Remarkable stability and cytostatic effect of a quercetin conjugate, 3,7-bis-O-pivaloxymethyl (POM) quercetin

Full text of DOI:10.1002/cmdc.201100478

DOI: 10.1002/cmdc.201100478
Remarkable Stability and Cytostatic Effect of a Quercetin Conjugate,
3,7-Bis-O-Pivaloxymethyl (POM) Quercetin
Mi Kyoung Kim, Kwang-Su Park, and Youhoon Chong*[a]
Quercetin (Q, 1) is a polyphenolic flavonoid that is readily
found in human diet. The tremendous growth in the study of
this bioactive compound has revealed numerous health-pro-
moting effects, such as antioxidant,[1] antiviral,[2,3] and anticanc-
er[4] activities. However, quercetin is unstable and undergoes
oxidative decomposition in aqueous solution.[5–7] As a result,
the bioactivity of quercetin is often limited by its stability, and
it is desirable to develop a method to increase quercetin stabil-
ity.
Many studies support the critical role of the free hydroxy
group in the 3 position in the oxidative decomposition of
quercetin.[8,9] In our previous study,[10] we introduced a pival-
oxymethyl (POM)[11,12] group to quercetin at this position and
observed remarkable stability of the resulting 3-O-POM-Q (2).
Quercetin–POM conjugate 2 showed very slow decomposition
(t1/2 =52 h) in a cell culture medium under conditions of high
oxidative stress. This remarkably stable quercetin–POM conju-
gate, however, failed to show cytostatic effects against various
cancer cell lines due to its inability to permeate the cell mem-
brane. Conversely, 7-O-POM-Q (3) with the POM group at-
tached to the hydroxy group in the 7 position of quercetin
showed much faster hydrolysis and/or decomposition in cell
culture medium (t1/2 =4 h) compared with 3-O-POM-Q (2).
However, unlike 3-O-POM-Q (2), 7-O-POM-Q (3) showed effi-
cient cellular uptake and intracellular conversion to quercetin
and its metabolites, which resulted in enhanced cytostatic ef-
fects against cancer cell lines compared with the parent com-
pound, quercetin.
We reasoned that the introduction of POM groups at both
the 3- and 7-hydroxys of quercetin would produce synergistic
effects to provide a stable quercetin–POM conjugate with effi-
cient cellular uptake and thereby significant cytostatic activity.
Herein, we report the synthesis and biological evaluation of
3,7-bis-O-POM-Q (4).
The synthesis of 4 was accomplished by nucleophilic substi-
tution of the selectively protected quercetin, quercetin diphe-
nylmethylketal (5), with excess amount of pivaloxymethyl
Scheme 1. Synthesis of 3,7-bis-O-POM-Q (4). Reagents and conditions:
iodide (POM-I) (Scheme 1).
a) Ph2CCl2, 1808C, 30 min, 35%; b) POM-I, K2CO3, acetone, RT, 4 h; c) H2, Pd/
C, THF/MeOH (1:1), RT, 12 h, 63%.
The stability of the quercetin–POM conjugates 2–4 was as-
sessed in phosphate buffered saline (PBS; pH 7.4) and Dulbec-
co’s modified eagle medium supplemented with fetal bovine
serum (cDMEM), and the results are summarized in Table 1 and
Figure 1.
The instability of quercetin in aerobic aqueous media such
as PBS (t1/2 =10 h, Table 1) and cDMEM (t1/2 <30 min, Table 1) is
well known.[10,13,14] In contrast, 3,7-bis-O-POM-Q (4) is as stable
as the previously reported quercetin–POM conjugates 2 and 3
(Table 1)[10] against hydrolytic cleavage or oxidative decomposi-
tion in PBS. More intriguingly, in cDMEM, 3,7-bis-O-POM-Q (4)
was even more stable (t1/2 =100 h, Table 1) than conjugates 2
(t1/2 =52 h, Table 1) and 3 (t1/2 =4 h, Table 1).[10] High-perfor-
mance liquid chromatography (HPLC) analysis of the cell cul-
[a] M. K. Kim, K.-S. Park, Prof. Y. Chong
Department of Bioscience and Biotechnology
Bio/Molecular Informatics Center, Konkuk University
Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Republic of Korea)
E-mail: chongy@konkuk.ac.kr
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100478.
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229

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its specific fluorescence (lex =488 nm, lem =500–540 nm).[15–18]
In our previous study,[10] we showed that detection of fluores-
cence inside the cell can be used as an efficient method to
evaluate the cellular uptake of quercetin and its conjugates.
Thus, after incubation of cells with 4, fluorescent staining was
observed in a confocal microscope at two different time points
(1 h and 12 h; Figure 2).
Table 1. Stability of quercetin and quercetin–POM conjugates in different
media.[a]
1
Half-life (t = ) [h]
2
Compd
PBS
cDMEM
Q (1)[b]
10
>24
>24
>24
<30 min
3-O-POM-Q (2)[b]
7-O-POM-Q (3)[b]
3,7-bis-O-POM-Q (4)
52
4
100[c]
[a] Phosphate-buffered saline (PBS); Dulbecco’s modified eagle medium
supplemented with fetal bovine serum (cDMEM). [b] Data taken from Ref-
erence [10]. [c] Conversion to 3-O-POM-Q (2) not to quercetin.
Figure 2. Confocal microscope images of fluorescent staining (lex =488 nm,
lem =500–540 nm) in HCT116 cells treated with 3,7-bis-O-POM-Q (4) in phos-
phate-buffered saline (3 mm) for 1 h (left panel) and 12 h (right panel).
Due to the hydrophobic nature of the POM moiety, 3,7-bis-
O-POM (4) is largely membrane-associated, which can be ob-
served by the fluorescence signal around the cell membrane
(Figure 2). However, intensified cytoplasmic fluorescence signal
observed 12 h after incubation (right panel, Figure 2) clearly
demonstrates the time-dependent cellular uptake as well as in-
tracellular localization of the quercetin derivatives.
The intracellular localization of 3,7-bis-O-POM-Q (4) and its
hydrolysis product 3-O-POM-Q (2) was also confirmed by HPLC
analysis of the cell lysate (Figure 3).
Knowing that 3-O-POM-Q (2) is not transported into the
cell,[10] it is clear that 3-O-POM-Q (2) found in the cell lysate is a
result of hydrolysis of intracellularly localized 4; no trace of 7-
O-POM-Q (3) or quercetin (i.e., metabolites) was observed. The
favorable properties associated with 3-O-POM-Q (2) and 7-O-
POM-Q (3), i.e., high stability and efficient cellular uptake, re-
spectively, are combined in 3,7-bis-O-POM-Q (4). However,
unlike 7-O-POM-Q (3), which was hydrolyzed to bioactive quer-
cetin,[10] 3,7-bis-O-POM-Q (4) did not convert to quercetin
inside the cell.
Figure 1. HPLC–UV chromatograms (340 nm) of 3,7-bis-O-POM-Q (4) after
incubation in cell-free cDMEM for a period of a) 1 h and b) 24 h.
ture media after 24 h (Figure 1b) showed less than 10% of
3,7-bis-O-POM-Q (4) was converted into its hydrolysis product,
3-O-POM-Q (2), but neither 7-O-POM-Q (3) nor quercetin was
observed in the HPLC chromatogram (Figure 1). The exception-
al stability of 4 in comparison with the mono-POM–quercetin
derivatives 2 and 3 suggests the synergistic effect of the POM
groups attached at the 3-hydroxy as well as the 7-hydroxy
group. Presumably, the two POM groups do not allow 3,7-bis-
O-POM-Q (4) to serve as a substrate for hydrolyzing enzymes
present in cDMEM.
Based on these results, we can conclude that the cellular
uptake of the POM-substituted quercetin derivatives depends
on the specific location of the POM group at the 7-hydroxy po-
sition. Therefore, rather than nonspecific passive transport, a
transporter-mediated active transport is the more plausible
mechanism involved in the selective cellular uptake of the
quercetin conjugates with POM groups at the 7-hydroxy posi-
tion.
The cytostatic effect of 3,7-bis-O-POM-Q (4) was next evalu-
ated. Cell lines derived from various types of cancer, including
breast (MCF-7), colon (HCT116), and prostate cancer (DU145),
as well as normal human diploid fibroblast cells (HS27) were
treated with quercetin and quercetin–POM conjugate 4, and
With the proven stability of 4 in cell culture media in hand,
we then evaluated its cellular uptake. Quercetin itself is not flu-
orescent but, upon binding to cellular target proteins, it exhib-
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ChemMedChem 2012, 7, 229 – 232

ure 4a–c). The lack of cytostatic effect by quercetin is not sur-
prising because it is known to be inactive in cell culture media
without stabilizing agent such as ascorbic acid.[5–7] However,
under the same conditions, the cells treated with 3,7-bis-O-
POM-Q (4) showed significantly reduced viability. The cytostatic
effect of 4 was specific to cancer cells, and the viability of the
normal human diploid fibroblast cell line (HS27) was unaffect-
ed by 4 (Figure 4d).
To compare the inhibitory mechanism of quercetin and 3,7-
bis-O-POM-Q (4) on cell proliferation, the effects of these com-
pounds on the cell cycle were investigated (Figure 5). Querce-
tin is known to arrest the cell cycle in the S/early G2 phase.[19]
As reported, 12 h after addition of quercetin, the percentage
of cells in the S phase increased with concurrent decrease of
cells in the G0/G1 as well as the G2/M phases (Figure 5b, d),
meaning that the S to G2/M progression was arrested. The
seemingly unrealistic cell cycle arrest by the unstable quercetin
(t1/2 <30 min in cell culture medium, Table 1) 12 h after incuba-
tion can be understood in the context of efficient cellular
uptake of quercetin followed by its intracellular metabolism.
Previously,[10] we observed quercetin and its metabolites (glu-
curonides, sulfates, and methyl derivatives) remaining in the
cytoplasm up to 12 h after incubation, and the sustained effi-
cacious level of quercetin and/or its metabolites inside the cell
must cause cell cycle arrest. In contrast, cells treated with quer-
cetin–POM conjugate 4 showed low populations in the S
phase but an increased percentage of cells in the G0/G1 phase
(Figure 5c, d), which can be attributed to cell cycle arrest in
the G0/G1 phase.
Figure 3. HPLC chromatograms of a) cell culture medium and b) cell lysate
after 12 h of incubation of MCF-7 cells with the quercetin conjugate 3,7-bis-
O-POM-Q (4).
the cell viabilities were estimated using an MTT assay
(Figure 4).
In order to demonstrate the stability of quercetin–POM con-
jugate 4 against extracellular oxidative stress, no antioxidant
additive was used. Under these assay conditions, quercetin did
not inhibit cell proliferation, whereas the 4 showed significant
cytostatic activity in all three cancer cell lines tested (Fig-
In conclusion, we have shown that 3,7-bis-O-POM-Q (4) ex-
hibits remarkable stability in cell culture medium and efficient
cellular uptake. Inside the cell, it is selectively converted into
the mono-hydrolysis product, 3-O-POM-Q (2), but is not hydro-
lyzed further to give the bioac-
tive parent compound, querce-
tin. However, intracellularly lo-
calized 4 and/or 2 exhibit signif-
icant inhibition of cell prolifera-
tion via a different mechanism
compared with that of querce-
tin. Taken together, this result
implies that stable quercetin–
POM conjugate 4 has potential
to serve as an anticancer agent.
Experimental Section
Full details of the synthesis of in-
termediate 5 and 3,7-bis-O-POM-
Q (4) are given in the Supporting
Information along with protocols
for the stability and biological
assays used in this study.
Figure 4. Viability of a) MCF-7, b) HCT116, c) DU 145, and d) HS27 cells treated with quercetin (1) [&] and 3,7-bis-
O-POM-Q (4) [~].
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Figure 5. Cell cycle analysis of MCF-7 cells by propidium iodide (PI) staining and flow cytometry after treatment with a) 1% DMSO, b) 100 mm of quercetin,
c) 30 mm of 3,7-bis-O-POM-Q (4) for 12 h. d) The percentage of cells in each phase of the cell cycle: pre-G1 (&), G0/G1 (&), S (&), G2/M (&).
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This work was supported by a Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korean Ministry of Edu-
cation, Science and Technology (MEST) (grant no. 2009-0071110),
and by a Priority Research Centers Program through the National
Research Foundation of Korea (NRF) also funded by MEST (pro-
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Keywords: cellular uptake · conjugates · cytostatic effects ·
quercetins · stability
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Received: October 14, 2011
Published online on November 18, 2011
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