Biological evaluation and SAR analysis of O-methylated analogs of quercetin as inhibitors of cancer cell proliferation

Article abstract of DOI:10.1002/ddr.21181

Preclinical Research Using a high-throughout screening approach, the anticancer activities of 16 O-methylated (OMe) analogs of quercetin were assessed. The structure-activity relationships showed that OMe moieties at the 4′ and/or 7 positions were important for maintaining inhibitory activities against the 16 cancer cell lines. Furthermore, when the OH groups at the 3′ and 4′ positions were both replaced by OMe moieties, anticancer activity was enhanced.

Full text of DOI:10.1002/ddr.21181

DRUG DEVELOPMENT RESEARCH 75 : 455–462 (2014)
Research Article
Biological Evaluation and SAR Analysis of O-Methylated
Analogs of Quercetin as Inhibitors of Cancer
Cell Proliferation
Zhi-Hao Shi,1,2 Nian-Guang Li,1* Yu-Ping Tang,1* Qian-Ping Shi,1 Hao Tang,1 Wei Li,1
Xu Zhang,1 Hai-An Fu,3 and Jin-Ao Duan1
1National and Local Collaborative Engineering Center of Chinese Medicinal Resources
Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine,
Nanjing, Jiangsu 210023, China
2Department of Organic Chemistry, China Pharmaceutical University, Nanjing,
Jiangsu 211198, China
3Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322,
United States
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
Using a high-throughout screening approach, the anticancer activities of 16 O-methylated
(OMe) analogs of quercetin were assessed. The structure–activity relationships showed that OMe moieties
at the 4' and/or 7 positions were important for maintaining inhibitory activities against the 16 cancer cell
lines. Furthermore, when the OH groups at the 3' and 4' positions were both replaced by OMe moieties,
anticancer activity was enhanced. Drug Dev Res 75 : 455–462, 2014.
© 2014 Wiley Periodicals, Inc.
Key words: structure–activity relationship; quercetin; methylation; analog; anticancer
INTRODUCTION
[Braganhol et al., 2006], U2.US/MTX300 (osteosar-
coma) [Xie et al., 2011], HeLa (cervical cancer) [Vidya
et al., 2010], CWR22Rv1 (prostate cancer) [Hsieh and
Wu, 2009], MDA-MB-453 (breast cancer) [Choi et al.,
2008], HT-29 (colorectal) [Priego et al., 2008], myeloid
leukemia [Duraj et al., 2005], and oral cancer [Kang
et al., 2010]. The doses of quercetin that exhibit
Cancer, characterized by the uncontrolled
growth of abnormal cells is projected as the primary
cause of death in the future [Sashidhara et al., 2010]
with growth in lung [Lv et al., 2012; Bejjanki et al.,
2013], head and neck [Mannelli and Gallo, 2012],
melanoma [Choi et al., 2011], breast [Labrie et al.,
1999], and cervical [Parkin, 2006] cancer incidence
rising globally. There is thus an urgent need to develop
more effective drugs.
*Correspondence to: Nian-Guang Li and Yu-Ping Tang,
National and Local Collaborative Engineering Center of Chinese
Medicinal Resources Industrialization and Formulae Innovative
Quercetin is found in abundance in onions, tea
[Scalbert and Williamson, 2000], apples, broccoli, Medicine, Nanjing University of Chinese Medicine, Nanjing,
Jiangsu 210023, China.
E-mail: linianguang@njutcm.edu.cn; yupingtang@njutcm.edu.cn
berries [Nijveldt et al., 2001], and red wine [Ramos,
2008] and is of interest due to its variety of biological
Received 18 April 2014; Accepted 29 May 2014
properties. Predominant among these are its anti-
cancer activities that have been shown in vitro in a
Published online in Wiley Online Library (wileyonlinelibrary
variety of cancer cell lines including U138MG (glioma) .com). DOI: 10.1002/ddr.21181
© 2014 Wiley Periodicals, Inc.

456
SHI ET AL.
OH
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
HO
O
H3CO
O
HO
O
HO
OH
O
HO
O
O
HO
HO
OCH3
OH
OH
OH
OH
OH
OCH3
OH
OH
OH
OCH3O
OH
OH
O
6
OH
OH
O
2
OH
O
4
OH
O
1
OH
OH
O
3
5
OH
OH
OH
OH
OCH3
OH
OH
OH
OH
OCH3
H3CO
O
HO
O
H3CO
O
HO
O
H3CO
O
O
OCH3
OCH3
OCH3
OCH3
OH
CH3O
O
10
CH3O
O
11
O
12
CH3O
O
7
O
8
O
9
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
H3CO
O
O
H3CO
H3CO
O
HO
O
H3CO
O
OH
OCH3
OCH3
OH
OCH3
OH
O
15
CH3O
O
17
OH
O
13
CH3O
O
14
OH
O
16
Fig. 1. Structures of quercetin (1) and series of methylated quercetin analogs (2–17).
antiproliferative effects in vitro range from 3 to 50 mM et al., 2012]. The preparation of 3'-O-methylquercetin
[Lamson and Brignall, 2000; Gibellini et al., 2011]. (2) is shown in Figure 2. Based on the different reac-
In vivo quercetin can prevent induced carci- tivity of the five hydroxy groups in quercetin following a
nogenesis, particularly in the colon [Murakami et al., specific sequence: 4' > 7 > 3 > 3' > 5 [Bouktaib et al.,
2008], and can inhibit melanoma growth, invasion, 2002], benzylation of quercetin with 3.0 equivalent of
and metastatic potential [Caltagirone et al., 2000]. benzyl bromide produced the tribenzylated product 18,
When administered in the diet, quercetin inhibited the then partial methylation of the free phenolic function at
initiation, growth, and/or dissemination of induced C-3' position in 18 with iodomethane (the C-5 position
tumors in animal models [Yang et al., 2001].
is less reactive) afforded 19 in 92% yield. Finally,
The anticancer effects of quercetin have been deprotection of the benzyl groups gave 3'-O-
attributed to its ability to interfere with tumor methylquercetin (2) in 90% yield.
vascularization via inhibition of endothelial cell growth
4'-O-Methylquercetin (3) was hemisynthesized
and migration [Igura et al., 2001; Tan et al., 2003]. from quercetin (1) (Fig. 2) relying on selective and suc-
Quercetin reduces the expression and activity of matrix cessive protections of the different quercetin phenolic
metalloproteinase-2, and inhibits constitutive endo- functions [Li et al., 2009]. Treatment of 1 with
thelial nitric oxide synthase [Chiesi and Schwaller, dichlorodiphenylmethane in diphenyl ether at 175°C
1995; Tan et al., 2003]. Replacement of OH groups [Li et al. 2007] afforded the desired product 20 in 86%
with O-methylated (OMe) moieties can enhance the yield. Subsequently, reaction of 20 with an excess of
metabolic stability of flavones while retaining chloromethyl methyl ether and K2CO3 in acetone
antiproliferative potency [Cai et al., 2009]. We have afforded 21 with a free phenolic hydroxy group at the
reported the synthesis of a series of monomethylated C-5 position. Under hydrogen conditions using 10%
(2–6), dimethylated (7–12), trimethylated (13–15), palladium on carbon as a catalyst, the benzophenone
tetramethylated (16), and pentamethylated (17) ketal was cleaved to selectively afford 22 in 95%
quercetins [Li et al., 2009, 2011; Shi et al., 2012]. In the yield with no side products detected by thin-layer
present study, the potential anticancer activity of these chromatography (TLC) analysis. Treatment of 22
16 OMe quercetin analogs (Fig. 1) were determined in with 1.2 equivalent. of iodomethane led selectively to
an in vitro human disease-oriented cancer cell line 23 in 92% yield with a methyl group at C-4' position.
using high throughout screening (HTS) method [Li Finally, hydrochloric acid–catalyzed removing of the
et al., 2012], including human lung cancers, melanoma, methoxymethyl-protecting group afforded 4'-O-
cervical, neck and head, and human breast cancer cells. methylquercetin (3) in 90% yield.
3-O-Methylquercetin (4) [Li et al. 2004] was syn-
thesized as summarized in Figure 2. Selective
MATERIALS AND METHODS
benzylation of rutin (24) with benzyl bromide and
Synthesis
hydrolysis of the glycosidic bond with HCl led mainly to
The 16 OMe analogs of quercetin were synthe- the formation of the tribenzylated product 25, then
sized as previously described [Li et al., 2009, 2011; Shi regioselective methylation of C3-OH afforded 26 in
Drug Dev. Res.

ANTICANCER ACTIVITY OF QUERCETIN ANALOGS
457
OH
OH
OBn
4'
OBn
HO
O
7
HO
HO
O
BnO
BnBr, K2CO3, DMF
O
BnO
MeI, K2CO3, DMF
O
OCH3
3' OH
OH
OCH3
Pd/C (10 wt%), H2, EtOH
25 °C
3
OH
OH
OH
OBn
5
OH
OBn
25 °C
25 °C
OH
O
2
O
1
OH
O
18
OH
O
19
OH
OH
O
O
O
O
Ph
Ph
Ph
Ph
O
HO
O
MOMO
O
MOMCl, K2CO3, acetone
Pd/C (10 wt %), H2, THF/EtOH
OMOM
Ph2CCl2, Ph2O
175 °C
OH
25 °C
reflux
OH
O
1
OH
O
OH
O
21
20
OH
OH
OCH3
OH
OCH3
OH
MOMO
O
MOMO
O
HO
O
MeI, K2CO3, DMF
HCl (1.0 M) in Et2O/CH2Cl2
25 °C
OMOM
OMOM
OH
25 °C
OH
O
22
OH
O
23
OH
O
3
OH
OH
OBn
OBn
OBn
OBn
OH
OH
HO
O
BnO
O
BnO
O
HO
O
Pd/C (10 wt %), H2, THF/EtOH
25 °C
MeI, K2CO3, DMF
25 °C
(1) BnBr, K2CO3, DMF, 40 °C
O
OH
OCH3
OCH3
OH
O
O
OH
O
25
OH
O
26
OH
O
4
(2) 36.5% HCl, 95% EtOH, 70 °C
OH
OH
O
O
OH
OH
OH OH
OH
24
OH
OH
O
O
Ph
Ph
Ph
Ph
BnO
O
BnO
O
HO
O
O
O
MeI, K2CO3, DMF
Pd/C (10 wt %), H2, THF/EtOH
25 °C
O
Ph
Ph
OBn
OBn
OBn
OH
OH
25 °C
HO
O
O
OH
OH
O
27
OCH3O
OCH3O
BnBr, K2CO3, DMF
29
5
OH
OH
OH
25 °C
+
O
O
Ph
Ph
Ph
Ph
OH
O
H3CO
O
HO
O
H3CO
O
20
O
O
MeI, K2CO3, DMF
Pd/C (10 wt %), H2, THF/EtOH
25 °C
OBn
25 °C
OH
O
6
O
28
OH
O
30
Fig. 2. Synthesis of 1 to 6.
95% yield; subsequently the cleavage of the benzyl benzyl groups under hydrogenation conditions using
group by hydrogenolysis gave 3-O-methylquercetin (4) 10% palladium on carbon as the catalyst afforded 6 in
in 91% yield.
5-O-Methylquercetin
93% yield.
The synthesis of the dimethylated quercetins
(5)
and
7-O-
methylquercetin (6) were synthesized from 20 as (7–11) is shown in Figure 3. Methylation of the free
shown in Figure 2. Treatment of 20 with benzyl hydroxy function at C-3' and C-5 positions in 18 with an
bromide and K2CO3 afforded two easily separated prod- excess of iodomethane gave 31, then the cleavage of the
ucts: the 3,7-dibenzyl (27) and the 3-benzyl (28) benzyl groups by hydrogenolysis on 10% palladium on
isomers. Reaction of 27 with iodomethane led to 29 in carbon afforded 7. 20 reacted with iodomethane in the
94% yield, then the benzophenone ketal and benzyl presence of K2CO3 afforded 32, then the 3,7-O-
groups were deprotected with 10% palladium on dimethylquercetin 8 was directly obtained in 93% yield
carbon as the catalyst afforded 5 in 92% yield. Treat- after hydrogenolysis of the benzophenone ketal using
ment of 28 with iodomethane led selectively to 30 in 10% palladium on carbon. Compound 22 reacted
93% yield with the desired methyl group at C-7 posi- with iodomethane leading to 33 in 92% yield with
tion, then the deprotection of benzophenone ketal and two methyl group at C-3' and C-4' positions, then the
Drug Dev. Res.

458
SHI ET AL.
OH
OBn
OH
OBn
HO
O
O
O
BnO
O
BnO
O
OCH3
OCH3
MeI, K2CO3, DMF
Pd/C (10 wt %), H2, THF/EtOH
25 °C
OH
7
OBn
OBn
31
25 °C
CH3 O
OH
OH
OH
O
O
CH3 O
O
O
18
OH
OH
O
O
O
O
Ph
Ph
Ph
Ph
HO
H3CO
H3CO
MeI, K2CO3, DMF
25 °C
Pd/C (10 wt %), H2, THF/EtOH
OH
OCH3
32
OCH3
8
25 °C
O
O
OH
O
O
OH
OH
O
O
20
OCH3
OCH3
OH
OCH3
HO
MOMO
MOMO
MeI, K2CO3, DMF
25 °C
OH
OCH3
HCl (1.0 M) in Et2O/CH2Cl2
OH
9
OMOM
22
OMOM
33
25 °C
O
O
OH
O
OBn
OBn
OH
OH
OBn
OBn
BnO
HO
O
O
HO
O
O
BnO
O
O
O
O
MeI, K2CO3, DMF
Pd/C (10 wt %), H2, THF/EtOH
OH
25
OCH3
10
OCH3
34
25 °C
25 °C
OH
CH3 O
CH3O
OH
OH
O
O
O
Ph
Ph
Ph
Ph
O
O
H3CO
H3CO
O
O
O
Pd/C (10 wt %), H2, THF/EtOH
MeI, K2CO3, DMF
OBn
28
OBn
35
OH
11
25 °C
25 °C
OH
O
CH3
CH
3O
Fig. 3. Synthesis of 7 to 11.
hydrolysis of the methoxymethyl group with HCl gave 9 methoxymethyl group with hydrochloric acid gave 14 in
in 91% yield. Methylation of the free hydroxy groups at 92% yield (Fig. 4). The trimethylquercetin 15 was syn-
C-3 and C-5 positions in 25 with an excess of thesized from rutin (24) in two steps including
iodomethane and K2CO3 gave 34 in 91%, and the methylation and hydrolysis of the glycosidic bond.
deprotection of the benzyl groups by hydrogenolysis on Treatment of 1 with excess iodomethane in the pres-
10% palladium on carbon afforded 10 in 91% yield. An ence of K2CO3 afforded a mixture of three products:
excess of iodomethane and K2CO3 reacted with 28 the trimethylated quercetin 13, the tetramethylated
afforded 35, then the deprotection of the two kinds of quercetin 16 and the pentamethylated quercetin 17
protecting group in 35 by hydrogenolysis on 10% (Fig. 4), which were easily separated by chromatogra-
palladium on carbon afforded 11 in 92% yield.
phy on silica gel.
The synthesis of dimethylated quercetin 12,
trimethylated quercetins (13–15), tetramethylated
quercetin (16), and pentamethylated quercetin (17) are
shown in Figure 4. Treatment of 1 with iodomethane
Biological Screening
Human lung cancers (A549, H157, H460, 1792,
in the presence of K2CO3 afforded a mixture of two H266, Hop62, 1299, 292G, and Calu1), melanoma
main products: the 4',7-dimethyl isomer 12 and the (LOX-IMVI and M14), cervical (Hela), neck and head
3,4',7-trimethyl isomer 13 (Fig. 4) that were easily (M4E), and human breast cancer (SKBR) were from
separated by chromatography on silica gel. Treat- American Type Culture Collection (ATCC, Manassas,
ment of 22 with excess iodomethane afforded 36 Virginia) grown in RPMI 1640 containing 10% fetal calf
in 90% yield with the desired methyl groups at C-3', serum (FCS), 100 UI/mL penicillin G, and 100 mg/
C-4' and C-5 positions. Then hydrolysis of the mL streptomycin. Dimethyl sulphoxide (DMSO) was
Drug Dev. Res.

ANTICANCER ACTIVITY OF QUERCETIN ANALOGS
459
OH
OCH3
OH
OCH3
OH
HO
O
O
H3CO
O
O
H3CO
O
O
OH
+
MeI, K2CO3, DMF
25 °C
OH
1
OH
12
OCH3
13
OH
OH
OH
OH
HO
OCH3
OCH3
OCH3
OCH3
OH
O
O
MOMO
MeI, K2CO3, DMF
25 °C
O
O
MOMO
O
O
OH
HCl (1.0 M) in Et2O/CH2Cl2
OH
14
OMOM
36
OMOM
22
25 °C
CH3O
CH3O
OH
OCH3
OCH3
OCH3
HO
O
H3CO
MeI, K2CO3, DMF
O
O
H3CO
36.5% HCl, 95% EtOH
O
OH
rutinosyl
OCH3
rutinosyl
O
O
37
OH
15
25 °C
70 °C
24
OH
O
OH
OH O
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H3CO
O
O
H3CO
O
O
H3CO
O
O
HO
O
O
OH
OH
+
+
MeI, K2CO3, DMF
OCH3
16
OCH3
17
OCH3
13
OH
1
25 °C
OH
CH3O
OH
OH
Fig. 4. Synthesis of 12 to 17.
purchased from Sigma Chemical Co. (St. Louis, MO), relative to control was calculated. The resulting solu-
and Alamar blue was from Promega (Madison, Wiscon- tions were measured at optical density 560 (OD560
)
sin). Cells were seeded into 384-well plates (Costar# and the values used to calculate the inhibitory rates
3712) (800–1000 cell/well or 20–25 cells/μL, 45 μL using the equation (OD560 [100%] − OD560 [com-
medium/well) using a liquid dispenser (Thermo Fisher pound]) / (OD560[100%] − OD560[blank]) ×100%, where
Multidrop Combi, Waltham, Massachusetts) in a bio- the 100% group contained no compound and the blank
safety cabinet. Plates were placed in an incubator over- group contained only the cells. The IC50 value was
night to allow for attachment and recovery. Compound defined as the concentration of compound that killed
plates were utilized and prepared to yield 10 mM of 50% of the total cell population as compared with
compound in DMSO (stock) by robot (Sciclone soft- control cells at the end of the incubation period and was
ware, San Francisco, California) to generate eight con- derived using OriginPro 7.5 software (OriginLab,
centrations with serial dilution; wells were reserved on Hampton, MA).
each plate for background and vehicle control (0.5%
DMSO). With the use of the liquid handling system, the
RESULTS AND DISCUSSION
following day the cells were treated with compounds for
72 h, at final concentrations of 50, 25, 12.5, 6.25, 3.125,
The results of anticancer activities of the 16
1.56, 0.78, and 0.39 μM in triplicate. A volume of 5 μL/ quercetin analogs against the growth of 16 human
well Alamar blue was transferred into the assay plates for cancer cell lines are shown in Table 1.
a final concentration of 10%. The plates were exposed to
5-O-Methylquercetin (5) generally had reduced
an excitation wavelength of 530 nm, and the emission at inhibitory activities against the 16 cancer cell lines as
560 nm was recorded to determine whether any of the compared with quercetin (1). 4'-O-methylquercetin (3)
test compounds fluoresced at the emission wavelength and 7-O-methylquercetin (6) were more potent in
and thus interfere with the assay.
inhibiting cancer cell growth inhibition than quercetin
Plates were returned to the incubator, and the (1), indicating that the methylation at the 4' or 7 posi-
fluorescence was read at 4 h. The percent viability was tions improved activity. The 4',7-dimethoxyquercetin
expressed as fluorescence counts in the presence of test (12), where the OH groups at the 4',7 positions were
compound as a percentage of that in the vehicle control. methylated, had stronger antiproliferative activity com-
The mean value and standard error for each treatment pared to quercetin (1), with its IC50 values against A549,
were determined, and the percentage of cell viability H157, H460, 1792, 1944, M14, SKBR and Hela were
Drug Dev. Res.

460
SHI ET AL.
3.07, 3.45, 2.75, 3.36, 2.86, 4.29, 8.30 and 1.49 μM
respectively.
Although 3-O-methylquercetin (4) showed less
antiproliferative potency compared with quercetin (1),
methylation of the OH groups at the 3 and 7 positions
resulted in the most potent compound 8 with an IC50
value of 0.46 μM against 1944; this compound also
showed potent cancer cell growth inhibition for A549,
H157, H460, 1792, Hop62, and 1299 with IC50 values
of 5.40, 12.97, 7.02, 19.67, 13.18, and 15.57 μM,
respectively. Methylation of the 3' OH (2) reduced
antiproliferative potency with this compound only
showing potent cancer cell growth inhibition for A549,
H157, H460, 1792, H266, Hop62, and Hela with IC50
values of 10.78, 20.52, 12.48, 33.36, 42.37, 23.34, and
17.93 μM, respectively, its IC50 values being greater
50 μM against the other cell lines. Methylation of the
3' and 4' OH resulted in 3',4'-dimethoxyquercetin (9),
which had potent cancer cell growth inhibition with
IC50 values of less than 10 μM against A549, H157,
H460, 1944, H522, 1299, 292G, M4E and SKBR, IC50
values of 14.32, 17.32, 13.04, 14.76, 14.08, 14.17 μM
against 1792, H266, Hop62, Calu1, LOX-IMVI, and
M14, respectively, and an IC50 value of 24.4 μM
against HeLa. These results indicate that the 3',4'-
arrangement of OMe residues is important for inhib-
iting cancer growth and that the OMe analogs may be
superior to their OH counterparts. Furthermore,
introduction of OMe moiety at the 5 position in 9 was
important for growth arrest of these cells; compound
14 showed comparable cancer cell growth inhibition
with IC50 values of less than 10 μM against A549,
H157, H460, 1944, 1299, LOX-IMVI, Hela, and
SKBR. Compound 15, where the OH groups at 3', 4',
7 positions were methylated, showed more potent
cancer cell growth inhibition compared with 3',4'-
dimethoxyquercetin (9), with IC50 values of less than
10 μM except for the 1944 and 292G cell lines.
Methylation of the OH group at position 3 in
compound 15 yielded tetramethylated quercetin 16
with IC50 values of 0.39 and 0.38 μM against LOX-
IMVI and M14, respectively, which warrant further
investigation. Pentamethylated quercetin (17) had
reduced antiproliferative activity compared with
quercetin (1) except for the cancer cell lines 1299,
292G, and SKBR where it was more active.
CONCLUSION
The present study provides an initial assessment
of the structure–activity relationships of methylated
quercetins as inhibitors of cancer proliferation.
Methylation at the 4' or (and) 7 positions was important
in maintaining inhibitory activities against the 16
Drug Dev. Res.

ANTICANCER ACTIVITY OF QUERCETIN ANALOGS
461
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quercetin, but not apigenin or luteolin, induced apoptosis in
human myeloid leukemia cells and their resistant variants.
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cancer cell lines while dimethylation enhanced acti-
vity. These findings suggest that methylation of the
OH groups quercetin warrants further evaluation for
compounds as potential lead compounds for novel
anticancer agents.
Gibellini L, Pinti M, Nasi M, Montagna JP, De Biasi S, Roat E,
Bertoncelli L, Cooper EL, Cossarizza A. 2011. Quercetin and
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29:4025–4032.
ACKNOWLEDGMENTS
This work was supported by National Natural
Science Foundation of China (81274058, 21302225),
the Program for New Century Excellent Talents by the
Ministry of Education (NCET-12-0741), 333 High-
level Talents Training Project Funded by Jiangsu
Province, Six Talents Project Funded by Jiangsu Prov-
ince (2013-YY-010), Technology Innovation Venture
Fund by Nanjing University of Chinese Medicine
(CX201301), Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education
Institutions, Jiangsu Collaborative Innovation Center
of Chinese Medicinal Resources Industrialization
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