Studies on chemical modification and biology of a natural product, gambogic acid (III): Determination of the essential pharmacophore for biological activity

Article abstract of DOI:10.1016/j.ejmech.2011.01.051

Caged 4-oxa-tricyclo[4.3.1.0^3,7]dec-2-one structural motifs are found in Garcinia natural products that demonstrate anti-tumor activity. Gambogic acid (GA, 1), the most abundant caged Garcinia xanthones, has been reported to be a promising anti-cancer agent. To identify the essential pharmacophore for its anti-tumor activity, a series of GA analogues that address potential key structural features for biological activity were synthesized, among which compound 11a displayed comparable in vitro anti-tumor activity as GA. Mechanistic studies on 11a determined that the compound induces apoptosis as well as arrests the G2/M phase of the cell cycle in HepG2 cells. The determination of the essential part of the scaffold found in GA to maintain anti-tumor effects, and the SAR based on the caged pharmacophore are reported and will provide key information for future anti-cancer drug development studies.

Full text of DOI:10.1016/j.ejmech.2011.01.051

European Journal of Medicinal Chemistry 46 (2011) 1280e1290
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studies on chemical modification and biology of a natural product, gambogic acid
(III): Determination of the essential pharmacophore for biological activity
,
,
Xiaojian Wang a 1, Na Lu b 1, Qian Yang a, Dandan Gong b, Changjun Lin a, Shenglie Zhang a, Meiyang Xi a,
,
a,b,
**
Yuan Gao b, Libing Wei b, Qinglong Guo b , Qidong You
*
a Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Caged 4-oxa-tricyclo[4.3.1.03,7]dec-2-one structural motifs are found in Garcinia natural products that
demonstrate anti-tumor activity. Gambogic acid (GA, 1), the most abundant caged Garcinia xanthones,
has been reported to be a promising anti-cancer agent. To identify the essential pharmacophore for its
anti-tumor activity, a series of GA analogues that address potential key structural features for biological
activity were synthesized, among which compound 11a displayed comparable in vitro anti-tumor
activity as GA. Mechanistic studies on 11a determined that the compound induces apoptosis as well as
arrests the G2/M phase of the cell cycle in HepG2 cells. The determination of the essential part of the
scaffold found in GA to maintain anti-tumor effects, and the SAR based on the caged pharmacophore are
reported and will provide key information for future anti-cancer drug development studies.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Article history:
Received 9 December 2010
Received in revised form
21 January 2011
Accepted 25 January 2011
Available online 3 February 2011
Keywords:
4-oxa-tricyclo[4.3.1.03,7]dec-2-one
Anti-tumor effects
Gambogic acid
HepG2 cell SAR studies
1. Introduction
[11], topoisomerase II [12], transferrin receptor [13] and stathmin
[14] which could explain part of the mechanism of action. More
GambogeresinsecretedbytheGarciniahanburyitreeinSoutheast
Asia is reported in traditional Chinese documents to possess diverse
biological effects [1]. Natural products isolated from gamboge resin
were classified as “caged xanthones” based on their unique 4-oxa-
tricyclo[4.3.1.03,7]dec-2-one ring system [2,3]. Gambogic acid (GA, 1),
the major ingredient from gamboges resin, is the most popular and
representative example. Preclinical investigation identified 1 as
a potent anti-tumor agent that inhibited cancer cell growth in vitro
and in vivo with minimal toxicity to normal cells. In addition, 1 pre-
vented cancer metastasis and angiogenesis [4e6], and has recently
finished phase II clinical trials in China.
recently, it was reported that GA covalently binds to the Cys179
residue of IKK protein thus mediating the suppression of lipo-
b
polysaccharide-induced activation of NF-kB in macrophages [15],
which established the role of 4-oxa-tricyclo[4.3.1.03,7]dec-2-one
ring system as Michael acceptor.
The topological structure of 1 can be divided into two parts:
a planar region that contains rings A, B, and C and a caged core D ring
system. Preliminary SAR studies indicated that modifications of
6-hydroxyl group, 8-ketone group, two isopentenyl groups and
30-carboxyl group are well tolerated, while the 9,10-double bond of
the a, b-unsaturated ketone is extremely important for the anti-tumor
The significant anti-tumor activity of 1 has attracted increasing
worldwide research interests. Biological studies revealed that the
mechanism of action of 1 is complex, involving apoptotic pro-
grammed cell death [5], cell cycle arrest [7], as well as activation of
T lymphocytes [8]. Other targets involved have also been reported
including the p53/mdm2 complex [9], bcl-2 family [10], surviving
activity of 1. Reduction of which leads to dramatically decreased
potency, demonstrating its essential role in the 4-oxa-tricyclo
[4.3.1.03,7]dec-2-one ring system for anti-tumor activity.
Recently, simplified GA derivatives have been identified to
exhibit partial anti-tumor activities, especially the basic caged
motif cluvenone (16) [16,18]. Based on these observations, a series
of analogues with a simplified planar region were synthesized in
order to further understand “caged” xanthones important for anti-
tumor activity. Analysis of the structural features of 1 resulted in
a strict “step by step” modification strategy (Scheme 1). In the first
step the A ring was completely removed from the core structure.
Secondly, the phenol group in the one position of the caged tricyclic
structure with the A ring removed was analyzed by transposition of
* Corresponding author. Tel./fax: þ86 25 83271440.
** Corresponding author. School of Pharmacy, China Pharmaceutical University,
Nanjing 210009, China. Tel./fax: þ86 25 83271351.
E-mail addresses: anticancerdrug@yahoo.cn (Q. Guo), youqidong@gmail.com
(Q. You).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.051

X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
1281
Scheme 1. Step by step simplified analogues of GA based on the caged ring system.
the phenolic group from C1 to the C2, C3 and C4 positions. Thirdly,
the phenolic group was removed from the A ring structure.
Furthermore, both A and B rings were completely cut from the core
structure. The SAR of the in vitro anti-tumor effects of these
compounds is discussed. Mechanistic studies for the most potent
compound 11a are reported which includes morphological obser-
vations, cell cycle and cell apoptosis evaluations.
benefit for the Claisen/DielseAlder rearrangement, the conversion
of 8a to bis(dimethylallyloxy) xanthone 9a was accomplished by
a two step procedure including propargylation of C5 and C6 phenols
with 2-chloro-2-methyl butyne followed by the removal of MOM
group in acidic medium. Compounds 9bed were obtained by
propargylation of the C5 and C6 phenols. Further reduction of the
alkynal groups using Lindlar catalyst gave the products 10aed.
Caged xanthone compounds 11aed were successfully obtained
though the expected Claisen/DielseAlder cascade reaction of 10aed
in DMF at 120 ꢀC for 1 h [20e22]. Deprotection of the MOM group in
11bed afforded the desired caged xanthone compounds 12bed,
respectively (Scheme 2).
2. Results and discussions
2.1. Chemistry
The cluvenone 16 was synthesized using previously reported
route [16,23]. Briefly, xanthone 13 was O-alkylated with 2-chloro-
2-methyl butyne in the presence of potassium carbonate and
a catalytic amount of copper iodide in acetone to afford alkyne 14.
Reduction of 14 with Lindlar catalyst produced 3,4-bis-(1,1-
dimethyl-allyloxy)-9H-xanthen-9-one (15). The desired targeted
compound 16 was obtained from Claisen/DielseAlder cascade
reaction of 15 in DMF at 120 ꢀC for 1 h (Scheme 3).
The preparation of caged compound 22 is outlined in Scheme 4.
An intramolecular Michael addition of commercially available
compound 17 was catalyzed under basic conditions to afford chro-
man-4-one (18). Demethylation of 18 followed by propargylation of
two hydroxyl groups led to ether compound 20. Lindlar-catalyzed
partial hydrogenation of alkyne 20 gave rise to intermediate 21. The
The synthetic route for xanthone-based caged compounds 11a
and 12bed are summarized in Scheme 2. Compounds 4aed were
obtained by aluminum trichloride catalyzed Friedel-Crafts acylation
of 2 with the corresponding dimethoxybenzoyl chloride (3aed) in
diethylether followed byan intramolecularcycloaddition in alkaline
conditions with 50e55% yield in two steps. Demethylation of 4aed
using 40% hydrobromic acid in acetic acid led to the formation of the
trihydroxyl xanthone 5aed. Reaction of 5aed in the presence
dichlorodiphenylmethane in diphenyl ether at 175 ꢀC afforded the
desired intermediates 6aed with the 5,6-dihydroxyl groups selec-
tively protected.[19] Chloromethyl methyl ether (MOM-Cl) was
employed to protect the remaining hydroxyl group. Subsequent
hydrogenolysis of the diphenylmethyl group afforded xanthone
compounds 8aed. Since the existence of C1-hydroxyl group was of
O
O
O
O
O
O
1
1
1
1
1
2
3
2
3
2
2
2
Cl
OMe
a,b
c
d
e
MeO
+
6
HO
3
MeO
MOMO
3
HO
6
6
6
MeO
OMe
OMe
3
O
O
O
OH
5
O
O
5
5
OMe
5
4
4
4a-d
4
4
4
OMe
O
O
OH
Ph
Ph
3a-d
2
5a-d
Ph
Ph
6a-d
7a-d
OH
O
OH
1
O
1
O
OH
O
O
j
1
g, h
i
6
6
O
O
O
O
O
5
5
O
O
O
O
1
9a
10a
11a
2
f
MOMO
6
3
OH
5
4
O
O
O
O
OH
1
1
O
O
O
O
O
O
2
3
2
3
1
1
8a-d
g
j
k
MOMO
MOMO
i
2
3
2
6
6
MOMO
HO
O
O
O
O
5
5
4
4
3
3a-8a C1 substituted
3b-8b C2 substituted
O
O
4
4
3c-8c
C3 substituted
9b-d
12b-d
10b-d
11b-d
3d-8d C4 substituted
9b-12b C2 substituted
9c-12c C3 substituted
9d-12d
C4 substituted
Scheme 2. The synthetic route for compound 11a and 12bed. Reagents and conditions: (a) AlCl3, Et2O, rt, 24 h; (b) 5 N NaOH, CH3OH, reflux, 36 h; (c) 40% HBr and HOAc mixed
solvent, reflux, 4 h; (d) Ph2CCl2, Ph2O, 175 ꢀC, 1 h; (e) MOM-Cl, NaH, DMF, 0 ꢀC, 3 h; (f) 10% Pd/C, THF/CH3OH (1:1), 45 ꢀC, 24 h; (g) ClC(CH3)2C^CH2, DBU, KI, CuI, CH3COCH3, reflux,
6 h; (h) 10% HCl, Et2O, rt, 4 h; (i) 10% Pd/BaSO4, CH3COOC2H5/C2H5OH (1:3), rt, 3 h; (j) DMF, 120 ꢀC, 1 h; (k) 1.0 mol/L HCl, Et2O/CH2Cl2 (1:1), 25 ꢀC, 4 h.

1282
X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
O
O
O
O
O
O
j
g
i
O
O
O
O
O
O
OH
O
O
OH
14
16
13
15
Scheme 3. The synthetic route for compound 16. Reagents and conditions: (g) DBU, KI, CuI, CH3COCH3, reflux, 6 h; (i) 10% Pd/BaSO4, CH3COOC2H5/C2H5OH (1:3), rt, 3 h; (j) DMF,
120 ꢀC, 1 h.
desired compound 22 was obtained from Claisen/DielseAlder
cascade reaction of 21 in DMF at 120 ꢀC for 1 h.
analysis (ꢁ400) presented significant morphological changes of
early apoptosis when treated with 11a. Identified by DAPI staining,
the bright nuclear condensation and the apoptotic bodies appeared
after treatment with 11a, while the untreated cells displayed
normal shape and clear skeleton (Fig. 1B).
2.2. Biological evaluations and SAR studies
2.2.1. Anti-proliferative activity and SAR studies of target
compounds
In detail, at 1
integrity, and the nuclei exhibited bright-condensed chromatin, at
M and 4 M concentration, typical apoptotic blebbing and
shrinkage were observed. Further quantitative assessment of 11a
on apoptosis induction was performed on flow cytometry using
fluorescein isothiocyanate Annexin V/Propidium Iodide (PI) double
staining assay. The percentage of apoptotic cells (Annexin Vþ/PI－
mM concentration, cell membrane still displayed
The anti-proliferative activities of the target compounds 11a,
12bed, 16 and 22 were assessed by the tetrazolium-based MTT
assay using human breast carcinoma MCF-7 cell line, human gastric
carcinoma BGC-823 cell line, human hepatocellular carcinoma
SMMC-7721 and HepG2 cell lines. Cancer cells were treated with
respective compounds for 24 h, and then cell viability was evalu-
ated through measurements of mitochondrial dehydrogenase
activity.
2
m
m
staining) in the control group was 3.0%. After treatment with 1
mM,
2
m
M and 4 M of 11a for 24 h, the early and median apoptotic cells
m
(lower right section of fluorocytogram) were increased to 8.1%,
18.9% and 23.3%, respectively, while the late apoptotic and necrotic
cells (upper right section of fluorocytogram) were increased
slightly (Fig. 2). These results suggested that 11a induced cell death
mainly via apoptosis rather than necrosis.
By analyzing the data in Table 1, it was evident that the struc-
tural feature relevant to anti-tumor effects of these caged xanthone
compounds embraced
a suitable planar conformation and
a hydroxyl group at the C1 position. The most active compound,
11a, possess equivalent in vitro anti-tumor effects as GA, while
12bed with a hydroxyl group at the C2, C3, and C4 position
respectively, as well as un-substituted cluvenone 16, were signifi-
cantly less effective than 11a, which suggest that C1 hydroxy group
is indispensable for the essential pharmacophore on GA. Xanthone
compound 22 with only C, D rings of GA was approximately 30e50
fold less active than GA, and about 4e15 fold less active than 11a.
The dramatically decreased activities revealed that a planar region
consisting of at least the B, C, D rings were essential for anti-
proliferative activity. These findings demonstrated that the
aromatic B ring acted as a rigid scaffold to support and restrict the
conformation of the caged ring system, and hydroxyl group at C1
position was also essential for anti-tumor potency.
2.2.3. The effect of 11a on cell cycle progression
Based on the significant cell growth inhibitory and apoptosis
inducing effect of 11a, the contribution of 11a on cell cycle
progression was further evaluated. Synchronized HepG2 cells
treated with 1 mM, 2 mM and 4 mM of 11a for 24 h were subjected to
flow cytometric analysis after DNA staining with PI. By analyzing
cell cycle distribution results shown in Fig. 3AeD, The fact was that
the exposure of HepG2 cells to gradient concentrations of 11a led to
a significant increase of cell population in G2/M phase accompa-
nied by a decrease in G1 phase compared with control. Cell cycle
distribution was summarized in histograms as shown in Fig. 3E.
These results indicated that the inhibitory and pro-apoptotic effects
of 11a were correlated with a pronounced cell cycle arrest in G2/M
phase in HepG2 cells, which were in accordance with previous
mechanistic studies of GA [17,24].
2.2.2. The effect of 11a on the induction of apoptosis
Compound 11a was evaluated for its influence on cell skeleton
by a morphological observation study. Under the inverted light
microscope (ꢁ200), incubation of 1
24 h resulted in phenotypic changes of HepG2 cells, such as
distortion, membrane blebbing and shrinkage, and large
m
M, 2
m
M and 4
m
M of 11a for
2.2.4. The influence of 11a on expression of bcl-2, bax and pro-
caspase-3 proteins
Preliminary explorations of the apoptotic induction mechanism
of 11a at the intra-cellular level were performed using western blot
technique. The influence of 11a on the expression level of Bcl-2,
bax, and pro-caspase-3 proteins on HepG2 cells were determined.
a
proportion of cells became round in shape and necrosis at high
concentrations, while cells in untreated group grew well and their
cytoskeletons were clear (Fig. 1A). The fluorescence microscopic
O
O
O
O
O
O
O
O
O
OH
O
O
O
p
O
O
m
o
l
n
O
O
O
OH
O
O
O
OH
O
19
17
18
20
21
22
Scheme 4. The synthetic route for compound 22. Reagents and conditions: (l) NaOEt, C2H5OH, reflux, 8 h; (m) AlCl3, C6H6, reflux, 5 h; (n) K2CO3, KI, CuI, CH3COCH3, reflux, 6 h; (o)
10% Pd/BaSO4, C2H5OH, rt, 2 h; (p) DMF, 120 ꢀC.

X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
1283
Table 1
irritation on vein vascular nor weight loss were observed in any of
the dose groups.
Anti-proliferative activity of caged compounds.a
Compound No.
IC50 ꢂ SD (
mM)
3. Conclusion
MCF-7
BGC-823
SMMC-7721
HepG2
11a
12b
12c
12d
16
22
9.16 ꢂ 0.11
20.6 ꢂ 1.77
12.4 ꢂ 1.85
19.4 ꢂ 1.05
13.9 ꢂ 1.31
68.3 ꢂ 3.47
2.19 ꢂ 0.20
3.59 ꢂ 0.095
32.5 ꢂ 0.95
17.6 ꢂ 1.17
19.3 ꢂ 1.64
20.3 ꢂ 0.7
8.06 ꢂ 0.087
20.0 ꢂ 1.3
12.3 ꢂ 0.9
11.7 ꢂ 1.11
10.5 ꢂ 0.95
147 ꢂ 6.56
3.20 ꢂ 0.36
2.37 ꢂ 0.055
12.3 ꢂ 0.6
23.4 ꢂ 0.89
14.3 ꢂ 1.22
6.77 ꢂ 0.12
95 ꢂ 5.56
In summary, we report a strict “step by step” simplification of
Gambogic acid. The xanthone-based compounds 11a, 12bed and 16
displayed in vitro anti-tumor effects against MCF-7, BGC-823,
SMMC-7721 and HepG2 cells, whereas the chromone-based
derivative 22 was substantially less effective. These results are
consistent with the tricyclic xanthone serving as an essential
skeleton that is required to maintain the observed inhibitory effect
on cancer cell growth. Among the above synthesized caged
xanthones, 11a was 5e8 fold more potent than 12bed and 16 in the
MTT assay, which indicated that hydroxyl group on C1 position is
essential for optimal anti-tumor activity. Preliminary mechanistic
studies showed that 11a was able to induce programmed cell death
and arrest cell cycle growth in the G2/M phase, accompanied by the
regulation of apoptotic related proteins and cellular caspase-3
activity, which is consistent with the apoptotic induction effects of
GA. Based on the promising anti-tumor effects in vitro and in vivo,
xanthone compound 11a is undergoing further evaluation as
a candidate for cancer chemotherapy.
81.1 ꢂ 2.52
2.35 ꢂ 0.07
Gambogic acid
2.40 ꢂ 0.78
a
The IC50 values were determined from eight different concentrations of
compounds at two-fold dilutions, and were the means of two separate experiments.
Bcl-2 protein is a suppressor of apoptosis while bax is an activator
of apoptosis. Caspase-3 is one of the most important proteins of the
caspase family involved in the activation of apoptosis. It is first
produced as the inactive pro-caspases-3 that is activated by caspase
9. Therefore, the decrease of pro-casepases-3 could be considered
as the signal of apoptosis. As shown in Fig. 4, after incubation with
1 mM, 2 mM and 4 mM of 11a for 24 h, the expression level of bcl-2
and pro-caspase-3 were decreased dramatically on HepG2 cells,
while the protein level of bax had no significant change compared
with control, which indicated the initiation of apoptosis. All the
above results showed that 11a could significantly inhibit cancer cell
growth through the apoptotic pathway.
4. Experimental protocols
4.1. General materials and methods
2.2.5. The influence of 11a on the activity of caspase-3
The potency of 11a as inducers of apoptosis was measured by
cellular caspase-3 colorimetric assay. As shown in Fig. 5, being
All reagents were purchased from commercial sources and were
used without further purification unless otherwise noted. Melting
points were determined by XT-4 melting point apparatus and were
reported without any correction. IR spectra were recorded on
a Nicolet Impact 410 spectrometer using KBr film. The 1H NMR and
13C NMR spectrawerecollected on Bruker AV-300 instruments using
deuterated solvents with tetramethylsilane (TMS) as internal stan-
dard. EI-MS was recorded on Shimadzu GCMS-2010 apparatus. Each
of the target compounds were purified by silica gel (60 Å, 70e230
mesh) column chromatography. Concentration and evaporation of
the solvent after reaction or extraction was carried out on a rotary
evaporator (Büchi Rotavapor) operated at reduced pressure.
treated with 1
mM, 2 mM and 4 mM of 11a for 24 h, the 3-fold
increase of cellular caspase-3 activity was observed, which means
that 11a induces a concentration-dependent increase in caspase-3
activity on HepG2 cells.
2.2.6. Efficacy of 11a on the growth of inoculated Heps in mice
Following the in vitro studies of 11a described above, we
determined the in vivo efficacy of 11a on the growth of hepatoma
solidity (Heps) in mice using GA as positive control. As shown in
Table 2, 11a displayed significant inhibitory effect on the growth of
inoculated Heps in mice with a dose dependent manner, while the
weight of tumor in control group was not changed. Besides, neither
Cancer cell lines involved in this study were obtained from Cell
Bank of Shanghai, Institute of Biochemistry and Cell Biology,
Fig. 1. Cell morphological assessment of 11a on HepG2 cells. (A) Light microscope detection (ꢁ200): (a) Control group; (b) 1
mM of 11a; (c) 2 mM of 11a; (d) 4 mM of 11a. (B)
Fluorescence microscope detection (ꢁ400): (a) Control group; (b) 1 M of 11a; (c) 2 M of 11a; (d) 4 M of 11a. The white arrows point the observed apoptotic bodies.
m
m
m

Fig. 2. Fluorescence-activated cell sorter analysis for 11a on HepG2 cells by Annexin-V/PI staining. Upper left: dead cells; Upper right: late apoptotic cells; Lower left: fully viable
cells; Lower right: early apoptotic cells. (A) Control group; (B) 1 M of 11a; (C) 2 M of 11a; (D) 4 M of 11a.
m
m
m
Fig. 3. Effect of 11a on cell cycle progression on HepG2 cells. (A) Control group; (B) 1 mM of 11a; (C) 2 mM of 11a; (D) 4 mM of 11a; (E) Quantitative cell cycle distribution of 11a.

X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
1285
Table 2
The inhibitory effect of iv administration of 11a on the growth of Heps transplanted
mice.
Group
Dose
(mg/kg)
Body weight (g)
Pre-dose After-dose
Weight of
tumor
(g)
Inhibitory
rate (%)
Control
11a
11a
0
12.5
25
19.80 ꢂ 1.48 26.90 ꢂ 3.41 2.01 ꢂ 0.40
20.20 ꢂ 1.99 26.30 ꢂ 4.62 1.71 ꢂ 0.33
0.00
14.92
20.20 ꢂ 1.14 29.40 ꢂ 3.37 1.36 ꢂ 0.37** 32.34
19.10 ꢂ 1.29 24.70 ꢂ 3.53 1.02 ꢂ 0.28** 49.25
20.90 ꢂ 1.29 27.60 ꢂ 3.37 1.10 ꢂ 0.27** 45.27
11a
50
Gambogic 20
acid
*P < 0.05，**P < 0.01 vs control.
(4.9 g, 0.12 mol) at 0 ꢀC. Then the reaction mixture was heated to
110 ꢀC for next 36 h. After cooled to 0 ꢀC, the mixture was acidified
with 2 mol/L HCl solution till pH ¼ 2e3. The precipitation was
formed, filtered, washed with cold water and dried to provide
4aed, respectively.
Fig. 4. Effect of 11a on the expression of bcl-2, bax, and pro-caspase-3 proteins on
HepG2 cells.
Chinese Academy of Sciences. Cells were cultured in 90% RPMI 1640
Medium (GIBCO, Invitrogen Corporation, NY) supplemented with
10% fetal bovine serum (Sijiqing, Zhejiang, China), 100 ug/mL
4.2.1.1. 1,5,6-Trimethoxy-9H-xanthen-9-one (4a). Yield: 55%; yellow
solid; mp: 158e159 ꢀC (lit.: 163 ꢀC). 1H NMR (Acetone-d6): 3.95
(s, 6H), 4.01 (s, 3H), 6.97 (d, J ¼ 0.6, 8.4 Hz, 1H), 7.14 (d, J ¼ 0.6,
8.4 Hz, 1H), 7.15 (d, J ¼ 9.0 Hz, 1H), 7.66e7.72 (m, 1H), 7.89 (d,
J ¼ 9.0 Hz, 1H). ESI-MS: m/z ¼ 287 [M þ H]þ, 309 [M þ Na]þ.
benzyl penicillin and 100
mg/mL streptomycin in a humidified
environment with 5% CO2 at 37 ꢀC. Samples containing >95%
Gambogic acid isolated from Gamboge resin according to the
protocols reported previously was used in the experiments. All the
tested compounds were dissolved in DMSO to a concentration of
0.01 mol/L and stored at ꢃ4 ꢀC.
4.2.1.2. 2,5,6-Trimethoxy-9H-xanthen-9-one
(4b). Yield:
50%;
yellow solid; mp: 155.2e156.5 ꢀC 1H NMR (CDCl3): 3.93e4.04
(m, 9H), 7.03 (d, J ¼ 9.0 Hz, 1H), 7.32 (dd, J1 ¼ 9.2 Hz, J2 ¼ 3.1 Hz, 1H),
7.52 (d, J ¼ 9.2 Hz,1H), 7.70 (d, J ¼ 3.1 Hz,1H), 8.11 (d, J ¼ 9.0 Hz,1H).
EI-MS: m/z ¼ 286 [M]þ, 271, 256.
4.2. Chemistry
4.2.1. General procedure for the synthesis of trimethoxy-9H-
xanthen-9-one (4aed)
4.2.1.3. 3,4,6-Trimethoxy-9H-xanthen-9-one (4c). Yield 53%; yellow
solid; mp: 120.2e121.2 ꢀC 1H NMR (Acetone-d6): 3.98e4.03
(m, 9H), 7.00 (dd, J1 ¼8.9 Hz, J2 ¼ 2.4 Hz,1H), 7.09 (d, J ¼ 2.4 Hz, 1H),
7.19 (d, J ¼ 9.0 Hz,1H), 7.95 (d, J ¼ 9.0 Hz,1H), 8.13 (d, J ¼ 8.9 Hz,1H);
EI-MS: m/z ¼ 286 [M]þ, 271, 256, 241.
To a solution of dimethoxylbenzoic acid (2.39 g, 13.14 mmol) in
dry dichlormethane (60 mL) was added oxalyl chloride (5.73 mL,
65.7 mmol). The mixture was stirred at room temperature for 24 h
and then concentrated under reduced pressure to afford dime-
thoxylbenzoyl chloride 3aed as colorless residue. To a solution of
3aed and 1,2,3-trimethoxybenzene (2, 2.43 g, 14.45 mmol) in
20 mL anhydrous ether, aluminum trichloride (5.26 g, 39.42 mmol)
was added at 0 ꢀC. The resulting mixture was stirred for 12 h at
room temperature under N2 protection. Then a mixture of 15%
hydrochloric acid and ethyl acetate (100 mL, V/V ¼ 1:1) were
added. The ethyl acetate layer was partitioned, washed with brine
(30 mL ꢁ 3), dried over magnesium sulfate and concentrated under
reduced pressure. The residue was suspended in a solution that
contained methanol (22.4 mL), water (14.9 mL) and sodium hydrate
4.2.1.4. 3,4,5-Trimethoxy-9H-xanthen-9-one (4d). Yield 50%; yellow
solid; mp: 130.2e131.5 ꢀC 1H NMR (DMSO-d6): 4.03e4.06 (m, 9H),
7.22 (d, J ¼ 9.0 Hz, 1H), 7.35 (t, J ¼ 8.0 Hz, 1H), 7.45 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 1.6 Hz, 1H), 7.78 (dd, J1 ¼ 8.0 Hz, J2 ¼ 1.6 Hz, 1H), 7.91 (d,
J ¼ 9.0 Hz, 1H). EI-MS: m/z ¼ 286 [M]þ, 271, 257, 243, 228.
4.2.2. General procedure for the synthesis of trihydroxy-9H-
xanthen-9-one (5aed)
To a stirred mixture of 40% hydrobromic acid (17 mL) in acetic
acid (34 mL) was added 4aed (500 mg, 1.75 mmol), the reaction
mixture was refluxed for 12 h at 120 ꢀC under N2 protection. After
cooled to 0 ꢀC, the reaction mixture was acidified till pH ¼ 3e4 with
10% NaOH solution, the precipitation was filtered, washed with ice
water and dried to provide 5aed, respectively.
4.2.2.1. 1,5,6-Trihydroxy-9H-xanthen-9-one (5a). Yield: 70%; white
solid; mp: >300 ꢀC (Lit.: >300 ꢀC). 1H NMR (DMSO-d6): 6.77 (dd,
J1 ¼ 0.87 Hz, J2 ¼ 8.3 Hz, 1H), 6.97 (d, J ¼ 8.8 Hz, 1H), 7.05 (dd,
J1 ¼ 0.87 Hz, J2 ¼ 8.3 Hz, 1H), 7.57 (d, J ¼ 8.8 Hz, 1H), 7.68
(t, J ¼ 8.3 Hz, 1H), 9.53 (s, 1H), 10.68 (s, 1H), 12.91 (s, 1H). EI-MS: m/
z ¼ 244 [M]þ.
4.2.2.2. 2,5,6-Trihydroxy-9H-xanthen-9-one (5b). Yield 80%; white
solid; mp: >300 ꢀC 1H NMR (DMSO-d6): 6.91 (d, J ¼ 8.8 Hz,1H), 7.26
(dd, J1 ¼ 9.0 Hz, J2 ¼ 3.0 Hz, 1H), 7.44 (d, J ¼ 3.0 Hz, 1H), 7.50
(d, J ¼ 9.0 Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 1H), 9.82 (s, 1H). EI-MS: m/
z ¼ 244 [M]þ, 216, 187, 142.
Fig. 5. The influence of 11a on caspase-3 activity o n HepG2 cells.

1286
X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
4.2.2.3. 3,4,6-Trihydroxy-9H-xanthen-9-one (5c). Yield 85%; white
solid; mp: >300 ꢀC 1H NMR (DMSO-d6): 6.83e6.93 (m, 3H), 7.50
(d, J ¼ 8.8 Hz, 1H), 8.00 (d, J ¼ 8.8 Hz, 1H), 9.28 (s, 1H), 10.31 (s, 1H),
10.77 (s, 1H). ESI-MS: m/z ¼ 243 [M－H]þ.
4.2.4.3. 9-(methoxymethoxy)-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]
xanthen-6-one (7c). Yield: 99%; yellow solid; mp: 179e181ꢀ
NMR (CDCl3): 3.44 (s, 3H), 5.21 (s, 2H), 6.91e6.96 (m, 2H), 7.10
(d, J ¼ 2.4 Hz, 1H), 7.32e7.36 (m, 6H), 7.54e7.59 (m, 4H), 7.82
(d, J ¼ 5.1 Hz, 1H), 8.17 (d, J ¼ 5.3 Hz, 1H). EI-MS: m/z ¼ 452 [M]þ,
375, 331, 165, 105.
C
1H
4.2.2.4. 3,4,5-Trihydroxy-9H-xanthen-9-one (5d). Yield 84%; white
solid; mp: >300 ꢀC 1H NMR (DMSO-d6): 6.93 (d, J ¼ 9 Hz, 1H),
7.22e7.31 (m, 2H), 7.53e7.60 (m, 2H), 9.82 (br, 3H); EI-MS: m/
z ¼ 244 [M]þ, 216, 187, 142.
4.2.4.4. 10-(methoxymethoxy)-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]
xanthen-6-one (7d). Yield: 90%; yellow solid; mp: 180e182ꢀ
C
1H
NMR (CDCl3): 3.64 (s, 3H), 5.38 (s, 2H), 7.02 (d, J ¼ 9 Hz, 1H),
7.24e7.30 (m, 3H), 7.39e7.45 (m, 5H), 7.50e7.54 (d, J ¼ 9 Hz, 1H),
7.62e7.68 (m, 4H), 7.92e8.00 (m, 1H). EI-MS: m/z ¼ 452 [M]þ, 375,
331, 165, 105.
4.2.3. General procedure for the synthesis of hydroxyl-2,2-diphenyl-
6H-[1,3]dioxolo[4,5-c] xanthen-6-one (6aed)
To a solution of 5aed (1 g, 4.46 mmol) in diphenyl ether (40 mL),
dichlorodiphenylmethane (1.4 mL,
7 mmol) was added. The
mixture was heated to 175 ꢀC for 4 h. After cooled to 60 ꢀC, the dark
mixture was poured into the petroleum (300 mL), the precipitation
was filtered and purified by column chromatography (eluent, PE/
EtOAc ¼ 8:1) to give 6aed, respectively.
4.2.5. General procedure for the synthesis of 5.6-dihydroxy-
methoxymethoxy- 9H-xanthen-9-one (8aed)
To a solution of 7aed (1 g, 2.2 mmol) in THF and methanol
mixed solvent (20 mL, V/V ¼ 1:1) was added 10% Pd/C (100 mg).
The reaction mixture was stirred under an atmosphere of hydrogen
for 24 h at room temperature and filtered through a plug of silica
gel, the filtration was concentrated and purified through a column
4.2.3.1. 7-Hydroxyl-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c] xanthen-6-
one (6a). Yield: 73%; yellow solid; mp: 203e205 ꢀC 1H NMR
(CDCl3): 6.72 (d, J ¼ 8.4 Hz, 1H), 6.90 (d, J ¼ 9.3 Hz, 1H), 6.93 (d,
J ¼ 9.3 Hz, 1H), 7.34 (m, 6H), 7.49 (t, J ¼ 9.3 Hz, 1H), 7.57 (m, 4H),
7.82 (d, J ¼ 8.4 Hz, 1H), 12.70 (s, 1H). EI-MS: m/z ¼ 408 [M]þ.
chromatography (eluent, PE/EtOAc
respectively.
¼
1:1) to afford 8aed,
4.2.5.1. 5,6-Dihydroxy-1-(methoxymethoxy)-9H-xanthen-9-one
(8a). Yield: 85%; green solid; mp: >300ꢀ 1H NMR (DMSO-d6):
4.2.3.2. 8-Hydroxyl-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]xanthen-6-
C
one (6b). Yield: 73.3%; yellow solid; mp: 238e240ꢀ
C
1H NMR
3.46 (s, 3H), 5.31 (s, 2H), 6.90 (d, J ¼ 8.25 Hz, 1H), 7.02 (dd,
J1 ¼ 8.8 Hz, J2 ¼ 2.2 Hz, 1H), 7.14 (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.2 Hz, 1H),
7.84 (dd, J1 ¼8.8 Hz, J2 ¼ 2.2 Hz,1H), 8.25 (d, J ¼ 8.25 Hz,1H). EI-MS:
m/z ¼ 288 [M]þ, 256, 244.
(CDCl3): 6.93 (d, J ¼ 9 Hz, 1H), 7.20 (d, J ¼ 9 Hz, 1H), 7.30e7.36 (m,
6H), 7.39 (d, J ¼ 9 Hz, 1H), 7.57 (m, 4H), 7.76 (d, J ¼ 9 Hz, 1H), 7.89
(s, 1H). EI-MS: m/z ¼ 408 [Mþ], 391, 331.
4.2.3.3. 9-Hydroxyl-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]xanthen-6-
4.2.5.2. 5,6-Dihydroxy-2-(methoxymethoxy)-9H-xanthen-9-one
(8b). Yield: 80%; white solid; mp: 181e183ꢀ C 1H NMR (DMSO-d6):
3.43 (s, 3H), 5.27 (s, 2H), 6.91 (d, J ¼ 8.7 Hz, 1H), 7.46e7.60 (m, 3H),
7.69 (d, 1H, J ¼ 3 Hz). EI-MS: m/z ¼ 288 [M]þ, 273, 258, 244.
one (6c). Yield: 61%; yellow solid; mp: 231e232ꢀ
C
1H NMR
(Acetone-d6): 6.94e6.98 (m, 2H), 7.1 (d, J ¼ 8.7 Hz, 2H), 7.47e7.50
(m, 6H), 7.66e7.69(m, 4H), 7.85(d, J ¼ 8.7 Hz, 1H,), 8.10(q, J ¼ 9 Hz,
1H); EI-MS: m/z ¼ 408 [M]þ, 331, 303, 165.
4.2.5.3. 5,6-Dihydroxy-3-(methoxymethoxy)-9H-xanthen-9-one
4.2.3.4. 10-Hydroxyl-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]xanthen-6-
(8c). Yield: 90%; white solid; mp: >300ꢀ 1H NMR (DMSO-d6):
C
one (6d). Yield 60%; yellow solid; mp: 248e249ꢀ
C
1H NMR
3.52 (s, 3H), 5.30 (s, 2H), 6.97 (d, J ¼ 8.8 Hz, 1H), 7.04e7.07
(q, J1 ¼ 2.2 Hz, J2 ¼ 8.8 Hz, 1H), 7.14 (d, J ¼ 2.2 Hz, 1H), 7.84 (d,
J ¼ 8.8 Hz, 1H), 8.25 (d, J ¼ 8.8 Hz, 1H). EI-MS: m/z ¼ 288 [M]þ, 258,
215, 187, 167.
(CDCl3): 6.20 (s, 1H), 6.99 (d, J ¼ 9 Hz, 1H), 7.22e7.35 (m, 3H),
7.40e7.43 (m, 6H), 7.60e7.63 (m, 4H), 7.82e7.84 (d, J ¼ 6 Hz, 1H),
7.96 (d, J ¼ 9 Hz, 1H). EI-MS: m/z ¼ 408 [M]þ, 331, 303, 165.
4.2.4. General procedure for the synthesis of methoxymethoxyl-2,2-
diphenyl-6H-[1,3] dioxolo[4,5-c]xanthen-6-one (7aed)
4.2.5.4. 3,4-Dihydroxy-5-(methoxymethoxy)-9H-xanthen-9-one
(8d). Yield: 79.6%; white solid; mp: >300ꢀ
C
1H NMR (DMSO-d6):
To a solution of 6aed (1.36 g, 3.3 mmol) in acetone (30 mL), was
added sodium hydride (160 mg, 6.6 mmol), the mixture was stirred
at 0 ꢀC for 30 min. Chloromethyl methyl ether (0.54 mL, 6.6 mmol)
was then added dropwise for 10 min. The mixture was stirred at
room temperature for 3 h and then poured into ice water. The
precipitation was filtered, washed with brine (2 ꢁ 30 mL) and dried
to afford 7aed, respectively.
3.60 (s, 3H), 5.41 (s, 2H), 6.95 (d, J ¼ 9 Hz,1H), 7.33 (t, J ¼ 8.4 Hz,1H),
7.57 (t, J ¼ 8.4 Hz, 2H), 7.78 (d, J ¼ 8.4 Hz, 1H). EI-MS: m/z ¼ 288
[M]þ, 271, 258, 243, 215, 187, 167.
4.2.6. 1-(methoxymethoxy)-5,6-bis(2-methylbut-3-yn-2-yloxy)-
9H-xanthen-9-one (9a)
To a solution of 8a (1.7 mmol) in acetonitrile (10 mL), potassium
iodide (288 mg, 1.7 mmol), DBU (1.2 mL, 8.16 mmol) and CuI (5 mg,
0.026 mmol) were added. The reaction mixture was stirred at room
temperature for 10 min, and then 2-chloro-2-methylbut-3-yne
(1.4 mL, 13.6 mmol) was added, the resulted mixture was stirred for
6 . Water (100 mL) and ethyl ether (20 mL) were added to the
mixture and acidified with 10% HCl solution till pH ¼ 3. The solution
was partitioned between ethyl acetate (50 mL) and water. The ethyl
acetate layer was dried over sodium sulfate and was concentrated
by rotary evaporation. The residue was purified through a column
chromatography (eluent, PE:EtOAc ¼ 1:1) to afford 9a as yellow
solid. Yield: 43%; mp: 138e140ꢀ C 1H NMR (CDCl3): 1.79 (s, 6H),1.83
(s, 6H), 2.28 (s,1H), 2.66 (s,1H), 6.79 (dd, J1 ¼8.1 Hz, J2 ¼ 0.6 Hz,1H),
6.95 (dd, J1 ¼ 8.1 Hz, J2 ¼ 0.6 Hz, 1H), 7.56 (t, J ¼ 8.1 Hz, 1H), 7.66 (d,
4.2.4.1. 7-(methoxymethoxy)-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]
xanthen-6-one (7a). Yield: 90%; yellow solid; mp: 138e140ꢀ
C
1H
NMR (CDCl3): 3.56 (s, 3H), 5.36 (s, 2H), 6.94 (d, J ¼ 8.4 Hz, 1H), 7.04
(dd, 1H, J1 ¼ 8.2 Hz, J2 ¼ 0.6 Hz, 1H), 7.17 (dd, J1 ¼ 8.2 Hz, J2 ¼ 0.6 Hz,
1H), 7.40 (m, 6H), 7.55 (dd, J1 ¼ 8.2H, J2 ¼ 0.6 Hz, 1H), 7.64 (m, 4H),
7.89 (d, J ¼ 8.4 Hz, 1H). EI-MS: m/z ¼ 452 [M]þ.
4.2.4.2. 8-(methoxymethoxy)-2,2-diphenyl-6H-[1,3]dioxolo[4,5-c]
xanthen-6-one (7b). Yield: 93%; yellow solid; mp: 160e162ꢀ
C
1H
NMR (CDCl3): 3.42 (s, 3H), 5.18 (s, 2H), 6.92 (d, J ¼ 8.4 Hz, 1H),
7.31e7.44 (m, 8H), 7.56e7.58 (m, 4H), 7.82 (d, J ¼ 3 Hz, 1H), 7.88
(d, J ¼ 8.4 Hz, 1H). EI-MS: m/z ¼ 452 [M]þ, 422, 407, 390, 375.

X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
1287
J ¼ 9 Hz, 1H), 7.98 (d, J ¼ 9 Hz, 1H), 12.71 (s, 1H). EI-MS: m/z ¼ 376
6.07e6.28 (m, 2H), 7.05 (d, J ¼ 9 Hz, 1H), 7.34 (m, 2H), 7.85 (m, 2H).
EI-MS: m/z ¼ 424 [M]þ, 409, 396, 381, 356, 341, 327, 288, 256, 244.
[M]þ, 310, 244.
4.2.8.3. 6-(methoxymethoxy)-5,6-bis(2-methylbut-3-en-2-yloxy)-
9H-xanthen-9-one (10c). Yield: 89%; yellow oil. 1H NMR (CDCl3):
1.49 (s, 6H), 1.52 (s, 6H), 3.44 (s, 3H), 4.94e5.18 (m, 4H), 5.22 (s, 1H),
6.07e6.27 (m, 2H), 6.95 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.1 Hz, 1H), 7.00e7.06
(m, 2H), 7.83 (d, J ¼ 9 Hz, 1H), 8.17 (d, J ¼ 8.7 Hz, 1H). EI-MS: m/
z ¼ 424 [M]þ 409, 381, 356, 327, 256, 244.
4.2.7. General procedure for the synthesis of xanthone compounds
9bed, 14
To a solution of 8bed, 13 (1.7 mmol) in acetonitrile (10 mL),
potassium iodide (288 mg, 1.7 mmol), DBU (1.2 mL, 8.16 mmol) and
CuI (5 mg, 0.026 mmol) were added. The reaction mixture was
stirred at room temperature for 10 min, then 2-chloro-2-methyl-
but-3-yne (1.4 mL, 13.6 mmol) was added and the resulted mixture
was stirred for 6 more hours. Water (50 mL) and EtOAc (20 mL) was
added to the mixture and stirred for 30 min. The organic layer was
separated, dried over sodium sulfate, and evaporated. The residue
was purified through column chromatography (eluent, PE/
EtOAc ¼ 8:1) to afford 9bed, 14 respectively.
4.2.8.4. 5-(methoxymethoxy)-5,6-bis(2-methylbut-3-en-2-yloxy)-
9H-xanthen-9-one (10d). Yield: 80%; yellow oil. 1H NMR (CDCl3):
1.51 (s, 6H), 1.56 (s, 6H), 3.51 (s, 3H), 4.90e5.18 (m, 4H), 5.33 (s, 1H),
6.13 (dd, J1 ¼ 10.6 Hz, J2 ¼ 17.7 Hz, 1H), 6.34 (dd, J1 ¼ 10.6 Hz,
J2 ¼ 17.7 Hz, 1H), 7.06 (d, J ¼ 9 Hz, 1H), 7.19 (m, 1H), 7.41 (d,
J ¼ 8.4 Hz, 1H), 7.86 (m, 2H). EI-MS: m/z ¼ 424 [M]þ, 409, 396, 381,
356, 341, 327, 288, 256.
4.2.7.1. 2-(methoxymethoxy)-5,6-bis(2-methylbut-3-yn-2-yloxy)-
9H-xanthen-9-one (9b). Yield: 50%; yellow oil. 1H NMR (CDCl3):
1.68 (s, 6H), 1.74 (s, 6H), 2.22 (s, 1H), 2.59 (s, 1H), 3.40 (s, 3H), 5.15
(s, 1H), 6.77 (d, J ¼ 8.4 Hz, 1H), 7.05 (d, J ¼ 8.4 Hz, 1H), 7.44 (d,
J ¼ 9 Hz, 1H), 7.81 (s, 1H), 7.97 (d, J ¼ 9 Hz, 1H). EI-MS: m/z ¼ 420
[M]þ, 405, 377, 354, 339, 309, 288, 244.
4.2.8.5. 3,4-bis(2-methylbut-3-en-2-yloxy)-9H-xanthen-9-one
(15). Yield: 85%; yellow oil. 1H NMR (CDCl3): 1.58 (s, 6H), 1.61
(s, 6H), 5.01e5.05 (d, J ¼ 12.1, 1H), 5.17e5.25 (m, 3H), 6.15e6.25 (dd,
J1 ¼ 17.7 Hz, J2 ¼ 11.1 Hz, 1H), 6.25e6.35 (dd, J1 ¼ 17.7 Hz,
J2 ¼ 11.1 Hz, 1H), 7.11e7.14 (d, J ¼ 9 Hz, 1H), 7.34e7.39 (t, J ¼ 7.5 Hz,
1H), 7.49e7.52 (d, J ¼ 5.1 Hz, 1H); 7.68e7.73 (t, J ¼ 9 Hz, 1H),
7.92e7.95 (d, J ¼ 9 Hz, 1H), 8.30e8.33 (d, J ¼ 9 Hz, 1H). EI-MS: m/
z ¼ 364 [M]þ.
4.2.7.2. 6-(methoxymethoxy)-3,4-bis(2-methylbut-3-yn-2-yloxy)-
9H-xanthen-9-one (9c). Yield: 45%; yellow solid; mp: 128e130ꢀ C 1H
NMR (CDCl3): 1.69 (s, 6H), 1.75 (s, 6H), 2.26 (s, 1H), 2.58 (s, 1H), 3.45
(s, 3H), 5.22 (s, 1H), 6.96 (d, J1 ¼ 8.7 Hz, J2 ¼ 2.1 Hz, 1H), 7.04 (d,
J ¼ 2.1 Hz, 1H), 7.54 (d, J ¼ 9 Hz, 1H), 7.97 (s, J ¼ 9 Hz, 1H), 8.18 (d,
J ¼ 8.7 Hz, 1H). EI-MS: m/z ¼ 420 [M]þ, 405, 377, 354, 339, 324, 309,
295, 288, 244.
4.2.9. General procedure for the synthesis of caged xanthone
compounds 11aed, 16
To a solution of 10aed, 15 (0.27 mmol) in DMF (2.0 mL) was
heated at 120 ꢀC for 1 h. The yellow reaction mixture was cooled to
25 ꢀC and was purified by column chromatography eluent, PE/
EtOAc ¼ 6:1 to afford caged xanthone 11aed and 16, respectively.
4.2.7.3. 5-(methoxymethoxy)-3,4-bis(2-methylbut-3-yn-2-yloxy)-
9H-xanthen-9-one (9d). Yield: 48%; yellow solid; mp: 125e127ꢀ
C
1H NMR (CDCl3): 1.56 (s, 6H), 1.91 (s, 6H), 2.22 (s, 1H), 2.67 (s, 1H),
3.56 (s, 3H), 5.34 (s, 2H), 7.25 (m, 1H), 7.49 (d, J ¼ 8.1 Hz,1H), 7.69 (d,
J ¼ 9 Hz, 1H), 7.97 (d, J ¼ 8.1 Hz, 1H), 8.04e8.07 (d, J ¼ 9 Hz, 1H). EI-
MS: m/z ¼ 420 [Mþ], 405, 377, 354, 339, 324, 309, 295.
4.2.9.1. Caged xanthone 11a. Yield: 62%; yellow solid; mp:
130e132ꢀ
C
1H NMR (CDCl3): 0.95 (s, 3H), 1.18e1.25 (m, 4H), 1.30
(s, 3H), 1.61 (s, 3H), 2.26 (dd, J1 ¼ 13.5 Hz, J2 ¼ 4.5 Hz, 1H), 2.37
(d, J ¼ 9.6 Hz, 1H), 2.54 (d, J ¼ 7.8 Hz, 2H), 3.44 (dd, J1 ¼ 6.9 Hz,
J2 ¼ 4.5 Hz, 1H), 4.34 (m, 1H), 6.43 (dd, J1 ¼ 8.1 Hz, J2 ¼ 0.9 Hz, 1H),
6.45 (dd, J1 ¼ 8.1 Hz, J2 ¼ 0.9 Hz, 1H), 7.32 (t, J ¼ 8.1 Hz, 1H), 7.41 (d,
J ¼ 9.6 Hz, 1H), 12.00 (s, 1H); 13C NMR (CDCl3): 16.68, 24.95, 25.49,
29.05, 29.14, 30.27, 46.98, 48.82, 83.53, 84.46, 90.02, 106.12, 107.38,
109.37, 118.64, 133.88, 134.90, 135.30, 138.75, 159.59, 162.90, 181.33,
202.69. EI-MS: m/z ¼ 380 [Mþ], 352; Anal. Calcd for C23H24O5 (%): C,
72.61; H, 6.36; Found: C, 72.60; H, 6.67.
4.2.7.4. 3,4-bis(2-methylbut-3-yn-2-yloxy)-9H-xanthen-9-one
(14). Yield 55%; white solid; mp: 136e137ꢀ C 1H NMR(CDCl3): 1.70
(s, 6H), 1.77 (s, 6H), 2.22 (s, 1H), 7.30 (t, J ¼ 7.5 Hz, 1H), 7.45 (d,
J ¼ 8.4 Hz, 1H), 7.58 (d, J ¼ 9.0 Hz, 1H), 7.62 (dd, J1 ¼ 7.5 Hz,
J2 ¼ 8.4 Hz, 1H), 7.80 (d, J ¼ 9.0 Hz, 1H), 8.26 (d, J ¼ 7.5 Hz, 1H). EI-
MS: m/z ¼ 442 [M]þ, 427, 376, 361, 324, 310, 281.
4.2.8. General procedure for the synthesis of xanthone compounds
10aed, 15
4.2.9.2. Caged xanthone 11b. Yield: 64%; yellow solid; mp:
148e150 ꢀC 1H NMR (CDCl3): 0.91 (s, 3H), 1.18e1.26 (m, 7H,), 1.64
(s, 3H), 2.26 (dd, J1 ¼ 13.5 Hz, J2 ¼ 4.5 Hz, 1H), 2.36 (d, J ¼ 9.6, 1H),
2.53 (d, J ¼ 6.6 Hz, 2H), 3.40 (s, 4H), 4.36 (m, 1H), 5.09 (s, 2H), 6.42
(d, J ¼ 9 Hz, 1H), 7.16 (dd, J1 ¼ 9 Hz, J2 ¼ 3 Hz, 1H), 7.47 (d, J ¼ 2.7 Hz,
1H). ESI-MS: m/z ¼ 425 [M þ H]þ.
To a solution of 9aed, 14 (0.53 mmol) in ethanol (10 mL) was
added 10% Pd/BaSO4 (23 mg). The mixture was stirred under an
atmosphere of hydrogen for 2 h at room temperature and filtered
through a plug of silica gel, the filtration was concentrated and
purified through column chromatography (eluent, PE/EtOAc ¼ 8:1)
to afford 10aed, 15, respectively.
4.2.9.3. Caged xanthone 11c. Yield: 63%; yellow solid; mp:
168e169ꢀ
C
1H NMR (CDCl3): 0.92 (s, 3H), 1.17e1.29 (m, 7H), 1.63
4.2.8.1. 1-(methoxymethoxy)-5,6-bis(2-methylbut-3-en-2-yloxy)-
9H-xanthen-9-one (10a). Yield: 89%; colorless oil. 1H NMR (CDCl3):
1.51 (s, 6H), 1.53 (s, 6H), 4.94e5.15 (m, 4H), 6.10e6.20 (m, 1H), 6.71
(dd, J1 ¼ 8.4 Hz, J2 ¼ 0.9 Hz, 1H), 6.86 (dd, J1 ¼ 8.4 Hz, J2 ¼ 0.9 Hz,
1H), 7.06 (d, J ¼ 9 Hz, 1H), 7.48 (t, J ¼ 8.4 Hz, 1H), 7.79 (d, J ¼ 9 Hz,
1H), 12.67 (s, 1H). EI-MS: m/z ¼ 380 [M]þ, 312, 244.
(s, 3H), 2.24 (dd, J1 ¼13.5 Hz, J2 ¼ 4.5 Hz,1H), 2.37 (d, J ¼ 9.6 Hz,1H),
2.51 (d, J ¼ 9.3 Hz, 2H), 3.36e3.44 (m, 4H), 4.36 (m, 1H), 5.15 (s, 2H),
6.57 (d, J ¼ 2.1 Hz, 1H), 6.64 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.1 Hz, 1H), 7.31
(d, J ¼ 6.9 Hz, 1H), 7.80 (d, J ¼ 8.7 Hz, 1H). EI-MS: m/z ¼ 424 [M]þ,
396, 381, 368, 353, 327, 299, 285, 257.
4.2.9.4. Caged xanthone 11d. Yield: 67%; yellow solid; m.p.
4.2.8.2. 2-(methoxymethoxy)-5,6-bis(2-methylbut-3-en-2-yloxy)-
9H-xanthen-9-one (10b). Yield: 90%; yellow oil. 1H NMR (CDCl3):
1.50 (s, 6H), 1.52 (s, 6H), 3.43 (s, 3H), 5.17 (s, 1H), 4.94e5.13 (m, 4H),
174e175ꢀ 1H NMR (CDCl3): 0.87 (s, 3H), 1.19e1.25 (m, 7H), 1.73
C
(s, 3H), 2.28 (dd, J1 ¼ 13.5 Hz, J2 ¼ 4.2 Hz, 1H), 2.39 (d, J ¼ 9.6 Hz,
1H), 2.51 (d, J ¼ 9.3 Hz, 2H), 3.41e3.51 (m, 4H), 4.34 (t, 1H), 5.17 (s,

1288
X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
2H), 6.89 (d, J ¼ 8.4 Hz, 1H), 7.22 (dd, J1 ¼ 8.4 Hz, J2 ¼ 6.9 Hz, 1H),
7.35 (d, J ¼ 6.9 Hz, 1H), 7.52 (d, J ¼ 6.9 Hz, 1H). ESI-MS: m/z ¼ 425
[M þ H]þ, 447 [M þ Na]þ.
yield 18 as white solid. Yield 94%; mp: 172e173ꢀ C 1H NMR (CDCl3):
1.48 (d, J ¼ 6.3 Hz, 3H), 2.56 (m, 2H), 3.79 (s, 3H), 3.84 (s, 3H),
4.51e4.58 (m, 1H), 6.55 (d, J ¼ 9 Hz, 1H), 7.57 (d, J ¼ 9 Hz, 1H). EI-
MS: m/z ¼ 222 [Mþ], 207, 181, 152.
4.2.9.5. Caged xanthone 16. Yield: 57%; yellow solid; mp:
124e126ꢀ
C
1H NMR (CDCl3): 0.84 (s, 3H), 1.15e1.30 (m, 7H), 1.66
4.2.12. 7,8-Dihydroxy-2-methychroman-4-one (19)
(s, 3H), 2.27 (dd, J1 ¼13.5 Hz, J2 ¼ 4.5 Hz,1H), 2.39 (d, J ¼ 9.6 Hz,1H),
2.55 (d, J ¼ 9.3 Hz, 2H), 3.41e3.47 (m, 1H), 4.35 (t, 1H), 6.97e7.02
(m, 2H), 7.36 (d, J ¼ 6.9 Hz,1H), 7.45 (dd, J1 ¼8.4 Hz, J2 ¼ 7.2 Hz, 1H),
7.87 (d, J ¼ 8.4 Hz, 1H); 13C NMR (CDCl3): 16.69, 25.14, 25.33, 29.12,
30.33, 30.93, 46.84, 48.83, 83.52, 84.62, 90.37, 118.10, 118.97, 119.14,
121.87, 126.99, 133.76, 134.78, 134.94, 136.21, 159.67, 176.52, 203.02.
EI-MS: m/z ¼ 364 [M]þ, 336, 321, 293, 267, 239, 225, 197. Anal. Calcd
for C23H24O4 (%): C, 75.80; H, 6.64; Found: C, 75.81; H 6.60.
To a solution of 18 (4.2 g) in benzene (40 mL), aluminum tri-
chloride was added in three portions. The mixture was refluxed for
5 h. After cooling, the reaction mixture was acidified till pH ¼ 1.
Ethyl acetate (50 mL) was added, and then the mixture was stirred
for 1 h. The organic layer was separated, dried over sodium sulfate
and evaporated. The product was purified by column chromatog-
raphy (eluent. PE/EtOAc ¼ 10:1) to yield compound 19 as light
yellow solid. Yield: 68%; mp: 264e265ꢀ C 1H NMR (CDCl3): 1.48 (d,
J ¼ 6 Hz, 3H), 2.60 (d, J ¼ 6.9 Hz, 2H), 4.51e4.58 (m, 1H), 5.25 (s, 1H),
7.71 (s, 1H), 6.57 (d, J ¼ 9 Hz, 1H), 7.37 (d, J ¼ 9 Hz, 1H). EI-MS: m/
z ¼ 194 [M]þ, 152.
4.2.10. General procedure for the synthesis of caged xanthone
compounds 12bed
To a solution of 11bed (50 mg) in dichlormethane (5 mL) and
ethyl ether (5 mL) was added 1 mL concentrated hydrochloric acid
at 0 ꢀC. The mixture was stirred at room temperature for 1 h. EtOAc
(5 mL) and water (20 mL) was added and stirred for 15 min. The
organic layer was separated, dried over sodium sulfate, and evap-
orated under reduced pressure. The residue was purified through
a column chromatography (eluent, PE/EtOAc ¼ 4:1) to give 12bed,
respectively.
4.2.13. 2-Methyl-7,8-bis(2-methylbut-3-yn-2-yloxy)chroman-4-
one (20)
To a solution of 19 (500 mg, 1.7 mmol) in acetone (10 mL),
potassium iodide (1.7 g, 10.3 mmol), potassium carbonate (1.4 g,
10.3 mmol) and CuI (25 mg, 0.13 mmol) were added. The reaction
mixture was stirred at room temperature for 10 min, then 2-chloro-
2-methylbut-3-yne (2.2 mL, 20.6 mmol) was added. The resulted
mixture was refluxed for 6 . Water (50 mL) and EtOAc (20 mL) was
added to the mixture and stirred for 30 min. The organic layer was
separated, dried over sodium sulfate, and evaporated. The residue
was purified through column chromatography (eluent, PE/
EtOAc ¼ 16:1) to afford 20 as yellow oil. Yield: 50%. 1H NMR (CDCl3):
1.56 (d, J ¼ 3 Hz, 3H), 1.63e1.67 (m, 12H), 2.27 (s, 1H), 2.56 (m, 3H),
4.42e4.54 (m, 1H), 7.18 (d, J ¼ 9 Hz, 1H), 7.54 (d, J ¼ 9 Hz, 1H). EI-
MS: m/z ¼ 326 [M]þ, 311, 283, 267, 260, 245.
4.2.10.1. Caged xanthone 12b. Yield: 85%; yellow solid; mp:
158e159ꢀ
C
1H NMR (CDCl3): 0.91 (s, 3H), 1.22e1.38 (m, 7H), 1.65
(s, 3H), 2.32 (dd, J1 ¼13.5 Hz,, J2 ¼ 4.5 Hz,1H), 2.43 (d, J ¼ 9.6 Hz,1H),
2.62(d, J¼ 9.3 Hz, 2H), 3.52(t, J ¼ 4.5 Hz,1H), 4.34(m,1H), 6.28(s,1H),
6.97 (d, J ¼ 9 Hz, 1H), 7.12 (dd, J1 ¼ 9 Hz,, J2 ¼ 3 Hz, 1H), 7.42
(d, J ¼ 6.9 Hz,1H), 7.50 (d, J ¼ 3 Hz,1H); 13C NMR (CDCl3): 16.84, 25.00,
25.35, 29.10, 29.14, 30.31, 46.93, 48.71, 83.58, 84.55, 90.09, 110.71,
118.90, 119.17, 119.51, 125.47, 133.98, 134.78,134.89, 150.81, 154.26,
177.11, 203.08. EI-MS: m/z ¼ 380 [M]þ, 352, 337, 283, 255, 213. Anal.
Calcd for C23H24O5 (%): C, 72.61; H, 6.36; Found: C, 72.33; H, 6.50.
4.2.14. 2-Methyl-7,8-bis(2-methylbut-3-en-2-yloxy)chroman-4-
one (21)
To a stirred solution of 20 (100 mg) in ethanol (20 mL) was
added 10% Pd/BaSO4 (10 mg). The reaction mixture was stirred
under an atmosphere of hydrogen for 2 h at room temperature. The
reaction mixture was filtered through a plug of silica gel, and the
residue was concentrated and purified through column chroma-
tography (eluent, PE/EtOAc ¼ 8:1) to afford 21 as brown oil. Yield:
80%. 1H NMR (CDCl3): 1.40e1.47(m, 15H), 2.52e2.55 (m, 2H),
4.39e4.49 (m, 1H), 4.90e5.12 (m, 4H), 6.02e6.19 (m, 2H), 6.65 (d,
J ¼ 9 Hz, 1H), 7.42 (d, J ¼ 9 Hz, 1H). EI-MS: m/z ¼ 330 [M]þ, 311, 287,
260, 245.
4.2.10.2. Caged xanthone 12c. Yield: 87%; yellow solid; mp:
197e199 ꢀC; 1H NMR (DMSO-d6): 0.96 (s, 3H), 1.13e1.33(m, 7H),
1.61 (s, 3H), 2.25 (dd, J1 ¼ 13.5 Hz, J2 ¼ 3.9 Hz, 1H), 2.42e2.53 (m,
3H), 3.45 (d, J ¼ 6.6 Hz, 1H), 4.3 (m, 1H), 6.40 (s, 1H), 6.56 (d,
J ¼ 9 Hz, 1H), 7.32 (d, J ¼ 6.9 Hz, 1H), 7.67(d, J ¼ 9 Hz, 1H); 13C NMR
(DMSO-d6): 16.58, 24.80, 25.18, 28.85, 28.87, 30.02, 46.18, 47.93,
83.05, 83.77, 90.22, 102.40, 111.34, 111.72, 118.56, 128.48, 133.07,
133.38, 134.29, 161.18, 165.05, 170.42, 203.24; EI-MS: m/z ¼ 379
[M ꢃ H]þ. Anal. Calcd for C23H24O5 (%): C, 72.61; H, 6.36; Found: C,
72.29; H, 6.54.
4.2.15. Caged xanthone 22
4.2.10.3. Caged xanthone 12d. Yield: 86%; yellow solid; mp:
To a solution of 21 (350 mg, 0.27 mmol) in DMF (4.0 mL) was
heated at 120 ꢀC for 1 h. The yellow reaction mixture was cooled to
25 ꢀC and the mixture was purified by column chromatography
(eluent, PE/EtOAc ¼ 6:1) to yield the caged xanthone 22. Yield: 45%;
203e205ꢀ C 1H NMR (CDCl3):
d 0.91 (s, 3H), 1.27e1.35(m, 7H), 1.73
(s, 3H), 2.36 (dd, J1 ¼13.5 Hz, J2 ¼ 4.5 Hz,1H), 2.45 (d, J ¼ 9.6 Hz,1H),
2.65 (m, 2H), 3.45 (m, 1H), 4.44 (m, 1H), 7.05e7.09 (m, 2H), 7.43
(d, J ¼ 6.9 Hz, 1H), 7.50 (t, J ¼ 7.2 Hz, 1H), 7.93 (d, J ¼ 7.5 Hz, 1H); 13C
NMR (CDCl3): 15.55, 24.31, 24.37, 27.92, 27.93, 29.97, 45.83, 48.06,
82.19, 83.35, 90.52, 117.04, 117.20, 118.25, 120.01, 120.94, 133.34,
133.73, 133.94, 143.81, 146.55, 175.10, 201.90. EI-MS: m/z ¼ 380
[M]þ, 352, 337, 309, 283, 255, 241, 213. Anal. Calcd for C23H24O5 (%):
C, 72.61; H, 6.36; Found: C, 72.60; H, 6.70.
mp: 155e156ꢀ
C
1H NMR (CDCl3): 1.18 (s, 3H), 1.24e1.36 (m, 4H),
1.42 (s, 3H), 1.51 (s, 3H), 1.54 (s, 3H), 2.09 (dd, J1 ¼18 Hz, J2 ¼ 16 Hz,
1H), 2.23e2.41 (m, 3H), 2.52e2.66 (m, 2H), 3.23e3.30 (m, 1H),
4.0e4.07 (m, 1H), 4.33 (m, 1H), 7.17 (d, J ¼ 7.2 Hz, 1H); 13C NMR
(CDCl3): 17.55, 20.66, 25.05, 27.39, 28.38, 28.42, 29.76, 44.4, 44.77,
45.74, 66.08, 82.37, 83.88, 86.58, 118.99, 133.06, 134.48, 136.18,
191.96, 203.33. ESI-MS: m/z ¼ 353 [M þ Na]þ. Anal. Calcd for
C20H26O4 (%): C, 72.70; H, 7.93; Found: C, 72.87; H, 7.93.
4.2.11. 7,8-Dimethoxy-2-methylchroman-4-one (18)
To a mixture of 17 (800 mg, 3.6 mmol) in ethanol (50 mL),
sodium ethoxide (4 g, 48.8 mmol) was added and heated to reflux
for 8 h. After cooling, the solution was filtered; the filtrate was
concentrated by rotary evaporation. The residue was purified
through a column chromatography (eluent, PE:EtOAc ¼ 8:1) to
4.3. MTT assay
Cell viabilities were measured by a colorimetric assay using 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT;

X. Wang et al. / European Journal of Medicinal Chemistry 46 (2011) 1280e1290
1289
Roche, Ltd.) as described previously. [25] Experiments carried out
4.8. Cellular caspase-3 colorimetric assay
in triplicate in a parallel manner for each concentration of target
compounds used and the results were presented as mean ꢂ SE.
Control cells were given only culture media. After incubation for
24 h, absorbance (A) was measured at 570 nm. Survival ratio (%)
was calculated using the following equation: survival ratio
(%) ¼ (Atreatment/Acontrol) ꢁ 100%. IC50 was taken as the concentra-
tion that caused 50% inhibition of cell viabilities and calculated by
the SigmaPlot software (Systat Software Inc., USA).
Caspase-3 activity was measured using the caspase-3 colori-
metric assay kit (KeyGen, Nanjing, China) according to the manu-
facturer’s protocol. Briefly, HepG2 cells were treated with 1
M and 4 L cell
M of 11a and incubated at 37 ꢀC for 24 h. 50
lysates were obtained, incubated for 20 min on ice, and centrifuged
at 10 000 rpm for 2 min at 4 ꢀC. Then 50
L supernatants, con-
taining 200 g total protein, were incubated with 5 L caspase-3
mM,
2
m
m
m
m
m
m
substrate (Ac-DEVD-pNA) at 37 ꢀC for 4 h. The absorbance intensity
was measured using a microplate reader (varioskan, Catalog
number 5250010) at wavelength 405 nm. The fold increase in
caspase-3 activity was determined by the ratio of ODtreared to
ODcontrol. All data in were presented as mean ꢂ SD. Statistical
significance was evaluated by t-test.
4.4. Cell morphological assessment
To detect morphological evidence of apoptosis, cell nuclei were
visualized following DNA staining with the fluorescent dye DAPI.
Briefly, cells were seeded at a concentration of 1 ꢁ 105 cells/well in
6-well tissue culture plates and treated with indicated concentra-
tion of 11a. At the end of incubation, the morphology of cells was
monitored under an inverted light microscope. Cells were then
fixed with 4% paraform for 20 min and washed with PBS, and then
incubated with DAPI (1 mg/ml) for 10 min. After washed with PBS,
cells were observed using fluorescent microscopy (Olympus, Japan)
with a peak excitation wavelength of 340 nm.
4.9. The in vivo tumor growth inhibition assay
Kunming mice with body weight of 18e22 g were transplanted
with hepatoma solidity (Heps) according to protocols of transplant
tumor research [26]. Twenty-four hours after tumor trans-
plantation, animals were weighed and randomly divided into five
groups. All test drugs were given through injections 24 h after
tumor transplantation. The treated groups were iv injected with
12.5, 25, or 50 mg/kg of 11a and 20 mg/kg of GA, respectively. The
negative control group received 0.9% normal saline. Treatments
were done at a frequency of iv one dose per two day for a total of
four treatments. All animals were killed 24 h after the fourth
treatments. All data in were presented as mean ꢂ SD. Statistical
significance was evaluated by t-test.
4.5. Annexin-V/PI double staining assay
Apoptosis-mediated programmed cell death was examined
using a double staining method with FITC-labeled Annexin-V/
propidium iodide (PI) apoptosis detection kit (KeyGen, Nanjing,
China) according to the manufacturer’s instructions. Flow cyto-
metric analysis was performed immediately after supravital stain-
ing. Data acquisition and analysis were performed in a Becton
Dickinson FACSCalibur flow cytometer using Cell Quest software.
Cells in early stages of apoptosis were Annexin-V positive; whereas,
cells that were both Annexin-V and PI positive were in the late stage
of apoptosis.
Acknowledgement
Financial support from National Natural Science Foundation of
China (No. 90713038, No. 21072231) and National Major Science
and Technology Project of China (Innovation and Development of
New Drugs, No.2008ZX09401-001 and No.2009ZX09501-003).
4.6. DNA content and cell cycle analyzed by flow cytometry
HepG2 cells were starved in serum-free medium for 24 h. After
treatment with 11a, cells were collected and fixed in 70% ethanol
overnight at 4 ꢀC. Fixed cells were washed with PBS, and re-sus-
pended in hypotonic PI staining solution (0.1% (w/v) sodium citrate,
0.1% (v/v) Triton X-100, 0.05 mg/ml PI, and 0.01 mg/ml RNase) for
15 min at 37 ꢀC in dark environment. The stained cells were
analyzed using FAScan laser flow cytometry (Becton Dickinson, San
Jose, CA).
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