Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein

Article abstract of DOI:10.1016/j.bmc.2016.12.034

Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents.

Full text of DOI:10.1016/j.bmc.2016.12.034

Accepted Manuscript
Quercetin-Glutamic Acid Conjugate with a Non-Hydrolysable Linker; A Novel
Scaffold for Multidrug Resistance Reversal Agents through Inhibition of P-
Glycoprotein
Mi Kyoung Kim, Yunyoung Kim, Hyunah Choo, Youhoon Chong
PII:
S0968-0896(16)31031-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.034
BMC 13461
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 October 2016
20 December 2016
21 December 2016
Please cite this article as: Kyoung Kim, M., Kim, Y., Choo, H., Chong, Y., Quercetin-Glutamic Acid Conjugate
with a Non-Hydrolysable Linker; A Novel Scaffold for Multidrug Resistance Reversal Agents through Inhibition
of P-Glycoprotein, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.12.034
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Submitted to Bioorganic & Medicinal Chemistry
Quercetin-Glutamic Acid Conjugate with a Non-Hydrolysable Linker; A Novel Scaffold
for Multidrug Resistance Reversal Agents through Inhibition of P-Glycoprotein
Mi Kyoung Kim,1, † Yunyoung Kim,1, † Hyunah Choo,2,3,* Youhoon Chong1,*
1Department of Integrative Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk
2
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;
3Department of Biological Chemistry, Korea University of Science and Technology, Youseong-gu,
Daejeon 305-350, Korea
†These two authors contributed equally on this work.
* To whom correspondence should be addressed
Hyunah Choo, Ph.D., E–mail: hchoo@kist.re.kr, Tel: +82–2–958–5157
Youhoon Chong, Ph.D., E–mail: chongy@konkuk.ac.kr, Tel: +82–2–2049–6100
Graphical Abstract
MDR-Reversal Activity
Keywords: Multidrug resistance (MDR) ∙ Cancer ∙ Quercetin ∙ Conjugate ∙ Reversal activity
1

ABSTRACT
Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to
reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents
through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable
nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis
product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-
glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to
evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we
prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-
hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay
were performed, and the non-hydrolysable quercetin conjugates showed significantly higher
activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not
as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note
that the structurally modified quercetin conjugates with a non-cleavable linker showed
significantly improved MDR-reversal activity compared with quercetin. Taken together, the
quercetin conjugates with appropriate structural modifications were shown to have a potential
to serve as a scaffold for the design of novel MDR-reversal agents.
2

1. Introduction
Multidrug resistance (MDR) renders cancer cells insensitive to anticancer agents, and it is
one of the major obstacles to the cancer chemotherapy1. Various mechanisms have been
proposed to be involved in cancer MDR and, among those, overexpression of P-glycoprotein
(Pgp) and thereby anticancer drug efflux is the most well-characterized cellular mechanism2-3.
There have been numerous attempts to block the Pgp-mediated anticancer drug efflux by
using chemical entities, but none is currently available for clinical use. First-generation
agents represented by cyclosporine and verapamil showed unacceptable toxicity4-5. The
ensuing second- (dexverapamil6, valspodar7-8, elacridar9, and biricodar10-12) and third-
(tariquidar13, zosuquidar14, laniquidar15, and ONT-09316) generation MDR-reversal agents
were obtained by structural modification of the first-generation molecules, but they suffered
from unpredictable pharmacokinetic behavior and toxicity. Therefore, new chemical entities
with favorable physicochemical, pharmacokinetic and safety profiles are urgently required for
development of novel MDR-reversal agents.
In this context, natural flavonoids with well-known safety profiles in addition to the MDR-
reversal activity have drawn special attention17-28. Previously, we have shown that the MDR-
reversal activity of quercetin (Q, Fig. 1), a natural flavonoid abundant in fruits and vegetables,
can be significantly enhanced through conjugation with glutamic acid (7-O-Glu-Q (1), Fig.
1)27. The quercetin conjugate 1 was designed as a prodrug and, as anticipated, it was
hydrolyzed to quercetin in the intracellular milieu (t1/2 = 9.3 h) to keep intracellular
concentration of quercetin high enough to show MDR-reversal activity27. Thus, the MDR-
reversal activity of 1 was attributed to its hydrolysis product, quercetin. On the other hand,
however, a functional assay for Pgp showed that the quercetin conjugate 1 binds to the drug-
binding cavity of Pgp to stimulate Pgp ATPase activity by 3.5-fold, which is significantly
3

higher than the quercetin-induced stimulation (1.8-folds)27. Also noteworthy is that, in Pgp
inhibition assay, 15-minute incubation of the Pgp-overexpressing MDR cells with 1 resulted
in effective inhibition of rhodamine 123 efflux27. Considering that 1 did not undergo
hydrolysis up to 6 h under the same incubation conditions, it is highly unlikely that the
hydrolysis product of 1, quercetin, was responsible for Pgp inhibition. Taken together, there
was ample reason to suspect that the intact quercetin-glutamic acid conjugate 1 has stronger
MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a
novel scaffold for MDR-reversal agents, we designed quercetin conjugates with a glutamic
acid attached at the 7-O position via a non-hydrolysable linker. Herein, we report synthesis
and biological evaluation of two non-hydrolysable quercetin-glutamic acid conjugates (2 and
3, Fig. 1).
Figure 1. Structures of quercetin (Q), 7-O-Glu-Q (1) and the title compounds (2 and 3)
2. Results and Discussion
2.1. Chemistry
4

In order to synthesize the title compounds (2 and 3), the 7-OH position of quercetin was
differentiated from other hydroxyl groups by using the well-known synthetic procedures29-30
,
and the 3,5,3′,4′-O-tetraacetylquercetin (4) was prepared in 73% yield from quercetin
(Scheme 1).
Scheme 1. Synthesis of title compounds 2 and 3
The intermediate 4, thus obtained, was converted into 7-O-methoxymethyl quercetin (5)
after protection of the 7-OH functionality with a methoxymethyl (MOM) group followed by
methanolysis of four acetyl groups at 3, 5, 3′, and 4′ positions. Treatment of 5 with excess
amount of BnBr in the presence of K2CO3 in acetone provided the corresponding 3,5,3′,4′-O-
tetrabenzyl ether which, after deprotection of the 7-O-MOM group under the acidic
conditions, was converted into the key intermediate 3,3′,4′-O-tribenzylquercetin (6) in 78%
5

yield. Nucleophilic substitution of 6 with N-(chloromethylcarbonyl)-Glu(OtBu)-(OtBu)
followed by successive deprotection of the benzyl and tert-butyl functionalities afforded the
desired product 2 in 34% combined yield. On the other hand, treatment of 6 with 1,2-
dibromoethane provided 7 in 68% yield, which, after substitution with H-Glu(OtBu)-
(OtBu)∙HCl followed by sequential removal of the benzyl and tert-butyl protecting groups,
provided another desired product 3 in 27% yield.
2.2. Intrinsic cytotoxicity and non-hydrolysable nature of 2 and 3
The synthesized quercetin-glutamic acid conjugates (2 and 3) were first evaluated for their
antioxidant activity, the characteristic bioactivity of quercetin. As it is well known that the
antioxidant activity of quercetin strongly depends on its B-ring catechol moiety31, the newly
synthesized quercetin conjugates with alkyl substituents at the 7-O positions did not show
any difference in DPPH radical scavenging activity compared with quercetin (data not
shown).
The toxicity of 2 and 3 in drug-sensitive human uterine sarcoma cell line MES-SA and
Pgp-overexpressed MDR MES-SA/Dx5 cells27-28, 32 were then determined but no significant
cytotoxicity was observed up to 100 μM concentration (data not shown). Next, the non-
hydrolysable nature of the title compounds (2 and 3) was confirmed. Thus, MES-SA and
MES-SA/Dx5 cells were incubated with 2 or 3, and the cell lysates were analyzed by HPLC.
No trace of quercetin was observed from the cell lysates but the title compounds 2 and 3
underwent slow decomposition with half-lives of 30 h and 56 h, respectively (Fig. 2).
6

Figure 2. HPLC chromatograms of MES-SA/Dx5 cell lysates after incubation with (a)
quercetin (Q), (b) 2, and (c) 3 for 0 h, 24 h and 48 h
2.3. Pgp-inhibitory activity
Pgp-inhibitory activity of the quercetin conjugates (2 and 3) was then evaluated.
Doxorubicin (DOX) was used as a fluorescent substrate of Pgp, and the assay was performed
by measuring fluorescence of DOX inside the MES-SA or MES-SA/Dx5 cell after combined
treatment of DOX with the quercetin conjugate (2 or 3)33. Flow cytometric analysis of
fluorescence from intracellularly localized DOX showed that significantly decreased
7

fluorescence in the Pgp-overexpressed Mes-SA/Dx5 cells were recovered by addition of the
Pgp inhibitor verapamil (5 μM) (Fig. 3). By the same token, the quercetin conjugates 2 and 3
(5 μM) showed similar effects albeit with a lower potency; 2 and 3 increased DOX
fluorescence by 2.3-fold and 2.1-fold, respectively, while verapamil showed 5.5-fold increase
(Fig. 3).
120
100
80
MES-SA
60
40
20
0
MES_SA/Dx5
DMSO
2
3
verapamil
Figure 3. Fluorescence from intracellularly localized doxorubicin (DOX) in MES-SA and
MES-SA/Dx5 cells in the absence (DMSO) and presence of the Pgp inhibitors (2, 3 and
verapamil) (5 μM). Results are presented as mean ± SD from three experiments
2.4. Pgp ATPase stimulation
Pgp inhibitors bind to one of the two binding sites (drug binding cavity and NTP binding
domain) inside Pgp and, depending on the binding site, they show either inhibition24 or
stimulation34 of Pgp-mediated ATP hydrolysis: compounds that bind to the NBD inhibit ATP
hydrolysis by Pgp24, while others that bind to the drug-binding cavity typically stimulate
ATPase activity of Pgp34. Therefore, evaluation of Pgp ATPase activity upon addition of the
Pgp inhibitor is known to be the method of choice for determining its binding site inside
Pgp24,26,27-28. In this study, effects of the quercetin conjugates (2 and 3) (100 μM) on the Pgp
ATPase activity were evaluated by Pgp-Glo™ assay (Promega, Madison, WI), and the results
8

are summarized in Fig. 4.
Figure 4. Fold increases in Pgp ATPase activity conferred by Pgp inhibitors (100 μM). VER;
verapamil. The means ± SD (error bars) of three independent experiments are shown.
Asterisks represent significant differences compared with the verapamil-stimulated Pgp
ATPase activity; p < 0.001.
Verapamil (100 μM), a known binder to the drug-binding site in TBD35, showed to stimulate
Pgp ATPase activity over the basal level by 5.4-fold (Fig. 4, “VER”). Compound 2 (or 3)
(100 μM) was also found to stimulate ATPase activity by 3.1-fold (or 2.8-fold). Even though
2 (or 3) is a weaker stimulator than verapamil, this result clearly indicates that, like verapamil,
2 (or 3) also binds to the drug-binding site in TBD rather than nucleotide binding domain
(NBD). This result is reminiscent of the previous reports that the substituted flavonoids
preferentially bind to the drug-binding site of Pgp19,24,26,27-28
.
2.5. MDR-reversal activity
The quercetin conjugates (2 and 3) with inhibitory activity against Pgp-mediated
9

doxorubicin efflux were then evaluated for their MDR-reversal activity (Fig. 5 and Table 1).
(a) (b)
Figure 5. Cell viabilities of MES-SA and MES-SA/Dx5 cells before and after combination of
doxorubicin (DOX) with (a) 2 and (b) 3. Verapamil (VER) was used as a positive control and
concentrations of VER, 2 and 3 were fixed at 5 μM. Assay was performed in triplicate
Table. 1 The MDR-reversal activity of quercetin (Que), quercetin conjugates (2 and 3), and
verapamil (VER) on the sensitivities of MES-SA/Dx5 to anticancer agentsa
Anticancer Agents
Doxorubicin
Actinomycin D
Vinblastine
EC50 (μM)
Paclitaxel
EC50 (μM)
EC50 (μM)b FRc
EC50 (μM)
FR
FR
─
FR
─
Noned 8.71 ± 0.35
─
14.52 ± 0.22
─
13.26 ± 0.18
4.82 ± 0.26*
0.51± 0.02*
0.82± 0.03*
0.24 ± 0.01
11.08 ± 0.12
4.46 ± 0.24*
0.35 ± 0.03*
0.64 ± 0.02*
0.20 ± 0.03
Que
2
4.48 ± 0.14* 1.94 4.75 ± 0.32*
0.34 ± 0.03* 25.6 0.30 ± 0.05*
0.39 ± 0.05* 22.3 0.71 ± 0.03*
3.06
48.4
20.5
80.7
2.75
26.0
16.2
55.3
2.48
34.6
17.3
55.4
3
VER
0.16 ± 0.02 54.4
0.18 ± 0.01
a Concentrations of the MDR-reversal agents were fixed at 5 μM.
b
Amounts of the anticancer agents required to reduce 50% viability of the drug-resistant
(MES-SA/Dx5) cells in the presence of the MDR-reversal agents. The means ± SD (error
bars) of three independent experiments are shown.
c FR = Fold Reversal = IC50 (untreated) / IC50 (treated with MDR-reversal agents)
10

d The row “None” denotes IC50 values of the anticancer agents obtained in the absence of the
MDR-reversal agents. Asterisks represent significant differences compared with these control
values; p < 0.01. The means ± SD (error bars) of three independent experiments are shown.
In drug-sensitive MES-SA cells, anticancer drug doxorubicin showed potent cytotoxic
activity with IC50 value of 0.067 μM (♦, Fig. 5a and 5b). In contrast, doxorubicin showed
significantly decreased activity in the Pgp-overexpressed MDR MES-SA/Dx5 cells with 130-
fold increase in IC50 value (IC50 = 8.71 μM) (■, Fig. 5a and 5b; Table 1). Upon combination
with verapamil (5 μM), however, doxorubicin showed dramatically increased cytotoxicity in
MES-SA/Dx5 cells, and its IC50 value (IC50 = 0.16 μM) was comparable to that obtained in
drug-sensitive MES-SA cells (●, Fig. 5a and 5b; Table 1). Quercetin also showed MDR-
reversal activity but the cytotoxicity of doxorubicin increased by only 1.94-fold upon
combination with quercetin (Table 1). The quercetin conjugates 2 and 3, however, exhibited
more potent MDR-reversal activity than quercetin, and they potentiated doxorubicin by 25.6-
fold (2) (▲, Fig. 5a and Table 1) and 22.3-fold (3) (▲, Fig. 5b and Table 1), respectively, in
MES-SA/Dx5 cells.
The quercetin conjugates 2 and 3 were also effective in reversing MDR upon combination
with other anticancer drugs such as actinomycin D, vinblastine and paclitaxel (Table 1). Thus,
in MES-SA/Dx5 cells, 5 μM of 2 potentiated cytotoxicity of these drugs by 48.4, 26.0 and
34.6-fold, respectively (Table 1). Compound 3 was less effective than 2, and it provided 20.5,
16.2 and 17.3-fold increases in the cytotoxic activity of actinomycin D, vinblastine and
paclitaxel, respectively (Table 1).
The MDR-reversal activity of 2 and 3 were further evaluated at various concentrations (0, 0.5,
1, 2.5, 5 and 10 μM), and their potencies were determined by the amounts required to reduce
IC50s of the anticancer agents by half in the drug-resistant MES-SA/Dx5 cells (EC50 values)27
11

(Fig. 6 and Table 2).
(a)
(b)
12
10
8
12
10
8
6
6
2
3
VER
VER
4
4
2
2
0
0
0
2.5
5
7.5
10
0
2.5
5
7.5
10
Concentration (uM)
Concentration (uM)
Figure 6. Concentration-dependent effects of (a) 2 and (b) 3 for lowering IC50s of
doxorubicin in MDR MES-SA/Dx5 cells. Verapamil (VER) was used as a positive control.
Assay was performed in triplicate
Table. 2 MDR-reversal potency (EC50) of the quercetin conjugates (2 and 3) in the drug-
resistant (MES-SA/Dx5) cells
EC50 (μM)a
Doxorubicin
2.2 ± 0.3*
2.8 ± 0.2*
0.7 ± 0.1
Actinomycin D
2.3 ± 0.2*
Vinblastine
2.5 ± 0.4*
2.4 ± 0.7*
1.0 ± 0.1
Paclitaxel
2.1 ± 0.3*
2.5 ± 0.2*
0.8 ± 0.1
2
3
2.7 ± 0.4*
Verapamil
0.9 ± 0.1
aAmount of the MDR-reversal agents required to reduce the IC50′s of the anticancer agents by
half in the drug-resistant MES-SA/Dx5 cell line. The means ± SD of three independent
experiments are shown. Asterisks represent significant differences compared with EC50 of
verapamil; p < 0.01.
As anticipated by the inhibitory activity against Pgp, both 2 (Fig. 6a) and 3 (Fig. 6b)
showed remarkable effects in reversing resistance toward doxorubicin with EC50 values of
2.2 μM and 2.8 μM, respectively (Table 2). Also, compounds 2 and 3 potently sensitized
12

MDR MES-SA/Dx5 cells to a broad spectrum of anticancer drugs (actinomycin D,
vinblastine and paclitaxel) with EC50 values of 2.1−2.8 μM (Table 2). Compared with
verapamil (EC50 = 0.7−1.0 μM), compounds 2 and 3 were 2.6-4 folds less potent, but it
should be noted that the substituted quercetins represent a novel scaffold for MDR-reversal
agents.
3. Conclusions
Based on our previous observation that the intact quercetin-glutamic acid conjugate might
have stronger MDR-reversal activity than quercetin, we prepared quercetin conjugates with a
glutamic acid attached at the 7-O position via a non-hydrolysable linker (2 and 3). Biological
activity of the quercetin conjugates (2 and 3) were assessed by Pgp inhibition assay, Pgp
ATPase assay, and MDR-reversal activity assay, and the non-hydrolysable quercetin
conjugates showed significantly higher activities compared with those of quercetin.
Unfortunately, however, the quercetin conjugates were not as effective as verapamil in Pgp-
inhibition and thereby reversing MDR. Nevertheless, it is worth to note that the structurally
modified quercetin conjugates with a non-cleavable linker showed significantly improved
MDR-reversal activity compared with quercetin. Thus, this study established that the
quercetin conjugate with appropriate structural modifications has a potential to serve as a
scaffold for the design of novel MDR-reversal agents. Further studies are warranted to
explore the molecular determinants for Pgp-inhibition of the quercetin conjugates and the
structure-activity relationships.
4. Experimental
4.1. Materials and General Methods
13

Chemicals were purchased from Sigma-Aldrich unless noted otherwise. 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. ESI mass
spectrometric data 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
1
13
for H NMR and at 100 MHz for C 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). Chemical shifts were
reported as parts per million (δ) relative to the solvent peak.
4.2. Synthesis of the title compounds
4.2.1. 2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-7-(methoxymethoxy)-4H-chromen-4-one
(5)
A mixture of potassium carbonate (269 mg, 2.1 mmol) and chloromethyl methyl ether (0.2
mL, 2.7 mmol) were added to a solution of 4 (1.0 g, 2.1 mmol) in acetone (20 mL). After
stirring for 4 h at room temperature, the reaction mixture was filtered and the filtrate was
concentrated under reduced pressure to give a pale yellow syrup. The crude product was
purified by column chromatography on silica gel (2:1 = Hexane:EtOAc) to give a yellow
powder (1.1 g, 2.1 mmol, 100% yield). The resulting methoxymethyl (MOM)-protected
quercetin (1.1 g, 2.1 mmol) was dissolved in NH3/MeOH (10 mL) and stirred at 0 oC for 4 h.
The reaction mixture was concentrated under reduced pressure, and the residue was purified
by column chromatography on silica gel (1:1 = Hexane:EtOAc) to afford 5 (692 mg, 2.0
1
mmol, 95% yield) as a yellow powder: H NMR (400 MHz, Acetone-d6) δ 12.34 (s, OH),
14

7.87 (d, J = 2.0 Hz, 1H, H2′), 7.74 (dd, J = 2.0, 8.4 Hz, 1H, H6′), 7.00 (d, J = 8.5 Hz, 1H, H5′),
6.77 (d, J = 2.0 Hz, 1H, H8), 6.41 (d, J = 2.0 Hz, 1H, H6), 5.34 (s, 2H, -CH2), 3.48 (s, 3H, -
CH3).
4.2.2. 3,5-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-7-hydroxy-4H-chromen-4-one (6)
A mixture of potassium carbonate (1.0 g, 8.4 mmol) and benzyl bromide (1.2 mL, 8.4 mmol)
were added to a solution of 5 (750 mg, 2.1 mmol) in DMF (10 mL). After stirring at 80 oC for
4 h, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure
to give pale yellow syrup, which was used for the next step without further purification. The
product obtained above was dissolved in 3N HCl, and the resulting solution was stirred at 40
oC for 4 h. The reaction mixture was concentrated under reduced pressure, and the residue
was purified by column chromatography on silica gel (2:1 = Hexane:Acetone) to give 6 (1.1
1
g, 1.6 mmol, 78% yield) as a yellow powder: H NMR (400 MHz, Acetone-d6) δ 12.84 (s,
OH), 7.84 (d, J = 2.1 Hz, 1H, H2′), 7.70 (dd, J = 2.1, 8.6 Hz, 1H, H6′), 7.54 (d, J = 7.4 Hz, 2H,
-ArH), 7.46 (d, J = 7.4 Hz, 2H, -ArH), 7.37-7.42 (m, 10H, -ArH), 7.29-7.34 (m, 6H, -ArH),
6.94 (d, J = 8.6 Hz, 1H, H5′), 6.50 (d, J = 2.0 Hz, 1H, H8), 6.28 (d, J = 2.0 Hz, 1H, H6), 5.20
(s, 2H, -CH2), 5.11 (s, 2H, -CH2), 5.04 (s, 2H, -CH2), 4.89 (s, 2H, -CH2).
4.2.3.
3-(Benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-7-(2-bromoethoxy)-5-hydroxy-4H-
chromen-4-one (7)
Potassium carbonate (83 mg, 0.6 mmol), 1,2-dibromoethane (0.1 mL, 0.3 mmol) and KI (1
mg) were added to a solution of 6 (200 mg, 0.3 mmol) in acetone (6 mL). After stirring at
room temperature for 4 h, the reaction mixture was filtered and the filtrate was concentrated
1
under reduced pressure to give 7 (153 mg, 0.2 mmol, 68% yield) as a dark yellow syrup: H
NMR (400 MHz, Acetone-d6) δ 12.84 (s, OH), 7.72 (d, J = 2.1 Hz, 1H, H2′), 7.62 (d, J = 7.5
15

Hz, 2H, -ArH), 7.54 (dd, J =2.1, 8.4Hz, 1H, H6′), 7.46 (d, J = 7.5 Hz, 2H, -ArH), 7.37-7.42
(m, 6H, -ArH), 7.28-7.36 (m, 5H, -ArH), 6.95 (d, J = 8.5 Hz, 1H, H5′), 6.64(d, J = 2.2 Hz, 1H,
H8), 6.43 (d, J = 2.2 Hz, 1H, H6), 5.28 (s, 2H, -CH2), 5.14 (s, 2H, -CH2), 4.96 (s, 2H, -CH2),
4.26 (t, J = 6.3 Hz, 2H, -CH2CH2), 3.63 (t, J = 6.3 Hz, 2H, -CH2CH2).
4.2.4. (S)-2-(2-((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)-
acetamido)pentanedioic acid (2)
Potassium
carbonate
(25
mg,
0.2
mmol)
and
(S)-di-tert-butyl
2-(2-
chloroacetamido)pentanedioate (50 mg, 0.2 mmol) were added to a solution of 6 (100 mg, 0.2
mmol) in DMF (4 mL). After stirring at room temperature for 6 h, the reaction mixture was
filtered and the filtrate was concentrated under reduced pressure to give a pale yellow syrup,
which was used for the next step without further purification. Under an atmosphere of
hydrogen gas (balloon), the degassed suspension of quercetin conjugate obtained above and
Pd/C (10% w/w, 15 mg) in a mixture of THF (3 mL) and MeOH (1 mL) was vigorously
stirred at room temperature for 8 h. The reaction mixture was filtered through a short celite
pad and the filtrate was recrystallized from CH2Cl2 to afford a yellow powder. The quercetin
conjugate, thus obtained, was treated with trifluoroacetic acid (1 mL) at 0 °C. After stirring at
0 °C for 4 h, the reaction mixture was concentrated under reduced pressure. The residue was
recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 2 (34 mg, 0.07
mmol, 34% yield) as a yellow solid: m.p. 262 oC; 1H NMR (400 MHz, Acetone-d6) δ 12.63 (s,
OH), 7.73 (d, J = 2.0 Hz, 1H, H2′), 7.58 (dd, J = 2.1, 8.4 Hz, 1H, H5′), 6.91 (d, J = 8.4 Hz, 1H,
H6′), 6.82 (d, J = 2.1 Hz, 1H, H8), 6.47 (d, J = 2.1 Hz, 1H, H6), 4.34-4.38 (m, 1H, -CH), 4.06
13
(s, 2H, -COCH2), 2.34-2.60 (m, 4H, -CH2CH2); C NMR (100 MHz, DMSO-d6) δ 178.0 (-
CH2CH2CO2H), 176.7 (C4), 174.6 (-CHCO2H), 168.8 (-OCH2CONH), 164.2 (C7), 155.8 (C5),
16

154.2 (C9), 148.4(C2), 148.0 (C4′), 145.5 (C3′), 136.5 (C3), 122.1 (C1′), 120.5 (C6′), 116.0
(C5′), 115.6 (C2′), 105.4 (C10), 98.6 (C6), 94.2 (C8), 68.2 (-OCH2CONH), 55.6 (α-CH), 30.5
(CH2CH2CO2H), 28.5 (CH2CH2CO2H); MS (ESI) m/z Found: 490.11 [M+H]+; Calcd for
C22H20NO12: 490.09.
4.2.5. (S)-2-((2-((2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-4H-chromen-7-yl)oxy)-
ethyl)amino)pentanedioic acid (3)
To a solution of 7 (100 mg, 0.2 mmol) in DMF (5 ml) were added H-Glu(OtBu)-OtBu∙HCl
(44 mg, 0.2 mmol) and DIPEA (0.1 mL, 0.3 mmol). After stirring at room temperature for 12
h, solvent was removed under reduced pressure to give a crude product, which was used for
the next step without further purification. Pd/C (10% w/w, 20 mg) was added to a solution of
crude product in a mixture of THF (3 mL) and MeOH (1 mL), and the reaction flask was
charged with hydrogen gas (balloon, 1 atm). After stirring at room temperature for 4 h, the
reaction mixtue was filtered through a Celite pad. The filtrate was concentrated under
reduced pressure and the residue was treated with trifluoroacetic acid (1 mL) at 0 °C. After
stirring at 0 °C for 4 h, the reaction mixture was concentrated under reduced pressure, and the
residue was recrystallized from a mixture of acetone (1 mL) and CH2Cl2 (10 mL) to give 3
(26 mg, 0.05 mmol, 27% yield) as a yellow solid: m.p. 230 oC; 1H NMR (400 MHz, Acetone-
d6) δ 12.64 (s, OH), 7.70 (d, J = 2.2 Hz, 1H, H2′), 7.56 (dd, J = 2.2, 8.4 Hz, 1H, H5′), 6.86 (d,
J = 8.4 Hz, 1H, H6′), 6.82 (d, J = 2.2 Hz, 1H, H8), 6.46 (d, J = 2.2 Hz, 1H, H6), 4.30-4.34 (m,
1H, -CH), 4.24 (t, J = 6.3 Hz, 2H, -OCH2), 3.61 (t, J = 6.3 Hz, 2H, -CH2N), 2.30-2.54 (m, 4H,
-CH2CH2CO2H); 13C NMR (100 MHz, DMSO-d6) δ 178.0 (-CH2CH2CO2H), 176.7 (C4),
174.6 (-CHCO2H), 165.4 (C7), 160.2 (C5), 158.2 (C9), 148.4(C2), 148.0 (C4′), 145.5 (C3′),
136.4 (C3), 122.1 (C1′), 120.5 (C6′), 116.0 (C5′), 115.6 (C2′), 105.4 (C10), 98.2 (C6), 92.4 (C8),
70.4 (-OCH2CH2NH), 67.2 (α-CH), 47.4 (-OCH2CH2NH), 30.5 (CH2CH2CO2H), 28.5
17

(CH2CH2CO2H); MS (ESI) m/z Found: 476.24 [M+H]+; Calcd for C22H22NO11: 476.11.
4.3. Biological Assay
4.3.1. Materials for Biological Studies
DMSO (dimethyl sulfoxide), MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium
bromide)], and verapamil were purchased from Sigma-Aldrich. Roswell Park Memorial
Institute (RPMI) 1640 media, penicillin, streptomycin and fetal bovine serum (FBS) were
purchased from HyClone Laboratories.
4.3.2. 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.
4.3.3. Cytotoxicity
Cytotoxicity
was
determined
by
MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, a yellow tetrazole) assay. 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 in complete RPMI-1640 and incubated for 24 h
(37 ˚C , 5% CO2). Quercetin (Q) and quercetin conjugates (2 and 3) dissolved in DMSO were
serially diluted (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM,), and the resulting
solutions were added to the media. After 72 h, cell viability was estimated by MTT assay.
18

Every assay was repeated three times.
4.3.4. Modulation of Doxorubicin Efflux
The quercetin conjugates 2 and 3 were screened for their ability to block the Pgp-dependent
doxorubicin efflux by flow cytometry analysis. Briefly, 24 h before the experiment, MES-
SA/Dx5 cells in the logarithmic phase of growth were seeded in 24-well plates at a density of
105 cells per well. Afterward, cells were incubated for 30 min at 37 °C in DMEM + 10% FBS
in the presence or absence of each quercetin conjugate (10 μM) or verapamil with
doxorubicin (2 μM). Finally, cells were washed twice with ice-cold PBS, trypsinized, and
resuspended in 0.2 mL of ice-cold PBS for immediate analysis. Fluorescence measurements
of individual cells were made with FACSCalibur (Becton Dickinson, USA).
4.3.5. 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), 2 (or 3) (100 μM), verapamil (100 μM), or verapamil
(100 μM) plus 2 (or 3) (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 signal-stabilization period
(20 min), luminescence was read on a Veritas microplate luminometer (Turner Designs, San
Francisco, CA). Pgp ATPase activity was presented as a drop in luminescence of samples
compared to that treated with Na3VO4.
4.3.6. Determining MDR-modulatory effect of the quercetin conjugates on the
19

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 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.001 μM, 0.0001
μM) of anticancer drugs (doxorubicin, vinblastine, paclitaxel, actinomycin) with or without
the MDR-reversal agents [quercetin conjugates (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
Survival % = (MDx – Mblank)/(Mcontrol – Mblank) x 100
Here, MDx presents light absorption values of cell, growth medium, anticancer drugs and
MDR-reversal agents, 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
IC50’s of anticancer drugs against MES-SA and MES-SA/Dx5 cells.
4.3.7. 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.
20

Acknowledgements
This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-
2014R1A1A2053495) and 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).
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