Synthesis of kojic acid-derived copper-chelating apoptosis inducing agents

Article abstract of DOI:10.1007/s00044-012-0094-y

Three classes of kojic acid derivatives were synthesized and examined for their antiproliferative activity against HeLa cells. Both 8b and 11 co-treated with copper ion exhibited synergistic effect on the HeLa cell growth inhibition with GI₅₀ v

Full text of DOI:10.1007/s00044-012-0094-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:995–1003
DOI 10.1007/s00044-012-0094-y
ORIGINAL RESEARCH
Synthesis of kojic acid-derived copper-chelating apoptosis
inducing agents
•
•
•
Yu-Hua Chen Pei-Jung Lu Christopher Hulme
Arthur Y. Shaw
Received: 10 November 2011 / Accepted: 14 May 2012 / Published online: 29 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Three classes of kojic acid derivatives were
synthesized and examined for their antiproliferative activ-
ity against HeLa cells. Both 8b and 11 co-treated with
copper ion exhibited synergistic effect on the HeLa cell
growth inhibition with GI50 values of 11.9 and 7.1 lM,
respectively. Flow cytometric analysis of HeLa cells
revealed that 11-Cu co-treatment induced the sub-G1 arrest
in a dose-dependent manner, suggesting that the growth-
inhibitory effect is attributed to DNA fragmentation.
Moreover, western blot of HeLa cells cytosolic extracts
displayed the cleavage of the 116-kDa protein poly(ADP-
ribose) polymerase and activation of caspase-3 by the
reduced level of the 32-kDa proenzyme, indicating that the
caspase-dependent apoptotic pathway was involved. We
further demonstrated that MAPK pathway regulators such
as ERK and p38 were activated in response to 11-Cu co-
treatment, suggesting that the intracellular oxidative stress
was dramatically stimulated by the copper ion. Taken
together, we have successfully synthesized kojic acid-
derived copper-induced apoptotic agents.
Keywords Kojic acid Á Apoptosis inducing agents Á
Copper-chelating effect Á Mitogen-activated protein
(MAP) kinases
Introduction
Kojic acid (1) (Fig. 1) is a metabolic compound produced by
several species of fungi, such as Aspergillus, Acetobacter, and
Penicillium(Yabuta, 1924). Kojic acidhas beenaddedtofoodas
an antioxidant, as a preservative to prevent formation of warm-
over flavor in beef, as a food additive for preventing enzymatic
discoloration of vegetables, crabs, and shrimps, and as a skin
lightening or bleaching agent in cosmetic preparations. More-
over, kojic acid has been demonstrated to exhibit bacteriostatic,
anti-inflammatory, insecticidal, antibiotic, cytotoxic, and anti-
tumor activities (Blumenthal, 2004; Bentley, 2006). Owing to its
versatile heterocyclic skeleton, many kojic acid derivatives have
shown to exhibit anticancer (Fickova et al., 2008; Yoo et al.,
2010), antifungal, antibacterial (Reddy et al., 2010), antimi-
crobial, and antiviral (Aytemir et al., 2010) activities.
On the other hand, kojic acid used as a skin lightening agent
is mainly attributed to its copper-chelating property for inhib-
iting tyrosinase activity. Tyrosinase is a copper-containing
enzyme responsible for the biosynthesis of the melanin pre-
cursor 3,4-dihydroxyphenylalanine (DOPA) (Cabanes et al.,
1994). Accordingly, kojic acid has shown to be a bidentate
ligand to chelate with copper ion (II) that forms a copper-kojate
dimmer as indicated in Fig. 1 (Toy and Smith, 1971).
Y.-H. Chen
Department of Chemistry, Tamkang University,
New Taipei City 251, Taiwan
P.-J. Lu
Institute of Clinical Medicine, National Cheng Kung University,
Tainan 701, Taiwan
C. Hulme Á A. Y. Shaw
Accordingly, biological activities of kojic acid derivatives
may not depend not only on their physicochemical properties
such as hydrophilicity but also on their metal-chelating
capability. On the basis of therapeutic indication, copper
chelators have revealed to demonstrate anticancer property.
For example, tetrathiomolybdate has been shown to suppress
Department of Pharmacology and Toxicology, College of
Pharmacy, The University of Arizona, Tucson, AZ 85721, USA
C. Hulme Á A. Y. Shaw (&)
BIO5 Oro Valley, The University of Arizona, 1580 E. Hanley
Blvd, Oro Valley, AZ 85737, USA
e-mail: shaw@pharmacy.arizona.edu
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Med Chem Res (2013) 22:995–1003
O
O
indicated, the C-5 hydroxyl group in kojic acid (1) was
selectively protected with benzyl (Bn) and p-methoxy-
benzyl (PMB) groups in the presence of potassium car-
bonate in DMF solution to afford 2 and 3 in 86 and 89 %
isolated yields, respectively. The C-7 hydroxyl group of 2
and 3 was subjected to undergo mesylation with meth-
anesulfonyl chloride (MsCl) in the presence of triethyl-
amine in DCM to afford 4a and 4b in 92 and 95 % isolated
yields, respectively. Both mesylate esters 4a and 4b con-
verted to bromide derivatives with sodium bromide in
DMF gave 5a (92 %) and 5b (87 %), respectively (Imaf-
uku et al., 1979). Arbuzov reaction of 5a and 5b with
triethylphosphite (P(OEt)3) in toluene at reflux gave
phosphonates 6a and 6b in 90 and 89 % isolated yields,
respectively. To synthesize ethylene-containing deriva-
tives, the Horner-Emmons reaction was employed in which
phosphonates 6a and 6b were treated with strong base
sodium hydride (NaH) in THF followed by the addition of
benzaldehydes to obtain 7a and 7b in 58 and 64 % isolated
yields, respectively. Finally, deprotection of both benzyl
and methyl groups in 7a was carried out by the treatment of
boron tribromide (BBr3) in DCM to give 8a in 63 % iso-
lated yield. On the contrary, PMB group in 7b was
removed by 10 % trifluoroacetic acid (TFA) in DCM to
obtain 8b (55 %).
OH
HO
O
O
O
Cu(II)
O
O
OH
O
OH
kojic acid (1)
Cu(II) kojate dimer
Fig. 1 Chemical structures of kojic acid (1) and Cu(II) kojate dimer
tumor growth, tumor metastases, and angiogenesis and is
being investigated in clinical trials (Brewer et al., 2000;
Redman et al., 2003). PDTC, a pyrrolidine derivative of
dithiocarbamate, is an ionophore capable of transporting
copper ion across cell membranes for inhibiting NF-jB sig-
naling pathway (Nobel et al., 1995; Burkitt et al., 1998). In
light of its copper-chelating property with tyrosinase, kojic
acid appears to be a potential candidate to develop novel
classes of metal-chelating antiproliferative agents. To the best
of our knowledge, growth inhibition of cancer cells in
response to the treatment of kojic acid derivatives in combi-
nation of copper ion has not yet been examined. To gain
insight into the anticancer activity of kojic acid-derived ana-
logs assisted by copper ion, herein we present the synthesis of
several classes of kojic acid derivatives for the evaluation of
copper ion co-treated antiproliferative effect on HeLa cells.
Apart from the ethylene group as a linker to generate 8a
and 8b, C-7 hydroxyl group was modified with an ether
linkage in which an aromatic ring was appended. As shown
in Scheme 2, bromides 5b and 5a were treated with the
corresponding 8-hydroxyquinoline 9 and resorcinol 12 in
the presence of K2CO3 in acetone to obtain 10 and 13 in 78
and 51 % isolated yields, respectively. Next, the removal
Results and discussion
Chemistry
As shown in Schemes 1 and 2, three classes of kojic acid
derivatives were synthesized for biological evaluations. As
O
O
O
O
HO
RO
RO
a
b
c
OH
OH
OMs
O
O
Kojic acid (1)
2
3
R = Bn
R = PMB
4a R = Bn
4b
R = PMB
OCH3
OCH3
O
O
O
7a R = Bn
Ar =
RO
RO
RO
O
d
e
P
Br
OEt
OEt
O
O
O
7b
5a R = Bn
R = PMB Ar =
6a R = Bn
6b
Ar
5b
R = PMB
R = PMB
O
O
HO
HO
g
f
7a
7b
O
O
OH
OH
8a
8b
Scheme 1 Synthesis of kojic acid-derived 7a, 7b, 8a and 8b
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Med Chem Res (2013) 22:995–1003
997
O
O
O
O
OH
N
HO
PMBO
a
b
5b
+
N
N
O
O
9
10
11
O
O
BnO
HO
c
HO
OH
a
5a
+
O
O
O
OH
O
OH
12
13
14
O
O
O
O
O
BnO
a
c
S
HO
5a
+
O
S
O
N
N
O
O
O
N
HN
N
S
N
16
15
17
Reagents: a) K2CO3, acetone; b) TFA, DCM, rt; c) BBr3, DCM, -20 oC to rt.
Scheme 2 Synthesis of kojic acid-derived 11, 14 and 17
Compound (25 µM)
Compound (25 µM) + CuCl2 (50 µM)
of p-methoxybenzyl group in 10 and benzyl group in 13
were carried out based on the above-mentioned conditions
to afford the corresponding products 11 and 14 in yields of
72 and 46 %, respectively. On the other hand, we also
introduced the piperazine moiety as a spacer to anchor the
kojic acid moiety contributed from 5a and an arylsulfo-
nylpiperazine 15 to afford 16 (52 %), followed by depro-
tection of the benzyl group to obtain the product 17 in
86 % yield.
140
120
100
80
60
40
20
0
DMSO KA
7b
8a
8b
11
14
17
Pharmacological Study
Fig. 2 Antiproliferation of HeLa cells in response to tested com-
pounds (25 lM) in the presence and absence of copper ion (50 lM)
We took advantage of HeLa cells as our working model to
examine the antiproliferative effect of kojic acid and its
derivatives with and without copper ion treatment. HeLa
cells were exposed to the tested compounds at 25 lM in
the presence of 10 % FBS DMEM medium for 48 h
treatment in the presence and absence of 50 lM copper
ion. The antiproliferation of tested compounds was
employed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide] assay. As shown in Fig. 2,
growth inhibition of HeLa cells was not observed by kojic
acid treatment with and without co-treated copper ion. This
result might be attributed to the poor hydrophobic property
of kojic acid for penetrating hydrophobic cell membrane.
The above-mentioned result could be in agreement with
those of 8a and 8b.
synergistic antiproliferative effect in the presence of copper
ion, suggesting that hydrophobicity might be a key factor
for antiproliferative activity. Moreover, exposure of HeLa
cells to PMB-protected 7b dramatically abolished the
copper-assisted antiproliferative effect in comparison to its
counterpart 8b, indicating that free 5-hydroxyl group along
with its adjacent carbonyl group play a pivotal role for
copper-chelating capability. Likewise, both 11 and 14
showed no activity in the absence of copper ion. Interest-
ingly, 11 exhibited synergistic effect on HeLa cell growth
inhibition in response to copper ion co-treatment with
95 % growth inhibition, while 14 did not show any effect
even in the presence of copper ion. Again, this result was
similar to that of 8a with poor hydrophobic property for
penetrating cell membrane. However, HeLa cells treated
with 17 in the presence of copper ion merely caused 35 %
growth inhibition which was not as significant as those of
As shown, neither 8a nor 8b exhibited antiproliferative
activity in the absence of copper ion. Nevertheless, 8b
significantly displayed antiproliferative effect in response
to copper ion co-treatment with 90 % growth inhibition.
Similar to that of kojic acid, 8a did not show any
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Med Chem Res (2013) 22:995–1003
8b and 11. Together, our data suggest that potentiation of
the antiproliferative effect assisted by copper addition is an
indicator of the ionophoric properties of a compound able
to raise intracellular copper concentrations to toxic levels.
Furthermore, more detailed evaluation of antiproliferative
activity indicated that in the presence of 50 lM copper ion,
both 8b and 11 exhibited potential synergistic effect on
HeLa cell growth inhibition with GI50 values of 11.9 and
7.1 lM, respectively (Table 1).
The mechanistic study of growth inhibition of HeLa
cells mediated by 11 in the presence of copper ion was
examined by flow cytometric analysis. As shown in Fig. 3,
exposure of HeLa cells to kojic acid (KA) at 25 lM did not
show any sub-G1 phase arrest either in the absence or
presence of 50 lM copper ion (Fig. 3c, d).
On the contrary, induction of the sub-G1 phase arrest in
HeLa cells was clearly observed in a dose-dependent
manner upon treatment of 11 at 5 and 15 lM in the pres-
ence of 50 lM copper ion with 11.85 and 48.56 %,
respectively (Fig. 3f, h). As a result, the growth-inhibitory
effect on HeLa cells mediated by 11 in the presence of
copper ion was attributable to the DNA fragmentation and
apoptotic cell death.
We further evaluated HeLa cells apoptosis induced by
11 in the presence of copper ion by western blotting assay.
As shown in Fig. 4, exposure of HeLa cells to KA and 11
at indicated concentration in the absence and presence of
copper ion for 24-h treatment showed that induction of
poly(ADP-ribose) polymerase (PARP) cleavage by 11 at
15 lM in the presence of 50 lM copper ion was clearly
observed. In addition, the disappearance of PARP at
89 kDa was in accordance with the activation of caspase-3,
as shown by the reduced level of the 32 kDa proenzyme,
suggesting that apoptosis mediated by 11 along with cop-
per ion was, in part, attributed to the caspase-dependent
pathway resulting in PARP cleavage and DNA fragmen-
tation. Moreover, the activation of mitogen-activated
Table 1 GI50 values of KA, 8b and 11 against HeLa cells
Entry
GIa50 (lM)
0
50 CuCl2 (lM)
KA
8b
11
a
ND
ND
64.5 3.3
11.9 1.9
7.1 2.5
15.2 % inhibition at 75 lM treatment
GI50 values are presented as the mean SEM (standard error of
the mean) from three separated experiments
Fig. 3 Flow cytometric
analysis of HeLa cells. Cells
were harvested after 24-h
treatment followed by fixation
and propidium iodide staining
before the flow cytometric
analysis. Sub-G1 phase
A
256
B
256
G1
DMSO
G2/M
CuCl2 (50 µM)
sub-G1
3.41%
S
3.22%
0
0
indicated the DNA
2n
4n
2n
4n
fragmentation and cell death.
Exposure of HeLa cells to kojic
acid (KA) at 25 lM did not
enhance sub-G1 phase arrest in
the absence and presence of
50 lM copper ion. The sub-G1
phase arrest was observed in a
dose-dependent manner upon
treatment of 11 at 5 lM and
15 lM in the presence of
D
C
256
256
KA (25 µM) + CuCl2 (50 µM)
KA (25 µM)
3.57%
2.16%
0
0
2n
4n
2n
4n
E
F
256
256
11 (5 µM) + CuCl2 (50 µM)
50 lM copper ion (11.58 and
48.56 %, respectively). a Cells
were treated with 0.5 % DMSO
as the control; b CuCl2 50 lM;
c Treatment of KA at 25 lM;
d Treatment of KA (25 lM) and
CuCl2 (50 lM); e Treatment of
11 at 5 lM; f Treatment of 11
(5 lM) and CuCl2 (50 lM);
g Treatment of 11 at 15 lM;
h Treatment of 11 (15 lM) and
CuCl2 (50 lM)
11(5 µM)
5.09%
5.85%
11.58%
48.56%
0
0
2n
4n
2n
4n
G
H
256
256
11(15 µM)
11 (15 µM) + CuCl2 (50 µM)
0
0
2n
4n
2n
4n
DNA contents
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Med Chem Res (2013) 22:995–1003
999
Experimental
Chemistry
Chemical reagents and organic solvents were purchased
from TCI and Alfa Aesar unless otherwise mentioned.
Nuclear magnetic resonance spectra (1H- and 13C-NMR)
were measured on a Bruker AC-300 instrument. Chemical
shifts (d) are reported in ppm relative to the TMS peak.
High resolution mass spectra (HRMS) were obtained by
FAB on a Jeol JMS-700 instrument. Flash column chro-
matography was performed using Merck Kiesegel 60 Art
(230–400 mesh).
5-(Benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (2)
To a stirred solution of kojic acid (1) (2.5 g, 17.6 mmol)
and K2CO3 (5.3 g, 38.4 mmol) in dry DMF (24 ml) benzyl
bromide (3.43 g, 20.09 mmol) was added; the reaction
mixture was heated at 50 °C for 2 h. The solvent was
removed under reduced pressure and the precipitate was
filtered, washed with ethyl acetate and brine, and evapo-
rated in vacuo to obtain crude 2. The crude product was
purified by silica gel chromatography to afford 2 (3.5 g,
86 %) mp 132–133 °C (lit. value 132 °C (Imafuku et al.,
1979)). 1H-NMR (CDCl3) d 4.45 (s, 2H), 5.05 (s, 2H), 6.51
(s, 1H), 7.36 (m, 5H), 7.51 (s, 1H) ppm.
Fig. 4 Induction of apoptosis and activation of MAPK pathway in
HeLa cells by 11 in the presence of copper ion after 24-h treatment.
The disappearance of 115-kDa poly(ADP-ribose) polymerase (PARP)
proteolysis was monitored by western blotting, which was in
accordance with the reduced level of the 32 kDa pro-caspase-3. The
phosphorylation of ERK and p38 was clearly observed upon
co-treatment of 11 and copper ion
protein (MAP) kinases such as ERK and p38 was clearly
observed due to the increasing of intracellular oxidative
stress mediated by copper ion. As a consequence, kojic
acid-derived 11 has demonstrated its metal-chelating
capability to shuttle copper ion into the cytosol for MAPK
pathway activation and induction of DNA fragmentation
and apoptosis.
2-(Hydroxymethyl)-5-(4-methoxybenzyloxy)-4H-
pyran-4-one (3)
The synthesis of 3 was similar to that of 2 except for the
1
replacement of BnBr with PMBCl. Yield: 89 %. H-NMR
Conclusions
(DMSO-d6) d 3.75 (s, 3H), 4.29 (s, 2H), 4.86 (s, 2H), 6.31
(s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.6 Hz,
2H), 8.14 (s, 1H) ppm. 13C-NMR(75 MHz,DMSO) d 55.5,
60.9, 112.4, 114.2, 127.8, 129.8, 141.8, 147.0, 159.9,
167.5, 158.7, 175.4 ppm.
In summary, in the present study, we synthesized three
classes of kojic acid derivatives and examined their
growth-inhibitory activity in the absence and presence of
copper ion against HeLa cells. We found that 8b and 11
exhibited dramatically enhanced antiproliferative activity
against HeLa cells in the presence of copper ion with GI50
values of 11.9 and 7.1 lM, respectively. Flow cytometric
analysis of HeLa cells revealed that 11 co-treated with
copper ion induced the sub-G1 arrest in a dose-dependent
manner, suggesting that the growth-inhibitory effect is
attributed to DNA fragmentation. Moreover, western
blot of HeLa cells cytosolic extracts prepared from 11
co-treated with copper ion demonstrated the cleavage of
116-kDa protein poly(ADP-ribose) polymerase (PARP),
which was in accordance with the activation of caspase-3
by the reduced level of the 32-kDa proenzyme. Taken
together, we have successfully synthesized kojic acid
derivatives as copper-assisted antiproliferative agents.
(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)methyl
methanesulfonate (4a)
To a stirred solution of 2 (1.16 g, 5 mmol) in dichloro-
methane (20 ml) methanesulfonyl chloride (0.565 g,
5 mmol) at 0 °C was added, triethylamine (1.02 g,
10 mmol) was added dropwise to the solution. After stir-
ring for 10 min, the mixture was diluted with dichloro-
methane (20 ml) and washed with brine (40 ml). The
organic layer was dried over MgSO4, concentrated in
vacuo, and purified by flash column chromatography to
afford 4a (1.42 g, 92 %). 1H-NMR (300 MHz, CDCl3)
3.89 (s, 3H), 4.95 (s, 2H), 5.09 (s, 2H), 6.52 (s, 1H),
7.38–7.39 (m, 5H), 7.57 (s, 1H) ppm. 13C-NMR (75 MHz,
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Med Chem Res (2013) 22:995–1003
Diethyl ((5-((4-methoxybenzyl)oxy)-4-oxo-4H-pyran-
2-yl)methyl)phosphonate (6b)
CDCl3) d 38.2, 56.1, 71.8, 115.5, 127.7, 128.5, 128.73,
135.3, 141.6, 147.4, 158.7, 173.9 ppm.
The synthesis of 6b was similar to that of 6a. Yield: 95 %.
1H-NMR(300 MHz,CDCl3) d 1.28(t, J = 7.1 Hz, 6H),
2.98(s, 1H), 3.05(s, 1H), 3.78(s, 3H), 4.11(m, 4H), 5.03(s,
2H), 6.34(s, 1H), 6.89(d, J = 6.7 Hz, 2H), 7.32(d,
J = 6.7 Hz, 2H), 7.57(s, 1H) ppm. d 16.2, 31.3, 38.4, 55.6,
71.9, 114.1, 115.8, 127.2, 129.7, 142.1, 147.3, 158.7,
160.3, 174.3 ppm.
(5-(4-Methoxybenzyloxy)-4-oxo-4H-pyran-2-yl)methyl
methanesulfonate (4b)
The synthesis of 4b was similar to that of 4a. Yield: 95 %.
1H-NMR(300 MHz,DMSO-d6) d 3.08(s, 3H), 3.80(s, 3H),
4.94(d, J = 5 Hz, 4H), 6.51(s, 1H), 6.87 (d, J = 3 Hz,
2H), 7.28(d, J = 3 Hz, 2H), 7.56(s, 1H) ppm. 13C-
NMR(75 MHz, DMSO-d6) d 38.4, 55.5, 65.3, 71.9, 114.3,
115.8, 127.6, 129.8, 142.0, 147.5, 158.8, 160.4, 174.2 ppm.
(E)-5-(Benzyloxy)-2-(3,4-dimethoxystyryl)-4H-pyran-
4-one (7a)
5-(Benzyloxy)-2-(bromomethyl)-4H-pyran-4-one (5a)
To a stirred solution of 5 (0.352 g, 1 mmol) and NaH
(0.08 g, 2 mmol, 60 % suspension in mineral oil) in dry
THF (20 ml) a solution of 3,4-dimethoxybenzaldehyde
(0.166 g, 1 mmol) in THF (5 ml) was added dropwise
under Ar atmosphere at 0 °C. After stirring at room tem-
perature for 1 h, the mixture was diluted with dichloro-
methane (20 ml) and washed with water (40 ml) and brine
(40 ml). The organic layer was dried over MgSO4, con-
centrated in vacuo, and purified by flash column chroma-
tography(ethyl acetate/dichloromethane = 1/9) to afford
To a stirred solution of 4a (0.93 g, 3 mmol) in DMF
(15 ml) NaBr (0.61 g, 6 mmol) was added, the mixture
was stirred at room temperature for 20 min. After filtering
the excess amount of NaBr, the filtrate was diluted with
ethyl acetate (50 ml) and washed with water (25 ml) and
brine (40 ml). The organic layer was dried over MgSO4,
concentrated in vacuo, and purified by silica gel chroma-
tography (ethyl acetate/dichloromethane = 3/7) to obtain
5a. Yield: 92 %. 1H-NMR(300 MHz,CDCl3) d 4.14(s, 2H),
5.08(s, 2H), 6.46(s, 1H), 7.35–7.40(m, 5H), 7.56(s, 1H)
ppm. 13C-NMR(75 MHz,CDCl3) d 26.4, 72.0, 115.1,
127.9, 128.6, 128.8, 135.6, 141.7, 147.4, 161.7, 174.4 ppm.
1
6a (0.211 g, 58 %). H NMR(300 MHz, CDCl3) d 3.9 (s,
6H), 5.11 (s, 2H), 6.35 (s, 1H), 6.52 (d, J = 16.1 Hz, 1H),
6.87 (d, J = 8.2 Hz, 1H), 7.02 (s, 1H), 7.07(d, J = 8.2 Hz,
1H), 7.27(d, J = 16.1 Hz, 1H), 7.33–7.44 (m, 5H), 7.52 (s,
1H) ppm. 13C NMR(75 MHz, CDCl3) d 56.1, 72.1, 109.4,
111.3, 113.3, 117.1, 122.01, 127.9, 128.4, 128.7, 136.2,
141.3, 147.0, 49.5, 150.9, 161.7, 175.1, 191.9 ppm.
5-(4-Methoxybenzyloxy)-2-bromomethyl-4H-pyran-
4-one (5b)
The synthesis of 5b was similar to that of 5a. Yield: 87 %.
1H-NMR(300 MHz, CDCl3) d 3.79(s, 3H), 4.13(s, 2H),
4.99(s, 2H), 6.44(s, 1H), 6.88(d, J = 6.7 Hz, 2H), 7.30(d,
J = 6.7 Hz, 2H), 7.54(s, 1H) ppm. 13C-NMR(75 MHz,
DMSO-d6) d 38.4, 55.5, 71.9, 114.1, 115.8, 127.2, 129.8,
142.1, 147.4, 158.7, 160.3, 174.3 ppm.
(E)-5-(Benzyloxy)-2-styryl-4H-pyran-4-one (7b)
Compound 7b was synthesized from the procedure
described for compound 7a. Yield (64 %). 1H-
NMR(300 MHz,CDCl3) d 5.11(s, 2H), 6.40 (s, 1H), 6.65 (d,
J = 16.1 Hz, 1H), 7.32 (d, J = 16.1 Hz, 1H), 7.34–7.50 (m,
10H), 7.51(s, 1H) ppm. 13C NMR(75 MHz,CDCl3) d 72.2,
114.0, 119.4, 127.7, 128.0, 128.6, 128.9, 129.1, 129.9, 135.0,
136.1, 136.4, 141.4, 147.2, 161.4, 175.2 ppm.
Diethyl (5-(benzyloxy)-4-oxo-4H-pyran-
2-yl)methylphosphonate(6a)
To a solution of 5a (1.47 g, 5 mmol) in toluene (10 ml)
triethylphosphite (1.66 g, 10 mmol) was added and the
mixture was stirred at reflux for 24 h. The solvent and
excess triethylphosphite were removed in vacuo and the
resulting oily residue was purified by flash column chro-
matography (ethyl acetate/dichloromethane = 4/1) to
afford 6a. Yield: 86 %. 1H-NMR(300 MHz,CDCl3) d
1.28(t, J = 7.1 Hz, 6H), 2.98(s, 1H), 3.05(s, 1H), 4.09(m,
4H), 5.04(s, 2H), 6.34(s, 1H), 7.27–7.38(m, 5H), 7.50(s,
1H) ppm. 13C-NMR(75 MHz,CDCl3) d 16.2, 31.3, 62.7,
71.7, 115.8, 127.7, 128.3, 128.6, 135.6, 141.6, 146.9,
159.3, 174.1 ppm.
(E)-2-(3,4-Dihydroxystyryl)-5-hydroxy-4H-pyran-
4-one (8a)
To a solution of 6a (0.364 g, 1.0 mmol) in DCM (20 ml)
boron tribromide (1.5 g, 6 mmol) at -20 °C was added;
the mixture was allowed to room temperature and stirred
for 24 h. The reaction mixture was diluted with methanol
(10 ml), washed with water (40 ml) and brine (40 ml). The
organic layer was dried over MgSO4, evaporated in vacuo,
and purified by purified by flash column chromatography
(ethyl acetate: methanol = 4/1) to afford 6a (0.155 g,
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Med Chem Res (2013) 22:995–1003
1001
63 %). 1H-NMR(300 MHz, DMSO-d6) d 6.43 (s, 1H), 6.73
(d, J = 16.1 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.92 (d,
J = 8.1, 1H), 7.02 (s, 1H), 7.20 (d, J = 16.1 Hz, 1H), 8.00
(s, 1H) ppm. 13C-NMR (75 MHz, CDCl3) d 110.9, 114.3,
115.9, 116.4, 120.6, 126.7, 135.7, 138.9, 145.7, 147.6,
161.9, 163.1, 174.1 ppm. HRMS (M ? 1)? calcd for
C13H11O5 247.0606, found 247.0607.
5-(Benzyloxy)-2-((3-hydroxyphenoxy)methyl)-
4H-pyran-4-one (13)
The synthesis of 13 was similar to that of 10 except for the
replacement of 5b and 9 with 5a and resorcinol 12,
respectively. Yield: 51 % yield. 1H-NMR (300 MHz,
DMSO-d6) d 4.93(s, 4H), 6.38(m, 3H), 6.49(s, 1H), 7.06(m,
1H), 7.39(s, 5H), 8.25(s, 1H) ppm. 13C-NMR (75 MHz,
DMSO-d6) d 65.0, 70.5, 102.1, 105.3, 108.8, 113.6, 128.1,
128.2, 128.4, 130.0, 136.0, 141.5, 146.9, 158.6, 158.6,
162.7, 172.9 ppm.
(E)-5-Hydroxy-2-styryl-4H-pyran-4-one (8b)
To a solution of 7b (0.304 g, 1 mmol) in 10 % trifluoro-
acetic acid in DCM (5 ml), the resulting mixture was
stirred at room temperature for 30 min. The solvent was
removed and the residue was purified by flash silica gel
chromatography (DCM ethyl acetate = 1/1) to give 8b
with 55 % yield. 1H-NMR (300 MHz, MeOH-d4) d 6.50 (s,
1H), 6.96 (d, J = 16.2 Hz, 1H), 7.35 (m, 1H), 7.40 (d,
J = 7.8 Hz, 2H), 7.47 (d, J = 16.2 Hz, 1H), 7.61 (d,
J = 7.8 Hz, 1H) ppm. 13C-NMR (75 MHz, MeOH-d6) d
102.8, 110.9, 119.0, 119.2, 120.0, 120.5, 121.2, 127.0,
128.1, 131.1, 154.6 ppm. HRMS (M ? 1)? calcd for
C13H11O3 215.0708, found 215.0713.
5-Hydroxy-2-((3-hydroxyphenoxy)methyl)-4H-pyran-
4-one (14)
The synthesis of 14 was similar to that of 8a with 46 %
yield. 1H-NMR (300 MHz, DMSO-d6) d 4.92(s, 2H),
6.38(m, 4H), 7.03(t, J = 9 Hz, 1H), 8.09(s, 1H), 9.20(s,
1H), 9.47(s, 1H) ppm. HRMS (M ? 1)? calcd for
C12H11O5 235.0606, found 235.0604.
5-(Benzyloxy)-2-((4-(phenylsulfonyl)piperazin-
1-yl)methyl)-4H-pyran-4-one (16)
5-(4-Methoxybenzyloxy)-2-((quinolin-
The synthesis of 16 was similar to that of 13 with 52 %
yield. 1H-NMR (300 MHz, DMSO-d6) d 2.57(m, 4H),
3.04(s, 4H), 3.33(s, 2H), 5.04(s, 2H), 6.36(s, 1H), 7.35(m,
5H), 7.48(s, 1H), 7.60(m, 3H), 7.52(d, J = 5 Hz, 2H)
ppm.13C-NMR (75 MHz,DMSO-d6) d 46.1, 52.3, 58.8,
72.0, 114.87, 127.9, 127.9, 128.5, 128.8, 129.3, 133.2,
135.4, 135.8, 141.7, 147.2, 163.9, 174.6 ppm.
8-yloxy)methyl)-4H-pyran-4-one (10)
To a solution of 5b (0.325 g, 1 mmol) in dry acetone
(7 ml) K2CO3 (0.256 g, 2 mmol) and 8-hydroxyquinoline
9 (0.145 g, 1 mmol) was added; the resulting mixture was
stirred at room temperature for 12 h. The reaction mixture
was filtered to remove K2CO3. The filtrate was evaporated
in vacuo to obtain the crude product. The crude product
was purified by flash chromatography (hexane: ethyl
acetate = 3/1) to afford 10. Yield: 78 %. 1H-NMR
(300 MHz, CDCl3) d 3.80(s, 3H), 5.00(s, 2H), 5.18(s,
2H), 6.62(s, 1H), 6.87(d, J = 9 Hz, 2H), 7.05(s, 1H),
7.30(d, J = 6 Hz, 2H), 7.42(m, 3H), 7.57(s, 1H), 8.15(s,
1H), 8.95(s, 1H) ppm.
5-Hydroxy-2-((4-(phenylsulfonyl)piperazin-
1-yl)methyl)-4H-pyran-4-one (17)
The synthesis of 17 was similar to that of 8a with 86 %
yield.1H-NMR (300 MHz, DMSO-d6) d 3.15(m, 4H),
3.62(m, 4H), 4.22(s, 2H), 6.55(s, 1H), 7.67(m, 5H), 8.09(s,
1H) ppm. 13C-NMR (75 MHz,DMSO-d6) d 43.2, 50.5,
54.9, 55.2, 117.2, 127.7, 129.7, 133.8, 140.5, 146.4, 156.3,
173.6 ppm. HRMS (M ? 1)? calcd for C16H19N2O5S
351.1015, found 351.1017.
5-Hydroxy-2-((quinolin-8-yloxy)methyl)-4H-pyran-4-
one (11)
To a solution of 10 (0.195 g, 0.5 mmol) in 10 % trifluo-
roacetic acid in DCM (5 ml), the resulting mixture was
stirred at room temperature for 30 min. The solvent was
removed and the residue was purified by flash silica gel
chromatography (DCM ethyl acetate = 1/1) to give 11
with 72 % yield. 1H-NMR (300 MHz, MeOH-d4) d 5.35(s,
2H), 6.72(m, 1H), 7.15(m, 1H), 7.62(m, 1H), 7.78(m, 2H),
7.83(m, 2H), 8.94(d, J = 9 Hz, 1H), 9.03(d, J = 6 Hz, 1H)
ppm. HRMS (M ? 1)? calcd for C15H12NO4 270.0766,
found 270.0758.
Cell culture
Cancer cells were purchased from the Bioresource Col-
lection and Research Center in Taiwan. Each cell line was
maintained in the standard medium and grown as a
monolayer in Dulbecco’s Modified Eagle Medium
(DMEM) containing 10 % fetal bovine serum, 2 mM glu-
tamine, 100 units/ml penicillin, and 100 g/ml streptomy-
cin. Cultures were maintained at 37 °C with 5 % CO2 in a
humidified atmosphere.
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Med Chem Res (2013) 22:995–1003
MTT assay for cell viability
terminated by the addition of 100 ll lysis buffer. For
Western blot analysis, the amount of proteins (50 lg) were
separated by electrophoresis in a 15 % SDS–PAGE and
transferred to a nitrocellulose membrane. After an over-
night incubation at 4 °C in TBST/5 % non-fat milk, the
membrane was washed with TBST three times and
immunoreacted with the monocolonal primary antibodies,
anti-poly-ADP-ribose polymerase(PARP) (1:500), anti-
pro-caspase-3 (1:1000), anti-phospho-ERK1/2 (1:1000),
anti-ERK1/2 (1:1000), anti-phospho-p38 (1:1000), anti-
p38 (1:1000), and anti-b-actin (1:1000) from Cell Signal-
ing Technology (Beverly, MA). After four washings with
TBST, the anti-mouse or anti-rabbit IgG (dilute 1:10,000)
was applied to the membranes for 1 h at room temperature.
The membranes were washed with TBST for 1 h and the
detection of signal was performed using an enhanced
chemiluminescence (ECL) detection reagents.
Cells were plated in 96-well microtiter plates at a density
of 5 9 103/well and incubated for 24 h. After that, cells
were treated with vehicle alone (control) or compounds
(drugs were dissolved in DMSO previously) at the con-
centrations indicated. Treated cells were further incubated
for 48 h. Cell survival is expressed as percentage of control
cell growth. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT, 2 mg/ml) dye reduction
assay in 96-well microplates were used. The assay is
dependent on the reduction of MTT by mitochondrial
dehydrogenases of viable cell to a blue formazan product,
which can be measured spectrophotometrically. Tumor
cells were incubated in each well with serial dilutions
of the tested compounds. After 2 days of incubation
(37 °C, 5 % CO2 in a humid atmosphere), 100 ll of MTT
(2 mg/ml in PBS) was added to each well and the plate was
incubated for a further 2 h (37 °C). The resulting formazan
was dissolved in 100 ll DMSO and read at 570 nm. The
percentage of growth inhibition was calculated by
the following equation: percentage growth inhibition =
(1-At/Ac) 9 100, where At and Ac represent the absor-
bance in treated and control cultures, respectively. The
drug concentration causing a 50 % cell growth inhibition
(GI50) was determined by interpolation from dose–
response curves. All determinations were carried out in
three separated experiments.
Acknowledgments Financial support from the National Science
Council of the Republic of China and Tamkang University to
A. Y. Shaw is gratefully acknowledged.
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