Synthesis and biological evaluation of phenyl substituted polyoxygenated xanthone derivatives as anti-hepatoma agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.020

A series of novel derivatives of phenyl substituted tetramethoxy xanthone were synthesized and evaluated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor hepatic cells. Among these derivatives, compound 6 was more potent than positive control 5-fluorouracil (5-Fu) on QGY-7703 and SMMC-7721 cells with IC₅₀ values of 6.27 μM, 7.50 μM and 15.56 μM, 14.55 μM, respectively. Furthermore, compounds 6, 14, 16, and 29 exhibited much better selectivity toward the normal hepatic cell line QSG-7701 than 5-Fu. Additionally, compound 6 significantly induced cell apoptosis in QGY-7703 cells. Our findings suggested that these phenylxanthone derivatives may hold promise as chemotherapeutic agents for the treatment of human HCC.

Full text of DOI:10.1016/j.ejmech.2013.08.020

European Journal of Medicinal Chemistry 69 (2013) 159e166
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of phenyl substituted
polyoxygenated xanthone derivatives as anti-hepatoma agents
,
,
Ming Dai a 1, Xing Yuan a 1, Jian Kang a, Zhi-Jun Zhu a, Rong-Cai Yue a, Hu Yuan c,
Bing-Yang Chen a, Wei-Dong Zhang a , Run-Hui Liu , Qing-Yan Sun
,
a,
b,
*
*
*
a Department of Phytochemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China
c School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel derivatives of phenyl substituted tetramethoxy xanthone were synthesized and eval-
uated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor he-
patic cells. Among these derivatives, compound 6 was more potent than positive control 5-fluorouracil
Received 11 May 2013
Received in revised form
8 August 2013
Accepted 12 August 2013
Available online 22 August 2013
(5-Fu) on QGY-7703 and SMMC-7721 cells with IC50 values of 6.27 mM, 7.50 mM and 15.56 mM, 14.55 mM,
respectively. Furthermore, compounds 6, 14, 16, and 29 exhibited much better selectivity toward the
normal hepatic cell line QSG-7701 than 5-Fu. Additionally, compound 6 significantly induced cell
apoptosis in QGY-7703 cells. Our findings suggested that these phenylxanthone derivatives may hold
promise as chemotherapeutic agents for the treatment of human HCC.
Keywords:
Xanthone
Anti-hepatoma
Ó 2013 Elsevier Masson SAS. All rights reserved.
Structureeactivity relationship
Cytotoxic selectivity
1. Introduction
xanthones in plants, prenylated xanthones [13e26] have also been
described to exhibit antitumor activity. Among these polyprenylated
Human hepatocellular carcinoma (HCC) is the sixth-most com-
mon cancer and the third leading cause of cancer-related mortality
worldwide [1,2]. Chemotherapy is one of the commonly used treat-
ment options, especially for patients with unresectable tumors.
Currently, there is no effective chemotherapy for HCC in humans, and
the use of conventional cytotoxic drugs, including cyclophospha-
mide, cisplatin, and fluorouracil, has not shown any improvement in
survival. Additionally, severe adverse effects are commonly observed
in patients with these treatments [3,4]. Therefore, development of
new chemotherapeutic agents will be of great significance.
Xanthones are a class of oxygen-containing heterocyclic com-
pounds that are widely distributed in nature [5] and exhibit a variety
of biological activities [6e8]. During the past several years, natural
xanthone has attracted increasing interest in the search for new
antitumor agents, such as monodictyxanthone analogs [9], a-man-
gostin [10,11], psorospermin [12]. Another naturally abundant type of
derivatives, some caged xanthone compounds were identified as a
potent anticancer agent, such as 7-hydroxy-forbesione, cantleyanone
B, cantleyanone C [25] and gambogic acid [26].
Phomoxanthones A and B (Fig. 1) [27], two novel xanthone di-
mers, which were isolated from the endophytic fungus Phomopsis
sp. BCC 1323, exhibited significant in vitro cytotoxicity. Further-
more, another type of compounds containing a biphenyl moiety,
such as honokiol and magnolol (Fig. 1), a biphenolic neolignan
isolated from Magnolia officinalis, has been reported to possess
cytotoxic activities against A459 cell line (human nonsmall lung
cell cancer), SK-MEL-2 (human melanoma), SK-OV-3 (ovarian
cancer), XF498 (CNS cell line), HCT-15 (colon cancer), and Hep-G2
(liver cancer) [28e31]. Professor Chen, designed and synthesized
a series of honokiol analogs and derivatives to investigate their
antiangiogenic activity by application of the transgenic zebrafish
screening model, antiproliferative and cytotoxic activity against
HUVECs, and three tumor cell lines by MTT assay [32]. Further
structureeactivity relationship (SAR) studies in his research sug-
gested that the orientation of the two allyl groups of the biphenyl
scaffold was responsible for the antiproliferative activity. Based on
these results, a series of phenyl substituted tetramethoxy xanthone
derivatives were synthesized and their potential cytotoxic activity
* Corresponding authors. Tel./fax: þ86 21 81871244.
E-mail addresses: wdzhangy@hotmail.com (W.-D. Zhang), lyliurh@126.com
(R.-H. Liu), sqy_2000@163.com (Q.-Y. Sun).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.020

160
M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
Fig. 1. Biphenyl moiety with promising antitumor activity.
against HCC cells was investigated. The effects of the prepared
compounds on HCC cell apoptosis were further evaluated. Herein,
the synthesis and biological evaluation of these derivatives are
reported.
22) at the ortho position was well tolerated, whereas the para-,
meta-substituted analogs (compounds 11, 31) and two fluoro
substituted analogs (compounds 9, 30, 33) led to almost total loss of
activity. It appears that the substitution with phenyl ring at 4 po-
sition of xanthone nuclear is essential for activity, replacement of
the phenyl ring with heterocyclic ring (compound 27) or no sub-
stitution (compound 4) led to dramatic loss of activity. To further
study the cytotoxic profile, the hit compounds (6, 12, 13, 14, 16, 22,
24, 26, 29 and 32) were selected for further cytotoxicity investi-
gation at different concentrations against three different types of
HCCs. In these tests, 5-fluorouracil (5-Fu, Sigma) was used as the
positive control. The IC50 values of individual compounds for the 5-
dose test against each HCC cell line are presented in Table 2. Most of
these compounds showed more potent activity against QGY-7703
and SMMC-7721 than HepG-2.
Among the 10 hit compounds, phenyl-substituted xanthone
analogs containing electron-withdrawing substituents on the
phenyl group (compounds 22) showed weaker cell growth inhibi-
tion than alkyl-substituted phenyl (compounds 12, 13, 14, 16, 29
and 32) or methoxy substituted phenyl (compounds 24 and 26)
analogs. This conclusion was consistent with the preliminary
screen results. Furthermore, no conclusive relationship was found
between the cytotoxic activity and the substituted position.
Among the derivatives tested, phenylxanthone 6 displayed the
most potent cytotoxicity against QGY-7703, Hep-G2 and SMMC-
2. Chemistry
The initial synthesis of the xanthone nucleus is illustrated in
Scheme 1. First, 2,3,4-thrimethoxybenzoyl chloride 2 was prepared
from the corresponding acid 1 in the presence of thionyl chloride,
and then reacted with 1,3,5-trimethoxybenzene to afford 2-hydroxy-
20,3,4,40,60-pentamethoxybenzophenone 3 in 65% yield. As already
described by Quillinan [33], a mono-demethylation occurred on the
ring ortho to the carbonyl function provided by the acid moiety.
Subsequent base-catalyzed cyclization of 3 led to 1,3,5,6-tetrame-
thoxy xanthone 4 in 83% yield along with methanol elimination.
Treatment of tetramethoxy xanthone 4 with 1.50 equiv of iodine in
DMF at room temperature provided xanthone 5 in satisfactory yield
(approximately 85% yield). Due to the long reaction time and
complicated operation, iodine was replaced with NIS to prepare
compound 5 without a loss in yield. With this key compound in hand,
title compounds 6e34 were accessed by Suzuki reaction using
palladium tetrakis(triphenylphosphine) as catalyst. The structures of
1
the synthesized target compounds were elucidated by H NMR, 13
C
NMR and MS. All spectral data were in accordance with assumed
structures.
7721 with IC50 values of 6.27
parison, the positive control 5-Fu had IC50 values of 15.66
12.48 M and 14.55 M, respectively. Adding alkyl groups on the
mM, 19.18
mM and 7.5
mM. For com-
mM,
m
m
3. Results and discussion
phenyl ring of compound 6 did not affect the cytotoxicity against
HCC (e.g., compounds 12, 13, and 29). However, the introduction of
a bulky substituent had a detrimental effect on inhibition potency,
as seen with isopropyl substituted 14 and tert-butyl substituted 16.
Methoxy is a strong electronic-donating group, but compared with
phenylxanthone 6, methoxy substituted phenylxanthones 24 and
26 displayed moderate to weak cytotoxicity toward all three HCCs,
presumably due to steric hindrance.
Cytotoxic selectivity against HCC cells is the most important
characteristic of the newly synthesized derivatives. Thus, the
inhibitory effect of active compounds 6, 12, 13, 14, 16, 22, 24, 26, 29
and 32 on the proliferation of non-tumor hepatic cells (QSG-7701)
was further examined. According to the data shown in Table 2, alkyl
substituted analogs (compounds 6, 14, 16, 29) exhibited better
All newly synthesized compounds were tested in vitro and
quantified as the percentage inhibition of treated QGY-7703 cells at
a single dose of 10 mM in comparison to untreated control cells
(Table 1). Of the 28 compounds tested, compounds 6, 12, 13, 14, 16,
22, 24, 26, 29 and 32 exhibited greater than 50% inhibition of QGY-
7703 cell growth at 10
mM. Compounds contained no group or
electron donating groups on substituted phenyl ring, such as alkyl
and methoxy showed more potent cytotoxic activity against QGY-
7703 cells. Decreased electron density on the aromatic ring there-
fore appeared to diminish activity, especially for the strongly
electron-withdrawing group at para position of substituted phenyl
ring, such as compounds 10, 15, 19, 20. Introduction of a weak
electron-withdrawing fluoro group on the phenyl ring (compound

M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
161
Scheme 1. Reagents and conditions: (a) thionyl chloride, rt, 2 h; (b) AlCl3/ether, rt, 8 h; (c) tetramethylammonium hydroxide, pyridine, water, reflux, 36 h; (d) I2 in DMF, rt, 2 days;
(e) NIS in CH2Cl2, rt, overnight; (f) phenylboronic acid, Na2CO3, DMEeH2O, 85 ꢀC, tetrakis(triphenylphosphine) palladium(0).
cytotoxic selectivity against the tumor cells and normal cells than
methoxy substituted analogs (24). However, fluoro substituted
compounds (22) and dimethoxy substituted analogs (26) had no
cytotoxic selectivity. Compared to 5-Fu, all these target compounds
presented a weak cytotoxic activity toward the non-tumor hepatic
cells.
To further explore the potential mechanisms of antiproliferative
effect induced by compound 6, annexin V-FITC binding analysis and
PI staining were performed to quantify cell apoptosis (Fig. 2). HCC
QGY-7703 cells were cultured for 48 h in the absence or presence of
4. Conclusions
In summary, a novel series of phenyl substituted tetramethoxy
xanthone derivatives were synthesized to survey the SAR of sub-
stituents on the phenyl ring. The SAR analysis indicated that: (i)
electronic effects in the phenyl ring played a crucial role in the
antiproliferative activity; and (ii) bulky substituents had a detri-
mental effect on the inhibition. Among the new compounds, 6, 14,
16, and 29 had selective inhibitory activity against HCC cells.
Furthermore, compound 6 induced a high frequency of apoptosis in
human HCC QGY-7703 cells. These derivatives, especially 6, pre-
sented better cytotoxic selectivity for HCC cells, suggesting their
potential in targeted chemotherapy for HCC. Further lead optimi-
zation and mechanism studies are thus worth pursuing.
compound 6 at different concentrations (3.75, 7.5, 15 and 30
mM).
Treatment with a low concentration (3.75 M) of compound 6 did
m
not alter the number of apoptotic cells compared to the control
group. In contrast, the apoptosis percentage increased significantly
after treatment with higher concentrations of compound 6. The
results demonstrated that compound 6-induced apoptosis of HCC
at least partially contributed to its antiproliferative effect.
5. Experimental section
5.1. Chemistry
Table 1
Inhibition rates of compounds 4, 6e33 against the QGY-7703 cell line at 10 mM.
5.1.1. Instruments
Compd
R
Yield
Inhibitory
ratio (%)
Nuclear magnetic resonance (NMR) spectra were recorded using
TMS as the internal standard in DMSO-d6 or CDCl3 with a Bruker
BioSpin GmbH spectrometer at 300 MHz and 600 MHz. When peak
multiplicities are reported, the following abbreviations are used:
s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet, br ¼ broadened,
dd ¼ doublet of doublets, dt ¼ doublet of triplets; ESI-MS was
recorded on Agilent 1100 LC/MSD (70 eV) spectrometers. HRMS
was recorded on a Waters Q-Tof micro. THF was distilled from so-
dium/benzophenone ketyl. Dichloromethane was distilled over
CaH2. All other reagents and starting materials were purchased and
4
6
7
8
e
e
14.13
66.45
14.16
19.73
11.22
0.53
13.94
77.14
68.11
52.67
5.23
H
91%
62%
55%
70%
75%
72%
85%
87%
88%
76%
88%
70%
72%
65%
60%
65%
65%
67%
80%
75%
88%
75%
50%
82%
72%
75%
92%
78%
30,50-Di-CF3
40-CHO
9
30,50-Di-F
40-F3CO
40-F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
40-CH3CH2
40-CH3
40-CH3(CH)CH3
40-CF3
40-C(CH3)3
40-CH3CO
40-CH3OCO
40-CH3SO2
40-CN
56.31
13.18
ꢁ5.48
1.59
Table 2
Effects of the target compounds on proliferation of human HCC cells and normal
hepatic cells.
Compd
IC50 (mM)
ꢁ1.84
12.97
52.38
ꢁ4.17
55.25
30.00
57.99
18.02
33.04
51.36
5.85
30-CHO
QGY-7703
HepG-2
SMMC-7721
QSG-7701
20-F
30-CN
6
6.27
22.55
14.05
26.14
33.13
39.56
11.94
31.90
13.26
24.58
15.66
19.18
27.99
24.60
32.46
39,21
48.85
28.69
45.00
5.00
7.50
16.35
3.15
>100
69.21
66.32
>100
>100
20.74
48.10
33.12
>100
90.68
0.60
30-OMe
30- CF3
12
13
14
16
22
24
26
29
32
5-Fu
30,50-Di-OMe
30-Pydidine
20-CF3O
30-CH3
20.11
18.15
26.76
10.56
31.47
14.82
16.99
14.55
30,40-Di-F
30-F
20,50-Di-CH3
20,50-Di-F
35.16
54.26
17.40
28.62
12.48

162
M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
used as received (Aldrich, TCI, Adamas). BL-GHX-V was purchased
from Shanghai Bilang Instrument Co., Ltd. Reactions were moni-
tored by analytical TLC using silica gel 60 F254 plates and spots were
visualized by UV light irradiation (254 nm). Flash column chro-
matography was performed by using silica gel (200e300 mesh). All
tested compounds showed ꢂ95% purity as determined by com-
bustion analysis or by high-performance liquid chromatography
(HPLC). HPLC conditions were as follows: CHIRALPAK ADH,
4.6 mm ꢃ 250 mm, 5% / 90% CH3CN/MeOH/0.1% acetic acid,
benzene (60 mL) was treated with 5.0 mL of thionyl chloride and
thoroughly stirred at room temperature. After 2 h, the solvent and
the excess reagent were removed under reduced pressure. The
residue, 2,3,4-trimethoxybenzoyl chloride was dissolved in anhy-
drous ether (80 mL), 1,3,5-trimethoxybenzene (2.20 g, 13.03 mmol)
and AlCl3 (5.0 g) were added. After stirring for 8 h at room tem-
perature, the mixture was hydrolyzed with ice-cold water (500 mL)
containing concentrated HCl (45 mL) and extracted with CH2Cl2.
Solvent removal and purification with gel-column chromatography
(CHCl3) gave yellow solid 2-hydroxy-20,3,4,40,60-pentamethox-
ybenzophenone (2.97 g, 8.53 mmol, 65%). The yellow solid (2.97 g,
8.53 mmol) was treated with pyridine (100 mL), H2O (50 mL) and
aqueous 10% tetramethylammonium hydroxide (45 mL). The
mixture was refluxed for 36 h, poured into ice, acidified with HCl,
15 min run, flow rate 0.7 mL/min, UV detection (
l
¼ 220 nm).
5.1.2. Synthesis of compound 4
5.1.2.1. 1,3,5,6-Tetramethoxy-9H-xanthen-9-one
(4,
C17H16O6).
2,3,4-Trimethoxybenzoic acid (2.78 g, 13.14 mmol) in anhydrous
Fig. 2. Effects of compound 6 on QGY-7703 cell apoptosis. QGY-7703 cells were treated with DMSO (control) or compound 6 at various concentrations for 48 h and apoptosis was
quantified using flow cytometry after staining with annexin V/PI. A: Flow cytometry analysis of apoptotic QGY-7703 cells; B: Quantitation of A. Data were presented as the
mean ꢄ SD for three independent experiments. *P < 0.05 versus control cells.

M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
163
and extracted with ether. Purification with gel-chromatography
(CHCl3) and recrystallization (CHCl3) gave colorless needle crys-
tals, 1,3,5,6-tetramethoxy xanthone (2.24 g, 7.08 mmol, 83%). 1H
J ¼ 8.2 Hz, 2H), 6.82 (d, J ¼ 9.0 Hz, 1H), 6.74 (s, 1H), 3.99 (s, 3H), 3.89
(s, 3H), 3.85 (s, 3H), 3.40 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d
(ppm), 194.8, 175.3, 163.6, 158.5, 157.9, 156.7, 150.5, 144.8, 140.8,
NMR (DMSO-d6, 300 MHz):
d
(ppm), 7.78 (d, J ¼ 9.0 Hz, 1H), 7.15 (d,
137.2, 136.9, 134.0, 130.7, 129.3, 123.0, 118.7, 115.8, 111.2, 111.0, 107.5,
94.0, 62.4, 61.6, 58.2, 51.6; HR-ESI-MS calcd for C24H20O7 [M þ H]þ
421.1282, found 421.1276.
J ¼ 9.1 Hz, 1H), 6.71 (d, J ¼ 2.4 Hz, 1H), 6.49 (d, J ¼ 2.4 Hz, 1H), 3.92
(s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H); 13C NMR (DMSO-d6,
150 MHz):
d (ppm), 174.9, 166.5, 163.3, 161.0, 158.3, 150.4, 137.3,
123.0, 119.0, 111.0, 107.7, 97.4, 95.2, 62.8, 58.2, 58.0, 57.9; HR-ESI-MS
5.1.5.4. 4-(3,5-Difluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-
calcd for C17H16O6 [M þ H]þ 316.0947, found 316.0949.
one (9). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.75
(d, J ¼ 8.9 Hz, 1H), 7.24 (t, J ¼ 9.4 Hz, 1H), 7.20e7.17 (m, 2H), 7.11 (d,
5.1.3. Synthesis of compound 5
J ¼ 9.2 Hz, 1H), 6.72 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.87 (s, 3H),
5.1.3.1. 4-Iodo-1,3,5,6-tetramethoxy-9H-xanthen-9-one
(5,
3.50 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm), 177.1, 163.7,
C
17H15IO6). Method I: A solution of compound 4 (1.11 g, 3.5 mmol),
162.1, 162.1, 161.0, 155.7, 155.2, 148.6, 140.2, 136.9, 118.8, 116.7, 113.4,
109.4, 109.4, 108.9, 107.5, 100.8, 90.2, 61.0, 56.7, 56.6, 56.2; HR-ESI-
MS calcd for C23H18O6F2 [M þ H]þ 428.1071, found 428.1053.
iodine (1.9 g, 7.5 mmol) in DMF (5 mL) was stirred under argon at
room temperature for 2 days, the reaction mixture was poured into
water (15 mL), followed by filtration to give a brown powder. The
brown powder was stirred in cyclohexene (100 mL) for 3 h to give a
light tan color powder 5 (1.31 g, 2.98 mmol, 85%) after filtration;
Method II: A solution of compound 4 (1.11 g, 3.5 mmol), NIS (1.18 g,
5.25 mmol) in CH2Cl2 (5 mL) was stirred under argon at room
temperature overnight, the reaction mixture was quenched with
1 M aqueous sodium hydroxide and extracted with CH2Cl2, the
combined organic layer was washed successively with brine and
dried over MgSO4, filtered and concentrated. The crude material
was purified by column chromatographed to yield light tan color
powder (1.31 g, 2.98 mmol, 85%). 1H NMR (DMSO-d6, 600 MHz):
5.1.5.5. 1,3,5,6-Tetramethoxy-4-(4-(trifluoromethoxy)phenyl)-9H-
xanthen-9-one (10). White solid; 1H NMR (DMSO-d6, 600 MHz):
d
(ppm), 7.79e7.69 (m, 1H), 7.52 (d, J ¼ 8.7 Hz, 2H), 7.42 (d,
J ¼ 8.3 Hz, 2H), 7.08 (d, J ¼ 9.1 Hz, 1H), 6.71 (s, 1H), 3.97 (s, 3H),
3.89e3.88 (m, 3H), 3.85 (s, 3H), 3.37 (s, 3H); 13C NMR (DMSO-d6,
150 MHz):
d (ppm), 175.3, 163.3, 163.2, 158.4, 157.3, 156.8, 150.4,
149.3, 137.1, 136.8, 133.6, 123.0, 122.9, 122.3, 121.7, 121.2, 118.7, 110.9,
107.4, 93.9, 62.1 (2C), 58.2 (2C); HR-ESI-MS calcd for C24H19F3O7
[M þ H]þ 477.1156, found 477.1170.
d
(ppm), 7.78 (d, J ¼ 9.0 Hz, 1H), 7.17 (d, J ¼ 9.0 Hz, 1H), 6.69 (s, 1H),
4.02 (s, 3H), 3.98 (s, 3H), 3.96 (s, 3H), 3.94e3.93 (m, 3H); 13C NMR
(DMSO-d6,150 MHz): (ppm),174.9,165.3,164.2,158.7,158.7,150.3,
5.1.5.6. 4-(4-Fluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-one
(11). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.73 (d,
d
J ¼ 8.9 Hz, 1H), 7.46e7.38 (m, 2H), 7.30e7.21 (m, 2H), 7.07 (d,
137.3, 123.4, 118.4, 111.4, 108.6, 94.3, 67.8, 63.0, 59.1, 58.4, 58.3; HR-
J ¼ 9.1 Hz, 1H), 6.69 (s, 1H), 3.96 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H),
ESI-MS calcd for C17H15IO6 [M þ H]þ 442.9986, found 442.9985.
3.42 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm), 175.3, 164.1,
163.3, 163.1, 162.5, 158.4, 156.9, 150.5, 137.2, 135.1, 135.1, 130.3,
123.0, 116.5, 116.4, 111.3, 110.9, 107.4, 93.9, 62.4 (2C), 58.2 (2C);
5.1.4. Synthesis of biaryls (general procedure to 6e33)
To a suspension of Pd(PPh3)4 (0.03 equiv) in anhydrous DME was
added the aryl bromide and the mixture was stirred for 10 min at rt.
To this solution were added sequentially the arylboronic acid
(1.5 equiv) in aqueous Na2CO3 (2 M solution, 2.0 equiv), and the
mixture was refluxed for 18 h, cooled, and subjected to filtration.
The filtrate was evaporated to dryness and the residue was treated
with saturated NaCl solution. Standard workup followed by column
chromatography gave the biaryl product.
HR-ESI-MS calcd for
411.1246.
C
23H19FO6 [M
þ
H]þ 411.1238, found
5.1.5.7. 4-(4-Ethylphenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-one
(12). White solid; 1H NMR (DMSO-d6, 600 MHz):
(ppm), 7.74 (d,
d
J ¼ 8.9 Hz, 1H), 7.62e7.58 (m, 2H), 7.27 (d, J ¼ 1.4 Hz, 2H), 7.07 (d,
J ¼ 9.1 Hz, 1H), 6.70 (s, 1H), 3.97 (s, 3H), 3.87 (s, 3H), 3.85e3.84 (s,
3H), 3.37 (br. s., 3H), 2.66 (q, J ¼ 7.7 Hz, 2H), 1.25e1.21 (t, 3H); 13C
NMR (DMSO-d6, 151 MHz):
d (ppm), 175.4, 163.4, 162.9, 158.3,
5.1.5. 1H and 13C NMR data of compounds 6e33
156.9, 150.5, 144.5, 137.3, 133.9, 133.4, 133.3, 133.0, 130.7, 130.6,
129.0, 122.9, 112.5, 110.8, 107.4, 94.0, 62.3, 58.2, 58.2, 58.1, 29.9,
17.7; HR-ESI-MS calcd for C25H24O6 [M þ H]þ 421.1646, found
421.1657.
5.1.5.1. 1,3,5,6-Tetramethoxy-4-phenyl-9H-xanthen-9-one
(6).
White solid; 1H NMR (DMSO-d6, 600 MHz):
d
(ppm), 7.74 (d,
J ¼ 8.9 Hz, 1H), 7.43 (dd, J ¼ 9.4, 5.4 Hz, 2H), 7.40e7.31 (m, 3H), 7.08
(d, J ¼ 9.1 Hz, 1H), 6.71 (s, 1H), 3.97 (s, 3H), 3.86 (s, 3H), 3.84 (d, 3H),
3.36 (s, 3H); 13C NMR (DMSO-d6, 151 MHz):
d
(ppm), 175.4, 163.3,
5.1.5.8. 1,3,5,6-Tetramethoxy-4-p-tolyl-9H-xanthen-9-one
(13).
163.0,158.3,156.9,150.5,137.2,134.1,133.1,129.6,128.9,122.9,118.7,
112.5, 110.9, 107.4, 93.9, 62.3, 58.1, 56.5, 56.2; HR-ESI-MS calcd for
C
White solid; 1H NMR (DMSO-d6, 600 MHz):
d
(ppm), 7.74 (d,
J ¼ 8.9 Hz, 1H), 7.26e7.22 (m, 4H), 7.07 (d, J ¼ 8.9 Hz, 1H), 6.70 (s,
23H20O6 [M þ H]þ 393.1338, found 393.1269.
1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.39 (s, 3H), 2.36 (s, 3H);
13C NMR (DMSO-d6, 151 MHz):
d (ppm), 175.3, 163.4, 162.9, 158.3,
5.1.5.2. 4-(3,5-Bis(trifluoromethyl)phenyl)-1,3,5,6-tetramethoxy-9H-
xanthen-9-one (7). White solid; 1H NMR (DMSO-d6, 600 MHz):
156.9,150.5,144.3,138.1,133.9,137.3,132.9,131.0,130.2,122.9,118.7,
112.4, 110.8, 107.4, 94.0, 62.4, 58.2, 58.2, 58.1, 22.8; HR-ESI-MS calcd
for C24H22O6 [M þ H]þ 406.1416, found 406.1428.
d
(ppm), 8.17 (s, 2H), 8.12 (s, 1H), 7.76 (d, J ¼ 9.0 Hz, 1H), 7.11 (d,
J ¼ 9.0 Hz, 1H), 6.74 (s, 1H), 3.99 (s, 2H), 3.90 (s, 2H), 3.86 (s, 1H),
3.37 (s, 2H); 13C NMR (DMSO-d6, 150 MHz):
(ppm), 177.1, 164.0,
d
5.1.5.9. 4-(4-Isopropylphenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-
161.0, 155.8, 155.7, 148.6, 137.9, 136.9, 133.0, 133.0, 128.8, 128.8,
122.4, 122.4, 119.5, 118.8, 116.7, 113.6, 108.9, 107.5, 96.3, 61.0, 56.7,
56.6, 56.2; HR-ESI-MS calcd for C25H18F6O6 [M þ H]þ 529.1080,
found 529.1086.
one (14). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.73
(d, J ¼ 8.9 Hz, 1H), 7.30e7.26 (m, 4H), 7.07 (d, J ¼ 9.1 Hz, 1H), 6.70 (s,
1H), 3.97e3.96 (m, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.32 (s, 3H), 2.97e
2.91 (m, 1H), 1.26e1.24 (m, 6H); 13C NMR (DMSO-d6, 151 MHz):
d
(ppm), 175.4, 163.4, 162.9, 158.3, 156.9, 150.5, 149.1, 137.2, 133.9,
5.1.5.3. 4-(1,3,5,6-Tetramethoxy-9-oxo-9H-xanthen-4-yl)benzalde-
133.3, 132.9, 130.7, 130.6, 127.5, 122.9, 118.7, 112.5, 110.9, 107.4, 93.9,
62.2, 58.2, 58.2, 58.1, 35.2, 25.9; HR-ESI-MS calcd for C26H26O6
[M þ H]þ 435.1802, found 435.1834.
hyde (8). White solid; 1H NMR (DMSO-d6, 600 MHz):
d
(ppm),10.07
(s, 1H), 7.98 (d, J ¼ 8.2 Hz, 2H), 7.76 (d, J ¼ 9.0 Hz, 1H), 7.65 (d,

164
M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
5.1.5.10. 1,3,5,6-Tetramethoxy-4-(4-(trifluoromethyl)phenyl)-9H-
xanthen-9-one (15). White solid; 1H NMR (DMSO-d6, 600 MHz):
150 MHz): d (ppm), 195.0, 175.3, 163.5, 163.2, 158.4, 156.8, 150.5,
139.5, 137.9, 137.1, 135.1, 134.5, 130.6, 130.0, 123.0, 118.7, 111.0, 107.4,
94.0, 62.3, 58.3, 58.2, 58.2; HR-ESI-MS calcd for C24H20O7 [M þ H]þ
421.1282, found 421.1280.
d
(ppm), 7.83 (d, J ¼ 8.9 Hz, 1H), 7.76 (m, 2H), 7.74 (m, 2H), 7.04 (d,
J ¼ 8.9 Hz, 1H), 6.50 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.84 (s, 3H),
3.70 (s, 3H); 13C NMR (DMSO-d6, 151 MHz):
(ppm), 177.1, 163.3,
d
161.4, 155.7, 154.1, 148.6, 140.5, 136.9, 129.9, 129.9, 129.0, 123.2,
123.2, 124.9, 118.8, 116.7, 114.0, 108.9, 107.6, 94.9, 61.0, 56.7, 56.6,
56.3; HR-ESI-MS calcd for C24H19F3O6 [M þ H]þ 461.1206, found
461.1209.
5.1.5.17. 4-(2-Fluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-
one (22). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.75
(d, J ¼ 9.0 Hz, 1H), 7.50e7.39 (m, 2H), 7.32e7.25 (m, 2H), 7.09 (d,
J ¼ 9.1 Hz, 1H), 6.73 (s, 1H), 3.99 (s, 3H), 3.89 (s, 3H), 3.86e3.83 (m,
3H), 3.38 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm), 175.2,
5.1.5.11. 4-(4-tert-Butylphenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-
163.8, 163.7, 158.4, 157.1, 150.4, 137.2, 135.3, 131.7, 131.7, 125.8, 123.0,
118.7, 117.2, 117.0, 111.0, 107.3, 105.9, 94.0, 62.2, 58.3, 58.2, 58.2; HR-
ESI-MS calcd for C23H19FO6 [M þ H]þ 411.1238, found 411.1242.
one (16). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.73
(d, J ¼ 8.9 Hz, 1H), 7.46e7.43 (m, 2H), 7.30e7.27 (m, 2H), 7.07 (d,
J ¼ 9.1 Hz, 1H), 6.71 (s, 1H), 3.97 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H),
3.36 (br. s., 3H), 1.33 (s, 9H); 13C NMR (DMSO-d6, 151 MHz):
d
(ppm),
5.1.5.18. 3-(1,3,5,6-Tetramethoxy-9-oxo-9H-xanthen-4-yl)benzoni-
175.4, 163.4, 162.9, 158.3, 157.0, 151.3, 150.5, 137.2, 132.7, 131.1 (2C),
126.3 (2C), 122.9, 118.7, 112.4, 110.8, 107.4, 93.9, 62.1, 58.2, 58.2, 58.1,
36.2, 33.1 (3C); HR-ESI-MS calcd for C27H28O6 [M þ H]þ 449.1959,
found 449.1937.
trile (23). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.95
(m, 1H), 7.84 (m, 1H), 7.75 (d, J ¼ 9.2 Hz, 1H), 7.66 (m, 2H), 7.11 (d,
J ¼ 9.2 Hz,1H), 6.74 (s,1H), 3.99 (s, 1H), 3.90 (s,1H), 3.87 (s,1H), 3.47
(s, 1H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm), 175.2, 164.1, 162.1,
158.7, 157.1, 150.5, 138.6, 136.9, 132.6, 131.6, 130.0, 128.3, 118.8, 117.4,
116.7, 116.5, 114.7, 108.9, 108.1, 94.0, 62.0, 58.7, 58.6, 58.3; HR-ESI-
MS calcd for C24H19NO6 [M þ H]þ 418.1285, found 418.1283.
5.1.5.12. 4-(4-Acetylphenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-one
(17). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 8.01 (s,
2H), 7.75 (d, J ¼ 8.9 Hz, 1H), 7.59e7.55 (m, 2H), 7.09 (d, J ¼ 8.9 Hz,
1H), 6.74 (s, 1H), 3.98 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.40 (s, 3H),
5.1.5.19. 1,3,5,6-Tetramethoxy-4-(3-methoxyphenyl)-9H-xanthen-9-
2.63 (s, 3H); 13C NMR (DMSO-d6, 151 MHz):
d
(ppm), 199.6, 175.3,
one (24). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.74
163.5, 163.2, 158.5, 156.7, 150.5, 137.4, 133.5, 133.4, 129.4, 123.0,
118.7, 111.5, 111.3, 111.0, 107.4, 94.0, 93.9, 66.9, 62.4, 61.6, 58.3, 58.2,
28.7; HR-ESI-MS calcd for C25H22O7 [M þ H]þ 435.1438, found
435.1428.
(d, J ¼ 8.9 Hz,1H), 7.36e7.32 (m,1H), 7.08 (d, J ¼ 9.1 Hz,1H), 6.95 (m,
1H), 6.94e6.93 (m, 2H), 6.70 (s,1H), 3.97 (s, 3H), 3.87 (s, 3H), 3.85 (s,
3H), 3.75 (s, 3H), 3.39e3.38 (m, 3H); 13C NMR (DMSO-d6, 150 MHz):
d
(ppm), 175.5, 163.3, 163.0, 160.7, 158.3, 156.8, 150.5, 137.2, 135.3,
130.6, 125.4, 122.9, 118.8, 118.7, 114.5, 112.4, 110.8, 107.4, 94.0, 62.3,
58.2, 58.2, 58.1, 56.9; HR-ESI-MS calcd for C24H22O7 [M þ H]þ
423.1438, found 423.1427.
5.1.5.13. Methyl
4-(1,3,5,6-tetramethoxy-9-oxo-9H-xanthen-4-yl)
(ppm),
benzoate (18). White solid; 1H NMR (DMSO-d6, 600 MHz):
d
8.03e8.01 (m, 2H), 7.75 (d, J ¼ 8.9 Hz, 1H), 7.57e7.55 (m, 2H), 7.09
(d, J ¼ 9.1 Hz, 1H), 6.73 (s, 1H), 3.98 (s, 3H), 3.88 (s, 3H), 3.88 (s, 3H),
5.1.5.20. 1,3,5,6-Tetramethoxy-4-(3-(trifluoromethyl)phenyl)-9H-
xanthen-9-one (25). White solid; 1H NMR (DMSO-d6, 600 MHz):
3.85 (s, 3H), 3.38 (s, 3H); 13C NMR (DMSO-d6, 151 MHz):
d (ppm),
175.3, 168.1, 163.5, 163.2, 158.4, 156.7, 150.4, 139.4, 137.2, 133.6,
130.7, 130.6, 130.4, 130.2, 123.0, 118.7, 111.2, 111.0, 107.4, 94.0, 62.4,
58.4,58.2, 58.2, 54.1; HR-ESI-MS calcd for C25H22O8 [M þ H]þ
450.1314, found 450.1322.
d
(ppm), 7.77 (m, 1H), 7.74 (d, J ¼ 8.9 Hz, 1H), 7.70e7.69 (m, 1H),
7.68e7.67 (m,1H), 7.08 (d, J ¼ 9.1 Hz,1H), 6.84e6.81 (m,1H), 6.72 (s,
1H), 3.98 (s, 1H), 3.89 (s, 1H), 3.85 (s, 1H), 3.47 (s, 1H); 13C NMR
(DMSO-d6, 150 MHz): d (ppm), 177.1, 163.8, 161.1, 155.7, 154.7, 148.5,
138.1, 136.9, 131.0, 130.4, 129.7, 126.5, 125.6, 122.3, 118.8, 116.7, 113.7,
108.9, 107.6, 95.7, 61.0, 56.7, 56.6, 56.3; HR-ESI-MS calcd for
C
5.1.5.14. 1,3,5,6-Tetramethoxy-4-(4-(methylsulfonyl)phenyl)-9H-
xanthen-9-one (19). White solid; 1H NMR (DMSO-d6, 600 MHz):
24H19F3O6 [M þ H]þ 461.1206, found 461.1194.
d
(ppm), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.75 (d, J ¼ 8.9 Hz, 1H), 7.70 (d,
J ¼ 8.4 Hz, 2H), 7.10 (d, J ¼ 9.1 Hz, 1H), 6.75 (s, 1H), 3.99 (s, 3H), 3.90
5.1.5.21. 4-(3,5-Dimethoxyphenyl)-1,3,5,6-tetramethoxy-9H-
(s, 3H), 3.86 (s, 3H), 3.39 (s, 3H), 3.28 (s, 3H); 13C NMR (DMSO-d6,
xanthen-9-one (26). White solid; 1H NMR (DMSO-d6, 600 MHz):
150 MHz):
d
(ppm), 175.3, 163.7, 163.1, 158.4, 156.9, 150.5, 141.4,
d
(ppm), 7.74 (d, J ¼ 9.1 Hz, 1H), 7.11 (d, J ¼ 9.2 Hz, 1H), 6.83e6.81
139.8, 137.2, 134.2, 128.2, 123.0, 118.6, 111.1, 110.7, 107.4, 94.0, 62.3,
58.3, 58.2, 58.1, 45.5; HR-ESI-MS calcd for C24H22O8S [M þ H]þ
471.1108, found 471.1105.
(m, 1H), 6.70 (s, 1H), 6.52 (m, 2H), 3.97e3.95 (m, 3H), 3.89e3.87 (m,
3H), 3.85 (s, 3H), 3.73 (s, 6H), 3.42 (s, 3H); 13C NMR (DMSO-d6,
150 MHz):
d (ppm), 175.4, 163.3, 163.0, 161.8, 158.3, 157.9, 156.8,
150.5, 144.8, 137.3, 135.9, 129.3, 122.9, 115.8, 111.2, 110.9, 107.3, 101.1,
93.9, 70.8, 62.2, 58.2, 58.1, 57.0, 57.0; HR-ESI-MS calcd for C25H24O8
[M þ H]þ 453.1544, found 453.1508.
5.1.5.15. 4-(1,3,5,6-Tetramethoxy-9-oxo-9H-xanthen-4-yl)benzoni-
trile (20). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm),
7.92e7.89 (m, 2H), 7.75 (d, J ¼ 8.9 Hz,1H), 7.65e7.63 (m, 2H), 7.10 (d,
J ¼ 9.1 Hz, 1H), 6.73 (s, 1H), 3.98 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H),
5.1.5.22. 1,3,5,6-Tetramethoxy-4-(pyridin-3-yl)-9H-xanthen-9-one
3.44 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d
(ppm), 175.2, 163.7,
(27). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 8.60e
163.1, 158.5, 156.7, 150.4, 139.5, 137.2, 134.4, 134.3, 133.4, 133.4,
123.0, 120.9, 118.7, 111.7, 111.0, 110.6, 107.4, 94.0, 62.4, 58.3, 58.3,
58.2; HR-ESI-MS calcd for C24H19NO6 [M þ H]þ 418.1285, found
418.1283.
8.60 (m,1H), 8.60e8.58 (m,1H), 8.56 (dd, J ¼ 1.5, 4.8 Hz,1H), 7.74 (d,
J ¼ 9.1 Hz, 1H), 7.50e7.46 (m, 1H), 7.08 (d, J ¼ 8.9 Hz, 1H), 6.72 (s,
1H), 3.97 (s, 3H), 3.89 (s, 3H), 3.85 (s, 3H), 3.42 (s, 3H); 13C NMR
(DMSO-d6, 150 MHz):
d (ppm), 175.3, 163.6, 163.4, 158.5, 157.0,
153.4, 150.4, 149.9, 140.6, 137.2, 130.1, 124.9, 123.0, 118.7, 111.0, 108.9,
107.4, 93.9, 62.4, 58.3, 58.2, 58.2; HR-ESI-MS calcd for C22H19O6N
[M þ H]þ 393.1212, found 393.1224.
5.1.5.16. 3-(1,3,5,6-Tetramethoxy-9-oxo-9H-xanthen-4-yl)benzalde-
hyde (21). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm),
10.05 (s, 1H), 7.96e7.95 (m, 1H), 7.92 (d, J ¼ 7.7 Hz, 1H), 7.77e7.74
(m, 2H), 7.70e7.66 (m, 1H), 7.09 (d, J ¼ 9.1 Hz, 1H), 6.74 (s, 1H), 3.99
(s, 4H), 3.89 (s, 3H), 3.85 (s, 3H), 3.35 (s, 3H); 13C NMR (DMSO-d6,
5.1.5.23. 1,3,5,6-Tetramethoxy-4-(2-(trifluoromethoxy)phenyl)-9H-
xanthen-9-one (28). White solid; 1H NMR (DMSO-d6, 600 MHz):

M. Dai et al. / European Journal of Medicinal Chemistry 69 (2013) 159e166
165
d
(ppm), 7.77 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 8.9 Hz, 1H), 7.49 (d,
medium were supplemented with 10% fetal bovine serum,
100 Units/mL penicillin/streptomycin at 37 ꢀC, 5% CO2.
J ¼ 2.5 Hz, 1H), 7.14 (m, 2H), 7.09 (d, J ¼ 9.2 Hz, 1H), 6.74 (s, 1H), 3.99
(s, 3H), 3.88 (s, 3H), 3.85e3.84 (m, 3H), 3.33 (br. s., 3H); 13C NMR
(DMSO-d6, 150 MHz): d (ppm), 175.1, 166.5, 163.8, 163.6, 158.4, 157.1,
5.3. Cell proliferation/viability assay
150.4, 149.0, 135.6, 133.9, 133.3, 131.6, 130.7, 130.6, 129.0, 123.0,
122.6, 118.6, 111.0, 93.7, 62.8, 62.0, 58.2, 58.2; HR-ESI-MS calcd for
Cell proliferation was measured using a Cell Counting Kit-8
(CCK-8) (Dojindo, Kumamoto, Japan) according to the manufac-
C
24H19F3O7 [M þ H]þ 477.1156, found 477.1170.
turer’s instructions. In brief, the cells (2 ꢃ 105 cells/well in 100
mL
5.1.5.24. 1,3,5,6-Tetramethoxy-4-m-tolyl-9H-xanthen-9-one
(29).
medium) were cultivated for 24 h in 96-well microplates in a hu-
midified 5% CO2 incubator at 37 ꢀC, then treated with varying
concentrations of compounds or 1% DMSO vehicle control. After
White solid; 1H NMR (DMSO-d6, 600 MHz):
d
(ppm), 7.74 (d,
J ¼ 8.9 Hz, 1H), 7.33e7.29 (m, 1H), 7.20e7.16 (m, 2H), 7.14 (d,
J ¼ 7.7 Hz, 1H), 7.07 (d, J ¼ 9.1 Hz, 1H), 6.70 (s, 1H), 3.97 (s, 3H), 3.86
(s, 3H), 3.85 (s, 3H), 3.38 (s, 3H), 2.33 (s, 3H); 13C NMR (DMSO-d6,
incubation for 48 h, 10 mL of CCK8 solution was added to each well,
then the samples were incubated for additional 3 h before the
absorbance was measured at 450 nm by an epoch microplate
spectrophotometer.
150 MHz):
d (ppm), 175.4, 163.3, 162.9, 158.3, 156.8, 150.5, 138.6,
137.3, 134.0, 133.6,130.1, 129.6, 129.5, 122.9, 118.7, 112.6, 110.9, 107.4,
93.9, 62.3, 58.2, 58.1, 58.0, 22.9; HR-ESI-MS calcd for C24H22O6
[M þ H]þ 406.1416, found 406.1428.
5.4. Flow cytometry analysis
5.1.5.25. 4-(3,4-Difluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-
For measuring apoptosis, QGY-7703 cells were seeded in six-
9-one (30). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm),
plates (5 ꢃ 105 cells/mL) for 24 h and then treated with com-
7.74 (d, J ¼ 8.9 Hz,1H), 7.56e7.45 (m, 2H), 7.27e7.22 (m,1H), 7.09 (d,
pound 6 at 3.75, 7.5, 15 and 30 mM. After 48 h, the cells floating in
J ¼ 9.1 Hz, 1H), 6.69 (s, 1H), 3.96 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H),
the medium were collected and the adherent cells were detached
with 0.05% trypsin, washed twice with cold PBS, and centrifuged at
1000 ꢃ g for 5 min at 4 ꢀC. Subsequently, QGY-7703 cells were
gently resuspended in the binding buffer. Thereafter, the cells were
stained using Annexin V-FITC/PI apoptosis detection kit (BD Bio-
sciences, San Jose, CA, USA). After incubation at room temperature
for 15 min in the dark, the apoptotic cells were immediately
analyzed by flow cytometry.
3.49 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm), 175.2, 163.4,
163.2, 158.5, 156.8, 150.5, 137.2, 131.4, 130.3, 123.0, 122.3, 122.2,
118.7, 118.6, 118.5, 111.0, 110.2, 107.4, 93.9, 62.3, 58.3, 58.2, 58.1; HR-
ESI-MS calcd for C23H18F2O6 [M þ H]þ 429.1144, found 429.1131.
5.1.5.26. 4-(3-Fluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-9-
one (31). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm), 7.75
(d, J ¼ 8.9 Hz, 1H), 7.49e7.45 (m, 1H), 7.28e7.24 (m, 1H), 7.24e7.22
(m, 1H), 7.21e7.18 (m, 1H), 7.09 (d, J ¼ 9.1 Hz, 1H), 6.71 (s, 1H), 3.97
(s, 3H), 3.89 (s, 3H), 3.86 (s, 3H), 3.43 (s, 3H); 13C NMR (DMSO-d6,
Acknowledgments
150 MHz):
d (ppm), 175.3, 163.4, 163.2, 158.5, 156.8, 150.5, 137.2,
This work was supported by the NCET Foundation, NSFC
(81230090) and partially supported by the Global Research
Network for Medicinal Plants (GRNMP) and King Saud University,
the Shanghai Leading Academic Discipline Project (B906), FP7-
PEOPLE-IRSES-2008 (TCMCANCER Project 230232), the Key labo-
ratory of drug research for special environments, PLA, the Shanghai
Engineering Research Center for the Preparation of Bioactive Nat-
ural Products (10DZ2251300) and the Scientific Foundation of
Shanghai, China (12401900801, 09DZ1975700, 09DZ1971500,
10DZ1971700). The National Major Project of China (2011ZX09307-
002-03) and the National Key Technology R&D Program of China
(2012BAI29B06) are also thanked for support.
131.4, 131.3, 129.4, 123.0, 120.0, 119.9, 118.7, 115.7, 111.1, 111.0, 107.4,
93.9, 62.3, 58.2, 58.2 (2C); HR-ESI-MS calcd for C23H19FO6 [M þ H]þ
411.1238, found 411.1249.
5.1.5.27. 4-(2,5-Dimethylphenyl)-1,3,5,6-tetramethoxy-9H-xanthen-
9-one (32). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm),
7.74 (d, J ¼ 8.9 Hz, 1H), 7.55e7.50 (m, 1H), 7.43 (d, J ¼ 8.1 Hz, 2H),
7.09 (d, J ¼ 9.1 Hz, 1H), 6.72 (s, 1H), 3.98 (s, 3H), 3.91e3.89 (m, 3H),
3.85 (s, 3H), 3.37 (s, 3H), 2.35 (s, 3H), 2.08 (s, 3H); 13C NMR (DMSO-
d6, 150 MHz):
d (ppm), 177.5, 161.4, 160.2, 155.7, 155.1, 149.3, 137.6,
136.5, 135.7, 134.7, 130.1, 127.8, 126.7, 118.8, 116.7, 114.1, 108.9, 107.4,
94.6, 61.0, 56.7, 56.6, 56.3, 21.3, 20.1; HR-ESI-MS calcd for C25H24O6
[M þ H]þ 420.1573, found 420.1586.
Appendix A. Supplementary data
5.1.5.28. 4-(2,5-Difluorophenyl)-1,3,5,6-tetramethoxy-9H-xanthen-
9-one (33). White solid; 1H NMR (DMSO-d6, 600 MHz):
d (ppm),
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.08.
020. These data include MOL files and InChiKeys of the most
important compounds described in this article.
7.74 (d, J ¼ 8.9 Hz, 1H), 7.35 (dd, J ¼ 6.8, 11.4 Hz, 1H), 7.17e7.12 (m,
2H), 7.10 (d, J ¼ 9.1 Hz, 1H), 6.72 (s, 1H), 3.98 (s, 3H), 3.91 (s, 3H),
3.86 (s, 3H), 3.46 (s, 3H); 13C NMR (DMSO-d6, 150 MHz):
d (ppm),
175.1, 164.1, 163.5, 159.1, 158.5, 157.5, 157.0, 150.3, 137.1, 123.1, 118.7,
118.5, 118.5, 118.4, 118.3, 111.1, 107.3, 104.8, 93.9, 62.2, 58.3, 58.3,
58.2; HR-ESI-MS calcd for C23H18F2O6 [M þ H]þ 429.1144, found
429.1167.
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