Synthesis of Antimicrobial Benzimidazole–Pyrazole Compounds and Their Biological Activities

The synthesis of new compounds with antimicrobial and antiviral properties is a central objective today in the context of the COVID-19 pandemic. Benzimidazole and pyrazole compounds have remarkable biological properties, such as antimicrobial, antiviral, antitumor, analgesic, anti-inflammatory, anti-Alzheimer’s, antiulcer, antidiabetic. Moreover, recent literature mentions the syntheses and antimicrobial properties of some benzimidazole–pyrazole hybrids, as well as other biological properties thereof. In this review, we aim to review the methods of synthesis of these hybrids, the antimicrobial activities of the compounds, their correlation with various groups present on the molecule, as well as their pharmaceutical properties.

Moreover, the literature mentions a series of benzimidazole-pyrazole hybrids with remarkable antimicrobial properties, and not only, antiviral activities, even anti-COVID-19 [51][52][53][54], in the context of the new pandemic, which has led us to current research, to study their synthesis methods, antimicrobial properties, structure-property relationships, and their biological activities.
In this review, we aim to review the various methods of synthesis of benzimidazolepyrazole hybrid compounds with antibacterial and antifungal properties, DNA-Gyrase inhibitors, topoisomerase IV inhibitors, as well as the other biological properties they possess, such as: antitumor, antioxidant, anti-inflammatory, analgesic, antiulcer ( Figure 1).

Benzimidazoles Substituted in the Position "1" with Pyrazole Moiety
Krishnanjaneyulu et al., synthesized a series of 1-substituted benzimidazoles with pyrazole moiety through a linker using a four-step strategy: benzimidazole synthesis, N-alkylation, condensation with aldehydes with the formation of chalcones 87a-87i and cyclization with the formation of the pyrazole nucleus, in compounds 88a-88i (Scheme 24) [78]. All compounds were evaluated for their antibacterial activity against four Gram-positive bacteria: Staphylococcus aureus ATCC 9144, Staphylococcus epidermidis ATCC 155, Micrococcus luteus ATCC 4698 and Bacillus cereus ATCC 11778, and three Gram-negative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 2853 and Klebsiella pneumoniae ATCC 11298. The antifungal activity of the compounds 88a-88i was evaluated against two fungi, Aspergillus niger ATCC 9029 and Aspergillus fumigatus ATCC 46645 (Table 10). It was found that compounds containing an electron-withdrawing group at the phenyl group attached to C-5 of pyrazole displayed superior antimicrobial activities than compounds possessing an electron-releasing group. The unsubstituted derivatives displayed moderate antimicrobial activity. The best antimicrobial activity was shown by compounds 88g and 88i, which in Table 10 is marked in green.

Benzimidazoles Substituted in the Position "4" ("7") with Pyrazole Moiety
Grilot et al., reported the synthesis of second-generation antibacterial benzimidazolepyrazoles from 2,3,6-trifluorobenzenamine in seven steps, as can be seen in Scheme 27 [81]. Compound 98a, with a 3-pyridine moiety at C5, which maintains a hydrogen bond with Arg136, showed a reasonable MIC against S. aureus (0.25 µg mL −1 ). It can be observed that introduction of a fluorine atom at C6 on pyrazole 98b, has no improved antibacterial potency (0.25 µg mL −1 against S. aureus), nor affinity for Gyrase B and Topoisomerase IV, as previously reported [80], but oral exposure was improved 2-fold, which led to the exclusive focus on C6-fluorobenzimidazole pyrazoles. The authors also studied the variation of MIC and the polarity of molecules with the introduction of the substituent in the "5" position, as seen in Table 11. Compound 98f showed a MIC against S. aureus of 0.125 µg mL −1 and could be improved slightly by the addition of a methyl group on the C5 substituent, as in 98g and its resolution yielded compounds 98h and 98i. The (S)-isomer 98i was 4-fold more potent than the (R)-isomer 98h against S. aureus and showed acceptable oral exposure, but compound 98h exhibited a high serum shift (16-fold).

Scheme 27.
Synthesis of benzimidazole-pyrazoles 98a-98i. Charifson et al., reported the synthesis of benzimidazole-pyrazoles 101-102 from 5-bromo-2-nitro-3-(1H-pyrazol-1-yl)benzenamine 99, using a Suzuki coupling reaction (Scheme 28) [82]. The compounds were found to be inhibitors of DNA Gyrase and Topoisomerase IV, with potent antibacterial activity. The results of the evaluation for enzymatic inhibition and antibacterial potency of the compounds are shown in Table 12. The superior enzymatic and antibacterial inhibitory activity of compound 102 vs. 101 is observed, due to the presence of the pyrimidine nucleus in the molecule.

Analgesic and Anti-Inflammatory Activity of Antimicrobial Benzimidazole-Pyrazole Compounds
Benzimidazole and pyrazole/pyrazoline are important nitrogen-containing heterocyclics in anti-inflammatory research [83,84]. In recent decades, there have been various studies on the anti-inflammatory activity of benzimidazole compounds.
Chikkula et al., reported the analgesic activity [65] of the compounds 39a-39i (Scheme 11). The analgesic activity of novel benzimidazole derivatives 39a-39i varied with reaction time. The compounds displayed moderate analgesic activity at 30 min of reaction time. In the second hour, the analgesic activity reached to peak level. Additionally, the presence of phenyl ring at the N-1 atom of pyrazole ring significantly increased analgesic activity. The anti-inflammatory activity of the synthesized compounds varies similarly to their analgesic activity. Compounds 39c, 39d and 39e had the best analgesic activity.
Arora et al., reported the synthesis of the compounds 104a-104e from chalcones 103a-103e (Scheme 29) [85]. The in vivo studies for analgesic activity were evaluated in albino mice by Eddy's hot plate method. The compound 104a showed mild activity at a dose of 100 and 200 mg kg −1 in the range of 7.38 ± 0.12-7.36 ± 0.15 at 90 min when compared with standard drug Diclofenac sodium at a dose of 5 mg kg −1 in the range of 9.45 ± 0.28 at 90 min. Additionally, the compound 104a showed slightly less moderate activity in the range of 53.03 and 59.09% at a dose of 100 and 200 mg kg −1 at a time interval of 3h of carrageenan challenge when compared to compound 104c (63.63%) as well as when compared with standard Diclofenac sodium at the same time period exhibited 69% of activity, respectively, at a dose of 100 mg kg −1 .

Benzimidazole-Pyrazole Compounds with Antitumor Activities
Kalirajan et al., [75] reported anticancer activity of compounds 80a-80h and 81a-81h against MCF7 human breast cell line by in vitro Sulforhodamine B assay (SRB assay) method. The compounds 80b, 81a and 81b have significant activity when compared with standard drug Doxorubicin (Adriamycin, ADR), with GI 50 values of 16.3 µg mL −1 , 16.0 µg mL −1 , and 17.1 µg mL −1 , respectively (Doxorubicin with GI 50 value <10 µg mL −1 ). El-Gohary and Shaaban [76] reported the antitumor activity of compounds 83a and 83b and the results are shown in Table 13. Compound 83a, exhibiting the highest in vitro antitumor activity, was evaluated for in vivo antitumor activity against EAC in mice. Additionally, compound 83a was assessed for in vitro cytotoxicity toward human normal lung fibroblast (W138) cell line employing MTT assay [86][87][88] and utilizing 5-fluorouracil as a standard cytotoxic drug. IC 50 value for 83a was determined 0.246 mM, therefore less cytotoxic than 5-fluorouracil (IC 50 = 0.051 mM). Additionally, results of the DNA-binding assay confirmed that antimicrobial and antitumor compound 83a exerts its biological activities through interaction with DNA. Bezimidazole-pyrazole 85 displayed eminent activity toward HCT-116 cell line [77] with IC 50 value of 5.41 µM close to that of 5-fluorouracil of 4.00 µM and lower than that measured for the previously prepared benzimidazoles [76,89]. Wang et al., synthesized a series of benzimidazole grafted benzsulfamide-containing pyrazole ring derivatives using a strategy in two steps: 1. synthesis of benzimidazolepyrazole hybrid by reaction of pirazolecarbaldehyde 105 with 1,2,-phenylenediamine and 2. reaction with sulfonyl chloride 107 with the hybrid 106 (Scheme 30) [90]. Compound 108 showed the most excellent inhibition against tubulin assembly (IC 50 = 1.52 µM) and in vitro growth inhibitory activity against a panel of four human cancer cell lines, IC 50 = 0.15, 0.21, 0.33 and 0.17 µM, respectively, for A549, Hela, HepG2 and MCF-7. Additionally, compound 108 validly induces A549 cell apoptosis, causes cell cycle arrest in the G2/M phase and disrupts the cellular microtubule network. Due to these promising results, along with molecular docking data, compound 108 is a potential anticancer agent. Shake et al., reported synthesis of 1-substituted benzimidazoles with pyrazole moiety 109a-109d (Figure 3), by cyclization of the corresponding chalcones with hydrazine hydrate in ethanol, and their antitumor and antiviral activities [91]. The in vitro cytotoxic screening of the compounds 109a-109d against four different cell lines is showed in Table 14. It can be seen that compound 109d has the best antitumor activity on all determined tumor lines.

Benzimidazole-Pyrazole Compounds as Antioxidants
Saundane et al., evaluated the scavenging effects of the compounds 31a-31c and 32a-32c [63] on the DPPH radical by Hatano's method [92]. The RSA (Radical Scavenging Activity) results suggested that the compound 31a exhibited good antioxidant activity of 71.95 and 72.43 % at the concentration of 100 µg mL −1 . Additionally, the reductive ability of synthesized compounds was assessed by the extent of conversion of Fe 3+/ ferricyanide complex to the Fe 2+/ ferrous form. The reductive ability results suggested that the compound 31a exhibited good reducing power ability at the concentration of 100 µg mL −1 .

Conclusions
This review summarizes the syntheses of benimidazole-pyrazole compounds with antimicrobial properties, as well as their biological activities mentioned in the literature. From the data presented, it can be concluded that hybrids with pyrazole moiety in position "4" ("7") possess the strongest antimicrobial properties. The presence of certain groups grafted on the benzimidazole and pyrazole nuclei, such as -COOCH 3 , -NHCO, -CHO, -CF 3 , -NO 2 , -CN, -F, -Cl, -OH, OCH 3 , -N(CH 3 ) 2 as well as other heterocycles in the molecule (pyridine, pyrimidine, thiazole, indole), increases the antimicrobial activity of the compounds [95]. Additionally, the binding linker between benzimidazole and pyrazole is important for their antimicrobial activity. Additionally, the antimicrobial activity is improved if the molecule contains linker groups such as carbonyl (CO), amide (NHCO), or other heteroatoms. We hope that this review will be a starting point for the synthesis of other benzimidazole-pyrazole hybrids with antimicrobial properties, which have much better bacterial and antifungal properties than those of antibiotics marketed or used in hospitals today.
Funding: This research received no external funding.

Acknowledgments:
The author is thankful to Department of Organic Chemistry, Biochemistry and Catalysis, for providing necessary facilities to carry out this research work.

Conflicts of Interest:
The authors declare no conflict of interest.