of prodrugs for which the onset of action is slower. Thus, there is an increased demand for producing the active form of drugs directly from benzimidazoles, and for this, researchers are paying attention to novel benzimidazole-derived therapeutics with lesser toxicity and better pharmacodynamic profile. In this case, PPIs containing benzimidazole ring might be modified, or the benzimidazole pharmacophore might be replaced by imidazole ring with the improved biological profile. The non-approved benzimidazole scaffolds for cancer therapy having eminent pharmacokinetics and safety profile might be repurposed to treat several life-threatening cancers with a lack of safe and effective treatment. Some anthelmintic drugs containing benzimidazole nuclei, such as flubendazole, albendazole, and mebendazole, could be considered to apply against various types of cancer in future investigations. Besides, stereochemistry could be regarded as to trace and explore more potent isomers of the existing approved benzimidazole derivatives because one enantiomer may exert distinct biochemical property than the other enantiomer.

CONCLUSION
Benzimidazole, an essential nitrogen-containing heterocyclic moiety, can be found in various therapeutically used compounds playing a vital role in treating many diseases. Many efforts have been given so far on the development of target-based benzimidazole derivatives, and the interest to produce new therapeutically active agents to treat different diseased conditions has grown noticeably during the last few years. However, this field has several challenges, especially to bring the numerous synthesized compounds into the clinical trial, which showed valuable pharmacological properties in different studies, and later to ensure their availability in the market and clinical practice. The present review has focused on the current status of benzimidazole moiety, emphasizing the SAR of different benzimidazole-based compounds explored by scientists around the world. So far this is the most inclusive and informative account about biological and therapeutic potential of benzimidazole derivatives. With a compilation of information from more than 250 latest pieces of literature, we aimed to aid the researchers, medicinal chemists, and drug designers with valuable and comprehensive knowledge and provide them the rationale to develop target-oriented and clinically useful benzimidazole-based molecules.