Water-Soluble and Cleavable Quercetin–Amino Acid Conjugates as

Article pubs.acs.org/jmc

Water-Soluble and Cleavable Quercetin−Amino Acid Conjugates as Safe Modulators for P‑Glycoprotein-Based Multidrug Resistance Mi Kyoung Kim,† Hyunah Choo,*,‡,§ and Youhoon Chong*,† †

Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea ‡ Center for Neuro-Medicine, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seoungbuk-gu, Seoul 136-791, Korea § Department of Biological Chemistry, University of Science and Technology, Youseong-gu, Daejeon 305-350, Korea ABSTRACT: Quercetin−amino acid conjugates with alanine or glutamic acid moiety attached at 7-O and/or 3-O position of quercetin were prepared, and their multidrug resistance (MDR)-modulatory effects were evaluated. A quercetin−glutamic acid conjugate, 7-O-Glu-Q (3a), was as potent as verapamil in reversing MDR and sensitized MDR MES-SA/Dx5 cells to various anticancer drugs with EC50 values of 0.8−0.9 μM. Analysis on Rh-123 accumulation confirmed that 3a inhibits drug efflux by Pgp, and Pgp ATPase assay showed that 3a interacts with the drug-binding site of Pgp to stimulate its ATPase activity. Physicochemical analysis of 3a revealed that solubility, stability, and cellular uptake of quercetin were significantly improved by the glutamic acid promoiety, which eventually dissociates from 3a to produce quercetin and quercetin metabolites in intracellular milieu. Taken together, potent MDRmodulating activity along with intracellular conversion into the natural flavonoid quercetin warrants development of the quercetin−amino acid conjugates as safe MDR modulators.

■

steroid (tamoxifen), and detergent (cremophor EL).9 However, as these first-generation Pgp inhibitors had not been specifically developed for inhibiting MDR, they suffered from lack of sensitivity and specificity to result in ineffectiveness or toxicity at the doses required to attenuate Pgp function.10 Investigations of less toxic and more potent Pgp inhibitors were then prompted by structural modification of the first-generation drugs to remove their non-MDR pharmacological effects, which led to identification of the second-generation MDR modulators such as dexverapamil,11 dexniguldipine,12 valspodar,13,14 and biricodar.15−17 Even though the side effects disappeared, clinical trials of the second-generation drugs were performed with little success due to their low potency as well as unpredictable pharmacokinetic interactions with chemotherapy, which elevated plasma concentration of the anticancer drugs beyond acceptable toxicity by limiting drug clearance and metabolism.18 Therefore, a need for a third generation of modulators with improved chemotherapeutic potentials was conceived and several compounds, including laniquidar,19 ONT-093,20 zosuquidar,21 elacridar,22 and tariquidar,23 were designed specifically for high transporter affinity and low pharmacokinetic interaction. These compounds exhibited effective MDR modulatory activity, high affinity and selectivity for target MDR transporter(s) at low nanomolar range, and subsequently low in vitro toxicity. However, recent phase III clinical trials of tariquidar and zosuquidar failed due to their toxicity and lack of

INTRODUCTION Cancer multidrug resistance (MDR), a term coined to represent cross-resistance or insensitivity of cancer cells to a wide spectrum of anticancer agents, remains as one of the important unsolved problems in cancer treatment.1 Investigations have revealed that MDR may have been mediated by numerous mechanisms operating at multiple events of cytotoxic action of the anticancer drugs. Among those, ATP-driven transmembrane transport of the structurally and functionally unrelated cytostatic agents thereby limiting them from reaching to therapeutic concentrations inside the cell is one of the best characterized MDR mechanisms.2 ATP-binding cassette (ABC) superfamily of membrane transporters which are overexpressed in malignant cells are known to be the key players in mediating MDR via ATP-dependent drug efflux mechanisms. Various transport proteins of the ABC superfamily have been characterized and they include P-glycoprotein (Pgp),3,4 multidrug resistance protein 1 (MRP1),5,6 and breast cancer resistance protein (BCRP).7 Ever since the pivotal roles of the ABC transporters in MDR were demonstrated, there has been intensive search for potent MDR modulators via inhibition of this class of membrane transporters, which led to the discovery of a group of Pgp inhibitors. The first-generation Pgp inhibitors represented by a calcium channel blocker verapamil8 consist of drugs already approved for other medical treatment, e.g., immunosuppressant (cyclosporine A), antibiotic (erythromycin), antimalarial (quinine), psychotropic phenothiazine (fluphenazine), indole alkaloid (reserpine), steroid hormone (progesterone), anti© 2014 American Chemical Society

Received: February 22, 2014 Published: August 14, 2014 7216

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

pharmacological effects.21 Taken together, the development of MDR-modulating agents has been hampered by lack of sensitivity, unacceptable toxicities, and unpredictable pharmacokinetic interactions. Therefore, investigation of new chemical entities with MDR modulatory activity as well as proven safety profiles is highly anticipated. In this regard, recent identification of natural products as potential MDR modulators24−31 is worth noting; because most of these natural compounds are essential components of human diet, they can be presumed to be low in toxicity even at higher doses. In particular, flavonoids, polyphenolic antioxidants widely distributed in the plant kingdom, drew our attention because dietary intakes of certain foods rich in flavonoids are known to be related with potential health benefits. Along with the well-known safety feature, various bioactivities associated with flavonoids such as antioxidant, anti-inflammatory, anticancer, and antiviral activities allow large amounts of daily human consumption in the forms of antioxidant supplements and complementary cancer therapy. Even though the antioxidant activity is the representative bioactivity of the flavonoids, we 32 and others33−44 have reported that the flavonoids or flavonoidrich plant extracts are able to alter cellular functions independently of their antioxidant potential. For example, as the A- (5-hydroxyl group) and C-ring (4-carbonyl functionality) moiety of flavonoids are supposed to mimic the adenine moiety of ATP (Figure 1), some flavonoids are known to serve

bind to the ATP-binding site of the ABC transporters to inhibit efflux of the drugs and/or drug conjugates.45 In this context, it is not surprising that there have been a number of attempts to evaluate MDR modulating activity of the flavonoids, which resulted in identification of flavonoids such as chrysin, quercetin, kaempferol, and dehydrosilybin as potential safe MDR modulators.46−52 By far the most successful approach to develop flavonoids with MDR modulatory activity was to use flavonoid dimers.53−55 Even though the mechanism of MDR modulatory activity of the flavonoid dimers has yet to be elucidated, structure−activity relationship study53−55 revealed a possible binding role of the flavonoid core structure to the target transporters as well as the importance of the bivalent binding of the two flavonoid monomers connected by poly(ethylene glycol) linkers. Apigenin homodimers are representative flavonoid dimers which have shown remarkable bivalent modulatory effect for both Pgp-mediated53 and MRP1mediated56 multidrug resistance. However, because of the highly hydrophobic nature, the flavonoid dimers were shown to be barely soluble in water, which did not allow in vivo animal model study.55 In addition, relatively high molecular weight (∼700) as well as loss of natural flavonoid structure due to incorporation of an uncleavable covalent spacer might raise other safety concerns during development of this class of MDR modulators. Thus, there is a compelling therapeutic need to use a flavonoid itself rather than its analogues in terms of making good use of the characteristic safety feature of the natural flavonoids. However, studies that support clinical uses of flavonoid are few, presumably because of its low biological activity and suboptimal physicochemical as well as pharmacokinetic properties including low solubility, low stability, poor cellular uptake,57−59 and fast metabolism.59−61 Previously, we have shown that both physicochemical properties and bioactivities of the flavonoid could be significantly improved via conjugation with a cleavable promoieties such as pivaloxymethyl (POM)62,63 or isopropyloxycarbonylmethoxy (POC)64 group to its 7-OH and/or 3OH position. Also confirmed was that the promoieties were cleaved in cellular milieu to release the parent drug, flavonoid, in time-dependent manner. On the basis of these observations, we intended to prepare a series of flavonoid conjugates for the

Figure 1. Comparison of structures of adenosine and flavonoids.

as inhibitors of the ATP-binding proteins such as protein kinases33−40 and ATPases41−44 via ATP-competitive binding. By the same mechanism, the flavonoids are also expected to

Figure 2. Structures of the quercetin (1) and its amino acid conjugates (2 and 3). 7217

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Quercetin−Amino Acid Conjugates (2−3)a

a Reagent and conditions: (a) Ph2CCl2, 180 °C; (b) Ac2O, Pyr, 70 °C; (c) PhSH, imidazole, NMP, 0 °C; (d) MOMCl, K2CO3, acetone, rt; (e) NH3/ MeOH, 0 °C; (f) BnBr, K2CO3, acetone, rt; (g) 3N HCl/THF, 40 °C; (h) 11a or 11b, DIPEA, DMF, rt; (i) H2, Pd/C, THF/MeOH, rt; (j) TFA, 0 °C; (k) (S)-N-Boc-Ala or (S)-N-Boc-Glu(OtBu), EDC, Oxyma, CH2Cl2, rt; (l) Boc2O, DMAP, CH2Cl2, rt; (m) bis-4-nitrophenyl carbonate, DIPEA, DMF, rt.

■

purpose of developing novel flavonoid MDR modulators with enhanced safety, water solubility, and MDR-modulating activity. In this proof-of-concept study, flavonoid quercetin, which is known to possess broad spectrum of biological activities including MDR modulation,49 was used as the flavonoid of choice. Also, two amino acids (alanine and glutamic acid), of which side chains greatly differ in polarity were used as promoieties, and introduced to 7-OH and/or 3OH of the quercetin scaffold through a carbamate and an ester linkage, respectively, to give the corresponding quercetin− amino acid conjugates (2−3, Figure 2). Herein, we report regioselective synthesis of six novel quercetin−amino acid conjugates, evaluation of their physicochemical properties such as solubility and stability, and their MDR modulatory activities.

RESULTS AND DISCUSSION I. Synthesis. For regioselective introduction of the amino acid functionalities into the quercetin scaffold, orthogonal protection of its four hydroxyl groups (3, 7, 3′, and 4′-OH) was required (Scheme 1). First, the catechol moiety of quercetin (1) was protected by reaction with 1,1-dichlorodiphenylmethane at 180 °C to give the corresponding diphenylmethylketal,65 of which three hydroxyl groups were acetylated with an excess amount of Ac2O in pyridine (93% yield). Treatment of the resulting triacetate 5 with PhSH and imidazole in NMP (N-methyl-2pyrrolidone) provided the 7-O-monodeacetylated product 6 in 90% yield.66 Direct coupling of this intermediate with (S)-N-(4nitrophenoxycarbonyl)alanine tert-butyl ester (11a) or (S)-N(4-nitrophenoxycarbonyl)glutamic acid di-tert-butyl ester (11b),67 obtained by reaction of the corresponding amino 7218

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

acid tert-butyl esters (10) with bis-4-nitrophenyl carbonate, to prepare the quercetin 7-carbamate was complicated due to concomitant loss of the acetyl groups under the basic reaction conditions. Thus, the 3,5-di-OAc functionalities of 6 were replaced by the base-stable ethereal protecting groups through sequential protection−deprotection strategy; protection of the free 7-OH of 6 with a methoxymethyl (MOM) group followed by ammonolysis of the remaining two acetyl groups at 3-O and 5O positions produced the key intermediate 7 in 93% yield. The 3-OH group of 7 was protected as benzyl ether, and the 7methoxymethyl ether linkage was then cleaved under acidic conditions to give 8 in 75% yield, which smoothly underwent coupling reactions with 11a or 11b to give the corresponding quercetin 7-carbamates (85% and 82% yield). Simultaneous hydrogenolysis of the diphenylmethylketal and the benzyl protecting groups followed by TFA-promoted cleavage of the tert-butyl esters provided the desired products 2a and 3a in 25% and 21% yield, respectively. On the other hand, the quercetin 3-esters (9) were obtained by EDC-coupling of 7 with (S)-N-Boc-Ala or (S)-N-Boc-Glu(OtBu) and subsequent removal of the 7-O-MOM functionality (72% yield for 9a and 68% yield for 9b). While removal of the diphenylmethylketal and the tert-butyl esters of 9a and 9b provided the free quercetin 3-esters 2b (14% yield) and 3b (17% yield), respectively, further coupling reactions of the N-Boc-protected 9 with 11a or 11b and ensuing deprotection reactions produced the quercetin 7-carbamoyl-3-esters 2c and 3c (13% and 18% yield). II. MDR Modulator Screening System. Drug-sensitive human uterine sarcoma cell line MES-SA and its drug-resistant counterpart MES-SA/Dx5, which is known to exert MDR through overexpression of Pgp,68 was used for screening MDR modulators. MES-SA/Dx5MDR cell line was derived from the sensitive cell line by prolonged in vitro exposure to doxorubicin,68 and overexpression of Pgp in this cell line was confirmed by Western blot. Figure 3 shows that the Pgp (relative molar mass, Mr = 170000) is expressed highly in MDR cell line (MES-SA/Dx5) but is not detected in the parental (MES-SA) cells.

actinomycin D, vinblastine, and paclitaxel, respectively (Figure 4a). In contrast, viability of the drug-resistant cell line (MESSA/Dx5) was shown to be only moderately affected by the anticancer drugs (IC50 = 8.20, 4.68, 4.90, and 4.66 μM) (Figure 4b), which correspond to 122-, 36-, 45-, and 48-fold decrease in cytotoxicity due to MDR conferred by overexpression of Pgp (Table 1). III. Evaluation of MDR Modulating Activity. MDR modulating activity of quercetin (1) and its amino acid conjugates (2a−3c) was estimated by two different methods: (i) measurement of restoring the cytotoxicity of a given chemotherapeutic drug toward the multidrug-resistant cancer cells (MES-SA/Dx5) in the presence of a quercetin conjugate69,70 and (ii) flow cytometric measurement of efflux of a specific fluorescent substrates of Pgp, rhodamine 123 (Rh123),71−74 out of MDR cells. III. A. In Vitro MDR Reversal Effect of Quercetin (1) and Quercetin−Amino Acid Conjugates (2a−3c). MDR modulatory effects of the title compounds were investigated by cotreatment of quercetin (1) or quercetin conjugates (2a−3c) with an anticancer drug and evaluation of restoration of its cytotoxicity in drug-resistant cancer cell line (MES-SA/Dx5). First, to evaluate if quercetin (1) and the synthesized quercetin conjugates (2a−3c) were cytotoxic per se, MTT assay was performed in two tumor cell lines [HCT116 (human colorectal carcinoma) and LNCaP (human prostate adenocarcinoma)] and in a normal cell line [HS27 (human foreskin fibroblast)]. Although viabilities of the tumor cell lines (HCT116 and LNCaP) were slightly decreased upon treatment with the quercetin conjugates at high concentrations (>50 μM), no significant intrinsic cytotoxicity associated with the quercetin conjugates was observed in all cell lines tested (IC50 > 100 μM) (data not shown). In particular, high cell viability (∼70%) was maintained in the normal cell line (HS27) even under high concentrations (100 μM) of the quercetin conjugates. Cytotoxicities of the quercetin conjugates were also evaluated in drug-sensitive MES-SA and MDR MES-SA/Dx5 cell lines. In MES-SA cell lines, it was confirmed that, at the concentration used to modulate MDR (5 μM), quercetin (1) and its conjugates (2a−3c) were not cytotoxic, and cytotoxic profiles of the anticancer agents were not affected by addition of quercetin or its conjugates. The same result was obtained when the well-known MDR-modulating agent verapamil, a positive control used in this study, was used in combination with the anticancer drugs (data not shown). In drug-resistant MES-SA/ Dx5 cell lines, however, verapamil restored the sensitivity of the resistant MES-SA/Dx5 cells to doxorubicin (Figure 5a). Thus, in combination with verapamil (5 μM), IC50 of doxorubicin against MES-SA/Dx5 was estimated to be 0.12 μM, which corresponds to 68.0-fold increased cytotoxicity compared with the result obtained with doxorubicin alone (Table 2). Addition of quercetin (1) (5 μM) also potentiated the cytotoxicity of doxorubicin against MES-SA/Dx5 but not to a significant degree (IC50 = 4.26 μM, fold-reversal = 1.92) (Figure 5a, Table 2). In contrast, the quercetin conjugates (5 μM) showed moderate to potent MDR-reversal activities depending on the type and position of the amino acid substituents attached on the quercetin scaffold (Figures 5b−d). Among the series, the conjugates with alanine and glutamic acid substituents at the 7O position of quercetin via an amide linkage, 2a and 3a, showed the most potent MDR-reversal activity (IC50 = 0.41 and 0.14 μM, fold-reversal = 20.0 and 58.6-fold, respectively) (Figure 5b, Table 2). In particular, quercetin−7-O-glutamic acid conjugate

Figure 3. Western blot to demonstrate the expression of the MDR protein (Pgp) in MES-SA/Dx5 (right lane) and its absence in MES-SA (left lane).

The MDR phenotype of MES-SA/Dx5 cell line was then confirmed by evaluating cytotoxicities of anticancer drugs such as doxorubicin, actinomycin D, paclitaxel, and vinblastine, which are subject to Pgp-mediated MDR.3,4 When the drugsensitive cancer cell line (MES-SA) was treated with anticancer drugs, the cell viabilities decreased in dose-dependent manners with IC50s of 0.067, 0.13, 0.11, and 0.098 μM for doxorubicin, 7219

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Figure 4. Comparative cytotoxicities of various anticancer drugs to (a) drug-sensitive MES-SA and (b) MDR MES-SA/Dx5 cell lines.

Table 1. Comparison of Cytotoxicities (IC50) of Anticancer Agents in Drug-Sensitive (MES-SA) and Drug-Resistant (MES-SA/ Dx5) Cell Lines doxorubicin IC50 (μM)a

fold-resistanceb

MES-SA MES-SA/Dx5

0.067 8.20 120

actinomycin D

vinblastine

paclitaxel

0.13 13.12

0.11 12.28

0.098 10.56

100

111

108

a

Amount of the anticancer agents required to reduce cell viability of the drug-sensitive (MES-SA) or drug-resistant (MES-SA/Dx5) cells by half. b Fold-resistance = IC50 (in MES-SA/Dx5)/IC50 (in MES-SA)

(7-O-Glu-Q, 3a) was found to be as effective as the positive control, verapamil, and potentiated the cytotoxic activity of doxorubicin against MES-SA/Dx5 cells with IC50 of 0.14 (foldreversal = 58.6, Table 2), which corresponds to 30.5-times increased MDR-reversal activity compared with that of quercetin. On the other hand, the quercetin conjugates with 3-O-alanyl and 3-O-glutamyl functionalities, 2b and 3b, showed moderate MDR-modulating activity (IC50 = 1.21 and 1.10 μM, respectively) (Figure 5c, Table 2) with slightly increased foldreversal (6.78- and 7.45-fold, respectively) (Figure 5c, Table 2) compared with that of quercetin (1.92, Table 2). Interestingly, introduction of the amino acid functionalities at both 7-O and 3-O positions (2c and 3c) resulted in MDR-modulating activity (IC50 = 0.78 and 0.71 μM, fold-reversal = 10.5- and 11.5-fold, respectively) (Figure 5d, Table 2) intermediate between those of 7-O-substituted (2a and 3a) and 3-O-substituted (2b and 3b) derivatives. The quercetin−7-O-glutamic acid conjugate (7-O-Glu-Q, 3a), which was most effective in reversing doxorubicinresistance in MES-SA/Dx5 cell line, also turned out to potentiate cytotoxicities of other anticancer agents in the same drug-resistant cell line (Figure 6). Thus, upon combination with 3a, IC50s of actinomycin D (Figure 6a), vinblastine (Figure 6b), and paclitaxel (Figure 6c) were decreased to 0.34, 0.33, and 0.32 μM, which were comparable to those obtained with cotreatment with verapamil (0.23, 0.24, and 0.22 μM, respectively). As with the previous doxorubicin case, MDR-modulating activity of quercetin (1) was estimated about 13.8−14.8-fold lower than those of 3a (Figures 6a−c). III. B. Concentration-Dependent Effect of the Quercetin− Glutamic Acid Conjugate 3a on Cytotoxicity of Anticancer Agents. As a quantitative measure of the MDR-reversal potency

of 3a, its concentration-dependent IC50-lowering effect was evaluated (Figure 7), and the amount of 3a required to reduce the IC50s of the anticancer agents by half in the drug-resistant MES-SA/Dx5 cell line was noted as its EC50 values (Table 3).53,55 In MES-SA/Dx5 cells, quercetin−glutamic acid conjugate 3a exhibited pronounced effects in modulating Pgp-mediated resistance toward doxorubicin, actinomycin D, vinblastine, and paclitaxel with EC50 values in submicromolar ranges between 0.8 and 0.9 μM (Table 3), which are comparable with those of verapamil (EC50 = 0.6−0.8 μM, Table 3). III. C. Impact of Stereochemistry of the Amino Acid on MDR Reversal Effect of the Quercetin−Glutamic Acid Conjugate 3a. Chirality of the amino acid promoieties could be associated with pharmacodynamic and pharmacokinetic aspects of the quercetin conjugates. Also, because of the cleavable nature of the quercetin−amino acid conjugates to release quercetin and free amino acids, the functional roles of the amino acid promoieties could be questioned. Thus, the MDR reversal effects of the quercetin−D-glutamic acid conjugate (ent-3a, Scheme 2) along with the natural (L-) and unnatural (D-) glutamic acids were evaluated and compared in Table 4. The enantiomer of 3a, quercetin−D-glutamic acid conjugate (ent-3a), was prepared using precisely the same synthetic procedure (Scheme 2). The compound ent-3a was also potent MDR modulator (fold-reversal = 52.2), albeit less so than the natural enantiomer 3a, and this result shows that the MDR modulatory activity of the quercetin−amino acid conjugates were not dependent upon the enantiomeric forms of the amino acid promoieties. On the other hand, the lack of MDR modulatory activity observed from the combination of doxorubicin with L- and D- glutamic acid 7220

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Figure 5. Cell viabilities of MES-SA/Dx5 cell lines after combination treatment of doxorubicin with (a) quercetin (1) and (b−d) its conjugates: (b) 2a/3a, (c) 2b/3b, and (d) 2c/3c. Verapamil was used as a positive control and concentrations of verapamil, quercetin (1), and quercetin conjugates (2a−3c) were fixed at 5 μM. Assay was performed in triplicate.

Table 2. Cytotoxicity (IC50) of Doxorubicin in Drug-Resistant (MES-SA/Dx5) Cell Lines upon Combination with Quercetin and Quercetin Conjugates (5 μM) modulator

none

verapamil

1

2a

2b

2c

3a

3b

3c

IC50 (μM)a fold-reversalb

8.20

0.12 68.0

4.26 1.9

0.41 20.0

1.21 6.78

0.78 10.5

0.14 58.6

1.10 7.45

0.71 11.5

a

Amount of doxorubicin required to reduce cell viability of the drug-resistant MES-SA/Dx5 cells by half. bFold-reversal = IC50 (doxorubicin alone)/ IC50 (combination treatment)

provides evidence that the amino acid promoieties dissociated from the quercetin−amino acid conjugates do not function as the active components. III. D. Flow Cytometric Measurement of Efflux of Rh-123. The potent MDR-reversal activity of 7-O-Glu-Q (3a) in Pgpoverexpressing MES-SA/Dx5 cell lines might result from its binding to Pgp to block efflux of the anticancer drugs from the cell. We tested this possibility by measuring the ability of 3a to modulate Pgp activity.71−74 Specifically, flow cytometry was used to measure the accumulation of Rh-123, a fluorescent Pgp

substrate, in the MES-SA/Dx5 cell line that was previously shown to overexpress Pgp (Figure 3). The cells were incubated with Rh-123 for a brief uptake period, then washed and incubated in the absence of Rh-123 for a 4 h efflux period. Flow cytometry was used to generate histograms representing Rh123 fluorescence within a population of cells (Figure 8). Addition of the known Pgp modulator, verapamil, led to an increase in the uptake and retention of Rh-123. Surprisingly, the quercetin conjugate 3a was nearly as potent as verapamil in increasing Rh-123 retention, demonstrating strong inhibitory 7221

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Figure 6. Comparison of IC50s of (a) actinomycin D, (b) vinblastine, and (c) paclitaxel in drug-resistant MES-SA/Dx5 cell lines after combination treatment with quercetin (1) and its conjugate 3a (5 μM). Verapamil was used as a positive control, and the assays were performed in triplicate. IC50 of each drug in drug-sensitive MES-SA cell line is given for demonstration of MDR-modulating activity.

Figure 7. Concentration-dependent effects of 3a and verapamil for lowering IC50s of (a) doxorubicin, (b) actinomycin D, (c) vinblastine, and (d) paclitaxel in drug-resistant MES-SA/Dx5 cells. Presented are typical experiments performed in triplicate.

Table 3. MDR Reversal Potency (EC50) of 3a in Drug-Resistant (MES-SA/Dx5) Cell Line EC50 (μM)

a

3a verapamil

doxorubicin

actinomycin D

vinblastine

paclitaxel

0.9 ± 0.2 0.8 ± 0.1

0.8 ± 0.1 0.7 ± 0.2

0.8 ± 0.3 0.6 ± 0.3

0.9 ± 0.2 0.7 ± 0.1

a Amount of the MDR modulators required to reduce the IC50s of the anticancer agents by half in the drug-resistant MES-SA/Dx5 cell line. The means ± SD of three independent experiments are shown.

others that bind to the nucleotide binding domain (NBD) of Pgp would inhibit ATP hydrolysis.53 To investigate the mechanism of Pgp inhibition by the quercetin−glutamic acid conjugate 3a, the effect of 3a (100 μM) on both Pgp ATPae activity and verapamil-induced ATPase activity was evaluated.53 The Pgp-Glo assay detects the remaining unmetabolized ATP as a luciferase-generated luminescent signal after incubation of

effect of 3a on the activity of Pgp in a concentration-dependent manner (Figure 8a,b). III. E. Effects of 3a on Pgp ATPase Activity. Compounds that interact with Pgp can be identified as stimulators or inhibitors of its ATPase activity; compounds that bind to the drug-binding cavity located within the transmembrane domain (TMD) of Pgp typically stimulate its ATPase activity,75 while 7222

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Scheme 2. Synthesis of Enantiomer of the Quercetin−Amino Acid Conjugate 3a (ent-3a)

Table 4. Comparison of Fold-Reversals Conferred by Combination of Doxorubicin with Quercetin Conjugate 3a, Its Enantiomer ent-3a, Natural Amino Acid (L-Glutamic Acid), and Unnatural Amino Acid (D-Glutamic Acid) modulatora

none

verapamil

3a

ent-3a

IC50 (μM)b fold-reversalc

8.20

0.12 68.0

0.14 58.6

0.16 52.2

L-glutamic

6.8 1.2

acid

D-glutamic

acid

7.2 1.1

a Concentrations of the modulators were fixed at 5 μM. bAmount of doxorubicin required to reduce cell viability of the drug-resistant MES-SA/Dx5 cells by half. cFold-reversal = IC50 (doxorubicin alone)/IC50 (combination treatment)

Figure 8. Inhibition of rhodamine 123 (Rh-123) efflux from Pgp-overexpressing MES-SA/Dx5 cells. MES-SA/Dx5 cells were pretreated with verapamil or the quercetin conjugate 3a followed by addition of Rh-123. Flow cytometry was used to generate histograms representing Rh-123 fluorescence within a population of cells. As a histogram peak shifts to the right on the x-axis, the intracellular fluorescence increases. The experiment was performed at two different concentrations of verapamil and the conjugate 3a: (a) 1 μM and (b) 5 μM.

it was a weaker stimulator than verapamil (3.5-stimulation fold, Figure 9). Interestingly, combination of verapamil and 3a resulted in reduced Pgp ATPase stimulation (4.0-stimulation fold, Figure 9) compared with the verapamil-treated samples, and this result might suggest that verapamil and 3a compete with each other for binding to the same or overlapping drugbinding sites inside TMD of Pgp. This result is more in line with the general observation that substituted flavonoids would preferentially bind to the steroid interacting region or drugbinding site of TMDs48,53,55 while the unsubstituted flavonoids interact bifunctionally with vicinal ATP- and steroid-binding sites of Pgp.77 IV. Physicochemical Properties of the Quercetin Conjugates. As the title compounds were designed specifically to serve as quercetin conjugates with water-soluble and cleavable amino acid functionalities, multiple physicochemical

ATP with the recombinant human Pgp in a cell membrane fraction. Accordingly, decreases in luminescence reflect ATP consumption by Pgp through stimulation of the Pgp ATPase activity. ATP consumption in the presence of Na3VO4, a selective Pgp inhibitor, is attributed to minor non-Pgp ATPase activities present in the membrane preparation, and the difference in luminescent signal between Na3VO4-treated samples and untreated samples represents the basal Pgp ATPase activity (Figure 9). Also, the difference in luminescent signal between Na3VO4-treated samples and samples treated with the test compound represents Pgp ATPase activity in the presence of the test compound (Figure 9). Verapamil is known to interact with the drug-binding site of Pgp to stimulate its ATPase activity,76 and it increased Pgp ATPase activity over the basal level by 4.6-fold (Figure 9). The quercetin−glutamic acid conjugate 3a (100 μM) also increased Pgp ATPase activity, but 7223

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

about 12 times more soluble than quercetin in pure water, which confirms superior solubility of 3a compared with that of quercetin in “real-life” conditions. IV. B. Stability. Previously, we reported low stability of quercetin particularly in cell culture media due to oxidative decomposition, and introduction of a promoiety to either 7-O or 3-O position of quercetin can significantly improve its stability profile.62−64 In this study, stabilities of quercetin (1) and the quercetin−amino acid conjugates (2a−3c) were assessed in PBS (phosphate buffered saline) and cRPMI (RPMI-1640 culture medium supplemented with 10% fetal bovine serum and 1% antibiotics) under cell-free conditions by HPLC analysis, and the result is summarized in Figure 11. Figure 9. Effects of 3a on Pgp ATPase activity. Pgp ATPase activity was presented as a decrease in luminescence of samples treated with the test compounds compared to the Na3VO4-treated samples. Data are expressed as mean ± SD from three independent experiments (VPR = verapamil, Que = quercetin, SF = stimulation-fold).

properties of the quercetin conjugates were evaluated and compared with those of quercetin. IV. A. Solubility. Solubilities of quercetin (1) and its amino acid conjugates (2a−3c) were estimated by measuring forward scattered light when a laser beam is directed through their PBS solutions (5% DMSO),78 and the results are summarized in Figure 10a. As anticipated by the polar nature of the amino acid promoieties, all the quercetin conjugates prepared in this study showed high aqueous solubility up to 400 μM, while the solubility of quercetin (1) sharply decreased at concentrations higher than 100 μM. It should be noted that, in the solubility study outlined above, the compounds were first dissolved in DMSO, followed by dilution in PBS (5% DMSO). Thus, to eliminate the cosolvent effect, the solubility of quercetin and its amino acid conjugates was evaluated in pure water.79 Briefly, the same molar quantities of quercetin (3.0 mg, 0.01 mmol) and 3a (4.8 mg, 0.01 mmol) were dissolved in water (1 mL) by vortexing, and the resulting solutions were centrifuged to remove undissolved material. The supernatant (100 μL) was taken, diluted with water (900 μL), and the UV−vis spectrum recorded (Figure 10b). On the basis of the observed absorbance and predetermined extinction coefficients (ε), it was estimated that the quercetin−glutamic acid conjugate 3a is

Figure 11. Half-lives (t1/2) of quercetin (1) and quercetin−amino acid conjugates (2a−3c) in cell culture media (cRPMI). *, p < 0.05, quercetin conjugates (2a−2b, 3a−3c) versus quercetin. **, p < 0.001, quercetin conjugate 2c versus quercetin. The means ± SD (error bars) of three independent experiments are shown.

As anticipated, quercetin (1) was least stable and showed fast decomposition with a half-life (t1/2) of 10.3 h, while the quercetin conjugates (2a−3c) were found intact even after 72 h incubation in PBS (data not shown). In cell-free cRPMI culture medium, quercetin underwent facile decomposition, leaving no trace on the HPLC chromatogram even after 30 min. Under the same cell culture conditions, the quercetin conjugates also underwent decomposition but their half-lives were significantly longer (2.5−9.3 h) than that of quercetin (0.4 h). Of particular

Figure 10. (a) Forward light scattering intensity of PBS solutions (5% DMSO) of quercetin (1) and quercetin−amino acid conjugates (2a−3c). (b) UV−vis spectroscopic comparison of solubility of quercetin (1, ■) and quercetin−glutamic acid conjugate 3a (◆) in pure water (with no cosolvent). 7224

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

Figure 12. Confocal images were taken to visually inspect cellular uptake of quercetin (1) and 7-O-Glu-Q (3a). The top row shows differential interference contrast (DIC) images of (a) MES-SA and (b) MES-SA/Dx5 cells exposed to 5 μM concentrations of quercetin (1, left panels) and 7O-Glu-Q (3a, right panels). The bottom row shows fluorescence images of (c) MES-SA and (d) MES-SA/Dx5 cells exposed to 5 μM concentrations of quercetin (1, left panels) and 7-O-Glu-Q (3a, right panels).

Figure 13. HPLC chromatograms of cell lysate after (a) 1 h, (b) 6 h, (c) 24 h, and (d) 48 h of incubation of MES-SA/Dx5 cells with 7-O-Glu-Q (3a). (Q, quercetin; Q-glu, quercetin glucuronide; Q-sul, quercetin sulfate). HPLC chromatograms of (e) the commercially available quercetin−3-Oglucuronide and (f) chemically synthesized quercetin sulfates.

interest is a positive correlation between the MDR-reversal activity (Table 2) and stability profiles (Figure 11) of the

Glu-Q (3a), which showed the most potent MDR-reversal activity, was most stable with a half-life of 9.3 h. IV. C. Intracellular Stability of 3a (7-O-Glu-Q). It is known that quercetin exhibits specific fluorescence (488 nmex/500− 540 nmem) upon intracellular localization followed by binding

quercetin conjugates: more stable quercetin conjugates tend to show more potent MDR-reversal activity. In particular, 7-O7225

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

to target proteins, and the fluorescence level depends on the degree of cellular uptake of quercetin.62,63,80,81 Therefore, cell fluorescence can be used as a surrogate marker for cell permeability as well as intracellular stability of quercetin or quercetin conjugates. After incubation of MES-SA and MESSA/Dx5 with quercetin (1) or 7-O-Glu-Q (3a) for 72 h, fluorescent staining was observed in a confocal microscope and the results are visualized in Figure 12. Upon treatment with quercetin (1), neither drug-sensitive (MES-SA) (Figure 12c, left panel) nor drug-resistant (MES-SA/Dx5) (Figure 12d, left panel) cell line showed fluorescence. In contrast, both MES-SA (Figure 12c, right panel) and MES-SA/Dx5 (Figure 12d, right panel) cells treated with 7-O-Glu-Q (3a) showed persistent fluorescence after 72 h of incubation. It is worth noting that, albeit at lower levels, the drug-resistant MES-SA/Dx5 cell line showed persistent fluorescence, which supports intracellular stability and accumulation of 3a even after 72 h. IV. D. Intracellular Metabolism of 3a (7-O-Glu-Q). Once inside the cell, the quercetin−amino acid conjugate 3a was designed to undergo hydrolysis to convert into quercetin. Thus, chemical species remaining in the intracellular compartment of MES-SA/Dx5 cells was analyzed by HPLC after incubation with 7-O-Glu-Q (3a), and the result is summarized in Figure 13. After short incubation (1 h) with 7-O-Glu-Q (3a), the native conjugate 3a was a sole compound which could be detected by HPLC analysis of the MES-SA/Dx5 cell lysate (Figure 13a). Intracellular conversion of 3a to quercetin (1) was first observed from the HPLC chromatogram of the cell lysate after 6 h of incubation (Figure 13b). The hydrolysis of 3a was slow but persistent, and both 7-O-Glu-Q (3a) and quercetin (1) were observed in the intracellular milieu of MES-SA/Dx5 cells even after prolonged incubation (24 and 48 h) (Figure 13c,d). In addition, quercetin metabolites such as quercetin glucuronide (Q-glu) and quercetin sulfate (Q-sul) were detected and identified by mass analysis62 from the samples obtained after extended incubation (Figures 13c,d). Q-glu and Q-sul coeluted with the authentic quercetin−3-O-glucuronide (Figure 13e) and the chemically synthesized quercetin sulfate82,83 (Figure 13f), respectively. Taken together, these results clearly show that the glutamic acid in 7-O-Glu-Q (3a) acts as a stable but cleavable promoiety to slowly dissociate from the quercetin for a prolonged period of time.

activity. Combined information on solubility, stability, and cellular uptake analyses showed that the glutamic acid promoiety in 7-O-Glu-Q (3a) enhanced solubility, stability, and cellular uptake of quercetin to maintain high intracellular level of 3a for a prolonged period of time, which provides a reasonable explanation for significantly enhanced MDRmodulating activity of 3a in comparison with quercetin. Also, HPLC analysis of the cell lysates confirmed that the quercetin− glutamic acid conjugate eventually undergoes metabolism to quercetin and quercetin metabolites in intracellular milieu. On the basis of the safe nature of quercetin, intracellular conversion of the quercetin conjugate 3a into quercetin and its metabolites warrants further development of the quercetin conjugate as a novel safe MDR modulator.

■

EXPERIMENTAL SECTION

General Methods for Chemistry. Chemicals were purchased from Sigma-Aldrich unless noted otherwise. H-Ala-OtBu·HCl and HGlu(OtBu)-OtBu·HCl were purchased from Tokyo Chemical Industry Co., Ltd. (TCI). H-D-Glu(OtBu)-OtBu·HCl, L-glutamic acid, and Dglutamic acid were obtained from Chem-Impex International Inc. TLC was performed on silica gel-60 F254 purchased from Merck. Column chromatography was performed using silica gel-60 (220−440 mesh) for flash chromatography. Mass spectrometric data (MS) were obtained by electron spray ionization (ESI). High resolution mass spectra (HRMS) were obtained at Korea Basic Science Institute (Daegu, Korea) and reported in the form of m/z (intensity relative to base peak = 100). Nuclear magnetic resonance spectra were recorded on Bruker 400 AMX spectrometer (Karlsruhe, Germany) at 400 MHz for 1H NMR and at 100 MHz for 13C NMR with tetramethylsilane as an internal standard. Chemical shifts were reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet). Coupling constants were reported in hertz (Hz). The chemical shifts were reported as parts per million (δ) relative to the solvent peak. The purity of the final compounds was determined by HPLC using the following method: Varian Polaris RP C18-A (250 mm × 4.6 mm, 5 μm particle size) column; gradient elution of acetonitrile in water for 60 min (5% for 0−8 min, 5%−10% for 5−15 min, 25% for 15−30 min, 25%−60% for 30−45 min, 60%−100% for 45−55 min, 100% for 55−60 min); flow rate of 1 mL/min; detection at two different wavelengths (340 and 254 nm). For all samples, 0.1% formic acid was added to water. All final biologically tested compounds were isolated in >97% purity as judged by HPLC. Acetic Acid 3,5-Diacetoxy-2-(2,2-diphenylbenzo[1,3]dioxol-5-yl)4-oxo-4H-chromen-7-yl Ester (5). Dichlorodiphenylmethane (8.5 mL, 44.3 mmol) was added to a stirred mixture of quercetin (1) (5 g, 14.8 mmol) in diphenyl ether (20 mL), and the reaction mixture was heated at 180 °C for 30 min. The mixture was cooled to room temperature, and petroleum ether (50 mL) was added to give a solid compound. Then the solid was filtered and purified by column chromatography (hexane:EtOAc = 4:1) to yield 2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-3,5,7-trihydroxy-4H-chromen-4-one (4.7 g, 10.1 mmol, 68% yield) as a yellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.18 (s, 1H, 5-OH), 7.89−7.92 (m, 2H, 2′-H, 6′-H), 7.63−7.69 (m, 5H, Ar-H), 7.45−7.49 (m, 5H, Ar-H), 7.19 (d, J = 8.5 Hz, 1H, 5′-H), 6.58 (s, 1H, 8-H), 6.28 (s,1H, 6-H). 13C NMR (100 MHz, DMSO- d6) δ 176.1 (4-C), 164.2 (7-C), 160.8 (5-C), 156.3 (9-C), 147.7 (3′-C), 146.8 (4′-C), 145.6 (2-C), 139.5 (Ar-C), 136.5 (3-C), 129.5 (Ar-C), 128.7 (CPh2), 125.8 (Ar-C), 125.3 (Ar-C), 123.1 (1′-C), 117.1 (6′-C), 108.9 (5′-C), 107.9 (2′-C), 101.1 (10-C), 98.4 (6-C), 93.7 (8-C). HRFABMS m/z Found: 467.1135 [M + H]+. Calcd for C28H19O7: 467.1056. To a solution of 2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-3,5,7trihydroxy-4H-chromen-4-one (1 g, 2.14 mmol) in anhydrous pyridine (10 mL) was added acetic anhydride (1 mL, 10.7 mmol) at room temperature. The reaction mixture was stirred for 6 h at 70 °C. After concentration, the crude product was purified by column chromatography (hexane:EtOAc = 2:1) on silica gel to give 5 (1.2 g, 2.0 mmol,

■

CONCLUSIONS In the quest to discover MDR modulators, natural flavonoid serves as a novel scaffold due to its proven safety profiles. However, clinical applicability of flavonoids is limited due to their relatively low activity as well as suboptimal physicochemical and pharmacokinetic properties. In this study, we tackled this problem by using flavonoid-amino acid conjugates. Thus, a series of quercetin−amino acid conjugates with alanine and glutamic acid moieties attached at 7-O and/or 3-O positions of quercetin were prepared and their MDR modulatory effects were evaluated. Among those, a quercetin−glutamic acid conjugate, 7-O-Glu-Q (3a), was as potent as verapamil in reversing MDR. In particular, 3a restored cytotoxicity of doxorubicin against the MDR cells with a fold-reversal of 58.6, which corresponds to 30.5 times increased MDR-reversal activity compared with that of quercetin. Flow cytometric analysis confirmed that 3a functions by inhibiting the drug efflux by Pgp, and Pgp ATPase assay showed that 3a interacts with the drug-binding site of Pgp to stimulate its ATPase 7226

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

93% yield) as white powder. 1H NMR (500 MHz, CDCl3) δ 7.57− 7.59 (m, 4H, 2′-H, 6′-H, Ar-H), 7.38−7.44 (m, 7H, Ar-H), 7.36 (s, 1H, Ar-H), 7.30 (d, J = 1.6 Hz, 1H, 8-H), 6.98 (d, J = 8.2 Hz, 1H, 5′H), 6.85 (d, J = 1.6 Hz, 1H, 6-H), 2.43 (s, 3H, −OCOCH3), 2.34 (s, 3H, −OCOCH3), 2.32 (s, 3H, −COCH3). 13C NMR (100 MHz, DMSO-d6) δ 169.5 (4-C), 169.1 (5-OCOCH3), 168.7 (7-OCOCH3), 168.1 (3-OCOCH3), 156.6 (7-C), 154.8 (9-C), 154.6 (2-C), 149.8 (5C), 149.4 (3′-C), 147.4 (4′-C), 139.5 (Ar-C), 133.0 (3-C), 130.0 (ArC), 129.0 (CPh2), 126.2 (Ar-C), 124.3 (Ar-C), 123.2 (1′-C), 117.9 (6′-C), 114.9 (10-C), 114.3 (6-C), 110.3 (5′-C), 109.7 (2′-C), 108.6 (8-C), 21.2 (5-OCOCH3), 21.1 (7-OCOCH3), 20.7 (3-OCOCH3). LC/MS (ESI) m/z Found: 593.31 [M + H]+. Calcd for C34H25O10: 593.14. Acetic Acid 5-Acetoxy-7-hydroxy-2-(2-methyl-2-phenylbenzo[1,3]dioxol-5-yl)-4-oxo-4H-chromen-3-yl Ester (6). To a stirred mixture of 5 (1.2 g, 2.0 mmol) and imidazole (27 mg, 0.4 mmol) in NMP (24 mL) was slowly added thiophenol (0.16 mL, 1.62 mmol) at 0 °C. The reaction mixture was stirred for 2 h at room temperature. The mixture was diluted with EtOAc and washed with 2 N HCl. The organic layer was concentrated under reduced pressure and dried over MgSO4. The crude product was purified by column chromatography on silica gel (hexane:EtOAc = 1:1) to give 6 (1 g, 1.8 mmol, 90% yield) as pale-yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.55−7.58 (m, 4H, 2′-H, 6′-H, Ar-H), 7.37−7.41 (m, 8H, Ar-H), 6.92 (d, J = 8.1 Hz, 1H, 5′-H), 6.63 (d, J = 2.2 Hz, 1H, 8-H), 6.50 (d, J = 2.2 Hz, 1H, 6-H), 2.35 (s, 3H, −OCOCH3), 2.29 (s, 3H, −OCOCH3). 13C NMR (100 MHz, DMSO-d6) δ 169.4 (4-C), 169.2 (5-OCOCH3), 168.4 (3OCOCH3), 163.1 (7-C), 158.0 (9-C), 154.0 (2-C), 150.6 (5-C), 149.3 (3′-C), 147.5 (4′-C), 139.6 (Ar-C), 132.7 (3-C), 130.1 (Ar-C), 129.1 (CPh2), 126.3 (Ar-C), 124.1 (Ar-C), 123.7 (1′-C), 118.0 (6′-C), 109.7 (5′-C), 109.6 (2′-C), 109.4 (10-C), 108.2 (6-C), 101.5 (8-C), 21.3 (5OCOCH3), 20.8 (3-OCOCH3). LC/MS (ESI) m/z Found: 549.32 [M − H]−. Calcd for C32H21O9: 549.13. 2-(2,2-Diphenylbenzo[d][1,3]dioxol-5-yl)-3,5-dihydroxy-7-(methoxymethoxy)-4H-chromen-4-one (7). To a solution of 6 (1 g, 1.8 mmol) in acetone (20 mL) was added K2CO2 (251 mg, 1.8 mmol) and chloromethyl methyl ether (0.2 mL, 2.7 mmol). The reaction mixture was stirred for 4 h at room temperature. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane:EtOAc = 2:1) to give 2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-7-(methoxymethoxy)-4-oxo-4H-chromene-3,5-diyl diacetate (891 mg, 1.5 mmol, 83% yield) as pale-yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.57−7.59 (m, 4H, 2′-H, 6′-H, Ar-H), 7.39−7.49 (m, 8H, Ar-H), 6.97 (d, J = 8.3 Hz, 1H, 5′-H), 6.88 (d, J = 2.3 Hz, 1H, 8-H), 6.70 (d, J = 2.3 Hz, 1H, 6-H), 5.14 (s, 2H, 7-OCHH2OCH3), 3.32 (s, 3H, 7-OCHH2OCH3), 2.42 (s, 3H, −OCOCH3), 2.30 (s, 3H, −OCOCH3). 13C NMR (100 MHz, DMSO-d6) δ 169.5 (4-C), 169.2 (5-OCOCH3), 168.3 (3OCOCH3), 163.5 (7-C), 157.9 (9-C), 154.2 (2-C), 150.4 (5-C), 149.4 (3′-C), 147.5 (4′-C), 139.6 (Ar-C), 132.9 (3-C), 130.1 (Ar-C), 129.1 (CPh2), 126.3 (Ar-C), 124.2 (Ar-C), 123.6 (1′-C), 118.0 (6′-C), 110.7 (5′-C), 110.6 (2′-C), 109.7 (10-C), 108.6 (6-C), 100.8 (8-C), 94.9 (7OCH2OCH3), 55.8 (7-OCHH2OCH3), 21.3 (5-OCOCH3), 20.8 (3OCOCH3). LC/MS (ESI) m/z Found: 595.21 [M + H]+. Calcd for C34H27O10: 595.15. A mixture of 2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-7-(methoxymethoxy)-4-oxo-4H-chromene-3,5-diyl diacetate (891 mg, 1.5 mmol) in satd NH3 in MeOH (10 mL) at 0 °C was stirred for 3 h at room temperature. After concentration under reduced pressure, the residue was purified by column chromatography on silica gel (hexane:acetone = 4:1) to give 7 (714 mg, 1.4 mmol, 93% yield) as yellow powder. 1H NMR (400 MHz, CDCl3) δ 11.68 (s, 1H, 5-OH), 7.78−7.80 (m, 1H, 6′-H), 7.59−7.61 (m, 6H, 2′-H, Ar-H), 7.34−7.44 (m, 5H, Ar-H), 7.09 (d, J = 8.3 Hz, 1H, 5′-H), 6.54 (d, J = 2.1 Hz, 1H, 8-H), 6.45 (d, J = 2.1 Hz, 1H, 6-H), 5.16 (s, 2H, 7-OCHH2OCH3), 3.34 (s, 3H, 7OCHH2OCH3). 13C NMR (100 MHz, DMSO-d6) δ 176.6 (4-C), 164.4 (7-C), 160.8 (5-C), 156.4 (9-C), 148.1 (3′-C), 147.1 (4′-C), 146.4 (2-C), 139.8 (Ar-C), 137.2 (3-C), 129.9 (Ar-C), 129.0 (CPh2), 126.1 (Ar-C), 125.5 (Ar-C), 123.5 (1′-C), 117.5 (6′-C), 109.3 (5′-C), 108.2 (2′-C), 104.6 (10-C), 98.5 (6-C), 94.8 (7-OCH2OCH3), 93.2

(8-C), 55.8 (7-OCHH2OCH3). LC/MS (ESI) m/z Found: 511.22 [M + H]+. Calcd for C30H23O8: 511.13. 3-(Benzyloxy)-2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4H-chromen-4-one (8). To a solution of 7 (714 mg, 1.4 mmol) in acetone (10 mL) was added K2CO2 (290 mg, 2.1 mmol) and benzyl bromide (0.3 mL, 2.1 mmol). The reaction mixture was stirred for 4 h at room temperature. After filtration, the filtrate was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. A mixture of the protected quercetin obtained above was dissolved in 3 N HCl (10 mL) and stirred for 4 h at 40 °C. After concentration under reduced pressure, the residue was purified by column chromatography on silica gel (hexane:acetone = 2:1) to give 8 (584 mg, 1.1 mmol, 75% yield) as yellow powder. 1H NMR (400 MHz, CDCl3) δ 7.60−7.65 (m, 4H, ArH), 7.58 (d, J = 1.8 Hz, 1H, 2′-H), 7.53 (dd, J = 1.8, 8.1 Hz, 1H, 6′-H), 7.41−7.48 (m, 11H, Ar-H), 6.89 (d, J = 8.1 Hz, 1H, 5′-H), 6.40 (d, J = 2.1 Hz, 1H, 8-H), 6.35 (d, J = 2.1 Hz, 1H, 6-H), 4.98 (s, 2H, 3OCHH2Ar). 13C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 163.2 (7-C), 162.0 (5-C), 157.0 (9-C), 156.9 (2-C), 149.3 (3′-C), 147.2 (4′C), 139.3 (Ar-C), 137.8 (Ar-C), 135.9 (3-C), 129.4 (Ar-C), 129.0 (CPh2), 128.4 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 126.3 (Ar-C), 124.2 (Ar-C), 124.1 (1′-C), 117.8 (6′-C), 109.0 (5′-C), 108.3 (2′-C), 105.6 (10-C), 99.4 (6-C), 94.2 (8-C), 74.7 (3-OCH2Ar). LC/MS (ESI) m/z Found: 557.38 [M + H]+. Calcd for C35H25O7: 557.56. (S)-2-(2,2-Diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4oxo-4H-chromen-3-yl 2-Amino-propanoate (9a). A mixture of 7 (600 mg, 1.2 mmol), EDC (559 mg, 3.6 mmol), Oxyma (256 mg, 1.8 mmol), and (S)-N-Boc-Ala (227 mg, 1.2 mmol) in CH2Cl2 (15 mL) was stirred at room temperature for 6 h. The mixture was purified by column chromatography on silica gel (hexane:CH2Cl2:acetone = 2:1:1) to give quercetin−3-O-alanine ester. To a solution of the quercetin−3-O-alanine ester in THF (10 mL) was added 3 N HCl, then the reaction mixture was heated at 40 °C for 3 h. The mixture was diluted with EtOAc and washed with brine. The organic layer was concentrated under reduced pressure and dried over MgSO4. The crude product was purified by column chromatography on silica gel (hexane:acetone = 1:1) to give 9a (464 mg, 0.9 mmol, 72% yield) as pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.58−7.59 (m, 2H, Ar-H), 7.57 (d, J = 1.8 Hz, 1H, 2′-H), 7.53 (dd, J = 1.8, 8.1 Hz, 1H, 6′H), 7.39−7.49 (m, 8H, Ar-H), 6.89 (d, J = 8.1 Hz, 1H, 5′-H), 6.50 (d, J = 2.1 Hz, 1H, 8-H), 6.26 (d, J = 2.1 Hz, 1H, 6-H), 4.15−4.22 (m, 1H, α-CH), 1.51 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 168.1 (3-OCO), 163.2 (7-C), 162.0 (5-C), 157.0 (9-C), 156.9 (2-C), 149.3 (3′-C), 147.2 (4′-C), 137.8 (Ar-C), 133.9 (3-C), 130.4 (Ar-C), 129.1 (CPh2), 126.3 (Ar-C), 124.2 (Ar-C), 124.1 (1′-C), 117.9 (6′-C), 109.2 (5′-C), 108.5 (2′-C), 105.6 (10-C), 99.4 (6-C), 94.2 (8-C), 51.0 (α-C), 18.1 (β-C). LC/MS (ESI) m/z Found: 538.32 [M + H]+. Calcd for C31H24NO8: 538.14. (S)-5-tert-Butyl 1-(2-(2,2-Diphenylbenzo[d][1,3]dioxol-5-yl)-5,7dihydroxy-4-oxo-4H-chromen-3-yl) 2-Aminopentanedioate (9b). A mixture of 7 (600 mg, 1.2 mmol), EDC (559 mg, 3.6 mmol), Oxyma (256 mg, 1.8 mmol), and (S)-N-Boc-Glu(OtBu) (364 mg, 1.2 mmol) in CH2Cl2 (15 mL) was stirred at room temperature for 6 h. The mixture was purified by column chromatography on silica gel (hexane:CH2Cl2:acetone = 2:1:1) to give quercetin−3-O-glutamic acid ester. To a solution of the quercetin−3-O-glutamic acid ester in THF (10 mL) was added 3 N HCl, then the reaction mixture was heated at 40 °C for 3 h. The mixture was diluted with EtOAc and washed with brine. The organic layer was concentrated under reduced pressure and dried over MgSO4. The crude product was purified by column chromatography on silica gel (hexane:acetone = 1:1) to give 9b (531 mg, 0.8 mmol, 68% yield) as pale-yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.58−7.59 (m, 2H, Ar-H), 7.57 (d, J = 1.8 Hz, 1H, 2′H), 7.53 (dd, J = 1.8, 8.1 Hz, 1H, 6′-H), 7.39−7.49 (m, 8H, Ar-H), 6.89 (d, J = 8.1 Hz, 1H, 5′-H), 6.50 (d, J = 2.1 Hz, 1H, 8-H), 6.26 (d, J = 2.1 Hz, 1H, 6-H), 4.38−4.42 (m, 1H, α-CH), 2.35−2.59 (m, 4H, βCH2, γ-CH2), 1.48 (s, 9H, tBu-H). 13C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 172.5 (CO2C(CH3)3), 168.0 (3-OCO), 163.2 (7-C), 162.0 (5-C), 157.0 (9-C), 156.9 (2-C), 149.3 (3′-C), 147.2 (4′-C), 137.8 (Ar-C), 133.4 (3-C), 130.4 (Ar-C), 129.1 (CPh2), 126.3 (Ar-C), 7227

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

7.73 (d, J = 2.0 Hz, 1H, 2′-H), 7.58 (dd, J = 2.0, 8.4 Hz, 1H, 6′-H), 6.91 (d, J = 8.5 Hz, 1H, 5′-H), 6.82 (d, J = 2.0 Hz, 1H, 8-H), 6.47 (d, J = 2.0 Hz, 1H, 6-H), 4.36−4.41 (m, 1H, α-CH), 2.32−2.58 (m, 4H, βCH2, γ-CH2). 13C NMR (100 MHz, DMSO-d6) δ 178.0 (CO2H), 176.7 (4-C), 172.6 (CO2H), 161.8 (5-C), 160.8 (7-C), 156.0 (9-C), 154.4 (7-OCO), 148.4 (2-C), 146.3 (4′-C), 145.5 (3′-C), 136.6 (3-C), 122.1 (1′-C), 120.5 (6′-C), 116.0 (5′-C), 115.6 (2′-C), 105.4 (10-C), 104.6 (6-C), 103.2 (8-C), 55.6 (α-C), 30.5 (γ-C), 26.5 (β-C). HRFABMS m/z Found: 476.0838 [M + H]+. Calcd for C21H18NO12: 476.0752. HPLC: retention time of 9.90 min, >98% pure at 340 and 254 nm. (S)-2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3yl 2-Aminopropanoate (2b). Palladium on charcoal (10% w/w, 20 mg) was added to a stirred solution of 9a (200 mg, 0.4 mmol) obtained above in THF (2 mL) and MeOH (2 mL) under hydrogen atmosphere. The suspension was stirred for 4 h and then filtered through a plug of Celite and eluted with acetone (100 mL). The filtrate was concentrated under reduced pressure and was recrystallized from CH2Cl2 to afford yellow powder. The tert-butyl-amino acid quercetin 3-ester obtained above was treated with trifluoroacetic acid (3 mL) at 0 °C. The reaction mixture was stirred for 2 h at 0 °C and concentrated under reduced pressure. The residue was recrystallized from a mixture of acetone (0.5 mL) and CH2Cl2 (5 mL) to give 2b (22 mg, 0.06 mmol, 14% yield) as pale-yellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.63 (s, 1H, 5-OH), 7.68 (d, J = 2.0 Hz, 1H, 2′H), 7.60 (dd, J = 2.1, 8.5 Hz, 1H, 6′-H), 6.96 (d, J = 8.5 Hz, 1H, 5′-H), 6.52 (d, J = 2.1 Hz, 1H, 8-H), 6.28 (d, J = 2.1 Hz, 1H, 6-H), 4.15−4.22 (m, 1H, α-CH), 1.51 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 168.0 (3-OCO), 164.7 (7-C), 161.6 (5-C), 156.7 (9-C), 156.5 (2-C), 146.3 (4′-C), 145.5 (3′-C), 134.8 (3-C), 121.7 (1′-C), 120.8 (6′-C), 116.2 (5′-C), 115.8 (2′-C), 105.3 (10-C), 99.1 (6-C), 94.1 (8-C), 51.0 (α-C), 18.1 (β-C). HR-FABMS m/z Found: 374.0836 [M + H]+. Calcd for C18H16NO8: 374.0798. HPLC: retention time of 11.32 min, >97% pure at 340 and 254 nm. (S)-4-Amino-5-((2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo4H-chromen-3-yl)oxy)-5-oxo-pentanoic Acid (3b). Palladium on charcoal (10% w/w, 30 mg) was added to a stirred solution of 9b (300 mg, 0.5 mmol) obtained above in THF (3 mL) and MeOH (3 mL) under hydrogen atmosphere. The suspension was stirred for 4 h and then filtered through a plug of Celite and eluted with acetone (100 mL). The filtrate was concentrated under reduced pressure and was recrystallized from CH2Cl2 to afford yellow powder. The tert-butylamino acid quercetin 3-ester obtained above was treated with trifluoroacetic acid (3 mL) at 0 °C. The reaction mixture was stirred for 2 h at 0 °C and concentrated under reduced pressure. The residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 3b (37 mg, 0.09 mmol, 17% yield) as pale-yellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.63 (s, 1H, 5-OH), 7.68 (d, J = 2.0 Hz, 1H, 2′-H), 7.60 (dd, J = 2.1, 8.5 Hz, 1H, 6′-H), 6.96 (d, J = 8.5 Hz, 1H, 5′-H), 6.52 (d, J = 2.1 Hz, 1H, 8-H), 6.28 (d, J = 2.1 Hz, 1H, 6H), 4.38−4.42 (m, 1H, α-CH), 2.35−2.59 (m, 4H, β-CH2, γ-CH2). 13 C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 177.2 (CO2H), 168.1 (3-OCO), 164.7 (7-C), 161.6 (5-C), 156.7 (9-C), 156.5 (2-C), 146.3 (4′-C), 145.5 (3′-C), 134.4 (3-C), 121.7 (1′-C), 120.8 (6′-C), 116.2 (5′-C), 115.8 (2′-C), 105.3 (10-C), 99.1 (6-C), 94.1 (8-C), 52.1 (αC), 32.5 (γ-C), 28.5 (β-C). HR-FABMS m/z Found: 432.0904 [M + H]+. Calcd for C20H18NO10: 432.0852. HPLC: retention time of 9.92 min, >97% pure at 340 and 254 nm. (S)-2-((((3-(((S)-2-Aminopropanoyl)oxy)-2-(3,4-dihydroxyphenyl)5-hydroxy-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)propanoic Acid (2c). Compound 9a (300 mg, 0.6 mmol) was dissolved in CH2Cl2 (10 mL) and treated with DMAP (73 mg, 0.6 mmol) and Boc2O (130 mg, 0.6 mmol). After stirring for 2 h at room temperature, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (hexane:acetone = 3:1) to give (S)-2-(2,2-diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 2-((tertbutoxycarbonyl)amino)propanoate as a pale-yellow oil (318 mg, 0.5 mmol, 83% yield). (S)-N-(4-Nitrophenoxycarbonyl)alanine tert-butyl ester 11a (155 mg, 0.5 mmol) was dissolved in 3 mL of dry DMF

124.2 (Ar-C), 124.1 (1′-C), 117.9 (6′-C), 109.2 (5′-C), 108.5 (2′-C), 105.6 (10-C), 99.4 (6-C), 94.0 (8-C), 82.0 (C O2C(CH3)3), 50.1 (αC), 32.2 (γ-C), 30.5 (β-C), 28.5 (CO2C(CH3)3). LC/MS (ESI) m/z Found: 652.42 [M + H]+. Calcd for C37H34NO10: 652.21. (S)-tert-Butyl 2-(((4-Nitrophenoxy)carbonyl)amino)propanoate (11a). H-Ala-OtBu·HCl (11a) (500 mg, 2.8 mmol) was dissolved in anhydrous THF (20 mL). Bis(4-nitrophenyl) carbonate (837 mg, 2.8 mmol) and N,N-diisopropylethylamine (1 mL, 5.5 mmol) were added to this solution, and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. (S)-Di-tert-butyl 2-(((4-Nitrophenoxy)carbonyl)amino)pentanedioate (11b). H-Glu(OtBu)-OtBu·HCl (11b) (500 mg, 1.7 mmol) was dissolved in anhydrous THF (20 mL). Bis(4-nitrophenyl) carbonate (514 mg, 1.7 mmol) and N,N-diisopropylethylamine (0.6 mL, 3.4 mmol) were added to this solution, and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. (S)-2-((((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)propanoic Acid (2a). (S)-N-(4Nitrophenoxycarbonyl)alanine tert-butyl ester 11a (279 mg, 0.9 mmol) was dissolved in 4 mL of dry DMF containing 0.2 mL (0.9 mmol) of DIPEA. A solution of 8 (500 mg, 0.9 mmol) in 2 mL of the same solvent was added, and the mixture was stirred room temperature for 6 h. The resulting solution was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. The degassed suspension of quercetin conjugate obtained above in a mixture of THF (3 mL) and MeOH (3 mL) and Pd/C (10% w/w, 65 mg), under an atmosphere of hydrogen gas (balloon), was vigorously stirred for 4 h at room temperature. The reaction mixture was filtered through a short Celite pad and was recrystallized from CH2Cl2 to afford yellow powder. The tert-butyl-amino acid quercetin carbamate obtained above was treated with trifluoroacetic acid (3 mL) at 0 °C. The reaction mixture was stirred for 4 h at 0 °C and concentrated under reduced pressure. The residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 2a (94 mg, 0.2 mmol, 25% yield) as paleyellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H, 5-OH), 7.73 (d, J = 2.0 Hz, 1H, 2′-H), 7.58 (dd, J = 2.0, 8.4 Hz, 1H, 6′-H), 6.91 (d, J = 8.5 Hz, 1H, 5′-H), 6.82 (d, J = 2.0 Hz, 1H, 8-H), 6.47 (d, J = 2.0 Hz, 1H, 6-H), 4.12−4.21 (m, 1H, α-CH), 1.50 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 176.7 (4-C), 172.6 (CO2H), 161.8 (5C), 160.9 (7-C), 156.0 (9-C), 154.2 (7-OCO), 148.4 (2-C), 146.3 (4′C), 145.5 (3′-C), 136.6 (3-C), 122.1 (1′-C), 120.5 (6′-C), 116.0 (5′C), 115.6 (2′-C), 105.4 (10-C), 104.6 (6-C), 103.2 (8-C), 54.5 (α-C), 17.2 (β-C). HR-FABMS m/z Found: 418.0722 [M + H]+. Calcd for C19H15NO10: 418.0696. HPLC: retention time of 11.32 min, >98% pure at 340 and 254 nm. (S)-2-((((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)pentanedioic Acid (3a). (S)-N-(4Nitrophenoxycarbonyl)glutamic acid di-tert-butyl ester 11b (382 mg, 0.9 mmol) was dissolved in 4 mL of dry DMF containing 0.2 mL (0.9 mmol) of DIPEA. A solution of 8 (500 mg, 0.9 mmol) in 2 mL of the same solvent was added, and the mixture was stirred room temperature for 6 h. The resulting solution was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. The degassed suspension of the quercetin conjugate obtained above and Pd/C (10% w/w, 76 mg) in a mixture of THF (5 mL) and MeOH (5 mL) was vigorously stirred under an atmosphere of hydrogen gas (balloon) for 4 h at room temperature. The reaction mixture was filtered through a short Celite pad and was recrystallized from CH2Cl2 to afford yellow powder. The tert-butyl-amino acid quercetin carbamate obtained above was treated with trifluoroacetic acid (4 mL) at 0 °C. The reaction mixture was stirred for 4 h at 0 °C and concentrated under reduced pressure. The residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 3a (90 mg, 0.2 mmol, 21% yield) as paleyellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H, 5-OH), 7228

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

γ-C), 28.5 (3-β-C), 26.6 (7-β-C). HR-FABMS m/z Found: 605.1256 [M + H]+. Calcd for C26H25N2O15: 605.1177. HPLC: retention time of 7.89 min, >97% pure at 340 and 254 nm. (R)-2-((((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)carbonyl)amino)pentanedioic Acid (ent-3a). (R)-N(4-Nitrophenoxycarbonyl)glutamic acid di-tert-butyl ester (ent-11b, 200 mg, 0.5 mmol) was dissolved in 2 mL of dry DMF containing 0.1 mL (0.5 mmol) of DIPEA. A solution of 8 (278 mg, 0.5 mmol) in 2 mL of the same solvent was added, and the mixture was stirred room temperature for 6 h. The resulting solution was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. The degassed suspension of quercetin conjugate obtained above and Pd/C (10% w/w, 42 mg) in a mixture of THF (3 mL) and MeOH (3 mL) was vigorously stirred under an atmosphere of hydrogen gas (balloon) for 4 h at room temperature. The reaction mixture was filtered through a short Celite pad and was recrystallized from CH2Cl2 to afford yellow powder. The tert-butyl-amino acid quercetin carbamate obtained above was treated with trifluoroacetic acid (3 mL) at 0 °C. The reaction mixture was stirred for 4 h at 0 °C and concentrated under reduced pressure. The residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give ent-3a (47 mg, 0.1 mmol, 20% yield) as paleyellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.67 (s, 1H, 5-OH), 7.71 (d, J = 2.0 Hz, 1H, 2′-H), 7.55 (dd, J = 2.0, 8.4 Hz, 1H, 6′-H), 6.90 (d, J = 8.5 Hz, 1H, 5′-H), 6.81 (d, J = 2.0 Hz, 1H, 8-H), 6.46 (d, J = 2.0 Hz, 1H, 6-H), 4.35−4.41 (m, 1H, α-CH), 2.32−2.57 (m, 4H, βCH2, γ-CH2). 13C NMR (100 MHz, acetone-d6) δ 178.1 (CO2H), 176.5 (4-C), 172.5 (CO2H), 161.6 (5-C), 160.7 (7-C), 156.1 (9-C), 154.2 (7-OCO), 148.4 (2-C), 146.1 (4′-C), 145.3 (3′-C), 136.5 (3-C), 121.8 (1′-C), 120.4 (6′-C), 116.2 (5′-C), 115.5 (2′-C), 105.2 (10-C), 104.5 (6-C), 103.1 (8-C), 55.5 (α-C), 30.6 (γ-C), 26.5 (β-C). HRFABMS m/z Found: 476.0764 [M + H]+. Calcd for C21H18NO12: 476.0751. HPLC: retention time of 9.90 min, >98% pure at 340 and 254 nm. Materials for Biological Studies. DMSO (dimethyl sulfoxide), MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide)], rhodamine-123, and verapamil were purchased from SigmaAldrich. Roswell Park Memorial Institute (RPMI) 1640 media, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from HyClone Laboratories. The precoated PAMPA plates were manufactured by BD Bioscience Discovery Labware (Bedford, MA) using a polyvinylidene fluoride (PVDF) 96-well filter plate with 0.4 μm pore size. The 100 mm cell culture plate was purchased from Dishes Biousing. Bio-Rad DC protein assay kit was purchased from Bio-Rad. Polyvinylidene difluoride (PVDF) membranes were purchased from GE Healthcare. Anti-MDR1 Antibody (clone JSB-1) and goat antimouse IgG antibody (Peroxidase Conjugated, H+L) were purchased from Merck Millipore. β-Actin was purchased from Santa Cruz Biotech. Cell Lines. MES-SA, a human uterine sarcoma cell line, was grown in monolayer. Resistant MES-SA/Dx5 cell line was isolated by stepwise selection upon culture with increasing concentrations of doxorubicin (DOX). Briefly, MES-SA/Dx5 cells were treated with DOX started from a sub-IC50 concentration, 5 μM. The medium was changed every other day with an increment of 5 μM DOX each time. Within 2 weeks, the MES-SA/Dx5 cells acquired DOX resistance and were cultured in a medium containing 5 μM DOX thereafter. Cells were harvested and analyzed for Pgp expression by Western blot analysis. Western Blot Analysis of Pgp Expression. Doxorubicinsensitive MES-SA cells or doxorubicin-resistant MES-SA/Dx5 cells were harvested, washed with PBS, and lysed with lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40, 10 μg/mL PMSF, protease and phosphatase inhibitor cocktail (Sigma)] for 1 h at 4 °C with occasional vortexing. Protein extracts were collected after centrifugation at 16000g for 20 min. Protein concentration was determined using a Bio-Rad DC protein assay kit. Equal amounts of protein extracts were resolved on 8% or 10% SDS-polyacrylamide gel followed by transfer onto polyvinylidene difluoride (PVDF) membranes. The

containing 0.08 mL (0.5 mmol) of DIPEA. A solution of (S)-2-(2,2diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4-oxo-4H-chromen3-yl 2-((tert-butoxycarbonyl)amino)propanoate (318 mg, 0.5 mmol) in 2 mL of the same solvent was added, and the mixture was stirred room temperature for 6 h. The resulting solution was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. The degassed suspension of quercetin conjugate obtained above and Pd/C (10% w/w, 65 mg) in a mixture of THF (3 mL) and MeOH (3 mL) was vigorously stirred under an atmosphere of hydrogen gas (balloon) for 4 h at room temperature. The reaction mixture was filtered through a short Celite pad and was recrystallized from CH2Cl2 to afford yellow powder. The quercetin 7-carbamoyl-3-ester obtained above was dissolved in CH2Cl2 (5 mL). To this solution, trifluoroacetic acid (3 mL) was added at 0 °C, and the solution was stirred for 2 h at 0 °C. The resulting solution was concentrated under reduced pressure, and the residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 2c (38 mg, 0.08 mmol, 13% yield) as pale-yellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.65 (s, 1H, 5-OH), 7.71 (d, J = 2.3 Hz, 1H, 2′-H), 7.62 (dd, J = 2.1, 8.5 Hz, 1H, 6′-H), 6.98 (d, J = 8.5 Hz, 1H, 5′-H), 6.83 (d, J = 2.1 Hz, 1H, 8-H), 6.49 (d, J = 2.0 Hz, 1H, 6H), 4.12−4.22 (m, 2H, α-CH), 1.51 (s, 3H, CH3), 1.50 (s, 3H, CH3). 13 C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 172.6 (CO2H), 168.1 (3-OCO), 161.8 (5-C), 160.9 (7-C), 157.0 (9-C), 154.5 (7-OCO), 154.1 (2-C), 146.3 (4′-C), 145.5 (3′-C), 133.8 (3-C), 121.8 (1′-C), 120.8 (6′-C), 116.2 (5′-C), 115.8 (2′-C), 105.4 (10-C), 104.6 (6-C), 103.2 (8-C), 54.3 (7-α-C), 50.5 (3-α-C), 17.1 (7-β-C), 18.0 (3-β-C). HR-FABMS m/z Found: 489.1092 [M + H]+. Calcd for C22H21N2O11: 489.1067. HPLC: retention time of 9.80 min, >97% pure at 340 and 254 nm. (S)-2-((((3-(((S)-2-Amino-4-carboxybutanoyl)oxy)-2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H -chromen-7-yl)oxy)carbonyl)amino)pentanedioic Acid (3c). Compound 9b (300 mg, 0.5 mmol) was dissolved in CH2Cl2 (10 mL) and treated with DMAP (61 mg, 0.5 mmol) and Boc2O (109 mg, 0.5 mmol). After stirring for 2 h at room temperature, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (hexane:acetone = 3:1) to give (S)-5-tert-butyl 1-(2-(2,2diphenylbenzo[d][1,3]dioxol-5-yl)-5,7-dihydroxy-4-oxo-4H-chromen3-yl) 2-((tert-butoxycarbonyl)-amino)pentanedioate as a pale-yellow oil (334 mg, 0.4 mmol, 89% yield). (S)-N-(4-Nitrophenoxycarbonyl)glutamic acid di-tert-butyl ester 11b (155 mg, 0.4 mmol) was dissolved in 4 mL of dry DMF containing 0.08 mL (0.4 mmol) of DIPEA. A solution of (S)-5-tert-butyl 1-(2-(2,2-diphenylbenzo[d][1,3]dioxol-5yl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl) 2-((tert-butoxycarbonyl)amino)pentanedioate (334 mg, 0.3 mmol) in 3 mL of the same solvent was added, and the mixture was stirred room temperature for 6 h. The resulting solution was concentrated under reduced pressure to give pale-yellow syrup, which was used for the next step without further purification. The degassed suspension of quercetin conjugate obtained above and Pd/C (10% w/w, 29 mg) in a mixture of THF (3 mL) and MeOH (3 mL) was vigorously stirred under an atmosphere of hydrogen gas (balloon) for 4 h at room temperature. The reaction mixture was filtered through a short Celite pad and was recrystallized from CH2Cl2 to afford yellow powder. The quercetin 7-carbamoyl-3ester obtained above was dissolved in CH2Cl2 (5 mL). To this solution, trifluoroacetic acid (3 mL) was added at 0 °C, and the solution was stirred for 2 h at 0 °C. The resulting solution was concentrated under reduced pressure, and the residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 3c (54 mg, 0.09 mmol, 18% yield) as pale-yellow solid. 1H NMR (400 MHz, acetone-d6) δ 12.65 (s, 1H, 5-OH), 7.71 (d, J = 2.3 Hz, 1H, 2′H), 7.62 (dd, J = 2.1, 8.5 Hz, 1H, 6′-H), 6.98 (d, J = 8.5 Hz, 1H, 5′-H), 6.83 (d, J = 2.1 Hz, 1H, 8-H), 6.49 (d, J = 2.1 Hz, 1H, 6-H), 4.37−4.41 (m, 2H, α-CH), 2.32−2.4.0 (m, 8H, β-CH2, γ-CH2). 13C NMR (100 MHz, DMSO-d6) δ 178.8 (4-C), 178.0 (3-CO2H, 7-CO2H), 172.6 (7CO2H), 168.1 (3-OCO), 161.8 (5-C), 160.8 (7-C), 157.0 (9-C), 154.4 (7-OCO), 154.1 (2-C), 146.3 (4′-C), 145.5 (3′-C), 134.4 (3-C), 121.7 (1′-C), 120.8 (6′-C), 116.2 (5′-C), 115.8 (2′-C), 105.4 (10-C), 104.5 (6-C), 103.2 (8-C), 55.6 (7-α-C), 52.2 (3-α-C), 32.5 (3-γ-C), 30.5 (77229

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

membranes were blocked overnight at 4 °C with 5% nonfat dried milk in TBS-T (TBS with 0.05% Tween-20) and then incubated with mouse monoclonal JSB-1 antihuman Pgp antibody (dilution 1:1000) and mouse monoclonal AC-15 anti-β-actin antibody, followed by incubation with horseradish peroxidase (HRP)-conjugated goat antimouse IgG secondary antibody (dilution 1:10000). Protein bands were detected using ECL Pico Western blotting detection reagents (Pierce, Rockford, IL, USA). Cytotoxicity. Cytotoxicity was determined by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay. Doxorubicin-sensitive MES-SA cells and doxorubicinresistant MES-SA/Dx5 cells were seeded (5 × 103 cells per well) in tissue-cultured COSTAR clear bottom 96-well plate in complete RPMI-1640 and incubated for 24 h (37 °C, 5% CO2). Quercetin (1) and quercetin conjugates (2a−3c) dissolved in DMSO were serially diluted (100, 10, 1, 0.1, 0.01, 0.001 μM), and the resulting solutions were added to the media. After 72 h, cell viability was estimated by MTT assay. Every assay was repeated three times. Determining MDR-Modulatory Effect of the Quercetin Conjugates on the Sensitivities of MES-SA and MES-SA/Dx5 to Anticancer Drugs. Doxorubicin-sensitive MES-SA cells and doxorubicin-resistant MES-SA/Dx5 cells were seeded (5 × 103 cells per well) in tissue-cultured COSTAR clear bottom 96-well plate. The cultures were incubated at 37 °C in 5% CO2 for 24 h. To the plates, 100 μL of the solution (final concentrations: 100, 10, 1, 0.1, 0.05, 0.01, 0.001, 0.0001 μM) of anticancer drugs (doxorubicin, vinblastine, paclitaxel, actinomycin) with or without the modulator [quercetin conjugate (5 μM, 1% DMSO) or verapamil (5 μM, 1% DMSO)] in the complete RPMI-1640 medium was added, and the cells were incubated for 72 h. To the control well, 1% DMSO in 100 μL of the growth medium was added and the cells were incubated for 72 h. To the blank well, which contains no cell, 100 μL of the growth medium was added. The growth medium was removed, and cell viability was estimated by MTT assay. Optical absorbance at 570 nm was then recorded with a microplate reader (SpectraMax M2e, Molecular Devices). Every assay was repeated three times. The relative survival rates were calculated according to the equation

ATPase activity was presented as a drop in luminescence of samples compared to that treated with Na3VO4. Solubility Test. Nephelometry. Stock solutions of quercetin and the quercetin conjugates were prepared at 0.5, 2.5, 5, 10, 20, 30, 40, and 50 mM in 5% DMSO and then serially diluted with 99% phosphate buffered saline (PBS, pH 7.4) to final concentrations of 5, 25, 50, 100, 200, 300, 400, and 500 μM. The volume of the test compound in each 96-well plate was set to be 200 μL, and the solubility was measured by the NEPHELOstar laser based microplate nephelometer which checks the solubility of compounds by measuring forward light scattering in microplates. All raw data were processed using the BMG LABTECH NEPHELOstar Galaxy Evaluation software. UV−vis Spectroscopy. The direct measurement of water solubility of quercetin and quercetin−glutamic acid conjugate 3a was performed using a UV−vis spectrophotometer: Stock solutions of 3a and quercetin were prepared by vortexing 4.8 mg (10 μmol) of 3a and 3.0 mg (10 μmol) of quercetin, respectively, in 1 mL of deionized water in an Eppendorf tube. Both samples were centrifuged at 13000 rpm for 2 min to eliminate any undissolved material. Then 100 μL of each stock solution was diluted with 900 μL of deionized water and thoroughly mixed. The UV−vis absorbance of the diluted 3a and the quercetin solution were recorded. Intracellular Localization Test (Confocal Microscopy). Doxorubicin-sensitive MES-SA cells and doxorubicin-resistant MES-SA/ Dx5 cells were seeded in poly-L-lysine-coated coverslip in 35 mm dishes (105 cells/well) and incubated for 1 day (37 °C, 5% CO2). The cells were treated with quercetin 1 (5 μM) or quercetin conjugate 3a (5 μM) for 72 h and then quickly washed with PBS. After addition of fresh PBS (1 mL), specimens were observed with FV-1000 spectral (Olympus, USA) at 488 nmex/520 nmem using 40× objective lens. Cellular Uptake and Intracellular Stability. Doxorubicinresistant MES-SA/Dx5 cells was seeded in tissue-cultured COSTAR clear bottom 6-well plates (105 cells/well) and incubated for 24 h (37 °C, 5% CO2). Quercetin 1 (10 μM) and quercetin conjugate 3a (10 μM) were treated for 6 and 48 h. Cells were trypsinized, collected, and sonicated with Vibra-Cell VCX-130 (SONICS, USA). After filtration, samples were analyzed by HPLC (Agilent, USA), and fractions of interest were collected, lypolized, and analyzed by mass spectroscopy (MALDI-TOF, Bruker Daltonics, USA). Statistical Analysis. Data represent the means of at least three separate experiments. Statistical analysis was performed using Student’s t test. A value of p < 0.05 was considered significant.

survival% = (MDx − Mblank )/(Mcontrol − Mblank )× 100 Here, MDx presents light absorption values of cell, growth medium, anticancer drugs, and modulator, Mcontrol presents light absorption values of cell and growth medium, and Mblank presents light absorption values of growth medium alone. The relative survival rates, thus calculated, were plotted against concentrations of anticancer drugs to define the IC50s of anticancer drugs against MES-SA and MES-SA/ Dx5 cells. Functional Aspect of Pgp on Cells Treated with Reversal Agents. Detection of Pgp activity using rhodamine-123 efflux. Doxorubicin-sensitive MES-SA cells and doxorubicin-resistant MESSA/Dx5 cells were washed twice with PBS and suspended at a density of 106 cell/mL in RPMI medium. After treatment with quercetin (5 or 10 μM), quercetin conjugate 3a (5 or 10 μM), verapamil (5 or 10 μM), and control (1% DMSO) for 15 min (37 °C, 5% CO2), rhodamine-123 (final concentration: 0.05 μg/mL) was added to the medium. Cells were incubated for 1 h at 37 °C and then analyzed using FACSCalibur (Becton Dickinson, USA). Pgp ATPase Assay. Pgp ATPase activity was measured with the Pgp-Glo assay system with human Pgp membrane by following the manufacturer’s instructions (Promega, Co. USA). The assay relies on the ATP dependence of the light-generating reaction of firefly luciferase. Briefly, 25 μg of Pgp membrane was incubated at 37 °C with either Na3VO4 (100 μM), solvent control (0.1% DMSO), quercetin (100 μM), 3a (100 μM), verapamil (100 μM), or verapamil (100 μM) plus 3a (100 μM). The ATPase reaction was initiated by addition of 5 mM MgATP and followed by incubation for 40 min at 37 °C. The reaction was stopped, and the remaining unmetabolized ATP was detected as a luciferase-generated luminescence signal by addition of ATP detection reagent. Following a room-temperature signalstabilization period (20 min), luminescence was read on a Veritas microplate luminometer (Turner Designs, San Francisco, CA). Pgp

■

AUTHOR INFORMATION

Corresponding Authors

* For H. C.: phone, +82-2-958-5157; E-mail, [email protected]. *For Y. C.: phone, +82-2-2049-6100; Fax, +82-2-454-8217; Email, [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2013R1A1A2006455), by a grant from the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093824), and by a grant of the Korean Health Technology R&D project, Ministry of Health & Welfare, Republic of Korea (A111698). Additional funding was provided by the Korea Institute of Science and Technology (KIST) Institutional Program (2E24510). 7230

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

■

Article

(17) Yanagisawa, T.; Newman, A.; Coley, H.; Renshaw, J.; Pinkerton, C. R.; Pritchard-Jones, K. BIRICODAR (VX-710; Incel): an effective chemosensitizer in neuroblastoma. Br. J. Cancer 1999, 80, 1190−1196. (18) Baer, M. R.; George, S. L.; Dodge, R. K.; O’Loughlin, K. L.; Minderman, H.; Caligiuri, M. A.; Anastasi, J.; Powell, B. L.; Kolitz, J. E.; Schiffer, C. A.; Bloomfield, C. D.; Larson, R. A. Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: cancer and Leukemia Group B Study 9720. Blood 2002, 100, 1224−1232. (19) van Zuylen, L.; Sparreboom, A.; van der Gaast, A.; van der Burg, M. E.; van Beurden, V.; Bol, C. J.; Woestenborghs, R.; Palmer, P. A.; Verweij, J. The orally administered P-glycoprotein inhibitor R101933 does not alter the plasma pharmacokinetics of docetaxel. Clin. Cancer Res. 2000, 6, 1365−1371. (20) Newman, M. J.; Rodarte, J. C.; Benbatoul, K. D.; Romano, S. J.; Zhang, C.; Krane, S.; Moran, E. J.; Uyeda, R. T.; Dixon, R.; Guns, E. S.; Mayer, L. D. Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance. Cancer Res. 2000, 60, 2964−2972. (21) Cripe, L. D.; Uno, H.; Paietta, E. M.; Litzow, M. R.; Ketterling, R. P.; Bennett, J. M.; Rowe, J. M.; Lazarus, H. M.; Luger, S.; Tallman, M. S. Zosuquidar, a novel modulator of P-glycoprotein, does not improve the outcome of older patients with newly diagnosed acute myeloid leukemia: a randomized, placebo-controlled trial of the eastern cooperative oncology group 3999. Blood 2010, 116, 4077− 4085. (22) Lagas, J. S.; van Waterschoot, R. A.; van Tilburg, V. A.; Hillebrand, M. J.; Lankheet, N.; Rosing, H.; Beijnen, J. H.; Schinkel, A. H. Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment. Clin. Cancer Res. 2009, 15, 2344− 2351. (23) Fox, E.; Bates, S. E. Tariquidar (XR9576): a P-glycoprotein drug efflux pump inhibitor. Expert Rev. Anticancer Ther. 2007, 7, 447−459. (24) Choi, B. H.; Kim, C. G.; Lim, Y.; Shin, S. Y.; Lee, Y. H. Curcumin down-regulates the multidrug-resistance mdr1b gene by inhibiting the PI3K/Akt/NF kappa B pathway. Cancer Lett. 2008, 259, 111−118. (25) Cheung, J. Y.; Ong, R. C.; Suen, Y. K.; Ooi, V.; Wong, H. N.; Mak, T. C.; Fung, K. P.; Yu, B.; Kong, S. K. Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. Cancer Lett. 2005, 217, 203−211. (26) Qian, F.; Wei, D.; Zhang, Q.; Yang, S. Modulation of Pglycoprotein function and reversal of multidrug resistance by (−)-epigallocatechin gallate in human cancer cells. Biomed. Pharmacother. 2005, 59, 64−69. (27) Fong, W. F.; Wang, C.; Zhu, G. Y.; Leung, C. H.; Yang, M. S.; Cheung, H. Y. Reversal of multidrug resistance in cancer cells by Rhizoma Alismatis extract. Phytomedicine 2007, 14, 160−165. (28) Limtrakul, P.; Siwanon, S.; Yodkeeree, S.; Duangrat, C. Effect of Stemona curtisii root extract on P-glycoprotein and MRP-1 function in multidrug-resistant cancer cells. Phytomedicine 2007, 14, 381−389. (29) Li, L.; Pan, Q.; Sun, M.; Lu, Q.; Hu, X. Dibenzocyclooctadiene lignans: a class of novel inhbitors of multidrug resistance-associated protein 1. Life Sci. 2007, 80, 741−748. (30) Zhang, S.; Yang, X.; Coburn, R. A.; Morris, M. E. Structure− activity relationships and quantitative structure−activity relationships for the flavonoid-mediated inhibition of breast cancer resistance protein. Biochem. Pharmacol. 2005, 70, 627−639. (31) Ma, Y.; Wink, M. Lobeline, a piperidine alkaloid from Lobelia can reverse Pgp dependent multidrug resistance in tumor cells. Phytomedicine 2008, 15, 754−758. (32) Cho, S. Y.; Kim, M. K.; Mok, H.; Choo, H.; Chong, Y. Separation of quercetin’s biological activity from its oxidative property through bioisosteric replacement of the catecholic hydroxyl groups with fluorine atoms. J. Agric. Food Chem. 2012, 60, 6499−6506. (33) De Azevedo, W. F., Jr.; Mueller-Dieckmann, H. J.; SchulzeGahmen, U.; Worland, P. J.; Sausville, E.; Kim, S. H. Structural basis

ABBREVIATIONS USED MDR, multidrug resistance; ABC, ATP-binding cassette; Pgp, P-glycoprotein; MRP1, human multidrug resistance associateprotein 1; BCRP, breast cancer resistance protein; ATPases, ATP hydrolases; POM, pivaloxymethyl; POC, isopropyloxycarbonylmethoxy; NMP, N-methyl-2-pyrrolidone; MOM, methoxymethyl; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide; Rh-123, rhodamine 123; TMD, transmembrane domain; NBD, nucleotide binding domain; PBS, phosphate buffered saline; DMSO, dimethyl sulfoxide; Q, quercetin; Qglu, quercetin glucuronide; Q-sul, quercetin sulfate

■

REFERENCES

(1) Gottesman, M. M.; Pastan, I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu. Rev. Biochem. 1993, 62, 385−427. (2) Gottesman, M. M. Mechanisms of cancer drug resistance. Annu. Rev. Med. 2002, 53, 747−754. (3) Gottesman, M. M.; Pastan, I.; Ambudkar, S. V. P-glycoprotein and multidrug resistance. Curr. Opin. Gen. Dev. 1996, 6, 610−617. (4) Bellamy, W. T. P-Glycoprotein and multidrug resistance. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 161−183. (5) Cole, S. P.; Bhardwaj, G.; Gerlach, J. H.; Mackie, J. E.; Grant, C. E.; Almquist, K. C.; Stewart, A. J.; Kurz, E. U.; Duncan, A. M.; Deeley, R. G. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992, 258, 1650−1654. (6) Hipfner, D. R.; Deeley, R. G.; Cole, S. P. Structural, mechanistic and clinical aspects of MRP1. Biochim. Biophys. Acta 1999, 1461, 359− 376. (7) Doyle, L. A.; Yang, W.; Abruzzo, L. V.; Krogmann, T.; Gao, Y.; Rishi, A. K.; Ross, D. D. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl. Acad. Aci. U. S. A. 1998, 95, 15665−15670. (8) Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 1981, 41, 1967−1972. (9) Ford, J. M.; Hait, W. N. Pharmacology of drugs that alter multidrug resistance in cancer. Pharmacol. Rev. 1990, 42, 155−199. (10) Lampidis, T. J.; Krishan, A.; Planas, L.; Tapiero, H. Reversal of intrinsic resistance to adriamycin in normal cells by verapamil. Cancer Drug Delivery 1986, 3, 251−259. (11) Pirker, R.; FitzGerald, D. J.; Raschack, M.; Frank, Z.; Willingham, M. C.; Pastan, I. Enhancement of the activity of immunotoxins by analogues of verapamil. Cancer Res. 1989, 49, 4791−4795. (12) Hofmann, J.; Wolf, A.; Spitaler, M.; Bock, G.; Drach, J.; Ludescher, C.; Grunicke, H. Reversal of multidrug resistance by B85935, a metabolite of B859-35, niguldipine, verapamil and nitrendipine. J. Cancer Res. Clin. Oncol. 1992, 118, 361−366. (13) Gruol, D. J.; Zee, M. C.; Trotter, J.; Bourgeois, S. Reversal of multidrug resistance by RU 486. Cancer Res. 1994, 54, 3088−3091. (14) Twentyman, P. R.; Bleehen, N. M. Resistance modification by PSC-833, a novel nonimmunosuppressive cyclosporin A. Eur. J. Cancer 1991, 27, 1639−1642. (15) Germann, U. A.; Ford, P. J.; Shlyakhter, D.; Mason, V. S.; Harding, M. W. Chemosensitization and drug accumulation effects of VX-710, verapamil, cyclosporin A, MS-209 and GF120918 in multidrug resistant HL60/ADR cells expressing the multidrug resistance-associated protein MRP. Anticancer Drugs 1997, 8, 141− 155. (16) Germann, U. A.; Shlyakhter, D.; Mason, V. S.; Zelle, R. E.; Duffy, J. P.; Galullo, V.; Armistead, D. M.; Saunders, J. O.; Boger, J.; Harding, M. W. Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro. Anticancer Drugs 1997, 8, 125−140. 7231

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 2735−2740. (34) Zapata-Torres, G.; Opazo, F.; Salgado, C.; Munoz, J. P.; Krautwurst, H.; Mascayano, C.; Sepulveda-Boza, S.; Maccioni, R. B.; Cassels, B. K. Effects of natural flavones and flavonols on the kinase activity of Cdk5. J. Nat. Prod. 2004, 67, 416−420. (35) Ferriola, P. C.; Cody, V.; Middleton, E., Jr. Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure− activity relationships. Biochem. Pharmacol. 1989, 38, 1617−1624. (36) Hagiwara, M.; Inoue, S.; Tanaka, T.; Nunoki, K.; Ito, M.; Hidaka, H. Differential effects of flavonoids as inhibitors of tyrosine protein kinases and serine/threonine protein kinases. Biochem. Pharmacol. 1988, 37, 2987−2992. (37) Akiyama, T.; Ishida, J.; Nakagawa, S.; Ogawara, H.; Watanabe, S.; Itoh, N.; Shibuya, M.; Fukami, Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 1987, 262, 5592−5595. (38) Sicheri, F.; Moarefi, I.; Kuriyan, J. Crystal structure of the Src family tyrosine kinase Hck. Nature 1997, 385, 602−609. (39) Robinson, M. J.; Corbett, A. H.; Osheroff, N. Effects of topoisomerase II-targeted drugs on enzyme-mediated DNA cleavage and ATP hydrolysis: evidence for distinct drug interaction domains on topoisomerase II. Biochemistry 1993, 32, 3638−3643. (40) Zyma, V. L.; Miroshnichenko, N. S.; Danilova, V. M.; En Gin, E. Interaction of flavonoid compounds with contractile proteins of skeletal muscle. Gen. Physiol. Biophys. 1988, 7, 165−175. (41) Di Pietro, A.; Conseil, G.; Perez-Victoria, J. M.; Dayan, G.; Baubichon-Cortay, H.; Trompier, D.; Steinfels, E.; Jault, J. M.; de Wet, H.; Maitrejean, M.; Comte, G.; Boumendjel, A.; Mariotte, A. M.; Dumontet, C.; McIntosh, D. B.; Goffeau, A.; Castanys, S.; Gamarro, F.; Barron, D. Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters. Cell. Mol. Life Sci. 2002, 59, 307−322. (42) Hirano, T.; Oka, K.; Akiba, M. Effects of synthetic and naturally occurring flavonoids on Na+, K+-ATPase: aspects of the structure− activity relationship and action mechanism. Life Sci. 1989, 45, 1111− 1117. (43) Thiyagarajah, P.; Kuttan, S. C.; Lim, S. C.; Teo, T. S.; Das, N. P. Effect of myricetin and other flavonoids on the liver plasma membrane Ca2+ pump. Kinetics and structure−function relationships. Biochem. Pharmacol. 1991, 41, 669−675. (44) Murakami, S.; Muramatsu, M.; Tomisawa, K. Inhibition of gastric H+,K+-ATPase by flavonoids: a structure−activity study. J. Enzyme Inhib. 1999, 14, 151−166. (45) Di Pietro, A.; Godinot, C.; Bouilant, M. L.; Gautheron, D. C. Pig heart mitochondrial ATPase: properties of purified and membranebound enzyme. Effects of flavonoids. Biochimie 1975, 57, 959−967. (46) Chieli, E.; Romiti, N.; Cervelli, F.; Tongiani, R. Effects of flavonols on P-glycoprotein activity in cultured rat hepatocytes. Life Sci. 1995, 57, 1741−1751. (47) Comte, G.; Daskiewicz, J. B.; Bayet, C.; Conseil, G.; ViorneryVanier, A.; Dumontet, C.; Di Pietro, A.; Barron, D. C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance. J. Med. Chem. 2001, 44, 763−768. (48) Critchfield, J. W.; Welsh, C. J.; Phang, J. M.; Yeh, G. C. Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism. Biochem. Pharmacol. 1994, 48, 1437−1445. (49) Scambia, G.; Ranelletti, F. O.; Benedetti Panici, P.; De Vincenzo, R.; Bonanno, G.; Ferrandina, G.; Piantelli, M.; Bussa, S.; Rumi, C.; Cianfriglia, M.; Mancuso, S. Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target. Cancer Chemother. Pharmacol. 1994, 34, 459−464. (50) Castro, A. F.; Altenberg, G. A. Inhibition of drug transport by genistein in multidrug-resistant cells expressing P-glycoprotein. Biochem. Pharmacol. 1997, 53, 89−93.

(51) Zhang, S.; Morris, M. E. Effects of the flavonoids biochanin A, morin, phloretin, and silymarin on P-glycoprotein-mediated transport. J. Pharmacol. Exp. Ther. 2003, 304, 1258−1267. (52) Jodoin, J.; Demeule, M.; Beliveau, R. Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols. Biochim. Biophys. Acta 2002, 1542, 149−159. (53) Chan, K.-F.; Zhao, Y.; Burkett, B. A.; Wong, I. L. K.; Chow, L. M. C.; Chan, T. H. Flavonoid dimers as bivalent modulators for Pglycoprotein-based multidrug resistance: synthetic apigenin homodimers linked with defined-length poly(ethylene glycol) spacers increase drug retention and enhance chemosensitivity in resistant cancer cells. J. Med. Chem. 2006, 49, 6742−6759. (54) Chan, K.-F.; Zhao, Y.; Chow, T. W. S.; Yan, C. S. W.; Ma, D. L.; Burkett, B. A.; Wong, I. L. K.; Chow, L. M. C.; Chan, T. H. Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: Structure−activity relationship. ChemMedChem 2009, 4, 594−614. (55) Chan, K.-F.; Wong, I. L. K.; Kan, J. W. Y.; Yan, C. S. W.; Chow, L. M. C.; Chan, T. H. Amine linked flavonoid dimers as modulators for P-glycoprotein-based multidrug resistance: structure−activity relationship and mechanism of modulation. J. Med. Chem. 2012, 55, 1999−2014. (56) Wong, I. L. K.; Chan, K.-F.; Tsang, K. H.; Lam, C. Y.; Zhao, Y.; Chan, T. H.; Chow, L. M. C. Modulation of multidrug resistance protein 1 (MRP1/ABCC1)-mediated multidrug resistance by bivalent apigenin homodimers and their derivatives. J. Med. Chem. 2009, 52, 5311−5322. (57) Boulton, D. W.; Walle, U. K.; Walle, T. Fate of the flavonoids quercetin in human cell lines: chemical instability and metabolism. J. Pharm. Pharmacol. 1999, 51, 353−359. ́ (58) Van der Woude, H.; Gliszczyńska-Swigło, A.; Struijs, K.; Smeets, A.; Alink, G. M.; Rietjens, I. M. C. M. Biphasic modulation of cell proliferation by quercetin at concentrations physiologically relevant in humans. Cancer Lett. 2003, 200, 41−47. (59) De Boer, V. C. J.; de Goffau, M. C.; Arts, I. C. W.; Hollman, P. C. H.; Keijer, J. SIRT1 stimulation by polyphenols is affected by their stability and metabolism. Mech. Ageing Dev. 2006, 127, 618−627. (60) O’Leary, K. A.; Day, A. J.; Needs, P. W.; Mellon, F. A.; O’Brien, N. M.; Williamson, G. Metabolism of quercetin−7- and quercetin−3glucuronides by an in vitro hepatic model: the role of human βglucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism. Biochem. Pharmacol. 2003, 65, 479−491. (61) Spencer, J. P.; Kuhnle, G. G.; Williams, R. J.; Rice-Evans, C. Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites. Biochem. J. 2003, 372, 173−181. (62) Kim, M. K.; Park, K.-S.; Lee, C.; Park, H. R.; Choo, H.; Chong, Y. Enhanced stability and intracellular accumulation of quercetin by protection of the chemically or metabolically susceptible hydroxyl groups with a pivaloxymethyl (POM) promoiety. J. Med. Chem. 2010, 53, 8597−8607. (63) Kim, M. K.; Park, K.-S.; Chong, Y. Remarkable stability and cytostatic effect of a quercetin conjugate, 3,7-bis-O-pivaloxymethyl (POM) quercetin. ChemMedChem 2012, 7, 229−232. (64) Cho, S. Y.; Kim, M. K.; Park, K.-S.; Choo, H.; Chong, Y. Quercetin−POC conjugates: differential stability and bioactivity profiles between breast cancer (MCF-7) and colorectal carcinoma (HCT116) cell lines. Bioorg. Med. Chem. 2013, 21, 1671−1679. (65) Chen, L.; Li, J.; Luo, C.; Liu, H.; Xu, W.; Chen, G.; Liew, O. W.; Zhu, W.; Puah, C. M.; Shen, X.; Jiang, H. Binding interaction of quercetin−3-beta-galactoside and its synthetic derivatives with SARSCoV 3CL(pro): structure−activity relationship studies reveal salient pharmacophore features. Bioorg. Med. Chem. 2006, 14, 8295−8306. (66) Li, M.; Han, X.; Yu, B. Facile synthesis of flavonoid 7-Oglycosides. J. Org. Chem. 2003, 68, 6842−6845. (67) Safavy, A.; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R.; Bonner, J. A. Design and development of watersoluble curcumin conjugates as potential anticancer agents. J. Med. Chem. 2007, 50, 6284−6288. 7232

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233

Journal of Medicinal Chemistry

Article

(68) Wesolowska, O.; Paprocka, M.; Kozlak, J.; Motohashi, N.; Dus, D.; Michalak, K. Human sarcoma cell lines MES-SA and MES-SA/Dx5 as a model for multidrug resistance modulators screening. Anticancer Res. 2005, 25, 383−390. (69) Pearce, H. L.; Safa, A. R.; Bach, N. J.; Winter, M. A.; Cirtain, M. C.; Beck, W. T. Essential features of the P-glycoprotein pharmacophore as defined by a series of reserpine analogs that modulate multidrug resistance. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 5128− 5132. (70) Mi, Q.; Cui, B.; Lantvit, D.; Reyes-Lim, E.; Chai, H.; Pezzuto, J. M.; Kinghorn, A. D.; Swanson, S. M.; Pervilleine, F. A new tropane alkaloid aromatic ester that reverses multidrug resistance. Anticancer Res. 2003, 23, 3607−3615. (71) Aszalos, A.; Weaver, J. L. Estimation of drug resistance by flow cytometry. Methods Mol. Biol. 1998, 91, 117−122. (72) Wang, E. J.; Casciano, C. N.; Clement, R. P.; Johnson, W. W. In vitro flow cytometry method to quantitatively assess inhibitors of Pglycoprotein. Drug Metab. Dispos. 2000, 28, 522−528. (73) Lee, J. S.; Paul, K.; Alvarez, M.; Hose, C.; Monks, A.; Grever, M.; Fojo, A. T.; Bates, S. E. Rhodamine efflux patterns predict Pglycoprotein substrates in the National Cancer Institute drug screen. Mol. Pharmacol. 1994, 46, 627−638. (74) Wang, E. J.; Casciano, C. N.; Clement, R. P.; Johnson, W. W. Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem. Biophys. Res. Commun. 2001, 289, 580−585. (75) Ambudkar, S. V.; Dey, S.; Hrycyna, C. A.; Ramachandra, M.; Pastan, I.; Gottesman, M. M. Biochemical, cellular, and pharmacological aspects of the multidrug transpoter. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 361−398. (76) Shepard, R. L.; Winter, M. A.; Hsaio, S. C.; Pearce, H. L.; Beck, W. T.; Dantzig, A. H. Effect of modulators on the ATPase activity and vanadate nucleotide trapping of human P-glycoprotein. Biochem. Pharmacol. 1998, 56, 719−727. (77) Conseil, G.; Baubichon-Cortay, H.; Dayan, G.; Jault, J.-M.; Barron, D.; Di Pietro, A. Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 9831− 9836. (78) Bevan, C. D.; Lloyd, R. S. A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates. Anal. Chem. 2000, 72, 1781−1787. (79) Dolai, S.; Shi, W.; Corbo, C.; Sun, C.; Averick, S.; Obeysekera, D.; Farid, M.; Alonso, A.; Banerjee, P.; Raja, K. “Clicked” sugar− curcumin conjugate: modulator of amyloid-β and tau peptide aggregation at ultralow concentrations. ACS Chem. Neurosci. 2011, 2, 694−699. (80) Nifli, A.-P.; Theodoropoulos, P. A.; Munier, S.; Castagnino, C.; Roussakis, E.; Katerinopoulos, H. E.; Vercauteren, J.; Castanas, E. Quercetin exhibits a specific fluorescence in cellular milieu: a valuable tool for the study of its intracellular distribution. J. Agric. Food Chem. 2007, 55, 2873−2878. (81) Mukai, R.; Shirai, Y.; Saito, N.; Yoshida, K.-I.; Ashida, H. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope. Cytotechnology 2009, 59, 177−182. (82) Jones, D. J. L.; Jukes-Jones, R.; Verschoyle, R. D.; Farmer, P. B.; Gescher, A. A synthetic approach to the generation of quercetin sulfates and the detection of quercetin 3′-O-sulfate as a urinary metabolite in the rat. Bioorg. Med. Chem. 2005, 13, 6727−6731. (83) Quercetin sulfates were prepared by reacting quercetin with 10fold molar excess of sulfur trioxide-N-triethylamine complex in dioxane, and an extract of the mixture was analyzed by HPLC. On the basis of mass spectrometric analysis, the peak at 21.9 min was identified as a quercetin monosulfate.

7233

dx.doi.org/10.1021/jm500290c | J. Med. Chem. 2014, 57, 7216−7233