Synthesis of methylated quercetin derivatives and their reversal activities on P-gp- and BCRP-mediated multidrug resistance tumour cells

Article abstract of DOI:10.1016/j.ejmech.2012.05.026

Three methylated quercetins and a series of O-3 substituted 5,7,3′,4′-tetra-O-methylated quercetin derivatives have been synthesized and evaluated on the modulating activity of P-gp, BCRP and MRP1 in cancer cell lines. Compound 17 (with a 2-((4-methoxybenzoyl)oxy)ethyl at O-3) is the most potent P-gp modulator. Three derivatives, compound 9 (3,7,3′,4′-tetra-O-methylated quercetin), compound 14 (with a 2-((3-oxo-3-(3,4,5trimethoxyphenyl)prop-1-en-1-yl)oxy)ethyl at O-3) and compound 17, consistently exhibited promising BCRP-modulating activity. Interestingly, compound 17 was found to be equipotent against both P-gp and BCRP. Importantly, these synthetic quercetin derivatives did not exhibit any inherent cytotoxicity to cancer cell lines or normal mouse fibroblast cell lines. These quercetin derivatives can be employed as safe and effective modulators of P-gp- or BCRP-mediated drug resistance in cancer.

Full text of DOI:10.1016/j.ejmech.2012.05.026

European Journal of Medicinal Chemistry 54 (2012) 413e422
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of methylated quercetin derivatives and their reversal activities
on P-gp- and BCRP-mediated multidrug resistance tumour cells
,
,c,
Jian Yuan a 1, Iris L.K. Wong b 1, Tao Jiang a, Si Wen Wang a, Tao Liu a, Bin Jin Wen a,
Larry M.C. Chow b
, Biao Wan Sheng
,
c
,
a,
*
**
a Key Laboratory of Marine Drugs, Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology,
School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
b Department of Applied Biology and Chemical Technology and the State Key Laboratory for Chirosciences, The Hong Kong Polytechnic University,
Hung Hom, Hong Kong SAR, China
c State Key Laboratory in Chinese Medicine and Molecular Pharmacology, Shenzhen, China
a r t i c l e i n f o
a b s t r a c t
Three methylated quercetins and a series of O-3 substituted 5,7,30,40-tetra-O-methylated quercetin deriv-
atives have been synthesized and evaluated on the modulating activity of P-gp, BCRP and MRP1 in cancer
cell lines. Compound 17 (with a 2-((4-methoxybenzoyl)oxy)ethylat O-3) is the most potent P-gp modulator.
Three derivatives, compound 9 (3,7,30,40-tetra-O-methylated quercetin), compound 14 (with a 2-((3-oxo-3-
(3,4,5trimethoxyphenyl)prop-1-en-1-yl)oxy)ethyl at O-3) and compound 17, consistently exhibited
promising BCRP-modulating activity. Interestingly, compound 17 was found to be equipotent against both
P-gp and BCRP. Importantly, these synthetic quercetin derivatives did not exhibit any inherent cytotoxicity
to cancer cell lines or normal mouse fibroblast cell lines. These quercetin derivatives can be employed as
safe and effective modulators of P-gp- or BCRP-mediated drug resistance in cancer.
Article history:
Received 23 December 2011
Received in revised form
1 April 2012
Accepted 17 May 2012
Available online 4 June 2012
Keywords:
Quercetin
Methylated quercetin derivatives
Multidrug resistance (MDR)
Ó 2012 Elsevier Masson SAS. All rights reserved.
P-gp
BCRP
MRP1
1. Introduction
their structural similarities, the substrate binding modes and
bindingsites of thethreeproteinsarenotwell characterized. Thereis
Multidrug resistance (MDR) in cancer has been a major obstacle
to successful cancer chemotherapy. An important mechanism for
MDR is the enhanced cellular effluxof anticancer agents due to over-
expression of ATP-binding cassette (ABC) transporter proteins [1].
Among the 48 ABC transporters identified so far, P-glycoprotein (P-
gp, ABCB1), multidrug resistance-related protein 1 (MRP1, ABCC1)
and breast cancer resistance protein (BCRP, ABCG2) are three main
efflux transporters that have been shown to be associated with MDR
[2]. The three ABC proteins consist of transmembrane domains
(TMDs) and nucleotide-binding domains (NBDs) [3e10]. Despite
no common “pharmacophore” that can be identified as an inhibitor
of these three ABC transporters [3]. Nevertheless, different inhibi-
tors or modulators of ABC multidrug efflux pumps have been iden-
tified either by serendipitous discovery, combinatorial chemistry
and rational drug design or based on the known structure of these
transporters [11e13]. There are three generations of P-gp inhibitors
including calcium channel blockers [14e16], calmodulin antagonists
[17,18], cyclic peptides, steroids, and some synthetic compounds
such as elacridar, tariquidar, biricodar, S-9788 (shown in Fig. 1),
ontogen, zosuquidar, and purine derivatives [3,19e23]. Pan-
toprazole, fumitremorgin C and its derivatives (eg. Ko143) are
specific ABCG2 inhibitors [3,24]. The third generation P-gp inhibi-
tors of elacridarand tariquidaralso modulateABCG2 activity [25,26].
Fewer MRP1 inhibitors have been identified. The Leukotriene C4
(LTC4) analogue, MK571, S-decylglutathione and probenecid have
been described as MRP1 modulators [3,27,28]. All of these three ABC
drug transporters can interact with the modulator biricodar [3].
It has been demonstrated that non-toxic natural flavonoids such
as kaempferol, quercetin, baicalein, myricetin, fisetin, morin and
* Corresponding author. Tel.: þ86 532 82032712; fax: þ86 532 82033054.
** Corresponding author. Department of Applied Biology and Chemical Tech-
nology and the State Key Laboratory for Chirosciences, The Hong Kong Polytechnic
University, Hung Hom, Hong Kong SAR, China. Tel.: þ86 852 34008662; fax: þ86
852 23649932.
E-mail addresses: bclchow@polyu.edu.hk (L.M.C. Chow), biaowan@ouc.edu.cn
(B. Wan Sheng).
1
These two authors contribute equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.05.026

414
J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
Fig. 1. Inhibitors of ABC transporters.
epigallocatechin gallate may reverse MDR [29]. Quercetin (Fig. 1)
can modulate P-gp-, BCRP-, or MRP1-mediated drug resistance
[30e34], and preliminary SAR studies of flavonoids as MDR
modulating agents were carried out by Conseil et al. [35]. However,
the concentration of quercetin in the experiment was still high. For
quercetin derivatives on the three main MDR-associated ABC
efflux transporters, P-gp, BCRP, and MRP1, was also investigated.
2. Results and discussion
example, in one study, addition of 100
mM of quercetin could result
2.1. Chemistry
in an increased daunomycin accumulation (201.8 ꢀ 16.4%) in the P-
gp overexpressed resistant cell line MCF-7/ADR, which was
We have previously found that permethyl epigallocatechin gallate
was more potent than pentamethyl epigallocatechin (Fig. 1) in
modulating P-gp in LCC6MDR cells [37], suggesting that substitutions
at C-3 position of quercetin may be important in P-gp modulating
activity. Here, we tested such hypothesis by synthesizing and evalu-
ating a series of 3-substitued 5,7,30,40-tetra-O-methylated quercetins,
such as compounds 7e17, 19, 21, and 23 (shown in Schemes 1e3
respectively) for their P-gp modulating activities. Based on the pub-
lished method [39], rutin was methylated by methyl iodide to
generate tetramethylated rutin 5, followed by acidolysis of 5 to give
5,7,30,40-tetra-O-methylated quercetin 6 (Scheme 1). Spectroscopic
data of compound 6 was identical to that reported in literature [39].
Compound 6 was refluxed with 2-bromoethanol in the presence of
potassium carbonate to give compound 7 which was also used as
intermediate in the preparation of 3-substitued 5,7,30,40-tetra-O-
methylatedquercetins. Rutinwasalsomethylated by methyl iodide in
the presence of NaH to produce permethylated rutin 8 (Scheme 1)
[41]. Compounds 9 and 10 were effectively obtained via the reaction
of quercetin with methyl iodide in DMF in the presence of potassium
carbonate (Scheme 1). The reaction conditions were milder than
reported [42]. When four equivalents of methyl iodide were used in
thereaction, itafforded21.2% of 9and35.6%of10. Ifmethyliodidewas
increased to ten equivalents, it produced trace of 9 and 57.6% of 10.
Further modifications of 5,7,30,40-tetra-O-methylated quercetin
6 at O-3 were described in Scheme 2. Esterification of 5,7,30,40-tetra-
O-methylated quercetin 6 with 3,4,5-trimethoxybenzoic acid cata-
lyzed by EDCI afforded compound 11. Intermediate 6 was coupled
comparable to 100
quercetin pentamethyl ether (at 20
m
M of verapamil (229.4 ꢀ 17.6%). Interestingly,
m
M) was found to have shown
potent P-gp modulating activity [31,36]. Other methylated phenolic
compounds like permethyl epigallocatechin gallate (Fig. 1) [37],
permethyl ningalin B (Fig. 1) [38] and its synthetic analogues (eg.
compounds 1 and 2 in Fig. 1) [39] also displayed potent P-gp-
modulating activities. These results suggest that methylation of
some natural phenolic compounds could improve P-gp modulating
activity, and multi-methoxy substituted phenyl rings may be an
important pharmacophore for reversing MDR. Moreover, we
introduce a basic nitrogen on flavonoids in order to increase their
lipophilic properties, Ferte et al. synthesized a series of flavonoid
(including diosmetin, diosmin and the flavanone hesperetin)
derivatives containing a N-benzylpiperazine side chain at O-5, O-7,
or O-30, and investigated their anti-MDR ability [40]. At 5
mM, it was
found that most compounds (such as compounds 3 and 4 shown in
Fig. 1) potentiated doxorubicin cytotoxicity on resistant K562/DOX
MDR cells [40]. These compounds were also able to increase the
intracellular accumulation of JC-1, a fluorescent molecule recently
described as a probe of P-glycoprotein-mediated MDR [40]. In the
present work, we aimed to synthesize a novel series of methylated
quercetin derivatives and 5,7,30,40-tetra-O-methylated quercetin
derivatives containing a heterocycle or a multi-methoxyphenyl
group in the O-3 substituents, and to investigate their anti-
MDR activity affected by different side chain positions from the
literature [40]. The modulating selectivity of the synthesized

J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
415
Scheme 1. Synthetic routes of compounds 6, 7, 8, 9 and 10. Reagents and conditions: a) CH3I, K2CO3, DMF, rt, 2 d; b) HCl, 95% ethanol, reflux, 2 h; c) BrCH2CH2OH, acetone, K2CO3,
reflux, 12 h; d) CH3I, NaH, DMF, rt, 15 h.
with 3,4,5-trimethoxybenzyl methanesulfonate to give compound
12, and reacted with 2-bromo-1-(3,4-dimethoxyphenyl)ethanone
to produce compound 13. As shown in Scheme 2, esterification of
intermediate 7 with (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid,
3,4,5-trimethoxybenzoic acid, 3,4-dimethoxybenzoic acid, or 4-
methoxybenzoic acid provided compounds 14, 15, 16, and 17
respectively.
In order to investigate heterocycle-containing quercetin derivatives,
three new compounds were prepared and the synthetic routes were
shown in Scheme 3. Coupling of compound 6 with 9-(2-bromoethyl)-
9H-purin-6-amine 18 [43], 9-(2-bromoethyl)-1,3-dimethyl-1H-purine-
2,6(3H,9H)-dione 20 [44], or 6-(2-bromoethoxy)-N-(3-chloro-4-
fluorophenyl)-7-methoxy-3,4-dihydroquinazolin-4-amine 22 [45]
provided compounds 19, 21, and 23 respectively.
2.2. Biological evaluation
2.2.1. P-Gp-, BCRP- and MRP1-modulating activities of quercetin
derivatives
Here, we designed and synthesized a novel series of methylated
quercetin derivatives (containing various side chains at O-3) and
tested for their P-gp-, BCRP- and MRP1-modulating activities.
Almost all quercetin derivatives exhibited no cytotoxicity towards
LCC6, LCC6MDR or normal mouse fibroblast cells (L929) with IC50
higher than 100
mM (Table 1). The only slightly toxic compound is
compound 12, with IC50 towards L929 at 17.6
mM.
We employed four drug-resistant cell lines in this study. These
include (1) a P-gp-transfected human breast cancer cell line,
LCC6MDR which displayed 90.7-fold resistance to paclitaxel
Scheme 2. Synthetic routes of compounds 11e17. Reagents and conditions: a) EDCI, DMAP, CH2Cl2, 3,4,5-trimethoxybenzoic acid, rt, overnight; b) K2CO3, DMF, 3,4,5-
trimethoxybenzyl methanesulfonate, rt, 12 h; c) K2CO3, DMF, 2-bromo-1-(3,4-dimethoxyphenyl)ethanone, rt, 12 h; d)EDCI, DMAP, CH2Cl2, (E)-3-(3,4,5-trimethoxy-phenyl)acrylic
acid, rt, overnight; e) EDCI, DMAP, CH2Cl2, 3,4,5-trimethoxybenzoic acid, 3,4-dimethoxybenzoic acid, or 4-methoxybenzoic acid, rt, overnight.

416
J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
Scheme 3. Synthetic routes of compounds 19, 21, and 23. Reagents and conditions: a) K2CO3, DMF, 60 ꢁC, 12 h.
(IC50 ¼ 145.1 ꢀ 8.2 nM) compared with parental LCC6 cells
(IC50 ¼ 1.6 ꢀ 0.3 nM) (Table 2), (2) a BCRP-transfected human
embryonic kidney cell line, HEK293/R2, which was 30.8-fold more
resistant to topotecan (IC50 ¼ 486.9 ꢀ 34.6 nM) than parental
HEK293/pcDNA3.1 cell line (IC50 ¼ 15.8 ꢀ 1.5 nM) (Table 2), (3)
a mitoxantrone-selected breast cancer cell line MCF7-MX100 in
which BCRP was found to be overexpressed and exhibited 117.5-fold
The MDR reversal activity of quercetin derivatives were listed in
Table 3.
Quercetin and its derivatives were categorized into five sub-
groups depending on their structures (Table 3 and Fig. 2). In
group A, quercetin and rutin (at 1.0 mM) did not exhibit any P-gp-
(RF of quercetin ¼ 0.8 and RF of rutin ¼ 0.9) or MRP1-modulating
activity (RF of quercetin ¼ 1.1 and RF of rutin ¼ 1.2). They also
displayed no BCRP-reversal activity in HEK293/R2 (RF of
quercetin ¼ 1.3 and RF of rutin ¼ 1.3) or very weak BCRP-reversal
activity in MCF7-MX100 cell lines (RF of quercetin ¼ 4.7 and RF
of rutin ¼ 4.3). These data suggest that quercetin and rutin are poor
chemosensitizers and are not specific for the ABC transporters.
Permethylated rutin 8, being more hydrophobic than rutin, dis-
played no P-gp- (RF of 8 ¼ 1.0) or MRP1-modulating activity (RF of
resistance to topotecan (IC50 ¼ 37.6 ꢀ 2.7
mM) compared to wild-
type MCF7 (IC50 ¼ 0.32 ꢀ 0.07 M) (Table 2), (4) a MRP1-
m
transfected ovarian cancer cell line, 2008/MRP1 which was 6.7-fold
more resistant to DOX (IC50 ¼ 417.8 ꢀ 65.4 nM) than parental 2008/
P cells (IC50 ¼ 62.5 ꢀ 5.1 nM) (Table 2). Verapamil is a known P-gp and
MRP1 inhibitor, whereas Ko143 was a BCRP-specific modulator. Here,
we included these positive controls in the cell proliferation assay.
8 ¼ 1.4) at 1.0
mM. With respect to the BCRP-modulating activity,
compound 8 displayed similar potency (RF ¼ 1.5 in HEK293/R2 and
RF ¼ 5.3 in MCF7-MX100) as rutin (RF ¼ 1.3 in HEK293/R2 and
RF ¼ 4.3 in MCF7-MX100). These data indicate that methylation of
Table 1
In vitro cytotoxicity IC50 values (
L929 cells.
mM) of quercetin derivatives to LCC6, LCC6MDR and
Cpdsa
LCC6
LCC6MDR
L929
6
7
8
9
10
11
12
13
14
15
16
17
19
21
23
26
Quercetin
Rutin
>100
>100
>100
>100
>100
>79
14.5 ꢀ 3.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Table 2
Cells lines used to study P-gp-, BCRP- and MRP1-modulating activities.
Cell lines
IC50 towards
>100
>100
Paclitaxel (nM)
145.1 ꢀ 8.2
148.0 ꢀ 13.9
1.6 ꢀ 0.3
RF
87.4 ꢀ 6.0
46.6 ꢀ 20.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
74.1 ꢀ 10.4
17.6 ꢀ 1.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
LCCMDR
1.0
1.0
LCC6MDR þ DMSO
LCC6
90.7
RF
Topotecan (nM)
486.9 ꢀ 34.6
534.7 ꢀ 25.7
15.8 ꢀ 1.5
HEK293/R2
1.0
0.9
HEK293/R2 þDMSO
HEK293/pcDNA3.1
30.8
Topotecan (mM)
MCF7-MX100
MCF7-MX100 þ DMSO
MCF7
37.60 ꢀ 2.7
1.0
1.1
33.00 ꢀ 4.0
0.32 ꢀ 0.07
DOX (nM)
117.5
RF
2008/MRP1
417.8 ꢀ 65.4
420.0 ꢀ 24.0
62.5 ꢀ 5.1
1.0
1.0
6.7
2008/MRP1 þ DMSO
IC50 were determined by treating LCC6, LCC6MDR or L929 cells with different
concentrations of quercetin derivatives. L929 is a mouse fibroblast cell line. Each
experiment has been repeated 3 times with the data presented as mean ꢀ standard
error of mean.3.
2008/P
Four cell lines were used in this study (refer to text for details). 0.1% DMSO was
added to control for the solvent effect. All modulators used in this study were dis-
solved in DMSO and used at a final concentration of 1.0 mM, which was equivalent to
a
All compounds were dissolved in DMSO and the highest percentage of DMSO
used was 1% at which no toxicity towards cancer cell lines and L929 was observed.
0.1% DMSO.

J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
417
Table 3
Structural characteristics and MDR reversal activity of quercetin derivatives.
Gp. Cpds
Substituents at O-3
Types of linker
LCC6MDR
HEK293/R2
MCF7-MX100
2008/MRP1
Paclitaxel IC50
(nM)
RF Topotecan IC50
(nM)
RF Topotecan IC50
RF DOX IC50
(nM)
RF
5.4
(mM)
Verapamil
Ko143
Quercetin OH
43.9 ꢀ 5.2
3.3
77.0 ꢀ 3.0
24.0 ꢀ 1.9
20.3 0.5 ꢀ 0.1
1.3 8.1 ꢀ 1.6
1.3 8.7 ꢀ 2.3
1.5 7.1 ꢀ 0.6
3.3 6.3 ꢀ 3.0
3.9 25.6 ꢀ 13.2
13.0 2.0 ꢀ 0.6
8.7 4.8 ꢀ 0.8
5.8 3.9 ꢀ 0.8
11.3 7.9 ꢀ 1.3
1.4 27.7 ꢀ 9.0
14.0 4.1 ꢀ 0.6
75.2
A
B
None
None
None
None
None
None
None
Carbonyl
Methylene
Methylenecarbonyl
174.8 ꢀ 25.9
167.2 ꢀ 33.9
149.4 ꢀ 43.8
83.0 ꢀ 6.7
0.8 368.8 ꢀ 152.9
0.9 361.9 ꢀ 137.5
1.0 318.2 ꢀ 133.7
1.7 146.8 ꢀ 85.1
1.4 124.2 ꢀ 8.6
3.7 37.3 ꢀ 2.0
1.9 55.9 ꢀ 4.1
2.1 84.5 ꢀ 27.2
9.6 43.0 ꢀ 6.2
1.3 349.4 ꢀ 68.3
6.0 34.7 ꢀ 3.4
4.7 367.0 ꢀ 54.8 1.1
4.3 345.1 ꢀ 61.8 1.2
5.3 298.6 ꢀ 29.7 1.4
6.0 608.4 ꢀ 70.6 0.7
1.5 497.1 ꢀ 87.9 0.8
19.3 575.0 ꢀ 123.1 0.7
7.9 554.0 ꢀ 100.5 0.8
9.6 465.9 ꢀ 60.9 0.9
4.7 345.5 ꢀ 60.4 1.2
1.4 481.4 ꢀ 75.0 0.9
Rutin
8
Dissacharide
Methylated dissacharide
OH
6
7
2-Hydroxyethyl
105.6 ꢀ 9.9
39.6 ꢀ 1.7
9
Methoxy
10
11
12
13
14
Methoxy
76.8 ꢀ 2.6
C
Trimethoxybenzoyl
Trimethoxybenzyl
Dimethoxyphenyl-2-oxo-ethyl
(E)-2-((3-oxo-3-(3,4,5-
Trimethoxy phenyl)prop-
1-en-1-yl)oxy)ethyl
67.7 ꢀ 6.4
15.1 ꢀ 1.3
115.1 ꢀ 15.1
D
Ethyleneoxycarbonylvinyl 24.2 ꢀ 2.2
9.1 287.0 ꢀ 1.0
1.5
15
16
2-((3,4,5-Trimethoxybenzoyl)oxy) Ethyleneoxycarbonyl
ethyl
46.0 ꢀ 10.5
22.8 ꢀ 2.1
3.2 102.7 ꢀ 12.3
6.4 105.5 ꢀ 2.8
4.7 18.9 ꢀ 5.8
4.6 5.6 ꢀ 0.8
2.0 376.5 ꢀ 73.2 1.1
6.7 359.0 ꢀ 9.6 1.2
2-((3,4-Dimethoxybenzoyl)oxy)
ethyl
Ethyleneoxycarbonyl
17
19
21
23
2-((4-Methoxybenzoyl)oxy)ethyl Ethyleneoxycarbonyl
12.9 ꢀ 1.2
129.1 ꢀ 19.7
122.2 ꢀ 12.8
99.7 ꢀ 9.9
11.3 40.2 ꢀ 8.2
1.1 410.9 ꢀ 72.6
1.2 355.4 ꢀ 55.0
1.5 113.2 ꢀ 18.1
12.1 5.2 ꢀ 0.6
1.2 32.9 ꢀ 7.1
1.4 27.0 ꢀ 3.0
4.3 31.5 ꢀ 6.8
7.3 272.4 ꢀ 18.4 1.5
1.2 509.8 ꢀ 91.4 0.8
1.4 408.4 ꢀ 32.7 1.0
1.2 438.3 ꢀ 22.3 1.0
E
Ethylꢂene linked heterocycle
Ethylene linked heterocycle
Ethylene linked heterocycle
ethyleneoxy
ethyleneoxy
ethyleneoxy
IC50 value was determined for paclitaxel, topotecan and DOX with 1.0
a low concentration of quercetin derivatives, no cytotoxic effect on the cell lines was observed. Relative fold (RF) ¼ (IC50 without modulator)/(IC50 with 1.0
N ¼ 2e4 independent experiment and values were presented as mean ꢀ standard error of mean. Verapamil was used at 20 M for testing MRP1-modulating activity.
m
M of quercetin derivatives using LCC6MDR, HEK293/R2, MCF7-MX100 and 2008/MRP1 cells. At such
mM modulator).
m
carbohydrate side chain has no impact on P-gp-, MRP1- and BCRP-
modulating activities.
MX100). These data suggest that the type of linkers between the
terminal phenyl ring and methylated quercetin at O-3 position is an
important factor for determining the BCRP-modulating activities of
quercetin derivatives. No MRP1-modulating activity was found in
compounds 11, 12 or 13.
In group D, compounds 14 (RF ¼ 6.0), 15 (RF ¼ 3.2), 16
(RF ¼ 6.4), and 17 (RF ¼ 11.3) all possessed promising P-gp
modulating activities. Compounds 15, 16 and 17 have mono, di and
tri methoxy groups on the terminal phenyls respectively. This
result suggests that the compound containing fewer methoxy
groups on the terminal phenyl ring would exhibit stronger P-gp-
modulating activity. Compound 14 (RF ¼ 6.0), containing a longer
ethyleneoxycarbonylvinyl linker and an extra C]C bond, was
Among the alkylated quercetins in group B, compound 9
(3,7,30,40-tetra-O-methylated quercetin) at 1.0
mM showed a rela-
tively higher P-gp (RF ¼ 3.7) and BCRP- (RF ¼ 13.0 in HEK293/R2
and RF ¼ 19.3 in MCF7-MX100) modulating activities than 6
(5,7,30,40-tetra-O-methylated quercetin) (RF ¼ 1.7 in LCC6MDR,
RF ¼ 3.3 in HEK293/R2 and RF ¼ 6.0 in MCF7-MX100) and 10
(quercetin pentamethyl ether) (RF ¼ 1.9 in LCC6MDR, RF ¼ 8.7 in
HEK293/R2 and RF ¼ 7.9 in MCF7-MX100). It demonstrates that 5-
O-non-methylation and 3-O-methylation contribute significantly
to P-gp- and BCRP-modulation activities. Compound 7, having a 2-
hydroxyethyl substitution at O-3 of compound 6, also has weak P-
gp- (RF ¼ 1.4) and BCRP- (RF ¼ 3.9 in HEK293/R2 and RF ¼ 1.5 in
MCF7-MX100) modulating activities. None of the compounds in
group B displayed MRP1-modulating activity as all RF values were
close to 1.0.
found to display
compound 15 (RF
eneoxycarbonyl linker.
a
¼
stronger P-gp-modulating activity than
3.2) which possessed only the ethyl-
Consistent with P-gp-modulation, the number of methoxy
groups on terminal phenyl ring also has a strong effect on BCRP
modulating activity. Compound 17 with the monomethoxy group
(RF ¼ 12.1 in HEK293/R2 and RF ¼ 7.3 in MCF7-MX100) exhibited
stronger BCRP-activity than compound 15 with trimethoxy groups
(RF ¼ 4.7 in HEK293/R2 and RF ¼ 2.0 in MCF7-MX100). Here,
compound 14 with longer linker and an extra C]C bond (RF ¼ 14.0
in HEK293/R2 and RF ¼ 9.1 in MCF7-MX100) showed a higher
BCRP1-modulating activity than compound 15 (RF ¼ 4.7 in
HEK293/R2 and RF ¼ 2.0 in MCF7-MX100) possessing a shorter
linker. Once again, we found that linker length between the
terminal phenyl ring and the methylated quercetin plays an
important role in P-gp- and BCRP-modulating activity. None of
quercetin derivatives in this series can modulate MRP1.
In group E, compounds 19, 21, and 23 contained various
nitrogen-containing heterocycles were designed and synthesized
in order to investigate varied side rings. These three compounds,
however, exhibited no significant P-gp-, BCRP- or MRP1-
modulating activities. Only compound 23 displayed a moderate
RF of 4.3 in BCRP-overexpressed HEK293/R2 cell line.
In group C, 5,7,30,40-tetra-O-methylated quercetin 6 was modi-
fied at O-3 to give the three compounds 11, 12, and 13. Compound
12 (RF ¼ 9.6), having a 3,4,5-trimethoxyphenyl group linked by
a methylene at O-3, displayed very potent P-gp-modulating activity
than the parent compound 6 (RF ¼ 1.7). Unlike compound 12, both
11 and 13, which possessed a carbonyl group in their linkers, gave
low P-gp-modulating activity (RF of 11 ¼ 2.1 and RF of 13 ¼ 1.3).
These data indicate that phenyl ring connected to the quercetin at
O-3 with a methylene linker but not carbonyl linker can enhance P-
gp-modulating activity.
Consistently, an improved BCRP-modulating activity of
compound 12 (RF ¼ 11.3 in HEK293/R2) was also noted in HEK293/
R2 as compared to compound 6 (RF ¼ 3.3 in HEK293/R2).
Compound 13 with a methylenecarbonyl linker has no BCRP-
modulating activity in both HEK293/R2 (RF ¼ 1.4) and MCF7-
MX100 (RF ¼ 1.4). Compound 11, with a carbonyl group in the
linker, can retain a moderate BCRP-modulating activity in both
HEK293/R2 (RF ¼ 5.8) and MCF7-MX100 (RF ¼ 9.6) as compared to
the compound 6 (RF ¼ 3.3 in HEK293/R2 and RF ¼ 6.0 in MCF7-

418
J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
Fig. 2. Structure of synthetic quercetin derivatives.
Of all quercetin derivatives tested, compound 17 (containing
monomethoxy substituted phenyl linked by an ethyl-
different potencies against P-gp, BCRP and MRP1. It seems that our
quercetin derivative exhibit higher specificity for P-gp and BCRP
than the MRP1.
a
eneoxycarbonyl group) displayed the highest P-gp-modulating
activity (RF ¼ 11.3). This activity was about 3.4-fold higher than that
of verapamil (RF ¼ 3.3). With respect to BCRP modulation, three
quercetin derivatives (compound 9, 14 and 17) displayed the
highest BCRP-modulating activity with RF ranging from 7.3 to 19.3.
These activities are still relatively low compared to Ko143.
3. Conclusions
In summary, we demonstrated that synthetic quercetin deriva-
tives exhibited promising P-gp- and BCRP-mediated MDR reversal
activities without any inherent cytotoxicity to cancer cell lines or
normal mouse fibroblast cell lines. The number of methoxy
substituents on the terminal phenyl ring and the type of linkers at
O-3 side chain are two key structural features for determining P-gp-
modulating activity of quercetin derivatives. Compound 17 with
a 3-methoxybenzoyloxyethyl at O-3 showed the highest P-gp-
modulating activity. Compound 9 (3,7,30,40-tetra-O-methylated
None of the quercetin derivatives displayed any MRP1-
modulating activity at 1.0
mM, suggesting that our quercetin
derivatives are less specific for MRP1 transporter as compared to
the P-gp and BCRP transporters. Verapamil is a weak MRP1
modulator. At a high concentration (20 mM), verapamil completely
reversed DOX resistance in 2008/MRP1 (RF ¼ 5.4 in Table 3). It is
likely that MRP1 modulator might have its own special pharma-
cophore. The structure of MRP1 is highly similar to P-gp, but with
an extra transmembrane domain (TM0) at its N-terminal [3,5,7]. It
is known that substrate selectivity of the ABC transporters mark-
edly differs. P-gp substrates are neutral or mildly positive lipophilic
compounds, whereas MRP1 is able to transport lipophilic anions.
MRP1 has a broad substrate specificity including glutathione S-
conjugates, glucuronide conjugates, sulphate conjugates, anti-
cancer drugs, heavy metals and organic anions etc [46e50]. This
wide difference in substrate specificity of various ABC transporters
may explain why our quercetin derivatives markedly exhibit
quercetin)
or
compound
14
(with
a
3,4,5-
trimethoxyphenylacryloyloxyethyl at O-3) or compound 17 dis-
played promising BCRP-modulating activity. Interestingly,
compound 17 was found to be equipotent against P-gp and BCRP. It
might be a good MDR reversal agent because of its dual-modulating
activities. The present study demonstrates that synthetic quercetin
derivatives can be employed as safe and effective modulators of P-
gp- or BCRP-mediated drug resistance in cancer cells. However,
none of the synthesized quercetin derivatives displayed any MRP1-
modulating activity at 1.0 mM. The reason of less specific for MRP1

J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
419
transporter as compared to the P-gp and BCRP transporters is under
investigated.
44.2% yield). Mp 65e67 ꢁC; 1H NMR (CDCl3, 600 MHz)
d
: 7.73 (d,
J ¼ 1.6 Hz, 1H), 7.59 (dd, J ¼ 8.2, 1.6 Hz, 1H), 6.85 (d, J ¼ 8.8 Hz, 1H),
6.37 (d, J ¼ 2.2 Hz, 1H), 6.24 (d, J ¼ 2.2 Hz, 1H), 5.66 (d, J ¼ 7.14 Hz,
1H), 4.56 (s, 1H), 3.88, 3.86, 3.85, 3.78, 3.56, 3.53, 3.41, 3.32, 3.24,
3.12 (10s, 30H, 10 ꢂ OCH3), 3.66 (q, J ¼ 4.9 Hz, 1H), 3.31e3.25 (m,
3H), 3.22e3.21 (m, 2H), 3.17e3.13 (m, 2H), 3.90e3.84 (m, 2H),
2.89e2.84 (m, 2H), 0.98 (d, J ¼ 6.1 Hz, 3H); 13C NMR (CDCl3,
4. Experimental section
4.1. General
Starting materials and reagents were purchased from commer-
cial suppliers and were used without further purification. 9-(2-
Bromoethyl)-9H-purin-6-amine (18) [43], 9-(2-bromoethyl)-1,3-
dimethyl-1H-purine-2,6(3H,9H)dione (20) [44], or 6-(2-bromo-
ethoxy)-N-(3-chloro-4-fluorophenyl)-7-methoxy-3,4-
150 MHz) d: 173.1, 163.9, 160.9, 158.6, 153.8, 150.7, 148.1, 135.9,
123.2, 121.8, 112.1, 110.4,109.1, 100.3, 97.3, 95.7, 92.2, 85.9, 84.3, 81.8,
80.7, 79.8, 74.3, 67.6, 66.6, 60.8, 60.5, 59.7, 58.5, 57.4, 56.2, 56.0, 55.8,
55.7, 17.5; HRMS calcd for (C37H50O16 þ H)þ 751.3177, found
751.3201.
dihydroquinazolin-4-amine (22) [45] were prepared following the
methods in literature. Anhydrous methylene chloride was distilled
under nitrogen from CaH2. Anhydrous DMF was distilled under
vacuum from CaH2. Reaction flasks were flame-dried under
a stream of N2. All reactions were monitored by thin-layer chro-
matography (TLC), on aluminium sheets (Silica gel 60-F254, E.
Merck). Compounds were visualized by UV light. Flash chroma-
tography was carried out using silica-gel 60 (200e300 mesh). Silica
gel chromatography solvents were of analytical grade. Melting
points were recorded on a micro melting point apparatus MP-500D
and were uncorrected. 1H NMR (600 MHz) and 13C NMR (150 MHz)
spectra were measured with TMS as an internal standard when
CDCl3 or CD3OD were used as a solvent. Chemical shifts are
4.1.3. 2-(3,4-Dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chr
omen-4-one (9) and 2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-
4H-chromen-4-one (10)
Method 1: A solution of quercetin (100 mg, 0.33 mmol) in DMF
(10 mL) and anhydrous potassium carbonate (69 mg, 0.50 mmol)
was treated with methyl iodide (187 mg,1.32 mmol) at 0 ꢁC, and the
solution was stirred at room temperature for 12 h until TLC showed
that the reaction had been completed. The mixture was poured into
ice-water (30 mL), and the solution was extracted with ethyl
acetate, and then the extract was washed with saturated aqueous
NaHCO3 and brine, and then was dried over anhydrous MgSO4. The
solvent was evaporated and the residue was purified by flash
chromatography on silica gel to afford the desired compounds 9
(25.1 mg, 21.2% yield) and 10 (43.7 mg, 35.6% yield).
expressed in
d (ppm) and coupling constants (J) in Hz. High-
resolution (ESI) MS spectra were recorded using a QTOF-2 Micro-
mass spectrometer. In addition to NMR and High-resolution (ESI)
MS, HPLC analysis was used to determine the purity (>95%) of the
compounds. Compounds were dissolved in methanol (1.5 mL). A
reversed phase Diamonsil C18 (2) (4.6 ꢂ 150 mm) column attached
to a Gilson 322 pump coupled to a Gilson UVevis-152 detector was
Method 2: When 10 equivalents methyl iodide were used in the
above reaction, trace of compound 9 and 57.6% of 10 were obtained.
The melting point of compounds 9 and 10 were identical to the
literature [32].
Compound 9: mp 156e157 ꢁC; 1H NMR (CDCl3, 600 MHz)
d:
used. Each sample was injected at a volume of 20
with methanol and the flow rate was 1 mL/min.
mL and eluted
12.65 (s, 1H), 7.74 (dd, J ¼ 8.7, 2.3 Hz), 7.69 (d, J ¼ 1.8 Hz,1H), 6.99 (d,
J ¼ 8.7 Hz,1H), 6.45 (d, J ¼ 1.9 Hz,1H), 6.36 (d, 2.3 Hz,1H), 3.98, 3.97,
3.88, and 3.86 (4s, 12H); 13C NMR (CDCl3, 150 MHz)
d: 178.8, 165.5,
4.1.1. 2-(3,4-Dimethoxyphenyl)-3-(2-hydroxyethoxy)-5,7-dime
thoxy-4H-chromen-4-one (7)
162.1, 156.8, 155.9, 151.4, 148.8, 139.0, 123.0, 122.3, 111.3, 110.9, 106.1,
97.9, 92.3, 60.3, 56.1, 56.0, 55.9; HRMS calcd for (C19H18O7 þ H)þ
359.1131, found 359.1145. Compound 10: mp 150e152 ꢁC; 1H NMR
A mixture of compound 6 (1.00 g, 2.80 mmol), 2-bromoethanol
(600 mg, 4.20 mmol), anhydrous K2CO3 (773 mg, 5.60 mmol) and KI
(100 mg, 0.60 mmol) in DMF (20 mL) was heated with stirring at
60 ꢁC for overnight. Then H2O (100 mL) was added and the resulting
mixture was extracted with ethyl acetate. The organic layer was
dried over anhydrous MgSO4. The solvent was evaporated to
dryness, and the resulting residue was purified by flash chroma-
tography to afford the title compound 7 (974 mg, 86.5% yield). Mp
(CDCl3, 600 MHz)
(d, J ¼ 2.2 Hz,1H), 6.30 (d, J ¼ 1.6 Hz, 1H), 3.93e3.92 (m, 9H), 3.87 (s,
3H) 3.84 (s, 3H); 13C NMR (CDCl3, 150 MHz)
: 178.9, 165.5, 162.1,
d: 7.68e7.66 (m, 2H), 6.94 (d, J ¼ 8.8 Hz, 1H), 6.46
d
156.8, 155.7, 148.8, 145.6, 139.3, 123.7, 121.7, 114.5, 110.5, 106.2, 97.9,
92.2, 60.3, 56.1, 55.9; HRMS calcd for (C20H20O7 þ H)þ 373.1287,
found 373.1264.
120e122 ꢁC; 1H NMR (CDCl3, 600 MHz)
d: 7.71e7.69 (m, 2H), 6.98
4.1.4. 2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chro
(d, J ¼ 8.8 Hz, 1H, H), 6.52 (d, J ¼ 2.2 Hz, 1H), 6.37 (d, J ¼ 2.2 Hz, 1H),
men-3-yl 3,4,5-trime-thoxybenzoate (11)
4.65 (br s,1H, eOH), 3.98 (t, J ¼ 3.8 Hz, 2H), 3.97, 3.96, 3.95, 3.91 (4 s,
A
mixture of compound
trimethoxycinnamic acid (178 mg, 0.84 mmol), 1-ethyl-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC$HCl,
6 (200 mg, 0.56 mmol), 3,4,5-
12H, 4 ꢂ OCH3), 3.79 (t, J ¼ 3.8 Hz, 2H); 13C NMR (CDCl3,150 MHz)
d:
174.9, 164.3, 161.0, 158.9, 153.7, 151.1, 148.9, 139.8, 129.9, 121.8, 111.1,
110.9, 109.0, 96.1, 92.5, 75.0, 61.7, 56.5, 55.9; HRMS calcd for
(C21H22O8 þ H)þ 403.1393, found 403.1372.
161 mg, 0.84 mmol), 4-dimethylaminopyridine (DMAP, 102 mg,
0.84 mmol) in anhydrous CH2Cl2 (20 mL) was stirred at room
temperature for overnight. Then the solution was washed by
saturated aqueous Na2CO3, saturated aqueous NH4Cl, and brine in
sequence. The organic layer was dried by anhydrous MgSO4. The
solvent was evaporated to dryness, and the residue was purified by
flash chromatography to afford the title compound 11 (264 mg,
4.1.2. 2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-3-(((2R,3R,4S,5R,6R)-
3,4,5-trimethoxy-6-((((2S,3R,4R,5S,6S)-3,4,5-trimethoxy-6-
methyltetrahydro-2H-pyran-2-yl)oxy)-methyl)tetrahydro-2H-pyran-
2-yl)oxy)-4H-chromen-4-one (8)
Under an N2 atmosphere, to a solution of rutin (1.00 g,
1.60 mmol) and sodium hydride (0.58 g, 24.00 mmol) in DMF
(20 mL) methyl iodide (4.50 g, 32.00 mmol) was added at 0 ꢁC. The
solution was stirred for 6 h. The solution was poured into ice-water
(100 mL), and the resulting mixture was extracted with chloroform.
The organic layer was dried over anhydrous MgSO4. The solvent
was evaporated to dryness, and the resulting residue was purified
by flash chromatography to afford the title compound 8 (651 mg,
85.4% yield). Mp 108e110 ꢁC; 1H NMR (CDCl3, 600 MHz)
d: 7.52 (dd,
J ¼ 2.22, 8.82 Hz, 1H), 7.45 (s, 1H), 7.41 (d, J ¼ 1.68 Hz, 1H), 6.91 (d,
J ¼ 8.82 Hz, 1H), 6.54 (d, J ¼ 2.22 Hz, 1H), 6.35 (d, J ¼ 2.22 Hz, 1H),
3.92, 3.90, 3.89, 3.88, 3.79 (5s, 21H, 7 ꢂ OCH3); 13C NMR (CDCl3,
150 MHz) d: 170.6, 164.4, 163.9, 161.3, 159.2, 153.3, 153.0, 151.3,
148.8, 142.9, 134.0, 123.9, 122.4, 121.7, 110.9, 110.7, 108.8, 107.8, 96.1,
92.7, 61.0, 56.3, 56.0, 55.9; HRMS calcd for (C29H28O11 þ H)þ
553.1710, found 553.1701.

420
J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
4.1.5. 2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-3-((3,4,5-trimethox
(CDCl3, 150 MHz) d: 173.8, 165.9, 164.0, 160.9, 158.7, 152.8, 152.8,
ybenzyl)oxy)-4H-chromen-4-one (12)
150.8, 148.4, 142.1, 139.7, 125.0, 123.0, 122.0, 111.1, 110.5, 109.3, 106.9,
95.8, 92.4, 69.9, 64.3, 60.9, 56.4, 56.2, 55.9, 55.8, 55.7; HRMS calcd
for (C31H32O12 þ H)þ 597.1972, found 597.1994.
A
solution of compound 6 (200 mg, 0.54 mmol), 3,4,5-
trimethoxybenzyl methanesulfonate (232 mg, 0.81 mmol), K2CO3
(149 mg, 1.1 mmol) in DMF (10 mL) was stirred in room tempera-
ture for 12 h. The solution was poured into ice-water, and the
precipitate was filtrated, and purified by flash chromatography to
afford the title compound 12 (252 mg, 86.7%). Mp 90e92 ꢁC; 1H
4.1.9. 2-((2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chr
omen-3-yl)oxy)ethyl 3,4-dimethoxybenzoate (16)
Using the same procedure for the preparation of compound 14,
but with 3,4-dimethoxybenzoic acid as the starting material, the
titled compound 16 was prepared. Yield 84.2%; mp 181e183 ꢁC; 1H
NMR (CDCl3, 600 MHz)
d
: 7.64 (d, J ¼ 2.22 Hz, 1H), 7.58 (dd, J ¼ 2.16,
8.76 Hz, 1H), 6.89 (d, J ¼ 8.82 Hz, 1H), 6.57 (s, 2H), 6.46 (d,
J ¼ 2.22 Hz, 1H), 6.31 (d, J ¼ 2.16 Hz, 1H), 4.99 (s, 2H), 3.94, 3.90,
3.86, 3.77, 3.75, 3.71 (6s, 21H, 7 ꢂ OCH3); 13C NMR (CDCl3, 150 MHz)
NMR (CDCl3, 600 MHz)
d
: 7.67 (dd, J ¼ 1.62, 8.22 Hz, 1H), 7.58 (d,
2.16 Hz, 1H), 7.45e7.43 (m, 2H), 6.78 (d, J ¼ 8.82 Hz, 1H), 6.66 (d,
J ¼ 8.76 Hz, 1H), 6.47 (d, J ¼ 2.22 Hz, 1H), 6.33 (d, J ¼ 2.22 Hz, 1H),
4.52e4.47 (AB, J ¼ 14.88, 5.52 Hz, 4H), 3.95, 3.92, 3.89, 3.87, 3.85,
d: 174.1, 163.9, 158.9, 153.3, 153.0, 150.7, 148.4, 139.5, 137.7, 132.7,
123.5, 121.8, 111.9, 110.5, 109.4, 106.1, 106.0, 95.8, 92.5, 74.2, 60.8,
56.5, 56.0, 55.8; HRMS calcd for (C29H30O10 þ H)þ 539.1917, found
539.1931.
3.78 (6s, 18H, 6 ꢂ OCH3); 13C NMR (CDCl3, 150 MHz)
d: 173.9, 166.1,
164.0, 161.0, 158.8, 152.9, 150.7, 148.5, 139.7, 123.7, 123.2, 122.5,
122.1, 112.0, 111.3, 110.5, 110.1, 95.8, 92.5, 70.0, 64.0, 56.4, 56.1, 56.0,
55.8, 55.7; HRMS calcd for (C30H30O11 þ H)þ 567.1866, found
567.1860.
4.1.6. 2-(3,4-Dimethoxyphenyl)-3-(2-(3,4-dimethoxyphenyl)-2-oxo
ethoxy)-5,7-dime- thoxy-4H-chromen-4-one (13)
A solution of compound 6 (200 mg, 0.56 mmol), 2-bromo-1-
(3,4-dimethoxy- phenyl)ethanone (217 mg, 0.84 mmol), K2CO3
(154.6 mg, 1.12 mmol) in DMF (10 mL) was stirred at room
temperature for overnight, the solution was poured into ice-water
(30 mL). The precipitate was filtrated, and purified by flash chro-
matography to afford the title compound 13 (231 mg, 76.8% yield).
4.1.10. 2-((2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chr
omen-3-yl)oxy)ethyl 4-methoxybenzoate (17)
Using the same procedure for the preparation of compound 14,
but with 4-methoxybenzoic acid as the starting material, the titled
compound 17 was prepared. Yield 83.4%; mp 107e109 ꢁC; 1H NMR
Mp 222e224 ꢁC; 1H NMR (CDCl3, 600 MHz)
d
: 7.88 (s, 1H), 7.76 (d,
(CDCl3, 600 MHz)
d
: 7.76 (d, J ¼ 8.76, 2H), 7.68 (dd, J ¼ 1.62, 8.22 Hz,
J ¼ 8.3 Hz, 1H), 7.73 (d, J ¼ 8.3 Hz, 1H), 7.54 (s, 1H), 6.92 (d,
J ¼ 8.22 Hz, 1H), 6.84 (d, J ¼ 8.82 Hz, 1H), 6.50 (s, 1H), 6.33 (s, 1H),
5.44 (s, 2H), 3.94, 3.91, 3.89 (3s, 18H, 6 ꢂ OCH3); 13C NMR (CDCl3,
1H), 7.58 (d, J ¼ 1.62 Hz, 1H), 6.81 (d, J ¼ 8.82 Hz, 2H), 6.67 (d,
J ¼ 8.82 Hz, 1H), 6.47 (d, J ¼ 2.22 Hz, 1H), 6.33 (d, J ¼ 2.16 Hz, 1H),
4.48 (s, 4H), 3.95, 3.88, 3.85, 3.83, 3.78 (5s, 15H, 5 ꢂ OCH3); 13C NMR
150 MHz)
d: 193.1, 173.9,164.0, 160.9, 158.8,153.6, 152.7,151.0,149.1,
(CDCl3, 150 MHz) d: 174.0, 166.1, 164.0, 163.3, 161.0, 158.8, 150.7,
148.6, 139.5, 127.9, 123.3, 123.0, 121.7, 111.8, 110.6, 110.2, 110.1, 109.2,
95.9, 92.5, 73.9, 56.5, 56.2, 56.1, 55.9, 55.9; HRMS calcd for
(C29H28O10 þ H)þ 537.1760, found 537.1777.
148.5, 139.6, 131.6, 123.2, 122.3, 122.2, 113.4, 111.3, 110.5, 109.4, 95.8,
92.5, 70.1, 63.8, 56.4, 56.0, 55.8, 55.7, 55.4; HRMS calcd for
(C29H28O10 þ H)þ 537.1760, found 537.1772.
4.1.7. (E)-2-((2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-
chromen-3-yl)oxy)-ethyl 3-(3,4,5-trimethoxyphenyl)acrylate (14)
A mixture of compound 7 (200 mg, 0.48 mmol), 3,4,5-
trimethoxycinnamic acid (229 mg, 0.96 mmol), 1-ethyl-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC$HCl,
184 mg, 0.96 mmol), 4-dimethylaminopyridine (DMAP, 117 mg,
0.96 mmol) in anhydrous CH2Cl2 (20 mL) was stirred at room
temperature for overnight. Then the solution was washed by
saturated aqueous Na2CO3, saturated aqueous NH4Cl, and brine
in sequence. The organic layer was dried by anhydrous MgSO4.
The solvent was evaporated to dryness, and the residue was
purified by flash chromatography to afford the title compound 14
(261 mg, 87.5% yield). Mp 152e154 ꢁC; 1H NMR (CDCl3, 600 MHz)
4.1.11. 3-(2-(6-Amino-9H-purin-9-yl)ethoxy)-2-(3,4-dimethoxyph
enyl)-5,7-dimethoxy-4H-chromen-4-one (19)
A
mixture of compound 6 (200 mg, 0.48 mmol), 9-(2-
bromoethyl)-9H-purin-6-amine 18 (140 mg, 0.58 mmol), K2CO3
(133 mg, 0.96 mmol) in anhydrous DMF (20 mL) was stirred at 60 ꢁC
for overnight. The solution was poured into ice-water, and the
precipitate was filtrated, and purified by flash chromatography to
afford the title compound 19 (203 mg, 81.4%). Mp 267e269 ꢁC; 1H
NMR (CD3COOD, 600 MHz) d: 8.47 (s, 1H), 8.34 (s, 1H), 7.32 (d,
J ¼ 2.22 Hz, 1H), 7.29 (dd, J ¼ 2.16, 8.76 Hz, 1H), 6.73 (d, J ¼ 8.82 Hz,
1H), 6.63 (d, J ¼ 2.16 Hz, 1H), 6.44 (d, J ¼ 2.22 Hz, 1H), 4.61e4.59 (m,
2H), 4.56e4.54 (m, 2H), 3.90, 3.88, 3.86, 3.78 (4s, 12H, 4 ꢂ OCH3);
13C NMR (CD3COOD, 150 MHz)
d: 174.7, 165.3, 160.8, 155.1, 152.8,
d
: 7.72 (d, J ¼ 2.22 Hz, 1H), 7.66 (dd, J ¼ 2.22, 8.82 Hz, 1H), 7.44
151.5, 148.6, 148.0, 143.6, 138.8, 122.2, 122.1, 111.2, 110.6, 108.4, 96.2,
92.7, 68.9, 55.7, 55.6, 55.4, 55.3, 44.7; HRMS calcd for
(C25H25N5O7 þ H)þ 520.1832, found 520.1817.
(d, J ¼ 16.96, 1H), 6.83 (d, J ¼ 8.76 Hz, 1H), 6.71 (s, 2H), 6.49
(d, J ¼ 2.16 Hz, 1H), 6.33 (d, J ¼ 2.22 Hz, 1H), 6.16 (d, J ¼ 15.96,
1H), 4.42 (s, 4H), 3.95, 3.92, 3.90, 3.88, 3.87, 3.71 (6s, 21H,
7 ꢂ OCH3); 13C NMR (CDCl3, 150 MHz)
d
: 173.9, 166.7, 164.0, 161.0,
4.1.12. 9-(2-((2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-H-
chromen-3-yl)oxy)ethyl)-1,3-dimethyl-1H-purine-2,6(3H,9H)-
dione (21)
158.9, 153.4, 153.1, 150.9, 148.4, 144.8, 140.1, 139.5, 129.9, 123.2,
121.9, 116.9, 112.0, 110.6, 109.3, 105.3, 95.9, 92.5, 70.10, 63.68,
61.0, 56.4, 56.2, 56.1; HRMS calcd for (C33H34O12 þ H)þ 623.2128,
found 623.2106.
Following the procedure for the preparation of compound 19,
but with 9-(2-bromo-ethyl)-1,3-dimethyl-1H-purine-2,6(3H,9H)-
dione 20 as starting material, the titled compound 21 was prepared.
4.1.8. 2-((2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-
chromen-3-yl)oxy)ethyl 3,4,5-trimethoxybenzoate (15)
Following the procedure for the preparation of compound 14,
but with 3,4,5-trimethoxybenzoic acid as starting material,
compound 15 was obtained. Yield 85.0%; mp 80e82 ꢁC; 1H NMR
Yield 84.6%; mp 263e265 ꢁC; 1H NMR (CDCl3, 600 MHz)
d: 7.87 (s,
1H), 7.38 (d, J ¼ 1.62 Hz, 1H), 7.31 (s, 1H), 6.79 (d, J ¼ 8.28 Hz, 1H),
6.48 (s, 1H), 6.36 (s, 1H), 4.70 (t, J ¼ 4.83 Hz, 2H), 4.35 (t, J ¼ 4.38 Hz,
2H), 3.97, 3.91, 3.89, 3.80, 3.58, 3.35 (6s, 18H, 6 ꢂ OCH3); 13C NMR
(CDCl3, 150 MHz) d: 173.8, 164.2, 161.0, 158.9, 155.2, 153.6, 151.6,
(CDCl3, 600 MHz)
d
: 7.63 (d, J ¼ 8.28 Hz,1H), 7.53 (s,1H), 7.17 (s, 2H),
151.0, 148.9, 148.6, 142.5, 139.7, 122.8, 121.6, 111.1, 110.4, 106.6, 96.0,
92.62, 69.95, 56.5, 56.0, 55.9, 55.9, 47.6, 29.8, 27.9; HRMS calcd for
(C28H28N4O9 þ H)þ 565.1934, found 565.1941.
6.61 (d, J ¼ 8.82 Hz,1H), 6.43 (s,1H), 6.28 (s,1H), 4.51 (s, 2H), 4.43 (s,
2H), 3.89, 3.85, 3.84, 3.82, 3.81, 3.74 (6s, 21H, 7 ꢂ OCH3); 13C NMR

J. Yuan et al. / European Journal of Medicinal Chemistry 54 (2012) 413e422
421
4.1.13. 3-(2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxy
quinazolin-6-yl)oxy)-ethoxy)-2-(3,4-dimethoxyphenyl)-5,7-
dimethoxy-4H-chromen-4-one (23)
at 37 ꢁC. After 5 days, the % of survival or viability was determined
by MTS according to procedures reported previously [51]. These
results were represented as mean ꢀ standard error of mean. IC50
values were calculated from the doseeresponse curves of MTS
assays (Prism 4.0).
Following the procedure for the preparation of compound 19,
but
with
6-(2-bromoethoxy)-N-(3-chloro-4-fluorophenyl)-7-
methoxy-3,4-dihydroquinazolin-4-amine 22 as starting material,
the titled compound 23 was prepared. Yield 64.5%; mp 127e129 ꢁC;
Acknowledgement
1H NMR (DMSO-d6, 600 MHz)
d: 9.45 (s, 1H), 8.49 (s, 1H), 8.10 (dd,
J ¼ 2.76, 6.60 Hz, 1H), 7.79e7.77 (m, 1H), 7.68 (d, J ¼ 2.16 Hz, 1H),
7.66 (s, 1H), 7.62 (d, J ¼ 1.68 Hz, 1H), 7.43 (t, J ¼ 9.36 Hz, 1H), 7.14 (s,
1H), 6.84 (d, J ¼ 8.76 Hz, 1H), 6.69 (d, J ¼ 2.22 Hz, 1H), 6.49 (d,
J ¼ 2.22 Hz, 1H), 4.48 (s, 2H), 4.34 (s, 2H), 3.89, 3.84, 3.73, 3.69 (4s,
This project was funded by National Natural Science Foundation
of China (NSFC 81172926), General Research Fund (B-Q16G) of the
Research Grant Council of Hong Kong, and Special Fund for Marine
Scientific Research in the Public Interest of China (201005024).
15H, 5 ꢂ OCH3); 13C NMR (DMSO-d6, 150 MHz)
d: 172.7,164.2,160.8,
158.7,156.5, 154.9, 153.1,152.8, 152.7,151.0,148.6,148.4,147.4,139.5,
137.3, 123.9, 122.9, 122.7, 122.1, 117.1, 117.0, 111.8, 111.3, 109.1, 109.04,
107.6, 102.6, 96.4, 93.5, 70.1, 68.7, 56.6, 56.6, 56.2, 55.8, 55.8; HRMS
calcd for (C36H31ClFN3O9 þ H)þ 704.1811, found 704.1820.
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