Quercetin analogs with high fetal hemoglobin-inducing activity

Article abstract of DOI:10.1007/s00044-019-02412-7

β-Thalassemia is the major health problems in developing countries, when affected patients and healthy carriers are numerous, resulting a total absence or severe decrease in the production of β-globin chains. The use of chemical agents for increasing the production of fetal hemoglobin (HbF) by reactivating γ-globin gene to balance excess α-globin chains is an alternative therapeutic approach. Therefore, the search for molecules exhibiting the property of inducing γ-globin gene expression is of great interest. In this report, we discovered that quercetin (1), the major flavonoid isolated from the heartwoods of the medicinal plant Anaxagorea luzonensis promoted the expression of γ-globin gene. Chemical modification of 1 to fourteen methyl ether analogs (2?15) was conducted. The structures of these compounds were established on the basis of their spectroscopic data and by comparison with those of the reported values. The parent flavonoid and its chemically modified analogs were investigated for their γ-globin gene induction for the first time. The parent compound 1 exhibited less induced γ-globin gene expression than cisplatin and hemin, the positive controls. 3,4′-Di-O-methylquercetin (7), the modified analog, significantly enhanced γ-globin gene expression with 2.6-fold change at 8 μM, which was slightly higher than cisplatin and hemin. Moreover, compounds 1 and 7 displayed less cytotoxic activity against K562::Δ^Gγ^AγEGFP cells than cisplatin. Structure-activity relationship (SAR) study revealed that the methoxyl groups at the 3- and 4?-positions and the free hydroxyl group at the 7-position are required for strong HbF-inducing activity.

Full text of DOI:10.1007/s00044-019-02412-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02412-7
ORIGINAL RESEARCH
Quercetin analogs with high fetal hemoglobin-inducing activity
Wachirachai Pabuprapap1 Yanisa Wassanatip2 Pichit Khetkam1 Waraluck Chaichompoo1
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Sukanya Kunkaewom1 Pongpan Senabud1 Janejira Hata1 Ratchanaporn Chokchaisiri3 Saovaros Svasti2
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1
Apichart Suksamrarn
Received: 29 March 2019 / Accepted: 19 July 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
β-Thalassemia is the major health problems in developing countries, when affected patients and healthy carriers are
numerous, resulting a total absence or severe decrease in the production of β-globin chains. The use of chemical agents for
increasing the production of fetal hemoglobin (HbF) by reactivating γ-globin gene to balance excess α-globin chains is an
alternative therapeutic approach. Therefore, the search for molecules exhibiting the property of inducing γ-globin gene
expression is of great interest. In this report, we discovered that quercetin (1), the major flavonoid isolated from the
heartwoods of the medicinal plant Anaxagorea luzonensis promoted the expression of γ-globin gene. Chemical modification
of 1 to fourteen methyl ether analogs (2−15) was conducted. The structures of these compounds were established on the
basis of their spectroscopic data and by comparison with those of the reported values. The parent flavonoid and its
chemically modified analogs were investigated for their γ-globin gene induction for the first time. The parent compound 1
exhibited less induced γ-globin gene expression than cisplatin and hemin, the positive controls. 3,4′-Di-O-methylquercetin
(7), the modified analog, significantly enhanced γ-globin gene expression with 2.6-fold change at 8 μM, which was slightly
higher than cisplatin and hemin. Moreover, compounds 1 and 7 displayed less cytotoxic activity against K562::ΔGγAγEGFP
cells than cisplatin. Structure-activity relationship (SAR) study revealed that the methoxyl groups at the 3- and 4ʹ-positions
and the free hydroxyl group at the 7-position are required for strong HbF-inducing activity.
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Keywords Quercetin Methylated quercetin Structure-activity relationship β-Thalassemia Hemoglobin F
Introduction
the β-globin gene (HBB) on chromosome 11 (Cao and
Galanello 2010). It is identified by the absence or decreased
production of β-globin chain synthesis in human erythroid
cells. The continued production of normal levels of the α-
globin chain exceeds the binding capacity of the β-globin
chain, leading to globin chain imbalance. Excess α-globin
chains precipitate in erythroid progenitor cells, resulting in a
buildup of reactive oxygen species, causing ineffective
erythropoiesis (Liu et al. 2013). The main cores of therapy
in β-thalassemia patients now are a regular schedule blood
transfusion supplemented with iron chelating agents. The
only curative treatment is stem cell transplantation. How-
ever, it has some limitations such as it is hard to find HLA
match donor and high cost (Rund and Rachmilewitz 2005).
In recent years, the use of fetal hemoglobin (HbF, α2γ2)
inducing drugs is regarded as the appropriate therapy for
hemoglobinopathies. HbF expression in β-thalassemia will
decrease the accumulation and precipitation of α-globin
chains, and thus reduces the unproductive erythropoiesis
(Fard et al. 2013), resulted in better conditions and
β-Thalassemia is one of the major types of thalassemia that
is hereditary blood disorders characterized by anomalies in
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02412-7) contains supplementary
material, which is available to authorized users.
* Apichart Suksamrarn
asuksamrarn@yahoo.com
s_apichart@ru.ac.th
1
Department of Chemistry and Center of Excellence for Innovation
in Chemistry, Faculty of Science, Ramkhamhaeng University,
Bangkok 10240, Thailand
2
Thalassemia Research Center, Institute of Molecular Biosciences
and Department of Biochemistry, Faculty of Science, Mahidol
University, Bangkok 10400, Thailand
3
Department of Chemistry, School of Science, University of
Phayao, Phayao 56000, Thailand

Medicinal Chemistry Research
Scheme 1 Synthesis of methylated quercetin analogs 2−15. Reagents
and conditions: a (1) CH3I (1.0 eqv.), K2CO3 (1.25 eqv.), acetone, r.t.,
2 h; (2) CH3I (0.5 eqv.), r.t., 1 h; b (1) CH3I (2.0 eqv.), K2CO3 (2.5
eqv.), acetone, r.t., 2 h; (2) CH3I (1.0 eqv.), r.t., 1 h; c (1) CH3I (3.0
eqv.), K2CO3 (3.75 eqv.), acetone, r.t., 2 h; (2) CH3I (1.5 eqv.), r.t.,
1 h; d (1) K2CO3 (10.0 eqv.), acetone, reflux, 30 min.; (2) CH3I
(excess), reflux, 24 h
transfusion independence of the patients. Accordingly, the
stimulation of γ-globin chain production is an alternative
treatment for β-thalassemia and sickle cell anemia patients.
Although the pharmacological agents for HbF induction,
such as 5-azacytidine, cisplatin and hydroxyurea, have been
studied for many years, they have low efficiency and high
toxicity (El-Beshlawy et al. 2009). Due to these limitations,
the research of new types of pharmacological agents that
can induce HbF with greater potency and less toxicity
seems to be necessary.
Plant-derived natural products have long been and will
continue to be extremely important as sources of medicinal
agents and models for design, synthesis, and structural
modification of novel substances for treating diseases. In
many cases, it was found that analogs of compounds
derived from structural modification exhibited higher bio-
logical activity. However, research on the therapeutic
effects of plants on thalassemia has not much been reported.
We previously discovered that the diterpenoids from the
aerial parts of Curcuma comosa enhanced HbF-induction
potency using a K562 reporter cell line with the enhanced
green fluorescence protein (EGFP) gene under the control of
a
Gγ-globin promoter (Chokchaisiri et al. 2010). We have
also found that the curcuminoids isolated from Curcuma
longa, particularly the modified analog hexahy-
drobisdemethoxycurcumin, showed potential γ-globin
enhancement (Chaneiam et al. 2013). Quercetin (3,5,7,3ʹ,4ʹ-
pentahydroxyflavone, 1) (Scheme 1) is a ubiquitous bio-
flavonoid present in most of the plants, fruits, and vege-
tables (D’Andrea 2015). Previous phytochemical
investigations revealed the presence of quercetin in many
plant species including Anaxagorea luzonensis, which
contained a large quantity of this flavonoid (Gonda et al.
2000). Quercetin (1) is wide pharmacologically important in
terms of anti-inflammatory (Li et al. 2016), anticancer
(Zheng et al. 2012), α-glucosidase inhibitory (Li et al.
2009a) activities, and is a beneficial powerful antioxidant
(Boots et al. 2008).
In our preliminary investigation on phytochemicals with
HbF induction from Thai medicinal plants, we found that
quercetin (1) isolated as the major constituent from the
active crude EtOAc extract of the heartwood of A. luzo-
nensis enhanced HbF expression. However, the activity of
this compound was less than those of cisplatin and hemin,

Medicinal Chemistry Research
the positive controls. Investigation on the chemical mod-
ification of quercetin (1) for HbF induction has not been
reported. We therefore decided to modify the structure of
compound 1 to analogs and the methyl ether analogs were
chosen for this study. This report describes the isolation of
compound 1 from A. luzonensis and structural modification
of compound 1 to the methyl ether analogs 2−15, and
evaluation of HbF-inducing activity of the parent and the
synthesized analogs.
CH2Cl2−MeOH and MeOH with increasing amounts of the
more polar solvent to give two subfractions (E10.1−E10.2).
Subfraction E10.2 (5.0 g) upon standing, a yellow solid
separated out, which was rechromatographed eluting under
isocratic condition of 6% MeOH in CH2Cl2 to yield 1
(3.5 g) as pale yellow powder.
Quercetin (1): pale yellow powder; m.p. >300 °C
(MeOH) (lit. m.p. 315.0 °C); IR νmax 3356, 3090, 2956,
1
1654, 1431, 1090, 881, 817, 786 cm−1; H NMR (CD3OD,
400 MHz,): δ = 7.71 (1H, d, J = 2.0 Hz, H-2′), 7.62 (1H,
dd, J = 8.5, 2.0 Hz, H-6′), 6.88 (1H, d, J = 8.5 Hz, H-5′),
6.37 (1H, d, J = 1.8 Hz, H-8), 6.18 (1H, d, J = 1.8 Hz, H-6);
13C NMR (CD3OD, 100 MHz,): δ = 177.8 (C, C-4), 166.1
(C, C-7), 163.0 (C, C-5), 158.7 (C, C-9), 149.2 (C, C-4′),
148.5 (C, C-2), 146.7 (C, C-3′), 137.7 (C, C-3), 124.6 (C,
C-1′), 122.1 (CH, C-6′), 116.7 (CH, C-2′), 116.5 (CH, C-
5′), 104.5 (C, C-10), 99.7 (CH, C-6), 94.9 (CH, C-8); HR-
TOF-MS (ESI+): m/z 325.0307 [M + Na]+ (calcd for
C15H10O7Na, 325.0318). The physical and NMR spectral
data were in accordance with those reported in the literature
(Mouffok et al. 2012).
Materials and methods
General
Merck pre-coated silica gel 60 F254 plates were used for
TLC. Spots on TLC were detected under UV light and by
spraying with anisaldehyde-H2SO4 reagent followed by
heating. Column chromatography was carried out using
Merck silica gel 60 (<0.063 mm and 0.063−0.200 mm).
The solvents used were redistilled commercial grade for
chromatography and analytical grade for synthesis.
Melting points were determined using an electrothermal
melting point apparatus and were uncorrected. IR spectra
were obtained using a Perkin-Elmer FT-IR Spectrum
Synthesis of quercetin methyl ether analogs
Preparation of mono- and di-O-methylated analogs of
quercetin (1)
1
spectrophotometer. H and 13C NMR spectra were recor-
ded on a Bruker ASCEND 400 spectrometer, operating at
400 (1H) and 100 (13C) MHz. HR-ESI-TOF-MS spectra
were measured with a Bruker micrOTOF QII mass
spectrometer.
To a mixture of quercetin (1) (200 mg, 0.66 mmol) and
anhydrous K2CO3 (114 mg, 0.82 mmol) in acetone (20 mL)
was added CH3I (40 µL, d = 2.28 g/mL, 0.66 mmol) drop-
wise. The mixture was kept stirring at room temperature for
2 h, and the progress of the reaction was monitored by TLC.
A second lot (20 µL, 0.33 mmol) of CH3I was added and
stirring was continued for another 1 h. Water (50 mL) was
added and the mixture was extracted with EtOAc. The
combined organic layer was washed with water, dried over
anhydrous Na2SO4, and the solvent was evaporated in
vacuo. The crude products were chromatographed on
Sephadex LH-20 column eluting with MeOH to give three
fractions (A1−A3). Fraction A1 was subjected to column
chromatography over silica gel eluted under isocratic con-
dition of 100% CH2Cl2 to yield the di-O-methyl ethers 4
(34 mg, 25%) and 5 (26 mg, 19%). Fraction A3 was
repeatedly chromatographed over silica gel eluted under
isocratic condition of CH2Cl2−MeOH (10:0.05) to furnish
the mono-O-methyl ethers 2 (39 mg, 30%) and 3 (25 mg,
19%), based on the unrecovered starting material, and the
starting material 1 (80 mg).
Plant material
The heartwoods of A. luzonensis were purchased from
Tai-an-jan herbal store, Bangkok, Thailand, in February
2013. A voucher specimen (Apichart Suksamrarn, No. 079)
is deposited at the Faculty of Science, Ramkhamhaeng
University.
Extraction and isolation of quercetin
The dried heartwoods of A. luzonensis (10.0 kg) were milled
and macerated successively with n-hexane and EtOAc to
yield, after evaporation of the solvents under reduced
pressure, the n-hexane (110.0 g) and EtOAc (995.0 g)
extracts, respectively.
The EtOAc extract (990.0 g) was fractionated by column
chromatography, using a gradient solvent system of n-
hexane, n-hexane−EtOAc, EtOAc, EtOAc−MeOH and
MeOH with increasing amounts of the more polar solvent.
The eluates were examined by TLC, and ten combined
fractions (E1−E10) were obtained. Fraction E10 (130.1 g)
was subjected to column chromatography, using CH2Cl2,
7-Mono-O-methylquercetin (2): yellow amorphous solid;
IR νmax 3449, 3211, 2952, 2848, 1661, 1417, 1096, 932,
1
814, 786 cm−1; H NMR (CD3OD, 400 MHz,): δ = 7.75
(1H, d, J = 2.1 Hz, H-2′), 7.65 (1H, dd, J = 8.4, 2.1 Hz,
H-6′), 6.86 (1H, d, J = 8.4 Hz, H-5′), 6.58 (1H, d, J =

Medicinal Chemistry Research
2.1 Hz, H-8), 6.30 (1H, d, J = 2.1 Hz, H-6), 3.88 (3H, s, 7-
OCH3); 13C NMR (CD3OD, 100 MHz,): δ = 177.4 (C, C-4),
166.9 (C, C-7), 162.2 (C, C-5), 158.0 (C, C-9), 148.8 (C, C-
4′), 146.2 (C, C-3′), 137.5 (C, C-2), 130.6 (C, C-3), 124.0
(C, C-1′), 121.6 (CH, C-6′), 116.1 (CH, C-5′), 116.0 (CH,
C-2′), 105.3 (C, C-10), 98.4 (CH, C-6), 92.6 (CH, C-8),
56.3 (CH3, 7-OCH3); HR-TOF-MS (ESI+): m/z 339.0473
[M + Na]+ (calcd for C16H12O7Na, 339.0475). The NMR
spectral data were compatible to those reported previously
(Saewan et al. 2011).
(CH, C-5′), 106.0 (C, C-10), 98.1 (CH, C-6), 92.5 (CH, C-
8), 60.2 (CH3, 3-OCH3), 56.0 (CH3, 7-OCH3); HR-TOF-
MS (ESI+): m/z 353.0648 [M + Na]+ (calcd for
C17H14O7Na, 353.0631). The NMR spectral data were in
agreement with those of the reported values (Valesi et al.
1972; Wang et al. 1989).
Preparation of di- and tri-O-methylated analogs of
quercetin (1)
3-Mono-O-methylquercetin (3): yellow amorphous solid;
IR νmax 3308, 3096, 2980, 2841, 1587, 1439, 1032, 1002,
Compound 1 (200 mg, 0.66 mmol) was methylated in the
same manner to the preparation of compounds 2−5 except
that more amounts of anhydrous K2CO3 (228 mg,
1.65 mmol) and CH3I (80 µL, 1.32 mmol) were used, fol-
lowed by further addition of another lot (40 µL, 0.66 mmol)
of CH3I. After the work-up, the crude products were sepa-
rated on Sephadex LH-20 column eluting with 10% CH2Cl2
in MeOH to give five fractions (B1−B5). Fraction B1 was
purified by column chromatography over silica gel eluted
under gradient solvent system of hexane−EtOAc (5:2 to
100% EtOAc) to afford the tri-O-methyl ethers 8 (43 mg,
27%) and 9 (50 mg, 32%), based on the unrecovered
starting material. Fractions B3 and B4 were rechromato-
graphed twice over silica gel eluted under gradient solvent
system of EtOAc−hexane−CH2Cl2 (3:2:2 to 100% EtOAc)
to yield the di-O-methyl ethers 4 (24 mg, 20%), 5 (20 mg,
13%), 6 (17 mg, 11%), 7 (8 mg, 5%), based on the unrec-
overed starting material, and the starting material 1 (64 mg).
3,3′-Di-O-methylquercetin (6): yellow amorphous solid;
IR νmax 3414, 3266, 2917, 2850, 1658, 1433, 1156, 930,
1
823, 787 cm−1; H NMR (CD3OD, 400 MHz,): δ = 7.60
(1H, d, J = 1.6 Hz, H-2′), 7.51 (1H, dd, J = 8.4, 1.6 Hz, H-
6′), 6.88 (1H, d, J = 8.4 Hz, H-5′), 6.37 (1H, d, J = 2.4 Hz,
H-8), 6.17 (1H, d, J = 2.4 Hz, H-6), 3.76 (3H, s, 3-OCH3);
13C NMR (CD3OD, 100 MHz,): δ = 179.9 (C, C-4), 165.9
(C, C-7), 163.0 (C, C-5), 158.3 (C, C-9), 157.9 (C, C-2),
149.9 (C, C-4′), 146.4 (C, C-3′), 139.4 (C, C-3), 122.8 (C,
C-1′), 122.2 (CH, C-6′), 116.4 (CH, C-2′), 116.3 (CH, C-
5′), 105.7 (C, C-10), 99.7 (CH, C-6), 94.6 (CH, C-8), 60.4
(CH3, 3-OCH3); HR-TOF-MS (ESI+): m/z 339.0478 [M +
Na]+ (calcd for C16H12O7Na, 339.0475). The NMR spectral
data were in accordance with those of the reported values
(Lee et al. 2003).
7,4′-Di-O-methylquercetin (4): yellow amorphous solid;
IR νmax 3403, 3178, 2945, 2847, 1655, 1432, 1110, 1006,
915, 819, 789 cm−1
;
1H NMR (CDCl3 + 5 drops of
CD3OD, 400 MHz,): δ = 7.78 (1H, dd, J = 8.6, 2.1 Hz, H-
6′), 7.74 (1H, d, J = 2.1 Hz, H-2′), 6.94 (1H, d, J = 8.6 Hz,
H-5′), 6.46 (1H, d, J = 2.0 Hz, H-8), 6.33 (1H, d, J =
2.0 Hz, H-6), 3.94 (3H, s, 4′-OCH3), 3.85 (3H, s, 7-OCH3);
13C NMR (CDCl3 + 5 drops of CD3OD, 100 MHz,): δ =
174.7 (C, C-4), 165.0 (C, C-7), 159.9 (C, C-5), 156.1 (C, C-
9), 156.1 (C, C-2), 147.9 (C, C-4′), 144.9 (C, C-3′), 135.4
(C, C-3), 123.3 (C, C-1′), 120.2 (CH, C-6′), 113.2 (CH, C-
2′), 109.9 (CH, C-5′), 103.4 (C, C-10), 97.2 (CH, C-6), 91.5
(CH, C-8), 55.3 (CH3, 4′-OCH3), 55.2 (CH3, 7-OCH3); HR-
TOF-MS (ESI+): m/z 353.0645 [M + Na]+ (calcd for
C17H14O7Na, 353.0631). The NMR spectral data were in
agreement with those reported in the literature (Haraguchi
et al. 1992).
1
873, 834 cm−1; H NMR (CDCl3 + 5 drops of CD3OD,
400 MHz,): δ = 7.65 (1H, d, J = 2.0 Hz, H-2′), 7.61 (1H,
dd, J = 8.4, 2.0 Hz, H-6′), 6.99 (1H, d, J = 8.4 Hz, H-5′),
6.38 (1H, d, J = 2.0 Hz, H-8), 6.23 (1H, d, J = 2.0 Hz, H-6),
3.93 (3H, s, 3′-OCH3), 3.79 (3H, s, 3-OCH3); 13C NMR
(CDCl3 + 5 drops of CD3OD, 100 MHz,): δ = 177.9 (C, C-
4), 162.8 (C, C-7), 160.9 (C, C-5), 156.1 (C, C-9), 155.1 (C,
C-2), 147.6 (C, C-4′), 145.7 (C, C-3′), 137.8 (C, C-3),
121.8 (CH, C-6′), 121.5 (C, C-1′), 113.9 (CH, C-5′), 110.2
(CH, C-2′), 104.5 (C, C-10), 98.1 (CH, C-6), 93.3 (CH, C-
8), 59.4 (CH3, 3-OCH3), 55.3 (CH3, 3′-OCH3); HR-TOF-
MS (ESI+): m/z 353.0620 [M + Na]+ (calcd for
C17H14O7Na, 353.0631). The NMR spectral data were in
accordance with those reported in the literature (Al-Dabbas
et al. 2006).
3,7-Di-O-methylquercetin (5): yellow amorphous solid;
IR νmax 3397, 3177, 2947, 2849, 1657, 1496, 1110, 916,
1
820, 790 cm−1; H NMR (CDCl3 + 5 drops of CD3OD,
400 MHz,): δ = 7.64 (1H, d, J = 2.2 Hz, H-2′), 7.56 (1H,
dd, J = 8.4, 2.2 Hz, H-6′), 6.90 (1H, d, J = 8.4 Hz, H-5′),
6.61 (1H, d, J = 1.6 Hz, H-8), 6.33 (1H, d, J = 1.6 Hz, H-6),
3.88 (3H, s, 7-OCH3), 3.79 (3H, s, 3-OCH3); 13C NMR
(CDCl3 + 5 drops of CD3OD, 100 MHz,): δ = 179.1 (C, C-
4), 165.9 (C, C-7), 161.6 (C, C-5), 157.3 (C, C-2), 157.1 (C,
C-9), 148.6 (C, C-4′), 145.1 (C, C-3′), 138.8 (C, C-3),
121.9 (C, C-1′), 121.7 (CH, C-6′), 115.7 (CH, C-2′), 115.6
3,4ʹ-Di-O-methylquercetin (7): yellow amorphous solid;
IR νmax 3443, 3273, 3078, 2941, 2841, 1651, 1436, 1089,
915, 880, 795 cm−1
;
1H NMR (CDCl3 + 5 drops of
CD3OD, 400 MHz,): δ = 7.64 (1H, dd, J = 8.5, 2.0 Hz, H-
6′), 7.61 (1H, d, J = 2.0 Hz, H-2′), 6.91 (1H, d, J = 8.5 Hz,
H-5′), 6.34 (1H, d, J = 2.0 Hz, H-8), 6.21 (1H, d, J =
2.0 Hz, H-6), 3.92 (3H, s, 4′-OCH3), 3.77 (3H, s, 3-OCH3); 13
C
NMR (CDCl3 + 5 drops of CD3OD, 100 MHz,): δ = 178.2

Medicinal Chemistry Research
(C, C-4), 163.5 (C, C-7), 160.9 (C, C-5), 156.4 (C, C-9),
155.3 (C, C-2), 148.9 (C, C-4′), 145.1 (C, C-3′), 138.3 (C,
C-3), 122.5 (C, C-1′), 120.9 (CH, C-6′), 114.1 (CH, C-2′),
110.2 (CH, C-5′), 104.7 (C, C-10), 98.5 (CH, C-6), 93.6
(CH, C-8), 59.7 (CH3, 3-OCH3), 55.5 (CH3, 4′-OCH3); HR-
TOF-MS (ESI+): m/z 353.0626 [M + Na]+ (calcd for
C17H14O7Na, 353.0631). The NMR spectral data were in
accordance with the reported values (Kwon and Kim 2003).
3,7,4ʹ-Tri-O-methylquercetin (8): yellow amorphous
solid; IR νmax 3360, 3003, 2935, 2843, 1645, 1498, 1091,
added and stirring was continued for another 1 h. After the
work-up, the impure product was crystallized with MeOH
to yield the tetra-O-methyl ether 14 (71 mg, 30%), based on
the unrecovered starting material. The filtrate was con-
centrated and separated on Sephadex LH-20 column eluting
with 30% CH2Cl2 in MeOH to give seven fractions (C1
−C7). The combined fraction (fractions C2−C5) was pur-
ified by column chromatography using hexane−CH2Cl2
−EtOAc (10:1:1 to 100% EtOAc) as eluent to afford the tri-
O-methyl ethers 8 (38 mg, 17%), 9 (47 mg, 21%), and 10
(25 mg, 11%), based on the unrecovered starting material.
Purification of fraction C6 using Sephadex LH-20 column
eluting with 100% MeOH followed by column chromato-
graphy twice eluted under isocratic condition of hexane
−CH2Cl2−MeOH (2:4:0.05) furnished the tri-O-methyl
ethers 11 (2 mg, 1%), 12 (14 mg, 6%) and 13 (2 mg, 1%),
based on the unrecovered starting material and the starting
material 1 (5 mg).
924, 814, 767 cm−1
;
1H NMR (CDCl3 + 5 drops of
CD3OD, 400 MHz,): δ = 7.57 (1H, d, J = 2.0 Hz, H-2′),
7.57 (1H, dd, J = 8.8, 2.0 Hz, H-6′), 6.87 (1H, d, J =
8.8 Hz, H-5′), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.24 (1H, d, J
= 2.0 Hz, H-6), 3.86 (3H, s, 4′-OCH3), 3.77 (3H, s, 7-
OCH3), 3.71 (3H, s, 3-OCH3); 13C NMR (CDCl3 + 5 drops
of CD3OD, 100 MHz,): δ = 178.6 (C, C-4), 165.3 (C, C-7),
161.2 (C, C-5), 156.6 (C, C-9), 156.1 (C, C-2), 149.6 (C, C-
4′), 145.7 (C, C-3′), 138.7 (C, C-3), 122.8 (C, C-1′), 121.0
(CH, C-6′), 114.7 (CH, C-2′), 110.6 (CH, C-5′), 105.6 (C,
C-10), 97.7 (CH, C-6), 92.0 (CH, C-8), 59.9 (CH3, 3-
OCH3), 55.6 (CH3, 7-OCH3), 55.6 (CH3, 4′-OCH3); HR-
TOF-MS (ESI+): m/z 367.0785 [M + Na]+ (calcd for
C18H16O7Na, 367.0788). The NMR spectral data were in
agreement with those of the reported values (Karimova
et al. 2015).
3,7,3′-Tri-O-methylquercetin (10): yellow amorphous
solid; IR νmax 3359, 3087, 2973, 2843, 1645, 1461, 1091,
957, 825, 796 cm−1
;
1H NMR (CDCl3 + 5 drops of
CD3OD, 400 MHz,): δ = 7.65 (1H, d, J = 1.7 Hz, H-2′),
7.62 (1H, dd, J = 8.4, 1.7 Hz, H-6′), 6.97 (1H, d, J =
8.4 Hz, H-5′), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.31 (1H, d,
J = 2.0 Hz, H-6), 3.93 (3H, s, 3′-OCH3), 3.83 (3H, s, 7-
OCH3), 3.79 (3H, s, 3-OCH3); 13C NMR (CDCl3 + 5 drops
of CD3OD, 100 MHz,): δ = 178.0 (C, C-4), 164.7 (C, C-7),
160.9 (C, C-5), 156.0 (C, C-9), 155.5 (C, C-2), 147.9 (C, C-
4′), 145.9 (C, C-3′), 138.1 (C, C-3), 121.5 (C, C-1′), 122.0
(CH, C-6′), 114.1 (CH, C-5′), 110.4 (CH, C-2′), 105.3 (C,
C-10), 97.2 (CH, C-6), 91.6 (CH, C-8), 59.5 (CH3, 3-
OCH3), 55.4 (CH3, 3′-OCH3), 55.2 (CH3, 7-OCH3); HR-
TOF-MS (ESI+): m/z 367.0781 [M + Na]+ (calcd for
C18H16O7Na, 367.0788). The NMR spectral data were in
agreement with those reported in the literature (Al-Dabbas
et al. 2006).
7,3′,4′-Tri-O-methylquercetin (9): yellow amorphous
solid; IR νmax 3347, 3089, 2974, 2846, 1646, 1438, 1091,
887, 814, 795 cm−1
;
1H NMR (CDCl3 + 5 drops of
CD3OD, 400 MHz,): δ = 7.74 (1H, dd, J = 8.6, 1.9 Hz, H-
6′), 7.69 (1H, d, J = 1.9 Hz, H-2′), 6.91 (1H, d, J = 8.6 Hz,
H-5′), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.28 (1H, d, J =
2.0 Hz, H-6), 3.90 (3H, s, 3′-OCH3), 3.89 (3H, s, 4′-OCH3),
3.81 (3H, s, 7-OCH3); 13C NMR (CDCl3 + 5 drops of
CD3OD, 100 MHz,): δ = 175.2 (C, C-4), 165.4 (C, C-7),
160.3 (C, C-5), 156.5 (C, C-9), 150.5 (C, C-4′), 148.5 (C,
C-3′), 145.7 (C, C-2), 135.8 (C, C-3), 123.2 (C, C-1′),
121.3 (CH, C-6′), 110.6 (CH, C-5′), 110.3 (CH, C-2′),
103.8 (C, C-10), 97.6 (CH, C-6), 91.9 (CH, C-8), 55.8,
55.7, 55.7 (3 × CH3, 7-OCH3, 3′-OCH3, 4′-OCH3); HR-
TOF-MS (ESI+): m/z 367.0798 [M + Na]+ (calcd
C18H16O7Na, 367.0788). The NMR spectral data were in
accordance with those reported in the literature (Dong et al.
1999; Shi et al. 2008).
5,7,3ʹ-Tri-O-methylquercetin (11): yellow amorphous
solid; IR νmax 3426, 3009, 2974, 2841, 1612, 1496, 1125,
1009, 814, 790 cm−1 1H NMR (CDCl3 + 5 drops of
;
CD3OD, 400 MHz,): δ = 7.77 (1H, d, J = 1.0 Hz, H-2′),
7.67 (1H, br d, J = 8.3 Hz, H-6′), 6.96 (1H, d, J = 8.3 Hz,
H-5′), 6.50 (1H, br s, H-8), 6.30 (1H, br s, H-6), 3.93 (3H,
s, 3′-OCH3), 3.91 (3H, s, 5-OCH3), 3.86 (3H, s, 7-OCH3);
13C NMR (CDCl3 + 5 drops of CD3OD, 100 MHz,): δ =
171.9 (C, C-4), 164.3 (C, C-7), 160.3 (C, C-5), 158.6 (C,
C-9), 147.3 (C, C-4′), 146.6 (C, C-3′), 142.6 (C, C-2),
137.2 (C, C-3), 122.9 (C, C-1′), 120.9 (CH, C-6′), 114.6
(CH, C-5′), 110.3 (CH, C-2′), 106.1 (C, C-10), 95.6 (CH,
C-6), 92.3 (CH, C-8), 56.1 (CH3, 3′-OCH3), 55.9 (CH3, 5-
OCH3), 55.7 (CH3, 7-OCH3); HR-TOF-MS (ESI+): m/z
367.0786 [M + Na]+ (calcd for C18H16O7Na, 367.0788).
This is the first report on the spectroscopic data of this
compound.
Preparation of higher methylated analogs of quercetin (1)
To a mixture of quercetin (1) (200 mg, 0.66 mmol) and
anhydrous K2CO3 (342 mg, 2.47 mmol) in acetone (20 mL)
was added CH3I (120 µL, d = 2.28 g/mL, 1.98 mmol)
dropwise. The mixture was kept stirring at room tempera-
ture for 2 h and the progress of the reaction was monitored
by TLC. A second lot (60 µL, 0.99 mmol) of CH3I was

Medicinal Chemistry Research
3,3ʹ,4ʹ-Tri-O-methylquercetin (12): yellow amorphous
solid; IR νmax 3475, 3090, 2980, 2845, 1659, 1460, 1093,
Permethylation of quercetin (1)
916, 867, 767 cm−1
;
1H NMR (CDCl3 + 5 drops of
A mixture of quercetin (1) (15 mg, 0.05 mmol), anhydrous
potassium carbonate (70 mg, 0.5 mmol) and acetone (6 mL)
was heated under reflux for 30 min and the reaction mixture
was then cooled to room temperature. CH3I (2 mL, excess)
was added and the reaction mixture was then refluxed for
24 h. The reaction mixture was poured into cold water and
the mixture was extracted with EtOAc; the solution was
concentrated in vacuo and left to stand. The solid that
separated out was filtered and washed with MeOH to give
compound 15 (21.8 mg, 84%).
3,5,7,3ʹ,4ʹ-Penta-O-methylquercetin (15): orange amor-
phous solid; IR νmax 2997, 2936, 2841, 1712, 1600, 1106,
895, 862, 767 cm−1; 1H NMR (CDCl3, 400 MHz,): δ = 7.24
(1H, d, J = 2.1 Hz, H-2′), 7.21 (1H, dd, J = 8.2, 2.1 Hz, H-
6′), 6.50 (1H, d, J = 8.2 Hz, H-5′), 6.02 (1H, d, J = 2.2 Hz,
H-8), 5.86 (1H, d, J = 2.2 Hz, H-6), 3.48 (9H, s, 5-OCH3,
3′-OCH3, 4′-OCH3), 3.42 (3H, s, 7-OCH3), 3.39 (3H, s, 3-
OCH3); 13C NMR (CDCl3, 100 MHz,): δ = 173.9 (C, C-4),
163.8 (C, C-7), 160.8 (C, C-5), 158.6 (C, C-9), 152.4 (C, C-
2), 150.6 (C, C-4′), 148.5 (C, C-3′), 141.0 (C, C-3), 123.2
(C, C-1′), 121.5 (CH, C-6′), 111.0 (CH, C-2′), 110.6 (CH,
C-5′), 109.3 (C, C-10), 95.6 (CH, C-6), 92.3 (CH, C-8),
59.8 (CH3, 3-OCH3), 56.3 (CH3, 3′-OCH3), 55.9 (CH3, 4′-
OCH3), 55.8 (CH3, 5-OCH3), 55.7 (CH3, 7-OCH3); HR-
TOF-MS (ESI+): m/z 395.1125 [M + Na]+ (calcd for
C20H20O7Na, 395.1101). The NMR spectral data were in
accordance with those reported in the literature (Sutthanut
et al. 2007).
CD3OD, 400 MHz,): δ = 7.68 (1H, dd, J = 8.5, 1.8 Hz,
H-6′), 7.64 (1H, d, J = 1.8 Hz, H-2′), 6.94 (1H, d, J =
8.5 Hz, H-5′), 6.36 (1H, brs, H-8), 6.22 (1H, brs, H-6), 3.92
(3H, s, 4′-OCH3), 3.91 (3H, s, 3′-OCH3), 3.78 (3H, s, 3-
OCH3); 13C NMR (CDCl3 + 5 drops of CD3OD,
100 MHz,): δ = 178.6 (C, C-4), 163.6 (C, C-7), 161.4 (C,
C-5), 156.8 (C, C-9), 155.7 (C, C-2), 151.2 (C, C-4′), 148.6
(C, C-3′), 138.7 (C, C-3), 122.8 (C, C-1′), 122.1 (CH, C-6′),
111.1 (CH, C-2′), 110.7 (CH, C-5′), 105.1 (C, C-10), 98.8
(CH, C-6), 94.0 (CH, C-8), 60.1 (CH3, 3-OCH3), 55.9 (CH3,
3′-OCH3), 55.9 (CH3, 4′-OCH3); HR-TOF-MS (ESI+): m/z
367.0827 [M + Na]+ (calcd for C18H16O7Na, 367.0788).
The NMR spectral data were in accordance with those of
the reported values (Daskiewicz et al. 2005).
5,3′,4ʹ-Tri-O-methylquercetin (13): yellow amorphous
solid; IR νmax 3351, 3009, 2945, 2844, 1611, 1457, 1054,
1008, 816, 790 cm−1
;
1H NMR (CDCl3 + 0.1 mL of
CD3OD, 400 MHz,): δ = 7.76 (1H, d, J = 2.0 Hz, H-2′),
7.74 (1H, dd, J = 8.4, 2.0 Hz, H-6′), 6.95 (1H, d, J =
8.4 Hz, H-5′), 6.47 (1H, d, J = 1.9 Hz, H-8), 6.29 (1H, d, J
= 1.9 Hz, H-6), 3.91 (3H, s, 3′-OCH3), 3.89 (3H, s, 4′-
OCH3), 3.89 (3H, s, 5-OCH3); 13C NMR (CDCl3 + 0.1 mL
of CD3OD, 100 MHz,): δ = 172.3 (C, C-4), 163.1 (C, C-7),
160.9 (C, C-5), 158.9 (C, C-9), 150.3 (C, C-4′), 148.7 (C,
C-3′), 142.6 (C, C-2), 137.3 (C, C-3), 124.0 (C, C-1′),
120.9 (CH, C-6′), 111.0 (CH, C-5′), 110.6 (CH, C-2′),
105.3 (C, C-10), 95.8 (CH, C-6), 95.3 (CH, C-8), 56.0
(CH3, 4′-OCH3), 56.0 (CH3, 3′-OCH3), 55.9 (CH3, 5-
OCH3); HR-TOF-MS (ESI+): m/z 367.0798 [M + Na]+
(calcd for C18H16O7Na, 367.0788). The NMR spectral data
were in accordance with those of the reported in the lit-
erature (Dong et al. 1999).
Cell line and treatment
K562::ΔGγ-AγEGFP, a stable reporter assay, containing
EGFP coding sequence in-frame replacement of the Gγ- and
Aγ-globin coding sequence under the control of Gγ-globin
promoter in the human-globin cluster was used for screen-
ing of γ-globin gene inducer (Vadolas et al. 2004). These
cells were cultured in RPMI 1640 supplemented with 20%
fetal bovine serum and incubated at 37 °C in 5% CO2
incubator. Cells were treated with the tested compounds at
different concentrations (0.1−30 µM). EGFP expression
was analyzed by flow cytometer after 5 days of treatment.
The untreated cells and 0.5% DMSO were used as the
negative control, and 5 µM cisplatin and 5 µM hemin were
used as positive control. The mean fluorescent intensity of
EGFP expression was measured and used for calculation of
the fold change response to treated and untreated cells. Cell
viability was analyzed by the propidium iodide (PI) staining
method. Percentage of PI stained cells as dead cells were
used to calculate the cell viability. The results of the tested
compounds were compared with untreated cells by paired-T
test to analyze statistical significance in the fold change of
3,7,3ʹ,4ʹ-Tetra-O-methylquercetin (14): white powder;
m.p. 159.5−160.5 °C (lit m.p. 158-159 °C); IR νmax 3153,
3004, 2946, 2910, 2839, 1653, 1429, 1093, 953, 820,
1
771 cm−1; H NMR (CDCl3, 400 MHz,): δ = 12.57 (1H, s,
5-OH), 7.69 (1H, dd, J = 8.5, 1.7 Hz, H-6′), 7.64 (1H, d,
J = 1.7 Hz, H-2′), 6.94 (1H, d, J = 8.5 Hz, H-5′), 6.40 (1H,
d, J = 2.0 Hz, H-8), 6.30 (1H, d, J = 2.0 Hz, H-6), 3.92 (3H,
s, 4′-OCH3), 3.92 (3H, s, 3′-OCH3), 3.83 (3H, s, 7-OCH3),
3.80 (3H, s, 3-OCH3); 13C NMR (CDCl3, 100 MHz,): δ =
178.6 (C, C-4), 165.3 (C, C-7), 161.8 (C, C-5), 156.6 (C, C-
9), 155.8 (C, C-2), 151.2 (C, C-4′), 148.6 (C, C-3′), 138.8
(C, C-3), 122.7 (C, C-1′), 122.1 (CH, C-6′), 111.0 (CH, C-
2′), 110.7 (CH, C-5′), 105.9 (C, C-10), 97.7 (CH, C-6), 92.1
(CH, C-8), 60.1 (CH3, 3-OCH3), 55.9 (CH3, 3′-OCH3), 55.9
(CH3, 4′-OCH3), 55.7 (CH3, 7-OCH3); HR-TOF-MS (ESI
+): m/z 381.0953 [M + Na]+ (calcd for C19H18O7Na,
381.0944). The physical and NMR spectral data were in
agreement with those of the reported data (Chu et al. 2004).

Medicinal Chemistry Research
EGFP expression and the percentage of cell viability.
P value ≤ 0.05 was considered as statistical significance.
HR-TOFMS (ESI+) consistent with the molecular formula
C16H12O7. The presence of a three-proton singlet signal at
δH 3.87 and δC 56.3 in the 1H and 13C NMR spectra,
respectively, indicated that mono-O-methylation has taken
place. The position of the methyl ether function has been
proven to be at the 7-position by 2D NMR experiments. The
heteronuclear multiple bond correlation, a long range
proton-carbon coupling (HMBC) spectra showed correla-
tions between 7-OCH3 (δ 3.87) and C-7 (δ 166.9). In
addition, the rotating-frame Overhauser enhancement
spectroscopy, a proton-proton space relationship (ROESY)
experiments revealed the correlations between 7-OCH3 and
H-8 (δ 6.57), thus confirming that methylation has taken
place at the 7-hydroxyl group. By the same analogy, the
structure of the mono-O-methyl ether 3 was established as
3-mono-O-methylquercetin.
Results and discussion
Chemistry
HbF-inducing drugs are promising alternative therapies for
β-thalassemia. Our preliminary investigation on phyto-
chemicals with HbF induction revealed that quercetin (1),
the major constituent of the heartwood of A. luzonensis,
enhanced HbF expression. Since the activity of this com-
pound was lower than the positive controls, cisplatin and
hemin, we therefore decided to modify the structure 1 to its
analogs. We previously discovered that number of free
hydroxyl group played an important role in HbF induction
(Chokchaisiri et al. 2010; Chaneiam et al. 2013). We
therefore planned to prepare analogs of compound 1 with a
varying number of free hydroxyl groups and the methyl
ether analogs are the analogs of choice. The strategy of this
work is to synthesize analogs with varying numbers of
methyl ether functions at different positions of the molecule
of 1.
In the previous reports, a number of mono-O-methyl-
quercetins have been synthesized by selective methylation
of compound 1 (Jurd 1958; Li et al. 2009b). 7-O-Methyl-
quercetin (2) was synthesized from 1 by a four-step trans-
formation (Li et al. 2011). Moreover, the direct treatment of
the flavonoid 1 with methylating agents in the presence of
bases has been explored to yield the corresponding
methylated analogs with varying number of methyl ether
functions (Jurd 1962; Bouktaib et al. 2002; Juvale et al.
2013; Karimova et al. 2015). Since we need a variety of
methylated analogs of compound 1 for our HbF-inducing
evaluation, the strategy was to prepare as many methylated
analogs as possible. Our preliminary investigation on
methylation with different reagents and conditions revealed
that methylation of compound 1 using methyl iodide in
acetone in the presence of anhydrous potassium carbonate
gave rise to methyl ether analogs with varying numbers and
positions. We therefore used this reagent in our work.
Reaction of quercetin (1) with CH3I in acetone and in the
presence of anhydrous K2CO3 at room temperature (ca. 32 °C),
using 1.0 equivalence of CH3I and 1.25 equivalences of
K2CO3, followed by addition of another half equivalence of
CH3I, afforded the mono-O-methyl ether analogs 2 and 3 in
30% and 19% yields, and the di-O-methyl ether analogs 4
and 5 in 25% and 19% yields, respectively, based on the
unrecovered starting material 1. The structure of the ether 2
was established by spectroscopic data, especially those of
1H and 13C NMR data and mass spectral data. Thus, com-
pound 2 showed the [M + Na]+ ion at m/z 339.0473 in the
Similar synthesis procedure was performed, but using 2
equivalences of CH3I and 2.5 equivalences of anhydrous
K2CO3, followed by additional 1.0 equivalence of CH3I, to
yield the di-O-methyl ether analogs 4, 5, 6 and 7 in 20%,
13%, 11%, and 5% yields, respectively, and the tri-O-
methyl ether analogs 8 and 9 in 27% and 32% yields,
respectively, based on the unrecovered starting material 1.
The structure of the di-O-methyl ether 4 was elucidated
based on spectroscopic data including 1H, 13C and 2D NMR
and mass spectra. Compound 4 displayed the [M + Na]+
ion at m/z 353.0645 in the HR-TOFMS (ESI+) compatible
1
with the molecular formula C17H14O7. The H NMR data
were similar to those of the parent compound 1. The sig-
nificant difference was the presence of two methoxyl (7-
OCH3 and 4′-OCH3) signals at δ 3.85 and δ 3.94 which
correlated in HMBC spectra to C-7 (δ 165.0) and C-4′ (δ
147.9), respectively. In addition, the positions of two
methoxyl groups were also confirmed by the ROESY
experiments. Thus, 7-OCH3 proton signal showed cross
peaks with H-6 (δ 6.33) and H-8 (δ 6.46) and 4′-OCH3
proton signal also showed cross peak with H-5′ (δ 6.94).
Compound 4 was thus concluded as 7,4′-di-O-methyl-
quercetin. By the same analogy, the structures of the di-O-
methyl ethers 5−7 were established as 3,7-di-O-methyl-
quercetin, 3,3′-di-O-methylquercetin, and 3,4′-di-O-
methylquercetin, respectively.
In order to obtain the additional tri-O-methyl ether ana-
logs and tetra-O-methyl ether analogs, the methylation
procedure was performed in similar manner to that of the
synthesis of the mono-O-methyl ether analogs 2 and 3, but
using 3.0 equivalences of CH3I and 3.75 equivalences of
K2CO3, followed by additional 1.5 equivalences of CH3I, to
yield the tri-O-methyl ether analogs 8, 9, 10, 11, 12 and 13
in 17%, 21%, 11%, 1%, 6%, and 1% yields, respectively,
and the tetra-O-methyl ether analog 14 in 30% yield. The
molecular formula of compound 8 was determined as
C18H16O7 from its HR-TOFMS (ESI+) at m/z 367.0785

Medicinal Chemistry Research
[M + Na]+. The 1H and 13C NMR data of 8 indicated that a
methoxyl group should be placed at the 3-, 7-, and 4′-
positions, which was also confirmed by a detailed analysis
of the HMBC and ROESY experiments by the same ana-
logy to that of compound 4. Thus, the structures of the
synthesized tri-O-methyl ethers were identified as 3,7,4′-tri-
O-methylquercetin (8), 7,3′,4′-tri-O-methylquercetin (9),
3,7,3′-tri-O-methylquercetin (10), 5,7,3′-tri-O-methylquer-
cetin (11), 3,3′,4′-tri-O-methylquercetin (12) and 5,3′,4′-tri-
O-methylquercetin (13). It should be noted that the unpre-
cedented tri-O-methyl ether analogs 11 and 13 were
obtained as the minor products. It is uncommon to find
products arising from methylation at the least active 5-
hydroxyl group while the hydroxyl groups at other positions
(3- and 4′-positions for compound 11 and 3- and 7-positions
for compound 13) are free.
The penta-O-methyl ether analog was the last methyl
ether analog to be synthesized. By performing similar pro-
cedure to those of the foregoing reactions, but using excess
CH3I and the reaction was conducted under refluxing
acetone, it was surprisingly found that only small quantity
of the penta-O-methyl ether analog 15 was yielded. Most of
the product was found to be the tetra-O-methyl ether analog
14. However, the required penta-O-methyl ether analog 15
was eventually successfully synthesized in 84% yield by
preheating compound 1 with K2CO3 in acetone under
reflux, followed by addition of excess CH3I to the cooled
reaction mixture followed by heating the reaction mixture
under reflux.
Biological activity
The crude extracts of A. luzonensis were screened for HbF
enhancing activity by using the K562::ΔGγAγEGFP reporter
cells. This cell line has previously been shown to respond to
known HbF inducers (Vadolas et al. 2004). The result
revealed that the crude EtOAc extract exhibited enhanced
EGFP expression when compared with negative control,
cells treated with 0.5% DMSO (data not shown). This result
prompted us to screen individual isolated compound from
this extract. Interestingly, we found that the natural quer-
cetin (1), the major flavonoid isolated from the crude EtOAc
extract, enhanced γ-globin promotor activity in a dose-
dependent manner (1.4 0.1 fold change) with 79.2% cell
viability at 30 µM when compared with cells treated with
0.5% DMSO (see Table 1). However, this result was less
active than the positive controls, cisplatin and hemin. We
therefore decided to investigate whether quercetin analogs
would give rise to higher EGFP expression and a number of
methyl ether analogs 2−15 were therefore synthesized. All
Table 1 The effects of quercetin
Compound
Substituent
Effective dose
EGFP
fold change
% Cell
viability
(1) and its analogs 2−15 on
EGFP expression from the
K562::ΔGγ-AγEGFP reporter
assaya and cell viability
(μM)b
3
5
7
3′
4′
1
OH
OH
OH
OH
OH
OH
OH
OH
OH
30
30
20
1.4 0.1
1.0 0.1
1.8 0.0
0.9 0.1
1.0 0.1
1.2 0.0
2.6 0.2
1.3 0.1
1.5 0.2
0.8 0.1
1.1 0.1
1.2 0.1
1.0 0.2
1.1 0.1
1.2 0.1
1.3 0.1
1.0 0.0
2.5 0.7
2.4 0.9
79.2 5.1
83.7 2.2
77.4 3.5
92.4 1.4
90.0 1.6
93.4 4.0
68.9 4.4
86.7 2.9
90.1 1.7
89.8 2.8
93.5 2.8
88.8 1.2
89.0 3.4
91.8 2.3
90.8 4.1
88.9 2.8
91.9 1.4
64.7 9.1
76.2 15.2
2
OCH3 OH
OH OH
3
OCH3 OH
OH OH
4
OCH3 OH
OCH3 OH
OCH3 20
5
OCH3 OH
OCH3 OH
OCH3 OH
OCH3 OH
OH
10
6
OH
OH
OCH3 OH
OH OCH3
30
8
7
8
OCH3 OH
OCH3 10
9
OH
OH
OCH3 OCH3 OCH3 10
10
OCH3 OH
OCH3 OCH3 OH
OCH3 OCH3 OCH3 OH
10
30
11
OH
12
OCH3 OH
OH
OCH3 OCH3 30
OCH3 OCH3 10
13
OH
OCH3 OH
14
OCH3 OH
OCH3 OCH3 OCH3 10
15
OCH3 OCH3 OCH3 OCH3 OCH3 10
Untreated cell
DMSO (0.5%)
Cisplatinc
Heminc
–
–
5
5
aData represent the mean SD of three independent experiments
bConcentrations are those giving the highest HbF-inducing potency
cCisplatin and hemin were used as positive controls

Medicinal Chemistry Research
analogs were also evaluated for HbF-inducing activity. The
results indicated that compound 7 presented the highest
HbF induction against cell line with 2.6 0.2 fold change at
8 μM. The EGFP expression of compound 7 was slightly
highly active compared with the positive controls, cisplatin
(2.5 0.7 fold change) and hemin (2.4 0.9 fold change) at
5 μM (Table 1). Through the investigation of EGFP
expression using a series of quercetin and analogs with
different chemical structures, relationship between the
structure and the HbF-inducing effect could be drawn from
this study. On comparing the activity of compound 1 with
that of compound 2, it is suggested that the methylation of 1
at the 7-position resulted in decreased HbF induction. On
the other hand, methylation of 1 at the 3-position to the
analog 3 enhanced its activity. Further methylation at the 4′-
position of the 3-methyl ether analog 3 to the 3,4′-dimethyl
ether analog 7 strongly enhanced its HbF-inducing potency,
suggesting that methylation at the 4′-position also enhanced
the activity. Additional methylation at the 7-position to the
3,7,4′-trimethyl ether analog 8 resulted in a considerable
decrease in activity, suggesting that structural requirements
for high HbF-inducing ability are the presence of 3- and 4′-
methoxyl groups and that the 7-hydroxyl group should be
free. Comparison of the activity of the 3,4′-dimethyl ether
analog 7 with that of the 3,3′,4′-trimethyl ether analog 12
indicated that methylation at the 3′-position resulted in
decrease in activity. Polymethylation of compound 1
decreased its activity.
The cytotoxicity of compounds 1−15 was also exam-
ined. At the concentration that gave the highest EGFP-
inducing potency, cell viability of most compounds was
more than 70% (Table 1). Compound 7 shows the most
toxic with 68.9 4.4% cell viability. Cytotoxic effect of
compounds 1 and 7 was not significant difference from that
of hemin. When compared with cisplatin, compounds 1 and
7 showed less toxic against K562::ΔGγAγEGFP cells. There
are reports about cytotoxic effect of quercetin (1) in several
cancers including breast, lung, nasopharyngeal, kidney,
colorectal, prostate, pancreatic, and ovarian cancers (Rauf
et al. 2018). Quercetin exhibits direct proapoptotic effects
on tumor cells and thus can inhibit the progress of numerous
human cancers. While cisplatin is a well-known che-
motherapeutic drug that used to treat a number of cancers,
its chemotherapy is also associated with substantial side
effects that include hepatotoxic, nephrotoxic, cardiotoxic,
neurotoxic, and/or hematotoxic damage (Dasari and
Tchounwou 2014). The cytotoxic effect of quercetin is
significantly less than cisplatin treatment in the same cell
line with comparable concentration which is consistent with
this study (Daker et al. 2012; Zhang et al. 2015). Although
the anticancer effect of quercetin has been documented in
numerous in vitro and in vivo studies that involved several
cell lines and animal models, the high toxic effect of
quercetin against cancer cells is accompanied with little or
no side effects or harm to normal cells (Rauf et al. 2018).
The K562::ΔGγAγEGFP cell is an erythroleukemia cell line.
Thus the cytotoxic effect of quercetin on K562::ΔGγAγEGFP
might also be higher than that in normal cells. However,
there was no previous report on biological effect and
cytotoxicity of compound 7. Hydroxyurea, the only US
Food and Drug Administration approved drug for HbF
inducer, was originally approved as an antineoplastic drug
for use in multiple cancers including melanoma, ovarian
cancer, and chronic myeloid leukemia. Hydroxyurea has
severe side effects including leukopenia, neutropenia,
thrombocytopenia, and skin rash (Ballas et al. 2013).
However, the dose used for treatment of β-thalassemia and
sickle cell anemia patients is lower than that of cancers.
This indicated that although compounds 1 and 7 exhibit
slightly cytotoxic effect on K562 cells, they may serve as
natural product-based alternative lead compounds with
HbF-inducing agents for the treatment of β-thalassemia
patients.
The regulation of γ-globin gene expression is upon
several cellular mechanisms both genetic and epigenetic
regulation. One of the proposed mechanisms is oxidative
stress defensive pathway due to the existence of antioxidant
responsive element (ARE) in promoter of γ-globin gene
(Macari and Lowrey 2011). The ARE sequence is the
specific region in promoter of cell stress response genes
which interact with the transcription factor, nuclear factor
erythroid 2-related factor 2 (NRF2), and induced cellular
defense system from oxidative stress (Zhang 2006). Several
compounds that activate the NRF2/ARE pathway such as
angelicin (Lampronti et al. 2003) and resveratrol (Rodrigue
et al. 2001) have been shown to induce γ-globin gene
expression (Kode et al. 2008; McMahon et al. 2001).
Moreover, tert-butylhydroquinone induced γ-globin gene
expression in K562 cell via NRF2/ARE pathway (Macari
et al. 2011). Quercetin (1), which is an antioxidative stress
polyphenol compound, induced NQO1 gene expression
through the NRF2/ARE signaling pathway (Tanigawa et al.
2007). Therefore, the flavonoid 1 and its analogs may
induce γ-globin gene expression by signaling through the
NRF2/ARE pathway.
Conclusion
In summary, structural modification of natural quercetin (1)
isolated from A. luzonensis heartwood to 14 O-methyl ether
analogs was achieved. All compounds were evaluated for
HbF-inducing activity. The present study demonstrated for
the first time that 3,4′-di-O-methylquercetin (7), the struc-
turally modified quercetin, exerted potent HbF induction
2.6-fold change, which is slightly greater than the positive

Medicinal Chemistry Research
controls, cisplatin and hemin. This compound strongly
exhibited EGFP expression with low cellular toxicity.
Quercetin (1) and its methylated analog 7 could be regarded
as a potential lead HbF-inducing molecule derived from
natural products for β-thalassemia drug development.
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Acknowledgements This work was supported by The Thailand
Research Fund (grant nos. DBG5980003 and DBG6180030) and the
Center of Excellence for Innovation in Chemistry (PERCH-CIC),
Office of the Higher Education Commission (OHEC), Ministry of
Education. WP acknowledges a scholarship from Science Achieve-
ment Scholarship of Thailand, OHEC, Ministry of Education.
Compliance with ethical standards
Jurd L (1958) Plant polyphenols. V. selective alkylation of the 7-
hydroxyl group in polyhydroxyflavones. J Am Chem Soc
80:5531–5536
Conflict of interest The authors declare that they have no conflict of
interest.
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Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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