138 Anti-Cancer Agents in Medicinal Chemistry, 2009, 9, 138-161 1871-5206/09 $55.00+.00 �� 2009 Bentham Science Publishers Ltd. Quercetin and Its Derivatives: Synthesis, Pharmacological Uses with Special Emphasis on Anti-Tumor Properties and Prodrug with Enhanced Bio-Availability Ketan V. Hirpara*, Pawan Aggarwal, Amrita J. Mukherjee, Narendra Joshi and Anand C. Burman Chemical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad - 201 010, U.P. India Abstract: Cancer is one of the leading causes of death in the world. American Cancer Society reported 12 million new cases of malig- nancy diagnosed worldwide in 2007, with 7.6 million people dying from the disease. Plant-derived molecules have played an important role in cancer chemotherapy. Many cytotoxic plant-derived molecules such as vinblastine, vincristine, navelbine, etoposide, teniposide, taxol, taxotere, topotecan and irinotecan have been approved as anticancer drugs. Flavonoids, a plant-derived molecule has shown to regulate proliferation and cell death pathways leading to cancer. Some Flavonoids have already entered in clinical trials, among them Quercetin is emerging as prospective anticancer drug candidates and its prodrug QC12 has entered in phase-I clinical studies. In this re- view authors have tried to cover in brief but comprehensive way, the chemistry related to synthesis and uses of ���Quercetin & its deriva- tives��� with special emphasis on the anticancer properties. Key Words: Anticancer, flavanoid, glycoside, quercetin, QC12, synthesis. OVERVIEW Cancer is one of the leading causes of death in the world. American Cancer Society reported 12 million new cases of malig- nancy diagnosed worldwide in 2007, with 7.6 million people dying from the disease [1]. As per World Health Organization (WHO), over 40% of all cancers can be prevented. Others can be detected early, treated and cured. Even with late stage cancer, the suffering of patients can be relieved with good palliative care. More than 70% of all cancer deaths occur in low and middle-income countries where resources available for prevention, diagnosis and treatment of cancer are limited or non-existent. Based on projections, cancer deaths will continue to rise with an estimated 9 million people dy- ing from cancer in 2015 and 11.4 million dying in 2030 [2]. Flavonoids have been shown to regulate proliferation and cell death pathways leading to cancer. Generally, flavonoids are bio- logical pigments providing colors from red to blue in flowers, fruit and leaves. Besides being biological pigment to plants, flavonoids have important roles in the growth and development of plants pro- tection against UV-B radiation forming antifungal barriers antimi- crobial, insecticidal and oestrogenic activities plant reproduction. As per structural patterns Flavonoids are usually classified into six main classes. Firstly, Flavone such as baicalein, apigenin second, Flavonol such as fisetin, Quercetin third, Flavanone such as eri- odictyol, hesperidin fourth, Flavanol such as (+)catechin, (- )epicatechin fifth, Anthocyanidin such as cyaniding, apigeninidin and sixth, Isoflavine such as daidzin, prunetin [3]. Few reviews on quercetin are available describing its pharma- cological and biological uses. Flavonoids are also important for human health. Like vitamins, these compounds are not produced endogenously by the body and must be supplied either through the diet or nutritional supplements. Quercetin, a Flavonoid, has been the subject of dozens of scientific reports over the past 30 years. It has shown the greatest activity among the flavonoids studied in experimental models. Quercetin is frequently used therapeutically O HO O OH OH O O O O OH O O O OH O+ OH O O Flavone Flavonol Flavanone Flavanol (catechin) Anthocyanidin Isoflavone Quercetin (1) 2 3 4 5 6 7 8 2' 3' 4' 5' 6' A B C OH OH Fig. (1). Structure of major classes of flavonoids and Quercetin (1). *Address correspondence to this author at the Chemical Research, Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad - 201 010, U.P. India Tel: +91-120-4378501 Fax: +91-120-4376902 E-mail: ketanvh@dabur.com

Quercetin and Its Derivatives Anti-Cancer Agents in Medicinal Chemistry, 2009, Vol. 9, No. 2 139 in allergic conditions, including asthma and hay fever, eczema and hives. Additional clinical uses include treatment of gout, pancreati- tis and prostatitis, which are also, in part, inflammatory conditions. The common link is its ability to mediate production and manufac- ture of pro-inflammatory compounds [4]. Quercetin possesses a variety of pharmacological activities and in order for further clinical application it is important to evaluate its safety. A review by Okamoto T on ���Safety of Quercetin for Clinical Application��� emphasized on safety of Quercetin and concluded that quercetin is genotoxic to salmonella but its safety upon human ap- plication is approved [5]. Formica J. V. and Regelson W. compiled the biology or sparing effects of Quercetin on Cardiovascular system in a review, ���Review of the biology of Quercetin and related bioflavonoids���. Quercetin and other flavonoids have been shown to modify Eicosanoid Bio- synthesis (antiprostanoid and anti-inflammatory responses), protect low-density lipoprotein from oxidation (prevent atherosclerotic plaque formation), prevent platelet aggregation (antithrombic ef- fects) and promote relaxation of cardiovascular smooth muscle (antihypertensive, antiarrhythmic effects). In addition, flavonoids have been shown to have antiviral and carcinostatic properties. However, flavonoids are poorly absorbed from the gut and are sub- jected to degradation by intestinal microorganisms. The role of flavonoids may transcend their presence in food. The activity of flavonoids as inhibitors of reverse transcriptase suggested a place for these compounds in the control of retrovirus infections, such as acquired immunodeficiency syndrome (AIDS) [6]. Davis W L et al. in their review concluded on anti-cancer activ- ity, including cell cycle regulation, interaction with type II estrogen binding sites and tyrosine kinase inhibition. Quercetin appears to be associated with little toxicity when administered orally or intrave- nously. Much in vitro and some preliminary animal and human data indicate quercetin inhibits tumor growth [7]. Harwood M et al. carried out a critical examination of the sci- entific literature associated with the safety of quercetin. Results of numerous genotoxicity and mutagenicity, short- and long-term animal, and human studies were reviewed in the context of quer- cetin exposure in vivo. The results of in vitro studies, which consis- tently demonstrated quercetin-related mutagenicity to the absence of carcinogenicity in vivo, the mechanisms that lead to the apparent in vitro mutagenicity and that ensure absence of quercetin toxicity in vivo [8]. Flavonoids like Quercetin (1) have already entered into clinical trials. It is emerging as prospective anticancer drug candidate. It���s prodrug QC12 (175 or 176) has entered in Phase-I clinical studies [3]. In this review the authors cover in brief but comprehensive way, the chemistry related to synthesis, Structural-Activity Rela- tionship (SAR) and pharmacological uses of ���Quercetin (1) & it���s derivatives��� with special emphasis on the anticancer properties. INTRODUCTION Quercetin (1) is widely distributed in the plant kingdom and is the most abundant of the flavonoid molecules. Quercetin (1), [2- (3,4 - dihydroxyphenyl) - 3,5,7 - trihydroxy - 4H-1-benzopyran-4-one], is a yellow to greenish crystalline powder melting at 302��C and is found in many often-consumed foods including apple, red onion, broccoli, tea, berries, and brassica vegetables, as well as many seeds, nuts, flowers, barks, and leaves. It is present at an average level of 10 mg/kg. Higher concentration can even be found in some common vegetable like onion (300 mg/kg) [9]. Initially, Quercetin was discovered as a Mutagen but subsequent studies by Prof. Terence Leighton (University of California at Berkeley) found it to be a powerful natural anti-cancer agent [10]. Further, studies were performed by different groups of scien- tists and found that Quercetin (1) was very efficient antioxidant and also appeared to be active in many diseases related to ageing like cancer, cardiovascular and neurodegenerative [9]. It has been found to inhibit production of heat shock proteins in several malignant cell lines, including breast cancer [11], leukemia [12] and colon cancer [13]. Quercetin's anti-tumor action appears diverse and in- cludes inhibition of inoculated cancer cells, chemically and virally induced cancers, leukemia and ovarian cancer [10]. It shows the ability to modulate the metabolism of carcinogens through inhibi- tion and/or induction of enzymes involved in the biotransformation of these carcinogens [14, 15]. Several mechanisms for the anti- proliferative effect of quercetin have been proposed, including the induction of DNA strand breakage, cell cycle arrest and/or apopto- sis [16-18]. It has shown to induce Estrogen Receptor II (ER II) expression in both type I estrogen receptor positive (ER+) and type I estrogen receptor negative (ER-) human breast cancer cells. The induction of ER II allows for greater growth inhibition of ER- cells with quercetin treatment [19]. Quercetin (1) (248 ��M) was found to down regulate expression of mutant p53 protein into nearly unde- tectable levels in human breast cancer cell lines, but lower concen- trations gave less reduction [20]. The inhibition of expression of p53 was found to arrest the cells in the G2-M phase of the cell cy- cle. This down regulation was found to be much less in cells with an intact p53 gene [21]. Mutations of p53 are among the most common genetic abnormalities in human cancers [22]. Quercetin (1) was the first tyrosine kinase inhibitor tested in a human phase I trial [23]. The concentration at which tumor cell growth was inhib- ited by 50 percent inhibitory concentration (IC50) ranged from 7 nM to just over 100 ��M. The in vivo studies IC50 value of quercetin ranges between 0.45���59 ��M against several malignant cell lines such as lung (non-small cell lung), leukemia (14 AML, 4 ALL lines, CML line K562), gastric (HGC-27, NUGC-2, MKN-7, MKN- 28), breast (MCF-7, MDA-MB-435, MDA-MB-468), ovarian (OVCA 433) and melanoma (MNT1, M10, M14) [24]. Gulati et al. checked antiproliferative effect of Quercetin in cancer cells. Anti- proliferative effect was mediated via inhibition of the PI3K- Akt/PKB pathway and found that treatment with Quercetin (25 ��M) for 0.5, 1 and 3 h completely suppressed the cancer cell growth and constitutively activated Akt/PKB phosphorylation at Ser-473 in HCC1937 cells [25]. Elio A. Soria et al. checked effects of Quercetin and silymarin on arsenite-induced cytotoxicity in two human breast adenocarci- noma cell lines [26]. Dae-Hee Lee et al. investigated the effect of Quercetin on the apoptotic pathway in a human prostate cell line (LNCaP) [27]. Boots AW et al. checked in vitro and ex vivo anti- inflammatory activity of quercetin in healthy volunteers and con- cluded that quercetin increases antioxidant capacity in vivo and displays anti-inflammatory effects in vitro, but not in vivo or ex vivo in the blood of healthy volunteers [28]. STRUCTURE-ACTIVITY RELATIONSHIPS There is very little understanding about a possible relationship between the flavonoid structure and their anticancer activity. The scientists carried out various studies, but they could not draw clear Structure-Activity Relationships (SAR) regarding apoptotic and anti-proliferative effects [29]. The possible SAR studies agree to the necessary presence of carbonyl group at C-4 position and the double bond between C-2 and C-3 in the C ring. A- and B- ring substitutions were much more difficult to interpret with respect to anti-proliferative activity. It was difficult to draw a clear conclusion on substitution profile but still, substitutions such as 5,7-dihydroxy 5,7-dihydroxy-6-methoxy or 5,6,7-trihydroxy in ring A and 3���, 4���- dihydroxy or 3���, 4���-dihydroxy-5���-methoxy in ring B were consid- ered as activating substitutions [30]. According to another study, the effect on 3-hydroxylation is unclear and the presence of at least three adjacent methoxy groups in the molecule can confer a more potent anti-proliferative effect [31]. J. B. Daskiewicz et al. screened a library of 42 natural and synthetic flavonoids for their effect on cell proliferation and apoptosis in a human colonic cell line (HT- 29) and found that Flavones and Flavonols showed greater anti-

140 Anti-Cancer Agents in Medicinal Chemistry, 2009, Vol. 9, No. 2 Hirpara et al. proliferative activity than Chalcones and Flavanones. They further assessed the effects of hydroxylation, methoxylation and /or C- alkylation at various positions in the A- and B- rings in flavonoids. With respect to substitutions, C-isoprenylation was the most effec- tive at C-8 position where as substitution with longer chains such as geranyl showed increase in anti-proliferative potential [32]. Classification Based on the success of Quercetin in cancer chemotherapy, these chemical classes are now being explored. A prodrug of Quercetin, QC12 has already reached in advanced clinical tri- als. It was, therefore, rationale to review the progress made on Quercetin as antitumor agents. In the present review, we have complied the synthesis of most potent derivatives of Quercetin along with its activity. Quercetin derivatives have been divided in following catego- ries: (a) Protection of Quercetin- Benzylation, Acetylation, Catechol protection. (b) Synthesis of methylated Quercetin derivatives- Rhamnetin, Iso- rhamnetin, Azaleatin, Tamarixetin. (c) Synthesis of Quercetin derivatives to improve activity- Malo- nate, Thiazolidinedione, [60] Fullerene, Chalcone. (d) Synthesis of glycosylated Quercetin derivatives to improve solubility and activity-D-Galactose, D-Xylose, D-Arabinose, D- Glucose, L-Fucose, (2���-galloyl)- -L-arabinopyranoside, Sopho- rotrioside, -D-glucuronide, Calabricoside A, (e) Synthesis of quercetin amino acid derivatives to improve solu- bility and bioavailability- QC12. CHEMISTRY Quercetin (1) is a polyphenolic Flavonoid, which has a common flavone nucleus, composed of two benzene rings linked through a heterocyclicpyrone ring. It is known to exhibit wide range of bio- logical activities. Its skeleton is the main part of the drug Flavopiri- dol, which is now under clinical trials [33]. So, to further explore the biological activity of Quercetin, a number of researchers have synthesized quercetin analogues based on total synthesis and hemi- synthesis including its prodrugs. Hemi synthesis relies on direct reactions of quercetin, which depends on the relative reactivity of its different positions [34]. L. Jurd observed that direct alkylation of Quercetin (1) with limited quantities of alkyl halide gave a mixture of unchanged Quercetin (1) and highly O-alkylated products. When Quercetin penta-acetate (2) was treated with excess of methyl iodide and po- tassium carbonate in dry acetone, only one acetyl group was re- placed and rahmnetin tetra-acetate (3) was obtained, which further on deacetylation provided Rhamnetin (4). Similarly, when Quer- cetin penta-acetate (2) was treated with benzyl chloride and potas- sium carbonate in dry acetone, monobenzyl-Quercetin tetra-acetate (5) was formed which further on deacetylation provided monoben- zyl-Quercetin (6). Compound (6) was methylated to give a mono- benzyl-tetra-O-methyl-Quercetin (7). Debenzylation of this methyl ether gave a tetra-O-methyl-Quercetin (8) as shown in Scheme 1 [35, 36]. Higher boiling solvents like methyl ethyl ketone may be used to replace the acetyl groups located elstwhere on the flavone nucleus. 7-O-benzyl-Quercetin tetra-acetate (5) was reacted with benzyl chloride in dry methyl ethyl ketone to give a tetra-O-benzyl-Quer- cetin monoacetate (9), which further on deacetylation furnished tetra-O-benzylquercetin (10) as shown in Scheme 1 [35, 36]. During benzylation of Quercetin (1), the hydroxyl groups at positions 3, 5 and 7 were benzylated and the fourth benzyl group was located at either 3���- or 4���-position. The spectral data strongly supported the benzylation at 4���-position in comparison to 3���-posi- tion. Partial benzylation of Quercetin penta-acetate (2) furnished the acetates of 7-O-benzyl-Quercetin (5), 4���,7-di-O-benzyl-Quercetin (11), 3,4���,7-tri-O-benzyl-Quercetin (12) or 3,4���,5,7-tetra-O-benzyl- Quercetin (9). The order of replacement of acetoxy groups attached to the flavone nucleus, therefore, lies in the order 7 4��� 3 5 3��� [36]. Kubota and Perkin had earlier reported that Quercetin-3,3���,4���,7- tetra-acetate (13) on methylation with diazomethane, gave 5-O- methyl-Quercetin tetra-acetate (14). It was also reported that Quer- cetin-3,3���,4���,7-tetra-acetate (13) and Quercetin-3,3���,4���-tri-acetate (15) when reacts with methyl iodide and potassium carbonate in anhydrous acetone, yielded 5-O-methyl-Quercetin tetra-acetate (14) and 5,7-di-O-methylQuercetin tri-acetate (16), respectively, as de- scribed in Scheme 2. However, benzylation of the same does not furnish its corresponding 5-O-benzyl compound [37, 38]. But L. Jurd et al. found that benzylation of Quercetin-3,3���,4���,7-tetra- acetate (13) gave 7-O-BenzylQuercetin tetra-acetate (5) which they attributed to the meta migration. The meta migrations were closely similar to Perkin���s ortho migrations [39]. L. Jurd found that the methylation or benzylation or allylation of Quercetin penta-acetate (2) in anhydrous acetone results in the replacement of acetyl groups by alkyl or benzyl at 7- position (5) and to lesser extent at 7 and 4���-position (11), which on deacetyla- tion provided 6 and 17, respectively, as shown in Scheme 1 and 3. Methylation of fully acetylated flavonol in a mixture of acetone and methanol, however, preferentially methylates hydroxyl groups at 7, 4��� and 3��� positions. A wide variety of naturally occurring partial methyl ethers have been prepared by these reactions, for example, preparation of Ombuin (18) and Quercetin-7,4���,3���-tri-O-methyl (19) as shown in Scheme 3. In the similar way, compound 11 on selec- tive methylation and deacetylation gave compound 20. Compound 20 on debenzylation provided Iso-rhamnetin (21) [40]. M. Bouktabil et al. found that most of in vitro studies were done on Quercetin (1) itself but not on it���s three non-degradable metabolites (methylated, sulfonated and glucuronidated Quercetin), which were found in plasma analysis of rat and pig. For further studies M. Bouktabil et al. synthesized O-monomethylated ana- logues of Quercetin through sequential protection of the different phenolic function of Quercetin (1) and also developed general methodology for hemi synthesis of the O-monomethyl Quercetin isomers on the basis of difference in reactivity of hydroxy groups in Quercetin (1). Authors have synthesized 5-O-methylquercetin (22, Azaleatin), 3���-O-methyl quercetin (21, Iso-Rhamnetin) described in Scheme 4 and 4���-O-methylquercetin (23, Tamarixetin), 3-O-methyl- quercetin (24), 7-O-methylquercetin (4, Rhamnetin) described in Scheme 5. Quercetin (1) reacted with benzyl bromide (3.5 equiv) in presence of K2CO3 and DMF, yielded a mixture of 3,3���,4���,7 tetra- benzyl (60% yield) (25) and 3,4,7 tri-benzyl (20% yield) (26) and trace amount of penta-benzyl Quercetin (27). The positions of ben- zyl group were confirmed by NOE spectroscopy. Reaction of tetra- benzylated Quercetin (25) with excess methyl iodide in presence of K2CO3 in DMF gave compound 28 which was deprotected using Pd(OH)2 in ethanol-THF at room temperature to yield 5-O-methyl- Quercetin (22, Azaleatin). Methylation of tribenzylQuercetin (26) using methyl iodide, K2CO3 in DMF gave compound 29. Finally, deprotection of benzyl group using Pd(OH)2 in ethanol-THF under hydrogen at room temperature afforded the targeted compound 3���- O-methylQuercetin (21, iso-rhamnetin) as shown in Scheme 4 [41]. M. Bouktabil et al. further synthesized 4���-O-methylQuercetin (23, Tamarixetin) and 7-O-methyl Quercetin (4, Rhamnetin) by initial protection of B-ring followed by reactions at other hydroxy groups. 3���-,4���-Hydroxy groups of Quercetin (1) were selectively protected by catechol ring using dichlorodiphenylmethane, K2CO3 in acetonitrile at 180��C to provide the protected product 30, which was benzylated using benzyl bromide, K2CO3 in DMF to provide B-ring protected-3,5,7-tribenzylated Quercetin (31). 3,7-dibenzyla- ted Quercetin (32) was obtained by deprotection of catechol ring