Advances in Chalcones with Anticancer Activities

Send Orders for Reprints to [email protected] Recent Patents on Anti-Cancer Drug Discovery, 2015, 10, 97-115 97 Advances in Chalcones with Anticancer Activities

Chandrabose Karthikeyana, Narayana S. H. Narayana Moorthya,b, Sakthivel Ramasamyc, Uma Vanamc, Elangovan Manivannand, Devarajan Karunagaranc and Piyush Trivedia,* aSchool of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar, Bhopal, MP, 462036, India; bDepartment of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 687, Rua de Campo Alegre, Porto, 4169-007, Portugal; cCancer Biology Laboratory, Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences Building, Indian Institute of Technology Madras, Chennai-600036 (T.N.), India; dSchool of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore 452 017, MP, India

Received: January 7, 2014; Accepted: July 21, 2014; Revised: July 29, 2014 Chandrabose Karthikeyan Abstract: Chalcones are naturally occurring compounds exhibiting broad spectrum biological activities including anticancer activity through multiple mechanisms. Literature on anticancer chalcones highlights the employment of three pronged strategies, namely; structural manipulation of both aryl rings, replacement of aryl rings with heteroaryl scaffolds, molecular hybridization through conjugation with other pharmacologically interesting scaffolds for enhancement of anticancer properties. Methoxy substitutions on both the aryl rings (A and B) of the chalcones, depending upon their positions in the aryl rings appear to influence anticancer and other activities. Similarly, heterocyclic rings either as ring A or B in chalcones, also influence the anticancer activity shown by this class of compounds. Hybrid chalcones formulated by chemically linking chalcones to other prominent anticancer scaffolds such as pyrrol[2,1-c][1,4]benzodiazepines, benzothiazoles, imidazolones have demonstrated synergistic or additive pharmacological activities. The successful application of these three pronged strategies for dis- covering novel anticancer agents based on chalcone scaffold has resulted in many novel and chemically diverse chalcones with potential therapeutic application for many types of cancer. This review summarizes the concerted efforts expended on the design and development of anticancer chalcones recorded in recent literature and also provides an overview of the patents published in this area between 2007 and 2014 (WO2013022951, WO201201745 & US2012029489). Keywords: Anticancer, anticancer chalcones, antioxidant, antiproliferative activity, chalcones, patents.

INTRODUCTION Chalcones are open-chain flavonoids, biosynthesized in a variety of plant species [1]. Chemically, chalcones are 1,3- diphenyl-2-propen-1-ones, which consist of two aromatic rings connected by a three-carbon ,-unsaturated carbonyl system [1]. Chalcone is an exceptional chemical template having multifarious biological activities, which include anticancer [2, 3], anti-inflammatory [4-6], antioxidant [7], antim- icrobial, anti-tubercular [8], anti-HIV [9], antimalarial [10] and anti-allergic activities [11]. For a comprehensive overview of a broad spectrum pharmacological activities displayed by chalcones, the readers are directed to excellent reviews by Dimmock et al. [12], Batovska et al. [13] and Sahu et al. [14]. The anticancer activity of chalcones has been investigated extensively by both the academia and the industry for more than three decades [15]. However, only a few such reports describe the in-depth mechanistic basis of the cytotoxic or anti-proliferative activity exhibited by the chalcones. Chalcones exert cytotoxic activities through multiple mechanisms which include cell cycle disruption, angiogenesis Fig. (1). Schematic diagram for the cell cycle arrest by chalcones.

*Address correspondence to this author at the School of Pharmaceutical Sci- inhibition, tubulin polymerization inhibition, apoptosis in- ences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar, Bhopal, MP, 462036, India; Tel: +91-755-2678883; Fax: +91- duction and blockade of nuclear factor-kappa B (NF-B) 755-2742001; E-mail: [email protected]; [email protected] signaling pathway (Fig. (1)) [1, 2, 16, 17].

H C OR3 O O 2 CH3 CH3 R2O OR1 OH HO H3CO OH

(1) Licochalcone A (2) Xanthohumol R1 = CH3, R2 = H, R3 = H (3) Xanthoangelol

OH O (6) OH O OH H OH HO O O

O OH OH HO OH H (4) 4-hydroxyderricin O OH (5) Butein (6) Garcinol OH O OH R HO O HO OH OH HO OH OH HO O O HO HO O (7) Flavokawain A R = OCH3 (8) Flavokawain B R = H OH O O HO OH O (9) Flavokawain C R = OH HO (10) Hydroxy Safflor Yellow A (11) Broussochalcone A

OH O O O H3CO OH

OCH3 (H3C)2N HO OCH3 O O OCH3O OCH3 O (12) Dimethylaminochalcone (13) Cardamonin (14) HTMC (15) Pyranochalcone from Millettia pachycarpa Fig. (2). Structures of the naturally derived chalcones.

Further, chalcones have also been demonstrated to inhibit class of chalcones are licochalcones, xanthohumol, butein, kinases essential for tumor cell survival and proliferation like etc. (Fig. (2)). epidermal growth factor receptor (EGFR), vascular endothe- Licochalcone A (1) (LA), an oxygenated chalcone found lial growth factor receptor-2 (VEGFR-2) and B-Raf (BRAF) in the roots of the Chinese liquorice (Glycyrrhiza uralensis), kinase [18-20]. There are a number of reviews that had dealt has been demonstrated to possess many bioactive properties with the pharmacological and chemical basis of the antican- including anti-parasitic, estrogenic, antimalarial and antitu- cer activities exhibited by the chalcones [15, 21-23]. How- mor activities [24-26]. LA was also reported to elicit growth ever, the present perspective focuses on the medicinal chem- control and induction of apoptosis in androgen-independent istry strategies employed for the design and development of p53-null PC-3 prostate cancer cells [25, 27]. Xanthohumol anticancer chalcones citing the examples reported in recent (2), a prenylated chalcone isolated from the hop cones (Hu- literature and patents (International, United States and Japa- mulus lupulus L.) is suggested to exhibit broad spectrum nese patent databases) published in the last seven years anticancer properties against different types of human cancer (2007 to 2014). The biological signiﬁcance of these chal- cells primarily through inhibition of the proliferation and cones alongwith their therapeutic potential against different induction of human cancer cell apoptosis [27, 28]. Xantho- types of cancer is also summarized herein. angelol (3), a natural chalcone isolated from stem exudate of Angelica keiskei, induced apoptotic cell death by activation NATURALLY OCCURRING CHALCONES of caspase-3 in neuroblastoma and leukemia cells through a An overall review of the recent literatures reveals a con- mechanism that does not involve Bax/Bcl-2 signal transduc- tinuous search for naturally occurring chalcones with po- tion [29]. 4-Hydroxyderricin (4), another chalcone found in tent anticancer properties and a novel mechanism of action. the roots of Angelica keiskei also induced apoptotic cell Consequently, many naturally occurring chalcones with death in leukemia cells (HL60) via both the death receptor- potent anticancer efficacy against a variety of cancer cell mediated pathway and the mitochondrial pathway by, at least lines have been found. Some prominent examples of this in part, topoisomerase II inhibition [30]. Furthermore, the Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 99 chalcone also exhibited excellent cytotoxic activity against mainly focused on both the phenyl rings (A and B) Fig. (3). four human tumor cell lines (HL60 (leukemia), CRL1579 Similar to naturally occurring chalcones, hydroxy and (melanoma), A549 (lung) and AZ521 (stomach). Butein (3, methoxy groups in specific positions on the phenyl rings fa- 4, 2',4'-tetrahydroxychalone) (5), a plant polyphenol, isolated vors the anticancer activity exhibited by these compounds. from the stems of Rhus verniciflua has been shown to inhibit However, there are also reports which indicate that other human colon adenocarcinoma cell proliferation [31]. An- groups such as dimethylamino, aryl/heteroarylrings either other study by Kim et al. in human leukemia HL-60 cell line fused or substituted are also tolerated. suggested that butein induces apoptosis in HL-60 cells via A recent study found 1,3-diphenyl propenone (16) to be positive modulation of caspase-3 activity associated with potent anti-angiogenic compared to heteroaryl chalcones 3- downregulation of Bcl-2 expression and upregulation of Bax phenyl-1-thiophen-2-yl-propenone (PhT2P) (17), 3-phenyl- expression [32]. Garcinol (6), a tri-isoprenylated hydroxy- 1-thiophen-3-yl-propenone (PhT3P) (18) and 1-furan-2-yl-3- chalcone extracted from the plant Garcinia indica, has been phenyl-propenone (FPhP) (19). The study also established shown to possess anti-inflammatory and anticarcinogenic chalcone as a multitargeted kinase inhibitor with inhibitory activities [33]. The mechanism by which garcinol elicits activity against VEGF receptor tyrosine kinase (weak) and these activities involves modulation of arachidonic acid me- several other receptor tyrosine kinases including Tie-2, epi- tabolism by blocking the phosphorylation of cytosolic dermal growth factor (EGF) receptor, EphB2, fibroblast phoshpolipase A2 (cPLA2) and by decreasing inducible NO growth factor (FGF) receptor 3 and insulin-like growth fac- synthase (iNOS) protein level mediated through inhibition of tor-1 (IGF-1) receptors. Signal Transducers and Activators of Transcription (STAT1) Methoxylated chalcones are structurally similar to com- activation [33, 34]. bretastatin A-4-5 and colchicine due to their similar spatial Flavokawain A, B, and C (7-9) in kava extracts have orientation between two aromatic rings (A and B). Similar to been shown to possess strong antiproliferative and apoptotic combretastatin and colchicine, methoxylated chalcones bind effect in human bladder cancer cells [35]. Flavokawain A to the tubulin effectively (20) [39], hence show prominent causes loss of mitochondrial membrane potential and release anticancer activity against various cancer cell lines. Studies of cytochrome C into the cytosol in an invasive bladder can- on these methoxylated chalcones suggested that the number cer cell line T24. Hydroxy safflor yellow A (10) (HSYA), a and the position of methoxy substituents on the aromatic pigment from the flower petals of Carthamus tinctorius, has rings appeared to be critical for cytotoxicity. The antimitotic an inhibitory effect on the blood vessel growth of trans- effect of chalcones is dependent on the substitutions at the B- planted gastric adenocarcinoma [1]. Xi et al. described that ring, especially in 2-, 4-, and 6-positions. In case of di- downregulation of the VEGF and basic fibroblast growth methoxylated derivatives, the methoxy groups should pref- factor (bFGF) mRNA expression as the underlying mecha- erably be linked to carbons 2 and 6 in the B ring [40]. How- nism for anti-angiogenic activity exhibited by HSYA [36]. ever, in case of the trimethoxylated chalcones, methoxylation th Other natural chalcones such as broussochalcone A (11), at 3, 4 and 5 position of the B ring is favored. 3,4,5- dimethylaminochalcones (12) and cardamonin (13) have Trimethoxychalcones have been shown to elicit prominent been reported to possess anti-inflammatory and anticancer anticancer activity against five cancer cell lines (ACHN activities [24]. 2-Hydroxy-2,3,4,6-tetramethoxychalcone (human renal cell adenocarcinoma Panc 1 (human pancreatic (14) (HTMC), a natural chalcone from a medicinal plant carcinoma), Calu 1 (human non-small cell lung carcinoma), Caesalpinia pulcherrima showed potent in vitro cytotoxicity H460 (human non cell lung carcinoma), HCT116 (human selectively against cell lines derived from human lung cancer colon carcinoma)) and their inhibitory activities were found [37]. HTMC was found to cause G1 phase cell-cycle arrest in to be more or less similar with flavopiridol and gemcitabine. Furthermore, studies by Srinivasan et al. show that ((E)-1- A549 lung adenocarcinoma cells. Ye et al. isolated a novel (3,4,5-trimethoxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en- pyranochalcone (15) from Millettia pachycarpa, which 1-one) (21) also potently inhibits NF-B activation in lung showed strong cytotoxic effects against several cancer cell cancer cells (A549) via inhibition of IKK and/or interleukin- lines [38]. 1 receptor-associated kinase 4 (IRAK4) [2]. Another study by Tu et al. revealed that 2,5-dimethoxylchalcone derivatives SYNTHETICALLY DERIVED CHALCONES (22 and 23) exhibited signiﬁcant cytotoxic effects on two hu- The successful implication of naturally occurring chal- man cancer cell lines, NTUB1 (human bladder cancer cell cones as probable anticancer agents has inspired numerous line) and PC3 (human prostate cancer cell line) [41]. synthetic efforts to develop novel synthetic chalcones with Few unconventional chalcones which differ in the substi- anticancer properties. Such efforts generally deploy a three tution pattern or basic structural framework have also been pronged strategy, namely; structural manipulation of both aryl reported for anticancer activity (Fig. (3)). Lorenzo and co- rings, replacement of aryl with heteroaryl/alicyclic/steroidal workers reported a series of adamantyl arotinoids (24) scaffolds and molecular hybridization through conjugation (AdArs) structurally related to MX781 (25) with strong with other pharmacologically interesting scaffolds for en- growth inhibitory activity against prostate (PC-3) and breast hancement of anticancer properties. Diphenyl-2-propen-1- (MDA-MB-468) carcinoma cells [42]. The anti-proliferative ones (also known as chalcones) being the simplest are the activity shown by AdArs correlated with increased inhibition most widely studied chalcones for anticancer properties. of recombinant IKK in vitro, suggesting that the activity Structural manipulations in diphenyl-2-propen-1-ones were might be related to the inhibition of IKK/NFB signaling. 100 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al.

A B S R2 O O R1 R3 O (16) DPhP (17)PhT2P B O R4 HO ro H3CO A S R5 HO O O OH O O O O (18) PhT3P (19) FPhP Aryl/Heteroaryl Chalcones (20) Methoxylated Chalcones (21)

O R O H (22), R = N O O O COOH O O OR (23), R = N (24) 2',5'-dimethoxychalcones

O N CF O R1 = CH3 2 3 O O R2 = H R = Cl 3 O O OH NO O O R 2 O O 3 O COOH O R (25) MX781 1 R2 (26) (27) SD400

O O R1 O R2

(28) Biaryl Chalcones (29) Biphenyl Chalcones Fig. (3). Structure of synthetically derived chalcones.

Zhang et al. recently reported a series of unconventional A series of biaryl chalcones (28) which combines a basic chalcones, which incorporate a 2,6-dinitro-4-(trifluoromethyl) chalcone framework with a biphenyl moiety has also been phenoxy moiety in the ortho position of the phenyl ring B as found to exhibit significant NF-kB inhibitory activity and potential inhibitors of tubulin [43]. Among the reported cytotoxicity against a panel of cancer cell lines [17]. Further compounds, 3-(3-chloro-2-(2,6-dinitro-4-(trifluoromethyl)phen- exploration on this class of chalcones revealed that the oxy)phenyl)-1-p-tolylprop-2-en-1-one (26) showed the most biphenyl chalcones (29) preferentially inhibited the growth potent antiproliferative activity against MCF-7 and A549 cell of the aggressive human breast cancer cell lines, MDA-MB- -1 lines with IC50 values of 0.03 and 0.95gmL respectively. 231 in the range of 4.4 - 30M [46]. The mode of action of The compound also inhibited the polymerization of tubulin these compounds was established to be apoptosis, cell cycle -1 with IC50 of 1.42gmL . arrest and loss of mitochondrial membrane potential [47]. Ducki and co-workers while studying a novel series of - methyl chalcones structurally analogous to combretastatin CHALCONES WITH HETEROARYL MOIETIES A4 (CA4) for anticancer and antimitotic activity (SD400 A plethora of reports employing heteroaryl groups either (27)). The chalcone SD400 was also able to inhibit tubulin in ring A or B is recorded in the literature (Fig. (4)). A series assembly at a higher degree than the reference compounds of novel thiazolyl-chalcones was found to exhibit potent CA4 (IC50 tubulin = 3.3M) and SD400 (IC50 tubulin = growth inhibitory activity on four human cancer cell lines 3.7M) [44, 45]. (BGC-823, PC-3, NCI-H460 and BEL-7402) [48]. In vivo Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 101

N O Br O NO2 O R2 O S S S X R R1 O O X = S/O O O N O (30) R1 = 3,4-Cl; R2 = 3,4,5-OCH3 R (34) R = H/CH (31) R1 = 3-Cl, 4-F; R2 = 3,4,5-OCH3 (32) X = O and R = 4-Cl (33) 3 (35)Indolyl Chalcones Thiazolyl–chalcones Thiophenyl and Furanyl Chalcones Thiophenyl Chalcones

R O O O O O O O O N

O HN O

O O N N O N H (40), R = H N R JAI-51 (36) (39) MIPP Indole Retinoid derivatives (37) (38)

O O O O

R R O R1 (43), Ar = N H C N O 3 H S X N Cl N Br (44), Ar = S N R (45), R = 4-Cl Ar (46), R = 4- OCH3 1 (42) (41) R = CF3, R = 3, 4 di OMe & X = CH O (47), R = 4- OH Imidazopyridine/pyrimidine-chalcones Quinolyl-thienyl Chalcones 3-phenylquinolinylchalcone derivatives Quinolone Chalcones

O O O N OH O R O

N R O O N O O N H H (49), R = 4- OCH 3 O O (50), R = 2,5-OCH3 (48) (51), R = 3,4-OCH3 (53) (54), R = 2,3,4-OCH3 (52), R = 2 4-OCH3 Quinoxaline-derived chalcones Coumarin Chacones 3-cinnamoyl-4-hydroxy-2H-chromen-2-ones.

R1O

O O O O O R2 O O O O

A B R O 1 2 O O O N (59), R = H, R = N (C2H5)2 O 1 2 (60), R = CH3CO, R = N (C2H5)2 (71) (55) (56), R = 3 NO2 5-hydroxyaurone derivatives Methoxylated aurones Millepachine derivatives (57),R = 4 NO2 (58), R = 4 CN Chromanochalcones Fig. (4). Structures of chalcones with heteroaryl moieties. antitumor activity of two compounds (30 and 31) was evalu- yl)prop-2-en-1-one (34) demonstrated higher cytotoxicity ated in ICR mice bearing sarcoma 180 tumors and the results than other chalcones in cell morphology, live/dead and MTT indicated that the compounds 30 and 31 have moderate assays. Additionally, compound 33 was also found to induce in vivo activity with 22-25% tumor weight inhibition. Solo- apoptosis in flow cytometry annexin V assay. mon et al. compared the antiproliferative activity of aryl Indolyl chalcones with a 3,4,5-trimethoxy substitution in thiophenyl chalcones with aryl furanyl chalcones (Fig. (4)) in phenyl ring A (35) were reported to exhibit significant cyto- breast cancer cells [49]. Their results suggested that the thio- toxic activity against pancreatic cancer cell lines [51]. An- phene chalcones effectively inhibited the proliferation of other study by Martel-Frachet and co-workers established MDA-MB231 while the furan chalcones exhibited potent an- 1-(N-methylindolyl)-3-phenylpropenones (36) specifically tiproliferative activity on MDA-MB468 breast cancer cells. methoxylated analogues as effective anticancer agents acting Compound ((E)-1-(4-chlorophenyl)-3-(5-(4-methoxyphenyl) 32 on bladder carcinoma cells [52]. N-Methyl-1-indolyl chal- furan-2-yl)prop-2-en-1-one) was considered to be most po- cones (37) have also been shown to exhibit potent inhibitory tent in the series since its antiproliferative activity was 3 to activity on breast cancer resistance protein (BCRP/ABCG2), 7-fold higher on cancer cells than non-cancer cells. which binds and extrudes the anticancer drugs out of the A recent report by de Vasconcelos et al. studied the cyto- cells, thereby creating drug resistance [53]. JAI-51, N-methyl toxic and apoptotic effects of six chalcone derivatives incor- indolylchalcone (38) inhibited proliferation of glioblastoma porating a thiophene ring on human colon adenocarcinoma cells through microtubule depolymerization. In vivo investi- cells [50]. Chalcones, 3-(4-bromophenyl)-1-(thiophen-2- gations performed on C57BL/6 mice bearing GL26 glioblas- yl)prop-2-en-1-one (33) and 3-(2-nitrophenyl)-1-(thiophen-2- toma xenografts showed that the compound induced a delay 102 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al. in tumor onset [54]. Another series of indolyl chalcones (39, arrest. Furthermore, compound 50 was also found to inhibit 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-ones AKT activation, allied to the stimulation of ERK MAP-kinase. (MIPP) was reported as inducers of methuosis in glioblas- Coumarins are an interesting class of compounds en- toma and other types of cancer cells [55]. dowed with broad spectrum antitumor activities, hence chal- Gurkan-Alp et al. studied anticancer properties of the cones employing coumarinyl moiety as a substitute for ring novel (E)-3-(5-substituted-1H-indol-3-yl)-1-(5,5,8,8-tetrame- A or B have been reported in literature for anticancer proper- thyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one deri- ties. Sashidhara et al. synthesized and evaluated a series of vatives (Fig. (4)) (40), which combines both indole and reti- coumarin-chalcone hybrids for their in vitro cytotoxicity noid components in a chalcone structural framework [56]. against a panel of four human cancer cell lines and normal Most of the novel indole retinoid compounds studied were fibroblasts (NIH3T3) [62]. Compound 53 was found to be found to possess the antiproliferative capacity in liver, breast the most promising in the series with 30-fold more selectiv- and colon cancer cell lines. ity towards C33A (cervical carcinoma) cells over normal fibroblast NIH3T3 cells with an IC50 value of 3.59M. A novel series of isatin linked chalcones (Fig. (4)) incorporating pharmacophoric elements of isatins and chalcones was Patel and co-workers evaluated a series of 4-hydroxy reported for antibreast cancer activity by Karthikeyan et al. coumarinyl chalcones (Fig. (4)) for anti-breast cancer activ- [57]. Chalcones with imidazo[2,1-b]pyridine/pyrimidines (Fig. ity using breast cancer cell lines (MDA-MB231, MDA- (4)) as ring B have been shown to possess promising anti- MB468, MCF7) and one non-cancerous breast epithelial cell cancer activity against NCI panel of cancer cells [58]. More line (184B5) [63]. Their results indicated that coumarinyl specifically, compound 41 which possesses a trifluoromethyl chalcones especially compound 54 with 2,3,4-trimethoxy substituent in ring B and a 3,4,5-trimethoxy substitution in substitution in aryl ring B showed comparable potency to the ring A exhibited good antiproliferative potency at submi- reference drug cisplatin as well as a two-fold higher selectiv- cromolar concentrations against leukemia, lung, colon, ity for the breast cancer cell lines than 184B5 cells. breast, ovarian and melanoma cancer cells. Wang et al. synthesized 38 derivatives of Millepachine, a Rizvi et al. discovered a series of quinolyl-thienyl chal- naturally occurring pyranochalcone isolated from Millettia pachycarpa and evaluated their in vitro and in vivo antipro- cones (42) as potential VEGFR-2 kinase inhibitors through structure based virtual screening protocols [19]. One of the liferative activities against a panel of six cancer cell lines (HepG2, K562, SK-OV-3, HCT116, HT29 and SW620) identified quinolyl-thienyl chalcones exhibited in vitro [64]. Their synthetic efforts were mainly focused on optimiz- VEGFR-2 kinase inhibitory activity and inhibition of HUVEC ing the substitution in the aryl rings and among the different proliferation at nanomolar concentrations. 3-Phenylquinolinyl- substitutions tried, compound 55 which incorporates a di- chalcone derivatives (Fig. (4)) with potent antiproliferative ethyl amino group in the para position of phenyl ring B ef- activity against a panel of six cancer cell lines were reported fectively inhibited tubulin polymerization in HepG2 cells by Tseng et al. [59]. Two compounds in the series; (E)-3-(3- and induced the HepG2 cell cycle arrest at the G2/M phase (4-methoxyphenyl)quinolin-2-yl)-1-phenylprop-2-en-1-one in a concentration-dependant manner. Murthy et al. studied (43) and (E)-1-(5-bromothiophen-2-yl)-3-(3-(4-methoxyphenyl) the antiproliferative activity of a novel class of chromano- quinolin-2-yl)prop-2-en-1-one ( ) were found to exhibit 44 chalcones (Fig. (4)) against the human cancer cell lines of most potent anticancer activities. diverse origin [65]. Structure activity relationship studies Abonia et al. recently reported a series of novel quino- suggested that substitutions on the aryl ring B significantly line-2-one based chalcones (Fig. (4)) and their anticancer affected the potency. Three compounds 56, 57 and 58 with activity against NCI panel of 60 different human tumor cell electron withdrawing groups (NO2 and CN) in aryl ring B lines [60]. Four compounds among the nine compounds were found to display excellent cytotoxic profile by inhibit- screened for anticancer activity (45-48) exhibited the good ing greater than 90% cell proliferation in HL-60 and Caco-2 antiproliferative potency. Compound 48 displayed a remark- cells at 50M concentration. Mechanistic studies revealed able activity against 50 human tumor cell lines and inhibited that these three compounds induced G0/G1 cell cycle arrest the proliferation of most of cancer cells at submicromolar and apoptosis, which was accompanied by loss of mitochon- concentrations. An acute toxicity study indicated that com- drial membrane potential, DNA fragmentation and nuclear - morphology in HL-60 cells. pound 48 was well tolerated intraperitoneally (150mgkg 1dose-1) by athymic nude mice. A series of quinoxaline- A series of 4-substituted 5-hydroxyaurone derivatives derived chalcones shown in Fig. (4), structurally analogous (Fig. (4)) was studied for their inhibitory activities against to selective PI3Kg inhibitor AS605240 was reported by the proliferation of endothelial cells and two cancer cell lines Mielcke and co-workers for potent inhibitory activity on by Cheng et al. [66]. They demonstrated that two most active glioma cell lines from human and rat origin (U-138 MG and compounds 59 and 60 effectively inhibited in vitro endothe- C6, respectively) [61]. The results of their study revealed lial cell motility and tube formation, which are basic proper- that four chalcones (49-52), which incorporate methoxy ties of endothelial cells for angiogenesis. An independent groups at A-ring, displayed higher potency against glioma study by Sim et al. established methoxylated aurones (61) as cells. Flow cytometry studies demonstrated that the com- promising compounds associated with low toxicities and pound 50 induced apoptosis in C6 cells by causing G1 phase potent modulatory effects on the ABCG2 efflux protein [67]. Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 103

CHALCONE HYBRIDS Kamal and co-workers also reported a series of novel chalcone linked imidazolones (Fig. (5)) with potent anti- “Molecular hybridization” has been one of the success- breast cancer activity [76]. Some of chalcone-imidazolone fully applied strategies for the design, and development of conjugates (70-72) showed good anticancer activity with new and efficient chemotherapeutic agents [68]. Molecular GI50 values ranging from 1.26 to 13.9μM. The chalcone- hybridization involves the combination of two distinct imidazolone hybrids were shown to induce G2/M cell cycle pharmacophores or chemical entities by either linking or arrest at 10μM. However, increasing the concentration of fusing each other to form new hybrid moieties [68, 69]. these conjugates to 30μM, caused accumulation of cells in The selection of the pharmacophores or chemical moieties sub G1 phase (i.e., G0 phase) indicating the apoptosis induc- is based on their known bioprofiles and it is expected that ing nature of these conjugates. Further investigations of the these hybrid molecules might exhibit synergistic or additive mechanism of apoptosis induction revealed that the conju- pharmacological activities [70, 71]. Several structural gates caused telomere initiated DNA damage which involved modifications to the chalcone template, particularly conju- both senescence like growth arrest and apoptosis [77]. gation of pharmacologically important groups to either A or B phenyl ring have been shown to enhance their biologi- A recent report by the same group described the synthesis cal profiles (Fig. (5)). of a series of chalcone-amidobenzothiazole conjugates (Fig. (5)) and their anticancer activities [78]. The chalcone- Schobert et al. studied three chalcone-Pt(II) complex amidobenzothiazole conjugates exhibited potent activity and conjugates comprising a combretastatin A-4 analogous chal- the IC50 of two potential compounds (73 and 74) against dif- cone covalently linked to dichlorido(6-aminomethylnicotinate) ferent cancer cell lines were in the range of 0.85-3.3 μM. platinum complex either directly or by means of a spacer for Flow cytometric analysis revealed that these compounds their anti-proliferative activity on a panel of 21 tumor cell induced cell cycle arrest at G2/M phase in A549 cell line lines from 9 entities [72]. The parent chalcone 62 and the leading to caspase-3 dependent apoptotic cell death. The directly linked conjugate 63 exhibited excellent antiprolifera- tubulin polymerization assay (IC50 of 73 was 3.5μM and 74 tive activities, similar in magnitude (average logIC50 values was 5.2μM) and immuofluorescence analysis showed that of 7.3M (62) and 7.0M (63)) and cell line speciﬁcity but these compounds effectively inhibited microtubule assembly slightly different in the mechanism of apoptosis induction. at both molecular and cellular levels in A549 cells. Further, Chalcones (phenyl-butenones and diaryl-propenones) annexin staining also suggested that these compounds in- conjugated with the tricyclic planar pyrroloquinoline nucleus duced cell death by apoptosis. (Fig. (5)) were studied for their anticancer properties on Yang et al. reported a novel series of dihydroartemisinin three human tumor cell lines [73]. A noticeable cytotoxic derivatives incorporating a substituted chalcone moiety effect was observed for all pyrroloquinoline-conjugated linked by either ether or ester (75) and their cytotoxicity compounds, specifically compounds 64-67, particularly against human leukemia HL-60 and mouse lymphoma P388 against human melanoma cell line JR8 (IC50 1.2 - 3.3M). cells [79]. These dihydroartemisinin derivatives were shown Linear flow dichroism experiments on the conjugates con- to exhibit greater anti-proliferative and cytotoxic effects in firmed that the pyrroloquinoline nucleus as an efficacious both cell lines than the dihydroartemisinin. Dihydroartemis- carrier for intercalative complexation with DNA. The ability inin chalcones linked by ether were found to have more cyto- of pyrroloquinoline derivatives to intercalate between the toxic than dihydroartemisinin chalcones linked by ester with base pairs appeared to inhibit the relaxation of supercoiled apoptosis induction abilities. DNA by topoisomerase II with no significant DNA cleavage. Mourad et al. reported a group of nitric oxide (NO) do- A new class of C8-linked pyrrolo[2,1-c][1,4]benzodia- nating chalcone derivatives prepared by binding amino chal- zepine chalcone conjugates (68) was synthesized by Kamal cones with different NO-donating moieties including; nitrate and co-workers employing a solid-phase synthetic protocol esters (76 and 77), furoxans (78) and oximes (79) [80]. Anti- [74]. These novel hybrid chalcones incorporate a pyr- cancer activity screening of these target compounds revealed rolo[2,1-c][1,4]benzodiazepines (PBDs) moiety conjugated that the selected NO-donating compounds exhibited mild to to the chalcone through ether linkage. Some of these chal- strong cytotoxic activity. The NO/chalcone hybrids (76 and cone-PBD conjugates showed significant DNA-binding af- 77) were found to exhibit remarkable activity against differ- finity and potential anticancer activity in a number of human ent types of cancer cell lines, especially against the colon and cancer cell lines. Another report by the same group also ex- melanoma cells. plored 1,2,3-triazole ring side-armed with alkane spacers for Ruan and coworkers reported a series of novel resveratrol linking the chalcones with pyrrolobenzodiazepine moiety derivatives (Fig. (5)) possessing chalcone moiety as antimi- [75]. These chalcone-PBD conjugates (69) showed promis- totic agents [81]. Among the twenty-three resveratrol- ing in vitro anticancer activity ranging from < 0.1 to 2.92 chalcone derivatives synthesized by them, compound 80 M. These PBD-conjugates were found to induce G1 cell exhibited the most potent in vitro anticancer activity against cycle arrest with effect on G1 cell cycle regulatory proteins HepG2, B16-F10, and A549 cell lines with IC50 values of such as cyclin D1 and Cdk4. These conjugates were also 0.2, 0.1, and 1.41 gmL-1, respectively. Compound 80 was shown to exhibit inhibitory effects on NF-kB, Bcl-XL pro- also found to exhibit significant tubulin polymerization in- -1 teins that play a vital role in breast cancer cell proliferation. hibitory activity (IC50 = 2.6gmL ). 104 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al.

OCH3 O O OCH3

H3CO OH O OCH3 O H3CO OCH3 O OCH3 N (62) Pt H2N Cl (63) Cl

Chalcone-Pt(II) complex conjugates

O OH O O N H (64), R = H n H R N O O N (65), R = HN O O HN S H (66), R = OH O O N H n N O O N (67), R = NO2 O O n = 1 -3 Pyrroloquinoline-linked Butenones and Chalcones (68) Pyrrolobenzodiazepine–Chalcone Conjugates

N H R N N O N O O n N O NH R1 O O N O O R n = 1-3 H N N N N O N HO (70), R = 4 -OH-3-OCH3; R1 = H R (71), R = 4 -OH-3-OCH3; R1 = 4-OCH3 O O (72), R = 4 -OH-3-OCH3; R1 = 4 -Cl O Chalcone linked Imidazolones (69) 1,2,3- triazolo-Chalcone-Pyrrolobenzodiazepine Conjugates O

R R O O N CH O H 3 O O O N S X H H3C O O O H O O H X = -C(=O)-/-CH2- O O

(73), R = H CH3 (74), R = 6 -OCF3 Chalcone-Benzothiazole Conjugates (75) Dihydroartemisinin linked Chalcones

O R O N NO2 (76), R = 4Cl H (77), R = 3', 4'- OCH2O- O O O O O O O N R R O O O O O O (78) N N H O R O O R' O O R (81), R = 2', 5'- CH , R' = H O 3 N H N (80) (79) OH

Nitric Oxide Donating Chalcone Hybrids Resveratrol Linked Chalcones Bifendate-Chalcone Hybrids

O O O R F COOH R1 R O O NH N N R N N N 2 O N N

(82), R = H (86), R = OCH3 (83), R = 3,4,5 -OCH3 R1 = 4-OCH3 (84), R = 4-OCH3 R2 = C6H11 (85), R = 3', 4'- OCH2O-

N-4-Piperazinyl-Ciprofloxacin-Chalcone Hybrids 1,2,3-Triazole tethered Beta-lactam-chalcone bifunctional hybrids Fig. (5). Structures of chalcone hybrids. Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 105

O CHAL

O R3 O R7 H3CO R5 R1 R4 X R8 H3CO R2 R6 Linker OCH 3 OCH3 Chalcone Covalent Binding Portion O S O (87) CMAC1 LIN O COV

O O H CO 3 H3CO O O H CO O 3 H3CO OCH3 OCH3 S OCH3 O O O Cl (88) CMAF1 (89) Fig. (6). Modified chalcones.

A series of bifendate-chalcone hybrids, (Fig. (5)) was de- O signed and synthesized by Gu and co-workers with an objec- O OCH3 tive to find novel effective P-glycoprotein (P-gp) inhibitors O H OCH3 [82]. The compound 81 was found to be the most active as it OCH3 (90) DMU-135 had little intrinsic cytotoxicity (IC50 > 200 μM), and could increase the accumulation of Rhodamine 123 in K562/A02 Fig. (7). 3,4-Methylenedioxy-substituted chalcones. cells more potently than bifendate and verapamil (VRP) by inhibiting P-gp efflux function. Abdel-Aziz et al. reported a Rose and Hartman in 2007 patented some modified chal- novel N-4-piperazinyl-ciprofloxacin-chalcone hybrids (Fig. cones (CHAL) incorporating a covalent bonding portion (5)), for their anticancer and topoisomerase I and II inhibitory (COV) and an optional linker portion (LIN) as antimitotic activities [83]. Compound 82, a 4-amino chalone, linked to agents (Fig. ( )) [85]. They postulated that introduction of a ciprofloxacin was found to exhibit broad-spectrum of antitu- 6 covalent bonding portion in the chalcone scaffold either di- mor activity without pronounced selectivity, while compound rectly or through a linker will enhance its tubulin binding revealed high selectivity towards the leukemia subpanel 83 affinity and also circumvent multidrug resistance (MDR) with selectivity ratio of 6.71 at GI level. Furthermore, com- 50 associated with conventional antimitotic chalcones. Thirty pounds and showed remarkable topoisomerase II inhibi- 84 85 seven chalcones designed and synthesized on the basis of tory activity compared to etoposide and comparable topoi- aforementioned rationale were evaluated for growth inhibi- somerase I inhibitory activity to camptothecin. tory activity against NCI 60 cell line panel. However, the Singh et al. reported the synthesis of novel 1,2,3-triazole growth inhibitory activity was only disclosed for three modi- tethered -lactam-chalcone bifunctional hybrids (Fig. (5)) fied chalcones (CMAC-1 (87), CMAF-1 (88), 3-[3-(2- [84], they suggested that the presence of a cyclohexyl sub- chloroacryloyloxy)-4-methoxyphenyl]-1-(3,4,5-trimethoxy- stituent at N-1 of -lactam ring and methoxy substituents, phenyl) propenone (89)). Among the three, the chalcone preferably ortho on ring A and para on ring B on chalcones CMAC-1 was found to exhibit potent growth inhibitory ac- markedly improved the anticancer profiles of the synthesized tivity against a variety of cancer cell lines (leukemia, colon, scaffolds with the most potent of the tested compound (86) breast, lung, ovarian, prostate and CNS) at less than 50 nM exhibiting an IC50 value of < 1, 67.1, < 1 and 6.37M against concentrations. A-549 (lung), PC-3 (prostate), THP-1 (leukemia), and Caco-2 Butler and Potter in a patent publication in 2009 dis- (colon) cell lines, respectively. closed a series of 3,4-methylenedioxy-substituted chalcones (Fig. (7)) as potent antiproliferative agents [86]. The 3,4- CHALCONES IN RECENT PATENTS methylenedioxy-substituted chalcones were claimed to selec- As evident from the reviewed literature, chalcones and its tively inhibit the proliferation of tumor cells expressing cy- derivatives have been extensively studied for anti- tochrome P450 1B1 (CYP1B1) enzyme with relatively no or proliferative and cytotoxic activity against many types of lesser effect on normal cells which do not express CYP1B1. These chalcones were tested for cell growth inhibition in a cancer. This over surge of interest in the anticancer potential MTT assay against non-induced MCF-7 cells and TCDD of chalcones has translated into many patents. The present (Dioxin) induced MCF-7 cells (expresses CYP1B1). Non- article reviews a selection of chalcones patented in the period induced MCF-7 cells in culture are metabolic analogous to from 2007 to early 2014 for anticancer activity through dif- normal cells that do not express CYP1B1, whilst TCDD- ferent mechanisms. 106 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al. induced MCF-7 cells express the CYP1B1 enzyme as is pre- fluorescent microscopical studies revealed that compound 91 sent in fresh human tumors. Therefore the cytotoxicity of (4-tetrahydropyrrolylcarbamoyl-2,5-dimethoxychalcone) compounds in non-induced MCF-7 cells correlates to the induced the microtubule bundle formation in NTUB1 cells cytotoxicity of compounds against normal cells, whilst the and mimicked the effect of paclitaxel. cytotoxicity of compounds against TCDD-induced MCF-7 O cells correlated to the cytotoxicity of compounds against real tumors that express CYP1B1. The results of this assay OCH3 R showed that compound DMU-135 (90) is 65-fold more toxic to tumor cells than to normal cells. The tumor selective cytotoxic activity of DMU-135 was further corroborated in a cell OCH O 3 HN culture based assay using advanced breast cancer cell line (91), R = H MDA-468 and normal breast cell line MCF-10A. The results (92), R = N of this assay demonstrated that compound DMU-135 is 120- fold more toxic to cancer cells than to normal cells whilst the Fig. (8). 2, 5-Dimethoxychalcone derivatives. clinically used anticancer agent doxorubicin was found to be 10-fold more toxic to normal cells than to cancer cells. Konieczny and co-workers patented a series of novel Lin et al. in a US patent (US20110306775) reported chalcone derivatives of general structures (93-96 in Fig. (9)) some 2, 5-dimethoxychalcone derivatives (Fig. (8)) with as cytotoxic agents useful for the treatment of neoplastic microtubule-targeted anticancer activity (tubulin polymeriz- diseases [88]. Over 178 chalcone derivatives were synthe- ing agent) on NTUB1 and PC3 cell lines [87]. The 2, 5- sized and tested for in vitro cytotoxic activity in a MTT as- dimethoxychalcone derivatives reported in the patent (repre- say against A549, HCT-116 and HeLa cancer cell lines and sented by “Fig. (8) incorporates a 2,5-dimethoxy substitu- the results showed that many compounds were cytotoxic to tion in the aryl ring A and 4-carboxyl group or its derivatives the studied cancer cell lines at submicromolar to nanomolar in aryl ring B. Among the eighteen compounds synthesized concentrations. In addition to in vitro cytotoxicity, these and evaluated for cytotoxicity, the compounds with pyr- compounds were also shown to exert in vivo anticancer ac- rolidine (91; IC50 = 1.26 and 0.53M) and N, N-isopropyl tivity in mice with HCT116-luc2 cancer cell line. (92; IC50 = 1.97 and 1.58M) substitution on R possessed significant anticancer activity than the reference compound Kamal et al. from Council of Scientific and Industrial Research has patented a series of chalcone linked to pyr- cisplatin (IC50s 3.27 and 4.56M) against NTUBI and PC3 cancer cell lines. Flow cytometric analysis of the effects of rolo[2,1-c][1,4]benzodiazepine hybrids (Fig. (10)) as poten- these two compounds on cell cycle progression showed that tial anticancer agents [89]. The hybrid compounds were syn- these molecules induced G1 phase arrest accompanied by an thetically derived by either linking substituted chalcones or increase in apoptotic cell death in NTUB1 cells and S/G1 quinolinyl chalcones to pyrrolo[2,1-c][1,4]benzodiazepines phase arrests accompanied by an increase of apoptotic cells by means of carbon chain linker. The hybrid compounds in PC3 cell lines respectively. Additionally, immuno- were tested against human tumor cell lines derived from

O X O OR O n(H C) n(H2C) X 2 Y S S OR Y m(O) m(O) O (94) (93)

OR O OR X X n(H2C) O Y O Y n(H2C) S O O (O)m

(95) (96)

R is H, C1 - C6-alkyl, carboxy-C1 - C6-alkyl, -(CH2)p-NR1R2 or O(CH2)3NH(CO)CF3 Y is -CH = CH-, -O-, -S- or -CH = N- X is H, halogen atom, OH, -C1 - C6-alkoxy, carboxy-C1- C6-alkoxy, di(C1 - C6-alkyl)amino-C1- C6-alkoxy, O-PO(OH)2, or NO2 or NH2 m = 0, 1 or 2; n = 1 or 2, p = 2 to 6; q = 4 or 5

Fig. (9). Novel chalcones patented by Konieczny et al. Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 107

R1 O O O O N

H R O n O N

(97)

R1 O O O O N

H N R2 O n O N

(98)

R = H, OH or F; R = OH, methyl, ethyl or phenyl; R = H or methyl; n = 1 - 4 1 2

Fig. (10). Pyrrolobenzodiazepine-chalcone hybrids.

O different cancer types, leukemia, non-small-cell lung, colon, O NH CNS, melanoma, ovarian, prostate, and renal breast cancer. N Aryl or Heteroaryl The resultant in vitro growth inhibitory data suggested that 7- R methoxy-8-{n-[4-1-(2 or 4-substituted phenyl)-3-(4-hydroxy- 3-methoxyphenyl)-2-propen-1-one]alkyl}-oxy}-(11aS)-1,2,3, (99) 11a-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-ones (97) showed Imidazolone-chalcone hybrids. selective and potent growth inhibition against various cancer Fig. (11). cell lines in comparison to 7-methoxy-8-{n[4-1-(3-methoxyphenyl)-1-(2,4-alkyl-3-quinolyl)-2-propen-1-one]alkyl}-oxy}- R O (11aS)-1,2,3,11a-5H-pyrralo[2,1c][1,4]ben-zodiazepin-5-one R = H or CH N S 3 R = Aryl or Napthyl or Biphenyl or Heteroaryl (98). Thermal denaturation studies of chalcone linked to pyr- R2 2 N R3 = Aryl or CF3 rolo[2,1-c][1,4]benzodiazepine hybrids using calf thymus R3 DNA (CT-DNA) showed that the compounds possess good DNA binding affinity. (100) Imidazolone-chalcone derivatives, (Fig. (11)) (99) with Fig. (12). Imidazothiazole-chalcone hybrids. promising anticancer potential were disclosed in a series of patents by the same group in 2011 [90]. The patent sought to In 2012, the same group has patented a series of 2-phenyl design and develop novel hybrid chalcones with promising benzothiazole (101) and amidobenzothiazole chalcone hybrids anticancer activity by incorporating two pharmacophores (102) as potential anticancer agents against human cancer cell (chalcones and imidazolones) well known for antimitotic and lines [92, 93]. The benzothiazole/amidobenzothiazole hybrids anticancer activities. Selected compounds among the re- described in the patent include olefin, chalcone, pyrazoline, ported imidazolone-chalcones exhibited significant cytotoxic pyrazole, isoxazoline and isoxazoles linked to 2-phenyl ben- activity against fifty three human cancer cell lines in a sul- zothiazoles/amidobenzothiazole with aliphatic chain length forhodamine B (SRB) assay. variations with and without piperazine (Fig. (13)). In vitro Another patent from the same group described a series of cytotoxicty studies of selected compounds of the series against imidazothiazole-chalcone hybrids (Fig. (12)) (100) as poten- seven human tumor cell lines derived from six cancer types tial anticancer agents [91]. These novel chalcones hybrids suggested that both benzothiazole and amidobenzothiazole represented by the structure given in Fig. (12) incorporate a hybrids exhibited significant cytotoxic activity against differ- substituted imidazothiazole as ring “B” and a substituted ent human tumor cell lines. phenyl/napthyl/biphenyl or heteroaryl as ring “A” in the chalcone scaffold. The imidazothiazole-chalcone hybrids A series of novel 1-adamantyl chalcones (Fig. (14)) was were tested against sixty human tumor cell lines derived reported for treatment of breast cancer and/or other prolifer- from nine cancer types (leukemia, non-small cell lung can- ating disorders by Anderson et al. [94]. These 1-adamantyl cer, colon cancer, CNS cancer, melanoma, ovarian cancer, chalcones have been tested for growth inhibitory activity renal cancer, prostate cancer and breast cancer) in an SRB against two different types of breast cancer cell lines (MCF-7 assay as per NCI protocol. Five among the 180 compounds and MDA-MB435) and a non-cancerous breast epithelial cell reported in the patent exhibited a wide spectrum of activity line (MCF-10). The results showed that out of the listed compounds, five compounds ( ) significantly in- against sixty cell lines in nine cell panels, with GI50 value of 103-107 < 9 M. creased the cell death of both human breast cancer cell lines 108 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al.

R O O G O O N R S G X NH S R1 R (101) 2 (102)

R = H or OCH3

R, R1, R2 = H, alkyl, alkoxy, halo, haloalkyl, X = (CH2) or N N n2 n1 halomethoxy, nitro

n, n1 & n2 = 2-6

G =

H3CO

H3CO or H3CO OCH3 OCH OCH3 H3CO 3 OCH3

O O N N N N H3CO H3CO or or H3CO OCH3 H3CO OCH3 OCH3 OCH3

N O N O H3CO H3CO or or H3CO OCH3 H3CO OCH3 OCH3 OCH3 Fig. (13). 2-Phenyl benzothiazole and amidobenzothiazole-chalcone hybrids.

MCF-7 (ER-positive), and MDA-MB435 (ER-negative), o without altering the viability of normal breast epithelial cell R line MCF-10. The anticancer activity exhibited by these compounds against both MCF-7 and MDA-MB435 cell lines was found to be better than some known anti-breast cancer (103) R = pyrid-2-yl agents (e.g., Tamoxifen, Doxirubicin and Genestein). Fur- (104) R = pyrid-3-yl thermore, mechanistic studies with compound 103 on MCF- (105) R = pyrid-4-yl 7 and MDA-MB435 cell lines indicated that this compound (106) R = 6-methylpyrid-2-yl (107) might be exerting its anticancer effects against MCF-7 and R = quinol-4-yl MDA-MB435 cells by blocking the growth stimulatory ef- Fig. (14). 1-Adamantyl chalcones. fects of EGF and/or TGF-.

Synthesis and use of “B” ring aminoalkyl substituted chal- O OH cones (bushrachalcones) as anticancer and antimalarial agents have been patented in 2011 [95]. The bushrachalcones were H3CO evaluated for antiplasmodial activity against chloroquine sen- N sitive strain of the Plasmodium falciparum parasite and the results revealed that some compounds possessed good antimalarial activity (IC50 < 3M) against the parasite. The cytotoxic activity of the compounds was tested against TK, UACC-62 (108) Bushrachalcone W and MCF-7 cell lines in a MTT assay. Compound 108 (bus- Bushrachalcones. rachalcone W) with a piperidin-1-yl-methyl substitution at the Fig. (15). para position of phenyl ring “B” was found to be the most potent with IC50 values 20.26, 5.52 and 5.83M against TK, A composition containing extracts of Angelica keiskei UACC-62 and MCF-7 cell lines respectively (Fig. (15)). comprising chalcone and isoflavones has been shown to in- Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 109

O O R1

HN OH R X R2 O S O

X = NH or O (109) TSAHC R = Alkyl or Aryl sulphonyl

R = R = H or OH 1 2 Fig. (16). Chalcones containg sulfonyl or sulfonamide groups.

R5 R6 O

n(R7) (R8)m R2 R3 R1 R4

Vinylogous chalcone derivatives

O OCH3 O OCH3

O OCH3 OCH3

(110) (111) Fig. (17). Vinylogous chalcone derivatives. duce adiponectin production and useful for treatment of cir- A novel class of vinylogous chalcone derivatives, (Fig. culatory diseases, diabetes and cancer [96]. (17)) was reported for the treatment of cancer, particularly Researchers at Gyeonsang National University and Seoul malignant hematological diseases/disorders in WO2012013725 National University have developed a method for screening [98]. Researchers at Medizinische Universität Wien identi- anticancer compounds based on various biochemical events fied a novel lead molecule (110) with promising anticancer induced by the expression of TM4SF5 [97]. TM4SF5 is a potential during their attempts to improve the anticancer po- transmembrane glycoprotein of the transmembrane 4 L6 tency of chalcones by structural modification concurrent superfamily, a branch of the tetraspanin family and highly with in vitro tests to evaluate their cytotoxic potential in dif- expressed in many types of cancers including pancreatic, ferent cancer and non-cancer cell lines. Optimization of the hepatic and colon. They have also reported the screening of a lead molecule 110 through an extensive SAR study resulted in the highly cytotoxic compound 111. Both the compounds series of chalcones containing a sulfonyl (SO3-) or a sul- were tested for antiproliferative activity against a variety of fonamide (SO2NH-) group for the anticancer effects on TM4SF5-expressing cells (Fig. (16)). The screening results cancer cell lines (HUVEC, colon cancer (HCT116, SW430, HT29) and melanoma (518A2)). The lead molecule 110 was revealed that chalcones containing OH, NH2 or OCH3 substituent on the A ring does not display any activity against found to inhibit the growth of all the five cell lines at nano- TM4SF5. However, the introduction of a sulfonyl group into molar concentrations ranging from 100 to 500, whereas a chalcone confers anticancer activity, inhibiting TM4SF5- compound 111 exhibited profound growth inhibitory potency mediated events. These chalcones have also been shown to (< 50nM) against SW80 and 518A2 cells. Cell viability stud- possess TM4SF5 antagonistic activity and potential for ies using compounds 110 and 111 on cancer cell lines repre- TM4SF5-mediated tumorigenesis. A representative chalcone senting different hematological malignancies indicated that derivative TSAHC (109) inhibited the activity of matrix the compound 111 showed better cytotoxicity ( 50nM) than metalloproteins (MMPs), and also exhibited TM4SF5 an- the lead molecule 110 in almost all cancer cells except for tagonistic activity likely through targeting the extracellular Jurkat and K562 cells. An evaluation of apoptosis of lym- region of TM4SF5, which is important for protein-protein phoma cells (SU-DHL-6 andSU-DHL-9 NHL) promoted by interactions. In vivo studies revealed that intraperitoneal ad- the compound 110 and 111 was also performed by dual ministration of [4'-(p-toluenesulfonylamino)-4-hydroxy chal- staining method with annexin V-fluorescein isothiocyanate cone] (TSAHC) at a dose of 50 mg/kg in rats decreases (FITC)/propidium iodide (PI). The results indicated that both (88%) the tumor size without displaying any significant toxic the compounds induce apoptosis in these cancer cells a con- effects. centration dependent manner. Cell cycle analysis by flow 110 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al.

O HO O OH HO O OH OH HO OH OH HO

(113) Okanin (112) Melanoxetin Fig. (18). Xanthine oxidase inhibitors found in ethanolic extracts of Acacia species. cytometry also indicated that the cytotoxic properties of the constituents which include leridal chalcone (114) (Fig. (19)), compounds may be mediated by the inhibition of cell divi- flavonoids and other compounds (lignoceric acid, petiveral, sion and the induction of apoptosis. Furthermore, the com- myricitrin, etc.). The isolated bioactive fraction was shown pounds 110 and 111 were also evaluated for their cytotoxic to induce cell death in tumor cells by different pathways; potential against primary chronic lymphocytic leukemia acting on cytoskeleton by inducing cell cycle arrest in G2 (CLL) cells and in a co-culture model of CLL cells and phase followed by induction of apoptosis by either mito- stromal cells using the Cell Titer-Blue Cell Viability Assay. chondria dependent or independent mechanisms. They have Fludarabine and cyclophosphamide, which are standard also claimed that the bioactive fraction in combination with therapeutic drugs in CLL were used as controls. The results any immunostimulant can produce the phenotypic and/or demonstrated that compounds 110 and 111 showed higher functional maturation of the dendritic cells. cytotoxicity than the control drugs fludarabine and cyclo- OH O phosphamide against both CLL models. The impact of the OHC compounds on healthy cells was also assessed using normal human hematopoietic progenitor cells in a colony forming H3CO OH assay and peripheral blood mononuclear cells (PBMC) in a Cell Titer-Blue Cell Viability Assay. The results demon- (114) strated that the normal cells are less affected than the malignant cells and both the compounds have a favorable thera- Fig. (19). Leridal chalcone. peutic range with the preferential killing of malignant cells.

Chang and co-workers described the synthesis and devel- O opment of a new fluorescence dye of diamino-chalcone with potential application as an embryonic stem cell probe for cell F3C OCH3 imaging analysis for various diseases [99]. Researchers from National Taiwan University described (115)2'-CF & 2-OCH a composition for inhibiting xanthine oxidase comprising an 3 3 effective amount of the extracts from Acacia species [100]. Fig. (20). Trifluoromethylated chalcones. Ethanolic extracts from various parts of Acacia confusa were separated in liquid-liquid fraction with ethyl acetate, n-butyl A series of chalcones containing a methoxy substitution alcohol and water to generate ethyl acetate fraction, n-butyl on the phenyl ring A and trifluoro methyl substitution on the alcohol fraction and water fraction. Each fraction was further phenyl ring B (Fig. (20)) was claimed to be the activators of evaluated for xanthine oxidase inhibitory activity and the nuclear factor erythroid-2 related factor-2 (Nrf2) with poten- results showed that the ethyl acetate fraction showed better tial therapeutic applications in diseases, disorders, or condi- xanthine oxidase inhibitory activity. Eight major compounds tions associated with Nrf2-regulated pathways such as auto- mostly flavanoids (3,7,8,3,4-pentahydroxyflavone (Melan- immune disease, bone marrow transplant for leukemia and oxetin), 7,8,3,4-tetrahydroxyflavone, 7,8,3,4-tetrahydroxy- related cancers, bone marrow deficiencies, inborn errors of 3-methoxyflavone (Transilitin), 3,7,8,3-tetrahydroxy-4-meth- metabolism and other immune disorders, etc. [102]. The oxyflavone, 7,8,3-trihydroxy-3,4-dimethoxyflavone, 7,3,4- chalcones were evaluated for their ability to activate the ex- trihydroxyflavone, 7,3,4-trihydroxy-3-methoxyflavone and pression of Nrf2-regulated cytoprotective genes and for cyto- a chalcone (3,4,2,3,4-pentahydroxy trans-chalcone (Okanin)) toxicity in human lung epithelial cells. An in depth SAR were isolated from the ethyl acetate extract and among them, analysis revealed that the position of CF3 substitution in ring melanoxetin (112) and okanin (113) exhibited potent inhibi- B of the chalcone was crucial for the activity and ortho CF3- tory activity on xanthine oxidase greater than standard drug substituted chalcones were the most active and non cytotoxic allopurinol (Fig. (18)). in the series. Eight chalcones with promising Nrf2 activation Gomez et al. in US20120294897 described a method for potency and no cytotoxicity were further tested for in vivo the treatment of cancer via the administration of a bioactive potency to activate Nrf2 using mouse models. The results fraction of Petiveria alliacea with antitumor activity [101]. revealed that the compound 115 to be the most potent in- The bioactive fraction comprises multiple phytochemical ducer of Nrf2 activity in vivo. Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 111

O O O

O O

O O HO

(116)Coumarin chalcone hybrid (117) Chalcone

O O

(118) Coumarin Fig. (21). Coumarin/chalcone hybrids.

O O N Ar Ar' N

Ar = 2-pyridyl or 3-pyridyl or 4-pyridyl Ar' = 3-pyridyl or 2-quinolinyl or 1-napthyl or (119) substituted (2-Cl or 2-NHCOCH ) phenyl 3 Fig. (22). Aza chalcones.

Certain coumarin/chalcone hybrids have been patented as tumor efficacy of compound 119 was also tested in five in anticancer agents useful for the treatment or prevention of three tumor models (mouse Lewis lung carcinoma xenografts cervical carcinoma, oral squamous cell carcinoma or lung or grown in C57/Blk6 mice, MDA-MB231 human breast adeno- prostate carcinoma or brain tumor [103]. A representative carcinoma tumors established in Balb/c nude mice and HL-60 coumarin chalcone hybrid (116) was shown to elict better leukemia cell xenografts in Balb/c nude mice). The results of activity than individual chalcone (117), coumarin (118), and the in vivo studies demonstrated that compound 119 exhibits their combination (117 + 118 (1: 1 molar ratio)) against the cytostatic activity in Lewis lung carcinoma and HL-60 human cervical cancer cell line (C33A) and also non-toxic to xenograft models, while encompassing an early cytotoxic ef- the fibroblast cells NIH3T3 (IC50 > 150 M) (Fig. (21)). The fect followed by cytostatic activity in MDA-MB231 xenograft compound also exhibited better in vivo anticancer efficacy model. In a recent patent published in 2012, Carmeliet et al. on human tumor xenografts in SCID mice bearing HeLa claimed that the aza chalcones either alone or in conjunction human cervical cancer cells than the reference compound with siRNAs directed against PFKFB3 would be useful for adriamycin. diseases which involves pathological angiogenesis [105, 106]. Chesney et al. in 2009 disclosed seven aza chalcones as A chalcone ( having 2-hydroxyl and 4-methoxy inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-bisphos- 120) substitution in phenyl A and B respectively (Fig. (23)) was phatase 3 (PFKFB3) with anti-neoplastic activities (Fig. (22)) shown to possess great therapeutic potential on anti- [104]. Extensive biological studies (in vitro and in vivo) were atherosclerosis by acting as peroxisome proliferator activated performed using compound 119 establishing its PFKFB3 receptor gamma (PPAR ) inducer, P44/42 mitogen activated inhibitory activity and antiproliferative activity on trans- protein kinase (MAPK) inhibitor and cell cycle blocker formed Jurkat cells expressing inducible FLAG-PFKFB3, leukemia (K562, Jurkat, HL-60), breast adenocarcinoma [107]. More importantly, the chalcone exhibited no toxicity in human aortic smooth muscle cells (HASMCs). Addition- (MDA-MB231), Lewis lung carcinoma (HeLa, A549) and ally, the chalcone also demonstrated a synergistic effect with melanoma (CRL -11174) cells. Compound 119 was shown to decrease intracellular Fru-2, 6-BP, 2-DG uptake, and lac- the PPAR ligand (rosiglitazone) to inhibit cell proliferation tate secretion in cellular assays using Jurkat cells, suggesting and the upregulation of cyclin D1, cyclin D3, interleukin-1 that the inhibition of cellular proliferation might be through (IL-1) and interleukin-6 (IL-6) induced by oxidized low disruption of energetic and anabolic metabolism. The anti- density lipoprotein (Ox-LDL). 112 Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 Karthikeyan et al.

O dominantly influence the anticancer activities elicited by simple chalcones. Hetroaryl moieties especially indole or quinoline either as ring A or B appear to enhance the cyto- OH OCH3 toxic activity of chalcones against many types of cancer cells. Furthermore, conjugations of chalcones with other (120) biologically active moieties have yielded novel hybrid chalcones with enhanced and synergistic anticancer properties. Fig. (23). (E)-1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2- Chalcones have been shown to exert cytotoxic activity en-1-one. against many cancer cells through multiple mechanisms which include cell cycle disruption, angiogenesis inhibition, CURRENT & FUTURE DEVELOPMENTS tubulin polymerization inhibition, apoptosis induction and The present review concludes that the chalcones; even blockade of NF-B signaling pathway and inhibition of cell after three decades of extensive research still remains to be cycle regulatory kinases. Recent literature also establishes an interesting scaffold for anticancer drug discovery owing chalcones as inhibitors of breast cancer resistance protein to their broad spectrum activity against many types of can- and P-gp hence may have an additional therapeutic use as cer. The anticancer properties of chalcones are mainly influ- MDR reversal agents in cancer chemotherapy. All the reports enced by the substitutions on the two aryl rings of chalcone presented in this review demonstrate chalcones as a ‘privi- molecule and their substitution patterns (Fig. (24)). Litera- leged scaffold’ for drug discovery efforts targeting cancer. ture on anticancer chalcones highlights employment of a Given the quantum of work reported in the literature on anti- three pronged strategy, namely; structural manipulation of cancer chalcones and the number of patents filed by aca- both aryl rings, replacement of aryl rings with heteroaryl demic and industrial sectors on novel anticancer agents scaffolds, molecular hybridization through conjugation with based on chalcones, it seems inevitable that novel anticancer other pharmacologically interesting scaffolds for enhance- chalcones with desired potency, selectivity, and in vivo effi- ment of anticancer properties. Methoxy substitutions on both cacy will ultimately emerge for clinical use in the near fu- the phenyl rings (A and B) and their substitution pattern pre- ture. Heterocyclic rings OH or OCH or Methylene dioxy 3 Antiproliferative activity Anticancer activity on various cell lines O Di or tri OCH3 groups Anticancer Kinase inhibitory activity

R B A R1 2

Halogens Aryl/Heteroaryl rings and/or Anticancer substitutions Napthyl, phenyl, biphenyl, Aryl/Heterocyclic rings and/or as substitutions heterocycles, etc. N-Methyl imidazole, Amidobenzothiazole, N-Methyl indolyl, Anticancer activity on various tetrahydro pyrrolyl carbamoyl: cell lines Microtubule depolymerizing agent Coumarin, thiazolyl, N-Methyl indolyl: Anticancer, Antibreast Cancer Piperidine, 1-Adamantyl: Antibreast cancer Imidazothiazole, Napthyl, Furanyl, Thiophene, Phenyl quinolone, Quinoxaline, Pyrazinoisoquinoline, Pyrroloquinidine, Triazole tethered beta lactam, Pyrrolobenzodiazipine: Anticancer Dihydro artemisinin: Anticancer (Apoptosis)

Fig. (24). Summary of SAR of chalcone derivatives.

Anticancer Activities of Chalcones Recent Patents on Anti-Cancer Drug Discovery, 2015, Vol. 10, No. 1 113

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