Concise synthesis of 5-methoxy-6-hydroxy-2-methylchromone-7-O- and 5-hydroxy-2-methylchromone-7-O-rutinosides. Investigation of their cytotoxic activities against several human tumor cell lines

Article abstract of DOI:10.1021/jo102325s

The synthesis of two novel 2-methylchromone-7-O-rutinosides is reported, and the in vitro biological activities of these compounds and their synthetic precursors have been investigated on the basis of their cytotoxicity against several human tumor cell lines. The synthesis features early stage assembly of the acidic labile glycosidic bond between sugar and 2-methylchromone aglycon under phase transfer catalyzed glycosidation conditions, whereas all the other standard glycosylation conditions specific to a wide array of rutinosyl donors bearing different anomeric leaving groups (e.g., SPh, OC(NH)CCl₃, Br, OH groups) failed to furnish any detectable products.

Full text of DOI:10.1021/jo102325s

NOTE
pubs.acs.org/joc
Concise Synthesis of 5-Methoxy-6-hydroxy-2-methylchromone-7-O-
and 5-Hydroxy-2-methylchromone-7-O-rutinosides. Investigation of
Their Cytotoxic Activities against Several Human Tumor Cell Lines
Baolin Wu,† Wenpeng Zhang,† Zhonghua Li,‡ Li Gu,†,§ Xin Wang,‡ and Peng George Wang*,†
†Departments of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, United States
‡College of Pharmacy and State Key Laboratory of Element-Organic Chemistry, Nankai University, Tianjin 300071, People's Republic of
China
§National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan,
Shangdong 250100, People's Republic of China
S Supporting Information
b
ABSTRACT: The synthesis of two novel 2-methylchromone-7-O-rutinosides is reported,
and the in vitro biological activities of these compounds and their synthetic precursors have
been investigated on the basis of their cytotoxicity against several human tumor cell lines.
The synthesis features early stage assembly of the acidic labile glycosidic bond between sugar
and 2-methylchromone aglycon under phase transfer catalyzed glycosidation conditions,
whereas all the other standard glycosylation conditions specific to a wide array of rutinosyl
donors bearing different anomeric leaving groups (e.g., SPh, OC(NH)CCl3, Br, OH
groups) failed to furnish any detectable products.
lavonoids, a class of polyphenolic compounds ubiquitous
in plants, are emerging as a potentially important new class of
As depicted in Scheme 1, 5-methoxy-6,7-dihydroxy-2-methyl-
chromone aglycone (4) was synthesized on the basis of literature
precedures4 commencing from the commercially available Vis-
nagin, which was first oxidized with chromic acid to destroy the
furan ring, furnishing the intermediate formylchromone, fol-
lowed by subsequent hydrogen peroxide induced rearrangement
in alkaline solution to afford chromone 4.5 Subsequent glycosy-
lation of 4 with 2,3,4,6-tetra-O-acetyl-R-D-glucosyl bromide (5)
in a biphasic reaction media (CHCl3/H2O) in presence of the
phase transfer catalyst Bu4NBr and K2CO3 as a base at 60 °C for
20 h afforded compound 6 in 56% yield along with a small
amount of diglycosylated byproduct. The regioselectivity of the
glycosylation was confirmed by the NOE relationship between
the anomeric proton and H8 phenyl proton (Scheme 1). Of note
in this transformation is that our original attempts for convergent
assembly of this type of glycosidic linkage using per-O-benzoy-
lated rutinosyl donors (e.g., rhamnopyranosyl-R-(1f6)-gluco-
pyranosyl disaccharides) as coupling partners which bear
different standard anomeric leaving groups such as Br, OH,
SPh, and OC(NH)CCl3 to couple with 6-hexonyl-protected 4
(for the purpose of increasing its solubility6) were not successful
under the corresponding standard glycosylation conditions (data
not shown). Such difficulties in glycosylation resembles Lin-
hardt’s recent work7 for their synthesis of quercetin 3-sophoro-
trioside, in which the convergent assembly of the target molecule
via glycosylation of the sophorotriosidic donor with protected
F
pharmaceutical lead substrates which have been shown to
possess a wide spectrum of biological activities spanning
from antiallergic, anti-inflammatory, antioxidant, antimutagenic,
and anticarcinogenic to modulation of enzymatic activities.1
Most prominent of these are their potential roles serving as
promising antitumor agents, and much of the recent research
efforts have been directed toward flavonoid-based anticancer
drug development and elucidation of their mechanism of action
against tumor cell growth, due to their attractively low side
effects on normal cells even at high dosage.2 In 2009, McCloud
and co-workers isolated the two new 2-methylchromone-
7-O-rutinosides 1 and 3 from Crossosoma bigelovii, which they
reported exhibit potent in vitro cytotoxicity against A-547,
MCF-7, and HT-29 tumor cell lines at a micromolar concentra-
tion level (GI50 = 0.4-6 μM), while, interestingly, no activity was
observed with their nonglycosylated aglycone counterpart.3
Intrigued by these bioactivities, we decided to set out to develop
a straightforward and concise synthetic strategy to access
these relatively simple yet biologically remarkable agents, with
the purpose of gleaning insight into the structure-activity
relationship. Herein we disclose our synthetic results for 5-meth-
oxy-6-hydroxy-2-methylchromone-7-O-rutinoside (2), an iso-
meric form of 1, and natural 5-hydroxy-2-methylchromone
7-O-rutinoside (3). Additionally, the in vitro antiproliferative
activities of the synthetic Intermediates 2 and 3 against MCF-7,
LS174T, and K562 human cancer cell lines have been
assayed.
Received: November 23, 2010
Published: March 02, 2011
r
2011 American Chemical Society
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|

The Journal of Organic Chemistry
NOTE
Scheme 1. Synthesis of 2-Methylchromone-7-O-glucopyra-
noside (8)
Scheme 2. Synthesis of 5-Methoxy-6-hydroxy-2-methylchro-
mone-7-O-rutinoside (2)
tetra-O-acetyl-R-D-glucosyl bromide (5) with 11 has proven to be
extremely sluggish, requiring addition of another aliquot of
bromide donor after 24 h of stirring at 60 °C to drive the reaction
to completion. The glycosylated product 12 was obtained in 46%
yield after 2 days, with concomitant formation of a significant
amount of hemiacetal arising from the hydrolysis of glycosyl
bromide 5. In order to circumvent this problem, we have screened
various PTC catalysts and finally were delighted to find that the
commercially available PTC catalyst Aliquat 336 could dramati-
cally accelerate the reaction to completion within 18 h, affording
12 cleanly with remarkably improved 74% yield without the need
of extra addition of bromide 5 during the course of the reaction.
The exclusive regioselective glycosylation at the 7-OH position
could be explained by the strong hydrogen bond formation
between 5-OH and the adjacent carbonyl group (Scheme 3).
Analogous to the preparation of 7, compound 13 was synthe-
sized in 86% yield on the basis of a one-pot three-step sequence
quercetin was also not successful, highlighting a common
challenge in a quick entry into flavonoid glycosides of such types
via a convergent strategy. Next, subsequent conversion of
compound 6 to compound 7 was carried out in a one-pot
three-step sequence: (1) Zemplꢀen deacetylation using catalytic
amount of NaOMe in methanol, (2) 1,4-diazabicyclo-
[2.2.2]octane (DABCO) promoted tritylation8 of the resultant
primary OH group, and (3) recapping of the remaining hydroxyl
groups as acetates. It should be noted that in the tritylation step
extremely low conversion (<20%) was observed under conven-
tional conditions (trityl chloride/pyridine) even after long reac-
tion times (72 h). Removal of the trityl protecting group also
proved not to be trivial. We found that the glycosidic bond of 7
was susceptible to cleavage even under mild acidic detritylation
conditions such as using 1% I2 in methanol at elevated tempera-
ture (60 °C).9 Extensive experimentation disclosed that Sabitha’s
recently developed protocol10 using bismuth trichloride as a
detritylation agent was the method of choice by which the
desired product 8 could be obtained cleanly in 65% yield after
48 h of stirring at room temperature in dry CH3CN, albeit with
the recovery of some unreacted starting material (Scheme 1).
Glycosylation of per-O-benzoyl-R-D-glucopyranosyl trichlor-
oacetimidate (9a) with 8 took place uneventfully in dichloro-
methane with the promotion of TMSOTf, affording 10a in 60%
yield as a single isomer. The reaction yield was subsequently
optimized to 92% simply by swapping protecting groups of the
trichloroacetimidate 9a, with the reaction executed under other-
wise identical conditions. Final global removal of the protecting
groups using catalytic NaOMe in anhydrous methanol at room
temperature for several hours afforded the target compound 2 in
quantitative yield (Scheme 2).
(Zemplꢀen deacetylation
selective tritylation of primary OH
f
group acetylation of remaining OH groups). Interestingly, the
f
5-OH group was resistant to the acetylation reaction, as evi-
denced by a downfield proton signal resonating at 12.67 ppm in
1H NMR spectrum of 13 in CDCl3 at 500 MHz, obviously due to
the hydrogen bond formation between 5-OH and 4-carbonyl
oxygen. Bismuth trichloride catalyzed detritylation of 13 in
anhydrous acetonitrile at room temperature for 48 h smoothly
delivered the desired product 14 in 75% yield. Next, addition of
catalytic TMSOTf into a mixture of trichloroacetimidate 9b and
14 in anhydrous CH2Cl2 in the presence of activated molecular
sieves at -20 °C, followed by a slow increase of the reaction
temperature over 3 h, afforded glycosylated product 15 in 68%
yield as a single isomer. Global removal of the acetyl protecting
groups of 15 using a catalytic amount of NaOMe in dry methanol
at room temperature furnished 3 in quantitative yield. All
spectroscopic data of 3 (NMR, optical rotation, HRMS) were
identical with those of natural 3, as reported by McCloud and co-
workers3 (Scheme 3).
Synthesis of the natural 2-methylchromone rutinoside 3 was
conducted by a route similar to that for 2, commencing with 5,7-
dihydroxy-2-methylchromone (11), which was readily prepared
on a gram scale using the Kostanecki-Robinson synthesis from
2,4,6-trihydroxyacetonphenone on the basis of the literature
protocol.11 Next, NBu4Br-catalyzed glycosylation of 2,3,4,6-
Driven by the purpose of gleaning insight into the structure
and activity relationship (SAR) of these synthetic compounds,
we have performed a systematic bioassay to evaluate the cell
toxicity of the target glycosides 2 and 3 and their respective
synthetic precursors against three tumor cell lines, including
MCF-7, a breast cancer cell line, LS174T, a colon cancer cell line,
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NOTE
Scheme 3. Synthesis of 5-Hydroxy-2-methylchromone-7-O-
rutinoside (3)
stirring was continued for another 12 h, after which another portion of
2,3,4,6-tetra-O-acetyl-R-D-glucosyl bromide (0.53 g, 1.29 mmol) was added
and the stirring was continued for 24 h at 50 °C. The reaction mixture was
cooled to room temperature, diluted with CHCl3 (100 mL), washed with
cold 1 N HCl solution and brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by column chromatography on
silica gel (toluene/ethyl acetate, 3/1) to isolate the desired product 12 (0.63
g, 1.2 mmol) in 46% yield.
Method 2 (using Aliquat 336). To a stirred solution of 2,3,4,6-tetra-
O-acetyl-R-D-glucosyl bromide (5; 0.86 g, 2.09 mmol) and 5,7-dihy-
droxy-2-methylchromone (11; 0.20 g, 1.05 mmol) in a mixed solution of
CHCl3 and H2O (10 mL/10 mL) was added K2CO3 (0.29 g, 2.09
mmol), followed by Aliquat 336 (0.21 g, 0.50 mmol). The resulting
reaction mixture was stirred at 50 °C for 18 h before it was cooled to
room temperature and diluted with CHCl3 (50 mL). The organic
solution was washed with cold 1 N HCl solution and brine, dried over
anhydrous Na2SO4, and concentrated. Column chromatographic
purification using the same conditions as for method 1 afforded the
title product 12 (0.40 g, 0.78 mmol) in 74% yield. 1H NMR (CDCl3, 500
MHz): δ 12.65 (s, 1H), 6.42 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz, 1H),
6.02 (s, 1H), 5.24-5.32 (m, 2H), 5.11-5.15 (m, 2H), 4.25 (dd, J = 6.0,
12.5 Hz, 1H), 4.18 (dd, J = 3.0, 12.5 Hz, 1H), 3.93 (m, 1H), 2.32 (s, 3H),
2.10 (s, 3H), 2.044 (s, 3H), 2.041 (s, 3H), 2.02 (s, 3H). 13C NMR
(CDCl3, 125 MHz): δ 182.4, 170.5, 170.1, 169.4, 169.2, 167.3, 162.2,
161.8, 157.6, 108.9, 106.5, 99.7, 98.1, 95.3, 77.3, 72.6, 72.4, 70.9, 68.2, 61.9,
20.6, 20.5, 20.4. ESI HRMS: m/z calcd for C24H26O13Na [M þ Na]þ
545.1271, found 545.1259.
Representative Detritylation Procedure. 7-O-(2,3,4-Tri-O-
acetyl-β-D-glucopyranosyl)-5-hydroxy-2-methylchromone
(14). To a stirred solution of compound 13 (0.14 g, 0.19 mmol) in dry
CH3CN (10.0 mL) was added BiCl3 (0.12 g, 0.38 mmol) at room
temperature. After 6 h of stirring at room temperature, another aliquot of
BiCl3 (0.12 g, 0.38 mmol) was added and the stirring was continued for
an additional 12 h before the reaction was quenched with aqueous
saturated NaHCO3 solution. The mixture was filtered through Celite
and washed with ethyl acetate. The filtrate was concentrated under
reduced pressure, and the residue was partitioned between ethyl acetate
(50.0 mL) and water (50.0 mL). The organic solution was washed with
brine, dried, and concentrated. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate, 1/1) to isolate the
desired product 14 (67 mg, 0.14 mmol) in 75% yield, while recovering
the unreacted starting material (11 mg, 0.015 mmol). 1H NMR (CDCl3,
500 MHz): δ 12.67 (s, 1H), 6.41 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.0 Hz,
1H), 6.04 (s, 1H), 5.36 (t, J = 9.5 Hz, 1H), 5.14-5.28 (m, 3H), 3.67-
3.85 (m, 3H), 2.69 (t, J = 6.5 Hz, 1H), 2.37 (s, 3H), 2.10 (s, 3H), 2.07
(s, 3H), 2.06 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ 182.5, 170.2,
170.0, 169.2, 167.4, 162.2, 161.8, 157.7, 109.0, 106.4, 99.5, 98.0, 95.0,
77.2, 74.8, 72.5, 71.0, 68.3, 61.1, 20.63, 20.60, 20.58, 20.5. ESI HRMS:
m/z calcd for C22H24O12Na [M þ Na]þ 503.1165, found 503.1183.
Representative Rhamnosylation of 2-Methylchromone Glu-
cosides. 7-O-[2,3,4-Tri-O-acetyl-r-L-rhamnosyl-(1f6)-2,3,4-tri
-O-acetyl-β-D-glucopyranosyl]-5-hydroxy-2-methylchromone
(15). A mixture of compound 14 (25.0 mg, 0.054 mmol), 2,3,4-tri-O-
acetyl-R-L-rhamnosyl trichloroacetimidate (9b; 56.0 mg, 0.11 mmol), and
activated 4 Å molecular sieves (100 mg) in anhydrous CH2Cl2 (4.0 mL)
was stirred at room temperature for 1 h before it was cooled to -20 °C.
One equivalent of TMSOTf was then added, and the resulting reaction
mixture was stirred for 3 h, during which time the reaction temperature
was raised to 0 °C. The reaction mixture was then quenched with aqueous
NaHCO3 solution, diluted with CH2Cl2 (50 mL), and filtered through
Celite. The filtrate was washed with brine, dried over anhydrous Na2SO4,
and concentrated. The residue was purified by column chromatography
on silica gel (hexane/ethyl acetate, 1/1.2) to isolate the title compound 15
(27 mg, 0.037 mmol) in68% yield. 1H NMR (CDCl3, 500 MHz):δ 12.69
and K562, a leukemia cancer cell line with the well established
MTS assay, wherein the anticancer drug doxorubicin was used as
a positive control. Specifically, all the cancer cell lines were
treated by the compounds at various concentrations (0.01, 0.1,
1.0, 10, 100 μM) for 4 days, followed by monitoring of inhibition
of the cell growth by MTS assay, in which the growth ratio of
treated cells was calculated by comparison with the nontreated
cell control. Much to our surprise, although compounds 10b and
15 in the MCF-7 cell line and compounds 12 and 15 in the K562
cell line showed some inhibition at 100 μM concentration, none
of the other tested compounds showed any significant inhibitory
effect against these cancer cells, even at high concentration (see
Figures 1-3 in the Supporting Information). Most strikingly, the
result obtained for compound 3 in our assays is in sharp contrast
with that by McCloud and co-workers, where they have demon-
strated that 3 has a 0.8 μM value of GI50 against the MCF-7 cell
line.3 Such a bioassay discrepancy underlines the notion that the
cytotoxicity of this type of compounds may be cell-type depen-
dent and/or the compounds themselves are inherently inactive.
’ EXPERIMENTAL SECTION
Representative Procedure for Phase Transfer Catalyzed
Glycosylation of 2-Methylchromone Aglycon with Glycosyl
Bromide using either TBAB or Aliquat 336. 7-O-(2,3,4,6-
Tetra-O-acetyl-β-D-glucopyranosyl)-5-hydroxy-2-methyl-
chromone (12). Method 1 (using TBAB). To a stirred solution of
2,3,4,6-tetra-O-acetyl-R-D-glucosyl bromide (5; 1.28 g, 3.10 mmol) and 5,7-
dihydroxy-2-methylchromone (11; 0.5 g, 2.60 mmol) in a mixed solution of
CHCl3 and H2O (20 mL/20 mL) was added K2CO3 (0.36 g, 2.59 mmol),
followed by Bu4NBr (0.84 g, 2.59 mmol). The resulting mixture was stirred
at 50 °C for 12 h under an argon atmosphere; then one portion of 2,3,4,6-
tetra-O-acetyl-R-D-glucosyl bromide (0.53 g, 1.29 mmol) was added. The
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NOTE
(s, 1H), 6.45 (d, J = 2.0 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 6.06 (s, 1H),
5.25-5.34 (m, 4H), 5.19 (d, J = 8.0 Hz, 1H), 5.13 (t, J = 9.5 Hz, 1H), 5.03
(t, J = 9.5 Hz, 1H), 3.90-3.94 (m, 1H), 3.84 (dd, J = 6.5, 9.5 Hz, 1H), 3.79
(dd, J = 3.0, 12.0 Hz, 1H), 3.67 (dd, J = 6.0, 12.0 Hz, 1H), 2.38 (s, 3H),
2.09 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.17 (d, J = 6.5 Hz,
3H). 13C NMR (CDCl3, 125 MHz): δ 182.6, 170.2, 170.0, 169.8, 169.7,
169.4, 169.2, 167.5, 162.3, 161.8, 157.8, 108.9, 106.7, 100.1, 98.1, 98.0,
94.7, 77.2, 73.6, 72.5, 71.0, 70.9, 69.4, 69.0, 68.8, 66.7, 66.2, 20.8, 20.73,
20.68, 20.61, 20.58, 20.4, 17.3. ESI HRMS: m/z calcd for C34H40O19Na
[M þ Na]þ 775.2061, found 775.2052.
’ REFERENCES
(1) (a) Clifford, M.; Brown, J. E. In Flavonoids: Chemistry, Bioche-
mistry and Applications; Andersen, Ø. M., Markham, K. R., Eds.; Taylor
& Francis: Boca Raton, FL, 2006; pp 319-370. (b) Dangles, O.; Dufour,
C. In Recent Advances in Polyphenol Research; Daayf, F., Lattanzio, V., Eds.;
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Mojzis, J. Recent Prog. Med. Plants 2008, 21, 105. (d) Lopez-Lazaro, M.
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Litwinienko, G. J. Org. Chem. 2009, 74, 2699. (g) Gilbert, E. R.; Liu, D.
Curr. Med. Chem. 2010, 17, 1756.
(3) Klausmeyer, P.; Zhou, Q.; Scudiero, D. A.; Uranchimeg, B.;
Melillo, G.; Cardellina, J. H.; Shoemaker, R. H.; Chang, C.-j.; McCloud,
T. G. J. Nat. Prod. 2009, 72, 805. The structure of 3 was not drawn
correctly in the reference.
(4) (a) Sch€onberg, A.; Badran, N.; Starkowsky, N. A. J. Am. Chem.
Soc. 1955, 77, 1019. (b) Bodendiek, S. B.; Mahieux, C.; Hansel, W.;
Wulff, H. Eur. J. Med. Chem. 2009, 44, 1838. (c) Sch€onberg, A.; Badran,
N.; Starkowsky, N. A. J. Am. Chem. Soc. 1955, 77, 5390. (d) Fillion, E.;
Dumas, A. M.; Kuropatwa, B. A.; Malhotra, N. R.; Sitler, T. C. J. Org.
Chem. 2005, 71, 409.
(5) The alternative approach to constructing 6,7-dimethylated4 using
the method recently developed by Fillion and co-workers5a via a
trifluoroacetic acid promoted annulation reaction between 3,4,5-tri-
methoxyphenol and 5-(1-methoxyethylidene) Meldrum’s acid afforded
only 5,6,7-trimethoxy-4-methylcoumarin instead of the desired 5,6,7-
trimethoxy-2-methylchromone on the basis of our careful NMR analysis
of the product. These two types of compounds could be unequivocally
differentiated by their diagnostic conjugated carbonyl carbon’s chemical
shift, where the chromone carbonyl carbon usually resonates around
175-185 ppm while that of coumarin moves upfield at 155-165 ppm.5b,c
We observed in the 13C NMR spectrum of the product that the carbonyl
carbon’s chemical shift was 160.9 ppm. Additionally, both 1H and
13C NMR spectra of the product were in full agreement with that of the
reported 5,6,7-trimethoxy-4-methylcoumarin, which was synthesized via
methylation of 5,7-dimethoxy-6-hydroxy-4-methylcoumarin. For related
references, see: (a) Agrawal, P. K. Carbon-13 NMR of Flavonoids; Elsevier
Science: Amsterdam, 1989. (b) Breitmaier, E.; Voelter, W. Carbon-13
NMR spectroscopy; VCH: Weinheim, Germany, 1987. (c) Parmar, V. S.;
Sharma, N. K.; Husain, M.; Watterson, A. C.; Kumar, J.; Samuelson, L. A.;
Cholli, A. L.; Prasad, A. K.; Kumar, A.; Malhotra, S.; Kumar, N.; Jha, A.;
Singh, A.; Singh, I.; Himanshu; Vats, A.; Shakil, N. A.; Trikha, S.;
Mukherjee, S.; Sharma, S. K.; Singh, S. K.; Kumar, A.; Jha, H. N.; Olsen,
C. E.; Stove, C. P.; Bracke, M. E.; Mareel, M. M. Bioorg. Med. Chem. 2003,
11, 913.
Representative Global Removal of Protecting Groups of
2-Methylchromone Rutinosides. 5-Hydroxy-7-O-β-rutino-
syl-2-methylchromone (3). To a stirred solution of compound 15
(20 mg, 0.027 mmol) in anhydrous methanol (4.0 mL) was added several
drops of freshly prepared 1 M NaOMe solution in methanol at room
temperature. The reaction mixture was stirred at room temperature for 1 h
before it was neutralized with Amberlyst-15 ion-exchange resin until the pH
value was around 7. The resin was filtered off and washed with methanol
several times. The collected filtrate was concentrated under reduced
pressure, affording a residue which was purified by a short silica gel column
chromatograph (CH2Cl2/MeOH, 4/1) to isolate the final product 3 (12
mg, 0.027 mmol) in quantitative yield. [R]D24 = -50.0° (c 0.1, MeOH).
1H NMR (DMSO-d6, 500 MHz): δ 12.78 (s, 1H), 6.64 (d, J = 2.0 Hz, 1H),
6.38 (d, J = 2.0 Hz, 1H), 6.24 (s, 1H), 5.44 (d, J = 4.5 Hz, 1H), 5.23 (t, J =
5.5 Hz, 1H), 5.00 (d, J = 7.0 Hz, 1H), 4.71 (d, J = 5.0 Hz, 1H), 4.60 (d, J =
4.5 Hz, 1H), 4.52 (s, 1H), 4.48 (d, J = 5.5 Hz, 1H), 3.87 (d, J = 10.0 Hz,
1H), 3.67 (br s, 1H), 3.59 (t, J = 8.0 Hz, 1H), 3.25-3.49 (m, 4H), 3.09-
3.19 (m, 2H), 2.42 (s, 3H), 1.10 (d, J= 6.5 Hz, 3H). 13CNMR(CDCl3, 125
MHz): δ 182.0, 168.7, 162.7, 161.0, 157.5, 108.2, 105.1, 100.7, 99.8, 99.6,
94.4, 76.4, 75.6, 73.0, 72.1, 70.7, 70.2, 69.9, 68.3, 66.4, 20.0, 17.8. ESI HRMS:
m/z calcd for C22H28O13Na [M þ Na]þ 523.1422, found 523.1468.
MTS Assay. The cytotoxicity of synthesized compounds was
evaluated by MTS assays using three cancer cell lines: MCF-7, K562,
and LS174T. The cancer cells were cultured in DMEM (Gibco) with
10% FBS (Gibco) (MCF-7, K562) or 20% FBS (LS174T) at 37 °C with
5% CO2. Briefly, a total of 2000 (LS174T) or 4000 (MCF-7, K562) cells
for each well were cultured overnight in a 96-well plate. Then, the
compounds of interest or Doxorubicin was added to each well at varying
concentrations (0.01, 0.1, 1, 10, 100 μM). After 4 days, MTS (3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium, 0.2 mg/mL) and PMS (phenazine methosulfate, 2.5
μM) were added to the cell culture and the cells were incubated at 37 °C
for 2-3 h. The absorbance of formazan (the metabolite of MTS by
viable cells) was measured at 490 nm to quantify the viable cells. The
growth ratio of treated cells was calculated by comparing the absorbance
to that of the nontreated cells.
’ ASSOCIATED CONTENT
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: wang.892@osu.edu.
’ ACKNOWLEDGMENT
We thank the NIH (No. R01AI083754) for financial support
of this work.
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dx.doi.org/10.1021/jo102325s |J. Org. Chem. 2011, 76, 2265–2268