Original article
Synthesis of methylated quercetin derivatives and their reversal activities on P-gp- and BCRP-mediated multidrug resistance tumour cells
Graphical abstract
Synthesized methylated quercetin derivatives showed potent P-gp and BCRP-mediated multidrug resistance reversal activity.
Highlights
► A series of quercetin derivatives with various O-3 substituents were synthesized. ► They have not any inherent cytotoxicity to cancer and normal mouse fibroblast cells. ► O-3 substituent played a key role for determining P-gp- and BCRP-modulating activity. ► Compound 17 was equipotent against both P-gp and BCRP.
Introduction
Multidrug resistance (MDR) in cancer has been a major obstacle to successful cancer chemotherapy. An important mechanism for MDR is the enhanced cellular efflux of anticancer agents due to over-expression of ATP-binding cassette (ABC) transporter proteins [1]. Among the 48 ABC transporters identified so far, P-glycoprotein (P-gp, ABCB1), multidrug resistance-related protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) are three main efflux transporters that have been shown to be associated with MDR [2]. The three ABC proteins consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) [3], [4], [5], [6], [7], [8], [9], [10]. Despite their structural similarities, the substrate binding modes and binding sites of the three proteins are not well characterized. There is no common “pharmacophore” that can be identified as an inhibitor of these three ABC transporters [3]. Nevertheless, different inhibitors or modulators of ABC multidrug efflux pumps have been identified either by serendipitous discovery, combinatorial chemistry and rational drug design or based on the known structure of these transporters [11], [12], [13]. There are three generations of P-gp inhibitors including calcium channel blockers [14], [15], [16], calmodulin antagonists [17], [18], cyclic peptides, steroids, and some synthetic compounds such as elacridar, tariquidar, biricodar, S-9788 (shown in Fig. 1), ontogen, zosuquidar, and purine derivatives [3], [19], [20], [21], [22], [23]. Pantoprazole, fumitremorgin C and its derivatives (eg. Ko143) are specific ABCG2 inhibitors [3], [24]. The third generation P-gp inhibitors of elacridar and tariquidar also modulate ABCG2 activity [25], [26]. Fewer MRP1 inhibitors have been identified. The Leukotriene C4 (LTC4) analogue, MK571, S-decylglutathione and probenecid have been described as MRP1 modulators [3], [27], [28]. All of these three ABC drug transporters can interact with the modulator biricodar [3].
It has been demonstrated that non-toxic natural flavonoids such as kaempferol, quercetin, baicalein, myricetin, fisetin, morin and epigallocatechin gallate may reverse MDR [29]. Quercetin (Fig. 1) can modulate P-gp-, BCRP-, or MRP1-mediated drug resistance [30], [31], [32], [33], [34], and preliminary SAR studies of flavonoids as MDR modulating agents were carried out by Conseil et al. [35]. However, the concentration of quercetin in the experiment was still high. For example, in one study, addition of 100 μM of quercetin could result in an increased daunomycin accumulation (201.8 ± 16.4%) in the P-gp overexpressed resistant cell line MCF-7/ADR, which was comparable to 100 μM of verapamil (229.4 ± 17.6%). Interestingly, quercetin pentamethyl ether (at 20 μM) was found to have shown potent P-gp modulating activity [31], [36]. Other methylated phenolic compounds like permethyl epigallocatechin gallate (Fig. 1) [37], permethyl ningalin B (Fig. 1) [38] and its synthetic analogues (eg. compounds 1 and 2 in Fig. 1) [39] also displayed potent P-gp-modulating activities. These results suggest that methylation of some natural phenolic compounds could improve P-gp modulating activity, and multi-methoxy substituted phenyl rings may be an important pharmacophore for reversing MDR. Moreover, we introduce a basic nitrogen on flavonoids in order to increase their lipophilic properties, Ferte et al. synthesized a series of flavonoid (including diosmetin, diosmin and the flavanone hesperetin) derivatives containing a N-benzylpiperazine side chain at O-5, O-7, or O-3′, and investigated their anti-MDR ability [40]. At 5 μM, it was found that most compounds (such as compounds 3 and 4 shown in Fig. 1) potentiated doxorubicin cytotoxicity on resistant K562/DOX MDR cells [40]. These compounds were also able to increase the intracellular accumulation of JC-1, a fluorescent molecule recently described as a probe of P-glycoprotein-mediated MDR [40]. In the present work, we aimed to synthesize a novel series of methylated quercetin derivatives and 5,7,3′,4′-tetra-O-methylated quercetin derivatives containing a heterocycle or a multi-methoxyphenyl group in the O-3 substituents, and to investigate their anti-MDR activity affected by different side chain positions from the literature [40]. The modulating selectivity of the synthesized quercetin derivatives on the three main MDR-associated ABC efflux transporters, P-gp, BCRP, and MRP1, was also investigated.
Section snippets
Chemistry
We have previously found that permethyl epigallocatechin gallate was more potent than pentamethyl epigallocatechin (Fig. 1) in modulating P-gp in LCC6MDR cells [37], suggesting that substitutions at C-3 position of quercetin may be important in P-gp modulating activity. Here, we tested such hypothesis by synthesizing and evaluating a series of 3-substitued 5,7,3′,4′-tetra-O-methylated quercetins, such as compounds 7–17, 19, 21, and 23 (shown in Scheme 1, Scheme 2, Scheme 3 respectively) for
Conclusions
In summary, we demonstrated that synthetic quercetin derivatives exhibited promising P-gp- and BCRP-mediated MDR reversal activities without any inherent cytotoxicity to cancer cell lines or normal mouse fibroblast cell lines. The number of methoxy substituents on the terminal phenyl ring and the type of linkers at O-3 side chain are two key structural features for determining P-gp-modulating activity of quercetin derivatives. Compound 17 with a 3-methoxybenzoyloxyethyl at O-3 showed the
General
Starting materials and reagents were purchased from commercial suppliers and were used without further purification. 9-(2-Bromoethyl)-9H-purin-6-amine (18) [43], 9-(2-bromoethyl)-1,3-dimethyl-1H-purine-2,6(3H,9H)dione (20) [44], or 6-(2-bromo-ethoxy)-N-(3-chloro-4-fluorophenyl)-7-methoxy-3,4-dihydroquinazolin-4-amine (22) [45] were prepared following the methods in literature. Anhydrous methylene chloride was distilled under nitrogen from CaH2. Anhydrous DMF was distilled under vacuum from CaH2
Acknowledgement
This project was funded by National Natural Science Foundation of China (NSFC 81172926), General Research Fund (B-Q16G) of the Research Grant Council of Hong Kong, and Special Fund for Marine Scientific Research in the Public Interest of China (201005024).
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These two authors contribute equally to this work.