Enhanced stability and intracellular accumulation of quercetin by protection of the chemically or metabolically susceptible hydroxyl groups with a pivaloxymethyl (POM) promoiety

Article abstract of DOI:10.1021/jm101252m

In order to increase stability of quercetin, its metabolically and chemically susceptible hydroxyl groups 7-OH and 3-OH respectively were transiently blocked with a pivaloxymethyl (POM) promoiety to provide two novel quercetin conjugates [7-O-POM-Q (2), 3

Full text of DOI:10.1021/jm101252m

J. Med. Chem. 2010, 53, 8597–8607 8597
DOI: 10.1021/jm101252m
Enhanced Stability and Intracellular Accumulation of Quercetin by Protection of the Chemically or
Metabolically Susceptible Hydroxyl Groups with a Pivaloxymethyl (POM) Promoiety
Mi Kyoung Kim,†,§ Kwang-Su Park,†,§ Chaewoon Lee,† Hye Ri Park,† Hyunah Choo,‡ and Youhoon Chong*,†
†Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu,
Seoul 143-701, Korea, and ‡Neuro-Medicine Center, Life/Health Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong,
Seongbuk-gu, Seoul 136-791, Korea. § These two authors contributed equally to this work.
Received June 28, 2010
In order to increase stability of quercetin, its metabolically and chemically susceptible hydroxyl groups
7-OH and 3-OH respectively were transiently blocked with a pivaloxymethyl (POM) promoiety to pro-
vide two novel quercetin conjugates [7-O-POM-Q (2), 3-O-POM-Q (3)]. In the absence of stabilizer
(ascorbic acid), the synthesized conjugates showed significantly increased stability in cell culture media
[t1/2 = 4 h (2), 52 h (3)] compared with quercetin (t1/2 < 30 min) and quercetin prodrug 1 (t1/2 = 0.8 h). In
addition, the quercetin conjugate 2 underwent efficient cellular uptake and intracellular levels of its
hydrolysis product, quercetin, were maintained up to 12 h. Stability and intracellular accumulation of 2
were demonstrated by its stabilizer-independent cytostatic effect and induction of apoptotic cell death.
Even though 3 was more stable than 2, it failed to penetrate cell membranes. However, the remarkable
stability of 3 warrants further investigation of quercetin conjugates with various promoieties at the
3-OH position.
Introduction
Quercetin (Figure 1), a polyphenolic flavonoid, is abundant
in human diet including apples, onions, grapes, red wine, and
green tea, and its various health promoting effects such as
antioxidant,1 antiviral,2,3 and anticancer4 activities have been
studied in detail. However, studies that support clinical uses of
quercetin are few presumably because of its unfavorable
Figure 1. Structures of quercetin and quercetin prodrug, 30-(N-
physicochemical as well as pharmacokinetic properties in-
carboxymethyl)carbomyl-3,40,5,7-tetrahydroxyflavone (QC12) (1),
with numbering system.
cluding oxidative degradation5-7 and fast metabolism.7-9
Pro-oxidative compounds in cell systems such as enzymes
7-hydroxyl groups as the preferential sites of conjugation.28-30
and transition metal cations are known to facilitate oxidative
Among the two hydroxyl groups, 30-OH has been actively
transformed into the corresponding esters31-39 or carba-
mates40-42 by conjugation with various promoieties, which
resulted in clinical investigation of quercetin-glycine carba-
mate, 30-(N-carboxymethyl)carbomyl-3,40,5,7-tetrahydroxy-
flavone (QC12) (1, Figure 1) as a potential anticancer agent.40
However, the quercetin conjugates with promoieties at the
catecholic hydroxyl groups (30-OH or 40-OH) suffer from
accelerated hydrolysis due to the “ortho effect” exerted by the
neighboring free -OH group31 to result in low stability in
aqueous media, and the half-life of 1 (QC12) in whole blood
was reported to be only 0.39 h.40 In contrast, only a limited
number of studies have been reported to introduce promoi-
eties at 7-OH with a cleavable linkage.32
degradation of quercetin.10-15 Oxidative degradation has also
been reported as the main cause of rapid elimination of quer-
cetin from the cultured human hepatocarcinoma cell line(Hep
G2).5 In spite of the lack of correct identification of the inter-
mediates associated with the oxidative degradation, there
seems to be a broad consensus that benzofuran-3(2H)-one
E (Figure 2) is one of the major degradation products16-21 that
is formed by nucleophilic attack of water to the initially formed
intermediate B (Figure 2)21 followed by equilibration. In this
respect, it is of particular interest that a free C3 hydroxyl is
reported to be essential for formation of the key intermediate
B,22 which suggests that transient protection of the C3 hydro-
xyl group with an adequate promoiety would stabilize quer-
cetin against the devastating oxidative degradation.
On the other hand, a promoiety attached at 30-OH or 7-OH
is anticipated to work as a removable metabolic blocker. After
in-depth investigation of the overall percentage of phase II con-
jugation23-27 at the various hydroxyl groups in the different
model systems, van der Woude et al. designated the 30- and
Taken together, as hydroxyl groups of quercetin such as 3-OH
and 7-OH are involved in oxidative degradation and metabo-
lism, respectively, transient protection of those hydroxyl groups
with a metabolically susceptible promoiety would provide safe
cell delivery followed by slow release of quercetin in intracellular
compartment to result in a sustained therapeutic effect.
In this study, pivaloxymethyl (POM), which has been suc-
cessfully exploited in antiviral drug of adefovir dipivoxyl,43,44
*To whom correspondence should be addressed. Phone: þ82-2-2049-
6100. Fax: þ82-2-454-8217. E-mail: chongy@konkuk.ac.kr
r
2010 American Chemical Society
Published on Web 11/19/2010
pubs.acs.org/jmc

8598 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kim et al.
Figure 2. Proposed mechanism for oxidative degradation of quercetin.21,22
Scheme 1. Synthesis of 7-O-POM-Q (2)a
Figure 3. Quercetin-POM conjugates (2 and 3) investigated in this
study.
was used as our promoiety of choice. With the proven resistance
to chemical hydrolysis, the POM group was expected to serve
as a good protecting group of the chemically or metabolically
susceptible hydroxyl groups of quercetin during its intracellu-
lar transit. Once localized inside the cell, the POM ester is
known to undergo esterase-catalyzed acyl cleavage to release
the parent drug molecule.43-46 Along with the role as a tran-
sient protecting group, the neutral lipophilic character of the
POM group is also expected to facilitate passive diffusion of
quercetin across the cell membrane.
a Reagents and conditions: (a) Ac2O, Pyr, 70 °C; (b) PhSH, NMP,
imidazole, 0 °C; (c) POM-I, K2CO3, acetone, room temp; (d) NH3,
MeOH, 0 °C.
Herein, wereportregioselectivesynthesisof two novelquer-
cetin conjugates with POM promoieties at 7-OH [7-O-POM-
Q (2), Figure 3] and 3-OH [3-O-POM-Q (3), Figure 3] and the
investigation of their physicochemical as well as biological
properties in comparison with quercetin.
pyridine provided quercetin peracetate 5, of which the 7-OAc
group was selectively removed upon treatment with a mix-
ture of PhSH, NMP, and imidazole49 to give 6 in 73% yield
(Scheme 1). Alkylation of 6 with POM-I followed by global
deprotection with methanolic ammonia provided the desired
7-O-POM-Q (2) in 84% yield.
Results
Regioselective introduction of the POM promoiety at the
3-O position required more tedious protection-deprotection
steps to provide a quercetin derivative with orthogonal pro-
tecting groups at the catechol functionality, 7-O position,
and 3,5-O-position (10, Scheme 2).
Thus, quercetin diphenylmethylketal (7, Scheme 2), obtained
by reaction of quercetin with 1,1-dichlorodiphenylmethane,
was peracetylated with an excess amount of Ac2O in pyridine
to give 8 in 93% yield. The 7-OAc group of 8 was selectively
removed by treatment with a mixture of PhSH, NMP, and
imidazole to give 9 (90% yield), which was reprotected with a
benzyl group to give the key intermediate 10 in 69% yield.
For selective introduction of the POM promoiety at the 3-O
position, the acetate functionality was removed by treatment
with methanolic ammonia to give 11 with free phenolic hy-
droxyl groups at 3 and 5 positions of quercetin (90% yield).
Intramolecular hydrogen bond between the carbonyl oxygen
Synthesis. The quercetin prodrug 1 (Figure 1) was pre-
pared by the method previously reported.47 Synthesis of
quercetin-POM conjugates were accomplished by nucleo-
philic substitution of appropriately protected quercetin de-
rivatives (6 and 11; Schemes 1 and 2) with pivaloxymethyl
iodide (POM-Ia). Thus, regioselective monodeacetylation of
the peracetylated quercetin48 (Scheme 1) or regioselective
protection of quercetin catechol moiety49 (Scheme 2) was the
key to the syntheses of novel quercetin conjugates, which was
attempted by slight modification of the literature conditions.
Treatment of quercetin with excess amount of Ac2O in
a Abbreviations: POM-I, pivaloxymethyl iodide; Ac2O, acetic anhy-
dride; PhSH, benzenethiol; NMP, N-methylpyrrolidone; PBS, phos-
phate buffered saline; DMEM, Dulbecco’s modified Eagle medium;
FBS, fetal bovine serum; PAMPA, parallel artificial membrane perme-
ability assay; Pgp, P-glycoprotein; MRP, multidrug resistance protein.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8599
Scheme 2. Synthesis of 3-O-POM-Q (3)a
a Reagents and conditions: (a) Ph2CCl2, 180 °C; (b) Ac2O, Pyr, 70 °C; (c) PhSH, NMP, imidazole, 0 °C; (d) BnBr, K2CO3, acetone, room temp;
(e) NH3/MeOH, 0 °C; (f) POM-I, K2CO3, acetone/DMF, room temp; (g) H2, Pd/C, THF/MeOH, room temp.
Table 1. Stability of Quercetin and Quercetin-POM Conjugates in PBS
and Complete DMEM
atom and 5-OH prohibits chemical reactions at the 5-OH,
which resulted in regioselective alkylation of 11 at 3-O posi-
half-life t1/2 (h)
tion upon treatment with POM-I. Simultaneous deprotec-
tion of the diphenylmethylketal and benzyl groups under
quercetin 1a 7-O-POM-Q (2) 3-O-POM-Q (3)
hydrogenolysis conditions afforded the desired 3-O-POM-Q
(3) in 67% yield.
buffer
pH 2.0
pH 7.4 (PBS)
7
10
3
10
7
>72
4
20
>72
52
cDMEM
<0.5
0.8
Stability. Quercetin is reported to have low stability in
PBS (pH 7.4) with a half-life of less than 10 h,50 which can be
ascribed to its fast auto-oxidation.51 The oxidation of quer-
cetin in PBS can be prevented by addition of antioxidants
such as ascorbic acid (1 mM),50,52,53 but because of the rein-
forced oxidative stress in cell culture media such as McCoy’s
5A, degradation of quercetin in cell system is known to be-
come 5 times faster even in the presence of antioxidants.10-15
In this study, no stabilizer was added to the incubation
media for the purpose of estimation of stabilities of the
quercetin conjugates in comparison with quercetin. Stabili-
ties of quercetin and the quercetin-POM conjugates were
assessed in various buffer solutions [pH 2.0 and pH 7.4
(PBS)] and DMEM culture media supplemented with FBS
under cell-free conditions (cell-free cDMEM), and the result
of HPLC analysis is summarized in Table 1.
a Half-life for hydrolysis of 1 to quercetin.
that the levels of 2 and 3 remained constant up to 72 h. In
acidic buffer solution (pH 2.0) and cell-free cDMEM, the
amount of 2 decreased to half at 7 and 4 h after incubation,
respectively, but the lack of quercetin in the incubation media
attributes this to direct oxidative decomposition of the quer-
cetin conjugate itself. In this context, it is noteworthy that
introduction of the POM carbamate at the noncatecholic and
chemically labile 3-OH group of quercetin provided the result-
ing conjugate 3 with near-optimal in vitro stability profile.
Neither chemical hydrolysis nor oxidative decomposition of 3
seems feasible (Table 1).
Solubility. Solubilities of quercetin, quercetin prodrug (1),
and quercetin-POM conjugates (2 and 3) in PBS, DMEM,
and cDMEM were estimated by measuring forward scattered
light when a laser beam is directed through the solution,54 and
the results are summarized in Table 2 and Figure 4. In order to
prevent decomposition of quercetin, analysis was performed
immediately after dissolving the solutes in PBS, but solubility
of quercetin in cell culture media was not amenable to estima-
tion because of its instability.
In line with literature,10-15,50 without added stabilizer,
quercetin showed a moderate to low stability profile with
half-lives (t1/2) of 7 h, 10 h, and less than 30min inacidicbuffer
solution (pH 2.0), PBS (pH 7.4), and cell-free cDMEM,
respectively (Table 1). Previously, the water-soluble quercetin
prodrug 1 was reported to be stable against in vitro hydrolysis
in water (t1/2 = 16.9 h) but unstable in blood with a half-life of
only 0.39 h.40 In our study, a similar trend was observed in the
hydrolysis of 1 to quercetin (Table 1). In contrast, the quer-
cetin conjugates 2 and 3 did not provide the hydrolysis pro-
duct, quercetin, in any conditions tested. As anticipated by
introduction of the carbamate linkage to the noncatecholic
hydroxyl groups of quercetin (7-OH and 3-OH), the resulting
quercetin conjugates 2 and 3 must have acquired resistance
against hydrolytic cleavage. In PBS, HPLC analysis showed
In PBS, quercetin was soluble up to 100 μM, after which
the solubility decreased abruptly (Table 2, Figure 4a, [). As
reported previously,40 the quercetin prodrug 1 was comple-
tely soluble in PBS as well as cell culture media, and no light
scattering caused by insoluble solute molecules was observed
up to 400 μM (Table 2, Figure 4, b). Because of the presence
of the lipophilic POM promoiety, limited solubility of the
quercetin conjugate 2 (Figure 4a, 2) in PBS was observed

8600 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kim et al.
(5 μM, Table 2). On the contrary, the conjugate 3 showed al-
most the same solubility profile as quercetin in PBS (Figure 4a,
9). The high solubility of 3-O-POM-Q (3) compared with
7-O-POM-Q (2) might be attributed to the bulky POM group
at 3-O position which shifts the planarity of the molecule to an
out-of-plane conformation to disrupt the crystal packing and
increase solubility. In DMEM without added FBS (Figure 4b),
no significant change in solubility was observed in comparison
with PBS (Figure 4a, Table 2). However, upon addition of FBS
to DMEM (Figure 4c), forward light scattering intensity from
the samples of both 2 and 3 showed significant decrease, which
may be attributed to interactions of the lipophilic POM
promoiety of the conjugates with the serum proteins in FBS.
Intracellular Localization. With proven solubility as well as
stability in cell-free culture media, intracellular localization
remains as the next biological barrier for the quercetin con-
jugates 2 and 3, which was visualized by fluorescence micro-
scopy. It is known that quercetin exhibits specific fluorescence
(488 nmex/500-540 nmex) upon internalization in cells due to
binding to intracellular target proteins.55-58 Therefore, dif-
ferent levels of fluorescence were anticipated depending on
efficiencies of cellular uptake of quercetin and its conjugates.
Following incubation of human colon cancer cells (HCT116)
with quercetin and the conjugate 2, fluorescent staining was
observed in a confocal microscope at two different time points
(1 and 12 h after incubation), and the results are visualized in
Figure 5.
After short incubation (1 h), quercetin (Figure 5a, left
panel) showed strong fluorescence which, however, did not
persist until 12 h (Figure 5a, right panel). Taking short half-
life of quercetin (<30 min, Table 1) in the extracellular media
into consideration, efflux of the internalized quercetin fol-
lowed by extracellular decomposition might provide a rea-
sonable explanation for this observation. 7-O-POM-Q (2)
also showed bright fluorescence signal upon incubation with
the cells for 1 h (Figure 5b, left panel) and the fluorescence
was maintained up to 12 h (Figure 5b, right panel). However,
unlike quercetin and 2, no significant fluorescence was ob-
served from the cells treated with the conjugate 3 regardless
of the incubation time (data not shown). The lack of intra-
cellular fluorescence was in good agreement with the low
PAMPA59 permeability of 3 (Table 3). Whereas quercetin
(5.6-15.9%) and 1 (40.3-55.1%) were observed in moder-
ate to high amounts in the acceptor plates after penetration
of the artificial membrane, the quercetin conjugate 3 could be
found only in the donor plate.
Table 2. Solubilities of Quercetin, Quercetin Prodrug (1), and Querce-
tin-POM Conjugates (2 and 3) in PBS and Cell Culture Media
Membrane permeability is much higher for more lipo-
philic molecules than for polar molecules because mole-
cules must pass through a highly nonpolar lipid bilayer
membrane. In this context, it is worth remembering the high
water-solubility of 3 in spite of the presence of the lipophilic
POM group (Table 2), and the polar nature of 3 might be
related to its low membrane permeability and thereby low
cellular uptake.
solubility (μM)
media
PBS
DMEM
quercetin
1
7-O-POM-Q (2)
3-O-POM-Q (3)
100
>400
>400
>400
5
50
100
100
NDa
NDa
cDMEM
200
>400
a ND: not determined because of instability of quercetin in cell culture
media.
Figure 4. Forward light scattering intensity of solutions of quercetin ([), 1 (b), 7-O-POM-Q (2, 2), and 3-O-POM-Q (3, 9) dissolved in
(a) PBS, (b) DMEM, and (c) cDMEM. In PBS (a), light scattering intensities from the solutions of quercetin ([) and 3-O-POM-Q (3, 9) are
almost the same and the data labels for the two compounds are overlapped.
Figure 5. Cellular uptake of quercetin and its conjugates visualized by fluorescence microscopy. HCT116 cells were treated with 5 μM
(a) quercetin and (b) 7-O-POM-Q (2) for 1 h (left panel) and 12 h (right panel). After a wash with PBS, fluorescent staining was observed in a
confocal microscope at 488 nmex/500-540 nmem
.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8601
Table 3. PAMPA Permeability: Percentage of Quercetin or Quercetin Conjugates Contained in the Donor and Acceptor Plate after Incubation for
1 and 5 h
percentage (%)a
quercetin
1
s7-O-POM-Q (2)
Db Ac
NDd NDd
NDd NDd
3-O-POM-Q (3)
incubation time (h)
Db
Ac
Db
Ac
Db
Ac
1
5
45
8
16
6
6
18
55
40
87
74
0
0
a Distribution of contents in each plate was calculated from the integrated HPLC peak area. b D: donor plate. c A: acceptor plate. d ND: not
determined because of low solubility of 7-O-POM-Q (2) in PBS at the concentration for PAMPA assay (50 μM).
Figure 6. HPLC chromatograms of (a) cell culture media after 1 h, (b) cell lysate after 1 h, (c) cell culture media after 12 h, and (d) cell lysate
after 12 h of incubation of MCF-7 cells with the quercetin conjugate 2. Chromatogram of 2 before incubation is presented as an inset in (a).
Expanded chromatogram of the cell lysate sampled 12 h after incubation was inserted in (d). Q: quercetin. Q-glu: quercetin glucuronide. Q-sul:
quercetin sulfate. Q-oxi (E): oxidized product of quercetin (compound E, Figure 2). Q-methyl: methyl quercetin.
Table 4. Quantitative Analysis of 7-O-POM-Q (2), Quercetin, and
Quercetin Metabolites Remaining in the Cell Culture Media and Cell
Addition of Pgp inhibitor (verapamil) or MRP inhibitor
(gemfibrozil) to the cell culture media did not have any effect
on accumulation of 3, and no intracellular fluorescence was
observed by confocal live-cell imaging. This result suggests
Lysate after Incubation of 2 in MCF-7 Cell Linea
percentage (%)b
after 1 h
after 12 h
that, along with the lack of passive permeation, active trans-
port followed by efflux mechanism of 3 is not in action, either.
Next, as another measure of the cellular uptake and efflux,
chemical species remaining in extracellular as well as intracel-
lular compartment were analyzed by HPLC after incubation
of human breast cancer cells (MCF-7) with the conjugate 2,
and the result is summarized in Figure 6 and Table 4.
culture
media
cell
lysate
culture
media
compd
cell lysate
7-O-POM-Q (2)
Q
Q metabolites
62
5
21
7
12
5
4
17
Q-Glu (6), Q-Sul (3),
Q-Oxi (2), Q-Me (1)
a Q: quercetin. Q-glu: quercetin glucuronide. Q-sul: quercetin sulfate.
Q-oxi (E): oxidized product of quercetin (compound E, Figure 2).
Q-methyl: methyl quercetin. b Percentage (%) of each content in culture
media and cell lysate was calculated from the integrated HPLC peak
area.
After short incubation (1 h) with 7-O-POM-Q (2), HPLC
analysis of the cell culture media (Figure 6a) showed the native
conjugate 2 with only 62% (Table 4) of its initial amount
(Figure 6a, insert) along with a small amount of quercetin (5%,
Table 4). On the basis of the lack of nonenzymatic hydrolysis
of 2 described above, identification of quercetin in the cell cul-
ture media suggests a series of events that includes cellular up-
take of 2, intracellular hydrolysis of 2 to quercetin, and efflux
of quercetin out of the cell. The HPLC chromatogram of the
cell lysate at the same detection point (1 h after incubation,
Figure 6b) supports this hypothesis with significant intracel-
lular accumulation of the conjugate 2 (21%, Table 4) along
with quercetin (7%, Table 4). Significant reduction of 2 in the
extracellular fluid after 12 h (Figure 6c, Table 4) was well cor-
related with its stability as well as cellular uptake profile. Once
again, extracellular quercetin level (5%, Table 4), kept steady
up to 12 h, (Figure 6c), provides support for the aforemen-
tioned intercellular trafficking and intracellular hydrolysis
mechanism of 2 and quercetin. Intracellularly, along with
quercetin (17%, Table 4), several minor peaks were observed
in the HPLC chromatogram (Figure 6d, insert), and they were

8602 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kim et al.
identified as either oxidized quercetin (Q-oxi, compound E
in Figure 2) or quercetin metabolites [quercetin-glucuronide
(Q-glu), quercetin-sulfate (Q-sul), and methylated quercetin
(Q-methyl)] by mass analysis (Figure 6d, insert). High intra-
cellular level (17%, Table 4) and low metabolism (1-4%,
Table 4) of quercetin, however, might be attributed to low
oxidative and metabolic capacity of MCF-7 cells, and the cell
type-specific intracellular accumulation of quercetin was pro-
ven by repeating the same experiment in metabolically compe-
tent hepatocytes (Huh-7 cell line). Incubation of the conjugate 2
in the Huh-7 cell line for 1 h and HPLC analysis of the extra-
cellular and intracellular contents provided almost the same
chromatograms as in Figure 6a and Figure 6b, respectively
(data not shown). However, presumably because of the in-
creased oxidative stress in the Huh-7 hepatoma cell line,60 the
quercetin conjugate 2 was not stable enough in this cell culture
system and started to decompose after 1 h. Therefore, 12 h
after incubation, quercetin was observed only at a trace level
and intracellular accumulation of the quercetin metabolites
such as Q-sul and Q-methyl was not significant (<5%).
Unlike 2, the extracellular amount of 3 remained constant
regardless of the cell type and incubation time. Also, HPLC
analysis of the cell lysate showed neither 3 nor quercetin
(metabolites). This result provides another piece of evidence
that, even though introduction of a POM promoiety at the
chemically susceptible 3-OH position provided remarkable
stability to the resulting quercetin conjugate 3 in cell culture
media, the 3-O-POM group failed to transport the resulting
conjugate 3 across the cell membrane.
lung,65 hepatoma,66 oral,67 and colon62 cancer but not in
normal cells.68 Viabilities of cancer cell lines such as breast
(MCF-7), colon (HCT116), and prostate cancer (DU145)
as well as normal human diploid fibroblast cell line (HS27)
treated with quercetin and quercetin conjugates (2 and 3) in
the absence of ascorbic acid were estimated, and the results
are summarized in Table 5.
Quercetin, because of the instability in cell culture media,
was not cytotoxic at all up to 100 μM (Table 5). In contrast,
because of the enhanced stability as well as efficient intra-
cellular transport, the conjugate 2 showed significant cyto-
static effects in three cancer cell lines tested (Table 5). On the
other hand, as expected by lack of intracellular accumula-
tion, 3-O-POM-Q (3) did not affect the cell viability at all
even at concentration of 100 μM (Table 2). Neither quercetin
nor its conjugates had effects on viability of normal diploid
cell line HS-27 (Table 5). Interestingly, 7-O-POM-Q (2) showed
preferential effect on DU145 with EC50 value of 10 μM
(Table 5).
The stability-limited cytotoxicity of quercetin was con-
firmed by addition of an antioxidative stabilizer, ascorbic
acid, to the cell culture media. As ascorbic acid shows cell type-
specific cytotoxicity in MCF-7 cell line,10 HCT116 was used as
a cell line of choice in this study (Figure 7). Whereas ascorbic
acid rescued the cytotoxic effect of quercetin (Figure 7a), it
had no effect on the viability of the cancer cells treated with
7-O-POM-Q (2, Figure 7b). In combination with the stabi-
lity profiles observed for quercetin and its conjugate 2, this
result clearly shows that resistance to oxidative decomposi-
tion is one of the key issues in determining the biological
effect of quercetin.
Cell Cycle Analysis. Antiproliferative effect of the con-
jugate 2 promoted our interest in its effect on cell cycle
distribution. Quercetin is known to arrest the cell cycle in
S/early G2 phase,40,62 and the same effect was anticipated by
2 which gives quercetin in the intracellular compartment.
Considering poor stability and thereby low antiproliferative
effect of quercetin, cells were treated with quercetin in higher
concentration (50 μM) compared with 2 (30 μM). Untreated
cells or cells treated with quercetin or 2 were submitted to cell
cycle analysis after 12 h of treatment, and the results are
shown in Figure 8.
By combination of information obtained from confocal
microscopy, PAMPA permeability, and HPLC analysis, it
can be concluded that 7-O-POM-Q (2) is efficiently absorbed
into the cell and undergoes intracellular conversion to quer-
cetin and its metabolites over a relatively long period. Unlike
2, 3-O-POM-Q (3) is neither localized into the cell nor hydro-
lyzed to quercetin.
Cell Viability. Studies have shown that quercetin has
antiproliferative effects61,62 and can induce apoptotic cell
death in several cancer cell lines such as leukemia,63 breast,64
Table 5. Effects of Quercetin and Its Conjugates (2 and 3) on Viability
(EC50) of Three Different Cancer Cell Lines (MCF-7, HCT116, and
DU145) and a Normal Cell Line (HS27)
In line with literature,62 cell cycle analysis showed a de-
crease in the percentage of cells in the G0/G1 phase (21.0%,
Figure 8d) with a concurrent increase in the S-phase (78.0%,
Figure 8d) when cells were exposed to 50 μM quercetin
(Figure 8b) compared to control (Figure 8a). Interestingly,
after exposureto the conjugate2 (Figure8c), the percentageof
cells in the G0/G1-phase decreased further (an approximately
EC50 (μM)
compd
MCF-7
HCT116
DU145
HS27
2
3
20
>100
>100
36
>100
>100
10
>100
>100
>100
>100
>100
quercetin
Figure 7. Effects of ascorbic acid on the viability of HCT116 cells treated with (a) quercetin and (b) 7-O-POM-Q (2).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8603
Figure 8. Cell cycle distribution of MCF-7 cells by propidium iodide staining and flow cytometry after treatment with (a) 1% DMSO,
(b) 50 μM of quercetin, and (c) 30 μM of 2 for 12 h. The percentage of cells in each phase is summarized in (d).
10% decrease in comparison with quercetin, Figure 8d), while
the number of cells in the sub-G1 phase increased (an approxi-
mately 120% increase when compared to quercetin, Figure 8d).
This result indicates that inhibition of cell proliferation by the
conjugate 2 could partly be attributed to an increase in the
number of apoptotic cells in the sub-G1 phase.
In this study, in order to increase stability of quercetin, the
metabolically (7-OH) and chemically (3-OH) susceptible hy-
droxyl groups of quercetin were transiently blocked with a
POM promoiety to provide the corresponding conjugates
7-O-POM-Q (2) and 3-O-POM-Q (3).
In extracellular fluid without added stabilizing factor, quer-
cetin was highly unstable to decompose rapidly (t1/2 < 30 min).
The water-soluble quercetin prodrug 1 also underwent fast
hydrolysis to quercetin (t1/2 = 0.8 h). In contrast, under the
same conditions, the half-life of 7-O-POM-Q (2) was increased
to 4 h. In addition, both confocal live-cell imaging and HPLC
analysis of the extracellular and intracellular contents after
incubation of cells with 7-O-POM-Q (2) revealed that the
quercetin conjugate 2 undergoes efficient cellular uptake, and
the intracellular levels of 2and its hydrolysis product, quercetin,
are maintained up to 12 h. Extracellular stability and facile
intracellular accumulation of the quercetin conjugate 2 were
demonstrated by its stabilizer-independent cytostatic effect and
induction of apoptotic cell death.
Discussion
Because of the chemical instability against oxidative stress,
quercetin decomposes in the cell culture media and cells
experience lower concentrations of quercetin than originally
added. As a result, simultaneous addition of ascorbic acid as
an antioxidant50,52,53 or refreshment of the media during in-
cubation is in general practice to suppress the devastating
oxidative degradation of quercetin. However, addition of
stabilizers such as ascorbic acid may lead to overestimation
oftheinvitrobiological activityofquercetin. In particular, the
cell-type-specific antiproliferativeeffect of ascorbicacid10,69,70
should be carefully taken into consideration before adding it
into the cell culture. Alteration of the quercetin structure
through metabolism also results in complication of its biolog-
ical activity as well as reduction in bioavailability.
On the other hand, as anticipated by protection of the chem-
ically susceptible 3-OH with a POM group, 3-O-POM-Q (3)
was stable in every condition tested up to 24 h. Unfortunately,

8604 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Kim et al.
however, the remarkably stable conjugate 3 failed to accumu-
late in the cell.
(m, 2H), 7.51 (d, J = 8.5 Hz, 1H), 6.94 (s, 1H), 6.65 (s, 1H),
2.33 (s, 6H), 2.30 (s, 6H).
Synthesis of [2-(3,4-Dihydroxyphenyl)-3,5-dihydroxy-4-oxo-
4H-chromen-7-yloxy]methyl Pivalate (2). Potassium carbonate
(88 mg, 0.64 mmol) and POM iodide (232 mg, 0.96 mmol) were
added to a solution of 6 (150 mg, 0.32 mmol) in acetone (8 mL).
The reaction mixture was stirred for 4 h at room temperature,
filtered, and concentrated under reduced pressure to give a pale
yellow syrup, which was used for the next step without further
purification. A mixtureof the quercetinconjugate obtainedabove
was dissolved in NH3/MeOH (12 mL) and stirred for 2 h at 0 °C.
After concentration under reduced pressure, the residue was puri-
fied by column chromatography on silica gel (1:1 = hexanes/
EtOAc) to afford 2 (112 mg, 0.27 mmol, 84% yield) as a yellow
powder: 1H NMR (400 MHz, DMSO-d6) δ 12.64 (s, 1H), 7.73
(d, J = 2.0 Hz, 1H), 7.58 (dd, J = 8.4, 2.0 Hz, 1H), 6.91 (d, J =
8.5 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H),
5.90 (s, 2H), 1.15 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ
176.7, 176.4, 161.8, 160.9, 156.0, 148.4, 148.1, 145.5, 136.6, 122.1,
120.5, 116.0, 115.6, 105.4, 98.6, 94.2, 84.8, 38.8, 26.9. LC/MS
(ESI) m/z found: 415.2 [M - H]- (loss of phenolic proton). Calcd
for C21H20O9: 416.11. HPLC:retention time of 12.28 min, >95%
pure at 340 and 254 nm.
Synthesis of 2-(2,2-Diphenylbenzo[1,3]dioxol-5-yl)-3,5,7-tri-
hydroxychromen-4-one (7). A mixture of quercetin (1) (5 g, 14.8
mmol) and dichlorodiphenylmethane (8.5 mL, 44.3 mmol) was
stirred for 30 min at 180 °C. The reaction mixture was taken with
CHCl3 andconcentrated under reduced pressure. The residue was
purified by column chromatography (4:1 = hexanes/EtOAc) on
silica gel to afford 7 (2.4 g, 5.2 mmol, 35% yield) as a yellow
powder. 1H NMR (400 MHz, acetone-d6) δ 12.18 (s, 1H), 7.89-
7.92 (m, 2H), 7.63-7.69 (m, 5H), 7.45-7.49 (m, 5H), 7.19 (d, J =
10.5 Hz, 1H), 6.58 (s, 1H), 6.28 (s,1H).
In conclusion, our results demonstrate that conjugation
of quercetin with a POM promoiety at C7 hydroxyl group (2)
can enhance stability and intracellular accumulation of the
corresponding quercetin conjugate as well as quercetin. Even
though 3-O-POM-Q failed to localize inside the cell, its high
solubility as well as remarkable stability in extracellular fluid
warrants further investigation of quercetin conjugates with
various promoieties at the 3-OH position.
Experimental Section
Materials and General Method. All chemicals including MTT
[(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)],
verapamil, and gemfibrozil were purchased from Sigma-Aldrich.
Dulbecco’s modified Eagle medium (DMEM), penicillin, strep-
tomycin, and fetal bovine serum (FBS) were purchased from
Invitrogen. The precoated PAMPA plates were manufactured
by BD Biosciences Discovery Labware (Bedford, MA) using a
polyvinylidene fluoride (PVDF) 96-well filter plate with 0.4 μm
pore size. Nuclear magnetic resonance spectra were recorded
on a Bruker 400 AMX spectrometer (Karlsruhe, Germany) at
400 MHz for 1H NMR and at 100 MHz for 13C NMR with tetra-
methylsilane as the internal standard. Chemical shifts are reported
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br
s (broad singlet). Coupling constants are reported in hertz (Hz).
The chemical shifts are reported as parts per million (δ) relative
to the solvent peak. All tested compounds were g95% purity,
as determined by reverse phase HPLC. HPLC was performed on
Agilent 1050 (Hewlett-Packard) equipment with variable wave-
length (VW) UV dectctor. The column was Polaris 5, C18-A
250 mm ꢀ 4.6 mm (Varian). Analytical conditions were as follows:
Gradient used was 25% acetonitrile in water containing 0.1%
formic acid (0-8 min), 25-35% acetonitrile in water containing
0.1% formic acid (8-18 min), 35% acetonitrile in water containing
0.1% formic acid (18-25 min), 35-80% acetonitrile in water
containing 0.1% formic acid (25-40 min), 80-100% acetonitrile
in water containing 0.1% formic acid (40-45 min), and 100% ace-
tonitrile in water containing 0.1% formic acid (45-50 min). Flow
was 1 mL/min. UV was detected at two different wavelengths (340
and 254 nm). 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 spectro-
metric data (MS) were obtained using a MALDI-TOF-TOF mass
spectrometer (Ultraflex III, Bruker Daltonik).
Chemistry. Synthesis of 2-(3,4-Diacetoxyphenyl)-4-oxo-4H-
chromene-3,5,7-triyl Triacetate (5). Acetic anhydride (12.5 mL,
132 mmol) was added to a solution of quercetin (1) (5 g, 16.5
mmol) in pyridine (35 mL). The reaction was monitored by
TLC. After starting material was consumed, the organic layer
was concentrated under reduced pressure. The crude compound
was purified by column chromatography on silica gel (4:1:1 =
hexanes/acetone/CH2Cl2) to afford 5 (4.5 g, 8.8 mmol, 54%
yield) as a white-yellow powder. 1H NMR (400 MHz, CDCl3) δ
7.72 (dd, J = 8.6, 2.0 Hz, 1H), 7.70 (d, J = 1.9 Hz, 1H), 7.35 (d,
J = 8.6 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 6.88 (d, J = 2.2 Hz,
1H), 2.43 (s, 3H), 2.34 (s, 12H).
Synthesis of 4-(3,5-Diacetoxy-7-hydroxy-4-oxo-4H-chromen-
2-yl)-1,2-phenylene Diacetate (6). Thiophenol (0.48 mL, 4.69
mmol) was slowly added to a stirred mixture of 5 (3 g, 5.85
mmol) and imidazole (80 mg, 1.17 mmol) in NMP (30 mL) at
0 °C. The reaction mixture was stirred for 2 h at room tempera-
ture. 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 compound was purified by
column chromatography on silica gel (2:1:1 = hexanes/acetone/
EtOAc) to give 6 (2 g, 4.25 mmol, 73% yield) as a white powder.
1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.83-7.80
Synthesis of Acetic Acid 3,5-Diacetoxy-2-(2,2-diphenylbenzo-
[1,3]dioxol-5-yl)-4-oxo-4H-chromen-7-yl Ester (8). Acetic anhy-
dride (1 mL, 10.7 mmol) was added to a solution of 7 (1 g, 2.14
mmol) in anhydrous pyridine (10 mL) at room temperature. The
reaction mixture was stirred for 6 h at 70 °C. After concentration,
the crude compound was purified by column chromatography
(2:1 = hexanes/EtOAc) on silica gel to give 8 (1.2 g, 2.0 mmol,
1
93% yield) as a white powder. H NMR (500 MHz, CDCl3) δ
7.57-7.59 (m, 4H), 7.38-7.44 (m, 7H), 7.36 (s, 1H), 7.30 (d, J =
1.6 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H), 6.85 (d, J = 1.6 Hz, 1H),
2.43 (s, 3H), 2.34 (s, 3H), 2.32 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 169.5, 169.1, 168.7, 168.1, 156.6, 154.8, 154.6, 149.8,
149.4, 147.4, 139.5, 133.0, 130.0, 129.0, 126.2, 124.3, 123.2, 117.9,
114.9, 114.3, 110.3, 109.7, 108.6, 21.2, 21.1, 20.7. LC/MS (ESI)
m/z found: 593.3 [M þ H]þ. Calcd for C34H24O10: 592.14.
Synthesis of 2-(2,2-Diphenylbenzo[d ][1,3]dioxol-5-yl)-7-hy-
droxy-4-oxo-4H-chromene-3,5-diyl Diacetate (9). Thiophenol
(0.16 mL, 1.62 mmol) was slowly added to a stirred mixture of 8
(1.2 g, 2.0 mmol) and imidazole (27 mg, 0.4 mmol) in NMP
(24 mL) 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
compound was purified by column chromatography on silica
gel (1:1 =hexanes/EtOAc) to give 9 (1 g, 1.8 mmol, 90% yield)
as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.55-7.58
(m, 4H), 7.37-7.41 (m, 8H), 6.92 (d, J = 8.1 Hz, 1H), 6.63 (d,
J = 2.2 Hz, 1H), 6.50 (d, J = 2.2 Hz, 1H), 2.35 (s, 3H), 2.29 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 169.4, 169.2, 168.4,
163.1, 158.0, 154.0, 150.6, 149.3, 147.5, 139.6, 132.7, 130.1, 129.1,
126.3, 124.1, 123.7, 118.0, 109.7, 109.6, 109.4, 108.6, 101.5, 21.3,
20.8. LC/MS (ESI) m/z found: 549.3 [M - H]- (loss of phenolic
proton). Calcd for C32H22O9: 550.13.
Synthesis of Acetic Acid 5-Acetoxy-7-benzyloxy-2-(2,2-dip-
henylbenzo[1,3]dioxol-5-yl)-4-oxo-4H-chro-men-3-yl Ester (10).
Potassium carbonate (251 mg, 1.8 mmol) and benzyl bromide
(0.32 mL, 2.7 mmol) was added to a solution of 9 (1 g, 1.8 mmol)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8605
in acetone (20 mL). The reaction mixture was stirred for 12 h at
room temperature. After filtration, the filtrate was concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel (2:1 = hexanes/EtOAc) to give 10
(800 mg, 1.25 mmol, 69% yield) as a pale yellow oil. 1H NMR
(400 MHz, CDCl3) δ 7.57-7.59 (m, 4H), 7.39-7.39 (m, 13H),
6.97 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 2.3 Hz, 1H), 6.70 (d, J =
2.3 Hz, 1H), 5.14 (s, 2H), 2.42 (s, 3H), 2.31 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 169.5, 169.2, 168.3, 163.1, 157.9, 154.2,
150.4, 149.4, 147.5, 139.6, 136.2, 132.9, 130.1, 129.1, 120.0, 128.8,
128.5, 126.3, 124.2, 123.6, 118.0, 110.6, 109.7, 108.6, 100.8, 70.9,
21.3, 20.8. LC/MS (ESI) m/z found: 641.3 [M þ H]þ. Calcd for
C39H28O9: 640.17.
Synthesis of 7-Benzyloxy-2-(2,2-diphenylbenzo[1,3]dioxol-5-yl)-
3,5-dihydroxy-chromen-4-one (11). A mixture of 10 (800 mg, 1.25
mmol) in NH3/MeOH (12 mL) at 0 °C was stirred for 2 h at room
temperature. After concentration under reduced pressure, the resi-
due was purified by column chromatography on silica gel (6:2:1 =
hexanes/CH2Cl2/EtOAc) to give 11 (624 mg, 1.12 mmol, 90%
yield) as a yellow powder. 1H NMR (400 MHz, CDCl3) δ 11.69
(s, 1H), 7.77-7.81 (m, 2H), 7.59-7.61 (m, 4H), 7.34-7.44 (m,
10H), 7.09 (d, J = 8.3 Hz, 1H), 6.60 (s, 1H), 6.54 (d, J = 2.1 Hz,
1H), 6.45 (d, J = 2.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ
176.6, 164.4, 160.8, 156.4, 148.1, 147.1, 146.4, 139.8, 137.2, 136.5,
129.9, 129.0, 128.9, 128.5, 128.2, 126.1, 125.5, 123.5, 117.5, 109.3,
108.2, 104.6, 98.5, 93.4, 70.3; LC/MS (ESI) m/z found: 557.3
[M þ H]þ. Calcd for C35H24O7: 556.15.
Solubility Test. Stock solutions of the quercetin conjugates
were prepared at 0.5, 2.5, 5, 10, 20, 30, 40, and 50 mM in 1%
DMSO and then serially diluted with either 99% phosphate
buffered saline (PBS, pH 7.4) buffer or Dulbecco’s modified
Eagle medium (DMEM) or DMEM with fetal bovine serum
(FBS 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.
Intracellular Localization Test (Confocal Microscopy). HCT-
116 cell was seeded in poly-L-lysine-coated coverslip in 35 mm
dishes (105 cells/well) and incubated for 1 day (37 °C, 5% CO2).
Quercetin (5 μM) and quercetin conjugates (5 μM) were pro-
vided for 1 and 12 h and then quickly washed with PBS and fixed
with 4% formaldehyde for 5 min followed by mounting with PBS.
Specimens were observed with FV-1000 spectral (Olympus, USA)
at 488 nmex/500-540 nmem using 40ꢀ objective lens.
PAMPA (Parallel Artificial Membrane Permeability Assay)
Study. The precoated PAMPA plate was warmed to room
temperature for 30 min. Quercetin conjugates were dissolved
in a solution of 95% PBS buffer solution (5% DMSO) to a final
concentration of 50 μM. The initial solutions were added to
the wells (300 μL/well) of the donor plate, and PBS buffer was
added to the wells (200 μL/well) of the acceptor plate. The acce-
ptor plate was placed on top of the donor plate, and the plate
assembly was incubated at room temperature for 1 and 5 h. After
incubation, the plates were separated and aliquots (100 μL) were
taken from each well plate and injected into the HPLC.
Synthesis of 2,2-Dimethylpropionic Acid 2-(3,4-Dihydroxy-
phenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yloxymethyl Ester
(3). Potassium carbonate (55 mg, 0.4 mmol) and POM iodide
(130 mg, 0.54 mmol) were added to a solution of 11 (200 mg, 0.36
mmol) in a mixture of acetone (7 mL) and DMF (4 mL). The
reaction mixture was stirred for 12 h at room temperature, filtered,
and concentrated under reduced pressure to give a pale yellow
syrup, which was used for the next step without further purifica-
tion. 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, 15 mg), under an atmosphere of hydrogen gas
(balloon), was vigorously stirred for 12 h at room temperature.
The reaction mixture was filtered through a short Celite pad and
purified by column chromatography on silica gel (2:1:1 =
hexanes/CH2Cl2/EtOAc) to afford 3 (100 mg, 0.24 mmol, 67%
Cellular Uptake and Intracellular Stability. MCF-7 cell was
seeded in tissue-cultured COSTAR clear bottom six-well plates
(105 cells/well) and incubated for 24 h (37 °C, 5% CO2). Quercetin
conjugates (10 μM) were provided for 12 h. The cells were washed
in PBS, trypsinized, and sonicated with Vibra-Cell VCX-130
(SONICS). After filtration, samples were analyzed by HPLC
(Agilent). Each peak was manually collected, lyophilized, and
analyzed by mass spectrometry (MALDI-TOF, Bruker Daltonics).
Cell Viability Study. Cells (MCF-7, HCT116, DU145, and
HS27) were seeded (5 ꢀ 103 cells/well) in tissue-cultured COST-
AR clear bottom 96-well plate in complete DMEM (Dulbecco’s
modified Eagle medium) and incubated for 1 day (37 °C, 5%
CO2). Prepared quercetin conjugates which dissolved in DMSO
were diluted into seven different concentrations (0.1, 0.5, 1, 5,
10, 50 μM) and added to the medium. After 24 h, cell viability
was estimated by MTT assay. Each experiment was performed
in triplicate and repeated three times.
Cell Cycle Distribution Analysis by FACS. MCF-7 cell was
seeded in 35 mm dishes (105 cells/well) and incubated for 12 h
(37 °C, 5% CO2). After treatment with quercetin (30 or 100 μM),
quercetin conjugates (30 μM), and control (1% DMSO) for 12 h
(37 °C, 5% CO2), cells were trypsinized, collected, washed
with cold PBS, and fixed in 70% EtOH at 4 °C for at least
30 min. The fixed cells were washed with cold PBS and stained
with 50 μL/mL of propidium iodide in the presence of 25 μg/mL
of RNase A. Cell cycle phase distribution was analyzed using
FACSCalibur (Becton Dickinson).
1
yield) as a yellow powder: H NMR (400 MHz, acetone-d6) δ
12.63 (s, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.5, 2.1 Hz,
1H), 6.96 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 6.28 (s, 1H), 5.80 (s,
2H), 0.88 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ 177.2,
177.0, 164.7, 161.6, 156.7, 156.5, 149.3, 145.5, 134.8, 121.7, 120.8,
116.2, 115.8, 104.3, 99.1, 94.1, 87.3, 38.4, 26.6. LC/MS (ESI) m/z
found: 415.2 [M - H]- (loss of phenolic proton). Calcd for
C21H20O9: 416.11. HPLC: retention time of 11.94 min, >97%
pure at 340 and 254 nm.
Stability in PBS Buffer. The synthesized quercetin conjugates
(50 μM) were added to PBS buffer (pH 7.4) and incubated at
37 °C. At different time points (0, 30, 60, 90, 120, 150, 180, 210,
240, 300, and 360 min), an aliquot (300 μL) of the reaction
mixture was taken out and injected into the HPLC equipped
with a C-18 reverse phase column; flow rate, 1 mL/min; detec-
tion, UV 340 nm; mobile phase, 0-8 min (10% aqueous ace-
tonitrile and 0.1% formic acid), 8-12 min (40% aqueous
acetonitrile and 0.1% formic acid), 12-15 min (90% aqueous
acetonitrile and 0.1% formic acid) and 15-20 min (10% aque-
ous acetonitrile and 0.1% formic acid).
Stability in DMEM Cell Culture Media. Quercetin conjugates
were dissolved in culture media to a final concentration of 50 μM
from a 10 mM stock solution in DMSO. The quercetin con-
jugate solutions were incubated at 37 °C and 5% CO2 in 24-well
plates. The media were harvested at different time points up to
24 h and stored at -80 °C until analysis. After thawing, the
samples were vortexed and analyzed with HPLC under the same
analysis conditions described above.
Acknowledgment. This work was supported by Priority
Research Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (Grant 2009-0093824)
and a grant from Biogreen 21 (Korea Ministry of Agriculture
and Forestry).
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Kim et al.
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