Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents

*Yousef Najajreh*

#### **Abstract**

Benzimidazole derivatives are known to act against a range of biological targets and thus gained clinical applications in a wide spectrum of diseases. Few examples of multitargeted benzimidazole derivatives that were reported during the last decade will be described in this chapter. Multitargeting agents for serving the polypharmacology approach to combat shortcomings of the main one-drug-one target main dogma will be briefly explored. In that context, the multitargeting benzimidazole derivatives gain a special attention. This includes discovery (hit-to-lead), structureactivity relationship (SAR), and binding mode of at least one lead (or hit) in each group. Special attention will be given to two structures dovitinib and AT9283 that are reported to exhibit potent in vitro and in vivo activities against a group of kinases and non-kinase target (as shown recently for dovitinib).

**Keywords:** benzimidazole, selective, cytotoxic, inhibitor, multitargeting, multikinase, polypharmacology, antiproliferative, quinolinone, carbamate, quinolinone, pyrazole, urea, aniline, anilinobenzimidazolylpyrimidine, chloroacetamide, amidine, binding, mode

#### **1. Introduction**

#### **1.1 Antiproliferative action of benzimidazoles**

Benzimidazole, a heterocyclic moiety comprising six-membered benzene ring fused with five-membered imidazole ring, containing molecules, was known for its ability to induce antiproliferative effects (named as antineoplastic, anticancer, or antitumor agents). Numerous structures were reported as effective inhibitors of cell growth and division, thus acting as antiviral, antibacterial, antifungal, anthelmintic (or antihelminthics), and anticancer agents. Over the years, several published scripts have reviewed the synthetic approaches, medicinal chemistry, SAR, bioactivities, and preclinical and clinical studies of such "gifted" fragment [1–8].

#### **1.2 Benzimidazoles act on numerous biological targets**

A wide range of activities and medical situations benzimidazole containing compounds have been used for. That includes antihypertensive [9–12], anti-inflammatory [13–15], antibacterial [16–18], antiviral [19–21], antifungal [22–24], antihelmintic [25–28], anticancer [29–32], antiulcer [33–35], antioxidant [36–38], and

psychoactive drugs [39]. And proton pump inhibitors [8, 33], anticoagulants [40, 41], immunomodulators [42], hormone modulators [43, 44], antidepressants [45], lipid level modulators [46–49], and antidiabetics [50–52] are partial list of therapeutic effects of benzimidazole containing comprising compounds. Benzimidazole derivatives exert their actions by interacting with vital biological targets including β-tubulin [52–55], DNA minor groove [56–58], serotonin receptors (5-hydroxytryptamine receptors; 5-HT) [59–62], histamine receptors 4 (H4H) [63], dopamine receptor 2 (D2R) [64], chemokine receptor (CXCR3) [65], interleukin 2-inducible T-cell kinase (ITK) [66], lymphocyte tyrosine kinase (Lck) [67], phosphatidylinositol 3-kinase (PI3K) [68], activated protein kinase (MEK1) [69, 70], anaplastic lymphoma kinase (ALK) [71], polo-like kinase 1 (PLK1) [72, 73], breakpoint cluster region-Abelson kinase (BCR-Abl) [74], casein kinase 2 (CK2) [75], telangiectasia and Rad3-related protein kinase (ATR) [76], tyrosine kinase receptors [fibroblast growth factor receptors (FGFR-1/FGFR-2/FGFR-3)], vascular endothelial growth factor receptor (VEGFR-1/VEGFR-2/VEGFR-3), platelet-derived growth factor receptor (PDGFR-α/PDGFR-β), stem cell factor receptor (c-KIT), FMS-like tyrosine kinase 3 (FLT3) [77], poly(ADP-ribose)polymerase-1 (PARP-1) [78–82], dihydroorotate dehydrogenase (DHODH) [83], topoisomerase 1 (TOPO1) [84], DNA and RNA polymerases [85–89], histone deacetylase 2 (HDAC2) and sirtuin [3, 90], antagonism of angiotensin 1 [2], neuropeptide Y binding [91], inhibition of proton pumps [8], DNA intercalating agents [92], inhibition of cyclin-dependent kinases (CDK) activity [93–96], activation of the p53 protein [97], etc. to mention part of the asserted cellular targets.

#### **1.3 Scope: benzimidazoles as emerging multitargeting agents**

The profound success in bringing into clinical application several kinase inhibitors as anticancer drugs made "kinase targeting" a central branch of targetable biomolecules during the past two decades. Nevertheless, the emerging of resistant tumors kinase-directed therapeutics and adverse side effects turned such promising "targeted therapeutics" into challenging field. In addition, it was noticed that lack of response to kinase inhibitors is accompanied by changes in signaling network composition through adaptive kinome reprogramming. Such reprogramming is believed to allow the tumor to escape effects of the drug and manifest resistance. In contrast to the "one-drug-one-target" approach, the "bitopic, that is, two drugs acting on one target" or the "dual, that is, one drug acting on two targets," "polypharmacology" which refers to a novel paradigm that purposes at "simultaneous modulation of more than two biological targets by a single drug" has been emerging as strategy to improve the efficacy and durability of clinical responses to therapies. In cancer treatment, polypharmacology is a result of the ability of "one drug" to simultaneously inhibit multiple cancer-driving targets. However, discovering inhibitors with an appropriate multitarget profile is a challenging task that necessitates a systemic deeper investigation accompanied by major clinical developments. Therefore, a strategy is required to identify single polypharmacological agents with the ability to target multiple cancer-promoting or -sustaining pathways that does not necessarily rely on inhibiting multiple kinases [98]. As a matter of fact, high ratio (~30%) of the FDA-approved kinome-targeting drugs were reported be multitargeted ones [99]. Actually, the first kinase inhibitor imatinib was approved as multitarget agent in a later stage (in addition to its primary target BCR-Abl, it inhibits stem cell factor receptor (c-KIT) and platelet-derived growth factor receptors A and B (PDGFRα and PDGFRβ) tyrosine kinases and human quinone reductase 2

**107**

**Figure 1.**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

*Multitargeting anticancer agents. (a) Multitargeting cytotoxic benzimidazole-based structures. 3-Benzimidazol-2-ylhydroquinolin-2-one based dovitinib [TKI258, CHIR258; (1)], N-cyclopropyl-N'-[3-[5-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]Urea [AT9283 (2)],* 

*(8), axitinib (9), and lenvatinib (10) and pazopanib (11)].*

*2-anilino-4-(benzimidazol-2-yl)pyrimidine based [2-anilino-4-(benzimidazol-2-yl)-pyrimidine 2-methoxy-5-{[4-(1-methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]amino}phenol (3)], α-haloacetamidebenzimidazolebased [2-chloro-N-(2-(p-tolyl)-1H-benzo[d]imidazol-5-yl)acetamide (4)], and amidine-benzimidazole based [2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d] imidazole (5)]; (b) FDA-approved multitargeting anticancer agents [sorafenib (6), regorafenib (7) sunitinib* 

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

*Chemistry and Applications of Benzimidazole and its Derivatives*

part of the asserted cellular targets.

**1.3 Scope: benzimidazoles as emerging multitargeting agents**

The profound success in bringing into clinical application several kinase inhibitors as anticancer drugs made "kinase targeting" a central branch of targetable biomolecules during the past two decades. Nevertheless, the emerging of resistant tumors kinase-directed therapeutics and adverse side effects turned such promising "targeted therapeutics" into challenging field. In addition, it was noticed that lack of response to kinase inhibitors is accompanied by changes in signaling network composition through adaptive kinome reprogramming. Such reprogramming is believed to allow the tumor to escape effects of the drug and manifest resistance. In contrast to the "one-drug-one-target" approach, the "bitopic, that is, two drugs acting on one target" or the "dual, that is, one drug acting on two targets," "polypharmacology" which refers to a novel paradigm that purposes at "simultaneous modulation of more than two biological targets by a single drug" has been emerging as strategy to improve the efficacy and durability of clinical responses to therapies. In cancer treatment, polypharmacology is a result of the ability of "one drug" to simultaneously inhibit multiple cancer-driving targets. However, discovering inhibitors with an appropriate multitarget profile is a challenging task that necessitates a systemic deeper investigation accompanied by major clinical developments. Therefore, a strategy is required to identify single polypharmacological agents with the ability to target multiple cancer-promoting or -sustaining pathways that does not necessarily rely on inhibiting multiple kinases [98]. As a matter of fact, high ratio (~30%) of the FDA-approved kinome-targeting drugs were reported be multitargeted ones [99]. Actually, the first kinase inhibitor imatinib was approved as multitarget agent in a later stage (in addition to its primary target BCR-Abl, it inhibits stem cell factor receptor (c-KIT) and platelet-derived growth factor receptors A and B (PDGFRα and PDGFRβ) tyrosine kinases and human quinone reductase 2

psychoactive drugs [39]. And proton pump inhibitors [8, 33], anticoagulants [40, 41], immunomodulators [42], hormone modulators [43, 44], antidepressants [45], lipid level modulators [46–49], and antidiabetics [50–52] are partial list of therapeutic effects of benzimidazole containing comprising compounds. Benzimidazole derivatives exert their actions by interacting with vital biological targets including β-tubulin [52–55], DNA minor groove [56–58], serotonin receptors (5-hydroxytryptamine receptors; 5-HT) [59–62], histamine receptors 4 (H4H) [63], dopamine receptor 2 (D2R) [64], chemokine receptor (CXCR3) [65], interleukin 2-inducible T-cell kinase (ITK) [66], lymphocyte tyrosine kinase (Lck) [67], phosphatidylinositol 3-kinase (PI3K) [68], activated protein kinase (MEK1) [69, 70], anaplastic lymphoma kinase (ALK) [71], polo-like kinase 1 (PLK1) [72, 73], breakpoint cluster region-Abelson kinase (BCR-Abl) [74], casein kinase 2 (CK2) [75], telangiectasia and Rad3-related protein kinase (ATR) [76], tyrosine kinase receptors [fibroblast growth factor receptors (FGFR-1/FGFR-2/FGFR-3)], vascular endothelial growth factor receptor (VEGFR-1/VEGFR-2/VEGFR-3), platelet-derived growth factor receptor (PDGFR-α/PDGFR-β), stem cell factor receptor (c-KIT), FMS-like tyrosine kinase 3 (FLT3) [77], poly(ADP-ribose)polymerase-1 (PARP-1) [78–82], dihydroorotate dehydrogenase (DHODH) [83], topoisomerase 1 (TOPO1) [84], DNA and RNA polymerases [85–89], histone deacetylase 2 (HDAC2) and sirtuin [3, 90], antagonism of angiotensin 1 [2], neuropeptide Y binding [91], inhibition of proton pumps [8], DNA intercalating agents [92], inhibition of cyclin-dependent kinases (CDK) activity [93–96], activation of the p53 protein [97], etc. to mention

**106**

#### **Figure 1.**

*Multitargeting anticancer agents. (a) Multitargeting cytotoxic benzimidazole-based structures. 3-Benzimidazol-2-ylhydroquinolin-2-one based dovitinib [TKI258, CHIR258; (1)], N-cyclopropyl-N'-[3-[5-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl]Urea [AT9283 (2)], 2-anilino-4-(benzimidazol-2-yl)pyrimidine based [2-anilino-4-(benzimidazol-2-yl)-pyrimidine 2-methoxy-5-{[4-(1-methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]amino}phenol (3)], α-haloacetamidebenzimidazolebased [2-chloro-N-(2-(p-tolyl)-1H-benzo[d]imidazol-5-yl)acetamide (4)], and amidine-benzimidazole based [2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d] imidazole (5)]; (b) FDA-approved multitargeting anticancer agents [sorafenib (6), regorafenib (7) sunitinib (8), axitinib (9), and lenvatinib (10) and pazopanib (11)].*

(NQO2)). Thus the question of how efficacious are selective and specific one-drugone-target-approved agents in treating advanced and metastatic cancer is still under evaluation [100–102].

This chapter will concisely provide a deeper insight into the benzimidazolecontaining structures that exhibit action on multiple cellular targets. Special focus will be drawn to the identification and discovery, the structural activity relationship, proposed binding and interaction, and mechanism of action of each group of compounds. Detailed synthetic procedures and preclinical and clinical studies are out of scope of the current chapter. The focus of this chapter will be on groups of compounds that had been unveiled as concurrently antagonizing multiple targets. Instead, this chapter will focus on five groups of compounds reported to possess cytotoxic activities by acting on multiple (see **Figure 1a**) compounds (1, 2, 3, 4, and 5) holding the potential to be administered as "polytherapies."

#### **2. Benzimidazole scaffold for multitargeting of cancer**

Multitargeting agent is defined as "a single chemical entity exerting action as a result of direct interactions on multiple biomolecular targets" [103]. Such agents can be beneficial in overcoming single (or dual)-targeting limitations including compromised effectiveness, severe side effects, emergence of resistant target mutants, and target non-related mutations. In addition, the efficacy of singlemolecular-targeted FDA-approved agents in treating brutal and mortal cancers (breast, colorectal lung, pancreatic, and prostate) is limited. Most tumors escape from the inhibition of any single chemotherapeutic agent, and thus one possible therapeutic strategy could be in (1) administering cocktails of highly selective inhibitors (combinational therapy) or (2) development of multitarget inhibitors that act on inhibiting concurrently multiple validated target in cancer cell initiating a concerted molecular response, leading to cell death. Multitargeting chemotherapeutics hold the potential of exhibiting synergistic or at least additive effects when compared to single-targeted ones. It is believed that advances in signaling cascades, networks and crosstalk, chemo- and bioinformatics, detailed three-dimensional structural information of target proteins, computational chemistry tools, proteomics, etc. will allow for designing novel multitarget inhibitors.

It has been realized that molecular targeted therapeutics are facing acquired resistance. Multitargeting approach is gaining increased attention especially when combating resistant cancer cells. Accumulated evidence showed that drug treatment aggravates "selective pressure" of evolutionary force exerted on tumor cells that leads to resistance.

Benzimidazole fragment is reported to be an integral part of multitargeted inhibitors. Such inhibitors challenge the dominant paradigm in drug discovery which deemed to design and develop bioactive agent with maximum selectivity and specificity to individual drug target. Such compounds hold the hope for a new avenue of combating disease cases that could not be cured with one inhibitor acting on single target such as cancer [104, 105].

#### **2.1 Benzimidazolylquinolinone: a scaffold for targeting multiple biomolecules**

#### *2.1.1 Discovery of dovitinib (TKI258, CHIR258)*

Dovitinib [(TKI258, CHIR258); 4-amino-5-fluoro-3-(5-(4-methylpiperazin-1-yl)-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (1)] was first designed and synthesized as vascular endothelial growth factor receptor (FEGFR) inhibitor in the

**109**

**Figure 3.**

**Figure 2.**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

context of developing targeted antiangiogenic treatments [106]. The compound was later reported as a multitargeted kinase inhibitor (by [107]) following the realization that its potent inhibitory effects on cancer cells are associated with action on other multiple key players in oncogenesis, development, and proliferation of cancer [107]. The commercially available 3-benzimidazol-2-ylhydroquinolin-2-one scaffold [benzimidazolylquinolinone for short from now on, **Figure 2** (13)] was identified using high-throughput screening (HTS) method and reported by Renhowe et al. (Novartis) as a potent (IC50 values < 0.1 μM) reversible ATP competitive inhibitor of VEGFR-2, FGFR-1, and PDGFRβ [106]. Due to desirable properties as low-molecular-weight compound exhibiting submicromolar activity, (12) was considered a good hit to start with. To overcome the undesirable physicochemical properties of (13) (low aqueous solubility), further optimization was needed that ended up with a drug-like compound (1). Determining the key structural features required for potent kinase inhibition, molecular modeling was employed. The assumption was that in quinolinone portion, both NH at position 1 and the carbonyl group, together with benzimidazole NH, form a donor-acceptor-donor motif that would most probably bind to the hinge region of the RTKs and should be preserved. To test this hypothesis, a systematic study was conducted through which hydrogen bond donors were masked by methyl group (CH3-) as shown in **Figure 3** (13a–c) and 14a and 14b. These changes led to significant loss in the potency against all three receptor tyrosine kinases (VEGFR-2, FGFR-1, and PDGFRβ RTKs). The dimethylated analogue (**Figure 3**, 14b) showed no kinase activity at a concentration as high as 25 μM. Interestingly, it was noticed that monomethylation seemed to affect the kinase selectivity profile as well. Introduction of a methyl on the benzimidazole NH (13b) had a more dramatic effect on VEGFR-2 affinity than the methylation at NH in position 1 of the hydroquinolin-2-one (13a). This underlies the importance

*Benzimidazolquinolinone-based multitargeting scaffold. (a) The basic skeleton of dovitinib (TKI258, CHIR258), 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one (12) with the two fragments quinolinone (blue) and benzimidazole (red) is indicated, (b) structures of commercially available starting "hit" (13) identified using* 

*Assessing the effect of the HBD and HBA on the activity of derivative of 3-benzimidazol-2-ylhydroquinolin-2-one. Methylated analogues of (13 and 14). The monomethylation caused a significant drop in the potency* 

*HTS, and the "lead" approved as a multitargeting drug dovitinib (1).*

*toward RTKs, while dimethylation aborted the RTKs' activity [106].*

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

#### *Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

context of developing targeted antiangiogenic treatments [106]. The compound was later reported as a multitargeted kinase inhibitor (by [107]) following the realization that its potent inhibitory effects on cancer cells are associated with action on other multiple key players in oncogenesis, development, and proliferation of cancer [107].

The commercially available 3-benzimidazol-2-ylhydroquinolin-2-one scaffold [benzimidazolylquinolinone for short from now on, **Figure 2** (13)] was identified using high-throughput screening (HTS) method and reported by Renhowe et al. (Novartis) as a potent (IC50 values < 0.1 μM) reversible ATP competitive inhibitor of VEGFR-2, FGFR-1, and PDGFRβ [106]. Due to desirable properties as low-molecular-weight compound exhibiting submicromolar activity, (12) was considered a good hit to start with. To overcome the undesirable physicochemical properties of (13) (low aqueous solubility), further optimization was needed that ended up with a drug-like compound (1). Determining the key structural features required for potent kinase inhibition, molecular modeling was employed. The assumption was that in quinolinone portion, both NH at position 1 and the carbonyl group, together with benzimidazole NH, form a donor-acceptor-donor motif that would most probably bind to the hinge region of the RTKs and should be preserved.

To test this hypothesis, a systematic study was conducted through which hydrogen bond donors were masked by methyl group (CH3-) as shown in **Figure 3** (13a–c) and 14a and 14b. These changes led to significant loss in the potency against all three receptor tyrosine kinases (VEGFR-2, FGFR-1, and PDGFRβ RTKs). The dimethylated analogue (**Figure 3**, 14b) showed no kinase activity at a concentration as high as 25 μM. Interestingly, it was noticed that monomethylation seemed to affect the kinase selectivity profile as well. Introduction of a methyl on the benzimidazole NH (13b) had a more dramatic effect on VEGFR-2 affinity than the methylation at NH in position 1 of the hydroquinolin-2-one (13a). This underlies the importance

#### **Figure 2.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

evaluation [100–102].

(NQO2)). Thus the question of how efficacious are selective and specific one-drugone-target-approved agents in treating advanced and metastatic cancer is still under

This chapter will concisely provide a deeper insight into the benzimidazolecontaining structures that exhibit action on multiple cellular targets. Special focus will be drawn to the identification and discovery, the structural activity relationship, proposed binding and interaction, and mechanism of action of each group of compounds. Detailed synthetic procedures and preclinical and clinical studies are out of scope of the current chapter. The focus of this chapter will be on groups of compounds that had been unveiled as concurrently antagonizing multiple targets. Instead, this chapter will focus on five groups of compounds reported to possess cytotoxic activities by acting on multiple (see **Figure 1a**) compounds (1, 2, 3, 4,

Multitargeting agent is defined as "a single chemical entity exerting action as a result of direct interactions on multiple biomolecular targets" [103]. Such agents can be beneficial in overcoming single (or dual)-targeting limitations including compromised effectiveness, severe side effects, emergence of resistant target mutants, and target non-related mutations. In addition, the efficacy of singlemolecular-targeted FDA-approved agents in treating brutal and mortal cancers (breast, colorectal lung, pancreatic, and prostate) is limited. Most tumors escape from the inhibition of any single chemotherapeutic agent, and thus one possible therapeutic strategy could be in (1) administering cocktails of highly selective inhibitors (combinational therapy) or (2) development of multitarget inhibitors that act on inhibiting concurrently multiple validated target in cancer cell initiating a concerted molecular response, leading to cell death. Multitargeting chemotherapeutics hold the potential of exhibiting synergistic or at least additive effects when compared to single-targeted ones. It is believed that advances in signaling cascades, networks and crosstalk, chemo- and bioinformatics, detailed three-dimensional structural information of target proteins, computational chemistry tools, pro-

and 5) holding the potential to be administered as "polytherapies."

**2. Benzimidazole scaffold for multitargeting of cancer**

teomics, etc. will allow for designing novel multitarget inhibitors.

It has been realized that molecular targeted therapeutics are facing acquired resistance. Multitargeting approach is gaining increased attention especially when combating resistant cancer cells. Accumulated evidence showed that drug treatment aggravates "selective pressure" of evolutionary force exerted on tumor cells

Benzimidazole fragment is reported to be an integral part of multitargeted inhibitors. Such inhibitors challenge the dominant paradigm in drug discovery which deemed to design and develop bioactive agent with maximum selectivity and specificity to individual drug target. Such compounds hold the hope for a new avenue of combating disease cases that could not be cured with one inhibitor acting

**2.1 Benzimidazolylquinolinone: a scaffold for targeting multiple biomolecules**

Dovitinib [(TKI258, CHIR258); 4-amino-5-fluoro-3-(5-(4-methylpiperazin-1-yl)-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (1)] was first designed and synthesized as vascular endothelial growth factor receptor (FEGFR) inhibitor in the

**108**

that leads to resistance.

on single target such as cancer [104, 105].

*2.1.1 Discovery of dovitinib (TKI258, CHIR258)*

*Benzimidazolquinolinone-based multitargeting scaffold. (a) The basic skeleton of dovitinib (TKI258, CHIR258), 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one (12) with the two fragments quinolinone (blue) and benzimidazole (red) is indicated, (b) structures of commercially available starting "hit" (13) identified using HTS, and the "lead" approved as a multitargeting drug dovitinib (1).*

#### **Figure 3.**

*Assessing the effect of the HBD and HBA on the activity of derivative of 3-benzimidazol-2-ylhydroquinolin-2-one. Methylated analogues of (13 and 14). The monomethylation caused a significant drop in the potency toward RTKs, while dimethylation aborted the RTKs' activity [106].*

of preventing the hydrogen bond donor (HBD). An opposite effect was noticed for FGFR-1, which indicates that despite the high homology of the two ATP-binding sites in the tow targets, selectivity opportunities still exist that are likely due to small changes in the shape of binding site. Such change in the shape can influence the accessibility of alternate binding poses of the monomethylated ligands (13a–13b and 14a in **Figure 1**) [106].

#### *2.1.1.1 Structure–activity relationship (SAR)*

The scaffold (13) was annotated by four rings (A–D). Modifications were introduced in a systemic manner. Once the basic structural components needed for affinity to targets of interest were understood, a study of the structure–activity relationship around the periphery of central 3-benzimidazol-2-ylhydroquinolin-2-one (13) scaffold was undertaken. Besides electrophilicity, nucleophilicity, bulkiness, steric hindrance, HBD versus HBA, and basicity, C4 of ring A was used for incorporation of moieties that might impart favorable physicochemical properties.

**SAR of ring B (C4)**: While removal of the hydroxyl group reduced the activity, its replacement with amine improved significantly affinity to RTK and also cell potency [EC50 of 0.078 μM (NH2 > OH > H)], suggesting an importance of the HBD at C4 of the hydroquinolin-2-one fragment. Thus, incorporation of larger substituents on the C4-NH of the hydroquinolin-2-one was explored and found to be tolerated (see compounds 15b and 15c, **Figure 4**). Not only substantial improvement in the solubility was attained when the substituents carried an additional basic nitrogen were introduced to this position, it was noticed that this position modulates the selectivity profile of this class of compounds. It was reported that both derivatives (12a) and (12b) exhibited enhanced potency against PDGFR than VEGFR-1 (3000-fold) and FGFR (>1500-fold). Large basic amines like aminoquinuclidine potentiate the derivative (15d) against CHK-1 and GSK-3.

#### **Figure 4.**

*Summary of structure–activity relationship (SAR) of 3-benzimidazol-2-ylhydroquinolin-2-one (1) [106].*

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*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

phosphorylation was dipped in endothelial cells treated with (1).

In summary, dovitinib (1), an antineoplastic benzimidazolylquinolinone derivative, inhibits multiple growth factor receptor tyrosine kinases important for tumor angiogenesis and tumor growth. Dovitinib is well established as type III–V receptor tyrosine kinase (RTK) inhibitor. Though it potently inhibits fibroblast growth factor receptors (FGFR-1/FGFR-2/FGFR-3), the compound also inhibits vascular endothelial growth factor receptor (VEGFR-1/VEGFR-2/VEGFR-3), platelet-derived growth factor receptor (PDGFR-α/β), stem cell factor receptor (c-KIT), FMS-like tyrosine kinase 3 (FLT3), and colony-stimulating factor receptor 1 (CSFR-1) emphasizing the nonspecific action of the drug [108]. The orally bioavailable lactate salt of (1) strongly binds to fibroblast growth factor receptor 3 (FGFR3) and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death. The activation of the abovementioned RTK in singularity or together is associated with cell proliferation

Dovitinib (TKI258, 1) is a highly potent, novel multitargeting receptor tyrosine kinase inhibitor with IC50 of 1, 2, 10, 8, 27, and 36 nM for FLT3, c-KIT, VEGFR-1/ VEGFR-2/VEGFR-3, PDGFRß, and CSFR −1, respectively. Due to its inhibitory effect of VEGFR1/VEGFR2, the compound displayed both antitumor and antian-

Trudel and colleagues reported that in addition to inhibiting the abovementioned TRKs (types II, IV, V), (1) potently inhibits wild-type (WT) FGFR3, F384 L-FGFR3 (IC50 = 25 nM), and FGFR3 mutants (IC50 = 70–90 nM for the various mutations) driven by B9 cells [107]. Additionally, same group reported that (1) inhibited the proliferation of multiple myeloma (MM) cells. When assessing its antiproliferative effect against U266 and 8226 cells, (1) displayed a potent inhibitory effect (IC50 ~ 90 nM) against KMS11 (FGFR3-Y373C), OPM2 (FGFR3- K650E) cells and IC50 ~ 550 nM KMS18 (FGFR3-G384D) [109]. (1) Exhibited exceedingly potent antiproliferative effect against acute myelogenous leukemia (AML) cells MV4;11 (mutant FLT3-ITD) compared to AML RS4;11(FLT3 WT) cells [EC50 = 13 nmol/L and EC50 = 315 nmol/L for MV4;11 and RS4;11, respectively, i.e., (~24-fold decrease in potency for FLT3 WT cells)]. Such results indicated that (1) exhibited far more potent activity against cells that are dependent on constitutively active FLT3-ITD. A similar conclusion was affirmed by Heise et al. by the notion that (CHIR258, 1) inhibited the proliferation of MOLM13 and MOLM14 that are FLT3-ITD mutant cells with EC50 ∼ 6 nmol/L similar to the ones with MV4;11 [109]. Besides the potent action of (1) against a wide range of RTK, its inhibitors' effect

ion fibroblast growth factor receptors in a variety of tumor xenograft models in athymic mice, including acute myeloid leukemia, multiple myeloma, and colon-

In conclusion, substitution at C4 position was revealed as critical to the activity of the benzimidazolylhydroquinolinone scaffold; however for RTK inhibitor program, the NH2 group was the optimal substituent at C4 as it avoided inhibition of these additional serine threonine kinases, which could complicate the pharmaco-

**SAR of ring D**: The overall structure–activity relationship (SAR) is summarized in **Figure 4**. Medicinal chemistry efforts were concluded in the selection of compound (1) as a candidate for further development. The compound (1) displayed exceedingly potent inhibitory effect when assessed against receptor protein kinases VEGFR-2, FGFR-1 and FGFR-3, PDGFRβ, VEGFR-1, VEGFR-2 and VEGFR-3, c-KIT, CSF-1R, and FLT-3 with IC50 values between 3 and 27 nM. Such activity is translated into antiproliferative action on cells that are VEGF-, FGF-, SCF-, CSF-, or PDGF-driven. Mechanistically, it was also indicated that VEGF-mediated ERK

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

logical application of these agents.

and survival in all cancer cell types.

and prostate-derived models was promising.

giogenic activities in vivo.

*Chemistry and Applications of Benzimidazole and its Derivatives*

and 14a in **Figure 1**) [106].

*2.1.1.1 Structure–activity relationship (SAR)*

of preventing the hydrogen bond donor (HBD). An opposite effect was noticed for FGFR-1, which indicates that despite the high homology of the two ATP-binding sites in the tow targets, selectivity opportunities still exist that are likely due to small changes in the shape of binding site. Such change in the shape can influence the accessibility of alternate binding poses of the monomethylated ligands (13a–13b

The scaffold (13) was annotated by four rings (A–D). Modifications were introduced in a systemic manner. Once the basic structural components needed for affinity to targets of interest were understood, a study of the structure–activity relationship around the periphery of central 3-benzimidazol-2-ylhydroquinolin-2-one (13) scaffold was undertaken. Besides electrophilicity, nucleophilicity, bulkiness, steric hindrance, HBD versus HBA, and basicity, C4 of ring A was used for incorpo-

ration of moieties that might impart favorable physicochemical properties.

nuclidine potentiate the derivative (15d) against CHK-1 and GSK-3.

*Summary of structure–activity relationship (SAR) of 3-benzimidazol-2-ylhydroquinolin-2-one (1) [106].*

**SAR of ring B (C4)**: While removal of the hydroxyl group reduced the activity, its replacement with amine improved significantly affinity to RTK and also cell potency [EC50 of 0.078 μM (NH2 > OH > H)], suggesting an importance of the HBD at C4 of the hydroquinolin-2-one fragment. Thus, incorporation of larger substituents on the C4-NH of the hydroquinolin-2-one was explored and found to be tolerated (see compounds 15b and 15c, **Figure 4**). Not only substantial improvement in the solubility was attained when the substituents carried an additional basic nitrogen were introduced to this position, it was noticed that this position modulates the selectivity profile of this class of compounds. It was reported that both derivatives (12a) and (12b) exhibited enhanced potency against PDGFR than VEGFR-1 (3000-fold) and FGFR (>1500-fold). Large basic amines like aminoqui-

**110**

**Figure 4.**

In conclusion, substitution at C4 position was revealed as critical to the activity of the benzimidazolylhydroquinolinone scaffold; however for RTK inhibitor program, the NH2 group was the optimal substituent at C4 as it avoided inhibition of these additional serine threonine kinases, which could complicate the pharmacological application of these agents.

**SAR of ring D**: The overall structure–activity relationship (SAR) is summarized in **Figure 4**. Medicinal chemistry efforts were concluded in the selection of compound (1) as a candidate for further development. The compound (1) displayed exceedingly potent inhibitory effect when assessed against receptor protein kinases VEGFR-2, FGFR-1 and FGFR-3, PDGFRβ, VEGFR-1, VEGFR-2 and VEGFR-3, c-KIT, CSF-1R, and FLT-3 with IC50 values between 3 and 27 nM. Such activity is translated into antiproliferative action on cells that are VEGF-, FGF-, SCF-, CSF-, or PDGF-driven. Mechanistically, it was also indicated that VEGF-mediated ERK phosphorylation was dipped in endothelial cells treated with (1).

In summary, dovitinib (1), an antineoplastic benzimidazolylquinolinone derivative, inhibits multiple growth factor receptor tyrosine kinases important for tumor angiogenesis and tumor growth. Dovitinib is well established as type III–V receptor tyrosine kinase (RTK) inhibitor. Though it potently inhibits fibroblast growth factor receptors (FGFR-1/FGFR-2/FGFR-3), the compound also inhibits vascular endothelial growth factor receptor (VEGFR-1/VEGFR-2/VEGFR-3), platelet-derived growth factor receptor (PDGFR-α/β), stem cell factor receptor (c-KIT), FMS-like tyrosine kinase 3 (FLT3), and colony-stimulating factor receptor 1 (CSFR-1) emphasizing the nonspecific action of the drug [108]. The orally bioavailable lactate salt of (1) strongly binds to fibroblast growth factor receptor 3 (FGFR3) and inhibits its phosphorylation, which may result in the inhibition of tumor cell proliferation and the induction of tumor cell death. The activation of the abovementioned RTK in singularity or together is associated with cell proliferation and survival in all cancer cell types.

Dovitinib (TKI258, 1) is a highly potent, novel multitargeting receptor tyrosine kinase inhibitor with IC50 of 1, 2, 10, 8, 27, and 36 nM for FLT3, c-KIT, VEGFR-1/ VEGFR-2/VEGFR-3, PDGFRß, and CSFR −1, respectively. Due to its inhibitory effect of VEGFR1/VEGFR2, the compound displayed both antitumor and antiangiogenic activities in vivo.

Trudel and colleagues reported that in addition to inhibiting the abovementioned TRKs (types II, IV, V), (1) potently inhibits wild-type (WT) FGFR3, F384 L-FGFR3 (IC50 = 25 nM), and FGFR3 mutants (IC50 = 70–90 nM for the various mutations) driven by B9 cells [107]. Additionally, same group reported that (1) inhibited the proliferation of multiple myeloma (MM) cells. When assessing its antiproliferative effect against U266 and 8226 cells, (1) displayed a potent inhibitory effect (IC50 ~ 90 nM) against KMS11 (FGFR3-Y373C), OPM2 (FGFR3- K650E) cells and IC50 ~ 550 nM KMS18 (FGFR3-G384D) [109]. (1) Exhibited exceedingly potent antiproliferative effect against acute myelogenous leukemia (AML) cells MV4;11 (mutant FLT3-ITD) compared to AML RS4;11(FLT3 WT) cells [EC50 = 13 nmol/L and EC50 = 315 nmol/L for MV4;11 and RS4;11, respectively, i.e., (~24-fold decrease in potency for FLT3 WT cells)]. Such results indicated that (1) exhibited far more potent activity against cells that are dependent on constitutively active FLT3-ITD. A similar conclusion was affirmed by Heise et al. by the notion that (CHIR258, 1) inhibited the proliferation of MOLM13 and MOLM14 that are FLT3-ITD mutant cells with EC50 ∼ 6 nmol/L similar to the ones with MV4;11 [109].

Besides the potent action of (1) against a wide range of RTK, its inhibitors' effect ion fibroblast growth factor receptors in a variety of tumor xenograft models in athymic mice, including acute myeloid leukemia, multiple myeloma, and colonand prostate-derived models was promising.

Recent studies reported the comparative activities of dovitinib against 16 colorectal cancer (CRC) cell lines (among them, 10 were KRAS or BRAF mutants). Results showed the affectivity of the drug in inhibiting the proliferation of majority of the cell lines excluding the ones harboring KRAS or BRAF mutants. However, when assessing the efficacy of the drug in vivo, it reduced the tumor growth in vivo regardless of the KRAS and BRAF mutation status. The drug exerted significant reduction of the xenograft size of both resistant cell lines (KRAS mutant LoVo cells but not in BRAF mutant HT-29) but without a detectable effect in the resistant mutant cell BRAF mutant HT-29 in vitro on s. Such results were explained by the multitarget action of the drug in which by acting on FGFR and FGFR together with VEGFR has been able to interfere with resistance mechanisms emerging from the synergistic interaction between the various signaling cascades in promoting neovascularization that is believed to be one resistance factor in renal cell carcinoma or pancreatic cancer [110, 111].

Dovitinib was selected to proceed ahead for preclinical and clinical trials. Several clinical trials have been conducted, and others are also underway with the drug and alone or in combination with several chemotherapeutic agents [112–118].

#### *2.1.2 Binding mode of dovitinib (CHIR258, 1) to FGFR-1*

Based on FGFR-1 crystal structure (PDB 2FGI) in conjunction with the information received from the X-ray structure of (1) with CHK1, a homology model for (1) complexed with VEGRF2 was constructed [106]. The model was helpful in guiding for the important interactions of (1) with active site. It was concluded that (1) participated in three hydrogen bonds to the hinge domain (Glu917 and Cys919). In addition A-ring makes a VDW interaction with the hydrophobic gatekeeper Val916 and was engaged in an S-H/π interaction with Cys1045. Leu840, Val848 (both in the P-loop and ceiling of the purine pocket), Ala866 (ceiling of the purine pocket), Val 899 (floor of the purine pocket), Phe918 (part of the hinge), Lys920, Gly922 (both in the lower hinge region), and Leu1035 (floor of the purine pocket) took part in hydrophobic interaction with (1). In the following studies, the X-ray structures of (1) complexed with native and with mutant FGFR1 and with FGFR4 were reported [119–122].

#### *2.1.2.1 Going beyond kinases*

Although dovitinib binds to several kinases at nanomolar concentrations, recent studies reported its inhibitory effect against cancer-related targets including topoisomerase I and II (Topo I and II) [123] and human recombinant bone morphogenetic protein (BMP)-2, indicating that the cell growth inhibitory activity and the anticancer activity of dovitinib may result, in part, from its ability to target Topo I and II in addition to the ability to inhibit multiple kinases [124]. A study disclosed dovitinib inhibition of BMP-2 enhanced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/Smad5/Smad8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and p38 **Figure 5**. An increase in the expression of mRNA of BMP-4, BMP-7, ALP, and osteocalcin (OCN) was noticed following treatment with (1). It was also noted that the potent stimulating

**113**

**Figure 5.**

*into solution [119].*

stage through activation of p53 protein.

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

effect of (1) on BMP-2-induced osteoblast differentiation suggests a potential repositioning for the use of (1) treatment of bone-related disorders [124]. In a recent study, Ye Zhang **et al**. initially used the central scaffold 3-(1*H*-benzimidazol-2-yl)quinolin-2(1*H*)-one (12) to explore that potential diversification of functional groups decorating (12). The compounds synthesized were assessed against HepG2 (human liver cancer cells), SK-OV-3 (human ovarian cancer cells), NCI-H460 (human large cell lung cancer cells), BEL-7404 (human liver cancer cells), and HL-7702 (human liver normal cells) cell lines. Initial studies showed that halogenated derivative [3-(6-chloro-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (15e) and 3-(6-bromo-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (15f) (see **Figure 4**)] exhibited better activity than 5-FU and cisplatin when assessed against HepG2, SKOV-3, NCI-H460, and BEL-7404 but not HL-7702. The authors postulated that (15e) and (15f) inhibit HepG2 proliferation by blocking the cells in G2/M

*(a) Cartoon representation of the crystallographic structure of complex of (1) to FGFR-1 (PDB 5 AM6); the binding site is depicted showing the kinase with residues interacting with the ligand in stick model, (b left) the main interactions between (1) and the kinase domain and (b right) the surface representation, with the surface colored by atom type (red, oxygen; blue, nitrogen; yellow, sulfur; gray, carbon/hydrogen). The donor-acceptordonor motif is shown to interact with the hinge region while the 1-methylpiperazine substituent on C5′ points* 

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

#### **Figure 5.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

*2.1.2 Binding mode of dovitinib (CHIR258, 1) to FGFR-1*

pancreatic cancer [110, 111].

[112–118].

[119–122].

*2.1.2.1 Going beyond kinases*

Recent studies reported the comparative activities of dovitinib against 16 colorectal cancer (CRC) cell lines (among them, 10 were KRAS or BRAF mutants). Results showed the affectivity of the drug in inhibiting the proliferation of majority of the cell lines excluding the ones harboring KRAS or BRAF mutants. However, when assessing the efficacy of the drug in vivo, it reduced the tumor growth in vivo regardless of the KRAS and BRAF mutation status. The drug exerted significant reduction of the xenograft size of both resistant cell lines (KRAS mutant LoVo cells but not in BRAF mutant HT-29) but without a detectable effect in the resistant mutant cell BRAF mutant HT-29 in vitro on s. Such results were explained by the multitarget action of the drug in which by acting on FGFR and FGFR together with VEGFR has been able to interfere with resistance mechanisms emerging from the synergistic interaction between the various signaling cascades in promoting neovascularization that is believed to be one resistance factor in renal cell carcinoma or

Dovitinib was selected to proceed ahead for preclinical and clinical trials. Several clinical trials have been conducted, and others are also underway with the drug and alone or in combination with several chemotherapeutic agents

Based on FGFR-1 crystal structure (PDB 2FGI) in conjunction with the information received from the X-ray structure of (1) with CHK1, a homology model for (1) complexed with VEGRF2 was constructed [106]. The model was helpful in guiding for the important interactions of (1) with active site. It was concluded that (1) participated in three hydrogen bonds to the hinge domain (Glu917 and Cys919). In addition A-ring makes a VDW interaction with the hydrophobic gatekeeper Val916 and was engaged in an S-H/π interaction with Cys1045. Leu840, Val848 (both in the P-loop and ceiling of the purine pocket), Ala866 (ceiling of the purine pocket), Val 899 (floor of the purine pocket), Phe918 (part of the hinge), Lys920, Gly922 (both in the lower hinge region), and Leu1035 (floor of the purine pocket) took part in hydrophobic interaction with (1). In the following studies, the X-ray structures of (1) complexed with native and with mutant FGFR1 and with FGFR4 were reported

Although dovitinib binds to several kinases at nanomolar concentrations, recent studies reported its inhibitory effect against cancer-related targets including topoisomerase I and II (Topo I and II) [123] and human recombinant bone morphogenetic protein (BMP)-2, indicating that the cell growth inhibitory activity and the anticancer activity of dovitinib may result, in part, from its ability to target Topo I and II in addition to the ability to inhibit multiple kinases [124]. A study disclosed dovitinib inhibition of BMP-2 enhanced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/Smad5/Smad8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and p38 **Figure 5**. An increase in the expression of mRNA of BMP-4, BMP-7, ALP, and osteocalcin (OCN) was noticed following treatment with (1). It was also noted that the potent stimulating

**112**

*(a) Cartoon representation of the crystallographic structure of complex of (1) to FGFR-1 (PDB 5 AM6); the binding site is depicted showing the kinase with residues interacting with the ligand in stick model, (b left) the main interactions between (1) and the kinase domain and (b right) the surface representation, with the surface colored by atom type (red, oxygen; blue, nitrogen; yellow, sulfur; gray, carbon/hydrogen). The donor-acceptordonor motif is shown to interact with the hinge region while the 1-methylpiperazine substituent on C5′ points into solution [119].*

effect of (1) on BMP-2-induced osteoblast differentiation suggests a potential repositioning for the use of (1) treatment of bone-related disorders [124]. In a recent study, Ye Zhang **et al**. initially used the central scaffold 3-(1*H*-benzimidazol-2-yl)quinolin-2(1*H*)-one (12) to explore that potential diversification of functional groups decorating (12). The compounds synthesized were assessed against HepG2 (human liver cancer cells), SK-OV-3 (human ovarian cancer cells), NCI-H460 (human large cell lung cancer cells), BEL-7404 (human liver cancer cells), and HL-7702 (human liver normal cells) cell lines. Initial studies showed that halogenated derivative [3-(6-chloro-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (15e) and 3-(6-bromo-1*H*-benzo[d]imidazol-2-yl)quinolin-2(1*H*)-one (15f) (see **Figure 4**)] exhibited better activity than 5-FU and cisplatin when assessed against HepG2, SKOV-3, NCI-H460, and BEL-7404 but not HL-7702. The authors postulated that (15e) and (15f) inhibit HepG2 proliferation by blocking the cells in G2/M stage through activation of p53 protein.

#### **2.2 Pyrazolbenzimidazols as multikinase inhibitors**

#### *2.2.1 Discovery of AT9283a*

In developing a selective potent aurora kinase inhibitor by employing fragmentbased discovery method, the pyrazol-4-yl urea benzimidazole derivative (AT9283, 21) was identified as a multitargeting kinase inhibitor. The pyrazolebenzimidazolebased clinical candidate (21) was optimized by Steven Howard and his colleagues following efficient structure-guided fragment to hit IC50 as low as 3 nM activity as a dual potent inhibitor toward Aurora A/Aurora B [125]. AT9283 was identified starting from the pyrazole-benzimidazole fragment (16) that was previously identified during the endeavor of developing cyclin-dependent kinase (CDK) inhibitors. Subsequent structure-based approach using CDK2 crystallographic structure led to the identification of the benzamide analogue (18). Throughout the process of developing CDK2 inhibitors, pyrazole-benzimidazole derivative was identified to act with high potency toward Aurora A. Starting with fragment, (18) demonstrated superior ligand efficiency (LE = 0.59) for Aurora A compared to CDK1 and CDK2 and also sufficient potency to allow detection in a conventional enzyme bioassay [125].

Aiming at optimizing, the "hit" (18) on the way to end up with a lead SAR is performed on the benzamide analogues. The team was aided by polyploid phenotype in HCT116 cells, as a functional assay that differentiates for Aurora A and Aurora B inhibition, combined with potency when screening for further analogues. Guided by the hypothesis that introducing a basic motif into fragment (18) will improve the potency of the compound, modifications were introduced successfully to 5- or 6-position of the benzimidazole without causing any clashes with the protein. In a further step, the morpholinomethyl motif was functionalized at position 5. Details grasped from the X-ray crystal structure of (19) complexed with Aurora A revealed that the pyrazolebenzimidazole motif is positioned in an excellent complementarity with the narrow region of the ATP pocket. A result directed the steps to follow in the design of the optimized structure (**Figure 6**). While retaining the 5-morpholinomethyl on the pyrazolebenzimidazole motif, the benzamide portion was subjected to modifications. Keeping in mind the need to keep the molecular weight while introducing increased flexibility on the glycine region, the amide was converted to urea (20). This strategy was fruitful when comprehending that the urea analogue (20) exhibited reduced plasma protein binding while maintaining in vitro activity against Aurora kinases.

In the following step, the X-ray structure of (20) complexed with Aurora A was solved and iterated a similar binding mode to the hinge region. To resolve a twisted conformation of the phenyl plane in regard to pyrazole-benzimidazole portion of the molecule, a fully reduced cyclohexyl and difluorophenyl groups were also introduced (compound (20a) and (20b), respectively). Adsorption, disposition, metabolism, and excretion (ADME) considerations lead to proposing cyclopropyl derivative (21). As an alternative to introducing additional heterocyclic moiety, aiming at reducing the lipophilicity of (20a) for improving the ADME, the size of cyclohexyl ring was reduced to cyclopropyl analogue resulting in compound (21) that exhibits high enzyme and cellular potency still with reduced both the molecular weight (MW) and lipophilicity (logD7.4 = 2.1, MW = 381). Compound (21) demonstrated potent inhibition of HCT116 colony formation (IC50 = 12 nM), a clean CYP450 profile (IC50 > 10 μM for CYP3A4, 2D6, 1A2, 2C9, 2C19), acceptable mouse plasma protein binding (81.5%), and good thermodynamic solubility (2.0 mg/mL at pH = 7.0 and 13 mg/mL at pH = 5.5).

Later, AT9283 (21) was shown to bind and potently inhibit a number of kinases including the Aurora kinases A and B (serine–threonine kinases that are known to play essential roles in mitotic checkpoint control during mitosis at IC50 ~ 3 nM),

**115**

**Figure 6.**

*benzimidazole motifs [125].*

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

Janus kinase 2 (JAK2) and JAK3 (1.2 and 1.1 nM, respectively), breakpoint cluster region-Abelson (BCR-Abl) T315I (4 nM), and mitogen-activated protein kinase kinase kinase 2 (MEKK2) with IC50 values of lower nanomolar (4.7–18 nM). This set of known kinases is known to play key roles in mitotic progress in cell cycle, induction of proliferation, evasion of apoptosis and tumor growth and thus considered vital targets to chemotherapeutic agents (see **Table 1**). Therefore, AT9283

*(a) Main steps in the identification and discovery of pyrazolebenzimidazole-based multitargeting agent AT9283 (21) using fragment-based identification starting from fragment (16). (b) The structure N-cyclopropyl-N'-[3-[5-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl] urea [AT9283 (21a)]. The "folded conformation" of (21b). Dotted lines to indicate the hydrophobic interaction between the cyclopropyl and* 

AT-9283 inhibits effective proliferation of cancer cells both in vitro and in vivo with and its effect is enhanced by with other agents (see **Table 2**) [127]. Henceforth T9283 proceeded to clinical trials including in children with relapsed or refractory acute leukemia, imatinib-resistant BCR-Abl-positive leukemic cells, and patients with relapsed or refractory multiple myeloma. Accumulative results indicate a need for optimizing the pharmacological profile on the way to overcome faced challenges

The activity in imatinib-resistant BCR-Abl chronic myelogeneous leukemia (CML) explained based on modeling which reiterated the assumption that AT-9283 is bound to the kinase domain in the "folded conformation" which allows the needed interactions with the hinge region without a clash between the cyclopropyl group and the isoleucine residue in the T315I mutant. The results obtained in

(21) is defined as multikinase (multitargeting) inhibitor [126].

in clinical application of the compound [127, 128].

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

#### **Figure 6.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

**2.2 Pyrazolbenzimidazols as multikinase inhibitors**

binding while maintaining in vitro activity against Aurora kinases.

(2.0 mg/mL at pH = 7.0 and 13 mg/mL at pH = 5.5).

In the following step, the X-ray structure of (20) complexed with Aurora A was solved and iterated a similar binding mode to the hinge region. To resolve a twisted conformation of the phenyl plane in regard to pyrazole-benzimidazole portion of the molecule, a fully reduced cyclohexyl and difluorophenyl groups were also introduced (compound (20a) and (20b), respectively). Adsorption, disposition, metabolism, and excretion (ADME) considerations lead to proposing cyclopropyl derivative (21). As an alternative to introducing additional heterocyclic moiety, aiming at reducing the lipophilicity of (20a) for improving the ADME, the size of cyclohexyl ring was reduced to cyclopropyl analogue resulting in compound (21) that exhibits high enzyme and cellular potency still with reduced both the molecular weight (MW) and lipophilicity (logD7.4 = 2.1, MW = 381). Compound (21) demonstrated potent inhibition of HCT116 colony formation (IC50 = 12 nM), a clean CYP450 profile (IC50 > 10 μM for CYP3A4, 2D6, 1A2, 2C9, 2C19), acceptable mouse plasma protein binding (81.5%), and good thermodynamic solubility

Later, AT9283 (21) was shown to bind and potently inhibit a number of kinases including the Aurora kinases A and B (serine–threonine kinases that are known to play essential roles in mitotic checkpoint control during mitosis at IC50 ~ 3 nM),

In developing a selective potent aurora kinase inhibitor by employing fragmentbased discovery method, the pyrazol-4-yl urea benzimidazole derivative (AT9283, 21) was identified as a multitargeting kinase inhibitor. The pyrazolebenzimidazolebased clinical candidate (21) was optimized by Steven Howard and his colleagues following efficient structure-guided fragment to hit IC50 as low as 3 nM activity as a dual potent inhibitor toward Aurora A/Aurora B [125]. AT9283 was identified starting from the pyrazole-benzimidazole fragment (16) that was previously identified during the endeavor of developing cyclin-dependent kinase (CDK) inhibitors. Subsequent structure-based approach using CDK2 crystallographic structure led to the identification of the benzamide analogue (18). Throughout the process of developing CDK2 inhibitors, pyrazole-benzimidazole derivative was identified to act with high potency toward Aurora A. Starting with fragment, (18) demonstrated superior ligand efficiency (LE = 0.59) for Aurora A compared to CDK1 and CDK2 and also sufficient potency to allow detection in a conventional enzyme bioassay [125]. Aiming at optimizing, the "hit" (18) on the way to end up with a lead SAR is performed on the benzamide analogues. The team was aided by polyploid phenotype in HCT116 cells, as a functional assay that differentiates for Aurora A and Aurora B inhibition, combined with potency when screening for further analogues. Guided by the hypothesis that introducing a basic motif into fragment (18) will improve the potency of the compound, modifications were introduced successfully to 5- or 6-position of the benzimidazole without causing any clashes with the protein. In a further step, the morpholinomethyl motif was functionalized at position 5. Details grasped from the X-ray crystal structure of (19) complexed with Aurora A revealed that the pyrazolebenzimidazole motif is positioned in an excellent complementarity with the narrow region of the ATP pocket. A result directed the steps to follow in the design of the optimized structure (**Figure 6**). While retaining the 5-morpholinomethyl on the pyrazolebenzimidazole motif, the benzamide portion was subjected to modifications. Keeping in mind the need to keep the molecular weight while introducing increased flexibility on the glycine region, the amide was converted to urea (20). This strategy was fruitful when comprehending that the urea analogue (20) exhibited reduced plasma protein

*2.2.1 Discovery of AT9283a*

**114**

*(a) Main steps in the identification and discovery of pyrazolebenzimidazole-based multitargeting agent AT9283 (21) using fragment-based identification starting from fragment (16). (b) The structure N-cyclopropyl-N'-[3-[5-(4-morpholinylmethyl)-1H-benzimidazol-2-yl]-1H-pyrazol-4-yl] urea [AT9283 (21a)]. The "folded conformation" of (21b). Dotted lines to indicate the hydrophobic interaction between the cyclopropyl and benzimidazole motifs [125].*

Janus kinase 2 (JAK2) and JAK3 (1.2 and 1.1 nM, respectively), breakpoint cluster region-Abelson (BCR-Abl) T315I (4 nM), and mitogen-activated protein kinase kinase kinase 2 (MEKK2) with IC50 values of lower nanomolar (4.7–18 nM). This set of known kinases is known to play key roles in mitotic progress in cell cycle, induction of proliferation, evasion of apoptosis and tumor growth and thus considered vital targets to chemotherapeutic agents (see **Table 1**). Therefore, AT9283 (21) is defined as multikinase (multitargeting) inhibitor [126].

AT-9283 inhibits effective proliferation of cancer cells both in vitro and in vivo with and its effect is enhanced by with other agents (see **Table 2**) [127]. Henceforth T9283 proceeded to clinical trials including in children with relapsed or refractory acute leukemia, imatinib-resistant BCR-Abl-positive leukemic cells, and patients with relapsed or refractory multiple myeloma. Accumulative results indicate a need for optimizing the pharmacological profile on the way to overcome faced challenges in clinical application of the compound [127, 128].

The activity in imatinib-resistant BCR-Abl chronic myelogeneous leukemia (CML) explained based on modeling which reiterated the assumption that AT-9283 is bound to the kinase domain in the "folded conformation" which allows the needed interactions with the hinge region without a clash between the cyclopropyl group and the isoleucine residue in the T315I mutant. The results obtained in

#### *Chemistry and Applications of Benzimidazole and its Derivatives*


#### **Table 1.**

*The inhibitory concentration 50% (IC50 of the "lead" (21)) [126].*


#### **Table 2.**

*IC50s are the mean of two or more independent determinations.*

refractory CML suggest that AT-9283 can be efficient in Ph + acute lymphoblastic leukemia (Ph + ALL). It is the distinct binding mode that allows AT-9283 in similar manner to MK-0457 and PHA-739358 to exhibit potent activity against imatinibresistant T315I mutant [127, 129].

#### *2.2.2 Binding mode of AT-9283 (21) to kinases*

Currently, there exist 11 X-ray resolved crystallographic structures of AT-9283 complexed with target proteins that are documented at the Protein Data Bank. They include aurora A, aurora B, mutant of aurora B, JAK2, and protein kinase A mutants as surrogate model for Aurora B. A closer look clarifies that in a similar manner to the binding of dovitinib, the benzene portion in benzimidazole fragment is pointing in an orientation toward the solvents' exposed opening of the binding site. The pyrazole and urea fragments took part in multiple HBA and HBD interactions with the hinge region of the enzyme. The morpholine basic amine is oriented toward the solvent and enhanced significantly the solubility of the compound in physiological pH.

The crystal structure of compound (21) complexed with Aurora A is shown in **Figure 7** [130]. The molecule is positioned at the ATP-binding site of the kinase. It is revealed the urea linker adopts a *cis*/*trans* configuration that results in the molecule having a "folded conformation." This same conformation was also observed in the

**117**

**Figure 7.**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

crystal structure of (21B) alone (**Figure 8**) and in DMSO. Such "folded conformation" was confirmed by NMR measurement. An NOE was observed between H3b/ H3b′ of the cyclopropyl ring and the H4 and H7 protons of the benzimidazole ring. This "folded conformation" was explained by the occurrence of additional stabiliza-

*(a) Cartoon representation of the crystallographic structure of complex pyrazol-4-yl urea AT9283 (21) complexed with JAK2 JH2 (PDB 5UT0); (b) the surface representation, with main interactions between (21) and the kinase domain, colored by atom type (red, oxygen; blue, nitrogen; yellow, sulfur; gray, carbon/ hydrogen). The donor-acceptor-donor motif is shown to interact with the hinge region, while morpholine* 

The crystallographic structures of complexes both dovitinib-FGFR-1 and AT-9283 –Aurora A, revealed that there is a co-planarity between the benzimidazole and the quinolin-2-one of dovitinib, and pyrazole motif in AT-9283. A tautomeric rearrangement of the double bond induces a restriction on the rotation around the connection between the two fragments in each case (see **Figure 8**). This indicate the

Recently AT-9283 was phase I/phase II trial of AT9283, a selective inhibitor of Aurora kinase in children with relapsed or refractory acute leukemia: challenges to

Employing virtual screening methods of PubChem database as a first step, selected support vector machine (SVM) virtual hits were evaluated by Lipinski's

tion due to a hydrophobic interaction between these two groups.

run early phase clinical trials for children with leukemia [131–137].

**2.3 α-Haloacetamidebenzimidazole derivatives as multitargeting agents**

favorite binding to the less rotatable conformer (21B).

*substituent on C5 points toward the solution [130].*

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

#### **Figure 7.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

refractory CML suggest that AT-9283 can be efficient in Ph + acute lymphoblastic leukemia (Ph + ALL). It is the distinct binding mode that allows AT-9283 in similar manner to MK-0457 and PHA-739358 to exhibit potent activity against imatinib-

**Inhibitory effect of AT9283 on tumor cell colony formation after 10–14 days treatment**

Abl(Q252H), DRAK1, FGFR1, FGFR1(V561 M), FGFR2(N549H), FGFR3, VEGFR1(Flt1),

Flt-3, PDGFR-α(D842V), PDK1, PKCμ, Rsk4, SRC(T341 M), VEGFR2

*The inhibitory concentration 50% (IC50 of the "lead" (21)) [126].*

**Origin Cell line IC50 (nM) p53 status Colon** HCT116 13 +

**Enzyme IC50 (nM)** Aurora A 52% I at

Aurora B 58% I at

JAK3 1.1 JAK2 1.2 Abl (T315I) 4 GSK3-β, FGFR2, VEGFR3 (Flt4), Mer, Ret, Rsk2, Rsk3, Tyk2, Yes 1–10

**Ovarian** A2780 7.7 + **Lung** A549 12 + **Breast** MCF7 20 + **Pancreatic** MIA-Pa-Ca-2 7.8 — *+ indicates expression of wild-type p53; − indicates no expression of p53 or that p53 is nonfunctional [126].*

HT-29 11 — SW620 14 —

3.0 nM

3.0 nM

10–30

Currently, there exist 11 X-ray resolved crystallographic structures of AT-9283 complexed with target proteins that are documented at the Protein Data Bank. They include aurora A, aurora B, mutant of aurora B, JAK2, and protein kinase A mutants as surrogate model for Aurora B. A closer look clarifies that in a similar manner to the binding of dovitinib, the benzene portion in benzimidazole fragment is pointing in an orientation toward the solvents' exposed opening of the binding site. The pyrazole and urea fragments took part in multiple HBA and HBD interactions with the hinge region of the enzyme. The morpholine basic amine is oriented toward the solvent and

The crystal structure of compound (21) complexed with Aurora A is shown in **Figure 7** [130]. The molecule is positioned at the ATP-binding site of the kinase. It is revealed the urea linker adopts a *cis*/*trans* configuration that results in the molecule having a "folded conformation." This same conformation was also observed in the

enhanced significantly the solubility of the compound in physiological pH.

**116**

**Table 2.**

**Table 1.**

resistant T315I mutant [127, 129].

*2.2.2 Binding mode of AT-9283 (21) to kinases*

*IC50s are the mean of two or more independent determinations.*

*(a) Cartoon representation of the crystallographic structure of complex pyrazol-4-yl urea AT9283 (21) complexed with JAK2 JH2 (PDB 5UT0); (b) the surface representation, with main interactions between (21) and the kinase domain, colored by atom type (red, oxygen; blue, nitrogen; yellow, sulfur; gray, carbon/ hydrogen). The donor-acceptor-donor motif is shown to interact with the hinge region, while morpholine substituent on C5 points toward the solution [130].*

crystal structure of (21B) alone (**Figure 8**) and in DMSO. Such "folded conformation" was confirmed by NMR measurement. An NOE was observed between H3b/ H3b′ of the cyclopropyl ring and the H4 and H7 protons of the benzimidazole ring. This "folded conformation" was explained by the occurrence of additional stabilization due to a hydrophobic interaction between these two groups.

The crystallographic structures of complexes both dovitinib-FGFR-1 and AT-9283 –Aurora A, revealed that there is a co-planarity between the benzimidazole and the quinolin-2-one of dovitinib, and pyrazole motif in AT-9283. A tautomeric rearrangement of the double bond induces a restriction on the rotation around the connection between the two fragments in each case (see **Figure 8**). This indicate the favorite binding to the less rotatable conformer (21B).

Recently AT-9283 was phase I/phase II trial of AT9283, a selective inhibitor of Aurora kinase in children with relapsed or refractory acute leukemia: challenges to run early phase clinical trials for children with leukemia [131–137].

#### **2.3 α-Haloacetamidebenzimidazole derivatives as multitargeting agents**

Employing virtual screening methods of PubChem database as a first step, selected support vector machine (SVM) virtual hits were evaluated by Lipinski's

**Figure 8.**

*Tautomeric rearrangement of multitarget inhibitors (1) and (21). (a) benzimidazole quinolin-2-one heterocyclic, and (b) benzimidazole pyrazole derivatives.*

rule of five. The compounds which passed Lipinski's rule of five were subject to further and more refined screening by using molecular docking. This sequential refinement led to the identification of 2-aryl benzimidazole group of derivatives as multitarget "EGFR, VEGFR-2, and PDGFR" inhibitors [138]. A mechanistic study reported by Jiang and colleagues displayed that (22) exhibited low to moderate micromolar IC50 against nine established breast cancer cell lines that are known to have variable expressing EGFR and HER2 (MDA-MB-468, BT-549, MDA-MB-231, HCC1937, T-47D, BT-474, MDA-MB-453, ZR-75-1, MCF-7, and MCF-10 A). Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, (24 and 25) exerts moderate inhibitory effect on growth of panel of breast cancer cell lines (IC50 values of 2–9 μM) and was reported to be more potent than lapatinib against MDA-MB-468, BT-549, MDA-MB-231, ZR-75- 1, and MCF-7. A correlation was observed between the level of HER2 and EGFR amplification and expression and the sensitivity toward (22). IC50 = 3.58 μM against BT-474 (high expression of HER2), whereas against MDA-MB-453 (lower levels of HER2 expression) IC50 = 4.91 μM. The activity against lower EGFR and HER2 expressing cell lines (ZR-75-1 and MCF-7), IC50 = 1.81–2.99 μM was explained by the assumption that (22) is able to act via other targets of EGFR and HER2 [139].

Docking the compounds into kinase domains revealed that (22) occupies the ATP-binding site of EGFR (PDB: 2J6M). The compound was able to form a hydrogen bond with amino acid MET 793 (N–H⋯O:2.485 Å), claimed to be an important binding site of EGFR. The difference in the activity between the two compounds against VEGFR2 was explained by the difference in hydrogen bonding using docking into VEGFR-2 kinase. It was shown that (22) formed two hydrogen bonds with amino acids CYS917 (N–H⋯Cl:2.484 Å) and ASP1044 (N–H⋯O:2.429 Å), whereas compound (23) formed only one hydrogen bond with ASP1044 (N–H⋯O:2.419 Å) [140].

The authors concluded that electron-withdrawing substituent residing at 2-aryl ring together with shorter aliphatic chain contributed to the cytotoxic potency and to the induction of apoptosis by such group of compounds in HepG-2 cell lines. Though reported as multitargeting agent, the activity of 2-chloro-N-(2-*p*-tolyl-1*H*-benzo[d]imidazol-5-yl)acetamide (22) exhibiting most potency could not be explained explicitly by docking alone. (22) encompasses a reactive alphahaloacetamide (see **Figure 9**) that is vulnerable to nucleophilic substitution by biological

**119**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

nucleophiles like thiols (-SH). Thus, a study to explore the formation of irreversible adducts with cellular proteins like kinases is recommended and hoped to uncover

*α-Haloacetamidebenzimidazole derivatives as multitargeting agents. 2-chloro-N-(2-p-tolyl-1H-benzo[d] imidazol-5-yl)acetamide (21), a novel 2-arylbenzimidazole derivative exhibited remarkable activity toward breast cancer. In a study reported by Jiang et al. (22 and 23) were virtually identified as multikinase EGFR and VEGFR inhibitor while (22) was identified as EGFR inhibitor and (23) as PDGFR inhibitor [140, 142].*

Anilinopyrimidines (**Figure 10**) displays a wide range of bioactivities. Asymmetric 2-anilinopyrimidines bearing 3-aminopropamides exhibit activity against epidermal growth factor receptor EGFR) [141]. 2-anilinopyrimidine derivatives bearing 4-piperidino substituents exhibited improved and selective activity against triple-negative breast cancer cell line MDA-MB-468 believed to be due to EGFR inhibition. Decorating the pyrimidine nucleus with different substituents at position 4 endowed the final derivatives (4-substituted-2-anilinopyrimidine) with activity as well as selective toward corticotropin-releasing factor (CRF) antagonists [142]. Having the anilino fragment at 2- together with thiazolyl at 4- of the pyrimidine core was reported to exert antagonistic effect of cyclin-dependent kinase-2 (CKD2) [143], and improved

**2.4 2-Anilino-4-(benzimidazol-2-yl)pyrimidines: a multikinase inhibitor** 

Bis-anilinopyrimidine was reported as potent and selective PAK1 inhibitor and as highly selective group I p21-activated kinase (PAK1) inhibitor [146]. Additionally, N-phenyl-N′-[4-(pyrimidin-4-ylamino)phenyl] urea derivatives (see (27) at **Figure 10**) exhibit selective inhibition to class III receptor tyrosine kinase subfamily [147]. Other symmetric 4,6-dianilinopyrimidines induce selective EGFR

Notably, introducing the benzimidazolyl moiety at position 4 of the 2 anilinopyrimidine core to produce 2-anilino-4-(benzimidazol-2-yl)-pyrimidines renders such group of compounds' activity against a wider range of kinases (see

of compounds that are based on the 2-anilino-4-(benzimidazol-2-yl)-pyrimidine scaffold (**Figure 10**, (30)) [142]. The most potent derivative exhibited antiproliferative activity for several cancer cell lines of the NCI panel in submicromolar concentrations. SAR study was concluded in indicating a basic correlation with the anilinopyrimidine fragment and the substitution pattern at the aniline moiety. It is worth mentioning that 2-anilinopyrimidine fragment (**Figure 10**, (30)) is found in a range of kinase inhibitors.

Renate Determann et al. reported the synthesis and in vitro activity of a small library

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

the principal mechanism of its wide action.

inhibitory activity toward CDK9 and (CDK2) [143–145].

**scaffold**

**Figure 9.**

inhibitions [148].

**Figure 10**).

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

**Figure 9.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

*heterocyclic, and (b) benzimidazole pyrazole derivatives.*

rule of five. The compounds which passed Lipinski's rule of five were subject to further and more refined screening by using molecular docking. This sequential refinement led to the identification of 2-aryl benzimidazole group of derivatives as multitarget "EGFR, VEGFR-2, and PDGFR" inhibitors [138]. A mechanistic study reported by Jiang and colleagues displayed that (22) exhibited low to moderate micromolar IC50 against nine established breast cancer cell lines that are known to have variable expressing EGFR and HER2 (MDA-MB-468, BT-549, MDA-MB-231, HCC1937, T-47D, BT-474, MDA-MB-453, ZR-75-1, MCF-7, and MCF-10 A). Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, (24 and 25) exerts moderate inhibitory effect on growth of panel of breast cancer cell lines (IC50 values of 2–9 μM) and was reported to be more potent than lapatinib against MDA-MB-468, BT-549, MDA-MB-231, ZR-75- 1, and MCF-7. A correlation was observed between the level of HER2 and EGFR amplification and expression and the sensitivity toward (22). IC50 = 3.58 μM against BT-474 (high expression of HER2), whereas against MDA-MB-453 (lower levels of HER2 expression) IC50 = 4.91 μM. The activity against lower EGFR and HER2 expressing cell lines (ZR-75-1 and MCF-7), IC50 = 1.81–2.99 μM was explained by the assumption that (22) is able to act via other targets of EGFR and

*Tautomeric rearrangement of multitarget inhibitors (1) and (21). (a) benzimidazole quinolin-2-one* 

Docking the compounds into kinase domains revealed that (22) occupies the ATP-binding site of EGFR (PDB: 2J6M). The compound was able to form a hydrogen bond with amino acid MET 793 (N–H⋯O:2.485 Å), claimed to be an important binding site of EGFR. The difference in the activity between the two compounds against VEGFR2 was explained by the difference in hydrogen bonding using docking into VEGFR-2 kinase. It was shown that (22) formed two hydrogen bonds with amino acids CYS917 (N–H⋯Cl:2.484 Å) and ASP1044 (N–H⋯O:2.429 Å), whereas compound (23) formed only one hydrogen bond with

The authors concluded that electron-withdrawing substituent residing at 2-aryl ring together with shorter aliphatic chain contributed to the cytotoxic potency and to the induction of apoptosis by such group of compounds in HepG-2 cell lines. Though reported as multitargeting agent, the activity of 2-chloro-N-(2-*p*-tolyl-1*H*-benzo[d]imidazol-5-yl)acetamide (22) exhibiting most potency could not be explained explicitly by docking alone. (22) encompasses a reactive alphahaloacetamide (see **Figure 9**) that is vulnerable to nucleophilic substitution by biological

**118**

HER2 [139].

**Figure 8.**

ASP1044 (N–H⋯O:2.419 Å) [140].

*α-Haloacetamidebenzimidazole derivatives as multitargeting agents. 2-chloro-N-(2-p-tolyl-1H-benzo[d] imidazol-5-yl)acetamide (21), a novel 2-arylbenzimidazole derivative exhibited remarkable activity toward breast cancer. In a study reported by Jiang et al. (22 and 23) were virtually identified as multikinase EGFR and VEGFR inhibitor while (22) was identified as EGFR inhibitor and (23) as PDGFR inhibitor [140, 142].*

nucleophiles like thiols (-SH). Thus, a study to explore the formation of irreversible adducts with cellular proteins like kinases is recommended and hoped to uncover the principal mechanism of its wide action.

#### **2.4 2-Anilino-4-(benzimidazol-2-yl)pyrimidines: a multikinase inhibitor scaffold**

Anilinopyrimidines (**Figure 10**) displays a wide range of bioactivities. Asymmetric 2-anilinopyrimidines bearing 3-aminopropamides exhibit activity against epidermal growth factor receptor EGFR) [141]. 2-anilinopyrimidine derivatives bearing 4-piperidino substituents exhibited improved and selective activity against triple-negative breast cancer cell line MDA-MB-468 believed to be due to EGFR inhibition. Decorating the pyrimidine nucleus with different substituents at position 4 endowed the final derivatives (4-substituted-2-anilinopyrimidine) with activity as well as selective toward corticotropin-releasing factor (CRF) antagonists [142]. Having the anilino fragment at 2- together with thiazolyl at 4- of the pyrimidine core was reported to exert antagonistic effect of cyclin-dependent kinase-2 (CKD2) [143], and improved inhibitory activity toward CDK9 and (CDK2) [143–145].

Bis-anilinopyrimidine was reported as potent and selective PAK1 inhibitor and as highly selective group I p21-activated kinase (PAK1) inhibitor [146]. Additionally, N-phenyl-N′-[4-(pyrimidin-4-ylamino)phenyl] urea derivatives (see (27) at **Figure 10**) exhibit selective inhibition to class III receptor tyrosine kinase subfamily [147]. Other symmetric 4,6-dianilinopyrimidines induce selective EGFR inhibitions [148].

Notably, introducing the benzimidazolyl moiety at position 4 of the 2 anilinopyrimidine core to produce 2-anilino-4-(benzimidazol-2-yl)-pyrimidines renders such group of compounds' activity against a wider range of kinases (see **Figure 10**).

Renate Determann et al. reported the synthesis and in vitro activity of a small library of compounds that are based on the 2-anilino-4-(benzimidazol-2-yl)-pyrimidine scaffold (**Figure 10**, (30)) [142]. The most potent derivative exhibited antiproliferative activity for several cancer cell lines of the NCI panel in submicromolar concentrations. SAR study was concluded in indicating a basic correlation with the anilinopyrimidine fragment and the substitution pattern at the aniline moiety. It is worth mentioning that 2-anilinopyrimidine fragment (**Figure 10**, (30)) is found in a range of kinase inhibitors.

#### **Figure 10.**

*Development of multitargeting 2-anilino-4-(benzimidazol-2-yl)-pyrimidine scaffold (30) starting from hinge binding compound 1,3-dimethyl-7-(1-methyl-1H-benzimidazol-2-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (31). 2-anilino-4-(benzimidazol-2-yl)-pyrimidine derivatives 2-methoxy-5- {[4-(1-methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]amino}phenol (33) most potent compound and 2-anilino-4-(benzimidazol-2-yl)-pyrimidine derivatives 2-hydroxy-5-{[4-(1-methyl-1H-benzimidazol-2-yl) pyrimidin-2-yl]amino}phenol (32))), 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamine (35), and 2-anilino-4-(thiazol-5-yl)pyrimidine (29).*

Based on high-throughput screening method radiometric protein kinase assay (33PanQinase® Activity Assay) [149], 11 recombinant cancer-related protein kinases (AKT1, ARK5, Aurora B, AXL, FAK, IGF1-R, MET, PLK1, PRK1, SRC, VEGF-R2) were screened by a library of compounds. Interestingly, four kinases (Aurora B, FAK, PLK1, and VEGF-R2) proved to be of particular sensitivity to the tested compounds (**Table 3**). This group of four kinases is involved in oncogenesis and maintenance of vital processes of cancer. Thus it is believed that their concerted inhibition could be useful in the treatment of various malignancies. It is worth noting the infectivity of most of tested compounds, including the active ones against AKT1 (shown in **Table 3**).

#### *2.4.1 2-Anilino-4-(benzimidazol-2-yl)pyrimidine-target interactions*

Though the authors did not report a prudent SAR, however, docking compound (33) to ATP-binding pocket of PLK1 (PDB 2OWB) helped rationalize the initial observations [142]. One main reflection highlighted the positioning of the anilinopyrimidine fragment in the hinge region, forming a pair of hydrogen bonds to Cys133. Methoxy (CH3O-) group at the position 2 of the aniline moiety forms a

**121**

**Table 3.**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

**AKT1 Aurora** 

**B**

**FAK PLK1 VEGF-R2**

>100 7 ± 2.3 10.4 ± 2.7 6.0 ± 0.1 7.5 ± 2.0

>100 6.0 ± 0.2 3.4 ± 0.8 1.2 ± 0.2 7.2 ± 0.3

>100 >100 92 >100 85

hydrogen bonding with the guanidine of Arg136 residing at the opening of the PLK1 ATP-binding pocket. The inactivity of derivatives with substituents bulkier than methoxy group (CH3O-) was explained partially by the clash with Leu59 and Arg136

Sorafenib >10 1.8 >10 >10 0.022 Sunitinib >10 1.5 1.6 >10 0.070

Silvana Raić-Malića and colleagues reported the synthesis of a group of benzimidazole amidine derivatives [150]. Specifically, compound (**Figure 11**, (36)) abrogated the activity of several protein enzymes including tissue transglutaminase (TGM2) and kinases like CDK9, sphingosine kinase 1(SK1), and p38 mitogenactivated protein kinase (p38 MAPK), whereas compound (37) did not have profound effect on CDK9 and TGM2 but showed moderate downregulation of SK1

A small library comprising 27 compounds was screened for the potency. Two of them, *p*-chlorophenyl-substituted 1,2,3-triazolyl derivatives of amidine *N*-isopropyl amidine (36) and imidazoline amidine (37), exhibited remarkable antiproliferative activities with IC50 of 0.05 and 0.06 μM in non-small cell lung

In their endeavor to look for potent inhibitors for treatment of non-small cell lung cancer, Silvana Raić-Malić and her team developed a group of benzimidazole amidine derivative that showed an inhibitory effect on several key players in cancer

at the pocket entrance indicating limited tolerance to variation at this region.

*Protein kinase inhibition by (32 and 33) compared pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (39) to* 

**2.5 Benzimidazolylamidines as multitargeting agents**

*standard multitargeting FDA-approved agents sorafenib and sunitinib.*

*Compound (33) exhibited activities that range between IC50 = 1.2 and 7.2 μM [142].*

cancer cells A54 and was defined as multitarget inhibitors.

and significant reduction in p38 MAPK.

*DOI: http://dx.doi.org/10.5772/intechopen.86249*


#### *Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

#### **Table 3.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

Based on high-throughput screening method radiometric protein kinase assay (33PanQinase® Activity Assay) [149], 11 recombinant cancer-related protein kinases (AKT1, ARK5, Aurora B, AXL, FAK, IGF1-R, MET, PLK1, PRK1, SRC, VEGF-R2) were screened by a library of compounds. Interestingly, four kinases (Aurora B, FAK, PLK1, and VEGF-R2) proved to be of particular sensitivity to the tested compounds (**Table 3**). This group of four kinases is involved in oncogenesis and maintenance of vital processes of cancer. Thus it is believed that their concerted inhibition could be useful in the treatment of various malignancies. It is worth noting the infectivity of most of tested compounds, including the active ones against AKT1 (shown in **Table 3**).

*Development of multitargeting 2-anilino-4-(benzimidazol-2-yl)-pyrimidine scaffold (30) starting from hinge binding compound 1,3-dimethyl-7-(1-methyl-1H-benzimidazol-2-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (31). 2-anilino-4-(benzimidazol-2-yl)-pyrimidine derivatives 2-methoxy-5- {[4-(1-methyl-1H-benzimidazol-2-yl)pyrimidin-2-yl]amino}phenol (33) most potent compound and 2-anilino-4-(benzimidazol-2-yl)-pyrimidine derivatives 2-hydroxy-5-{[4-(1-methyl-1H-benzimidazol-2-yl) pyrimidin-2-yl]amino}phenol (32))), 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamine (35), and 2-anilino-*

Though the authors did not report a prudent SAR, however, docking compound (33) to ATP-binding pocket of PLK1 (PDB 2OWB) helped rationalize the initial observations [142]. One main reflection highlighted the positioning of the anilinopyrimidine fragment in the hinge region, forming a pair of hydrogen bonds to Cys133. Methoxy (CH3O-) group at the position 2 of the aniline moiety forms a

*2.4.1 2-Anilino-4-(benzimidazol-2-yl)pyrimidine-target interactions*

**120**

**Figure 10.**

*4-(thiazol-5-yl)pyrimidine (29).*

*Protein kinase inhibition by (32 and 33) compared pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (39) to standard multitargeting FDA-approved agents sorafenib and sunitinib.*

hydrogen bonding with the guanidine of Arg136 residing at the opening of the PLK1 ATP-binding pocket. The inactivity of derivatives with substituents bulkier than methoxy group (CH3O-) was explained partially by the clash with Leu59 and Arg136 at the pocket entrance indicating limited tolerance to variation at this region.

#### **2.5 Benzimidazolylamidines as multitargeting agents**

Silvana Raić-Malića and colleagues reported the synthesis of a group of benzimidazole amidine derivatives [150]. Specifically, compound (**Figure 11**, (36)) abrogated the activity of several protein enzymes including tissue transglutaminase (TGM2) and kinases like CDK9, sphingosine kinase 1(SK1), and p38 mitogenactivated protein kinase (p38 MAPK), whereas compound (37) did not have profound effect on CDK9 and TGM2 but showed moderate downregulation of SK1 and significant reduction in p38 MAPK.

A small library comprising 27 compounds was screened for the potency. Two of them, *p*-chlorophenyl-substituted 1,2,3-triazolyl derivatives of amidine *N*-isopropyl amidine (36) and imidazoline amidine (37), exhibited remarkable antiproliferative activities with IC50 of 0.05 and 0.06 μM in non-small cell lung cancer cells A54 and was defined as multitarget inhibitors.

In their endeavor to look for potent inhibitors for treatment of non-small cell lung cancer, Silvana Raić-Malić and her team developed a group of benzimidazole amidine derivative that showed an inhibitory effect on several key players in cancer

#### **Figure 11.**

*(a) Hit compounds prepared and screened for multitarget action 2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole hydrochloride (36),2-(4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-N-isopropyl-1H-benzo[d]imidazole-6-carboximidamide (37); (b) summary of structure–activity relationship of benzimidazolylamidines [150].*

proliferation [150]. A recent study reported that synthesis of amidino 2-substituted benzimidazoles linked to 1,4-disubstituted 1,2,3-triazoles by applying microwave and ultrasound irradiation in click reaction and subsequent condensation of thus obtained 4-(1,2,3-triazol-1-yl)benzaldehyde with *o*-phenylenediamines. The study concluded the improved cytotoxic effect (within the nanomolar range; IC50 of 50 and 60 nM) against hepatocellular carcinoma cells. A follow-up study affirms the conclusion that when benzimidazole is conjugated to 1,2,3-triazole moiety, the hybrid exerts potent and selective antiproliferative effect against a panel of cell lines [non-small cell lung cancer (A549), ductal pancreatic adenocarcinoma (CFPAC-1), cervical carcinoma (HeLa), and metastatic colorectal adenocarcinoma (SW620) as well as on normal human lung fibroblasts (WI38)] with 5-fluorouracil (5FU) as a positive control. Two hits (36) and (37) (**Figure 11a**) demonstrated a potent activity at nM range (IC50 of 50 and 60 nM) against non-small cell lung cancer (A549). Interestingly, benzyl-substituted 1,2,3-triazolyl analogue of imidazoline (36) exhibited a remarkable and selective activity (IC50 = 0.07 μM) on A549 cell line. A mechanistic study performed on A549 cell line using Western blotting reinforced the belief that nature of aromatic substituent of 1-(1,2,3-triazolyl) and amidino moiety at C-5 position of benzimidazole ring is critical to the cytostatic activity of this group of compounds. In silico analysis supported the conception that (36) is bound slightly better than (37) to ATP-binding site of p38 MAPK, which correlates with observed decrement in the expression level of phospho-p38 MAPK displayed by (36). The importance of triazole was referred to its ability to form one H-bond with Met109 in the hinge region. Aminobenzimidazole group forms a number of HB with polar amino acids Glu71, Hid148, and Asp168 in the linker region. Phenyl moieties found on the hybrid both are placed in the hydrophobic environment. The phenyl connected to the triazole is assumed to participate in a π-π stacking with Phe169 (see **Figure 11**). The study reported (36) as a multitarget inhibitor since it abrogated the activity of several protein kinases including TGM2, CDK9, SK1, and p38 MAPK.

**123**

ing agent.

data available from public repositories.

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

According to the definition of Richard Morphy, the multitarget drugs are defined as "compounds that are designed to modulate multiple targets of relevance to a disease, with the overall goal of enhancing efficacy and/or improving safety"

Modulating the function of numerous biological molecules is a wellestablished pharmacological approach in medicine practice. Paracetamol, a traditional therapeutic used worldwide, is believed to induce its effects via action on multiple targets. Several psychoactive, serotonergic, cholinergic, and adrenergic agonists or antagonists exercise their actions on a wider range of singular

Apart from the alphachloroacetamidobenimidzoles (22), the groups of compounds reported so far in the literature as multitarget agents act in most cases on receptor tyrosine kinases (RTKs) as competitive ATP inhibitors. Those by virtue occupy the vicinity of ATP with the heteroaromatic system interactively buried in the purine portion pocket and interact with the hinge region of the kinase domain. The thiol (-SH)- π and the stacking π-π together with the hydrophobic interaction with the floor and the ceiling of the purine-binding regions are believed to do the required binding adjustment as kinase inhibitors. Crystallographic structure of dovitinib human FGFR1 revealed the occupancy of the purine-binding regions (part of the ATP-binding site) with the quinolinonebenzimidazole fragment, while the N-methylpiperazine attached to C5' at the phenyl part of the benzimidazole is pointing toward the opening and is exposed to the solution. Thus, it seems that benzimidazole portion is not interacting directly with the hinge region of the enzyme. Similar binding is noticed with AT9382. The pyrazolylbenzimidazole and the benzamide motif take part in HBD-

In the case of 2-anilino-4-(benzimidazole-2-yl)pyrimidine, the benzimidazole portion looks immersed deep in the purine-binding regions of the ATP-binding site participating in direct interactions via hydrogen bonding and hydrophobic interactions, while

the hydroxymethoxyaniline portion points towards the solvent exposed area.

Despite the imbedded potential, the multitarget activity of the reported benzimidazole-based scaffolds was identified serendipitously. In other words, none of the benzimidazole anticancer multitargeting agents seem to be identified in unforeseen manner, and in many ways they emerge with no intention to be designed initially. While adhering to the development of selective and specific agents, results accumulated afterword revealed multitarget action. For example, 3-benzimidazol-2-ylhydroquinolin-2-one scaffold [benzimidazolylquinolinone (**Figure 4**, (12)] was identified using high-throughput screening (HTS). AT9283 (**Figure 6**, (21)) was identified following fragment-based structural approach with the initial aim to develop an Aurora selective inhibitor, and later it was reported to act as multitarget-

It is hoped that the identification, discovery, and optimization of benzimidazolebased multitargeting anticancer agent will benefit from the "big data era" fueled by

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

**3.1 Multitargeting and polypharmacology**

HBA bridging with the hinge of the kinase domain.

(Morphy, Rankovic, 2005) [151].

biomolecular target.

**3.2 Lessons learnt**

*3.2.1 Discovery methods*

**3. Conclusion**

#### **3. Conclusion**

*Chemistry and Applications of Benzimidazole and its Derivatives*

proliferation [150]. A recent study reported that synthesis of amidino 2-substituted benzimidazoles linked to 1,4-disubstituted 1,2,3-triazoles by applying microwave and ultrasound irradiation in click reaction and subsequent condensation of thus obtained 4-(1,2,3-triazol-1-yl)benzaldehyde with *o*-phenylenediamines. The study concluded the improved cytotoxic effect (within the nanomolar range; IC50 of 50 and 60 nM) against hepatocellular carcinoma cells. A follow-up study affirms the conclusion that when benzimidazole is conjugated to 1,2,3-triazole moiety, the hybrid exerts potent and selective antiproliferative effect against a panel of cell lines [non-small cell lung cancer (A549), ductal pancreatic adenocarcinoma (CFPAC-1), cervical carcinoma (HeLa), and metastatic colorectal adenocarcinoma (SW620) as well as on normal human lung fibroblasts (WI38)] with 5-fluorouracil (5FU) as a positive control. Two hits (36) and (37) (**Figure 11a**) demonstrated a potent activity at nM range (IC50 of 50 and 60 nM) against non-small cell lung cancer (A549). Interestingly, benzyl-substituted 1,2,3-triazolyl analogue of imidazoline (36) exhibited a remarkable and selective activity (IC50 = 0.07 μM) on A549 cell line. A mechanistic study performed on A549 cell line using Western blotting reinforced the belief that nature of aromatic substituent of 1-(1,2,3-triazolyl) and amidino moiety at C-5 position of benzimidazole ring is critical to the cytostatic activity of this group of compounds. In silico analysis supported the conception that (36) is bound slightly better than (37) to ATP-binding site of p38 MAPK, which correlates with observed decrement in the expression level of phospho-p38 MAPK displayed by (36). The importance of triazole was referred to its ability to form one H-bond with Met109 in the hinge region. Aminobenzimidazole group forms a number of HB with polar amino acids Glu71, Hid148, and Asp168 in the linker region. Phenyl moieties found on the hybrid both are placed in the hydrophobic environment. The phenyl connected to the triazole is assumed to participate in a π-π stacking with Phe169 (see **Figure 11**). The study reported (36) as a multitarget inhibitor since it abrogated the activity of several protein kinases including TGM2, CDK9, SK1, and

*(36),2-(4-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-N-isopropyl-1H-benzo[d]imidazole-6-carboximidamide (37); (b) summary of structure–activity relationship of benzimidazolylamidines [150].*

*(a) Hit compounds prepared and screened for multitarget action 2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-(4,5-dihydro-1H-imidazol-2-yl)-1H-benzo[d]imidazole hydrochloride* 

**122**

p38 MAPK.

**Figure 11.**

#### **3.1 Multitargeting and polypharmacology**

According to the definition of Richard Morphy, the multitarget drugs are defined as "compounds that are designed to modulate multiple targets of relevance to a disease, with the overall goal of enhancing efficacy and/or improving safety" (Morphy, Rankovic, 2005) [151].

Modulating the function of numerous biological molecules is a wellestablished pharmacological approach in medicine practice. Paracetamol, a traditional therapeutic used worldwide, is believed to induce its effects via action on multiple targets. Several psychoactive, serotonergic, cholinergic, and adrenergic agonists or antagonists exercise their actions on a wider range of singular biomolecular target.

Apart from the alphachloroacetamidobenimidzoles (22), the groups of compounds reported so far in the literature as multitarget agents act in most cases on receptor tyrosine kinases (RTKs) as competitive ATP inhibitors. Those by virtue occupy the vicinity of ATP with the heteroaromatic system interactively buried in the purine portion pocket and interact with the hinge region of the kinase domain. The thiol (-SH)- π and the stacking π-π together with the hydrophobic interaction with the floor and the ceiling of the purine-binding regions are believed to do the required binding adjustment as kinase inhibitors. Crystallographic structure of dovitinib human FGFR1 revealed the occupancy of the purine-binding regions (part of the ATP-binding site) with the quinolinonebenzimidazole fragment, while the N-methylpiperazine attached to C5' at the phenyl part of the benzimidazole is pointing toward the opening and is exposed to the solution. Thus, it seems that benzimidazole portion is not interacting directly with the hinge region of the enzyme. Similar binding is noticed with AT9382. The pyrazolylbenzimidazole and the benzamide motif take part in HBD-HBA bridging with the hinge of the kinase domain.

In the case of 2-anilino-4-(benzimidazole-2-yl)pyrimidine, the benzimidazole portion looks immersed deep in the purine-binding regions of the ATP-binding site participating in direct interactions via hydrogen bonding and hydrophobic interactions, while the hydroxymethoxyaniline portion points towards the solvent exposed area.

#### **3.2 Lessons learnt**

#### *3.2.1 Discovery methods*

Despite the imbedded potential, the multitarget activity of the reported benzimidazole-based scaffolds was identified serendipitously. In other words, none of the benzimidazole anticancer multitargeting agents seem to be identified in unforeseen manner, and in many ways they emerge with no intention to be designed initially. While adhering to the development of selective and specific agents, results accumulated afterword revealed multitarget action. For example, 3-benzimidazol-2-ylhydroquinolin-2-one scaffold [benzimidazolylquinolinone (**Figure 4**, (12)] was identified using high-throughput screening (HTS). AT9283 (**Figure 6**, (21)) was identified following fragment-based structural approach with the initial aim to develop an Aurora selective inhibitor, and later it was reported to act as multitargeting agent.

It is hoped that the identification, discovery, and optimization of benzimidazolebased multitargeting anticancer agent will benefit from the "big data era" fueled by data available from public repositories.

#### *3.2.2 Shift in the paradigm*

Multitargeting can occur via three possible ways: acting on the same target, on different targets of the same pathway, or on different targets of different pathways. So far the benzimidazole derivatives that have been explored are reported to act as the third category "acting on different targets of different pathways." The focus has been so far on the kinome-relevant signaling key player with dovitinib widening the landscape to non-kinase targets. Broadening "multitargeting" concept to identify novel inhibitors with potency against key targets outside the human kinome necessitates treating complex diseases using "polypharmacology" gains special interest in resistant mutated spreadable cancers [151].

Despite the initial enthusiasm for the efficacy of molecular targeted therapeutics following the approval of imatinib, a small tyrosine kinase inhibitor targeting BCR-Abl, in chronic myeloid leukemia (CML) and trastuzumab, a monoclonal antibody against HER2, for treatment of metastatic breast cancer, scientists and clinicians were challenged by recurrent relapse due to cancer patients who developed drug resistance. In the case of RTKi, resistance can emerge as a result of selection for mutant sin in the target that renders the binding site inaccessible, reduced influx accompanied by enhance efflux, shift in metabolism and excretion of the drug, and the activation of alternative signaling pathways. Thus, the rationale for targeting drugs is shifting. In the last two decades, the main effort was aimed at developing highly specific inhibitors acting on single target. Now, there is a general agreement that molecules interfering simultaneously with multiple RTKs might be more effective than single-target agents. With the recent approval by the FDA of sorafenib, regorafenib, sunitinib, lenvatinib, and axitinib-targeting VEGFR, PDGFR, FLT-3, and c-KIT—more attention is drawn to broad-spectrum anticancer properties multikinase targeting drugs. Thus it is anticipated that more multitargeting agents will be getting into clinical trials and making their way to clinical application. It is hoped that identification, discovery, and optimization of benzimidazole-based multitargeting agents will benefit from the "big data era" fueled by the availability of big data, advances in technology, and artificial intelligence.

#### **Acknowledgements**

The author would like to acknowledge "Zamala" Program: University Fellowship Program in Palestinian Universities (http://www.taawon.org/ar/zamala) for the fund granted to make this research stay at London Regional Cancer Center, Western University Ontario, Canada, possible. Also, I would like to acknowledge my home institution Al-Quds University, Jerusalem-Palestine, for the support and encouragement. In addition, I wish to thank the editor and high value the editor's work, reading, and comments. The accomplishment of this chapter was made possible thanks to such support and help.

#### **Conflict of interest**

No conflict of interest exists.

#### **Notes/thanks/other declarations**

I would like to express my special thanks, gratitude and truthful appreciation to my dearest family: my wife Muna, my daughters Aseel and Layan and my sons

**125**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

Mulham and Majd for all the love, compassion, and support I received from them

This chapter is dedicated to the respectable memories of my mother Jaleelah and father Salem who died of old age and to the reminiscence of my dearest brother

Mohammed who left this world due to leukemia. Peace Be Upon Them All.

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

ADME adsorption, disposition, metabolism, excretion

AXL "anexelekto" receptor tyrosine kinase BCR-Abl breakpoint cluster region-Abelson kinase

FGFR-1/FGFR-2/FGFR-3 fibroblast growth factor receptors 1, 2, 3

IC50 half maximal inhibitory concentration

HER2 human epidermal growth factor receptor 2

CDK inhibition of cyclin-dependent kinases IGF-1R insulin-like growth factor 1 receptor ITK interleukin 2-inducible T-cell kinase

p38 MAPK p38 mitogen-activated protein kinase Ph + ALL Ph + acute lymphoblastic leukemia PI3K phosphatidylinositol 3-kinase

PARP-1 poly(ADP-ribose)polymerase-1

PDGFR-α/β platelet-derived growth factor receptor-α/β

MEKK2 mitogen-activated protein kinase kinase kinase 2

MDA-MB-468 human breast carcinoma cell lines

CML chronic myelogeneous leukemia DHODH dihydroorotate dehydrogenase

PRK1 actin-regulating kinase MEK1 activated protein kinase AML acute myeloid leukemia

ARK5 AMPK-related kinase 5 ALK anaplastic lymphoma kinase

ALP alkaline phosphatase

D2R dopamine receptor 2

FAK focal adhesion kinase

HB hydrogen bind HBD hydrogen bond donor HBA hydrogen bond acceptor 5-HTR hydroxytryptamine receptors

JAK-1/JAK-2/JAK-3 Janus kinase-1/2/3 LE ligand efficiency

PAK1 p21-activated kinase

PLK-1 polo-like kinase 1

nM nanomolar μM micromolar

Lck lymphocyte tyrosine kinase

HGF or MET hepatocyte growth factor H4H histamine receptors 4 HDAC2 histone deacetylase 2

CK2 casein kinase 2 CXCR3 chemokine receptor

tetrazolium bromide

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

**Acronyms and abbreviations**

all along.

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

Mulham and Majd for all the love, compassion, and support I received from them all along.

This chapter is dedicated to the respectable memories of my mother Jaleelah and father Salem who died of old age and to the reminiscence of my dearest brother Mohammed who left this world due to leukemia. Peace Be Upon Them All.


#### **Acronyms and abbreviations**

*Chemistry and Applications of Benzimidazole and its Derivatives*

resistant mutated spreadable cancers [151].

of big data, advances in technology, and artificial intelligence.

Multitargeting can occur via three possible ways: acting on the same target, on different targets of the same pathway, or on different targets of different pathways. So far the benzimidazole derivatives that have been explored are reported to act as the third category "acting on different targets of different pathways." The focus has been so far on the kinome-relevant signaling key player with dovitinib widening the landscape to non-kinase targets. Broadening "multitargeting" concept to identify novel inhibitors with potency against key targets outside the human kinome necessitates treating complex diseases using "polypharmacology" gains special interest in

Despite the initial enthusiasm for the efficacy of molecular targeted therapeutics following the approval of imatinib, a small tyrosine kinase inhibitor targeting BCR-Abl, in chronic myeloid leukemia (CML) and trastuzumab, a monoclonal antibody against HER2, for treatment of metastatic breast cancer, scientists and clinicians were challenged by recurrent relapse due to cancer patients who developed drug resistance. In the case of RTKi, resistance can emerge as a result of selection for mutant sin in the target that renders the binding site inaccessible, reduced influx accompanied by enhance efflux, shift in metabolism and excretion of the drug, and the activation of alternative signaling pathways. Thus, the rationale for targeting drugs is shifting. In the last two decades, the main effort was aimed at developing highly specific inhibitors acting on single target. Now, there is a general agreement that molecules interfering simultaneously with multiple RTKs might be more effective than single-target agents. With the recent approval by the FDA of sorafenib, regorafenib, sunitinib, lenvatinib, and axitinib-targeting VEGFR, PDGFR, FLT-3, and c-KIT—more attention is drawn to broad-spectrum anticancer properties multikinase targeting drugs. Thus it is anticipated that more multitargeting agents will be getting into clinical trials and making their way to clinical application. It is hoped that identification, discovery, and optimization of benzimidazole-based multitargeting agents will benefit from the "big data era" fueled by the availability

The author would like to acknowledge "Zamala" Program: University Fellowship

I would like to express my special thanks, gratitude and truthful appreciation to my dearest family: my wife Muna, my daughters Aseel and Layan and my sons

Program in Palestinian Universities (http://www.taawon.org/ar/zamala) for the fund granted to make this research stay at London Regional Cancer Center, Western University Ontario, Canada, possible. Also, I would like to acknowledge my home institution Al-Quds University, Jerusalem-Palestine, for the support and encouragement. In addition, I wish to thank the editor and high value the editor's work, reading, and comments. The accomplishment of this chapter was made possible

*3.2.2 Shift in the paradigm*

**Acknowledgements**

thanks to such support and help.

No conflict of interest exists.

**Notes/thanks/other declarations**

**Conflict of interest**

**124**

#### *Chemistry and Applications of Benzimidazole and its Derivatives*


#### **Author details**

Yousef Najajreh Anticancer Drugs Research Lab, Faculty of Pharmacy, Al-Quds University, Jerusalem, Palestine

\*Address all correspondence to: y.s.najajreh@gmail.com

© 2019 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.

**127**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

[10] Shah DI, Sharma M, Bansal Y, Bansal G, Singh M. Angiotensin II-AT1 receptor antagonists: Design, synthesis and evaluation of substituted carboxamido benzimidazole derivatives.

European Journal of Medicinal

[11] Sakamoto H, Ojima M, Kubo K, Fuse H, Tanaka M, Kohara Y, et al. In vitro antagonistic properties of a new angiotensin type 1 receptor blocker, Azilsartan, in receptor binding and function studies. Journal of Pharmacology and Experimental

[12] Zhang J, Liu X, Wang SQ, Liu GY, Xu WR, Cheng XC, et al. Identification of dual ligands targeting angiotensin II type 1 receptor and peroxisome proliferatoractivated receptor-γ by core hopping of telmisartan. Journal of Biomolecular

Structure and Dynamics. 2017

[13] Achar KCS, Hosamani KM,

Seetharamareddy HR. In-vivo analgesic and anti-inflammatory activities of newly synthesized benzimidazole derivatives. European Journal of Medicinal Chemistry. 2010

[14] Rathore A, Sudhakar R, Ahsan MJ, Ali A, Subbarao N, Jadav SS, et al. In vivo anti-inflammatory activity and docking study of newly synthesized benzimidazole derivatives bearing oxadiazole and morpholine rings. Bioorganic Chemistry. 2017

[15] Bukhari SNA, Lauro G, Jantan I, Chee CF, Amjad MW, Bifulco G, et al. Anti-inflammatory trends of new benzimidazole derivatives. Future

[16] Padalkar VS, Borse BN, Gupta VD, Phatangare KR, Patil VS, Umape PG, et al. Synthesis and antimicrobial activity of novel 2-substituted benzimidazole, benzoxazole and

Medicinal Chemistry. 2016

Chemistry. 2008

Therapeutics. 2010

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

[2] Bansal Y, Silakari O. The therapeutic journey of benzimidazoles: A review. Bioorganic & Medicinal Chemistry. 2012

[4] Walia R, Naaz SF, Iqbal K, Lamba HS. Benzimidazole derivatives—An overview. International Journal of Research in Pharmaceutical Chemistry. 2011

[5] Ahamad A, Pandurangan A, Rana K,

Benzimidazole: A short review of their antimicrobial activities. International Current Pharmaceutical Journal. 2012

[6] Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, et al. The biology and chemistry of antifungal agents: A review. Bioorganic

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[9] Sharma MC, Kohli DV, Sharmab S,

Sharma AD. Synthesis and antihypertensive activity of some new benzimidazole derivatives of 4′-(6-methoxy-2-substitutedbenzimidazole-1-ylmethyl)-biphenyl-2-carboxylic acid in the presences of BF3·OEt2. Der Pharmacia Sinica. 2010

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ATP adenosine triphosphate PDB Protein Data Bank

PTKs protein tyrosine kinase PTP protein tyrosine phosphatases c-KIT stem cell factor receptor FLT3 FMS-like tyrosine kinase 3

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TOPO1/TOPO2 topoisomerase 1/2 TKRs tyrosine kinase receptors

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Yousef Najajreh

Jerusalem, Palestine

provided the original work is properly cited.

\*Address all correspondence to: y.s.najajreh@gmail.com

© 2019 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,

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*Chemistry and Applications of Benzimidazole and its Derivatives*

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[116] Schäfer N, Gielen GH, Kebir S, Wieland A, Till A, Mack F, et al. Phase I trial of dovitinib (TKI258) in recurrent glioblastoma. Journal of Cancer Research and Clinical Oncology. 2016

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[118] Konecny GE, Finkler N, Garcia AA, Lorusso D, Lee PS, Rocconi RP, et al. Second-line dovitinib (TKI258) in patients with FGFR2-mutated or FGFR2 non-mutated advanced or metastatic endometrial cancer: A non-randomised, open-label, two-group, two-stage, phase 2 study. The Lancet Oncology. 2015

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2013

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[107] Trudel S, Li ZH, Wei E, Wiesmann M, Chang H, Chen C, et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma. Blood. 2005

[108] Pyo K-H, Cho BC, Kim H, Moon YW, Jang KW, Kang HN, et al. Antitumor activity and acquired resistance mechanism of dovitinib (TKI258) in RET-rearranged lung adenocarcinoma. Molecular Cancer

[109] Lopes De Menezes DE, Peng J, Garrett EN, Louie SG, Lee SH,

[110] Lee CK, Lee ME, Lee WS, Kim JM, Park KH, Kim TS, et al. Dovitinib (TKI258), a multi-target angiokinase inhibitor, is effective regardless of KRAS or BRAF mutation status in colorectal cancer. American Journal of Cancer

[111] Valverde A, Gomez-Espana A, Hernandez V, Jimenez J, Lopez-Sanchez LM, Cano MT, et al. The multi-targeted kinase inhibitor aee788 exerts antiproliferative effects in braf mutated colorectal cancer cells. Annals of

[112] André F, Bachelot T, Campone M, Dalenc F, Perez-Garcia JM, Hurvitz SA, et al. Targeting FGFR with dovitinib (TKI258): Preclinical and clinical data in breast cancer. Clinical Cancer Research.

[113] Angevin E, Lopez-Martin JA, Lin CC, Gschwend JE, Harzstark A, Castellano D, et al. Phase I study of dovitinib (TKI258), an oral FGFR,

Wiesmann M, et al. CHIR-258: A potent inhibitor of FLT3 kinase in experimental tumor xenograft models of human acute myelogenous leukemia. Clinical Cancer

Chemistry. 2009

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Research. 2005

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2013

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[122] Lesca E, Lammens A, Huber R, Augustin M. Structural analysis of the human fibroblast growth factor receptor 4 kinase. Journal of Molecular Biology. 2014

[123] Hasinoff BB, Wu X, Nitiss JL, Kanagasabai R, Yalowich JC. The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II. Biochemical Pharmacology. 2012

[124] A receptor tyrosine kinase inhibitor, dovitinib (TKI-258), enhances BMP-2-induced osteoblast differentiation in vitro. Molecules and Cells. 2016

[125] Howard S, Berdini V, Boulstridge JA, Carr MG, Cross DM, Curry J, et al. Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor with potent aurora kinase activity. Journal of Medicinal Chemistry. 2009

[126] Curry J, Angove H, Fazal L, Lyons J, Reule M, Thompson N, et al. Aurora B kinase inhibition in mitosis: Strategies for optimising the use of aurora kinase inhibitors such as AT9283. Cell Cycle. 2009

[127] Tanaka R, Squires MS, Kimura S, Yokota A, Nagao R, Yamauchi T, et al. Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells. Blood. 2010

[128] Santo L, Hideshima T, Cirstea D, Bandi M, Nelson EA, Gorgun G, et al. Antimyeloma activity of a multitargeted kinase inhibitor, AT9283, via potent Aurora kinase and STAT3 inhibition either alone or in combination with lenalidomide. Clinical Cancer Research. 2011

[129] Smyth T, Reule M, Yokota A, Ottmann OG, Nagao R, Tanaka R, et al. Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells. Blood. 2010

[130] Puleo DE, Kucera K, Hammarén HM, Ungureanu D, Newton AS, Silvennoinen O, et al. Identification and characterization of JAK2 pseudokinase domain small molecule binders. ACS Medicinal Chemistry Letters. 2017

[131] Moreno L, Marshall LV, Pearson ADJ, Morland B, Elliott M, Campbell-Hewson Q, et al. A phase I trial of AT9283 (a selective inhibitor of aurora kinases) in children and adolescents with solid tumors: A cancer research UK study. Clinical Cancer Research. 2015

[132] Qi W, Liu X, Cooke LS, Persky DO, Miller TP, Squires M, et al. AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B-cell lymphomas. International Journal of Cancer. 2012

[133] Vormoor B, Veal GJ, Griffin MJ, Boddy AV, Irving J, Minto L, et al. A phase I/II trial of AT9283, a selective inhibitor of aurora kinase in children with relapsed or refractory acute leukemia: Challenges to run early phase clinical trials for children with leukemia. Pediatric Blood & Cancer. 2017

[134] Foran JM, Ravandi F, O'Brien SM, Borthakur G, Rios M, Boone P, et al. Phase I and pharmacodynamic trial of AT9283, an aurora kinase inhibitor, in patients with refractory leukemia. Journal of Clinical Oncology. 2008

[135] Dent S, Chi K, Jonker D, Capier K, Simpson R, Chen E, et al. 512 NCIC CTG IND.181: Phase I study of AT9283 given as a weekly 24 hour infusion. European Journal of Cancer Supplements. 2010

[136] Jayanthan A, Cooper TM, Hoeksema KA, Lotfi S, Woldum E, Lewis VA, et al. Occurrence and modulation of therapeutic targets of Aurora kinase inhibition in pediatric acute leukemia cells. Leukemia & Lymphoma. 2013

[137] Duong JK, Griffin MJ, Hargrave D, Vormoor J, Edwards D, Boddy AV. A population pharmacokinetic model of AT9283 in adults and children to predict the maximum tolerated dose in children with leukaemia. British Journal of Clinical Pharmacology. 2017

[138] Li Y, Tan C, Gao C, Zhang C, Luan X, Chen X, et al. Discovery of benzimidazole derivatives as novel multi-target EGFR, VEGFR-2 and PDGFR kinase inhibitors. Bioorganic & Medicinal Chemistry. 2011

[139] Chu B, Liu F, Li L, Ding C, Chen K, Sun Q, et al. A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells. Cell Death & Disease. 2015

[140] Yun CH, Boggon TJ, Li Y, Woo MS, Greulich H, Meyerson M, et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: Mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell. 2007

[141] Han C, Wan L, Ji H, Ding K, Huang Z, Lai Y, et al. Synthesis and evaluation of 2-anilinopyrimidines bearing 3-aminopropamides as potential epidermal growth factor receptor inhibitors. European Journal of Medicinal Chemistry. 2014

[142] Determann R, Dreher J, Baumann K, Preu L, Jones PG, Totzke F, et al. 2-Anilino-4-(benzimidazol-2-yl) pyrimidines—A multikinase inhibitor scaffold with antiproliferative activity toward cancer cell lines. European Journal of Medicinal Chemistry. 2012

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[144] Shao H, Shi S, Huang S, Hole AJ, Abbas AY, Baumli S, et al. Substituted 4-(thiazol-5-yl)-2-(phenylamino) pyrimidines are highly active CDK9 inhibitors: Synthesis, X-ray crystal structures, structure-activity relationship, and anticancer activities. Journal of Medicinal Chemistry. 2013

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[146] McCoull W, Hennessy EJ, Blades K, Chuaqui C, Dowling JE, Ferguson AD, et al. Optimization of highly kinase selective bis-anilino pyrimidine PAK1 inhibitors. ACS Medicinal Chemistry Letters. 2016

[147] Gandin V, Ferrarese A, Dalla Via M, Marzano C, Chilin A, Marzaro G. Targeting kinases with anilinopyrimidines: Discovery of N-phenyl-N'-[4-(pyrimidin-4-ylamino) phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily. Scientific Reports. 2015

[148] Zhang Q, Liu Y, Gao F, Ding Q, Cho C, Hur W, et al. Discovery of EGFR selective 4,6-disubstituted pyrimidines

**137**

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.86249*

from a combinatorial kinase-directed heterocycle library. Journal of the American Chemical Society. 2006

[149] Von Ahsen O, Bömer U. Highthroughput screening for kinase inhibitors. Chembiochem. 2005

[150] Bistrović A, Krstulović L, Harej A, Grbčić P, Sedić M, Koštrun S, et al. Design, synthesis and biological evaluation of novel benzimidazole amidines as potent multi-target inhibitors for the treatment of nonsmall cell lung cancer. European Journal

of Medicinal Chemistry. 2018

and Therapy. 2011

[151] Morphy R, Rankovic Z. Designed multiple ligands. An emerging drug discovery paradigm. Journal of Medicinal Chemistry. 2005

[152] Ballas MS, Chachoua A. Rationale for targeting VEGF, FGF, and PDGF for the treatment of NSCLC. OncoTargets

*Benzimidazoles: From Antiproliferative to Multitargeted Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.86249*

from a combinatorial kinase-directed heterocycle library. Journal of the American Chemical Society. 2006

*Chemistry and Applications of Benzimidazole and its Derivatives*

[142] Determann R, Dreher J, Baumann K, Preu L, Jones PG, Totzke F, et al. 2-Anilino-4-(benzimidazol-2-yl) pyrimidines—A multikinase inhibitor scaffold with antiproliferative activity toward cancer cell lines. European Journal of Medicinal Chemistry. 2012

[143] Wang S, Meades C, Wood G, Osnowski A, Anderson S, Yuill R, et al. 2-Anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors: Synthesis, SAR analysis, X-ray crystallography, and biological activity. Journal of Medicinal

[144] Shao H, Shi S, Huang S, Hole AJ, Abbas AY, Baumli S, et al. Substituted 4-(thiazol-5-yl)-2-(phenylamino) pyrimidines are highly active CDK9 inhibitors: Synthesis, X-ray crystal structures, structure-activity

relationship, and anticancer activities. Journal of Medicinal Chemistry. 2013

[145] Wang S, Griffiths G, Midgley CA, Barnett AL, Cooper M, Grabarek J, et al. Discovery and characterization of 2-anilino-4-(thiazol-5-yl)pyrimidine transcriptional CDK inhibitors as anticancer agents. Chemistry &

[146] McCoull W, Hennessy EJ, Blades K, Chuaqui C, Dowling JE, Ferguson AD, et al. Optimization of highly kinase selective bis-anilino pyrimidine PAK1 inhibitors. ACS Medicinal Chemistry

[147] Gandin V, Ferrarese A, Dalla Via M, Marzano C, Chilin A, Marzaro G. Targeting kinases with anilinopyrimidines: Discovery of N-phenyl-N'-[4-(pyrimidin-4-ylamino) phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily. Scientific Reports.

[148] Zhang Q, Liu Y, Gao F, Ding Q, Cho C, Hur W, et al. Discovery of EGFR selective 4,6-disubstituted pyrimidines

Chemistry. 2004

Biology. 2010

Letters. 2016

2015

[135] Dent S, Chi K, Jonker D, Capier K, Simpson R, Chen E, et al. 512 NCIC CTG IND.181: Phase I study of AT9283 given as a weekly 24 hour infusion. European Journal of Cancer

[136] Jayanthan A, Cooper TM, Hoeksema KA, Lotfi S, Woldum E, Lewis VA, et al. Occurrence and modulation of therapeutic targets of Aurora kinase inhibition in pediatric acute leukemia cells. Leukemia &

[137] Duong JK, Griffin MJ, Hargrave D, Vormoor J, Edwards D, Boddy AV. A population pharmacokinetic model of AT9283 in adults and children to predict the maximum tolerated dose in children with leukaemia. British Journal of Clinical Pharmacology.

[138] Li Y, Tan C, Gao C, Zhang C, Luan X, Chen X, et al. Discovery of benzimidazole derivatives as novel multi-target EGFR, VEGFR-2 and PDGFR kinase inhibitors. Bioorganic &

[139] Chu B, Liu F, Li L, Ding C, Chen K, Sun Q, et al. A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells.

[140] Yun CH, Boggon TJ, Li Y, Woo MS, Greulich H, Meyerson M, et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: Mechanism of activation and insights into differential inhibitor sensitivity.

[141] Han C, Wan L, Ji H, Ding K, Huang Z, Lai Y, et al. Synthesis and evaluation of 2-anilinopyrimidines bearing 3-aminopropamides as potential epidermal growth factor receptor inhibitors. European Journal of

Medicinal Chemistry. 2014

Medicinal Chemistry. 2011

Cell Death & Disease. 2015

Cancer Cell. 2007

Supplements. 2010

Lymphoma. 2013

2017

**136**

[149] Von Ahsen O, Bömer U. Highthroughput screening for kinase inhibitors. Chembiochem. 2005

[150] Bistrović A, Krstulović L, Harej A, Grbčić P, Sedić M, Koštrun S, et al. Design, synthesis and biological evaluation of novel benzimidazole amidines as potent multi-target inhibitors for the treatment of nonsmall cell lung cancer. European Journal of Medicinal Chemistry. 2018

[151] Morphy R, Rankovic Z. Designed multiple ligands. An emerging drug discovery paradigm. Journal of Medicinal Chemistry. 2005

[152] Ballas MS, Chachoua A. Rationale for targeting VEGF, FGF, and PDGF for the treatment of NSCLC. OncoTargets and Therapy. 2011

**139**

**Chapter 8**

**Abstract**

60 cell line panel.

**1. Introduction**

Vacuolar (H+

Bisbenzimidazoles: Anticancer

Small molecule chemotherapeutic agents such as Imatinib, Gefitinib, and Erlotinib have played a significant role in the treatment of cancer. Although the unprecedented progress has been achieved in cancer treatment with these targeted agents, there is a strong demand for the development of selective and highly efficacious cancer drugs. V-ATPases are emerging as important target for the identification of novel therapeutic agents for cancer. Our screening and drug discovery processes have identified the bisbenzimidazole derivative (**RP-15**) as a potent anticancer V-ATPase inhibitor. In the present study, bisbenzimidazoles (compound-**25**, **RP-11** and **RP-15**) have been tested for proton-pump inhibition activity in human hepatoma cell line (Huh7.5). **RP-15** displayed comparable proton-pump inhibition activity to the standard Bafilomycin A1. We examined the antiproliferative activity of these analogs in two highly invasive and metastatic inflammatory breast cancer (IBC) cell lines (SUM 149PT and SUM190PT) along with Huh7.5. The compound-**25** (SUM190PT: IC50 = 0.43±0.11 μM) and its structural analog **RP-11** (SUM190PT: IC50 = 0.49±0.09 μM) have shown significant inhibition toward IBC cell lines. Additionally, **RP-11** and **RP-15** have demonstrated very good cytotoxicity toward the majority of cancer cell lines in the NCI

**Keywords:** bisbenzimidazoles, anticancer, V-ATPase, proton-pump, inhibitors

Since Paul Ehrlich's introduction of the concept of chemotherapy, development of chemotherapeutic agents for cancer over the past several decades has seen marvelous records of accomplishments [1, 2]. Cancer is one of the major health problems globally and is second leading cause of death in the USA [3, 4]. Cancer is a very complex disease and our understanding towards it has been advanced tremendously over the last six decades since the first human cancer cell line HeLa identified in 1952 [5]. Over the past few years, the search for new anticancer drugs has changed dramatically. Advances in the molecular nature of drug action, new technology and more recently market considerations have produced new approaches to cancer drug discovery [6]. Recent advances in molecular biology, high throughput screening

*Renukadevi Patil, Olivia Powrozek, Binod Kumar,* 

*William Seibel, Kenneth Beaman, Gulam Waris,* 

*Neelam Sharma-Walia and Shivaputra Patil*

)-ATPase Inhibitors

#### **Chapter 8**

## Bisbenzimidazoles: Anticancer Vacuolar (H+ )-ATPase Inhibitors

*Renukadevi Patil, Olivia Powrozek, Binod Kumar, William Seibel, Kenneth Beaman, Gulam Waris, Neelam Sharma-Walia and Shivaputra Patil*

#### **Abstract**

Small molecule chemotherapeutic agents such as Imatinib, Gefitinib, and Erlotinib have played a significant role in the treatment of cancer. Although the unprecedented progress has been achieved in cancer treatment with these targeted agents, there is a strong demand for the development of selective and highly efficacious cancer drugs. V-ATPases are emerging as important target for the identification of novel therapeutic agents for cancer. Our screening and drug discovery processes have identified the bisbenzimidazole derivative (**RP-15**) as a potent anticancer V-ATPase inhibitor. In the present study, bisbenzimidazoles (compound-**25**, **RP-11** and **RP-15**) have been tested for proton-pump inhibition activity in human hepatoma cell line (Huh7.5). **RP-15** displayed comparable proton-pump inhibition activity to the standard Bafilomycin A1. We examined the antiproliferative activity of these analogs in two highly invasive and metastatic inflammatory breast cancer (IBC) cell lines (SUM 149PT and SUM190PT) along with Huh7.5. The compound-**25** (SUM190PT: IC50 = 0.43±0.11 μM) and its structural analog **RP-11** (SUM190PT: IC50 = 0.49±0.09 μM) have shown significant inhibition toward IBC cell lines. Additionally, **RP-11** and **RP-15** have demonstrated very good cytotoxicity toward the majority of cancer cell lines in the NCI 60 cell line panel.

**Keywords:** bisbenzimidazoles, anticancer, V-ATPase, proton-pump, inhibitors

#### **1. Introduction**

Since Paul Ehrlich's introduction of the concept of chemotherapy, development of chemotherapeutic agents for cancer over the past several decades has seen marvelous records of accomplishments [1, 2]. Cancer is one of the major health problems globally and is second leading cause of death in the USA [3, 4]. Cancer is a very complex disease and our understanding towards it has been advanced tremendously over the last six decades since the first human cancer cell line HeLa identified in 1952 [5]. Over the past few years, the search for new anticancer drugs has changed dramatically. Advances in the molecular nature of drug action, new technology and more recently market considerations have produced new approaches to cancer drug discovery [6]. Recent advances in molecular biology, high throughput screening

(HTS), computer-aided drug design (CADD), and combinatorial chemistry technologies have allowed a combination of both knowledge around the drug receptor and large library screening to be used for anticancer drug discovery today [7–10].

As the understanding of human biology and new technologies progressed, the discovery and development process moved from a random pattern to a more predictable one. The development of a molecularly targeted anticancer drug has gained importance in recent years [11]. One of the important small molecule targeted therapy, Imatinib (Gleevec®), a tyrosine kinase inhibitor, achieved incredible advancement in cancer treatment [12–14]. Imatinib's success stimulated the scientists to develop variety of targeted anticancer agents including Gefitinib (Iressa™) and Erlotinib (Terceva®) for the treatment of different types of cancer patients (**Figure 1**). Targeted agents represented significant developments in cancer treatment and have increased the life expectancy of patients [15–18]. Despite the unprecedented progress achieved, the anticancer drug discovery research remains highly challenging and there is strong demand for the development of highly efficacious and safe anticancer drugs which can overcome cancer metastasis, and drug resistance.

Recent studies suggest that an acidic microenvironment in the tumor is responsible for cancer development, progression, and metastasis. Novel drugs that specifically target the mechanism by which V-ATPase lowers the pH of the tumor microenvironment are essential for cancer chemotherapy. Among the key regulators of the tumor, acidic microenvironment V-ATPases plays an important role in the regulation of the pH gradient. V-ATPases play a vital role in the maintenance of the tumor acidic microenvironment and are overexpressed in many types of metastatic cancers including breast cancer. V-ATPases are functionally expressed in plasma membranes of tumor cells and they have specialized functions in metastasis [19]. Recent research has demonstrated that the preferential expression of V-ATPase at the cell surface is important for the acquisition of invasiveness and the metastasis of breast cancer cells [19]. Therefore, V-ATPase is a potential target to investigate for metastatic breast cancer therapy. Discovery and development of easily synthesized,

**141**

**Figure 2.**

*Natural potent V-ATPase inhibitors.*

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

cancer.

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

structures of existing natural inhibitors.

*)-ATPase Inhibitors*

cost-effective, and potent small molecule drugs targeting V-ATPase are needed to evaluate the therapeutic potential of V-ATPase inhibitors in metastatic breast

The V-ATPases are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. The V-ATPase proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V1 is responsible for ATP hydrolysis and V0 provides the transmembrane proton channel [20–23]. The V-ATPases have been associated with cancer invasion, metastasis and drug resistance [19, 24–27]. Several preclinical studies have reported the anticancer effects of V-ATPase inhibitors [28–32]. V-ATPase inhibitors will be beneficial for cancer patients given either in combination with cytotoxic agents or dual-acting (anticancer and V-ATPase inhibitor) agents. Thus, V-ATPases are emerging as an important target for the identification of potential novel chemotherapeutic agents. Despite the clear involvement of V-ATPases in cancer, to date, therapeutic use of V-ATPase targeting small molecules have not reached the clinic. Natural products macrolide antibiotics, such as bafilomycin and concanamycin, potently inhibit V-ATPases [33–37] (**Figure 2**), but their use is complicated by non-specific effects on other targets. Moreover, these molecules have been difficult to synthesize in large quantities. Despite huge efforts by both academic and pharmaceutical industry medicinal chemists, development of useful V-ATPase inhibitors has been limited because of the complicated chemical

We have been actively involved in the design and development of novel small molecular agents for different types of cancers. Past few years, we have reported the chromene-, chromenopyridine-, and imidazoquinoline-based pharmacophores as initial lead anticancer drug candidates through screening and drug development process [38–40]. Notably, we have identified the highly potent microtubule targeting anticancer agent (**SP-6-27**) for ovarian cancer [41]. Since then our laboratory has been active in identifying anticancer agents with different mechanisms of action. In continuation of our drug discovery research, we recently initiated a collaborative effort on the V-ATPases as anti-cancer targets. Successful identification of new lead small molecule drugs for ovarian cancer by screening and drug development processes [41] inspired us to screen the library of compounds based on the literature of known V-ATPase inhibitors. We identified the bisbenzimidazole scaffold from screening process. Bisbenzimidazoles are nitrogen heterocycles with wide spectrum of biological activities. We

**Figure 1.** *Molecularly targeted clinically successful chemotherapeutic agents.*

*Chemistry and Applications of Benzimidazole and its Derivatives*

(HTS), computer-aided drug design (CADD), and combinatorial chemistry technologies have allowed a combination of both knowledge around the drug receptor and large library screening to be used for anticancer drug discovery today [7–10]. As the understanding of human biology and new technologies progressed, the discovery and development process moved from a random pattern to a more predictable one. The development of a molecularly targeted anticancer drug has gained importance in recent years [11]. One of the important small molecule targeted therapy, Imatinib (Gleevec®), a tyrosine kinase inhibitor, achieved incredible advancement in cancer treatment [12–14]. Imatinib's success stimulated the scientists to develop variety of targeted anticancer agents including Gefitinib (Iressa™) and Erlotinib (Terceva®) for the treatment of different types of cancer patients (**Figure 1**). Targeted agents represented significant developments in cancer treatment and have increased the life expectancy of patients [15–18]. Despite the unprecedented progress achieved, the anticancer drug discovery research remains highly challenging and there is strong demand for the development of highly efficacious and safe anticancer drugs which can overcome cancer metastasis, and

Recent studies suggest that an acidic microenvironment in the tumor is responsible for cancer development, progression, and metastasis. Novel drugs that specifically target the mechanism by which V-ATPase lowers the pH of the tumor microenvironment are essential for cancer chemotherapy. Among the key regulators of the tumor, acidic microenvironment V-ATPases plays an important role in the regulation of the pH gradient. V-ATPases play a vital role in the maintenance of the tumor acidic microenvironment and are overexpressed in many types of metastatic cancers including breast cancer. V-ATPases are functionally expressed in plasma membranes of tumor cells and they have specialized functions in metastasis [19]. Recent research has demonstrated that the preferential expression of V-ATPase at the cell surface is important for the acquisition of invasiveness and the metastasis of breast cancer cells [19]. Therefore, V-ATPase is a potential target to investigate for metastatic breast cancer therapy. Discovery and development of easily synthesized,

**140**

**Figure 1.**

*Molecularly targeted clinically successful chemotherapeutic agents.*

drug resistance.

cost-effective, and potent small molecule drugs targeting V-ATPase are needed to evaluate the therapeutic potential of V-ATPase inhibitors in metastatic breast cancer.

The V-ATPases are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. The V-ATPase proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V1 is responsible for ATP hydrolysis and V0 provides the transmembrane proton channel [20–23]. The V-ATPases have been associated with cancer invasion, metastasis and drug resistance [19, 24–27]. Several preclinical studies have reported the anticancer effects of V-ATPase inhibitors [28–32]. V-ATPase inhibitors will be beneficial for cancer patients given either in combination with cytotoxic agents or dual-acting (anticancer and V-ATPase inhibitor) agents. Thus, V-ATPases are emerging as an important target for the identification of potential novel chemotherapeutic agents. Despite the clear involvement of V-ATPases in cancer, to date, therapeutic use of V-ATPase targeting small molecules have not reached the clinic. Natural products macrolide antibiotics, such as bafilomycin and concanamycin, potently inhibit V-ATPases [33–37] (**Figure 2**), but their use is complicated by non-specific effects on other targets. Moreover, these molecules have been difficult to synthesize in large quantities. Despite huge efforts by both academic and pharmaceutical industry medicinal chemists, development of useful V-ATPase inhibitors has been limited because of the complicated chemical structures of existing natural inhibitors.

We have been actively involved in the design and development of novel small molecular agents for different types of cancers. Past few years, we have reported the chromene-, chromenopyridine-, and imidazoquinoline-based pharmacophores as initial lead anticancer drug candidates through screening and drug development process [38–40]. Notably, we have identified the highly potent microtubule targeting anticancer agent (**SP-6-27**) for ovarian cancer [41]. Since then our laboratory has been active in identifying anticancer agents with different mechanisms of action. In continuation of our drug discovery research, we recently initiated a collaborative effort on the V-ATPases as anti-cancer targets. Successful identification of new lead small molecule drugs for ovarian cancer by screening and drug development processes [41] inspired us to screen the library of compounds based on the literature of known V-ATPase inhibitors. We identified the bisbenzimidazole scaffold from screening process. Bisbenzimidazoles are nitrogen heterocycles with wide spectrum of biological activities. We

**Figure 2.** *Natural potent V-ATPase inhibitors.*

reported the focused set of bisbenzimidazoles as anticancer V-ATPase agents (**Figure 3**) [42]. Bisbenzimidazole derivatives (**RP-3**–**RP-15**) have been screened in selected human breast cancer (MDA-MB-231, MDA-MB-468, MCF-7) and ovarian cancer (cisplatin-sensitive A2780, cisplatin-resistant Cis-A2780 and PA-1) cell lines. Among this small set of bisbenzimidazoles, **RP-15** demonstrated high potency towards the epidermal growth factor receptor (EGFR) over expressed triple negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM). Very interestingly, **RP-15** is not toxic to normal breast epithelial cells. It is nearly 40 times less toxic in the normal breast epithelial cell line, MCF10A (IC50 = 1.62 ± 0.14 μM). Furthermore, the bisbenzimidazole derivatives (Compound-**25**, **RP-11** and **RP-15**) have demonstrated encouraging proton pump inhibition activity in MDA-MB-231. In particular our most efficacious anticancer analog **RP-15** has shown comparable proton pump inhibition activity to standard agent Bafilomycin A1.

In the present study, we selected and screened top two bisbenzimidazole derivatives (**RP-11** and **RP-15**) along with initial hit (compound **25**) for proton pump inhibition activity in human hepatoma cells, Huh7.5 using pH indicator Lysosensor Yellow/Blue DND-160. These compounds have also been screened for their antiproliferative activity using BrDU incorporation assay in selected inflammatory breast cancer (IBC) cell lines (SUM149PT and SUM190PT) along with Huh7.5 human hepatoma cancer cell line. Additionally, **RP-11** and

**143**

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

human cancer cell lines.

**2.1 Chemical synthesis**

**2. Methods**

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

*)-ATPase Inhibitors*

**RP-15** have been tested in NCI Developmental Therapeutics Program (DTP) nine major (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) 60

We recently reported the synthesis and detailed characterization of all these new bisbenzimidazoles [42]. In brief, we developed a fast and efficient synthetic one pot procedure to prepare all these analogs (**RP-3**–**RP-15**). Condensation of 4-(6-(4-methylpiperazin-1-yl)-1H, 30H-[2, 50-bibenzo [d]imidazol]-20-yl) phenol with substituted alkyl halides in the presence of cesium carbonate in dimethyl formamide (DMF). For the more detailed synthesis and spectral and analytical

**2.2 Proton pump inhibition activity in human hepatoma (Huh7.5) cell line**

We used Huh7.5 cell line for proton pump activity. Briefly, the Huh7.5 cells were cultured in DMEM media supplemented with 10% serum to a confluency of 80%. The Huh7.5 cells were treated with the compounds (Compound-**25**, **RP-11** and **RP-15**) at a concentration of 12 μM for 20 minutes followed by incubation with Lysosensor Yellow/Blue DND-160 (10 μM) for 10 minutes at 37°C. The cells

Cell proliferation ELISA BrdU colorimetric (assay no. 11647229001; Roche, Basel, Switzerland) was used to quantify cell proliferation by the measurement of BrdU incorporated during DNA synthesis. Cells from a 90% confluent T-25 flask were seeded 100 μL/well of 96-well plates and incubated overnight. Dimethyl Sulfoxide (DMSO) stock solutions of the compounds (Compound-**25**, **RP-11** and **RP-15**) were diluted in pure F-12 media and exposed to different concentrations for 24 and 48 hours. Each concentration and controls were done in triplicates. The mean ± standard deviation (S.D.) was calculated and shown on the graph with untreated cells serving as a negative control, 20 minutes after adding the substrate, the absorbance was read at 370 nm. The compound concentration that inhibited cell growth by 50% of the untreated control (IC50) was calculated from the dose response curves constructed by normalizing the data to percentages based of the negative control and a nonlinear regression analysis in GraphPad Prism Software 7 (GraphPad Software, San Diego, CA, USA). For the Huh7.5 cell line we used CellTiter-Glo Luminescent

The bisbenzimidazoles (**RP-11** and **RP-15**) have been tested for growth inhibition against 60 human cancer cell lines from the NCI's anticancer screening program. The NCI's screening procedure has been given in detail elsewhere [43–47] and

characterization of all these compounds please see Ref. [42].

were visualized under the microscope.

Cell Viability Assay kit (Promega, Madison, WI, USA).

presently DTP uses the sulforhodamine B (SRB) assay.

**2.4 The NCI 60 cell lines** *in vitro* **screening**

**2.3 Antiproliferative activity**

**Figure 3.** *Bisbenzimidazoles derivatives.*

**RP-15** have been tested in NCI Developmental Therapeutics Program (DTP) nine major (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) 60 human cancer cell lines.

## **2. Methods**

*Chemistry and Applications of Benzimidazole and its Derivatives*

activity to standard agent Bafilomycin A1.

reported the focused set of bisbenzimidazoles as anticancer V-ATPase agents (**Figure 3**) [42]. Bisbenzimidazole derivatives (**RP-3**–**RP-15**) have been screened in selected human breast cancer (MDA-MB-231, MDA-MB-468, MCF-7) and ovarian cancer (cisplatin-sensitive A2780, cisplatin-resistant Cis-A2780 and PA-1) cell lines. Among this small set of bisbenzimidazoles, **RP-15** demonstrated high potency towards the epidermal growth factor receptor (EGFR) over expressed triple negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM). Very interestingly, **RP-15** is not toxic to normal breast epithelial cells. It is nearly 40 times less toxic in the normal breast epithelial cell line, MCF10A (IC50 = 1.62 ± 0.14 μM). Furthermore, the bisbenzimidazole derivatives (Compound-**25**, **RP-11** and **RP-15**) have demonstrated encouraging proton pump inhibition activity in MDA-MB-231. In particular our most efficacious anticancer analog **RP-15** has shown comparable proton pump inhibition

In the present study, we selected and screened top two bisbenzimidazole derivatives (**RP-11** and **RP-15**) along with initial hit (compound **25**) for proton pump inhibition activity in human hepatoma cells, Huh7.5 using pH indicator Lysosensor Yellow/Blue DND-160. These compounds have also been screened for their antiproliferative activity using BrDU incorporation assay in selected inflammatory breast cancer (IBC) cell lines (SUM149PT and SUM190PT) along with Huh7.5 human hepatoma cancer cell line. Additionally, **RP-11** and

**142**

**Figure 3.**

*Bisbenzimidazoles derivatives.*

#### **2.1 Chemical synthesis**

We recently reported the synthesis and detailed characterization of all these new bisbenzimidazoles [42]. In brief, we developed a fast and efficient synthetic one pot procedure to prepare all these analogs (**RP-3**–**RP-15**). Condensation of 4-(6-(4-methylpiperazin-1-yl)-1H, 30H-[2, 50-bibenzo [d]imidazol]-20-yl) phenol with substituted alkyl halides in the presence of cesium carbonate in dimethyl formamide (DMF). For the more detailed synthesis and spectral and analytical characterization of all these compounds please see Ref. [42].

#### **2.2 Proton pump inhibition activity in human hepatoma (Huh7.5) cell line**

We used Huh7.5 cell line for proton pump activity. Briefly, the Huh7.5 cells were cultured in DMEM media supplemented with 10% serum to a confluency of 80%. The Huh7.5 cells were treated with the compounds (Compound-**25**, **RP-11** and **RP-15**) at a concentration of 12 μM for 20 minutes followed by incubation with Lysosensor Yellow/Blue DND-160 (10 μM) for 10 minutes at 37°C. The cells were visualized under the microscope.

#### **2.3 Antiproliferative activity**

Cell proliferation ELISA BrdU colorimetric (assay no. 11647229001; Roche, Basel, Switzerland) was used to quantify cell proliferation by the measurement of BrdU incorporated during DNA synthesis. Cells from a 90% confluent T-25 flask were seeded 100 μL/well of 96-well plates and incubated overnight. Dimethyl Sulfoxide (DMSO) stock solutions of the compounds (Compound-**25**, **RP-11** and **RP-15**) were diluted in pure F-12 media and exposed to different concentrations for 24 and 48 hours. Each concentration and controls were done in triplicates. The mean ± standard deviation (S.D.) was calculated and shown on the graph with untreated cells serving as a negative control, 20 minutes after adding the substrate, the absorbance was read at 370 nm. The compound concentration that inhibited cell growth by 50% of the untreated control (IC50) was calculated from the dose response curves constructed by normalizing the data to percentages based of the negative control and a nonlinear regression analysis in GraphPad Prism Software 7 (GraphPad Software, San Diego, CA, USA). For the Huh7.5 cell line we used CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA).

#### **2.4 The NCI 60 cell lines** *in vitro* **screening**

The bisbenzimidazoles (**RP-11** and **RP-15**) have been tested for growth inhibition against 60 human cancer cell lines from the NCI's anticancer screening program. The NCI's screening procedure has been given in detail elsewhere [43–47] and presently DTP uses the sulforhodamine B (SRB) assay.

#### **3. Results and discussion**

Inhibition of V-ATPase has shown the link between cell biophysical properties and proliferative signaling selectively in malignant hepatocellular carcinoma (HCC) cells, which provides a new strategy to combat HCC [48]. HCC is the third most common cause of cancer-related deaths worldwide. HCC is accounting for almost 90% of primary malignant hepatic tumors in adults. In continuation of our work on V-ATPase inhibition, we used Huh7.5 cells for the proton pump inhibition activity. We have performed proton pump inhibitory activity of selected bisbenzimidazole derivatives (Compound-**25**, **RP-11** and **RP-15**) in Huh7.5 cells using Lysosensor Yellow/Blue DND-160 protocol [49]. The DND-160 is a pH indicator and cellular compartments with acidic pH elicit yellow fluorescence when stained, while the destabilized compartments with higher pH elicit blue fluorescence.

The compound **RP-15** displayed maximum inhibition of the proton-pump activity of V-ATPase followed by compound-**25** and **RP-11**. The untreated cells showed the strong intensity of yellow fluorescence (converted to pseudo-green in the **Figure 4A**) while the cells treated with bisbenzimidazoles (Compound-**25**, **RP-11** and **RP-15**) showed the strong intensity of blue fluorescence representing varying degree of destabilization of pH due to impaired vacuolar ATPase activity (**Figure 4A** and **B**). Additionally, these compounds have been tested for their cytotoxicity towards Huh7.5 cells using the CellTiter-Glo Luminescent Cell Viability Assay. The IC50 were calculated based on the results obtained for these compounds treated for 24 hours only for Huh7.5 cells compared to breast and ovarian cancer cell lines where we treated all test compounds for 48 hours. Bisbenzimidazoles, **RP-11** and **RP-15** have demonstrated very moderate antiproliferative activity towards Huh7.5 cells for 24 hours (**Table 1**).

High potency of bisbenzimidazole analog (**RP-15**) against the EGFR over expressed TNBC cell line (MDA-MB-468) inspired us to explore the selected bisbenzimidazoles in other breast cancer cell lines for anticancer activity. We selected two IBC cell lines (triple negative SUM149PT and Het2 positive SUM190PT) for the *in vitro* screening process [50]. Both SUM149 and SUM190 cell lines have been established from primary IBC tumors. IBC is one of the highly invasive, metastatic and lethal variant of human breast cancer. Development of therapeutic targets and agents for IBC is still in very early stage and it represents an opportunity for medicinal chemists to develop novel (pre) clinical drug candidates.

*In vitro* screening of the bisbenzimidazoles (Compound-**25**, **RP-11** and **RP-15**) towards these inflammatory cell lines has shown encouraging results (**Figure 5** and **Table 1**). Very interestingly our initial hit, compound-**25** (SUM149PT: IC50 = 0.80 ± 0.08 μM; SUM190PT: IC50 = 0.43 ± 0.11 μM) and its structural analog **RP-11** (SUM149PT: IC50 = 0.91 ± 0.15 μM; SUM190PT: IC50 = 0.49 ± 0.09 μM) have shown very good inhibition, whereas our TNBC lead **RP-15** (SUM149PT: IC50 = 1.77 ± 0.08 μM; SUM190PT: IC50 = 2.08 ± 0.56 μM) has demonstrated moderate inhibition towards these IBC cell lines. The high potency shown by compound-**25** and **RP-11** towards IBC has given us more insights to develop new anticancer agents for it and we plan to explore the structure-activity relationship (SAR) studies based on the bisbenzimidazole scaffold in very near future.

The Development Therapeutic Program (DTP) of the National Cancer Institute's 60 human tumor cell lines screen was developed as an *in vitro* drug discovery tool. We submitted both compounds (**RP-11** and **RP-15**) to the NCI Developmental Therapeutics Program (DTP) anticancer drug screen. Both of them have been first tested for three cell lines (MCF-7 breast cancer; NCI-H460 large-cell lung cancer; SF-268 glioma) to advance to the 60 cell line screen. This pre-screen process eliminates the inactive compounds but preserves active agents for 60 cell line screening.

**145**

**Figure 4.**

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

*)-ATPase Inhibitors*

Both compounds have been advanced to 60 cell lines representing nine major cancers (leukemia, non-small cell lung, central nervous system, colon, melanoma, ovarian, renal, prostate, and breast). Compounds have been tested over a broad range of concentrations against every cell line in the panel (five 10 fold dilutions starting

*(A) Staining of acidic compartments: Yellow signal (converted to pseudo-green) represents acidic pH, while the blue color represents slightly acidic to neutral pH. Huh7.5 cells were treated with the compounds (Compound-25, RP-11 and RP-15) at a concentration of 12 μM and standard Bafilomycin A1 at* 

*bisbenzimidazoles (Compound-25, RP-11 and RP-15) along with positive control BafilomycinA1.*

*concentration of 2 μM for 20 minutes followed by incubation with Lysosensor Yellow/Blue DND-160 (10 μM) for 10 minutes at 37°C. The cells were visualized under the microscope. The DND-160 is a pH indicator and cellular compartments with acidic pH elicit yellow fluorescence when stained, while the destabilized compartments with higher pH elicit blue fluorescence. The expected yellow color showed yellowish green of the filters available in the microscope. (B) Fold change in overall acidification of Huh7.5 cells upon treatment with*  *Chemistry and Applications of Benzimidazole and its Derivatives*

Inhibition of V-ATPase has shown the link between cell biophysical properties and proliferative signaling selectively in malignant hepatocellular carcinoma (HCC) cells, which provides a new strategy to combat HCC [48]. HCC is the third most common cause of cancer-related deaths worldwide. HCC is accounting for almost 90% of primary malignant hepatic tumors in adults. In continuation of our work on V-ATPase inhibition, we used Huh7.5 cells for the proton pump inhibition activity. We have performed proton pump inhibitory activity of selected bisbenzimidazole derivatives (Compound-**25**, **RP-11** and **RP-15**) in Huh7.5 cells using Lysosensor Yellow/Blue DND-160 protocol [49]. The DND-160 is a pH indicator and cellular compartments with acidic pH elicit yellow fluorescence when stained, while the

The compound **RP-15** displayed maximum inhibition of the proton-pump activity of V-ATPase followed by compound-**25** and **RP-11**. The untreated cells showed the strong intensity of yellow fluorescence (converted to pseudo-green in the **Figure 4A**) while the cells treated with bisbenzimidazoles (Compound-**25**, **RP-11** and **RP-15**) showed the strong intensity of blue fluorescence representing varying degree of destabilization of pH due to impaired vacuolar ATPase activity (**Figure 4A** and **B**). Additionally, these compounds have been tested for their cytotoxicity towards Huh7.5 cells using the CellTiter-Glo Luminescent Cell Viability Assay. The IC50 were calculated based on the results obtained for these compounds treated for 24 hours only for Huh7.5 cells compared to breast and ovarian cancer cell lines where we treated all test compounds for 48 hours. Bisbenzimidazoles, **RP-11** and **RP-15** have demonstrated very moderate antiproliferative activity

High potency of bisbenzimidazole analog (**RP-15**) against the EGFR over expressed TNBC cell line (MDA-MB-468) inspired us to explore the selected bisbenzimidazoles in other breast cancer cell lines for anticancer activity. We selected two IBC cell lines (triple negative SUM149PT and Het2 positive SUM190PT) for the *in vitro* screening process [50]. Both SUM149 and SUM190 cell lines have been established from primary IBC tumors. IBC is one of the highly invasive, metastatic and lethal variant of human breast cancer. Development of therapeutic targets and agents for IBC is still in very early stage and it represents an opportunity for medici-

*In vitro* screening of the bisbenzimidazoles (Compound-**25**, **RP-11** and **RP-15**) towards these inflammatory cell lines has shown encouraging results (**Figure 5** and **Table 1**). Very interestingly our initial hit, compound-**25** (SUM149PT:

IC50 = 0.80 ± 0.08 μM; SUM190PT: IC50 = 0.43 ± 0.11 μM) and its structural analog **RP-11** (SUM149PT: IC50 = 0.91 ± 0.15 μM; SUM190PT: IC50 = 0.49 ± 0.09 μM) have shown very good inhibition, whereas our TNBC lead **RP-15** (SUM149PT: IC50 = 1.77 ± 0.08 μM; SUM190PT: IC50 = 2.08 ± 0.56 μM) has demonstrated moderate inhibition towards these IBC cell lines. The high potency shown by compound-**25** and **RP-11** towards IBC has given us more insights to develop new anticancer agents for it and we plan to explore the structure-activity relationship (SAR) studies based on the bisbenzimidazole scaffold in very near future.

The Development Therapeutic Program (DTP) of the National Cancer Institute's 60 human tumor cell lines screen was developed as an *in vitro* drug discovery tool. We submitted both compounds (**RP-11** and **RP-15**) to the NCI Developmental Therapeutics Program (DTP) anticancer drug screen. Both of them have been first tested for three cell lines (MCF-7 breast cancer; NCI-H460 large-cell lung cancer; SF-268 glioma) to advance to the 60 cell line screen. This pre-screen process eliminates the inactive compounds but preserves active agents for 60 cell line screening.

destabilized compartments with higher pH elicit blue fluorescence.

towards Huh7.5 cells for 24 hours (**Table 1**).

nal chemists to develop novel (pre) clinical drug candidates.

**3. Results and discussion**

**144**

#### **Figure 4.**

*(A) Staining of acidic compartments: Yellow signal (converted to pseudo-green) represents acidic pH, while the blue color represents slightly acidic to neutral pH. Huh7.5 cells were treated with the compounds (Compound-25, RP-11 and RP-15) at a concentration of 12 μM and standard Bafilomycin A1 at concentration of 2 μM for 20 minutes followed by incubation with Lysosensor Yellow/Blue DND-160 (10 μM) for 10 minutes at 37°C. The cells were visualized under the microscope. The DND-160 is a pH indicator and cellular compartments with acidic pH elicit yellow fluorescence when stained, while the destabilized compartments with higher pH elicit blue fluorescence. The expected yellow color showed yellowish green of the filters available in the microscope. (B) Fold change in overall acidification of Huh7.5 cells upon treatment with bisbenzimidazoles (Compound-25, RP-11 and RP-15) along with positive control BafilomycinA1.*

Both compounds have been advanced to 60 cell lines representing nine major cancers (leukemia, non-small cell lung, central nervous system, colon, melanoma, ovarian, renal, prostate, and breast). Compounds have been tested over a broad range of concentrations against every cell line in the panel (five 10 fold dilutions starting


*‡ Data from Ref. [42].*

*† The IC50 is calculated based on the results obtained from 24 hours drug treatment only.*

#### **Table 1.**

*Half maximal inhibitory concentration of novel bisbenzimidazole analogs in different cancer cell lines.*

**Figure 5.**

*The cell viability (%) of breast cancer cell lines (SUM190PT and SUM149PT) following the exposure of various concentrations of bisbenzimidazoles (Compound-25, RP-11 and RP-15) for 48 hours.*

**147**

**Figure 7.**

**Figure 6.**

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

*)-ATPase Inhibitors*

*Dose response curves derived from screening of compound* **RP-11** *(NSC: D-800436) in 60 cell line screen using nine major human cancer cell lines (leukemia, non-small cell lung cancer, colon cancer, CNS cancer,* 

*The mean graph representation of antitumor effects of compound RP-11 (NSC: D-800436). The GI50 (50% of growth inhibition), TGI (total growth inhibition) and LC50 (50% of lethal concentration) mean graphs are* 

*derived from the dose response curves using Figure 6 from the initial screening.*

*melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer).*

*Bisbenzimidazoles: Anticancer Vacuolar (H+ )-ATPase Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.85231*

#### **Figure 6.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

*The IC50 is calculated based on the results obtained from 24 hours drug treatment only.*

**SUM149PT SUM190PT MDA-MB-468‡ MCF10A‡ Cis-A2780‡ Huh7.5†**

**C-25** 0.80 ± 0.08 0.43 ± 0.11 0.72 ± 0.08 1.14 ± 0.13 3.95 ± 0.33 17.1 ± 0.85 **RP-11** 0.91 ± 0.15 0.49 ± 0.09 0.56 ± 0.05 1.55 ± 0.04 3.03 ± 0.18 17.0 ± 0.78 **RP-15** 1.77 ± 0.08 2.08 ± 0.56 0.04 ± 0.02 1.62 ± 0.14 1.34 ± 0.14 16.4 ± 0.65 **Baf A1** ND ND ND 0.036 ± 0.04 0.008 ± 0.01 ND

*Half maximal inhibitory concentration of novel bisbenzimidazole analogs in different cancer cell lines.*

**Compd. IC50 ± SD (μM)**

*ND: not determined.*

*Data from Ref. [42].*

*‡*

*†*

**Table 1.**

**146**

**Figure 5.**

*The cell viability (%) of breast cancer cell lines (SUM190PT and SUM149PT) following the exposure of* 

*various concentrations of bisbenzimidazoles (Compound-25, RP-11 and RP-15) for 48 hours.*

*Dose response curves derived from screening of compound* **RP-11** *(NSC: D-800436) in 60 cell line screen using nine major human cancer cell lines (leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer).*

#### **Figure 7.**

*The mean graph representation of antitumor effects of compound RP-11 (NSC: D-800436). The GI50 (50% of growth inhibition), TGI (total growth inhibition) and LC50 (50% of lethal concentration) mean graphs are derived from the dose response curves using Figure 6 from the initial screening.*

from 10<sup>−</sup><sup>4</sup> M concentration). **Figures 6** and **8** describe the dose response curves for compounds **RP-11** (NSC: D-800436) and **RP-15** (NSC: D-800437) respectively. From these dose response curves three end points were calculated (GI50: 50% of growth inhibition; TGI: total growth inhibition; LC50: 50% of lethal concentration). **Figures 7** and **9** demonstrate mean graph patterns for compound **RP-11** and **RP-15** respectively. Mean graphs are created for GI50, TGI, and LC50 by plotting positive and negative values termed as deltas generated from dose response curves. More sensitive cell lines are displayed as bars that project to the right of the mean, whereas the less sensitive cell lines are displayed with bars projected to the left. The length of each bar is proportional to the relative sensitivity of the agent with the mean determination.

Both bisbenzimidazole analogs, **RP-11** and **RP-15** demonstrated very good cytotoxicity towards the majority of cancer cell lines in the 60 cell line panel. Compound **RP-11** displayed growth inhibition and total growth inhibition to low micromolar range and is moderate towards LC50 for MCF7 (GI50: 0.32 μM, TGI: 11.8 μM and LC50: 88.7 μM), MDA-MB-468 (GI50: 1.42 μM, TGI: 4.16 μM and LC50: 28.2 μM) and MDA-MB-231 (GI50: 2.25 μM, TGI: 6.49 μM and LC50: 60.4 μM). Interestingly, it showed low micromolar range effects against other cell lines such as SR (GI50: 0.50 μM); NCI-H522 (GI50: 0.34 μM); COLO 205 (GI50: 0.37 μM); SF-268 (GI50: 0.58 μM); OVCAR-3 (GI50: 0.62 μM) and MDA-MB-435 (GI50: 0.62 μM) (**Table 2**). Compound **RP-15** shows similar behavior as **RP-11**. Compound **RP-15** exhibited GI50: 1.91 μM, TGI: 4.13 μM and LC50: 8.91 μM for the MDA-MB-468 cell line, whereas a similar trend is observed for the MDA-MB-231 cell line (GI50: 2.85 μM, TGI: 5.83 μM and LC50: 21.3 μM). Similarly, low micromolar growth inhibition was observed for other cell lines such as MDA-MB-435 (GI50: 1.97 μM), RXF

#### **Figure 8.**

*Dose response curves derived from screening of compound RP-15 (NSC: D-800437) in 60 cell line screen using nine major human cancer cell lines (leukemia, non small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer).*

**149**

**Figure 9.**

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

*)-ATPase Inhibitors*

*The mean graph representation of antitumor effects of compound RP-15 (NSC: D-800437). The GI50 (50% of growth inhibition), TGI (total growth inhibition) and LC50 (50% of lethal concentration) mean graphs are* 

**Cell line RP-11 (μM) RP-15 (μM) Cell line RP-11 (μM) RP-15 (μM)**

CCRF-CEM 1.02 >100 >100 2.72 7.34 92.8 SK-MEL-2 1.64 3.59 7.89 19.7 3.5 75.0 HL-60(TB) 1.62 91.8 >100 4.71 29.1 100 SK-MEL-28 0.478 2.21 6.08 3.03 9.43 40.1 K-562 0.91 >100 >100 2.24 4.45 – SK-MEL-5 0.351 1.88 5.06 12.7 27.6 59.8 MOLT-4 2.81 >100 >100 3.12 10.7 100 UACC-257 1.71 5.92 35.9 20.5 38.3 71.4 RPMI-8226 4.11 35.5 >100 2.86 7.44 100 UACC-62 0.36 2.02 6.37 17.3 40.1 93.1

Non-small cell Lung IGROV1 1.71 4.81 >100 7.69 31.6 100 A549/ATCC 8.05 35.2 >100 4.69 17.7 88.6 OVCAR-3 0.62 3.13 57.0 5.22 16.8 46.7 EKVX 6.56 46.3 >100 4.67 21.4 81.4 OVCAR-4 0.313 2.99 >100 10.1 25.5 64.0 HOP-62 1.56 14.5 >100 7.10 26.8 89.0 OVCAR-5 3.59 9.96 >100 2.96 9.96 33.8 HOP-92 2.16 9.71 >100 14.3 33.2 77.0 OVCAR-8 0.931 23.4 >100 4.69 18.6 81.5

NCI-H23 1.21 5.09 57.2 15.1 33.0 71.9 SK-OV-3 2.70 15.6 >100 18.0 36.0 72.0

SR 0.50 21.9 >100 2.90 8.05 100 Ovarian cancer

**GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50**

0.62 2.14 5.66 1.97 3.99 8.10

>100 >100 >100 35.7 >100 >100

MB-435

ADR-RES

*derived from the dose response curves using Figure 8 from the initial screening.*

Leukemia MDA-

NCI-H226 1.44 – >100 23.8 52.2 100 NCI/

#### *Bisbenzimidazoles: Anticancer Vacuolar (H+ )-ATPase Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.85231*


#### **Figure 9.**

*Chemistry and Applications of Benzimidazole and its Derivatives*

M concentration). **Figures 6** and **8** describe the dose response curves for

compounds **RP-11** (NSC: D-800436) and **RP-15** (NSC: D-800437) respectively. From these dose response curves three end points were calculated (GI50: 50% of growth inhibition; TGI: total growth inhibition; LC50: 50% of lethal concentration). **Figures 7** and **9** demonstrate mean graph patterns for compound **RP-11** and **RP-15** respectively. Mean graphs are created for GI50, TGI, and LC50 by plotting positive and negative values termed as deltas generated from dose response curves. More sensitive cell lines are displayed as bars that project to the right of the mean, whereas the less sensitive cell lines are displayed with bars projected to the left. The length of each bar is proportional to the relative sensitivity of the agent with the mean determination. Both bisbenzimidazole analogs, **RP-11** and **RP-15** demonstrated very good cytotoxicity towards the majority of cancer cell lines in the 60 cell line panel. Compound **RP-11** displayed growth inhibition and total growth inhibition to low micromolar range and is moderate towards LC50 for MCF7 (GI50: 0.32 μM, TGI: 11.8 μM and LC50: 88.7 μM), MDA-MB-468 (GI50: 1.42 μM, TGI: 4.16 μM and LC50: 28.2 μM) and MDA-MB-231 (GI50: 2.25 μM, TGI: 6.49 μM and LC50: 60.4 μM). Interestingly, it showed low micromolar range effects against other cell lines such as SR (GI50: 0.50 μM); NCI-H522 (GI50: 0.34 μM); COLO 205 (GI50: 0.37 μM); SF-268 (GI50: 0.58 μM); OVCAR-3 (GI50: 0.62 μM) and MDA-MB-435 (GI50: 0.62 μM) (**Table 2**). Compound **RP-15** shows similar behavior as **RP-11**. Compound **RP-15** exhibited GI50: 1.91 μM, TGI: 4.13 μM and LC50: 8.91 μM for the MDA-MB-468 cell line, whereas a similar trend is observed for the MDA-MB-231 cell line (GI50: 2.85 μM, TGI: 5.83 μM and LC50: 21.3 μM). Similarly, low micromolar growth inhibition was observed for other cell lines such as MDA-MB-435 (GI50: 1.97 μM), RXF

*Dose response curves derived from screening of compound RP-15 (NSC: D-800437) in 60 cell line screen using nine major human cancer cell lines (leukemia, non small cell lung cancer, colon cancer, CNS cancer,* 

*melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer).*

from 10<sup>−</sup><sup>4</sup>

**148**

**Figure 8.**

*The mean graph representation of antitumor effects of compound RP-15 (NSC: D-800437). The GI50 (50% of growth inhibition), TGI (total growth inhibition) and LC50 (50% of lethal concentration) mean graphs are derived from the dose response curves using Figure 8 from the initial screening.*



HT29 0.58 >100 >100 1.78 4.07 9.30 UO-31 29.4 >100 >100 21.3 41.8 82.3 SW-620 0.90 10.2 36.5 2.12 3.96 7.37 PC-3 2.19 24.5 >100 2.63 5.81 21.8 CNS cancer DU-145 1.13 9.59 >100 1.83 3.40 6.29 SF-268 0.58 5.66 62.9 6.58 25.4 81.0 Breast cancer SF-295 1.38 15.7 >100 2.34 6.22 48.4 MCF7 0.32 11.8 88.7 1.85 3.96 – SF-539 1.22 2.83 6.59 11.5 26.1 59.5 MDA-MB-231/ATCC 2.25 6.49 60.4 2.85 5.83 21.3 SNB-19 1.87 10.5 52.9 2.15 4.09 7.79 HS578T 2.94 15.9 >100 10.5 37.1 >100 SNB-75 0.49 3.14 15.2 3.46 18.0 47.0 BT-549 1.76 3.96 8.91 17.5 40.1 91.7 U251 0.66 10.2 36.5 2.16 3.81 6.74 T-47D 1.00 9.09 >100 14.2 41.1 >100 Melanoma MDA-MB-468 1.42 4.16 28.2 1.91 4.13 8.91 LOX IMVI 0.85 2.83 8.66 1.84 3.67 – MALME-3 M 0.16 1.55 4.61 12.2 27.2 60.4 M14 0.45 2.50 9.80 2.39 6.53 40.0

#### **Table 2.**

*The NCI 60 cancer cell line screening results.*

393 (GI50: 1.91 μM), HT29 (GI50: 1.78 μM), LOXIMVI (GI50: 1.84 μM), DU-145 (GI50: 1.83 μM) and KM12 (GI50: 1.91 μM) (**Table 2**). Overall, the NCI 60 cell line results are encouraging for both new bisbenzimidazole derivatives.

#### **4. Conclusions and future directions**

In summary, our screening and drug discovery processes have identified the bisbenzimidazole (**RP-15**) as a potent anticancer V-ATPase inhibitor for TNBC and **RP-11** as initial lead for the IBC. The compound **RP-15** showed maximum inhibition of the proton-pump activity which is comparable to our standard agent Bafilomycin A1. The *in vitro* antiproliferative activity of these bisbenzimidazole analogs (Compound-**25**, **RP-11** and **RP-15**) towards IBC cell lines revealed that compound-**25** and its structural analog **RP-11** could be possibly considered for further exploration in other IBC cell lines. Bisbenzimidazoles **RP-11** (NSC: D-800436) and **RP-15** (NSC: D-800437) have demonstrated very good cytotoxicity towards the majority of cancer cell lines in the NCI 60 cell line panel. Overall, our research identified efficacious and selective anticancer V-ATPase inhibitors for TNBC and

**151**

**Author details**

Renukadevi Patil1

Kenneth Beaman2

OH, USA

provided the original work is properly cited.

, Olivia Powrozek<sup>2</sup>

University of Medicine and Science, North Chicago, IL, USA

Franklin University of Medicine and Science, North Chicago, IL, USA

\*Address all correspondence to: shivaputra.patil@rosalindfranklin.edu

, Gulam Waris2

*Bisbenzimidazoles: Anticancer Vacuolar (H+*

testing in TNBC and IBC patients.

**Acknowledgements**

**Conflict of interest**

*DOI: http://dx.doi.org/10.5772/intechopen.85231*

*)-ATPase Inhibitors*

IBC. We will continue to explore the SAR with this exciting pharmacophore to identify the highly selective and potent V-ATPase inhibitors which will ultimately lead to the generation of investigational new drug (IND) candidates for the clinical

The screening of **RP-11** and **RP-15** against 60 human cancer cell lines of NCI's

development therapeutic program (DTP) is greatly acknowledged. Rosalind Franklin University of Medicine and Science University start-up grant to

The authors declare no conflict of interest, financial or otherwise.

NSW. National Institute of Health grant (DK106244) to GW.

© 2019 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,

, Binod Kumar2

1 Pharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin

2 Department of Microbiology and Immunology, Chicago Medical School, Rosalind

3 Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati,

, Neelam Sharma-Walia2

, William Seibel3

,

and Shivaputra Patil1

\*

*Bisbenzimidazoles: Anticancer Vacuolar (H+ )-ATPase Inhibitors DOI: http://dx.doi.org/10.5772/intechopen.85231*

IBC. We will continue to explore the SAR with this exciting pharmacophore to identify the highly selective and potent V-ATPase inhibitors which will ultimately lead to the generation of investigational new drug (IND) candidates for the clinical testing in TNBC and IBC patients.

#### **Acknowledgements**

*Chemistry and Applications of Benzimidazole and its Derivatives*

**Cell line RP-11 (μM) RP-15 (μM) Cell line RP-11 (μM) RP-15 (μM)**

NCI-H460 3.63 14.7 >100 2.12 3.98 7.44 786.0 1.64 3.81 8.86 2.70 8.33 >100 NCI-H522 0.34 2.36 >100 13.6 38.1 >100 A498 1.21 3.39 9.54 17.5 35.1 70.3 Colon cancer ACHN 10.7 >100 >100 9.20 22.8 54.2 COLO 205 0.37 3.05 >100 12.9 30.0 69.8 CAKI-1 2.25 >100 >100 4.15 15.7 47.2 HCC-2998 1.78 3.78 8.03 5.06 18.5 56.7 RXF 393 1.80 3.63 7.32 1.91 3.84 – HCT-116 0.36 2.46 >100 1.82 3.44 – SN12C 1.42 11.8 >100 4.00 15.4 66.2 HCT-15 38.7 >100 >100 12.1 28.1 65.4 TK-10 4.74 40.2 >100 18.2 38.8 83.0 HT29 0.58 >100 >100 1.78 4.07 9.30 UO-31 29.4 >100 >100 21.3 41.8 82.3

SW-620 0.90 10.2 36.5 2.12 3.96 7.37 PC-3 2.19 24.5 >100 2.63 5.81 21.8 CNS cancer DU-145 1.13 9.59 >100 1.83 3.40 6.29

SF-295 1.38 15.7 >100 2.34 6.22 48.4 MCF7 0.32 11.8 88.7 1.85 3.96 –

SNB-19 1.87 10.5 52.9 2.15 4.09 7.79 HS578T 2.94 15.9 >100 10.5 37.1 >100 SNB-75 0.49 3.14 15.2 3.46 18.0 47.0 BT-549 1.76 3.96 8.91 17.5 40.1 91.7 U251 0.66 10.2 36.5 2.16 3.81 6.74 T-47D 1.00 9.09 >100 14.2 41.1 >100

231/ATCC

MB-468

2.25 6.49 60.4 2.85 5.83 21.3

1.42 4.16 28.2 1.91 4.13 8.91

NCI-H322M 3.07 9.93 >100 11.6 23.8 48.8 Renal cancer

KM12 2.58 14.3 65.4 1.91 4.20 9.26 Prostate cancer

SF-268 0.58 5.66 62.9 6.58 25.4 81.0 Breast cancer

SF-539 1.22 2.83 6.59 11.5 26.1 59.5 MDA-MB-

Melanoma MDA-

LOX IMVI 0.85 2.83 8.66 1.84 3.67 – MALME-3 M 0.16 1.55 4.61 12.2 27.2 60.4 M14 0.45 2.50 9.80 2.39 6.53 40.0

*The NCI 60 cancer cell line screening results.*

**GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50**

393 (GI50: 1.91 μM), HT29 (GI50: 1.78 μM), LOXIMVI (GI50: 1.84 μM), DU-145 (GI50: 1.83 μM) and KM12 (GI50: 1.91 μM) (**Table 2**). Overall, the NCI 60 cell line results

In summary, our screening and drug discovery processes have identified the bisbenzimidazole (**RP-15**) as a potent anticancer V-ATPase inhibitor for TNBC and **RP-11** as initial lead for the IBC. The compound **RP-15** showed maximum inhibition of the proton-pump activity which is comparable to our standard agent Bafilomycin A1. The *in vitro* antiproliferative activity of these bisbenzimidazole analogs (Compound-**25**, **RP-11** and **RP-15**) towards IBC cell lines revealed that compound-**25** and its structural analog **RP-11** could be possibly considered for further exploration in other IBC cell lines. Bisbenzimidazoles **RP-11** (NSC: D-800436) and **RP-15** (NSC: D-800437) have demonstrated very good cytotoxicity towards the majority of cancer cell lines in the NCI 60 cell line panel. Overall, our research identified efficacious and selective anticancer V-ATPase inhibitors for TNBC and

are encouraging for both new bisbenzimidazole derivatives.

**4. Conclusions and future directions**

**150**

**Table 2.**

The screening of **RP-11** and **RP-15** against 60 human cancer cell lines of NCI's development therapeutic program (DTP) is greatly acknowledged. Rosalind Franklin University of Medicine and Science University start-up grant to NSW. National Institute of Health grant (DK106244) to GW.

#### **Conflict of interest**

The authors declare no conflict of interest, financial or otherwise.

#### **Author details**

Renukadevi Patil1 , Olivia Powrozek<sup>2</sup> , Binod Kumar2 , William Seibel3 , Kenneth Beaman2 , Gulam Waris2 , Neelam Sharma-Walia2 and Shivaputra Patil1 \*

1 Pharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA

2 Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA

3 Division of Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

\*Address all correspondence to: shivaputra.patil@rosalindfranklin.edu

© 2019 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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10.1074/jbc.M404638200

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type H+

**154**

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Section 4

Benzimidazole Derivatives

in Chemistry of Materials

Section 4
