Chalcones: Potential Anticancer Agents

*Adam McCluskey and Cecilia Russell*

## **Abstract**

Chalcones in their various guises have been considered either valid and critically important lead compounds in the development of novel anticancer agents or as pan assay interference compounds, PAINS. Medicinal chemistry is replete with exemplars from both "camps" progressing to clinical utility. Chalcones offer a simple starting point for the development of specific compounds with high levels of activity toward key biological targets. Chalcones have been shown to display a wide array of anticancer compounds. This chapter seeks to offer an overview of key examples in an effort to encourage further reading and research in development in this intriguing space.

**Keywords:** chalcones, biologically active, cancer, structure activity relationship data

### **1. Introduction**

Arguably, cancer represents one of the most serious threats to human health. Its incidence is on the rise, and while there have been an increasing number of new drugs and new targets over the past 50 or so years, it is still responsible for multiple deaths across the globe [1]. The advent of targeted therapies arguably commenced with the discovery and clinical use of the protein kinase inhibitor imatinib [2]. Since this first report, there have been multiple novel protein kinase inhibitor-based drugs entering clinical use [3]. More recently, there has been a significant shift in treatment paradigms to the use of mono-clonal antibodies, with this market predicted to be >\$US300 billion by 2030 [4]. Despite this, the survival rates for metastatic breast cancer (Stage IV, 5-year survival is <25%), for pancreatic cancer this is a more dire 7% [5]. Treatment of glioblastoma and other neurological cancers has not advanced in the past 3–4 decades [6, 7].

#### **2. Biological activity of chalcones**

Chalcones or analogues or derivatives of (*E*)-1,3-diphenyl-2-propene-1-one represent a very diverse array of molecules. This family of molecules are known to possess a myriad of biological activities spanning (but not limited to) antidiabetic, antimicrobial, antioxidant, anti-inflammatory, anticancer and chemopreventative properties [8]. A number of chalcones are in current clinical use, exemplified by the selected analogues shown in **Figure 1** and in other figures throughout this chapter.

Note that the breadth of the potential applications of chalcones in cancer is expansive and beyond the scope of this chapter, the intent here is to supply a snapshot of chalcones and their targets to encourage further exploration, by the reader, of this area [8–10].

biological system. Clearly, Michael acceptors are generally biologically active. Michael acceptor-type compounds are known to be involved in cell signaling cascades and in many cases these compounds are capable of forming covalent attachments to the sulfhydryl of cysteine or other thiols to obtain the Michael adduct **7** (**Figure 3**), which may play an important role in their biological activities [19–23]. Interestingly, the past reservations about small molecules forming covalent linkages with proteins are subsiding with a wide variety of targeted drugs operating via a covalent interaction mode [24]. This may lead to a resurgence in the examination of

Despite the PAINS expectation, there have been a large number of chalcones reported to elicit anticancer activity via specific cell signaling pathways. Of note are those analogues (**8**–**10**) that target the NF-kB pathway. It has been reported that

> ,40 ,50

with its NF-κB inhibitory activity. The reported mechanism of action requires interaction with the IKKb cysteines (46% inhibition; 10 μM) [23] proceeding via

A key feature of chalcone **9** is its ability to synergize with existing clinical treatments. As a combination therapy, **9** and the TNF-related apoptosis-inducing

Within the NF-κB activation pathway the Toll-like receptor 4 (TLR4) and myeloid differentiation 2 (MD2) regulate the downstream signal transduction, such as MAPK phosphorylation. In a LPS-acute lung injury model chalcone **10** inhibited the

The removal of purported PAINS is more frustrating with recent examples where promiscuous inhibitors were not removed or the filters demonstrated an

a JNK-mediated autophagy pathway triggering c-IAP (**Figure 4**) [25].

activity of MD2 reducing the inflammatory effects in this model [28].

*Michael addition of a chalcone (6a) with cysteine to form the Michael adduct 7.*

*Exemplar chalcones known to be NF-κB inhibitors [28].*

ligand (TRAIL) or cisplatin significantly enhanced its cytotoxicity in lung cancer cells. This effect is mediated via the suppression of cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), which in combination activate


chalcones as lead compounds.

*Chalcones: Potential Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.91441*

autophagy [26, 27].

**Figure 3.**

**Figure 4.**

**77**

the anticancer activity of 3-hydroxy-4,30

Despite the numerous examples of clinically used chalcones, they are often overlooked for lead development as a function of PAINS filtering [11]. We note the key role here of the medicinal chemist in understanding both the limitations of the lead scaffold, potential promiscuity and the nature of the biological screening conducted. If the scaffold limitations are understood, there is limited rationale in excluding a whole compound class, especially given the current utility of these analogues. However, vigilance is required in SAR examinations. We recommend the removal of PAINS filters from preliminary screening cascades and the introduction of robust orthogonal assay procedures to enable rapid identification of true lead compounds [12, 13]. In so doing, we believe that this will increase the attractiveness of chalcones as leads; potentially matching their use will greatly increase the attractiveness of chalcones as potential starting points for drug discovery [9, 14].

Historically, chalcones, for example **1**–**5**, have been used in a therapeutic environment for millennia. Typically, through the ingestion of plants and herbs, chalcones have been used in the treatment of a myriad of conditions, spanning but not limited to inflammation, diabetes, and the topic of this chapter, cancer [8, 15–18]. Metochalcone (**1**) and sofalcone (**2**) have been used in the treatment of ulcers and as mucoprotective agents, respectively (**Figure 1**) [15, 16].

Being able to switch between two chalconoid structures (**6a** and **6b**; **Figure 2**) in principle establishes two Michael acceptor possibilities for this class of compounds. It is important to recognize at this point that key researchers view these and molecules such as these as PAINS [11]. As such, caution should be used in determining absolute effects and ascribing them to specific compounds' actions in a

**Figure 1.**

*Chemical structures of selected clinically used chalcones: metochalcone (1), sofalcone (2), isoliquiritigenin, xanthohumol (4) and hesperidin methylchalcone (5).*

**Figure 2.**

*The interplay between the s-cis and s-trans chalconoid structural motifs available to simple chalcones.*

#### *Chalcones: Potential Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.91441*

Note that the breadth of the potential applications of chalcones in cancer is expansive and beyond the scope of this chapter, the intent here is to supply a snapshot of chalcones and their targets to encourage further exploration, by the reader, of this

Despite the numerous examples of clinically used chalcones, they are often overlooked for lead development as a function of PAINS filtering [11]. We note the key role here of the medicinal chemist in understanding both the limitations of the lead scaffold, potential promiscuity and the nature of the biological screening conducted. If the scaffold limitations are understood, there is limited rationale in excluding a whole compound class, especially given the current utility of these analogues. However, vigilance is required in SAR examinations. We recommend the removal of PAINS filters from preliminary screening cascades and the introduction of robust orthogonal assay procedures to enable rapid identification of true lead compounds [12, 13]. In so doing, we believe that this will increase the attractiveness of chalcones as leads; potentially matching their use will greatly increase the attractiveness of chalcones as potential starting points for drug discovery [9, 14]. Historically, chalcones, for example **1**–**5**, have been used in a therapeutic environment for millennia. Typically, through the ingestion of plants and herbs, chalcones have been used in the treatment of a myriad of conditions, spanning but not limited to inflammation, diabetes, and the topic of this chapter, cancer [8, 15–18]. Metochalcone (**1**) and sofalcone (**2**) have been used in the treatment of

ulcers and as mucoprotective agents, respectively (**Figure 1**) [15, 16].

Being able to switch between two chalconoid structures (**6a** and **6b**; **Figure 2**) in principle establishes two Michael acceptor possibilities for this class of compounds. It is important to recognize at this point that key researchers view these and molecules such as these as PAINS [11]. As such, caution should be used in determining absolute effects and ascribing them to specific compounds' actions in a

*Chemical structures of selected clinically used chalcones: metochalcone (1), sofalcone (2), isoliquiritigenin,*

*The interplay between the s-cis and s-trans chalconoid structural motifs available to simple chalcones.*

area [8–10].

*Translational Research in Cancer*

**Figure 1.**

**Figure 2.**

**76**

*xanthohumol (4) and hesperidin methylchalcone (5).*

biological system. Clearly, Michael acceptors are generally biologically active. Michael acceptor-type compounds are known to be involved in cell signaling cascades and in many cases these compounds are capable of forming covalent attachments to the sulfhydryl of cysteine or other thiols to obtain the Michael adduct **7** (**Figure 3**), which may play an important role in their biological activities [19–23]. Interestingly, the past reservations about small molecules forming covalent linkages with proteins are subsiding with a wide variety of targeted drugs operating via a covalent interaction mode [24]. This may lead to a resurgence in the examination of chalcones as lead compounds.

Despite the PAINS expectation, there have been a large number of chalcones reported to elicit anticancer activity via specific cell signaling pathways. Of note are those analogues (**8**–**10**) that target the NF-kB pathway. It has been reported that the anticancer activity of 3-hydroxy-4,30 ,40 ,50 -tetramethoxychalcone (**9**) correlates with its NF-κB inhibitory activity. The reported mechanism of action requires interaction with the IKKb cysteines (46% inhibition; 10 μM) [23] proceeding via a JNK-mediated autophagy pathway triggering c-IAP (**Figure 4**) [25].

A key feature of chalcone **9** is its ability to synergize with existing clinical treatments. As a combination therapy, **9** and the TNF-related apoptosis-inducing ligand (TRAIL) or cisplatin significantly enhanced its cytotoxicity in lung cancer cells. This effect is mediated via the suppression of cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), which in combination activate autophagy [26, 27].

Within the NF-κB activation pathway the Toll-like receptor 4 (TLR4) and myeloid differentiation 2 (MD2) regulate the downstream signal transduction, such as MAPK phosphorylation. In a LPS-acute lung injury model chalcone **10** inhibited the activity of MD2 reducing the inflammatory effects in this model [28].

The removal of purported PAINS is more frustrating with recent examples where promiscuous inhibitors were not removed or the filters demonstrated an

**Figure 3.** *Michael addition of a chalcone (6a) with cysteine to form the Michael adduct 7.*

**Figure 4.** *Exemplar chalcones known to be NF-κB inhibitors [28].*

oversensitivity toward key compound types. That is, these filters may reject non-promiscuous compounds [12, 29].

The use of bioisosteric replacements with chalcones has high prevalence. Commencing with 2,4,6-trimethoxychalcone (**11**) a simple H (**11**) to F (**12**) isosteric replacement effected a 2-fold potency increase against HeLa (cervical cancer), A498 (renal cancer), and HepG2 (hepatocellular carcinoma) cells, with retention of activity against the A549 (lung adenocarcinoma epithelial) and A375 (skin malignant melanoma) cell lines with IC50 values spanning 0.03–0.120 μM (**Table 1**) [30].

Molt-4 (human T-lymphocyte), CEM (human T-lymphocyte), L1210 (murine leukemia), and FM3A cell lines (murine mammary carcinoma) (**Table 2**) [31].

The biological activity of chalcones, and study thereof is not limited to the parent structure, but has recently expanded to encapsulate hybrid (chimeric) molecules. These chimeras combine the cytotoxicity of the parent chalcone (**15**) and the biological activity of the second drug. Multiple chimeric partners have been reported including antibiotics (ciprofloxacin, **16**) linking through the N-aryl piperazine moiety. This allows access to the known inhibition of human DNA topoisomerase II, itself a known anticancer drug target (**Figure 5**) [32]. The chalcone-ciprofloxacin hybrid (**17**) inhibits human DNA topoisomerase II with potent in vitro anticancer activity against myriad of cancer cell lines [33–36]. Chalcones themselves are known to inhibit several anticancer targets, including thioredoxin reductase [21], and tubulin polymerization [37, 38]. Based on this there was an expectation (upheld) that chimeric molecules possessing a N-aryl piperazine and chalcone moieties would show higher potencies in the cell lines examined. Indeed, with these molecules considerable synergy arising from the combination of both partners was observed. Of the analogues reported, hybrid **17** displayed the highest activity against cervical cancer (Hela; IC50 = 190 nM) and gastric cancer (SGC7901; IC50 = 410 nM) cells (**Figure 5**). These data compare favorably with that reported for cisplatin in the same cell lines with IC50 values of 20 and 12 μM, respectively [32]. The introduction of an active warhead has been accomplished through the synthesis of a α-bromoacryloylamido chalcones (**Figure 6**). Analogues of this nature are expected to act as covalent modifiers of their target protein [39]. Intriguingly, this combines the once thought of anathema of a covalent inhibitor with a compound classified as a PAINS [11, 24]. Yet, compounds **18** and **19** exhibit the highest activity against tumor cell growth (IC50 < 1 μM) and 10- to 100-fold increases in potency relative to the corresponding amide derivatives. Preliminary mechanism of action studies support apoptosis induction via mitochondrial

engagement and activation of caspase-3. The related amide-linked dithiocarbamatechalcone (**20**) also exhibited excellent growth inhibition against SK-N-SH cells, with an IC50 value of 2.03 μM, with negligible toxicity toward the normal GES-1 cell line (IC50 > 50 mM). However, this effect is via G0/G1 arrest and progression through apoptosis. The nature of the linking and pendant moieties affects the

*Molecular hybrid obtained from the combination of chalcones (red) and N-aryl piperazine moiety (blue) [32].*

**2.1 Chalcone hybrids**

*Chalcones: Potential Anticancer Agents DOI: http://dx.doi.org/10.5772/intechopen.91441*

compound mode of action [40].

**Figure 5.**

**79**

Isosteric replacements have not been limited to simple Grimm's isosteres, but they have been explored in nonclassical isostere space with the replacement of the central olefin with a small heterocyclic compound, such as the thiophene analogues shown in **Table 2**. These modified chalcones, for example, **14** developed from **13**, displayed good levels of cytotoxicity against a range of cancerous cell lines, with activities noted in the sub-μM to mid-nM range (0.160–0.510 μM) against HeLa,

**Table 1.**

*Effect of H to F bioisosteric modification on the cytotoxicity of a 2,4,6-trimethoxychalcone (11). IC50 values are expressed in μM.*

**Table 2.**

*Bioisosterism represented by the replacing the double bond of the enone (blue) with a thiophene (red) [31]. IC50 values are expressed in μM.*

Molt-4 (human T-lymphocyte), CEM (human T-lymphocyte), L1210 (murine leukemia), and FM3A cell lines (murine mammary carcinoma) (**Table 2**) [31].
