Various Biological Properties

#### **Chapter 3**

### Benzimidazole: Pharmacological Profile

*Mahender Thatikayala, Anil Kumar Garige and Hemalatha Gadegoni*

#### **Abstract**

Benzimidazole is a bicyclic heterocyclic aromatic compound in which benzene fused to imidazole moiety. Benzimidazole holds a vital role in the field of medicinal chemistry which possesses wide variety of pharmacological activities like antibacterial, anti cancer, antifungal, antileishmanial, anti tubercular, anti viral and anti malarial respectively, hence the benzimidazole moiety attracting the medicinal chemist to synthesize the different benzimidazole derivatives with wide variety of pharmacological activities. The book chapter mainly discussed the anti cancer, anti HIV, antileishmanial and anti tubercular activites of recently synthesized benzimidazole derivatives.

**Keywords:** benzimidazole, anti cancer, anti HIV, antileishmanial, anti tubercular

#### **1. Introduction**

Benzimidazole is bicyclic heterocyclic aromatic compound in which benzene ring fused to 4 and 5 position of imidazole ring, it contain two nitrogen atoms at 1 and 3 position exhibit both acidic and basic nature called amphotericin nature and exists in two equivalent tautomeric forms, when the hydrogen present at first position nitrogen atom possess acidic nature, when the hydrogen present at third position nitrogen atom possess basic nature (**Figures 1**) [1]. Benzimidazole is a very important important pharmacophore among all the heterocyclic compounds due to its important pharmacological activities like anti-Alzheimer [2], antibacterial [3], anti cancer [4], antidiabetic [5], antifungal [6], anti HIV [7], anti leishmanial [8], anti inflammatory [9], analgesic [9], anti malarial [10], anti microbial [11] and anti tubercular [12] activity, there are many benzimidazole derivatives are using to treat many diseases, few presently marketing drugs contain benzimidazole moiety are the bezitramide using as an analgesic, ridinilazole sing as antibacterial, the candesartan, mibefradil using as antihypertensive drugs, mebendazole, albendazole, thiabendazole, and flubendazole usng as antihelminthics, astemizole, bilastine using as antihistamines, pantoprazole, lansoprazole, esomeprazole, ilaprazole using as proton pump inhibitors, bendamustine, selumetinib, galeterone, pracinostat using as antitumor agents and enviradine, samatasvir, and maribavir using as antiviral agents (**Figures 2**) [13–17].

**Figure 1.** *Chemistry of benzimidazole [1].*

#### **2. Pharmacological profile of benzimidazole derivatives**

#### **2.1 Anti cancer activity**

In the year of 2019 Tahlan et al., reported the synthesis and anti cancer activity of the new benzimidazole derivatives, among all the derivatives the compound **1** (**Figure 3**) found to be best activity at IC50 value of 4.53 μM against the human colorectal cancer cell line [4], same authors in 2018 reported the compound **2** (**Figure 3**) showed best activity at IC50 value of 4.12 μM against the human colorectal carcinoma cell line (HCT116) [18], same year few authors reported the synthesis, anti anti cancer activity of the new benzimidazole derivatives, Aikman et al., reported the compound **3** (**Figure 3**) found to be best active compound at EC50 value of 5 2 μM against the melanoma (A375) cells [19], Mohamed et al., reported the compound **4** (**Figure 3**) showed best activity at IC50 value of 80,35, 72 μg/ml against the against human breast adenocarcinoma (MCF-7), human lung carcinoma (A549), human epitheloid cervix carcinoma (HELA) [20], Gohary et al., reported the compound **5** (**Figure 3**) showed significant activity at IC50value of 0.022, 0.014, 0.015 μM against the against liver cancer (HepG2), colon cancer (HCT-116), breast cancer (MCF-7) cells [21], in 2017 Wang et al., reported the synthesis, anti-cancer activity of the chrysin benzimidazole derivatives, the compound **6** (**Figure 3**) showed significant activity at IC50 values of 25.72 3.95 μM against MFC cells [22] and Yadav et al., reported the anti cancer activity of synthesized the 2-(*1H*-benzo[d]imidazol-2-ylthio)acetami do)-*N*-(substituted-4 oxothiazolidin-3-yl)acetamides, the compound **7, 8** (**Figure 3**) showed significant activity at IC50 value of 0.00005, 0.00012 μM/ml against HCT116 cell line [23], Onnis et al., reported the anti cancer activity of benzimidazolehydrazones, the compound **9** (**Figure 3**) showed excellent activity at IC50 value of 0.98 0.02 μM against human T-lymphoblastic leukemia (CEM) cells [24].

In 2015 few authors worked on synthesis of benzimidazole and evaluated the nti-cancer activit, the Gao et al., reported the compound **10** (**Figure 3**) showed good activity at IC50 value of 2.68 μM against K562 and HepG-2 cells [25], Kamal et al., reported the compound **11** (**Figure 3**) found to be best at IC50 value of 1.8 μM against *Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

**Figure 2.** *Structures of marketing drugs containing benzimidazole moiety [13–17].*

**Figure 3.** *Structures of effective anticancer compounds.*

most of the tumor cell lines [26], T.S. Reddy et al., reported the compounds **12**, **13** (**Figure 3**) showed best anti-cancer activity with IC50 values of 1.81, 0.83, 1.76, 1.13, 0.95, 1.57 μM against lung (A549), breast (MCF-7), cervical (HeLa) human tumor cell lines [27], Rodionov et al., reported the compound **14** (**Figure 3**) found to be good activity with 87% tumor growth inhibition against carcinoma75 [28], Sharma et al., reported the Compound **15** (**Figure 3**) showed maximum activity at GI50 values of 3.16, 2, 1.36 μM against colon cancer, CNS cancer and ovarian cancer [29] and Wang et al., reported the compound **16** (**Figure 3**) showed excellent activity at GI50 values of 2.4, 3.8, 5.1 μM against human lung adenocarcinoma cells (A549), human liver hapatocellular carcinoma (HepG2), human breast carcinoma cells (MCF-7) [30].

In 2014 Yoon et al., evaluated the anti cancer activity of synthesized novel benzimidazole derivatives, the compounds **17** (**Figure 3**) **18** (**Figure 4**) found to be good at IC50 value of 49.63, 46.33, 62.43, 42.30 μM against breast cancer cells (MCF-7), triplenegative breast cancer cells (MDA-MB-468) [31], same year Wang et al., reported anticancer activity of benzimidazole-2-urea derivates, the compound **19** (**Figure 4**) showed significant activity at IC50 value range of 0.006 to1.774 μM against the K562, A431, HepG2, Hela, MDA-MB-435S cancer cells [32], Salahuddin et al., reported the compound **20** (**Figure 4**) showed best anti cancer activity at a percentage growth of 36.23, 47.56 against Breast cancer (MDA-MB-468), Melanoma (SK-MEL-28) cells [33], Paul et al., reported the compound **21** (**Figure 4**) found to be good anticancer activity at GI50 values of 0.34, 0.31 μM against colon cancer cell lines, prostate cancer cell lines [34], Madabushi et al., reported the compound **22** (**Figure 4**) showed best anticancer activity at IC50 values of 5.2, 9.8, 12.3, 11.1 μM against A549, MCF7, DU145, HeLa human cancer cell lines [35] and Guan et al., reported the compound **23** (**Figure 4**) showed significant anticancer activity with IC50 values of 0.098, 0.15, 0.13 μM against SGC-7901, A-549, HT-1080 human cancer cell lines [36].

In 2013 Sharma et al., reported the anti cancer activity of synthesized the benzimidazole quinazoline hybrids, the compound **24** (**Figure 4**) found to be activity with percentage growth of inhibition of 98, 94.2, 94.3, 97.5 against leukemia (K-562, SR), colon (HT29), melanoma (LOX IMVI) human cancer cell lines [37], in the same year Husain et al., reported the synthesis and the anti cancer activity of benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines, the compound **25** (**Figure 4**) found to be maximum activity with growth inhibition with GI50 values ranging from 0.20 to 2.58 mM against eukemia cell lines [38], Nassan et al., reported the anti cancer activity of synthesized novel 1,2,3,4 tetrahydro[1,2,4]triazino[4,5-a] benzimidazoles, the compound **26** (**Figure 4**) showed excellent activity at IC50 value of 0.0390 μM against human breast adenocarcinoma cell line (MCF7) [39] and Hranjec et al., reported the anti cancer activity of synthesized the novel benzimidazole schiff bases, the compound **27, 28** (**Figure 4**) found to be significant activity at IC50 values of 4.73, 0.96, 3.24, 1.67 μM against HeLa, WI38 cell lines [40].

#### **2.2 Anti HIV activity**

In the year of 2020 Srivastava et al., reported the synthesis and anti HIV activity of the new benzimidazole derivatives, among all the derivatives the compound **29** (**Figure 5**) found to be best activity at IC50 value of 0.386 <sup>10</sup><sup>5</sup> <sup>μ</sup>M against HIV-1 [7], Iannazzo et al., reported the synthesis and anti HIV activity of the new benzimidazole derivatives, among all the derivatives the compound **30** (**Figure 5**) showed best activity at IC50 value of 0.09 μg/mL against HIV-1 [41], Yadav et al., reported the anti HIV activity of synthesized benzimidazole derivatives, in all the synthesized

*Benzimidazole*

**Figure 4.** *Structures of effective anticancer compounds.*

derivatives the compounds **31–34** (**Figure 5**) found to be best active compounds with more than 50% of RT inhibition at concentration of 20 μM against HIV-1 [42], same year Pan et al., evaluated the anti HIV activity of synthesized benzimidazoles, the compounds **35, 36** (**Figure 5**) found to be significant activity with IC50 values of 3.45, 58.03 nM against HIV-1 [43], Masoudi et al., synthesized the new benzimidazole derivatives, evaluated the anti HIV activity, among all the synthesized derivatives, compounds **37** (**Figure 5**) found to be significant activity at EC50 1.15 μg/mL against HIV-1 and HIV-2 [44].

*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

#### **2.3 Anti leishmanial activity**

M. Tonelli et al., reported the antileishmanial activity of newly synthesized benzimidazole derivatives, among all the derivatives compound **38** (**Figure 6**) found to be

**Figure 6.** *Structures of effective anti-leishmanial compounds.*

significant inhibition of promastigotes, amastigotes of *Leishmania tropica, Leishmania infantum* at IC50 values of 0.19, 0.34, 0.31 μM and compound **39** (**Figure 6**) inhibited promastigotes of *Leishmani infantum* at IC50 value of 3.70, 4.76 μM [8], Oh et al., reported the antileishmanial activity of newly synthesized benzimidazole derivatives, among all the derivatives compound **40, 41** (**Figure 6**) found to be most active against promastigotes, amastigotes of *Leishmania donavani* at EC50 values of 1.25, 3.05, 1.48 5.29 μM [45].

#### **2.4 Anti tubercular activity**

In the year of 2019 S. Manivannan et al., reported the synthesis anti tubercular activity of benzimidazole derivatives, among all the derivatives compound **42, 43** (**Figure 7**) showed best anti tubercular activity with MIC values of 6.5, 6.5, 12.5, 6.5, 12.5, 6.5 μg/mL against *Mycobaterium tuberculosis* H37Rv, drug-resistant, drugsusceptible strains [12], previous year Mohanty et al., reported the anti tubercular activity of synthesized the novel azo derivatives of benzimidazoles, in all the derivatives the compounds **44** (**Figure 7**) showed best activity at IC50 value of 0.119 μM/mL against *Mycobaterium tuberculosis* [46], before previous year Yadav et al., synthesized the benzimidazole derivatives, reported the anti tubercular activity the compounds **45–53** (**Figure 7**) at MIC value of 12.5 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [47]. In the year of 2015 Ramprasad et al., reported the synthesis, anti tubercular activity of the imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole derivatives, the compounds **54–60** (**Figures 7** and **8**) showed best activity at MIC value of 3.125 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv, Species192, Specis210 [48], same year Yoon et al., evaluated the anti tubercular activity of synthesized the new benzimidazole aminoesters, the compound **61** (**Figure 8**) showed best activity with IC50 value of 11.52 μM against *Mycobaterium tuberculosis* strains of H37Rv [49].

In the year of 2014 many authors reported the anti tubercular activity of synthesized the new benzimidazole derivatives, Gong et al., reported the compound **62**

*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

**Figure 9.** *Structures of effective anti-tubercular compounds.*

(**Figure 8**) found to be best activity at MIC value of 0.20, 0.049 μg/mL against nonreplicating *Mycobaterium tuberculosis* and replicating *Mycobacterium tuberculosis* [50], Hameed et al., reported the compound the compounds **63** (**Figure 8**) showed significant activity at MIC value of 0.19 μM against fluoroquinolone-resistant strains of *Mycobaterium tuberculosis* [51], Kalalbandi et al., reported the compounds **64–66** (**Figure 8**) showed good activity at MIC value of 3.12, 3.12, 1.6 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [52], Park et al., reported the compounds **67** (**Figure 8**) showed excellent activity at MIC value of 0.63 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [53] and Gobis et al., reported the compounds **68–71** (**Figure 8**) found to be better activity at MIC value of 0.75 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv, Spec. 192, Spec. 210 [54].

In the year of 2013 also many authors evaluated the anti tubercular activity of newly synthesized benzimidazole derivatives, Nandha et al., reported the compound **72** (**Figure 9**) showed best activity at MIC value of 12.5 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [55], Birajdara et al., reported the compound **73, 74** (**Figure 9**) showed good activity at MIC value of 6.25 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [56], Anand et al., reported the compounds **75, 76** (**Figure 9**) found to be significant activity at MIC value of 1.56 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [57], Awasthi et al., reported the compound **77** (**Figure 9**) showed better activity at MIC value of 0.06 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [58], Yoon et al., reported the compound **78** (**Figure 9**) showed best activity at MIC value of 0.115, 6.12 μM against *Mycobacterium tuberculosis* H37Rv and INH-resistant *Mycobacterium tuberculosis* [59] and Ranjith et al., reported the compounds **79–83** (**Figure 9**) showed excellent activity at MIC value of 1 μg/mL against *Mycobacterium tuberculosis* H37Rv [60].

In 2012 Patel et al., reported the anti tubercular activity of synthesized the benzimidazolyl-1,3,4-oxadiazol-2ylthio-*N*-phenyl(benzothiazolyl)acetamides, among all the synthesized derivatives, the compounds **84–86** (**Figure 9**) showed best activity at MIC value of 12.5 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [61],

**Figure 10.** *Structures of effective anti-tubercular compounds.*

*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

Sangani et al., reported the synthesis and anti tubercular activity of pyrido[1,2-a] benzimidazole derivatives of beta-aryloxyquinoline, among all the derivative, the compound **87** (**Figure 9**) found to be best active compound at MIC value of 6.25 μg/ mL against *Mycobaterium tuberculosis* strains of H37Rv compared with isoniazid, refampicin [62] and Gobis et al., reported the anti tubercular activity of new benzimidazoles, the compound **88** (**Figure 10**) showed best activity at MIC value of 3.1, 1.5, 3.1 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv, Species 192, Species 210 [63].

In 2011 few authors reported the anti tubercular activity of synthesized benzimidaoles, Saleshier et al., reported the compounds **89–91** (**Figure 10**) found to be best activity at 10, 100mcg/ml concentrations against *Mycobaterium tuberculosis* [64], Camacho et al., reported the compound **92** (**Figure 6**) showed best activity with MIC values of 12.5 μg/mL, 6.25 μg/mL against multidrug-resistant MDR, MTB strains [65], Kumar et al., reported the compound **93** (**Figure 6**) found to be better activity at MIC99values of 1.0 μM, 1.0 μM against *Mycobaterium tuberculosis* strains of H37Rv, W210, NHN 20, NHN335, NHN382, TN587 [66] and Pieroni et al., reported the compound **94** (**Figure 10**) showed excellent activity at MIC values of 0.5 μg/mL, 1.0 μg/mL, 8.0 μg/mL against *Mycobaterium tuberculosis* strains of H37Rv [67].

#### **3. Conclusions**

The benimidazole plays in important role in the field of medicinal chemistry, many of the marketing drugs contain benzimidazole moiety are using to illness. In recent medicinal chemistry research the benzimidazole derivatives are in continuous development with many pharmacological activities such as anti-cancer, anti-HIV, antileishmanial, anti-tubercular, anti-malarial, anti-inflammatory, anti-diabetic, and so on, to meet pharmacological requirement. The present literature may helpful to researcher, medicinal chemist, pharmacologist to design, to synthesize, to develop pharmacologically active benzimidazole derivatives with low toxicity in future.

#### **Acknowledgements**

All the authors are thankful to managements of respective colleges for providing facilities to carry out the work.

#### **Author contribution**

Mahender Thatikayala contributed the chemistry, anti cancer, anti leishmanial and anti tubercular activity of benzimidazoles. Anil Kumar Garige, Hemalatha Gadegoni contributed the chemistry and anti HIV activity of benzimidazoles.

#### **Funding**

No funding applicable.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Mahender Thatikayala<sup>1</sup> \*, Anil Kumar Garige<sup>2</sup> and Hemalatha Gadegoni<sup>3</sup>

1 Avanthi Institute of Pharmaceutical Sciences, Hyderabad, Telangana, India

2 Jayamukhi Institute of Pharmaceutical Sciences, Warangal, Telangana, India

3 Pathfinder Institute of Pharmacy Education and Research, Warangal, Telangana, India

\*Address all correspondence to: mahenderpharma395@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

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*Benzimidazole: Pharmacological Profile DOI: http://dx.doi.org/10.5772/intechopen.102091*

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#### **Chapter 4**

## Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update

*Gopakumar Kavya and Akhil Sivan*

#### **Abstract**

Benzimidazole, one of the finest classes of heterocyclic aromatic compounds have the characteristic structure of benzene fused with a five-membered imidazole ring. Despite being made their first appearance in the late 1870s, they are considered as a 'privileged molecule'. The applications of this wonder molecule range from medicinal chemistry to material science. Benzimidazole being a potent inhibitor for various enzymes has got therapeutic effects like anticancer, antimicrobial, anthelmintic, antioxidant, anticonvulsant, antifungal, anti-inflammatory, antiviral, antihistaminic, antipsychotic, etc. It has also made its existence in various branches of medical science *viz* ophthalmology, neurology, cardiology and more. The applications of benzimidazole are not only limited to the biological field but also expanded to the field of material chemistry as well. This chapter summarizes the pharmacological properties of benzimidazole, illustrated on numerous derivatives since 2016.

**Keywords:** anti-cancer agent, Benzimidazole, biological activity, N-heterocycle, medicinal chemistry, pharmacophore

#### **1. Introduction**

The benzimidazole nucleus is fairly unique among heterocyclic ring systems because of its outstanding structural similarity with various naturally occurring nucleotides [1]. In 1872, Hoebrecker synthesized the first benzimidazole molecule by the reduction of 2-nitro-4-methylacetanilide [2]. The biological significance is because its structure is similar to purines, and the importance of the applications depends on their abundance in most of the biologically active molecules. The discovery of the structure of vitamin B12 with 5, 6-dimethylbenzimidazole moiety in it, also elicited the search for benzimidazole - similar motifs for various pharmacological applications [3– 5]. Following this, various research groups have outlined the synthesis and applications of benzimidazole [6, 7]. Benzene when fused with imidazole results in the formation of benzimidazole (**1**), which can readily undergo tautomerization as shown in **Figure 1**.

The greater reactivity of the 2nd position towards various electrophiles and nucleophiles is the outcome of tautomerization. Many drugs contain the benzimidazole

**Figure 1.**

*Benzimidazole (compound 1) with its tautomeric forms.*

**Figure 2.** *Drugs (compound 2, 3, 4, 5 and 6) based on benzimidazole core.*

nucleus as a core unit and have a widespread application in the pharmaceutical field [8–14]. The presence of benzimidazole pharmacophore in the various branches of medical science is inexplicable. The therapeutic uses of benzimidazole include anticancer [15–20], antimicrobial [21–24], antiparasitic [25, 26], anti-inflammatory and analgesics [27–29], antiviral [30–32], and antiulcerative [33] activities and in fields like ophthalmology, neurology, endocrinology, etc. The first example of a benzimidazole that was clinically available was thiabendazole (**2**), which can be used as a fungicide and for antiparasitic purposes [34]. The 2, 6-disubstituted albendazole (**3**) and mebendazole (**4**) were used as anthelmintic or antiparasitic agents. The 1, 2 disubstituted benomyl (**5**) was shown to have antifungal and anticancer activities whereas 2-substituted lansoprazole (**6**) acted as a therapeutic agent for the reduced production of stomach acid and cardiac failures (**Figure 2**).

In this chapter, a plethora of benzimidazole analogs with different pharmacological properties such as anticancer, antibacterial, antifungal, antiviral, anticoagulant, antiinflammatory, antiparasitic, anthelmintic activity, etc. has been discussed.

#### **2. Benzimidazole and its pharmacological significance**

Benzimidazoles were initially used as a plant fungicide and veterinary anthelminthic. After the discovery and use of thiabendazole (**2**), these benzimidazole motifs were used in human beings as well. Since then, a wide variety of molecules

#### *Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

having the core structure as (**1**) were synthesized and found their application in the medical world as well as in the material domain.

Various substituted derivatives of (**1**) were showcased diversified therapeutic properties such as antiparasitic, anticancer, anthelmintic, antiproliferative, antioxidants, antimicrobials, anti-inflammatory, antivirals, anticoagulants, antihypertensive, anticonvulsant, antidiabetic, lipid level modulators, anti-HIV, immunomodulators, hormone modulators, proton pump inhibitors and antidepressants. They have also used a building block for various other therapeutic agents. Let us have a peep at some of the innumerable reports of pharmacological activities of benzimidazole.

#### **2.1 Anticancer activity**

Cytotoxicity of benzimidazole derivatives is well known and, recently Noha et al. reported compounds of benzimidazole which are *N*-(benzimidazothiazolone) acetamides (**7**) [35]. In vitro analyses provided the cytotoxic activity of (**7**) over HCT-116 colon cancer cells. The further detailed study delivered the topoisomerase I-β (Topo Iβ) and inhibiting activities against tubulin (**Figure 3**).

The role of benzimidazole analogs as potential metal-based DNA-sensor is unanimous. Fluorogenic differential/sequential Schiff base chemosensors which solely consists of benzimidazole derivatives (**8**), for detecting Cu2+, CN, P2O7 <sup>4</sup>, and Zn2+ ions in human cervical (HeLa) and breast cancer (MDA-MB-231 and MCF-7) cell lines were designed by Anbu et al. [36] (**Figure 3**).

Drug repurposing of benzimidazole compounds is generally considered for the reason that, it has antitumor activities. Florio and coworkers screened anthelmintics which are derivatives of benzimidazole [37]. Certain drugs like albendazole (**3**), flubendazole (**9**), oxibendazole (**10**) etc. are subjected to the evaluation of their pharmacokinetics and physicochemical properties (**Figure 3**). For the potential repurposing of the drugs in cancer therapy, a silico target prediction was used to access the pharmacology of these benzimidazole compounds.

#### **Figure 3.**

*Benzimidazole derivatives (compound 7, 8, 9 and 10) acts as cytotoxic agents, chemosensors and repurposed drugs.*

**Figure 4.**

N*-substituted benzimidazole derivatives (compound 11, 12, 13, 14 and 15) with antiproliferative activity.*

#### **Figure 5.**

*Benzimidazole derivatives (compound 16 and 17) for the inhibition of HSP90.*

Synthesis of *N*-substituted benzimidazole analogues (**11**–**15**) with an alkyl chain and a nitrogen-containing 5- or 6-membered ring increased the anticancer effects on human ovarian carcinoma (OVCAR-3) and human breast adenocarcinoma (MCF-7) cell lines, were reported by Hsieh et al. [38]. (2*E*)-1-(1-(3-morpholinopropyl)-1*H*-benzimidazol-2-yl)-3-phenyl-2-propen-1-one) (**11**) acts as the most potent antiproliferative drug and has got more advantages than the standard drug, cisplatin (**Figure 4**).

The stabilization of proteins in the cell is being coordinated by heat shock proteins (HSPs). HSP90 plays a major role in it. This can be reflected in cancer therapy. Neverdauskas et al. synthesized benzimidazole derivatives with resorcinol (**16**) and (**17**), as potential inhibitors for HSP90 (**Figure 5**) [39].

Benzimidazole is considered a privileged molecule in the medicinal world. Hernández-Romero et al. in 2021 synthesized first-row transition metal compounds which contain benzimidazole moieties (**18**–**21**) as ligands in them (**Figure 6**) [40]. The advancement of metallodrugs for the treatment of cancer has been rapidly evolving. The use of benzimidazole as mono-, di-, tri-, and tetradentate ligands with metals like Cu, Co, Zn, Ni, Mn, V, and Fe led to the formation of effective drugs for cancer therapy by increasing the cytotoxic and antiproliferative activity.

Bistrović et al. synthesized monocationic benzimidazoles (**22**) and (**23**), starting from *o*-phenylenediamines and benzaldehydes having 1,4-disubstituted-1,2,3-triazole *Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

**Figure 6.** *Benzimidazole derivatives (compound 18, 19, 20 and 21) as metallodrugs.*

motifs and studied its antiproliferative activities [41]. These compounds showed potent and selective activities that are cytostatic against non-small cell lung cancer (A549) in the low nM range and could be because of apoptosis and primary necrosis (**Figure 7**). Because of the presence of different amidino groups and aromatic substituents, these compounds showed a difference in their cytostatic activities in

**Figure 7.**

*Benzimidazole derivatives (compound 22, 23, 24, 25 and 26) with potential inhibition for lung, colon, and breast cancer.*

Western blot analysis. The enzyme p38 MAPK got inhibited by both the compounds as shown by *in silico* structural analysis.

The 1, 3-disubstituted benzimidazoles (**24**–**26**) were synthesized starting from *o*diphenylamine and their interactions with cancer cells proteins were examined using molecular docking studies (**Figure 7**) [42]. The biochemical assay of the synthesized compounds were compared by doing theoretical calculations whereas its biological activity was tested against proteins such as colon cancer antigen (ID 2HQ6) and breast cancer (ID 2AR9) by using molecular docking studies. These benzimidazolyl halides were found to be better against the protein molecules studied and of which (**26**) was found to be more potent in action among the given three.

Synthesis of imidazo[1,2-*a*]pyrazine appended benzimidazoles (**27**) and (**28**) was done, starting from 1,3-dibromobenzene and evaluated its anticancer activities on the inhibition of growth of NCI-60 human cancer cell lines [43]. The antiproliferative activity of these molecules is attributed to causing damage to the DNA of such cells. The planar geometry of these compounds also enhanced the intercalated binding with cancer cells DNA. The cytotoxicity evaluation of the compounds was also done against the human normal cell line (Hek293) and found to be very low with higher LC50 values (**Figure 8**).

Srour et al. in 2020 reported the formation of a novel class of 2-thiazol linked benzimidazoles (**29**) and studied its inhibiting action against epidermal growth factor receptor (EGFR) (**Figure 8**). The *in vitro* studies of the synthesized compounds using erlotinib as a standard drug revealed its suppression activity against EGFR PK inhibitors, which targets human breast cancer (MCF-7) cells. They have also exhibited a

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

#### **Figure 9.**

*Derivatives of benzimidazole (compound 31, 32 and 33) with anticancer activity against liver, lung, and gastric cancer cells.*

very low suppression percentage among normal cells indicating its diminished side effects when used as an antiproliferative drug [44].

Benzimidazole-tethered pyrazoles (**30**) have been synthesized in multi-steps by the condensation of phenylhydrazine with acetylphenones followed by cyclization, Vilsmeier-Haack formylation and Knoevenagel reactions (**Figure 8**) [45]. A study of anti-inflammatory and antioxidant activities of the benzimidazoles showed a marked improvement when compared with diclofenac sodium and ascorbic acid as standards respectively. The anticancer activity was shown against human pancreatic cancer cell line AsPCl (progenitor) and SW1990 (squamous) which was also visible in the better binding with B-cell lymphoma in docking studies.

Mn(I) and benzimidazole co-ligands (**31**) with potential photo-activated carbon monoxide releasing molecules (CORMs) were synthesized and their biological activities were studied (**Figure 9**) [46]. The CO releasing properties, as well as luminescence intensities of these complexes, differed with the extend of conjugation and with the degree of unsaturation present in the benzimidazole co-ligands. The bioimaging capabilities of these complexes were proved by the absorption of it by liver cancer cells (SK-Hep1) and human liver cells (HL-7702) under cellular fluorescence imaging tests. Complex (**31**) showed excellent anticancer activities among all the molecules synthesized.

Prosser et al. synthesized a Cu(II) complex of benzimidazole (**32**) and studied their anticancer properties [47]. This derivative that was revised at the non-coordinated nitrogen of the benzimidazole molecules, exhibited excellent cytotoxicity against A549 adenocarcinomic alveolar basal epithelial cells (**Figure 9**).

Research works concentrating on the effective therapeutic agent possessing antiproliferative activity for human gastric cancer paved the way to the discovery of yet another benzimidazole derivative (**33**) with quinoline copper-based complex (**Figure 9**) [48]. The complex ensures G2/M phase arrest, apoptosis, mitochondrial dysfunction etc. and thus provides effective cytotoxicity.

Aromatase inhibitors (AIs) are compounds that control estrogen-related diseases and hence breast cancer, as its concentration was found to be higher in such cases. Çevik et al. in 2020 synthesized some novel benzimidazole- triazolothiadiazine

**Figure 10.**

*Benzimidazole derivatives (compound 34, 35, 36, 37, 38 and 39) provide inhibition against breast, lung, and prostate cancer cells.*

libraries and examined its aromatase inhibition activities [49]. Initial screening of these compounds towards anticancer properties against breast cancer cell line (MCF-7) in humans, resulted in getting good results. Upon further subjecting it to *in vitro* aromatase enzyme inhibition studies, the compound (**34**).

among them was found to be almost equal in activity when compared with a reference drug letrozole (**Figure 10**).

The role of benzimidazole compounds in the treatment of breast cancer is exemplary. Gangrade et al. demonstrated the use of benzimidazole derivatives in the inhibition of Wnt/β-catenin signaling [50]. The upregulation of Wnt/β-catenin signaling in triple-negative breast cancer (TNBC), when compared to normal and other breast cancer subtypes, is inevitable. Benzimidazole compounds like SRI33576 (**35**) and SRI35889 (**36**) have a high cytotoxicity rate in TNBC cell lines. They are found to be active inhibitors of Wnt/β-catenin signaling and have therapeutic properties for treating TNBC (**Figure 10**).

Cheong and co-workers designed and synthesized benzimidazole methylcarbamate analogue (**37**) with enhanced water solubility [51]. The existed drugs that account for the treatment of metastatic cancers are not suitably aiding the circumstances. Poorly soluble benzimidazole methylcarbamate drugs, which are effective anthelmintics are subjected to functionalization with oxetane or an amine group to improve the solubility and then used as an active therapeutic agent for the treatment of metastatic cancers. Cytotoxicity towards prostate, lung, and ovarian cancers is exhibited by the novel oxetanyl substituted compound (**37**) (**Figure 10**).

Liang and co-workers synthesized selenium-containing benzimidazole derivatives through condensation of peptide coupling reagents and irradiation of microwaves [52]. These selenediazole derivatives were recognized as potent anticancer agents

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

against MDA-MB-231 and MCF-7 breast cancer cell lines. Compounds (**38**) and (**39**) showed greater cytotoxic activity towards triple-negative breast cancer cell line MDA-MB-231 (**Figure 10**).

Husain et al. prepared various derivatives of furanone appended benzimidazoles, which effectively contribute to cancer therapy [53]. Compound (**40**) was found active against DU145 and MCF7 whereas compound (**41**) has got excellent activity against MCF7, A549, and DU145 cell lines (**Figure 11**). They are potential cytotoxic agents than the standard drug doxorubicin.

Compounds (**42**) and (**43**) are *bis*-benzimidazole analogs that have been synthesized to account for cancer therapy under microwave irradiation [54]. The anticancer activity was studied with the help of.

**Figure 11.** *Benzimidazole analogs (compound 40, 41, 42, 43, 44, 45, 46 and 47) with anticancer activities.*

Molinspiration software and they possess high bioactivity scores. It was also found that they obey Lipinski's rule and could be emerged as a lead anticancer drug (**Figure 11**).

Shinde and co-workers used D-glucose as the precursor for the synthesis of ribofuranosyl nucleosides (**44**) and (**45**) (**Figure 11**). Evaluation of their anticancer activity was done using the MDA-MB-231 cell line [55].

Sireesha et al. designed and synthesized benzimidazole/benzoxazole-linked *β*-carbolines (**46**) by the condensation of two various anti-cancer fragments (**Figure 11**) [56]. With the assistance of MTT assay, these compounds were subjected for the anti-cancer screening against Colo-205 (colon), MCF-7 (breast), A2780 (ovarian), and A549 (lung) and found that these exhibits maximum anti-cancer activity with the *β*-carbolines hybrid.

Benzimidazole derivatives (**47**) with a pyrrolidine side chain can be effectively used to treat sorafenib resistance (SR) in hepatocellular carcinoma, was reported in 2019 [57]. Mode of action is through the inhibition of proliferation of SR cell lines by interrupting the phosphorylation of AKT, p70S6, and the downstream molecule RPS6 (**Figure 11**).

Synthesis of organoruthenium(II) complexes of benzimidazoles (**48**) and (**49**) was reported by Welsh and coworkers (**Figure 12**) [58]. Their anti-cancer activity was screened against triple-negative MDA-MB-231 and MCF-7 breast cancer cell lines, respectively. Among the synthesized compounds, (**48**) showed more potency and (**49**) showed comparable potency with the cisplatin, against the MCF-7 cell line.

#### **2.2 Antibacterial and antifungal activity**

Heterocyclic appended benzimidazoles were synthesized and their antibacterial and antifungal activities were tested [59]. The mechanism of action of these molecules was also examined by using docking studies with bacterial proteins such as DNA gyrase subunit B (DNAG) and penicillin-binding protein 1a (PBP1a). The compounds with thiazole and thiadiazole moieties (**50**) and (**51**) respectively, showed marked inhibitory activity against *Escherichia coli*, *Bacillus pumilus*, and *Staphylococcus aureus* bacteria (**Figure 13**).

Ajani et al. synthesized various *o*-substituted and 1, 2-disubstituted benzimidazoles and examined their antibacterial properties [60].

**Figure 12.**

*Organoruthenium benzimidazole derivatives (compound 48 and 49) with inhibition against breast cancer cell lines.*

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

**Figure 13.** *Benzimidazole derivatives (compound 50, 51, 52 and 53) with inhibitory activity against common bacteria.*

Benzene-1,2- diamine undergoes condensation reactions with anthranilic acid, 3, 5 dinitrophenylbenzoic acid, and phenylacetic acid, catalyzed by NH4Cl yielded the precursor molecules, which on reaction with electrophile-releasing agents produced the corresponding *o*-substituted and 1,2-disubstituted benzimidazoles (**52**) and (**53**), respectively (**Figure 13**). *In vitro* studies of these compounds showed a better activity with a low minimum inhibitory concentration (MIC) value.

1-aryl-substituted 1, 2, 3-triazole appended amidinobenzimidazoles linked *via* phenoxymethylene units (**54**) and (**55**) were synthesized and their anti-bacterial as well as anti-trypanosomal activities and DNA/RNA binding affinities, were studied [61]. Compound (**54**) showed a remarked inhibition against gram-positive bacteria whereas compound (**55**) showed inhibition against gram-negative bacteria. These compounds also showed binding affinities towards ctDNA. Compound (**56**) with *N*-isopropylamidine and *p*-methoxyphenyl-1,2,3-triazole units exhibited enhanced anti-trypanosomal activities against *T. brucei* and reduced toxicity towards mammalian cells (**Figure 14**).

A microwave-assisted, Ni(II) catalyzed novel preparation of 2,6-disubstituted and 1,2,6-trisubstituted benzimidazoles were achieved by Patel and his group (**Figure 15**) [62]. The *in-vitro* antimicrobial studies of the title compounds (**57**) against grampositive and gram-negative bacteria and fungal strains showed an improved activity exhibited by them when compared with ampicillin, a standard drug. Certain compounds show potent anti-mycobacterium tuberculosis activity, antimalarial activity, antioxidant activity, etc. All these activities were supported by better molecular docking scores and their pharmacokinetics were also examined by ADME-Tox descriptors.

A comparative antimycobacterial activity study of 2,5-disubstituted and 1,2,5-trisubstituted benzimidazoles was reported in 2020 [63]. The *in vitro* studies against *Mycobacterium tuberculosis* H37Rv strain revealed an increased activity correlated with lipophilicity for disubstituted compounds (**58**–**60**) than for trisubstituted ones because of the addition of a long hydrocarbon chain at position 1 in the latter (**Figure 15**).

A library of mono and disubstituted benzimidazoles were synthesized by applying different methodologies, i.e., by using the microwave, ultrasound (US), infrared (IR), simultaneous application of US and IR, and by conventional heating [64]. The antimicrobial and antifungal activities of these benzimidazole derivatives were then evaluated. It was found that some compounds such as (**61**) and (**62**), were proved to be a better substitute than the standard drugs trimethoprim sulfamethoxazole and miconazole for antimicrobial and antifungal activities, respectively (**Figure 16**).

Very recently, Khan et al. designed and synthesized pyrimidine-benzimidazole hybrids (**63**) using the revised Biginelli reaction and evaluated its potential inhibition of SARS-CoV-2 main protease and spike glycoprotein [65]. Investigation about the pharmacological properties resulted in biological evidence like antimicrobial and antifungal properties. The derivatives developed possess more affinity in binding and anti-SARS-CoV-2 activity than presently approved drugs (**Figure 16**).

Zha et al. demonstrated benzimidazole derivatives (**64**) and (**65**) as potent antibacterial agents (**Figure 16**) [66]. Properties like enzyme inhibition, DNA binding, and having a synergistic effect with existing antibiotics makes benzimidazole an active warrior against methicillin-resistance *Staphylococcus aureus* (MRSA).

**Figure 15.** *Benzimidazole derivatives (compound 57, 58, 59 and 60) with antibacterial and antimycobacterial activities.* *Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

**Figure 16.** *Potential benzimidazole-derived antibiotics (compound 61, 62, 63, 64 and 65).*

Claisen-Schmidt condensation of 2-acetylbenzimidazole and aldehydes followed by a series of steps resulted in the synthesis of benzimidazole derivative (**66**) [67]. They exhibit exceptional anti-microbial and anti-bacterial activities. The grafting of certain functional groups and the presence of pyridine, pyrimidine, indole, etc. improvises the anti-microbial activity (**Figure 17**).

Karaburun et al. described the multi-step synthesis of a series of benzimidazole-1,3,4-oxadiazole derivatives (**67**) which are prominent for their antifungal activities against *Candida* species [68]. The ergosterol inhibition power was proven *via* ergosterol quantification assay and the docking studies were performed on 14-*α*-sterol (**Figure 17**).

#### **Figure 17.**

*Benzimidazole derivatives (compound 66, 67, 68 and 69) with antimicrobial, antifungal, and antibacterial activities.*

Recently, Aroso and co-workers computationally designed benzimidazole derivatives through palladium-catalyzed reactions [69]. The reaction between 4-bromo-1,2 diaminobenzene and 2-nitrobenzaldehyde, followed by a couple of palladiumcatalyzed Suzuki–Miyaura and Buchwald-Hartwig amination cross-coupling reactions resulted in the formation of (**68**) and (**69**) (**Figure 17**). The importance of these benzimidazoles is that it has an inhibitory effect on *E. coli* DNA gyrase B.

Chen et al. designed flavonoid analogs (**70**) which consist of benzimidazole derivatives like 4*H*–chromen-4-one, which provides a remarkable anti-bacterial resistance against members of *Xanthomonas* and *Ralstonia solanacearum* (**Figure 18**) [70]. Molecular docking studies showed the curative and protective activity for the *Tobacco mosaic virus* (TMV). The inhibition rate value is high for these analogs when compared with other anti-viral agents.

Compound (**71**) synthesized by Gençer and co-workers were tested against *Candida* species through microdilution methods [71]. MTT assay and other various microbiological studies provided the antifungal profile with good and effective *in vitro* cytotoxic effects along with inhibition on ergosterol biosynthesis (**Figure 18**).

Synthesis of benzimidazole derivatives like triazinane (**72**) and oxidiazinanes (**73**) through the process of amino methylation with the aid of different aryl-*N*, *N*<sup>0</sup> unsymmetrical thioureas were designed by Gullapelli and co-workers (**Figure 18**) [72]. The antibacterial activity was evaluated by using suitable gram-positive and negative bacterial strains.

Wang et al. reported the synthesis of a series of benzimidazole moieties (**74**–**76**) with quinolone analogs which exhibited antibacterial and antifungal properties (**Figure 19**) [73]. The bioactive assay proved that the 2-fluorobenzyl derivative has got remarkable antimicrobial activities against the *P. aeruginosa* and *C. tropicalis*.

A novel, one-pot synthesis of 2-substituted benzimidazoles and Mannich bases (**77**–**80**) with potent antimicrobial activity was reported by Marinescu et al. [74].

Qualitative and quantitative antimicrobial bioassay of these benzimidazole derivatives showed activity against a broad spectrum of gram-positive and negative bacterial strains both in planktonic and adherent states. The presence of nucleophilic groups like -OH or -CH3 accounts for the microbicidal activity (**Figure 20**).

#### **Figure 18.**

*Derivatives of benzimidazoles (compound 70, 71, 72 and 73) with potential antibacterial, antifungal, and cytotoxic activities.*

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

**Figure 19.** *Quinolone analogs of benzimidazole (compound 74, 75 and 76) with antibacterial and antifungal activities.*

**Figure 20.** *Potent antimicrobial derivatives of benzimidazole (compound 77, 78, 79 and 80).*

Benzimidazoles moieties linked with *N*-acyl substituted indole (**81**–**86**) were demonstrated by Abraham et al. [75]. The assessment of antimicrobial activity was done against gram-negative and gram-positive bacteria like *Pseudomonas aeruginosa* (MTCC424), *Staphylococcus aureus* (MTCC 2940), *Escherichia coli* (MTCC 443), and *Enterococcus fecalis*. These compounds also account for the hindering of biofilm formation and then the effective growth of *Staphylococcus epidermis* (**Figure 21**). Along with this, an HRBC membrane stabilization test was carried out for the evaluation of the anti-inflammatory activity.

Antoci and co-workers synthesized *bis*-(imidazole/benzimidazole)-pyridine derivatives (**87**) through *N*-alkylation (**Figure 22**) [76]. The anti-TB activity of the compound is good to excellent against both replicating and nonreplicating Mtb. The derivatives are effective against drug-resistant Mtb and some possess a bactericidal approach.

The synthesis of naphthyl-substituted benzimidazole derivatives (**88**) and (**89**) was reported by Ersan et al. in 2020 [77]. The antimicrobial activity was screened and was found that (**89**) showed maximum potency against all gram-positive and gramnegative bacteria. Also, (**88**) actively functions as an antifungal agent. These derivatives also interact with active sites of *E. coli* and can be accounted for inhibition of *E. coli* topoisomerase I (**Figure 22**).

Sirim et al. designed and synthesized benzimidazole-acrylonitrile hybrid derivatives from benzene-1, 2-phenyleneamine and ethyl cyanoacetate followed by reaction with piperazines [78]. All the derived compounds exhibited anti-mycobacterial activity against *M. tuberculosis* H37Rv strain by microplate alamar blue assay (MABA). Compound (**90**) was found to be more effective than standard drugs like isoniazid, ciprofloxacin, rifampicin, etc. (**Figure 22**).

**Figure 21.**

N*-acyl substituted indole-linked benzimidazole derivatives (compound 81, 82, 83, 84, 85 and 86) as antimicrobial agents.*

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

#### **2.3 Antiparasitic activity**

Taman et al. evaluated the antischistosomal activity of newly synthesized benzimidazole-related compounds like NBTP-OH (**91**) and NBTP-F (**92**) [79]. The Suzuki-Miyaura coupling reaction of 5-formyl thiophen-2-ylboronic acid and 1-bromo-4-hydroxy benzene or 1-bromo-4-flouro benzene followed by a series of reactions resulted in the formation of compounds (**91**) and (**92**), respectively (**Figure 23**). To date, the treatment of schistosomiasis depended on Praziquantel (PZQ). The use of these two structurally related benzimidazole derivatives can be an alternative for PZQ. The *in vitro* schistosomicidal assay performed on adult worms gave the conclusion that they were considered dead through the destruction of tegument after two minutes of treatment with **91** and **92**.

Synthesis of 1, 3-disubstituted benzimidazol-2-ones (**93**) and (**94**) starting from *o*-phenylenediamine and urea, followed by the evaluation of its anti-trichinellosis efficacy was done [80]. It was found that the synthesized benzimidazole derivatives are more effective than the standard drug albendazole, which is the traditional drug used in the treatment against *Trichinella spiralis*. The estimation of antiparasitic activity was employed through the Campbell method. Selective binding of benzimidazole moiety with the *β*-tubulin of the parasite results in the destruction of the cell, followed by the death of the parasite. The in vitro activity of all the tested benzimidazole analogs increases with the concentration against the Trichinella spiralis (**Figure 23**).

Molecular docking studies and quantitative structure–activity relationship (QSAR) delivered that benzimidazole derivatives (**95**–**97**) can be used as cruzain inhibitors for the deadly Chagas disease [81]. The model compounds used displayed a high statistical consistency and a notable capability to predict the inhibiting sites (**Figure 24**). The scenario with Chagas disease is the unavailability of an effective treatment method. Clinical studies related to the Chagas disease and cruzain inhibitors have been on the

**Figure 24.** *Benzimidazole derivatives (compound 95, 96 and 97) as cruzain inhibitors for Chagas disease.*

account of research scientists. In short, benzimidazole derivatives can be used as a lead in the drug discovery of Chagas disease by acting against the recombinant cruzain enzyme.

Tonelli et al. designed and synthesized benzimidazole derivatives from benzene-1, 2-diamine and various acids followed by suitable functionalization and used it as a potent antileishmanial agent [82]. Benzimidazole derivatives were tested against *Leishmania tropica* and *L. infantum* and were found that compounds bearing the derivatives of 1-lupinyl were commonly more active than dialkylaminoalkyl derivatives and compounds (**98**) exhibited the highest potency among the synthesized compounds (**Figure 25**). The observed antileishmanial activity was a result of the interaction of benzimidazole derivatives with acidic components of the cell membrane leading to its destruction.

Exploration of the inhibitory activity of certain benzimidazole compounds like albendazole (**3**), ricobendazole (**99**), oxfendazole (**100**) etc. resulted in

**Figure 25.** *Compounds 98, 99 and 100 with antileishmanial and antiparasitic activities.*

acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition at nanomolar level [83]. This has got immense importance in therapy emerging for handling the resistance among anti-cholinergic factors and in antiparasitic treatment (**Figure 25**). The benzimidazole derivatives are selectively toxic towards helminths which are considered parasites. The inhibitive effect of benzimidazole derivatives on *β*-tubulin leads to disruption of function in helminths and results in its death.

#### **2.4 Antiviral activity**

Compound (**101**) has been synthesized in a multi-step process by Bessieres et al. in 2021 (**Figure 26**) [84]. A study about the inhibition of Ebola virus infection resulted in the design of a more potent and selective drug than the reference drug, Toremifene.

Ibba et al. demonstrated the role of benzimidazole derivatives (**102**) and (**103**), which are active inhibitory agents against *enterovirus* A71 (EV-A71), which is a major cause for foot-mouth disease (HFMD), herpangina, etc. [85]. Penetration and apoptosis assay concluded that the derivatives are capable to inhibit viral endocytosis through reduced viral attachment and penetration to the host cells (**Figure 26**).

Research for the inhibitory action of *chikungunya virus* (CHIKV) infection led to the discovery of benzimidazole-related antiviral agent which targets the nonstructural protein 4 (nsP4), was reported by Wada and co-workers [86]. One of the compounds (**104**), synthesized by them can effectively inhibit CHIKV by using M2295 residue in the nonstructural protein 4 (nsP4) and with the help of CHIKV replicons, it inhibits the RNA-dependent RNA-polymerase (RdRp) function of CHIKV (**Figure 26**).

#### **2.5 Other properties like antipsychotic, antidiabetic, anticoagulant activities, etc.**

*In vitro* and *in vivo* characteristic studies of benzimidazole acetamide derivatives (**105**) in the ethanol-induced neuro-degeneration model was performed by.

Imran et al. in 2021 [87]. The derivatives lowered the neurodegeneration and inflammation of neurons by down-regulating inflammatory cascades caused by oxidative stress (**Figure 27**).

The prominence of benzimidazole in the field of medicine is exceptional. Etazene (**106**), a benzimidazole opioid that has got strong analgesic activity, is used as a new psychoactive substance (**Figure 27**) [88]. Misuse of certain benzimidazole derivatives can create social crises too.

Tantray and co-workers studied psychiatric disorders like depression and acknowledged the fact that glycogen synthase kinase-3*β* (GSK-3*β*) dysfunction is a potential implication [89]. They designed and synthesized several 1,3,4-oxadiazole carboxamides linked to benzimidazoles (**107**) and assessed their *in vitro* GSK-3*β* inhibition. It was found that these molecules are having antidepressant activity (**Figure 27**).

Hussain et al. synthesized certain benzimidazole analogs (**108**) for the effective management of type-II diabetics [90]. The sulfonamide bearing 2-marcaptobenzimidazoles (**108**), possesses better *in vitro α*-amylase enzyme inhibitory activity while compared with the standard drug, acarbose (**Figure 27**).

Dabigatran is an effective drug having a benzimidazole core as the activity center and is used for the treatment of cardiovascular diseases because of its antithrombin as well as anticoagulant activities. Zhang et al. in 2020 enhanced the activity and bioavailability of dabigatran by adding methyl and methoxy groups into the benzene ring [91]. By studying the anticoagulant action and thrombin inhibition properties of compounds (**109**) in rats, proved the possibility of using these molecules as potential antithrombin drug candidates in the future (**Figure 27**).

**Figure 27.**

*Benzimidazole derivatives (compound 105, 106, 107, 108 and 109) with antipsychotic, antidiabetic, and anticoagulant activities.*

*Exploring the Versatility of Benzimidazole Scaffolds as Medicinal Agents: A Brief Update DOI: http://dx.doi.org/10.5772/intechopen.101942*

#### **3. Conclusions**

To sum up, benzimidazole is a chemical compound that belongs to the family of heterocyclic aromatic organic compounds. It is a potent biologically important molecule with a noticeable therapeutic activity. Applications of benzimidazole extend to medicinal chemistry. Several advanced research in this area also found out that the aforementioned compound has significant antimicrobial activities especially against many strains of viruses, fungus, bacteria, etc. It is also widely used in medicinal chemistry as an accepted drug against parasites and their allied infections. Benzimidazole is also used as an analgesic and anti-inflammatory agent. Recent studies have also created a lot of attention for the compound since it has an anti-carcinogenic activity like cytotoxicity and hence may become a viable cure for cancer in the future. The applications of benzimidazole cannot be marginalized. It has got a whole spectrum of medicinal agents. Benzimidazole has gained popularity in material science.

Apart from this, the multi-target capability of benzimidazole scaffolds has not been explored extensively. Being a versatile motif, benzimidazole could provide a plethora of novel multi-target ligands against various debilitated pathological conditions. The lack of comprehensive compilation about the SAR of many compounds and the various research reports stemmed the reason for less number of active benzimidazole compounds reaching the market. The existing design of benzimidazole derivatives can be further revised to accommodate potential multitargeting agents, thus enhancing and treating multifactorial disorders. This can be a breakthrough establishment in benzimidazole history.

In short, the importance of this imidazoline compound has been proved by the number of research papers getting published in a short period. This chapter is trying to narrate the formulation as well as execution of benzimidazoles in different fields of medicinal chemistry.

#### **Acknowledgements**

The authors would like to acknowledge the support given by the Department of Chemistry, Amrita Vishwa Vidyapeetham, Amritapuri campus for providing the necessary facilities to carry out research work.

#### **Author details**

Gopakumar Kavya and Akhil Sivan\* Department of Chemistry, Amrita Vishwa Vidyapeetham, Amritapuri, Kollam, Kerala, India

\*Address all correspondence to: akhilsivan@am.amrita.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide Derivatives Bearing Thiophene Moiety

*Vinayak Adimule, Pravin Kendrekar and Sheetal Batakurki*

#### **Abstract**

In the present investigation, novel amide derivatives of benzimidazole (**4a**-**f**) with different thiophene acids (**a**-**f**) coupled in the presence of 1-[Bis (dimethylamino) methylene]-1H-1, 2, 3-triazolo [4, 5-b] pyridinium 3-oxide hexafluorophosphate (HATU) reagent at room temperature and as-synthesized derivatives were characterized by (<sup>1</sup> H-NMR and 13C-NMR) proton and carbon magnetic resonance, and high-performance liquid chromatography (HPLC) analytical techniques. The amide derivatives were tested for *in vitro* antimicrobial and antifungal activity and ciprofloxacin was used as standard. The antifungal activity was tested with *Carbendazim* and *Fenbendazole* cell lines using *clotrimazole* standard drug. The results indicated the potential activity toward *S. bacillus* with compounds having IC <sup>50</sup> of **4** (**a**), **4 (b)**, **4 (d)** and **4 (e)** against antimicrobial strains with IC50 of **51.8** μm, **57.4** μm, **54.5** μm and **56.5** μm respectively. However, compounds **4 (a)**, **4 (c)** and **4 (d)** showed greater inhibitions against *Carbendazim* fungal cell line with IC50 of **22.9**, **26.8** and **28.8** μm. On the other hand IC50 values of the *Fenbendazole* for compounds **4** (**a**), **4**(**c**) and **4** (**d**) were found to be **12.7**, **10.2** and **12.7** μm, respectively. The thiophene-substituted benzimidazole amide derivatives are the potential candidate drug for antibacterial and antifungal activity.

**Keywords:** thiophene, antibacterial, antifungal, benzimidazole, amides, HATU

#### **1. Introduction**

In the present investigation, organic and medicinal chemistry involves the nitrogen containing heterocycles especially Benzimidazole derivatives [1, 2]. Benzimidazole has become nature and synthetic active structural part in the biological characteristics such as antibacterial [3], anticancer [4], antiviral [5], antifungal [6] and antioxidant [7] etc. It is widely accepted that the amidino moieties at the benzimidazole substituents terminal would generate numerous pathophysiological and biochemical processes in the human body. Additional biological activity can be found with the benzimidazole substituents carrying amide moiety and tetracyclic derivatives interaction with DNA is large enough to encounter the selectivity towards the drug molecules. Benzimidazole is indispensable nucleus for the discovery of the new biologically important molecules. Literature reports suggest that benzimidazole has potential interest in antimicrobial [8, 9] and anticancer agents [10, 11]. New class of oxadiazole and thiadiazoles containing thiophene and phenyl substituents reported for enhanced anticancer activity [12–15]. Benzimidazole nucleus structurally analogues to purine and its derivatives and exhibit the synthesis of nucleic acids. Several DNA molecules will be cleaved upon interaction with the benzimidazole moiety and inhibit the growth of the microbial strains [16–18]. The development of new antimicrobial agents remains on priority [19]. Furthermore, Bisbezimidazole derivatives found to be biologically active towards antibacterial [20], antimicrobial [21] and anticancer [22] activities. On the other hand, benzylvanilline benzimidazole [23] derivatives were found to cleave DNA and potent towards leukemic cell lines. Distance between the benzimidazole molecule and ester containing phenyl group is very important factor for the antifungal activity. Extending the spacer between the benzimidazole and ester or amide substituents become important factor for the increased antifungal activity. Antitumor activity depends on the distance between the benzimidazole and spacer link of the ester or amide molecules. Thiophene and their derivatives which are sulfur compounds widely studied for their antifungal activity [24–28]. Some of the thiophene containing derivatives such as thicyofen, ethaboxam, silthiopham and penthiopyrad were widely used in agriculture as antifungal compounds [29–30]. Recently certain amide, thiazole, 1, 3, 4-oxadiazoles has been designed, synthesized and studied for their enhanced anticancer properties [31–36]. Incorporation of the thiophene moiety would enhance the antifungal activity of the benzimdiazole derivatives.

Based on the above literature evidences, Author envisaged by constructing the novel benzimidazole containing thiophene derivative could increase the antimicrobial and antifungal activity of the synthesized compounds. All the synthesized derivatives were characterized by <sup>1</sup> H-NMR, 13C-NMR, LCMS spectroscopic studies and tested against microbial and fungal cell lines. The results indicated potential activity towards *S. bacillus* tested compound with IC <sup>50</sup> of **4** (**a**), **4 (b)**, **4 (d)** and **4 (e)** derivatives against antimicrobial strains with IC50 of **51.8** μm, **57.4** μm, **54.5** μm and **56.5** μm respectively. However, compounds **4 (a)**, **4 (c)** and **4 (d)** showed greater inhibitions against *Carbendazim* fungal cell line with IC50 of **22.9** μm **26.8** μm and **28.8** μm respectively. On the other hand IC50 values of the *Fenbendazole* for compounds **4** (**a**), **4**(**c**) and **4** (**d**) was found to be **12.7** μm, **10.2** μm and **12.7** μm respectively.

#### **2. Materials and methods**

All the chemicals, reagents and solvents are procured from Sigma-Aldrich, S-d fine chemicals Ltd., Spectrochem Ltd. and used without any further purification. Intermediate chemicals purchased directly (99.8% purity, thiophene-2-carboxylic acid, Sigma Aldrich India (a)), (98% purity, (thiophen-3-yl) acetic acid Sigma Aldrich India (b)), (99.1% purity, 3,6-dichloro-1-benzothiophene-2-carboxylic acid, Matrix Scientific, India (c)), (99.2% purity, 5-(4-fluorophenyl)thiophene-2-carboxylic acid.

Sigma Aldrich, India (d)), (98% purity, 4-bromo-5-chlorothiophene-2-carboxylic acid, Chem Src, China (e)), (99.2% purity, 3-chloro-1-benzothiophene-2-carboxylic acid, Sigma-Aldrich India (f)) and used without any further purifications. <sup>1</sup> H-NMR, 13C-NMR spectral analysis was carried out using 300 MHz-1.2 GHz consisting of cryostat with excellent thermal efficiency, available with high performance vibration isolator make from Bruker Ascend series. LCMS (liquid crystal mass spectrometry)

*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

Triple quadrapole series of GCMS-TQ8050 NX fitted with high efficient trace level detection. In order to prepare the culture medium, the synthesized compounds were dissolved in DMSO and diluted with culture broth solution to 1 mg/mL. Serial dilutions were made to reach up to the 10 mL of the final concentration. About 100 μL of the each of the solution were distributed to 96 well plates and the sterility control and growth control solutions were placed into it. About 5 μL of the test and the growth solutions were inoculated into the well plates. All the experiments were carried out in triplicate. Bacterial growth was detected former by optical density (ELISA reader, CLX800 Biotech Instruments) and after by addition of 20 μL of an INT alcoholic solution (0.5 mg/mL). 7 mm filter paper discs (Whatman, no. 3) were impregnated with 10 mL of each of the different dilutions [37, 38]. The discs were allowed to remain at RT until complete diluent evaporation and kept under refrigeration until ready to be used. Discs loaded with synthesized derivatives which were placed onto the surface of the agar. Commercial chloramphenicol discs and paper discs impregnated with 20 mL of diluents used to dilute concentration of the synthesized products were used as control.

#### **3. Experimental**

#### **3.1 Synthesis of 1***H***-benzimidazole-2-carboxylic acid**

In a 100 mL round bottom flask fitted with a reflux condenser, 2-methyl-1*H*-benzimidazole (5.69 g, 1.1 mole), KMnO4 (4 mole), K2CO3 (3 mole) were dissolved in 100 mL of absolute ethanol and 20 mL of water under stirring. The contents of the reaction mixture heated to 90 C for 5 h. The progress of the reaction was monitored by TLC (thinlayer chromatography) (ethyl acetate: hexane: 40:60). After completion of the reaction, contents of the RB flask cooled to room temperature (RT), filtered over celite bed, filtrate was concentrated to half of its initial volume, acidified with concentrated HCl to PH 2. Off white colored solid separated out was filtered, washed with cold water and dried. Yield: 5.2 g. The solid was taken to next step without any purification [39, 40].

#### **3.2 Synthesis of 1-(1***H***-benzimidazol-2-yl) methanamine**

In a 100 mL RB flask fitted with an inert nitrogen atmosphere, 1H-benzimidazole-2-carboxylic acid (5.2 g) dissolved in dry THF solvent and LiAlH4 (1 mole) were added under stirring at RT. Progress of the reaction was monitored by TLC, after completion of the reaction. LiAlH4 was quenched in carbonate solution. The reaction mixture was concentrated to remove the organic solvent and filtered. The product was extracted in ethyl acetate (100 mL 3 times), washed with Na2CO3 (25 mL 2 times), brine (25 mL 2 times) and dried over Na2SO4. Solid that are separated out was filtered in 10 mL of n-hexane solvent and obtained as 1-(1*H*-benzimidazol-2-yl) methanamine (**Figure 1**). Yield: 3.87 g.

#### **3.3 General procedure for the synthesis of benzimidazole amide derivatives**

In a 100 mL RB flask fitted with a condenser, 1-(1*H*-benzimidazol-2-yl) methanamine (1mole) added with different thiophene substituted acids (**a-f**), Dichloromethane (DCC) (10 mL), HATU (0.25mole), Triethyl amine (TEA) (1.5mole) and stirred at RT for 24 h, after completion of the reaction (monitored by

#### **Figure 1.**

*Synthetic pathway of Benzimidazole intermediates.*

TLC), solvent and other volatile reagents are evaporated, solid obtained was mixed with 60–120 silica gel and purified by column-chromatography (ethyl acetate: hexane: 30:70), Solids obtained after the solvent evaporation was used for the antimicrobial property studies [41].

#### **3.4 Characterization of the synthesized compounds (Benzimidazole 2, 3 and 4a-4f)**

1 H-NMR Spectrum of the compounds 2, 3, 4a to 4 f.

1 H-NMR, 13C-NMR and LCMS analysis of the synthesized compounds. Compound 2.

Yellow solid; m.p-157.8°C; Yield = 97.8%; LCMS-98.1%; <sup>1</sup> H-NMR: *δ* <sup>1</sup> H NMR: *δ* 7.04 (1H, ddd, *J* = 7.7, 6.9, 1.3 Hz), 7.23 (1H, ddd, *J* = 8.2, 6.9, 1.7 Hz), 7.77 (1H, ddd, *J* = 8.2, 1.3, 0.5 Hz), 7.90 (1H, ddd, *J* = 7.7, 1.7, 0.5 Hz). 13C-NMR: *δ* 13C NMR: δ 114.3 (1C, s), 118.4 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 137.9 (1C, s), 138.4 (1C, s), 150.9 (1C, s), 156.7 (1C, s) (**Figure 2**) (**Figures 3**–**6**).

**Figure 2.** *Synthesis of amide derivatives of Benzimidazoles.*

*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

**Figure 3.** *1 H-NMR of the spectrum of 2 and 3.*

**Figure 4.** *1 H-NMR of the spectrum 4a and 4b.*

**Figure 5.** *1 H-NMR of the compounds 4c and 4d.*

**Figure 6.** *1 H-NMR Spectrum of the compounds 4e and 4 f.*

*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

Compound 3.

Pale yellow solid; m.p-159.2°C; Yield = 98.8%; LCMS-98.7%;<sup>1</sup> H-NMR: δ <sup>1</sup> H NMR: *δ* 4.32 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.19 (1H, ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.59–7.75 (2H, 7.65 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz). 13C-NMR: *δ* 13C NMR: δ 49.1 (1C, s), 114.3 (1C, s), 118.4 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 137.9 (1C, s), 138.4 (1C, s), 150.9 (1C, s).

Compound 4a.

Pale yellow solid; m.p-163.4°C; Yield = 97.8%; LCMS- 98.1%;1 H NMR: *δ* 4.93 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.12–7.27 (2H, 7.19 (ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.21 (dd, *J* = 7.2, 5.0 Hz)), 7.60–7.83 (4H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz), 7.76 (dd, *J* = 5.0, 1.2 Hz), 7.76 (dd, *J* = 7.2, 1.2 Hz). 13C-NMR: *δ* 13C NMR: *δ* 44.7 (1C, s), 114.3 (1C, s), 118.4 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 131.0 (1C, s), 137.9 (1C, s), 138.4 (1C, s), 139.9 (1C, s), 150.9 (1C, s), 160.1 (1C, s).

Compound 4b.

Off white colored solid; m.p-171.8°C; Yield = 97.8%; LCMS- 98.3%; <sup>1</sup> H NMR: *δ* 3.88 (2H, s), 4.99 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.10–7.26 (2H, 7.16 (dd, *J* = 7.5, 5.0 Hz), 7.19 (ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.31–7.45 (2H, 7.37 (dd, *J* = 5.0, 1.3 Hz), 7.39 (dd, *J* = 7.5, 1.3 Hz), 7.59–7.76 (2H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz). 13C-NMR: *δ* 13C NMR: *δ* 29.2 (1C, s), 44.7 (1C, s), 114.3 (1C, s), 118.4 (1C, s), 126.6 (1C, s), 127.5 (1C, s), 127.8 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 137.9 (1C, s), 138.1 (1C, s), 138.4 (1C, s), 150.9 (1C, s), 172.7 (1C, s).

Compound 4c.

Pale Yellow Solid; m.p174.5°C; Yield = 99.4%; LCMS- 97.8%; <sup>1</sup> H NMR: *δ* 4.90 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.19 (1H, ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.42 (1H, dd, *J* = 8.6, 1.9 Hz), 7.60–7.76 (2H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz), 7.94–8.07 (2H, 8.00 (dd, *J* = 1.9, 0.5 Hz), 8.01 (dd, *J* = 8.6, 0.5 Hz)). 13C-NMR: *δ* 13C NMR: *δ* 44.7 (1C, s), 114.3 (1C, s), 118.4–118.5 (2C, 118.4 (s), 118.5 (s)), 127.0 (1C, s), 127.8 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 128.7 (1C, s), 130.3 (1C, s), 132.6 (1C, s), 136.6 (1C, s), 137.9 (1C, s), 138.4 (1C, s), 139.3 (1C, s), 150.9 (1C, s), 160.1 (1C, s).

Compound 4d.

Yellow solid; m.p-157.8°C; Yield = 97.8%; LCMS- 96.8%; <sup>1</sup> H NMR: *δ* 4.92 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.12–7.32 (3H, 7.19 (ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.25 (ddd, *J* = 8.9, 1.4, 0.5 Hz)), 7.40 (1H, d, *J* = 8.7 Hz), 7.60–7.82 (5H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.66 (d, *J* = 8.7 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz), 7.76 (ddd, *J* = 8.9, 1.5, 0.5 Hz). 13C-NMR: *δ* 13C NMR: δ 44.7 (1C, s), 114.3 (1C, s), 115.4 (2C, s), 118.4 (1C, s), 124.0 (1C, s), 127.8 (2C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s)), 129.6 (1C, s), 134.3 (1C, s), 137.8–138.0 (2C, 137.8 (s), 137.9 (s), 138.4 (1C, s), 150.9 (1C, s), 151.1 (1C, s), 160.1 (1C, s), 162.5 (1C, s).

Compound 4e.

Pale Yellow Solid; m.p-179.2°C; Yield = 95.1%; LCMS- 98.2%; <sup>1</sup> H NMR: *δ* 4.92 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.19 (1H, ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.60–7.76 (3H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.65 (s), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz). 13C-NMR: *δ* 13C NMR: *δ* 44.7 (1C, s), 110.5 (1C, s), 114.3 (1C, s), 118.4 (1C, s), 123.0 (1C, s), 128.0 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 134.4 (1C, s), 137.9 (1C, s), 138.4 (1C, s), 150.9 (1C, s), 160.1 (1C, s).

Compound 4f.

White colored solid; m.p-178.5°C; Yield = 94.3%; LCMS- 99.2%; <sup>1</sup> H NMR: *δ* 4.91 (2H, s), 6.94 (1H, ddd, *J* = 7.7, 7.6, 1.2 Hz), 7.19 (1H, ddd, *J* = 8.1, 7.6, 1.4 Hz), 7.40–7.59 (2H, 7.47 (ddd, *J* = 8.0, 7.8, 1.4 Hz), 7.52 (ddd, *J* = 8.5, 7.8, 1.6 Hz), 7.60–7.80 (3H, 7.66 (ddd, *J* = 8.1, 1.2, 0.5 Hz), 7.69 (ddd, *J* = 7.7, 1.4, 0.5 Hz), 7.74 (ddd, *J* = 8.0, 1.6, 0.5 Hz)), 8.47 (1H, ddd, *J* = 8.5, 1.4, 0.5 Hz). 13C-NMR: *δ* 13C NMR: δ 44.7 (1C, s), 114.3 (1C, s), 118.4–118.5 (2C, 118.4 (s), 118.5 (s), 122.5 (1C, s), 123.3 (1C, s), 128.1–128.3 (2C, 128.2 (s), 128.2 (s), 128.3–128.6 (2C, 128.4 (s), 128.5 (s), 132.6 (1C, s), 136.1 (1C, s), 136.6 (1C, s), 137.9 (1C, s), 138.4 (1C, s), 150.9 (1C, s), 160.1 (1C, s).

#### **4. Results and discussion**

#### **4.1 Antimicrobial activity of the synthesized derivatives**

Antibacterial and antifungal activities of the synthesized compounds were tested by Agar well diffusion method. One mL of the fresh bacterial or fungi was placed in the sterile petri dish. Cooled Muller Hi was placed over it and upon solidification. 5 μL of the compounds dissolved in DMSO solvent was added to respective wells. The concentration has been fixed as per the previous reported literature [42, 43]. The Agar well plates were incubated for 30 minutes and subsequently kept at 35°C for 24 h. Antimicrobial activity was detected by measuring the zone of inhibition (including the wells diameter) appeared after the incubation period. DMSO at a concentration of 10% was employed as a negative control. All tested samples and their antimicrobial activity were expressed in terms of minimum inhibitory concentration (MIC values) (**Tables 1**–**3**). Different concentrations of the test solution are prepared and zone of inhibition and MIC values compared with the standards used for the evaluation [44]. The MIC was considered as the lowest concentration which inhibited the growth of the respective microorganisms. All assays were performed in triplicate. DMSO was served as a control for all the synthesized samples. **Tables 1** and **2** depicts the minimum inhibitory concentration of the antibacterial and antifungal activity (**Table 3**) of the synthesized derivatives. The results indicated potential activity towards *S. bacillus* tested compound with IC50 of **4** (**a**), **4 (b)**, **4 (d)** and 4 **(e)** derivatives against antimicrobial strains with IC50 of **51.8** μm, **57.4** μm, **54.5** μm and **56.5** μm respectively. However, compounds **4 (a)**, **4 (c)** and **4 (d)** showed greater inhibitions against *Carbendazim* fungal cell line with IC50 of **22.9** μm **26.8** μm and **28.8** μm respectively. The compounds **4** (**a**), **4**(**c**) and **4** (**d**) tested for anti micro (IC50) of the *Fenbendazole* for compounds were found to be **12.7** μm, **10.2** μm and **12.7** μm respectively [45].

#### **4.2 Structure activity relationships (SAR)**

The SAR may be attributed from the presence of halo, phenyl and aliphatic linkage present in the benzimidazoles derivatives and deduced from the following points. The minimum inhibitory concentration of the Cl, Br substituted aliphatic amide linkage draw attention for the enhanced binding and increased solubility of the compounds with respect to the target sites. Compounds of benzimidazoles derivatives bearing most active one **4**(**a**), **4** (**c**) and **4** (**d**) as a lead compound to develop novel antimicrobial agent [46]. The appreciable antimicrobial activity of the synthesized Benzimidazole derivatives compared to the standard drugs show that only minor structural changes needed for the derivatives to improve the binding of the molecules. The excellent antimicrobial activity of the synthesized Benzimidazole derivatives compared to the standard drug indicated a fact that for developing novel antimicrobial agent based on synthesized Benzimidazole derivatives. The above results also


*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

> **Table 1.**

*Antimicrobial activity data of the synthesized compounds against* E. Coli *and* S. Bacillus*.*


#### **Table 2.**

*Antimicrobial activities of the compounds against* Staphylococcus A *and* Salmonella Typhi*.*


#### **Table 3.**

*Antifungal activity data of the synthesized compounds.*

*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

#### **Figure 7.**

*SAR of the novel derivatives of benzimidazoles containing thiophene moiety.*

indicated a fact that different structural requirements are necessary for a compound to show different activities.

SAR study of synthesized compounds showed that the presence of electronwithdrawing moieties phenyl thiophene substituted with Cl, Br groups adjacent to the amide linkage in the benzimidazoles enhanced ant mycobacterial activity [47]. Further SAR of most of the derivatives indicated the attachment of thiophene-2 carboxamide moieties at amide position of the benzimidazoles increased antibacterial activity and the presence of 3, 6-dichloro-1-benzothiophene (**compound 4c**) at 2nd position of Benzimidazole carboximide also important for antimicrobial activities [48, 49]. The presence of electron withdrawing Cl and Br groups at 3rd and 4th position (**compound 4f and 4e**) required for antimicrobial activity. However substitution of 4-fluorophenyl) thiophene at 5th position of benzimidazoles carboxamide moiety increases the solubility of the compound (**compound 4d**) as well as the presence of phenyl ring increases the antibacterial properties (**Figure 7**).

Further, (thiophen-2-yl) acetamide and thiophene-2-carboxamide (**compound 4a and 4b**) attached to the benzimidazoles moiety at 2nd position of the ring system results in basicity of the –NH2 (amine) linker and thus increases the Clog P values where, C is the concentration of the compounds and P is permeability. On the other hand **compound 4c** contains 3, 6-dichloro-1-benzothiophene-2-carboxamide group attached at 2nd position of the benzimidazoles moiety responsible for moderate antibacterial and antiviral activities. If the 3, 6 dichloro groups present in the opposite direction of the ring system results in enhanced bioavailability of the benzimidazoles derivatives.

#### **5. Conclusion**

In the present research, novel series of thiophene amide derivatives containing benzimidazole moiety has been synthesized by the reaction with HATU at room temperature. All the synthesized derivatives are characterized by <sup>1</sup> H-NMR, 13C-NMR and LCMS spectroscopic studies. Synthesized compounds are purified by columnchromatography, and tested for antimicrobial strains (antibacterial and antifungal). The cell lines used was *E. coli*, *S. bacillus*, *Staphylococcus Aures* and *Salmonella Typhi* and ciprofloxacin used as standard. The results indicated potential activity towards *S. bacillus* tested compound with IC50 of **4** (**a**), **4 (b)**, **4 (d)** and **4 (e)** derivatives against antimicrobial strains with IC50 of **51.8** μm, **57.4** μm, **54.5** μm and **56.5** μm respectively. However, compounds **4 (a)**, **4 (c)** and **4 (d)** showed greater inhibitions against *Carbendazim* fungal cell line with IC50 of **22.9** μm **26.8** μm and **28.8** μm respectively. Compared with the activity (IC50) of the *Fenbendazole* for compounds **4** (**a**), **4**(**c**) and **4** (**d**) was found to be **12.7** μm, **10.2** μm and **12.7** μm respectively. The newly synthesized derivatives find its potential application in antibacterial and antifungal cells and replacing the existing drug in the market.

### **Acknowledgements**

All the authors are thankful to corresponding author Dr. Pravin Kendrekar, School of Natural Sciences, University of central Lancashire, Preston PR12HE, United Kingdom for carrying out antibacterial and antifungal activity of the synthesized compounds and M. S. Ramaiah University of Applied Science, Karnataka, Bangalore for synthesis of the thiophene substituted benzimidazole derivatives.

### **Conflict of interest**

All the authors declare that they do not have any conflict of interest.

### **Data availability**

All the obtained data has been incorporated in the main manuscript and more data can be obtained from the corresponding author on request.

*Synthesis, Characterization and Antimicrobial Properties of Novel Benzimidazole Amide… DOI: http://dx.doi.org/10.5772/intechopen.104908*

### **Author details**

Vinayak Adimule<sup>1</sup> , Pravin Kendrekar<sup>2</sup> \* and Sheetal Batakurki<sup>3</sup>

1 Angadi Institute of Technology and Management (AITM), Belagavi, Karnataka, India

2 Centre for Smart Materials, School of Natural Sciences, University of Central Lancashire, Preston, United Kingdom

3 Department of Chemistry, MS Ramaiah University of Applied Sciences, Bangalore, Karnataka, India

\*Address all correspondence to: kkpravin@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[48] Umesh K, Rakesh N, Surendra KN, Sachin KS, Vivek G. Benzimidazole: Structure activity relationship and mechanism of action as antimicrobial gent. Research Journal of Pharmacy and Technology. 2017;**10**(7):2400-2414. DOI: 10.5958/0974-360X.2017.00425.5

*Benzimidazole*

[49] Brishty SR, Hossain MJ, Khandaker MU, Faruque MRI, Osman H, Rahman SMA. A comprehensive account on recent progress in pharmacological activities of benzimidazole derivatives. Frontiers in Pharmacology. 2021;**12**:762807. DOI: 10.3389/fphar.2021.762807

Section 3

## Spectral and Theoretical Study of Benzimidazole

#### **Chapter 6**

## Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles

*A. Antony Muthu Prabhu*

#### **Abstract**

This chapter discusses about the spectral and theoretical aspects of selected benzimidazole and 2-phenyl substituted benzimidazole molecules. The synthesis of these benzimidazoles was reported in many methods by the reaction between ophenylenediamine with formic acid, aromatic aldehydes and N-benzylbezene-1,2 diamine in presence of oxidant tert-butyl hydroperoxide (TBHP). The spectral analysis of these molecules mainly such as UV-visible, fluorescence in solvents will be included in this chapter and discussed about the absorption, fluorescence maximum, conjugation, transition. Further the optimized structure of these molecules will be given using Gaussian 09 W (DFT 6-31G method). And also will be discussed about structural parameters, highest occupied molecular orbital (HOMO) – lowest unoccupied molecular orbital (LUMO) energy energy values, natural bond orbital (NBO), molecular electrostatic potential map (ESP). Many benzimidazole molecules having tautomers in the structure will be explained with the help of theoretical parameters to describe the structural properties.

**Keywords:** benzimidazole, spectral properties, computational study, NBO, MSP

#### **1. Introduction**

This chapter will be discussed about the spectral and theoretical studies of benzimidazole and 2-phenyl substituted benzimidazoles. A series of benzimidazole molecules are very important heterocyclic compounds in organic chemistry with two nitrogen atoms in five membered ring fused with aromatic moiety having different nature in spectral and biological properties. Benzimidazoles which contain a hydrogen atom attached to nitrogen in the 1-position readily tautomerize. In this way, many benzimidazole molecules are synthesized from the basic moiety to involving in large applications especially in medicinal fields. This type of benzimidazole derivatives possess the many pharmaceutical properties such as antiviral [1], antitumor [2], antihistaminic [3], antimicrobial [4], antihelminthic [5, 6], anticancer [7], antifungal [8], antimicrobial [4], antibacterial [9], analgesic [10], anti-convulsant [11] and antiulcer [12] activity. Some of benzimidazole molecules are used as corrosion inhibitors for metals and alloy surfaces in industrial field [13–15].

Particularly, Albendazole, Mebendazole and Thiabendazole having benzimidazole moiety are widely used as anthelmintic drugs [16].

Some fluoroquinolones substituted benzimidazole derivatives have been reported by microwave assisted method. The synthesized compounds are reported to be the derivatives of Ciprofloxacin & Norfloxacin [17].

The structural studies of synthesized benzimidazole derivatives are characterized using the spectral techniques such as single crystal XRD, UV-visible, Infrared, <sup>1</sup> H NMR, 13C NMR etc. [18–21]. These main techniques are usually referred for characterizing many organic synthesized molecules to elucidate the presence of functional groups, conjugation and structural parameters. Particularly the absorption and fluorescence spectral properties of these benzimidazole derivatives have been changed with respect to the change in substitution in aromatic ring at o- and p-position [22– 28].

Another important application of benzimidazoles is involved to exhibit the excited state intra-molecular proton transfer reaction (ESIPT) [22, 25, 29–31]. Particularly, the presence of hydroxyl group in benzimidazole at 2-position in benzene ring is exhibited this process through intramolecular hydrogen bonding between the acidic protons (-OH, -NH2) and basic centers (=N-, -C=O) in same molecule. ESIPT process for benzimidazole molecules is observed through dual fluorescence in aqueous solvent, one a normal stoke shifted fluorescence band and second large stoke shifted fluorescence band. Absorption and emission spectral study of these molecules were reported in different solvents with changing polarity. Moreover, the 2(2<sup>0</sup> -hydroxyphenyl)benzimidazole molecule are studied the enhancement of ESIPT process in aqueous β-cyclodextrin through the formation of host-guest inclusion complex [32].

The density functional theory (DFT) studies give information regarding the structural parameters, the functional groups, orbital interactions and vibrational

*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

frequencies [33]. The DFT calculations with the hybrid exchange-correlation functional B3LYP (Becke's three parameter (B3) exchange in conjunction with the Lee–Yang–Parr's (LYP) correlation functional) which are especially important in systems containing extensive electron conjugation and/or electron lone pairs [34–36]. The HOMO–LUMO energy, MSP map and the Mullikan population analysis will be calculated for the studied molecules. The natural bond orbital (NBO) analysis will explains the most important orbital interactions in order to clarify general structural features. The excited state potential energy surface, excited state intramolecular proton transfer of 2-(20 -Hydroxyphenyl)benzimidazole was investigated by TD-DFT method in gas phase and in solvent [37–39]. Further the theoretical calculations of Methyl-6-Nitro-1H-Benzimidazole and 1-methyl-2-phenyl benzimidazole was reported [40, 41]. Many benzimidazole molecules were reported theoretically structural properties and vibrational spectra, HOMO-LUMO, NBO analysis by ab initio HF and DFT methods [42–45]. Theoretical calculations of many benzimidazole molecules in gas phase were analyzed for the structural investigation with the help of experimental results [20, 44, 46–49].

#### **2. Benzimidazole and 2-phenyl benzimidazoles**

The benzimidazole molecule without any substitution was synthesized by the simple reaction between o-phenylenediamine with formic acid in the presence of alkali like sodium hydroxide, potassium hydroxide etc., [50]. The synthesis procedure of benzimidazole is given briefly. 27 g of o-phenylenediamine and 17.5 g of 90% formic acid are taken a 250 ml round bottom flask, the reaction mixture is heated at 100°C on a water bath for 2 hours. After cooling this reaction mixture, the 10% sodium hydroxide solution is added slowly to the solution with constant rotation then the reaction mixture becomes alkaline. Crude benzimidazole is filterd off at the pumb and washed with 25 ml of cold water and the crude product is dissolved in 400 ml of boiling water. Then 2 g of decolourising carbon is added to the solution and heat for 20 minutes. Finally the benzimidazole is formed after filteration at the pumb. New series of benzimidazole and its derivatives were synthesized and characterized using spectral analysis and applied for biological properties [51, 52]. For example, the series of substituted benzimidazole were reported particularly in the 2-position and 1-position replacing the hydrogen atom by both small and large size molecules (**Figure 1**).

The substituted 2-phenyl benzimidazoles were synthesized from the condensation reaction between *o*-Phenylenediamine and substituted aromatic aldehydes in chloroform and in the presence of ammonium chloride as a catalyst [53]. This reaction carried out at room temperature using many ammonium salts like ammonium bromide (NH4Br), ammonium chloride (NH4Cl), ammonium fluoride (NH4F), ammonium sulphate ((NH4)2SO4) and ammonium carbonate ((NH4)2CO3). Aromatic aldehydes such as benzaldehyde, m-methyl benzaldehyde, p-methyl benzaldehyde,

#### **Figure 1.** *The reaction shows between o-phenylene diamine with formic acid in presence of sodium hydroxide.*

**Figure 2.**

*The reaction shows between o-phenylene diamine with aromatic aldehyde in chloroform and in presence of ammonium chloride.*

**Figure 3.** *Tandem oxidative cyclization of different N-substituted benzene-1,2-diamines.*

m-methoxy benzaldehyde, p-methoxy benzaldehyde, o-hydroxy benzaldehyde, phydroxy benzaldehyde, o-amino benzaldehyde, p-amino benzaldehyde are used for the preparation of 2-phenyl substituted benzimidazole molecules. 1,2 phenylenediamine is added to the stirred solution of aromatic aldehydes and NH4Cl in CHCl3. This reaction mixture is stirred for 4 hours at room temperature. After completion of the reaction, the phenyl substituted benzimidazoles are formed using Thin layer chromatography, column chromatography (**Figure 2**).

Thus 2-phenyl substituted benzimidazole derivative has been reported by another method and the reaction scheme shows in **Figure 3**. The oxidative dehydrative coupling reaction of N-benzylbenzene-1,2-diamine in the presence of oxidant tert-butyl hydroperoxide (TBHP) in solvent acetonitrile to give substituted 2-phenyl benzimidazoles [54]. N-benzylbenzene1,2-diamine 1.96 g, I2 0.25 g, TBHP 1.8 g, is added in a 25 ml round bottomed flask in acetonitirle solvent and stirred at room temperature. The product is purified by column chromatography and finally the phenyl substituted benzimidazole is formed at the end of reaction.

#### **3. Absorption and emission spectral study**

Thus, the molecular diagrams of studied benzimidazole derivatives are shown in**Figure 4**. In this section, the absorption and emission spectral study is discussed for the selected benzimidazoles in solvents. The absorption and emission maximum was observed at 273 nm, 279 nm and at 291 nm for benzimidazole [55, 56] and at 295 and 350 nm for 2 phenyl benzimidazole [57]. These maximum are similar to 2-(m-methylphenyl)benzimidazole, 2-(p-methylphenyl)benzimidazole, 2-(m-methoxyphenyl)benzimidazole, 2-(pmethoxyphenyl)benzimidazole. But the 2-(o-hydroxyphenyl)benzimidazole molecule is observed the specific property in the ground and excited states to describe the keto-enol tautomeric equilibrium through the excited state intramolecular proton transfer [58–65], which property already given in introduction part and theoretical study also done for ketoenol tautomer in solvent effect. The absorption maximum of 2-(o-hydroxyphenyl)benzimidazole was observed at 335, 318 and 295 nm and fluorescence maximum at 355 and 465 nm in DMSO [32]. The absorption and emission maximum was observed at 320, 285 and 240 nm and fluorescence maximum at 417 nm in water for 2-(o-aminophenyl)benzimidazole [66] and at 313, 255, and 207 nm and at 382 nm in water for 2-(p-aminophenyl) benzimidazole [67].

*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

**Figure 4.**

*It shows the molecular diagram of selected molecules by DFT B3LYP 6-31G method.*

#### **4. Theoretical study**

#### **4.1 HOMO-LUMO energy parameters**

Initial computational study of selected benzimidazoles has been investigated from the HOMO, LUMO energy diagram in gas phase using DFT B3LYP 6-31G method. The chemical stability of selected benzimidazoles is demonstrated with the help of explaining this energy diagram [68]. Generally, the HOMO energy picture represents the ability to donate an electron and LUMO energy picture represent the ability to obtain an electron. For selected benzimidazoles the electron density is completely localized on the whole ring for both orbitals which shows in **Table 1**. The higher HOMO-LUMO energy gap of benzimidazole (�5.56 eV) and lower HOMO-LUMO energy gap of 2-(o-aminophenyl)benzimidazole (�4.38 eV) is observed theoretically to describing the stability and reactivity with addition of substitution in the phenyl ring. If the substitution in the benzimidazole is clearly changed the HOMO-LUMO energy gap particularly the amino group at o-position in the phenyl ring with lower value.

The theoretical physical parameters of selected benzimidazoles are determined by electronic chemical potential (μ), electronegativity (χ), absolute hardness (η), softness (S) and electrophilicity (ω) values from the HOMO as ionization energy (IE) and LUMO as electron affinity (EA) using the following equations, respectively. These parameters can be calculated using the following Eqs. (1)–(5).

$$
\mu = (E\_{\text{HOMO}} + E\_{\text{LUMO}})/2 \tag{1}
$$

$$
\chi = -\mu \tag{2}
$$

$$\eta = (E\_{\text{HOMO}} - E\_{\text{LUMO}})/2 \tag{3}$$

$$\mathbf{S} = \mathbf{1}/\eta \tag{4}$$

$$
\rho = \mu^2 / 2\eta \tag{5}
$$

*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

**Table 1.** *HOMO, LUMO energy diagram and molecular electrostatic potential (MSP) diagram obtained by DFT B3LYP 6-31G method.*

Theoretically calculated absolute hardness and softness are observed in the range from 1.11 to 2.78 eV. The values of μ, χ, η, S, ω for the amino group at o- and p-position leads to lesser values due to the electron donating nature of amino group. Absolute hardness and softness are important properties to measure the molecular stability and reactivity. S has been known as an indicator of the overall stability of a chemical system. A hard molecule has a large energy gap and a soft molecule has a narrow energy gap. Soft molecules are more reactive than hard molecules because they could easily offer electrons to an acceptor. For the simplest transfer of electrons, adsorption could occur at the part of the molecule where softness has the highest magnitude and hardness has the lowest [69]. The absolute hardness and softness of Albendazole molecule (benzimidazole moiety) was reported in the values of 2.56 and 0.39 eV in the gas phase [70]. Further the parameter of electrophilicity index (ω) of a organic molecule gives the information about the binding ability with biomolecules [71–73].

The calculated dipole moment values of the methoxy substituent at m- and p- are higher than those of other derivatives. But the hydroxyl group at o-position is higher than that of p-position and in amino group at p-position is higher value than that of oposition. The compound that has the highest dipole moment value is 2-( p-methoxyphenyl)benzimidazole with the value of 4.91 D. 2-(p-hydroxyphenyl) benzimidazole has the lowest dipole moment among the studied compounds (1.88 D).

Theoretically calculated energy for selected benzimidazole molecules are investigated with respect to substitution at m- and p-position of methyl, methoxy and also oand p-position for hydroxyl and amino groups. More negative energy values are observed for 2-(m-methoxyphenyl)benzimidazole and 2-(p-methoxyphenyl)benzimidazole. Further the comparisons of substitutions at m-, p-positions and o-, pposition are not significantly changed in energy values in gas phase. Also thermodynamic parameters such as enthalpy, free energy and entropy are calculated theoretically at room temperature in the gas phase. All values of substituted benzimidazoles are higher than the parent benzimidazole (**Table 2**).

#### **4.2 Molecular electrostatic potential**

Another investigating theoretical study is molecular electrostatic potential map (MSP) and displayed in **Table 1**. The electrophiles tend to negative ESP and the nucleophiles tend to region of positive ESP. The molecular electrostatic potential was calculated with DFT B3LYP 6-31G level of theory. The negative regions (red) are mainly contained on the nitrogen atom (=N-) and oxygen atom from methoxy, hydroxyl groups while the positive regions (blue) for the proton from –N-H, -OCH3, - OH and -NH2 group.

#### **4.3 Natural bond orbital (NBO) analysis**

Further to study of the intramolecular interactions of selected benzimidazoles, the theoretical NBO is used to calculate the stabilization energy for -C-H, -N-H and lone pair electrons in heteroatoms using second-order perturbation theory [74]. Some calculations about NBO analysis for investigating the intra and intermolecular interactions of isolated organic molecules and inclusion complexes between organic molecules and cyclodextrins [75, 76] were reported theoretically. NBO 3.1 program is applied to perform the natural bond orbital (NBO) analysis [77, 78] in the Gaussian 09 W package at the DFT/B3LYP level. In this work, DFT B3LYP 6-31G method is applied to analysis of intramolecular interactions for selected benzimidazoles. The


*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

*The values of energy of HOMO, LUMO, HOMO-LUMO energy gap, structural parameters, dipole, energy, thermodynamic parameters of selected benzimidazoles obtainedB3LYP 6-31G method.*

 *by DFT*

stabilization energy E(2) for the donor and acceptor orbital delocalization is involved through the occupied Lewis-type (bond or lone pair) NBOs and formally unoccupied (antibond or Rydberg) non-Lewis NBOs within the molecules [79, 80]. The electron density of all atoms is noted in the selected benzimidazole molecules.

The second-order Fock matrix is carry out to evaluate the donor–acceptor interactions within the molecules to specify in conjugative π bonds and lone pair electrons through the NBO analysis [81]. The Eq. (6) is given below to estimate the stabilization energy E(2) for the donor and acceptor orbital delocalization within the molecule.

$$E^{(2)} = \Delta E\_{ij} = q\_i \frac{\mathbf{F}(\mathbf{i}, \mathbf{j})^2}{\varepsilon\_j \cdot \varepsilon\_i} \tag{6}$$

where *qi* is the donor orbital occupancy, ε<sup>i</sup> and ε<sup>j</sup> are diagonal elements (orbital energies), and *F*(*i*, *j*) is the off-diagonal NBO Fock matrix element [82–84]. This analysis reveals that the conjugative interaction, hyper-conjugative interaction, intra and intermolecular hydrogen bond in the same molecules and combine with other molecules are well described for the donor – acceptor orbitals.

Thus the interaction between π(N11-C12) and π\*(C4-C7) are reveals the hyperconjugative energy about 19.04, 20.00, 20.05, 19.99, 20.10, 20.13, 19.41, 20.11, 19.96, and 20.30 kJ/mol for selected benzimidazole and 2-phenyl benzimidazoles respectively. This interaction could be revealed that the delocalization occurs in five membered ring for benzimidazole, due to the presence of C=N-C. Similarly the conjugative π bonds in the phenyl ring shows maximum delocalization during the interaction with π\* acceptor bonds. It is evident from benzimidazoles that the πC2-C5, C3-C6 and C4-C7 delocalize more energy to the acceptor bond (π\* acceptor). The electron density of donor bonds decreases while the acceptor (π\*) bond electron density increases. Investigation of NBO analysis is described the stabilization energy for the conjugative interaction or charge transfer between the donor and acceptor bond orbitals [85, 86]. The interactions of π(C-C) with π\*(C-C), π\*(N-C) and π(N-C) with π\*(C-C) are more responsible for the conjugation of respective π\* bonds in benzimidazole and substituted 2-phenyl benzimidazole. The investigated molecules are divided into parts from the results of NBO analysis. One part is benzimidazole moiety and other benzene ring without and with substitutions. From the **Table 3** these conjugative interactions are formed with close stabilization energy in the range from 17.00 to 23.00 kcal/mol. The stabilization energy values for these interactions are agreed with literature values [87]. From the **Table 3**, the π\*(N11-C12) delocalizes the maximum energy to π(C4-C7) and (C15-C18) bond respectively for all benzimidazoles in the range from 54.62 to 190.30 kJ/mol. Similarly, the π\*(C18-C19) bond transfers the high energy about 247.57 kJ/mol to (C16-C20) bond for 2-(ohydroxyphenyl)benzimidazole. The second order perturbation energies associated with the hyperconjugative interactions in NBO basis confirms the presence of intermolecular interactions.

#### **4.4 Mulliken atomic charges**

Thus the important quantum mechanical calculations further applied to calculate the atomic charges for molecular system [88]. The charge distributions of all atoms present in benzimidazole molecules are calculated by the Mulliken method [89]. The Mulliken atomic charges of selected benzimidazole molecules are presented in


#### *Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*


**Table 3.** *Second order perturbation theory analysis of Fock matrix in NBO basis for selected benzimidazoles by DFT B3LYP 6-31G method.*

#### *Benzimidazole*


#### *Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*



*Benzimidazole*

*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

#### **Figure 5.**

*The computed Mulliken charges for all atoms in selected benzimidazoles.*

**Table 4** and shown in **Figure 5**. The Mulliken atomic charges were computed at the DFT B3LYP 6-31G method. The carbon atoms numbering C4, C7 and C12 are shown with the positive values except other carbon atoms in the whole system. These results expected that these carbon atoms are connected with electronegative nitrogen atoms in benzimidazole [90]. In benzimidazole, C7 atom is bonded with N13-H having high positive value (0.329 a.u.) and C4 atom with N11 atom having less positive value (0.032 a.u.). The other benzimidazole molecules are following the same trend. A positive charge of all the hydrogen atoms are displayed in **Table 4**, but H26 was gained maximum positive charge than the other hydrogen atoms, due to the presence of electronegative atom (O25) in o-hydroxy and p-hydroxyphenyl benzimidazole when compared with the hydrogen in amino group lesser values. The presence of three nitrogen atoms in o-amino and p-aminophenyl benzimidazole (N11 = 0.481 a. u., N13 = 0.804 a.u. and N25 = 0.721 a.u.) are shown in different environment because N13 atom more negative values.

#### **5. Conclusion**

Ten compounds of benzimidazole and 2-phenyl substituted benzimidazoles such as (1) benzimidazole, (2) 2-phenylbenzimidazole, (3) 2-(m-methylphenyl)benzimidazole, (4) 2-(p-methylphenyl)benzimidazole, (5) 2-(m-methoxyphenyl)benzimidazole, (6) 2-(p-methoxyphenyl)benzimidazole, (7) 2-(o-hydroxyphenyl) benzimidazole, (8) 2-(p-hydroxyphenyl)benzimidazole, (9) 2-(o-aminophenyl)benzimidazole and (10) 2-(p-aminophenyl)benzimidazole were selected to study for the spectral and theoretical properties. Synthesis of these molecules by many methods were discussed and given the reaction scheme. Then the absorption and fluorescence spectrum of all molecules were given with changing the wavelength respect to the substitution of groups in the benzene ring. The conjugation and maximum also involved and π-π\* transition possible in the absorption spectrum. Further, DFT method was used to determine the structural parameters, energy values, HOMO-LUMO energy gap, thermodynamic parameters, molecular electrostatic potential map for all molecules. NBO analysis was revealed the hyperconjugative interaction between bonding orbitals, lone pair orbitals and antibonding orbitals and also calculated the stabilization energy of selected bonded orbitals. Finally the computed Mulliken atomic charges was determined using DFT B3LYP 6-31G method.

#### **Author details**

A. Antony Muthu Prabhu Department of PG Chemistry, Aditanar College of Arts and Science, Tiruchendur, Tamilnadu, India

\*Address all correspondence to: antonyphdchem@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Spectral and Theoretical Studies of Benzimidazole and 2-Phenyl Substituted Benzimidazoles DOI: http://dx.doi.org/10.5772/intechopen.101966*

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### *Edited by Pravin Kendrekar and Vinayak Adimule*

*Benzimidazole* is a comprehensive survey of the known and new methods of benzimidazole synthesis, the spectral and theoretical aspects of existing benzimidazole derivatives, and the anticancer properties of a new class of benzimidazole derivatives. This book examines aspects and newer mechanisms of benzimidazoles containing heterocyclic moiety. Chapters report on anticancer properties of benzimidazole derivatives, novel methods of synthesis of benzimidazoles, versatile nature of the benzimidazoles, spectral and theoretical studies of benzimidazole derivatives, and medicinal importance and pharmacological aspects of benzimidazole derivatives.

### *Miroslav Blumenberg, Biochemistry Series Editor*

Published in London, UK © 2022 IntechOpen © monsitj / iStock

Benzimidazole

IntechOpen Series

Biochemistry, Volume 34

Benzimidazole

*Edited by Pravin Kendrekar* 

*and Vinayak Adimule*