Quercetin-POC conjugates: Differential stability and bioactivity profiles between breast cancer (MCF-7) and colorectal carcinoma (HCT116) cell lines

Article abstract of DOI:10.1016/j.bmc.2013.01.057

In the course of our ongoing efforts to develop novel quercetin conjugates with enhanced stability profiles, we introduced an isopropyloxycarbonylmethoxy (POC) group to 7-OH and/or 3-OH of quercetin and prepared three novel quercetin conjugates. The querc

Full text of DOI:10.1016/j.bmc.2013.01.057

Bioorganic & Medicinal Chemistry 21 (2013) 1671–1679
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quercetin–POC conjugates: Differential stability and bioactivity
profiles between breast cancer (MCF-7) and colorectal carcinoma
(HCT116) cell lines
Suh Young Cho a, Mi Kyoung Kim a, Kwang-su Park a, Hyunah Choo b, Youhoon Chong a,
⇑
a Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
b Center for Neuro-Medicine, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seoungbuk-gu, Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In the course of our ongoing efforts to develop novel quercetin conjugates with enhanced stability pro-
files, we introduced an isopropyloxycarbonylmethoxy (POC) group to 7-OH and/or 3-OH of quercetin and
prepared three novel quercetin conjugates. The quercetin–POC conjugates were stable up to 96 h in PBS
but slowly hydrolyzed with half-lives of 1–54 h in cell-free culture medium, which is reminiscent of the
stability profiles of the previously reported quercetin–POM (pivaloxymethyl) conjugates. However, the
quercetin–POC conjugates were more susceptible to passive transport, intracellular hydrolysis, and
metabolism in breast cancer (MCF-7) cell line compared with their POM congeners to result in low con-
centration of quercetin in this cell line and thereby low antiproliferative effect. In contrast, upon incuba-
tion with colorectal carcinoma HCT116 cells, the quercetin–POC conjugates were shown to undergo slow
hydrolysis and metabolism to maintain concentrations of the active quercetin species high enough to
exert enhanced cytotoxicity. Taken together, the quercetin–POC conjugates synthesized in this study
exhibited cell type-specific stability as well as bioactivity profiles, which warrants further investigation
into the underlying mechanisms and therapeutic potential.
Received 13 December 2012
Revised 18 January 2013
Accepted 27 January 2013
Available online 4 February 2013
Keywords:
Quercetin
Isopropyloxycarbonylmethoxy (POC)
Conjugate
Stability
Metabolism
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
In the course of our ongoing efforts to develop novel quercetin
conjugates with enhanced stability profiles, we attempted to in-
Quercetin (1, Fig. 1) has various biological properties including
antioxidant,1 antiviral,2,3 antibacterial,4 and anticancer5 activities.
However, quercetin suffers from low stability particularly in aque-
ous medium with high pH. The two phenolic hydroxyl groups of
quercetin, 7-OH and 3-OH, are known to be responsible for meta-
bolic6–8 and chemical9 instability of quercetin, respectively. In our
previous study,10,11 to enhance the physicochemical properties of
quercetin, we have prepared quercetin conjugates by introducing
a POM (pivaloxymethyl) moiety at 3-O and/or 7-O position of quer-
cetin (Fig. 1).
stall an isopropyloxycarbonylmethoxy (POC) group, which has
been successfully exploited in antiviral tenofovir disoproxil fuma-
rate.12 In this study, 7-OH and/or 3-OH of quercetin were blocked
with POC promoiety to provide three novel quercetin conjugates
[7-O-POC-Q (5), 3-O-POC-Q (6) and 3,7-bis-O-POC-Q (7)] (Fig. 2).
Herein, we report regioselective syntheses of novel quercetin–
POC conjugates (5, 6 and 7) and evaluation of their physicochemi-
cal as well as biological properties.
2. Results and discussion
Conjugation of quercetin with a POM promoiety at C7 hydroxyl
group resulted in increased stability as well as enhanced cell
permeability, and the resulting quercetin–POM conjugates 7-O-
POM-Q (2)10 and 3,7-bis-O-POM-Q (4)11 underwent efficient intra-
cellular conversion to quercetin and 3-O-POM-Q (3), respectively.
On the other hand, 3-O-POM-Q (3), obtained by introduction of
POM promoiety at 3-O position of quercetin, exhibited excellent
stability profile but, due to the lack of cell permeability, it failed
to accumulate inside the cell.
2.1. Syntheses of the quercetin–POC conjugates
Syntheses of the quercetin–POC conjugates were accomplished
by nucleophilic substitution of appropriately protected quercetin
derivatives (9, 10 and 14: Schemes 1 and 2) with iodomethyl iso-
propyl carbonate (POC-I). Thus, regioselective preparation of the
protected quercetin derivatives (9,13,18 1014,15,19 and 14) was the
key to syntheses of the quercetin–POC conjugates.
Preparation of 7-O-POC-Q (5) was started with peracetylation of
quercetin by treatment with excess amount of acetic anhydride
and pyridine (Scheme 1). Selective deacetylation of 7-OAc group
⇑
Corresponding author.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.057

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S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
Quercetin (1)
R1 = H, R2 = H
OH
O
3'
OH
7-O-POM-Q (2)
R1
R1 = H, R2
R1 = R2
=
, R2 = H
O
4'
B
R1O
O
C
O
O
3-O-POM-Q (3)
7
=
A
OR2
3
5
O
OH
O
=
3,7-bis-O-POM-Q (4)
O
Figure 1. Structures of quercetin (1) and quercetin–POM conjugates (2, 3 and 4).
OH
OH
OH
OH
O
O
O
O
O
O
HO
O
O
O
OH
O
O
O
OH
OH
7-O-POC-Q (5)
3-O-POC-Q (6)
OH
OH
O
O
O
O
O
O
O
O
O
OH
O
3,7-bis-O-POC-Q (7)
Figure 2. Quercetin–POC conjugates (5, 6 and 7) investigated in this study.
of the resulting quercetin peracetate (8)16,17 was accomplished by
transesterification with PhSH to give the key intermediate 9 (73%
yield),18 which, upon substitution reaction with POC-I followed
by deacetylation, provided the desired 7-O-POC-Q (5) in 91% yield.
The syntheses of 3-O-POC-Q (6) and 3,7-bis-O-POC-Q (7) were
summarized in Scheme 2. The catechol group of quercetin (1)
was protected by reaction with 1,1-dichlorodiphenylmethane at
180 °C to provide 10.19 Alkylation of the key intermediate 10 with
excess amount of POC-I followed by hydrogenolysis in the pres-
ence of Pd/C in a mixture of THF and MeOH provided the desired
product 3,7-bis-O-POC-Q (7) (66% yield). On the other hand, prep-
aration of 3-O-POC-Q (6) required orthogonal protection of the 7-,
30-, and 40-hydroxyl groups through tedious protection-deprotec-
tion chemistry. First, quercetin diphenylmethylketal (10) was per-
acetylated with excess amount of Ac2O in pyridine (93% yield) to
give 11 of which 7-OAc group was selectively deacetylated.18
The free 7-OH group of the resulting intermediate 12 was then
protected with a benzyl group to provide 13 in 69% yield. The
remaining acetyl functionalities in 13 (3-OAc and 5-OAc) were re-
moved by treatment with methanolic ammonia to give the key
intermediate 14 in 90% yield. Alkylation of 14 with POC-I followed
by hydrogenolysis of the benzyl as well as diphenylmethyl protect-
ing groups furnished the desired product 3-O-POC-Q (6) in 61%
combined yield.
OH
OAc
OH
OAc
HO
O
O
AcO
O
a
OH
OAc
2.2. Properties of the quercetin–POC conjugates
OH
OAc O
1
8
2.2.1. Stability
Quercetin (1) is known to undergo oxidative degradation in PBS
buffer (pH 7.4) with a half-life of 10 h.10 Moreover, in oxidizing
environment such as cell culture medium, quercetin is highly
unstable to decompose in less than an hour (t1/2 <0.5 h).10 Querce-
tin’s free hydroxyl groups were known to be responsible for its oxi-
dative decomposition10 and we have been anticipating that
transiently blocking the 3-OH and/or 7-OH of quercetin would in-
crease its stability against the oxidative stress.10,11 Thus, stabilities
of the POC-protected quercetin conjugates (5, 6, and 7) were mea-
sured by HPLC (Fig. 3) in PBS and cell-free cell culture medium
(cDMEM), and their half-lives (t1/2) were summarized in Table 1.
In general, stability profiles of the quercetin–POC conjugates
were almost the same as the previously reported quercetin–POM
conjugates;10,11 the quercetin–POC conjugates (5, 6 and 7), which
showed remarkable stability in PBS with half-lives longer than
b
OH
OAc
OH
OAc
RO
O
HO
O
c, d
OH
OAc
OH
O
OAc O
5
9
O
R=
O
O
Scheme 1. Synthesis of 7-O-POC-Q (5). Reagents and conditions: (a) Ac2o, Pyr,
70 °C; (b) PhSH, NMP, imidazole, 0 °C; (c) POC-I, K2CO3, acetone, rt; (d) NH3/MeOH,
0 °C.

S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
1673
OH
OH
HO
O
OH
OH
1
O
a
Ph
Ph
Ph
O
Ph
Ph
Ph
O
O
O
O
O
HO
O
AcO
O
HO
O
c
b
OH
OAc
OAc
OH
10
O
OAc O
OAc O
11
12
f,g
d
OH
Ph
Ph
Ph
Ph
OH
O
O
O
O
R2O
O
O
BnO
O
BnO
O
f, g
e
OR1
OH
OH
OAc
O
OH
14
O
OAc O
13
6
7
R1
=
, R2 = H
O
O
O
R1 = R2
=
O
O
Scheme 2. Syntheses of 3-O-POC-Q (6) and 3,7-bis-O-POC-Q (7). Reagents and conditions: (a) Ph2CCl2, 180 °C; (b) Ac2O, Pyr, 70 °C; (c) PhSH, NMP, imidazole, 0 °C; (d) BnBr,
K2CO3, acetone, rt; (e) NH3/MeOH, 0 °C; (f) POC-I, K2CO3, acetone/DMF, rt; (g) H2, pd/C, THF/MeOH, rt.
Figure 3. HPLC chromatograms of (a) quercetin (1, Q), (b) 7-O-POC-Q (5), (c) 3-O-POC-Q (6), and (d) 3,7-bis-O-POC-Q (7) in cell-free culture medium (cDMEM) after
incubation for 1 h (a and b), 54 h (c), and 24 h (d).
96 h (Table 1), started to decomposed in cell-free culture medium.
Among the series, 7-O-POC-Q (5) showed fastest decrease in con-
decomposition (Fig. 3a), 3,7-bis-O-POC-Q (7) was not subject to
decomposition but hydrolyzed into 3-O-POC-Q (6) (Fig. 3b). It
should also be pointed out that 3-O-POC-Q (6) was the most stable
quercetin–POC conjugate with high resistance to both hydrolysis
and decomposition (Fig. 3c and d).
centration (t1/2 = 1 h) followed by 3,7-bis-O-POC-Q (7) (t1/2
=
24 h). However, it should be noted that, in comparison with
7-O-POC-Q (5) which showed hydrolysis to quercetin and/or

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S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
Table 1
Table 2
Stability of quercetin (1) and quercetin–POC conjugates (5, 6 and 7)
Permeability of quercetin (1) and quercetin conjugates (5–7) across artificial
membranesa
Half-life, t1/2 (h)
Compd
LogPe (cm/s)
Quercetin
(1)
7-O-POC-Q
(5)
3-O-POC-Q
(6)
3,7-Bis-O-POC-Q
(7)
1 h
5 h
pH 7.4
(PBS)
cDMEMa
10
>96
1
>96
54
>96
24b
Quercetin (1)
ꢀ4.14 0.03
ꢀ4.13 0.02
ꢀ4.56 0.02
—
ꢀ4.44 0.05
ꢀ4.38 0.03
ꢀ5.01 0.24
—
7-O-POC-Q (5)
3-O-POC-Q (6)
3,7-Bis-O-POC-Q (7)b
<0.5
a
cDMEM: complete Dulbecco’s modified Eagle’s medium.
Half-life for hydrolysis to 3-O-POC-Q (6).
b
a
Assay was performed in triplicate.
b
Assay was not performed due to low solubility of 7 in PBS at the concentration
M).
for PAMPA (25
l
2.2.2. Solubility
passive permeability of 3-O-POC-Q (6) is worth mentioning because
the previously reported 3-O-POM-Q (3) was not permeable at all
through the artificial membrane.10
To evaluate solubility of the quercetin–POC conjugates (5, 6 and
7), forward light scattering was measured when a laser beam is di-
rected through the solutions of the quercetin conjugates in PBS and
cDMEM (Fig. 4).
Presumably due to the similar lipophilic nature of POM and
POC, solubility profiles of the quercetin–POC conjugates were also
reminiscent of those of the POM conjugates;10,11 the quercetin–
POC conjugates such as 5 and 7 showed relatively low aqueous sol-
ubility in PBS whereas 3-O-POC-Q (6) was highly soluble even at
high concentrations. However, all the quercetin conjugates were
2.2.4. Metabolite analysis
Combining profiles of stability, solubility, and membrane per-
meability, the quercetin–POC conjugates were anticipated to safely
cross the cell membrane. Once localized inside the cell, the querce-
tin conjugates were supposed to undergo hydrolytic cleavage to re-
lease quercetin. Also, in the cellular milieu, the released quercetin
would be subject to metabolic changes to the corresponding glucu-
ronide (Q-Glu), methyl ether (Q-Me), and sulfate (Q-Sul).24 For this
reason, analysis of the cellular contents was performed, which was
expected to provide information about intracellular metabolism of
the quercetin conjugates. Thus, two different human cancer cell
lines, MCF-7 (breast cancer) and HCT116 (colorectal carcinoma),
were treated with the quercetin conjugates (5–7) and, after incu-
bation for 1, 3, 6, and 12 h, cells were harvested and lysed. The cell
lysates were then analyzed by HPLC to identify the remaining
quercetin conjugates, its hydrolysis product (quercetin), and/or
quercetin metabolites, and the results were summarized in Table 3.
In breast cancer (MCF-7) cell line, all the quercetin conjugates
were shown to undergo facile conversion to quercetin as well as
the quercetin metabolites and, after incubation for 12 h, neither
quercetin nor quercetin metabolite was observed from the cell ly-
sate. As already reported,24 the quercetin glucuronide (Q-Glu) was
identified as the major metabolite in all quercetin conjugates,
which was shown to increase until 6 h of incubation. Interestingly,
3-O-POC-Q (6), which was highly resistant to hydrolysis to querce-
tin in cell-free culture medium (t1/2 = 54 h, Table 1), was also trans-
formed into quercetin with a half-life of 3 h, and this result clearly
indicates that cellular uptake followed by intracellular hydrolysis
of 6 was underway in MCF-7 cell line. By the same token,
completely soluble up to 100
(Fig. 4b).
lM concentration in cDMEM
2.2.3. Membrane permeability
Besides the aqueous solubility of a drug substance, its permeabil-
ity is a second fundamental parameter for controlling oral drug
absorption. One of the in vitro methods that can be used to estimate
the drug permeability is the parallel artificial membrane permeabil-
ity assay (PAMPA) which utilizes an artificial membrane immobi-
lized between a donor and an acceptor compartment.20 With
passively transported drugs, an excellent correlation between the
flux across the artificial membrane and the extent of absorption
has been demonstrated.21–23 Thus, passive transport of quercetin
(1) and its POC conjugates (5–7) were evaluated by PAMPA. Unfortu-
nately, due to low solubility in PBS (Table 1), 3,7-bis-O-POC-Q (7)
was not amenable to PAMPA. Briefly, quercetin (1) and the POC con-
jugates (5 and 6) were dissolved in donor plates and then the donor
and acceptor plates were incubated together for 1–5 h after which,
the plates were separated, and the concentration of compound in
the accepter compartment was determined by UV340 measurement.
The PAMPA data summarized in Table 2 shows that, in terms of pas-
sive permeability, 7-O-POC-Q (5) is as efficient as quercetin whereas
3-O-POC-Q (6) is less permeable than others. Nevertheless, the
a
b
60000
60000
1
5
5
6
50000
50000
6
7
7
40000
30000
20000
10000
0
40000
30000
20000
10000
0
0.5
5
25
50 100 200 300
0.5
5
25
50 100 200 300
concentration (µM)
concentration (µM)
Figure 4. Aqueous solubility of quercetin (1) and quercetin–POC conjugates (5, 6 and 7) in (a) PBS and (b) cDMEM measured by forward light scattering intensity. Quercetin’s
solubility was evaluated only in PBS due to its low stability in cell culture medium.

S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
1675
Table 3
Quantitative analysis of quercetin (1), quercetin conjugates (5–7), and quercetin metabolites (Q-Met)a in cell lysates after incubation of the quercetin conjugates (5–7) in breast
cancer (MCF-7) and colorectal carcinoma (HCT116) cell lines
Q-Conj.
Contents in cell lysates
Relative concentrationb
MCF-7
HCT116
1 hc
3 h
6 h
12 h
1 h
3 h
6 h
12 h
d
7-O-POC-Q (5)
7-O-POC-Q (5)
Quercetin (1)
Q-Glu
Q-Me
Q-Sul
3-O-POC-Q (6)
Quercetin (1)
Q-Glu
51
24
—
—
—
92
3
—
6
2
1
23
8
2
—
65
4
1
34
11
—
—
1
76
—
—
—
47
—
18
13
—
—
2
71
—
—
—
20
—
12
4
—
—
1
65
—
—
—
3
11
21
6
—
49
26
7
2
6
—
3
24
9
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
80
—
—
—
64
—
34
—
—
—
3-O-POC-Q (6)
4
21
27
2
1
—
1
6
22
1
Q-Sul
—
3,7-Bis-O-POC-Q (7)
3,7-Bis-O-POC-Q (7)
7-O-POC-Q (5)
3-O-POC-Q (6)
Quercetin (1)
Q-Glu
25
14
8
13
2
—
69
40
—
—
63
—
—
—
—
Q-Sul
—
1
—
—
a
b
c
Q-Glu = quercetin glucuronide, Q-Me = methyl quercetin, Q-Sul = quercetin sulfate.
Percentage (%) of each content in cell lysate calculated from integrated HPLC peak area.
Incubation time.
Not detected.
d
hydrolysis of 3,7-bis-O-POC-Q (7) into 7-O-POC-Q (5) and 3-O-
POC-Q (6), which was unamenable in cell-free culture medium
(t1/2 = 24 h for transformation into 6 only, Table 1), could also be
explained by the intracellular event. At this stage, it should be
pointed out that, in the same breast cancer (MCF-7) cell line, the
quercetin–POC conjugates were more susceptible to intracellular
hydrolysis followed by metabolism compared with their POM
congeners.10,11 Also noteworthy is that 3-O-POC-Q (6) underwent
facile hydrolysis and metabolic changes while neither of those
events has not been observed in the case of quercetin-3-O-POM
conjugates such as 3-O-POM-Q (3)10 and 3,7-bis-O-POM-Q (4).11
In sharp contrast to this, the quercetin–POC conjugates were
shown to undergo slow hydrolysis and low, if any, metabolism in
the presence of the colorectal carcinoma (HCT116) cells. In addi-
tion, contents of the lysed HCT116 cells incubated with the querce-
tin–POC conjugates were the same as those obtained from the
cell-free culture medium (Table 1). Thus, 7-O-POC-Q (5) under-
went hydrolysis to quercetin leaving no trace of the quercetin
metabolites. As was the case with the cell-free culture medium,
3-O-POC-Q (6) was stable up to 12 h and quercetin was not ob-
served. Likewise, after incubation with 3,7-bis-O-POC-Q (7), the ly-
sates of the HCT116 cells were shown to contain 7 and 3-O-POC-Q
(6), the only hydrolysis product in the cell-free culture medium
(Table 1). Taken together, unlike MCF-7, HCT116 cells did not affect
the hydrolysis pattern and metabolism of the quercetin–POC con-
jugates but expedited hydrolysis of 3,7-bis-O-POC-Q (7) (t1/2 ꢁ 3 h)
compared with that in cell-free conditions (t1/2 = 24 h, Table 1),
which indicates a series of intercellular and intracellular events
surrounding 7 and its hydrolysis product 6.
was chosen. As the quercetin conjugates showed different profiles
of hydrolysis and metabolism in breast cancer (MCF-7) and colo-
rectal carcinoma (HCT116) cell lines, comparison of the cytotoxic-
ity of the quercetin conjugates in those two cancer cell lines was
also expected to provide correlation between stability of the quer-
cetin conjugates and their bioactivity. Thus, after treating MCF-7
and HCT116 cell lines with quercetin and its POC-conjugates, via-
bility of these cells were measured, and the results were summa-
rized in Figure 5.
Interestingly, the quercetin conjugates showed cell line-depen-
dent cytotoxicity; in MCF-7 cell line, the quercetin conjugates were
as cytotoxic as quercetin (Fig. 5a) while 7-O-POC-Q (5) and 3,7-bis-
O-POC-Q (7) were found to be more cytotoxic than others in
HCT116 cell line (Fig. 5b). In MCF-7 cell line, all of the quercetin
conjugates underwent fast hydrolysis, metabolism, and decompo-
sition to result in low concentrations of the active species such
as quercetin and/or quercetin conjugates, which is in good accor-
dance with their low cytotoxic effects in this cell line. In contrast,
concentration of quercetin and/or quercetin conjugates was kept
relatively high in HCT116 cell line due to slow hydrolysis as well
as resistance to metabolism, and the enhanced cytotoxicity of 7-
O-POC-Q (5) and 3,7-bis-O-POC-Q (7) compared with quercetin
might be attributed to their stability in this cell line. However, it
should be noted that 3,7-bis-O-POC-Q (7) showed higher cytotox-
icity than 7-O-POC-Q (5) (Fig. 5b) even though the conjugate 7
did not produce quercetin (Table 3). Also noteworthy is that the
most stable quercetin conjugate 3-O-POC-Q (6), the only hydrolysis
product of 7 in HCT116 cell line (Table 3), did not show cytotoxic
effect (Fig. 5b). Taken together, these results clearly show that
3,7-bis-O-POC-Q (7) has intrinsic cytotoxicity which is higher than
that of 7-O-POC-Q (5) or quercetin.
Overall, the quercetin–POC conjugates turned out to have dif-
ferential susceptibility to hydrolysis as well as metabolism by
MCF-7 and HCT116 cell lines, which was not the case in the quer-
cetin–POM series.
3. Conclusion
2.2.5. Cytotoxicity
In this study, suboptimal stability of quercetin was tackled by
novel quercetin conjugates which were prepared by regioselective
introduction of the isopropyloxycarbonylmethoxy (POC) group at
3-OH and/or 7-OH of quercetin. Compared with quercetin, the
quercetin–POC conjugates showed significantly enhanced stability
in PBS and cell culture medium. In comparison with the previously
Previously described results obtained from PAMPA and cell ly-
sate analysis all together suggest possible cellular uptake followed
by intracellular hydrolysis of the quercetin–POC conjugates, and
evaluation of the bioactivity of the quercetin conjugates in cell le-
vel would support this hypothesis. Among various bioactivities
associated with quercetin, cytotoxicity against cancer cell lines
reported quercetin–POM conjugates, the
quercetin–POC

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S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
a
140
120
100
80
140
b
120
100
80
60
60
1
5
6
7
1
5
6
7
40
40
20
20
0
0
1
5
10
50
100
1
5
10
50
100
concentration (µM)
concentration (µM)
Figure 5. Viabilities of (a) MCF-7 and (b) HCT116 cell lines treated with quercetin (1), 7-O-POC-Q (5), 3-O-POC-Q (6), and 3,7-bis-O-POC-Q (7).
conjugates showed characteristic profiles in passive transport,
intracellular hydrolysis, metabolism, and bioactivity. In particular,
the quercetin–POC conjugates were more susceptible to passive
transport, intracellular hydrolysis, and metabolism in breast cancer
(MCF-7) cell line compared with their POM congeners to result in
low antiproliferative effect against this cancer cell line. In contrast,
upon incubation with colorectal carcinoma (HCT116) cells, the
quercetin conjugates such as 7-O-POC-Q (5) and 3,7-bis-O-POC-Q
(7) were shown to undergo slow hydrolysis and metabolism to
maintain concentrations of the quercetin conjugates as well as
their hydrolyzed products high enough to exert enhanced
cytotoxicity.
in water containing 0.1% formic acid (8–18 min), 35% acetonitrile
in water containing 0.1% formic acid (18–25 min), 35–80% acetoni-
trile in water containing 0.1% formic acid (25–40 min), 80–100%
acetonitrile in water containing 0.1% formic acid (40–45 min),
100% acetonitrile in water containing 0.1% formic acid
(45–50 min), 100–20% acetonitrile in water containing 0.1% formic
acid (50–54 min) and 20% acetonitrile in water containing 0.1%
formic acid (54–60 min). Flow rate was 1 mL/min. UV was detected
at 340 nm.
4.2. Syntheses of the quercetin–POC conjugates
Taken together, the quercetin–POC conjugates synthesized in
this study exhibited cell type-specific stability as well as bioactiv-
ity profiles, which warrants further investigation into the underly-
ing mechanisms and therapeutic potential.
4.2.1. Acetic acid 3,5-diacetoxy-2-(3,4-diacetoxy-phenyl)-4-oxo-
4H-chromen-7-yl ester (8)16,17
To a solution of quercetin (1) (5 g, 16.5 mmol) in pyridine
(35 mL) was added acetic anhydride (12.5 mL, 132 mmol), and
the reaction mixture was stirred at rt. The reaction was monitored
by TLC. After starting material was consumed, the reaction mixture
was concentrated under reduced pressure. The crude compound
was purified by column chromatography on silica gel (4:1:1 = hex-
ane/acetone/CH2Cl2) as eluent and was recrystallized from CH2Cl2
to afford 8 (4.5 g, 8.8 mmol, 54% yield) as off-white powder; mp
190 °C (rec. CH2Cl2) (lit.16 mp 190 °C); 1H NMR (400 MHz, CDCl3)
d (ppm) 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); 13C NMR (100 MHz, DMSO-d6) d (ppm)
169.1, 168.6, 168.2, 168.0, 167.8, 167.6, 156.2, 154.2, 153.2,
149.3, 144.3, 142.0, 133.0, 126.9, 126.5, 124.3, 123.6, 114.5,
113.9, 109.9, 20.7, 20.6, 20.2, 20.17, 20.0; HR-FABMS (m/z): Found:
512.1030 [M+H]+; Calcd for C25H20O12: 511.0951.
4. Experimental
4.1. Materials and general methods
All chemicals were purchased from Sigma–Aldrich. Dulbecco’s
modified eagle medium (DMEM), penicillin, streptomycin, and fe-
tal bovine serum (FBS) were purchased from Invitrogen. The pre-
coated PAMPA plate were manufactured by BD Bioscience
Discovery Labware (Bedford, MA) using a polyvinylidene fluoride
(PVDF) 96-well filter plate with 0.4 lm pore size. The 100 mm cell
culture plate was purchased from Dishes Biousing™. Nuclear mag-
netic resonance spectra were recorded on a Bruker 400 AMX spec-
trometer (Karlsruhe, Germany) at 400 MHz for 1H NMR and
100 MHz for 13C NMR with tetramethylsilane as the internal stan-
dard. Chemical shifts (d) are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br s (broad singlet). Coupling
constants are reported in hertz. The chemical shifts are reported as
parts per million (d) relative to the solvent peak. TLC was per-
formed on silica gel 60 F254 purchased from Merck. Column chro-
matography was performed using silica gel-60 (220–440 mesh)
for flash chromatography. Mass spectrometric data (MS) were ob-
tained by electronspray ionization (ESI). High resolution fast atom
bombardment (FAB) mass spectra were recorded using a JEOL JMS-
700 mass spectrometer at the Daegu center of KBSI, Korea. All
tested compounds were P95% purity, as determined by reverse
phase HPLC. HPLC was performed on Agilent 1050 (Hewlett–Pack-
ard) equipment with variable wavelength (VW) UV detector using
Polaris 5, C18-A 250 ꢂ 4.6 mm (Varian) column. Analytical condi-
tions were as follows: gradient used was 20–25% acetonitrile in
water containing 0.1% formic acid (0–8 min), 25–35% acetonitrile
4.2.2. Acetic acid 2-acetoxy-4-(3,5-diacetoxy-7-hydroxy-4-oxo-
4H-chromen-2-yl)-phenyl ester (9)13,18
To a stirred mixture of 8 (3 g, 5.85 mmol) and imidazole (80 mg,
1.17 mmol) in NMP (30 mL) was slowly added PhSH (0.48 mL,
4.69 mmol) at 0 °C. The reaction mixture was stirred for 2 h at rt.
The mixture was diluted with EtOAc and washed with 2 N HCl.
The organic layer was concentrated under reduced pressure and
dried over MgSO4. The crude product was purified by column chro-
matography on silica gel (2:1:1 = hexane/acetone/EtOAc) as eluent
and was recrystallized from CH2Cl2 to give 9 (2 g, 4.25 mmol, 73%
yield) as white powder; mp 204 °C (rec. CH2Cl2); 1H NMR
(400 MHz, DMSO-d6) d (ppm) 11.33 (s, 1H), 7.83–7.80 (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); 13C NMR (100 MHz, DMSO-d6) d (ppm); 169.0, 168.8,
168.3, 168.1, 168.0, 162.9, 157.6, 152.4, 150.2, 144.2, 142.2,
132.8, 127.4, 126.5, 124.5, 123.6, 109.4, 109.1, 101.0, 20.9, 20.5,

S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
1677
20.4, 20.3; HR-FABMS (m/z): Found: 471.0926 [M+H]+; Calcd for
23H18O11: 470.0847.
column chromatography on silica gel (hexane/EtOAc = 1:1) to give
12 (1 g, 1.8 mmol, 90% yield) as pale yellow oil; 1H NMR (400 MHz,
CDCl3) d (ppm) 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) d (ppm)
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:
549.32 [MꢀH]ꢀ; Calcd for C32H22O9: 550.13.
C
4.2.3. Carbonic acid 2-(3,4-dihydroxy-phenyl)-3,5-dihydroxy-4-
oxo-4H-chromen-7-yloxymethyl ester isopropyl ester (5)
To a solution of 9 (150 mg, 0.32 mmol) in acetone (8 mL) was
added K2CO3 (88 mg, 0.64 mmol) and POC iodide (234 mg,
0.96 mmol). The reaction mixture was stirred for 4 h at rt, filtered,
and concentrated under reduced pressure to give pale yellow syr-
up, which was used for the next step without further purification.
The degassed suspension of the quercetin conjugate obtained
above and Pd/C (15 mg) in a mixture of THF (3 mL) and MeOH
(3 mL), under an atmosphere of hydrogen gas (balloon), was vigor-
ously stirred for 12 h at rt. The reaction mixture was filtered
through a short celite pad and purified by column chromatography
on silica gel (hexane/EtOAc = 1:1) to afford 5 (122 mg, 0.29 mmol,
91% yield) as yellow powder; 1H NMR (400 MHz, DMSO-d6) d
(ppm) 12.63 (s, 1H), 7.73 (d, J = 2.0 Hz, 1H), 7.60 (dd, J = 8.5,
2.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.47
(d, J = 2.1 Hz, 1H), 5.95 (s, 2H), 4.87–4.80 (m, 1H), 1.24 (d,
J = 6.2 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) d (ppm) 176.4,
161.5, 160.9, 156.0, 153.2, 148.3, 148.1, 145.5, 136.6, 122.1,
120.6, 116.0, 115.6, 105.5, 98.7, 94.1, 87.2, 73.1, 21.7; LC/MS (ESI)
m/z Found: 417.20 [MꢀH]ꢀ; Calcd for C20H18O10: 418.09.
4.2.7. Acetic acid 5-acetoxy-7-benzyloxy-2-(2,2-diphenyl-
benzo[1,3]dioxol-5-yl)-4-oxo-4H-chro-men-3-yl ester (13)
To a solution of 12 (1 g, 1.8 mmol) in acetone (20 mL) was
added K2CO3 (251 mg, 1.8 mmol) and BnBr (0.32 mL, 2.7 mmol).
The reaction mixture was stirred for 12 h at rt. After filtration,
the filterate was concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel (hexane/
EtOAc = 2:1) to give 13 (800 mg, 1.25 mmol, 69% yield) as pale yel-
low oil; 1H NMR (400 MHz, CDCl3) d (ppm) 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) d (ppm) 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:
641.34 [M+H]+; Calcd for C39H28O9: 640.17.
4.2.4. 2-(2,2-Diphenyl-benzo[1,3]dioxol-5-yl)-3,5,7-trihydroxy-
chromen-4-one (10)14,15,19
A
mixture of quercetin (1) (5 g, 14.8 mmol) and dic-
4.2.8. 7-Benzyloxy-2-(2,2-diphenyl-benzo[1,3]dioxol-5-yl)-3,5-
dihydroxy-chromen-4-one (14)
hlorodiphenylmethane (8.5 mL, 44.3 mmol) was stirred for
30 min at 180 °C. The reaction mixture was taken with CHCl3 and
concentrated under reduced pressure. The residue was purified
by column chromatography (hexane/EtOAc = 4:1) on silica gel as
eluent and was recrystallized from CHCl3 to afford 10 (2.4 g,
5.2 mmol, 35% yield) as yellow powder; mp 222–224 °C (rec.
CHCl3) (lit.14 mp 222–224 °C); 1H NMR (400 MHz, acetone-d6) d
(ppm) 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);
13C NMR (100 MHz, DMSO-d6) d (ppm) 176.1, 164.2, 160.8, 156.3,
147.7, 146.8, 145.6, 139.5, 136.5, 129.5, 128.7, 125.8, 125.3,
123.1, 117.1, 108.9, 107.9, 101.1, 98.4, 93.7; HR-FABMS (m/z):
Found: 467.1135 [M+H]+; Calcd for C28H18O7: 466.1056.
A mixture of 13 (800 mg, 1.25 mmol) in sat’d NH3 in MeOH
(12 mL) at 0 °C was stirred for 2 h at rt. After concentration under
reduced pressure, the residue was purified by column chromatog-
raphy on silica gel (hexane/CH2Cl2/EtOAc = 6:2:1) to give 14
(624 mg, 1.12 mmol, 90% yield) as yellow powder; 1H NMR
(400 MHz, CDCl3) d (ppm) 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) d (ppm) 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: 557.32 [M+H]+; Calcd for C35H24O7:
556.15.
4.2.5. Acetic acid 3,5-diacetoxy-2-(2,2-diphenyl-
benzo[1,3]dioxol-5-yl)-4-oxo-4H-chromen-7-yl ester (11)
To a solution of 10 (1 g, 2.14 mmol) in anhydrous pyridine
(10 mL) was added acetic anhydride (1 mL, 10.7 mmol) at rt. The
reaction mixture was stirred for 6 h at 70 °C. After concentration,
the crude product was purified by column chromatography (hex-
ane/EtOAc = 2:1) on silica gel to give 11 (1.2 g, 2.0 mmol, 93% yield)
as white powder; 1H NMR (500 MHz, CDCl3) d (ppm) 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) d (ppm) 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: 593.31 [M+H]+; Calcd for
4.2.9. Carbonic acid 2-(3,4-dihydroxy-phenyl)-5,7-dihydro-xy-4-
oxo-4H-chromen-3-yloxymethyl ester isopropyl ester (6)
To a solution of 14 (200 mg, 0.36 mmol) in a mixture of acetone
(7 mL) and DMF (4 mL) was added K2CO3 (55 mg, 0.4 mmol) and
POC iodide (132 mg, 0.54 mmol). The reaction mixture was stirred
for 12 h at rt, filtered, and concentrated under reduced pressure to
give pale yellow syrup, which was used for the next step without
further purification. The degassed suspension of the quercetin con-
jugate obtained above and Pd/C (15 mg) in a mixture of THF (3 mL)
and MeOH (3 mL), under an atmosphere of hydrogen gas (balloon),
was vigorously stirred for 12 h at rt. The reaction mixture was fil-
tered through a short celite pad and purified by column chroma-
tography on silica gel (hexane/CH2Cl2/EtOAc = 2:1:1) to afford 6
(92 mg, 0.22 mmol, 61% yield) as yellow powder; 1H NMR
(400 MHz, acetone-d6) d (ppm) 12.62 (s, 1H), 7.63 (d, J = 2.2 Hz,
1H), 7.55 (dd, J = 8.5, 2.2 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 6.52 (d,
J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 5.72 (s, 2H), 4.58 (septet,
J = 6.3 Hz, 1H), 1.08 (s, 3H), 1.06 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) d (ppm) 177.2, 164.8, 161.6, 157.1, 156.8, 153.5, 149.2,
145.5, 134.6, 121.6, 120.7, 116.1, 115.8, 104.3, 99.2, 94.1, 92.1,
72.4, 21.4; LC/MS (ESI) m/z Found: 417.11 [MꢀH]ꢀ; Calcd for
C34H24O10: 592.14.
4.2.6. Acetic acid 5-acetoxy-7-hydroxy-2-(2-methyl-2-phenyl-
benzo[1,3]dioxol-5-yl)-4-oxo-4H-chromen-3-yl ester (12)
To a stirred mixture of 11 (1.2 g, 2.0 mmol) and imidazole
(27 mg, 0.4 mmol) in NMP (24 mL) was slowly added PhSH
(0.16 mL, 1.62 mmol) at 0 °C. The reaction mixture was stirred
for 2 h at rt. The mixture was diluted with EtOAc and washed with
2 N HCl. The organic layer was concentrated under reduced pres-
sure and dried over MgSO4. The crude product was purified by
C20H18O10: 418.09.

1678
S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
4.2.10. Carbonic acid 2-(3,4-dihydroxy-phenyl)-5-hydroxy-7-
isopropoxycarbonyloxymethoxy-4-oxo-4H-chrom-en-3-yl-
oxymethyl ester isopropyl ester (7)
dissolved in 95% PBS buffer solution (5% DMSO) to final concen-
trations of 25 M. The solutions were added to the wells
(300 L/well) of the donor plates, and PBS buffer was added to
the wells (200 L/well) of the acceptor plates. The acceptor plate
was placed on top of the donor plate, and the plate assembly was
incubated at rt for 1 and 5 h. After incubation, concentration of
the test compound in the acceptor, donor and reference wells
l
l
To a solution of 10 (232 mg, 0.32 mmol) in acetone (8 mL) was
added K2CO3 (88 mg, 0.64 mmol) and POC iodide (234 mg,
0.96 mmol). The reaction mixture was stirred for 4 h at rt, filtered,
and concentrated under reduced pressure to give pale yellow syrup,
which was used for the next step without further purification. The
degassed suspension of quercetin conjugate obtained above and
Pd/C (15 mg) in a mixture of THF (3 mL) and MeOH (3 mL), under
an atmosphere of hydrogen gas (balloon), was vigorously stirred
for 12 h at rt. The reaction mixture was filtered through a short celite
pad and purified by column chromatography on silica gel (hexane/
EtOAc = 1:1) to afford 7 (113 mg, 0.21 mmol, 66% yield) as yellow
powder; 1H NMR (400 MHz, CDCl3) d (ppm) 12.36 (s, 1H), 7.83 (d,
1.8 Hz, 1H), 7.54 (dd, J = 8.5, 1.8 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H),
6.64 (d, J = 2.1 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 4.93–5.03 (m, 1H),
4.54–4.64 (m, 1H), 1.33 (d, J = 6.3 Hz, 6H), 1.09 (d, J = 6.3 Hz, 6H);
13C NMR (100 MHz, DMSO-d6) d (ppm) 177.5, 162.0, 161.4, 157.8,
156.4, 153.5, 153.2, 149.5, 145.6, 135.0, 121.7, 120.5, 116.2, 115.8,
106.5, 99.5, 94.6, 90.1, 87.2, 73.1, 72.4, 21.7, 21.5; LC/MS (ESI) m/z
Found: 533.33 [MꢀH]ꢀ; Calcd for C25H26O13: 534.14.
l
was determined by transferring 100 lL of solution from each well
to a new 96 well plate and quantitating using the UV plate read-
er. The effective permeability coefficients Pe (cm/s) was calculated
using the following published equation:25
h
i
CAðtÞ
ꢀln 1 ꢀ
Cequilbium
CDðtÞ ꢂ VD þ CAðtÞ ꢂ VA
VD þ VA
Pe ¼
where Cequilibrium
¼
1
1
A
A ꢂ ðV þ V Þ ꢂ t
D
where A is the filter area (0.3 cm2), t is the incubation time (s),
VA and VD are, respectively, the volumes in the acceptor (0.2 mL)
and the donor (0.3 mL) wells, CA(t) and CD(t) are, respectively, the
concentration of the compound (mM) in the acceptor and donor
wells at time t.
4.6. Metabolite analysis
4.3. Stability test
Each of MCF-7 and HCT116 cell was seeded in tissue-cultured
COSTAR clear bottom six-well plates (2 ꢂ 105 cells/well) and incu-
bated for 24 h (37 °C, 5% CO2). After addition of the quercetin–POC
4.3.1. Stability in PBS buffer
The synthesized quercetin–POC conjugates (5, 6 and 7) (50
were added to PBS buffer (pH 7.4) and incubated at 37 °C. At different
time points (0, 1, 3, 6, 12, 24, 48, 96 h), an aliquot (100 L) of the incu-
lM)
conjugates (30 lM) to each well, the plates were incubated (37 °C,
l
5% CO2). At each time point (1, 3, 6 and 12 h), the cells were
washed by PBS, trypsinized, and sonicated with Vibra-Cell VCX-
130 (SONICS). After filtration, samples were analyzed by HPLC
(Agilent) equipped with a C-18 reverse phase column; flow rate,
1 mL/min; detection, UV 340 nm; mobile phase, 0–8 min (20–25%
aqueous acetonitrile and 0.1% formic acid), 8–18 min (25–35%
aqueous acetonitrile and 0.1% formic acid), 18–25 min (35% aque-
ous acetonitrile and 0.1% formic acid), 25–40 min (35–80% aqueous
acetonitrile and 0.1% formic acid), 40–45 min (80–100% aqueous
acetonitrile and 0.1% formic acid), 45–50 min (100% aqueous ace-
tonitrile and 0.1% formic acid), 50–54 min (100–20% aqueous ace-
tonitrile and 0.1% formic acid) and 54–60 min (20% aqueous
acetonitrile and 0.1% formic acid). Each peak was manually col-
lected, lyophilized, and analyzed by mass spectrometry (MALDI-
TOF, Bruker Daltonics).
bation mixture was taken out and the component were analyzed by
HPLC equipped with a C-18 reverse phase column; flow rate, 1 mL/
min; detection, UV 340 nm; mobile phase, 0–8 min (20–25% aqueous
acetonitrile and 0.1% formic acid), 8–18 min (25–35% aqueous aceto-
nitrile and 0.1% formic acid), 18–25 min (35% aqueous acetonitrile
and 0.1% formic acid), 25–40 min (35–80% aqueous acetonitrile and
0.1% formic acid), 40–45 min (80–100% aqueous acetonitrile and
0.1% formic acid), 45–50 min (100% aqueous acetonitrile and 0.1% for-
mic acid), 50–54 min (100–20% aqueous acetonitrile and 0.1% formic
acid) and 54–60 min (20% aqueous acetonitrile and 0.1% formic acid).
4.3.2. Stability in DMEM cell culture medium
Quercetin–POC conjugates (5, 6 and 7) were dissolved in culture
medium to a final concentration of 50 lM from a 10 mM stock solu-
tionin DMSO. The solutionswereincubatedat37 °C. Themediawere
collected at different time points (0, 1, 3, 6, 12, 24, 48, 96 h), vortexed
and analyzed by HPLC under the same analysis conditions described
above.
4.7. Cytotoxicity
Cells (MCF-7 and HCT116) were seeded (5 ꢂ 103 cells per well)
in tissue-cultured COSTAR clear bottom 96-well plate in cDMEM
and incubated for 1 day (37 °C, 5% CO2). Prepared solutions of
quercetin (1) and quercetin–POC conjugates (5, 6 and 7) in 1%
DMSO were diluted into 6 different concentrations (1, 5, 10, 50,
100, 250 lM) and added to the media. After 12 h, cell viability
was estimated by MTT assay. Every assay was repeated three
times.
4.4. Solubility test
Stock solution of the quercetin conjugates were prepared at 0.05,
0.5, 2.5, 5, 10 mM in 1–3% DMSO, and then serially diluted with
either PBS (pH 7.4, 97–99%) or cDMEM [DMEM with 10% fetal bovine
serum (FBS)], resulting final concentrations of 0.5, 5, 25, 50, 100, 200
(2% DMSO) and 300
each 96 well plate was set to be 100
l
M (3% DMSO). Volume of the test compound in
l
L, and the solubility was mea-
sured by the NEPHELOstar laser based microplate nephelometer
which checks the solubility of compounds by measuring forward
light scatter in microplates. All raw data were processed using the
BMG LABTECH NEPHELOstar Galaxy Evaluation software.
Acknowledgments
This research was supported by Priority Research Centers Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2012-0006686), by a Grant of the Korean Health Technology
R&D project, Ministry of Health & Welfare, Republic of Korea
(A111698), and by a Grant from Next Generation Bio-Green 21
PJ007982 (Korea Rural Development Administration).
4.5. Membrane permeability assay [PAMPA (parallel artificial
membrane permeability assay)]
The precoated PAMPA plate was warmed to rt for 30 min.
Quercetin (1) and quercetin–POC conjugates (5, 6 and 7) were

S. Y. Cho et al. / Bioorg. Med. Chem. 21 (2013) 1671–1679
1679
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