Studies on chemical structure modification and biology of a natural product, Gambogic acid (I): Synthesis and biological evaluation of oxidized analogues of gambogic acid

Article abstract of DOI:10.1016/j.ejmech.2008.09.034

Gambogic acid (GA), a natural product, exhibits high potency in inhibiting cancer cell growth through the effective induction of apoptosis. In order to investigate the structure-activity relationships of GA derivatives, 11 oxidized derivatives of GA were

Full text of DOI:10.1016/j.ejmech.2008.09.034

European Journal of Medicinal Chemistry 44 (2009) 2611–2620
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Studies on chemical structure modification and biology of a natural product,
Gambogic acid (I): Synthesis and biological evaluation of oxidized analogues
of gambogic acid
Jinxin Wang a, Li Zhao b, Yang Hu b, Qinglong Guo b, Lei Zhang a, Xiaojian Wang a, Nianguang Li a,
,b,
Qidong You a
*
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
b Key laboratory of Carcinogenesis and Intervention of Jiangsu Province, 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
Article history:
Received 29 May 2008
Received in revised form 3 September 2008
Accepted 8 September 2008
Available online 7 October 2008
Gambogic acid (GA), a natural product, exhibits high potency in inhibiting cancer cell growth through the
effective induction of apoptosis. In order to investigate the structure–activity relationships of GA
derivatives, 11 oxidized derivatives of GA were synthesized. Some of them showed strong inhibitory
effects on HT-29, Bel-7402, BGC-823, A549, and SKOV 3 cell lines. Moreover, in this paper the cellular
growth inhibitor 39-hydroxy-6-methoxy-gambogic acid methyl ester (10) was identified as a HepG2 cell
apoptosis inhibitor through Annexin-V/PI double staining assay and the expression of the related
apoptotic proteins (Bax and Bcl-2). Compound 10 may serve as a potential lead compound for the
development of new anticancer drugs. Further SAR studies of GA derivatives indicated that modification
of carbon–carbon double bond at C-32/33 or C-37/38 and of the methyl groups at C-39/C-35 can improve
antitumor activity.
Keywords:
Gambogic acid derivatives
Anti-tumor
Apoptosis inducer
Structure–activity relationship
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
growth of a wide variety of tumor cells, including human gastric
carcinoma SGC-7901 cells, MGC-803 cells, human lung carcinoma
Apoptosis, the controlled death of cells, is an important meta-
bolic process in the regulation of cell growth. If apoptosis is blocked
in the normal cell, disordered metabolism can cause the develop-
ment of tumors. The induction of apoptosis in tumor cells has
become a chief parameter in the characterization of anti-tumor
drugs [1]. Currently, many anticancer drugs used clinically are
known to kill tumor cells through the induction of apoptosis.
Therefore, the discovery and development of compounds that
induce apoptosis are highly desirable goals in the development of
therapeutically effective antineoplastic agents and may lead to new
anticancer agents.
Between 1980 and 2000, more than 70% of anticancer drugs
approved by the US Food and Drug Administration were based on
compounds derived from natural sources [2,3]. Gambogic acid (GA,
CAS No. 2752-65-0) is the principal active component of gamboge,
the resin from various Garcinia species, including Garcinia morella
and Garcinia hanburyi. Many modern pharmaceutical studies have
been focused on its extensive and potent anti-tumor activities
[4–6]. Our previous studies demonstrated that GA can inhibit the
SPC-A1 cells, and human hepatoma SMMC-7721 cells. The potent
anticancer activity (both in vitro and in vivo) of GA is mainly
attributed to its activation of the impaired apoptotic pathways in
cancerous cells via down-regulation of telomerase. Studies have
also indicated that GA suppresses the growth of human tumors as
a potent apoptosis inducer in these cancer cells [11–14]. Because
the exact cytotoxic mechanism was not fully understood, further
research has been conducted. GA induced apoptosis through
a different mechanism from the taxanes and vinca alkaloids, which
induced apoptosis through interaction with tubulin and G2/M cell-
cycle arrest [7]. Recent studies on the mechanism of GA action
showed that it binds to the transferrin receptor, inducing a unique
signal leading to rapid apoptosis of tumor cells [8]. It also serves as
a novel ligand for the transferrin receptor and potentiates tumor
necrosis factor (TNF)-induced apoptosis through modulation of the
nuclear factor-
that GA exerted its antiproliferative effect by inhibiting the cata-
lytic activity of Topo II , inhibiting telomerase activity by down-
kB signaling pathway [9]. We have recently reported
a
regulating the expression of the hTERT gene (a target of c-MYC
activity), inducing G2/M phase cell-cycle arrest via disturbance of
CDK7-mediated phosphorylation of CDC2/p34 [16]. Furthermore,
angiogenesis was inhibited through suppression of vascular
endothelial growth factor-induced tyrosine phosphorylation of
* Corresponding author. Department of Medicinal Chemistry, China Pharma-
ceutical University, Nanjing 210009, China. Fax: þ86 025 83271351.
E-mail address: youqidong@gmail.com (Q. You).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.034

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J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
KDR/Flk-1[10,13,15,17]. Moreover, the selective anticancer activity
of GA may also be related to its higher distribution and longer
retention time in tumor cells as compared to normal cells. Indeed,
our studies suggested that these simultaneous effects of GA may
contribute to a highly effective anticancer drug candidate for
hepatocellular carcinoma [18].
The elucidation of the new antitumor mechanism of GA and
identification of its binding site are good starting points for addi-
tional research. At present, the key goal is to create more structural
classes of new derivatives of GA for the study of its structure–
activity relationships (SARs) and for further exploration of the
binding site.
Herein, we describe the synthesis of the gambogic acid
analogues 1–11 (Fig. 1) and have evaluated them for induction of
apoptosis in several cancer cell lines. We also performed SAR
studies of gambogic acid as an inducer of apoptosis. We also include
preliminary investigations of a promising derivative, compound 10,
as a potent apoptosis inducer in HepG2 cells by detection of growth
inhibition, observation of morphological changes, and the expres-
sion of the related apoptotic proteins (Bax, Bcl-2 and Actin).
2. Results and discussion
2.1. Chemistry
GA contains the xanthone core and a unique 4-oxatricyclo
[4.3.1.03,7
]
decan-2-one ring system [19,20]. Previous reports
GA was isolated in overall yield of approximately 5% from
available gamboge resin. It was purified by converting the crude
gamboge resin extract into the pyridine salt, followed by recrys-
tallization [25]. To investigate the basic SAR of GA with respect to
the induction of apoptosis, we planned to regioselectively modify
the carbon–carbon double bond at C-32/33, C-37/38, and the single
methyl at C-39/C-35 into homologous functional groups, respec-
tively, through different oxidative and reaction conditions. The
syntheses of derivatives 1–11 are depicted in Schemes 1–5, starting
from GA.
[7,21–23] examining the SARs showed that the C]C of the a,b-
unsaturated ketone in GA is critical for its antitumor activity,
while the 6-hydroxy, 8-ketone and 30-carboxy groups could
tolerate a variety of modifications. Thus, the syntheses of simple
derivatives of GA were undertaken. Based on the particular
complexity of its skeleton and the strong biological significance of
GA, chemical modifications were focused on exploring the basic
pharmacophore of GA for antitumor activity and inducing apoptosis,
with the goal of discovering novel derivatives as potential anti-
cancer agents. However, in considering the modification of GA,
one must take into account its formidable functional groups,
which are sensitive to alkali or acid.
As reported [24], GA is rapidly metabolized in rat liver
microsomes and M1 is crucial for the elimination of GA. Cyto-
chrome P-4501A2 is the major rat CYP involved in the metabolism
of GA. The important metabolic products, the hydration metabolite
and epoxide metabolite of GA, may be more active compounds than
the parent drug itself. These results suggested us that oxidative
metabolism products of GA may lead to new compounds with
greater antineoplastic activity than GA. Therefore, we designed and
synthesized 11 oxidized analogues of GA which have been
designed and synthesized through modification of the carbon–
carbon double bonds at C-32/33 and C-37/38 or by converting the
single methyl groups at C-40 and C-34 into the epoxy or hydroxyl
group, respectively.
Reaction of GA with (Me)2CO in the presence of K2CO3 and MeI
produced the methyl ester of GA in 95% yield [23] followed by
epoxide formation with CH2Cl2 in the presence of m-CPBA for
a short reaction time, giving the corresponding compound 1 in 36%
yield (Scheme 1). The presence of the alkene proton at d 5.07 (C32-H)
in compound 1 suggested that the carbon–carbon double bond at C-
32/33 remained intact, because the carbon–carbon double bond at
C-32/33 was electronically deficient. Similarly, reaction of GA-
methyl ester and GA with m-CPBA for a longer reaction time
produced the GA derivatives 2 and 3 containing two epoxy groups at
C-32/33 and C-37/38. As discussed below, in GA derivatives 1–3, the
stereochemistry of C-37 and C-32 was confirmed from the respec-
tive spectral data.
Our attempts to use KMnO4/EtOH to prepare compound 4a
(Scheme 2), which contained di-hydroxyl groups at C-37/38, were
completely ineffective [26]. However, as demonstrated by spectral
COOR
30
O
COOMe
35
34
32
COOH
O
O
O
O
O
O
27
O
O
O
31
O
O
O
37
39 38
40
O
O
O
36
12
OR O
8
3
9
6
4
10
OMe
R=Me (2)
O
OH
R=H ( 3)
1
GA
R1=CH3, R2=OH, R3=H
R1=CHO, R2=OH,R3=H
R1=R2=CH2OH, R3=H
(5)
(6)
(7)
COOR3
R2
COOH
O
O
O
O
R1
O
O
O
O
O
O
R1=CH2OH,R2=H,R3=Me (10)
OR3
OH
OH
R1=CH2OCOCH3,R2=H,
R3=Me
OH
(11)
4
COOMe
COOMe
O
O
O
O
OH
OH
O
O
O
O
O
O
OH
OH
OMe
OMe
9
8
Fig. 1. Structures of gambogic acid (GA) and its oxidative analogues 1–11.

J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
2613
COOMe
COOMe
COOH
O
O
O
O
O
ii
i
O
O
O
O
O
O
O
O
O
O
OMe
OMe
OH O
1
GA-methyl ester
GA
ii
ii
COOH
COOMe
O
O
O
O
O
O
O
O
O
O
O
O
OH O
OMeO
2
3
Scheme 1. Reagents and conditions: (i) K2CO3, MeI, (Me)2CO, R.T. (ii) m-CPBA, CH2Cl2, R.T.
data, compound 4 with di-hydroxyl groups at C-9/10 was achieved.
Presumably, KMnO4, acting as a nucleophile, attacked the carbon–
signals at
cross-peak was observed in the ROESY spectrum between
(CH-31a), 3.13 (CH-31b) and 3.91 (CH-34).
d
3.31 (CH-31a),
d
3.13 (CH-31b) and 1.48 (CH-35). No
d
3.31
carbon double bond in the
a,
b-unsaturated ketone. This oxidation
d
d
reaction resulted in compound 4, but not compound 4a. The
observed selectivity can be rationalized by the carbon–carbon
double bond at C-9/10 being electronically more deficient than that
at C-38/40.
Conventional regioselective oxidations of C–H bonds require
neighboring activating groups. Very common in synthesis is the
oxidation of allylic methylene or methyl groups to yield allylic
Reaction of GA with SeO2 and t-BuOOH in CH2Cl2 produced GA
oxidative derivative 6 with an aldehyde group at C-39 and
a hydroxyl methyl group at C-35 in 11% yield [27–29]. Another
oxidative derivative of GA, compound 7 with hydroxy methyl
groups at C-39 and at C-34 was afforded in 35% yield.
The Sharpless asymmetric dihydroxylation (AD) of olefins is an
indispensable tool for contemporary organic synthesis. The ADs of
GA-methyl ester (in accordance with the literature precedence
[29]) provided compounds 8 (75% ee) and 9 (85% ee), of which the
di-hydroxyl groups were formed at C-37/38 in the presence of
alcohols or
a,b-unsaturated carbonyl compounds. The successful
reagents are selenium dioxide. In following substantial experiments,
under very similar reaction conditions, different usage amounts of
selenium dioxide resulted in homologous products 5–7, respectively.
The single methyl groups at C-34 and C-39 of GA were,
respectively, modified as depicted in Scheme 3. The synthesis of 5
was carried out starting from GA, prepared from SeO2 and t-BuOOH
by the oxidation of the methyl at C-34 of GA in 31% yield. Why was
the hydroxyl group located at C-34? The observed selectivity can be
rationalized by a hydrogen bond formed between the C-30 COOH
group and the oxygen of the oxidative reagent SeO2. The cross-
peaks observed in the HMBC spectrum of compound 5 between
a mixture of t-BuOH, H2O, AD-mix
b and methanesulfonyl amide
(Scheme 4). Also the ee values were determined by 1H NMR anal-
ysis of the corresponding Moster’s ester [36,37]. Why were the di-
hydroxyl groups added at C-37/38? A possible reason for the site
selectivity was related to the large steric hindrance from the
phthalazine class of ligands, (DHQD)2-PHAL, and (DHQ)2-PHAL, and
the electronic abundance at C-37/38.
In Scheme 5, oxidation of the methyl at C-39 of GA-methyl ester
with CH2Cl2 in the presence of SeO2 and t-BuOOH resulted in
compound 10 in 61% yield, which was reacted with DMAP and Ac2O
in CH2Cl2 to generate acetate 11. In this synthesis, changed usage
amount of selenium dioxide and reaction times resulted in targeted
product 10. Observed side product was much trace. The cross-peaks
observed in the HMBC spectrum of compound 10 between
d
3.31 (CH-31a),
d
3.13 (CH-31b) with 123.7 (C-32),
d
3.91 (CH-34)
5.05 (CH-37)
with 123.7 (C-32), as well as the correlated signals at
d
with 17.51 (C-40)/25.5 (C-39), confirmed the position of the
hydroxyl group at C-34 or C-35. The position of the hydroxyl group
at C-34 was determined by the ROESY correlations between the
COOH
O
O
O
O
O
COOH
i
OH
OH
O
OH
O
O
O
O
i
4
COOH
OH
GA
O
O
OH
OH
O
O
O
OH
4a
Scheme 2. Reagents and conditions: (i) KMnO4, EtOH, R.T.

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J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
potent than the reference compound. Compound 7 gave the most
M,
almost 20-fold more potent than GA. In ovarian cancer cell line
(SKOV3), five compounds (2, 3, 5, 6 and 10) were w1.6- to 4.0-fold
more potent than GA, while compound 1 exhibited the lowest IC50
value of 0.64 mM,w4.8-fold more potent than GA. In a human
colorectal cancer cell line (HT-29), seven compounds (1–3, 5, 6, 9,
and 11) are w2.1- to 3.0-fold more potent than the parent structure.
COOH
R2
COOH
interesting result in the BGC-823 cell line, with IC50 ¼ 0.23
m
O
O
O
O
R1
O
O
O
i
O
O
O
OH
OH
GA
R1=CH3, R2=OH,
R1=CHO, R2=OH,
R1=R2=CH2OH,
(5)
(6)
(7)
Compound 10 displayed the strongest cytotoxicity (IC50 ¼ 0.89
mM),
an almost 6.0-fold increase in potency.
Our newly synthesized compounds 1–11 exerted significant in
vitro inhibitory effects on some cell growth. Compounds 1 and 2
were the most effective analogues in all cell lines, and compound 3
was more potent than GA in all cell lines except Bel7402. It is
noteworthy that compounds 1–3 possessed one or two epoxy
groups at positions C-32/33 or C-37/38. Further SAR studies of the
analogues 5–7, 10 and 11 were pursued, wherein the methyl at C-39
or C-35 was modified into hydroxyl or aldehyde groups by oxida-
tion, respectively. The major findings are as follows: (i) compound
10 exhibited greater potency than GA in all cell lines; (ii) compared
to GA, compounds 5 and 6 showed better selectivity toward SKOV3
and HT-29 cell lines; (iii) compound 7 had much better selectivity
toward the BGC823 and Bel7402 cell lines; and (iv) compound 11, in
which the C-40 hydroxyl group was protected by an acetyl group,
showed evident selectivity toward the BGC823 and HT-29 cell lines.
Moreover, compound 4 showed less cytotoxic activities against
all cell lines than GA, confirming the earlier report that the 9, 10
Scheme 3. Reagents and conditions: (i) SeO2, t-BuOOH, CH2Cl2, R.T.
d
d
d
5.32 (CH-37) and 68.8 (C-39),
5.32 (CH-37) with 20.8 (C-40) as well as the correlated signals at
3.37 (CH-31a), 3.26 (CH-31b) with 121.9 (C-32) and 2.04 (CH-36)
d 3.95 (CH-39) with 20.8 (C-40),
d
d
with 125.3 (C-37), confirmed the position of the hydroxyl group at
C-39 or C-40. A cross-peak was observed in the ROESY spectrum
between
d 2.04 (CH-36) and 1.68 (CH-40), confirming that the
position of the hydroxyl group was at C-39.
In summary, we have regioselectively synthesized 11 novel
oxidative derivatives of GA and identified their structures by
spectral techniques.
2.2. Structure–activity relationship
Earlier SAR studies [7] had identified the 9, 10 carbon–carbon
double bond of the a,b-unsaturated ketone as critical for cytotox-
carbon–carbon double bond of the
a,b-unsaturated ketone is
important for biological activity [9]. Remarkably, the two hydroxyl
groups at C-37/38 in compounds 8 and 9 had opposite stereo-
chemistry and they displayed evident differences in cytotoxic
activities against all cell lines. Evidently the stereochemistry of GA
is important for biological activity. Additionally, analogues con-
taining an epoxy group at C-37/38 showed better cytotoxic activi-
ties than those containing two hydroxyl groups at the same
position.
icity and apoptosis-inducing activity, while the 30-carboxy and 6-
hydroxy were not important. We concentrated our synthetic efforts
on modifications of the carbon–carbon double bond at C-32/33, C-
37/38 and of the methyl groups at C-39/C-35, and the observed
structure and activity relationships are summarized in Table 1. The
cytotoxic activities of the oxidized derivatives 1–11 were deter-
mined in vitro by an MTT assay which estimates cell viability by the
remaining activity of mitochondrial enzymes [31]. In the A549,
Bel7402, HT-29, BGC-823, SKOV3 cell lines, the IC50 values for the
2.3. Characterization of compound 10 as a potent apoptosis inducer
parent compound, gambogic acid, ranged between 3.0 and 6.0 mM
(Table 1). The novel compounds exhibited considerable variation in
their cytotoxic activities against the same cell lines.
An anticancer leading compound was screened partly by its
proapoptotic ability on tumor cells. Currently, human hepatocel-
lular carcinoma (HCC) is the most common liver cancer in the world
[32] and discovery of HCC therapeutic agents with greater effect but
less toxicity has become one of the main assignments for most
pharmaceutical researchers. Inspired by the improved cytotoxicity
of compound 10 against the A549, Bel7402, HT-29, BGC-823, and
SKOV3 cell lines, especially the Bel7402 cell line, we sought to
characterize compound 10 as a potent apoptosis inducer in the
human hepatocellular carcinoma cell line HepG2. We evaluated
this analogue with an optimized chemical structure by measuring
growth inhibition, morphological changes, and the expression of
related apoptotic proteins (Bax, Bcl-2).
As a result of cytotoxicity screening, compounds 2, 7, and 10
were identified as having a greater effect in the human lung
carcinoma cell line (A549) than GA. Compounds 1 and 3 are w4.0-
fold more potent than GA. In contrast, compounds 5, 9, and 11 were
comparable to GA. In the human hepatoma cell line (Bel-7402),
compounds 1, 2, and 10 were two- to threefold more cytotoxic than
the parent compound. In human gastric carcinoma cell line
(BGC823), compounds 3 and 9 had similar cytotoxicity to GA,
whereas compounds 1, 10, and 11 were wthree- to sixfold more
COOMe
HO
O
O
2.3.1. Cell viability inhibition
Compound 10 inhibited HepG2 cells viability in a concentration-
dependent manner as measured by 24 h MTT assay, and its IC50 was
calculated as 2.35 mM. Over the range of 0–8.0 mM, the inhibitory
effect became obvious. (Fig. 2).
O
O
O
OH
i
OMe
COOMe
8
O
O
COOMe
O
O
O
ii
2.3.2. Cell morphological assessment
Fig. 3 shows that morphological changes of cells by optical
microscopy after treatment with three concentrations (1.0, 2.0, and
O
O
OH
O
O
O
OMe
OH
3.0 mM) of compound 10 for 20 h. After incubation with compound
GA-methyl ester
OMe
9
10, HepG2 cells were severely distorted and grew slowly. Some cells
became round in shape. The untreated cells displayed normal and
healthy shapes, as demonstrated by the clear skeletons.
Scheme 4. Reagents and conditions: (i) AD-mix-
AD-mix- , CH3SO2NH2, t-BuOH-H2O, R.T.
a, CH3SO2NH2, t-BuOH-H2O, R.T.; (ii)
b

J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
2615
COOMe
COOMe
COOMe
O
OAc
OH
O
O
O
O
O
O
ii
i
O
O
O
O
O
O
O
OMe
10
Scheme 5. Reagents and conditions: (i) SeO2, t-BuOOH, CH2Cl2, R.T.; (ii) DMAP, Ac2O, CH2Cl2, R.T.
OMe
OMe
11
GA-methyl ester
Furthermore, the result demonstrated that the inhibitory effects on
compound 10 for 20 h, the percentages of apoptotic cells were from
HepG2 cells were strongly related to dosage of 10.
4.3% (Control) to 12.9% (6.0 mM) (Fig. 6).
2.3.3. AOEB staining
Fluorescence microscopic analysis showed clear morphological
changes in the nucleolus, typical of early apoptosis in HepG2 cells
3. Conclusion
Although these experiments were preliminary, the result of SAR
investigation suggests that promising agents with the antitumor
activities may be obtained by the conversion of the carbon–carbon
double bond at C-32/33, C-37/38 and the methyl at C-39/C-35 into
the epoxy and hydroxyl groups, respectively. Compound 10
exhibited much more potent cytotoxic activities against A549,
Bel7402, HT-29, BGC-823, SKOV3 cancer cells lines than the refer-
ence compound gambogic acid, and the induction of apoptosis on
HepG2 cells may be related with the expressions of Bcl-2 and Bax.
One of the analogues, compound 10, may be a highly effective
anticancer drug candidate as a potent apoptosis inducer. Moreover,
the newly synthesized compounds 1–11 can provide abundant
materials for biological studies and identification of the binding site
of gambogic acid in further research. In brief, our results suggest
that gambogic acid and its oxidized derivatives may become
attractive molecules for the development of anticancer agents.
following treatment with compound 10 (1.0, 2.0, and 3.0 mM) for
a period of 18 h (Fig. 4). Untreated HepG2 cells were stained with
uniform green fluorescence which showed the chromatin equably
distributed in the nucleolus. After compound 10 treatment, the
cells displayed congregated chromatin and nucleolus pyknosis,
which emitting bright fluorescence, the early phenomena of
apoptosis.
2.3.4. Expressions of Bcl-2, Bax protein
As shown in Fig. 5, after incubation with compound 10 (1.0, 2.0
and 6.0 mM) for 20 h, the expression of Bcl-2 protein decreased and
that of Bax was unchanged as compared with control. The current
data proved that compound 10 could selectively induce apoptosis
of human hepatoma HepG2 cells and the lower expression of the
apoptotic Bcl-2 proteins. In previous studies [33–35], it was shown
that the ratio of Bcl-2/Bax might be one of the critical factors of
a cell’s threshold for undergoing apoptosis, and Bax could bind with
Bcl-2 and inhibit its function in suppression of apoptosis. So, the
observed decrease of Bc1-2/Bax illustrated a possible mechanism of
the induction of apoptosis. In our experiments, the ratio of Bc1-2/
4. Experimental protocols
4.1. Chemistry
Bax was 0.469 (control), 0.476 (1.0
mM), 0.478 (2.0 mM) and 0.251
4.1.1. General methods
(6.0 M). It was shown obviously that compound 10 could decrease
m
Melting points were measured with a Melt-Temp II instruments.
IR spectra were recorded on a Nicolet Impact 410 spectrometer. 1H
NMR and 13C NMR spectra were recorded on Bruker AV-300 or AV-
500 MHz instruments in CDCl3 using tetramethylsilane (TMS) as
the internal standard. EI-MS was recorded Shimadzu GC–MS 2050
apparatus; ESIMS was recorded on Agilent 1100 LC/MSD (70 eV)
spectrometers. HREIMS was recorded on a Waters Q-Tof micro.
the ratio of Bc1-2/Bax in HepG2 cells. The apoptosis induction of
compound 10 was confirmed in HepG2 cells. Furthermore, how
compound 10 works, and the other mechanisms which may
contribute to the changes of cell cycle, should be the subject of
further research. Further biological in vivo evaluation is underway.
Compound 10 may be
a promising and significant leading
compound for the treatment of cancer diseases in the future.
2.3.5. Annexin-V/PI double-staining assay
120.00%
100.00%
80.00%
60.00%
40.00%
20.00%
The percentage of apoptotic cells (Annexin-Vþ/PIꢀ) in the
control group was 4.3%. After treatment with 1 mM, 2 mM and 6 mM
Table 1
The cytotoxicity data of GA and its derivatives [IC50 (mM)].
Compound
Cell lines
A549
BGC823
SKOV3
HT-29
Bel7402
1
2
3
4
5
6
7
8
1.48 ꢁ 0.30 0.75 ꢁ 0.22 0.64 ꢁ 0.13 2.19 ꢁ 0.77 1.40 ꢁ 0.29
3.61 ꢁ 1.09 3.21 ꢁ 0.7
1.56 ꢁ 0.26 4.38 ꢁ 1.0
1.80 ꢁ 0.22 1.86 ꢁ 0.34 1.66 ꢁ 0.4
0.76 ꢁ 0.16 2.08 ꢁ 0.41 3.73 ꢁ 0.41
0.00%
74.2 ꢁ 7.4
29.9 ꢁ 5.1
6.00 ꢁ 1.2
34.7 ꢁ 6.2
33.1 ꢁ 5.8
8.00 5.60 3.92 2.74 1.92 1.34 0.94 0.66 0.00
5.87 ꢁ 1.41 4.89 ꢁ 0.98 1.61 ꢁ 0.31 2.66 ꢁ 0.42 4.01 ꢁ 0.86
Dose (µM)
20.5 ꢁ 3.8
4.24 ꢁ 1.2.
>100
6.75 ꢁ 1.41 1.84 ꢁ 0.34 2.08 ꢁ 0.60 15.0 ꢁ 2.9
0.23 ꢁ 0.04 3.43 ꢁ 0.80 21.6 ꢁ 4.2
2.32 ꢁ 0.6
Fig. 2. Inhibitory effects of 10 on primary cultured HepG2 cells. Data are shown as
>100
>100
>100
>100
mean ꢁ SD; n ¼ 3.0, 0.66, 0.94, 1.34, 1.92, 2.74, 3.92, 5.60 and 8.00
were added, respectively. After 24 h of incubation, the culture medium was removed
and MTT was added. Four hours later, the supernatant was discarded and 100 l of
DMSO was added to each well. The mixture was shaken and measured at 570 nm using
a Universal Microplate Reader.
mM of Compound 10
9
10
11
5.86 ꢁ 1.4
3.46 ꢁ 1.0
3.23 ꢁ 1.08 3.75 ꢁ 0.72 3.65 ꢁ 0.65
4.38 ꢁ 0.82 0.84 ꢁ 0.18 1.28 ꢁ 0.32 0.89 ꢁ 0.13 0.96 ꢁ 0.28
m
5.25 ꢁ 1.1
1.42 ꢁ 0.43 15.8 ꢁ 3.2
2.41 ꢁ 0.46 3.60 ꢁ 0.61
3.06 ꢁ 1.15 5.61 ꢁ 1.2 3.31 ꢁ 0.92
Gambogic acid 5.81 ꢁ 1.2
4.51 ꢁ 1.3

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J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
Fig. 3. Morphologic changes of cells observed under inverted microscope: HepG2 cells. A: Control group; B: 1.0 mM compound 10; C: 2.0 mM compound 10; D: 3.0 mM compound 10.
Chemical shifts are reported as
d
values (parts per million) relative
Column and thin-layer chromatography (CC and TLC, resp.) were
performed on silica gel (200–300 mesh) and silica gel GF254,
respectively, supplied by Qingdao Marine Chemical Factory. GA-
methyl ester was prepared by a literature method [23].
to solvent peak. Coupling constants are reported in hertz. The
multiplicity is defined by s (singlet), d (doublet), t (triplet), or m
(multiplet).
Fig. 4. Nucleolus morphologic changes observed by fluorescence microscope (400ꢂ) in HepG2 cells. A: Control group; B: 1.0
mM compound 10; C: 2.0 mM compound 10; D: 3.0 mM
compound 10.The early apoptotic cells are observed: the cells were stained green with AO, their membrane retained integrity, and the nuclei exhibited bright condensed chromatin
(B). Dead cells were stained equally red with EB. Typical apoptotic blebbing was obvious and late apoptotic cells were observed. Their nuclei exhibited condensed chromatin, and
they were stained red with EB; dead cells were stained equally red with EB.

J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
2617
Compound 2: Mp: 70–73 ꢃC. IR (film): 2928, 1713, 1708, 1621,
1587, 1461, 1381, 1144, 880 cmꢀ1. 1H NMR (CDCl3, 300 MHz):
7.46
d
(1H, d, J ¼ 6.9 Hz, H-10), 6.69 (1H, d, J ¼ 10.1 Hz, H-4), 6.02 (1H, m,
H-27), 5.58 (1H, d, J ¼ 10.1 Hz, H-3), 3.83 (3H, s, 6-OCH3), 3.45 (3H, s,
COOCH3), 3.43 (1H, m, H-11), 2.98 (2H, m, H-26), 3.15 (2H, m, H-31),
2.70 (1H, m, H-32), 2.89 (1H, m, H-37), 2.53 (1H, m, H-22), 2.31 (1H,
m, H-21a), 1.40 (1H, m, H-21b), 1.98 (2H, m, H-36), 1.77 (3H, s, H-
25), 1.72 (2H, m, H-20), 1.67 (3H, s, H-29), 1.48 (3H, s, H-34), 1.43
(3H, s, H-40), 1.47 (6H, br s, H-39, H-24), 1.94 (3H, s, H-35), 1.28 (3H,
s, H-19). ESI-MS: 689 [M þ H]þ, 711 [M þ Na]þ. HRMS (M þ Na) m/z
711.3130 (Calcd for C40H48O10 Na 711.3145).
Fig. 5. Effects of 10 on the expression of Bcl-2, Bax and Actin proteins. HepG2 cells
were treated with 1.0 M, 2.0 M, and 6.0 M compound 10 for 20 h as described in
Section 4. The ratio of Bc1-2/Bax was 0.469 (control), 0.476 (1.0 M), 0.478 (2.0 M)
and 0.251 (6.0 M). Proteins were precipitated, solubilized and separated by 10% SDS-
PAGE. Proteins were transferred to Immobilon-Pmembranes and quantitated by
immunoprecipitation and development with antibodies as described in Section 4.
m
m
m
m
m
m
4.1.3. (32,33), (37,38)-Di-epoxyl-gambogic acid 3
To a stirred solution of GA (110 mg, 0.18 mmol) in CH2Cl2 (20 ml)
at room temperature, was added m-CPBA (36 mg, 0.21 mmol). The
resulting mixture was stirred for 12 h at the room temperature. The
mixture was extracted with EtOAc and then the organic layer was
washed with aqueous saturated Na2CO3, H2O, and brine, respec-
tively, then dried over Na2SO4. After removal of the solvent, the
residue was subjected to silica gel column chromatography (pet-
rolum:EtOAc ¼ 4:1) to give rise to 3 (40 mg, 36%) as a buff-colored
oil. IR (film): 3452, 2969, 2927, 1731, 1713, 1708, 1681, 1586, 1460,
4.1.2. (37,38)-Epoxy-6-methoxyl-gambogic acid methyl ester 1 and
(32,33), (37,38)-di-epoxyl-6-methoxyl-gambogic acid methyl ester 2
To a stirred solution of GA-methyl ester (328 mg, 0.5 mmol) in
CH2Cl2 (20 ml) at room temperature, was added m-CPBA (104 mg,
0.6 mmol). The resulting mixture was stirred for 6 h at the room
temperature. The mixture was extracted with EtOAc and then the
organic layer was washed with aqueous saturated Na2CO3, H2O, and
brine, respectively, then dried over Na2SO4. After removal of the
solvent, the residue was subjected to silica gel column chroma-
tography (petroleum ether:EtOAc ¼ 4:1) to give rise to 1 (120 mg,
36%) as a buff-colored oil, and 2 (80 mg, 24%). Compound 1: Mp:
72–75 ꢃC. IR (film): 2969, 1731, 1708, 1621, 1586, 1460, 1379,
1379 cmꢀ1.1H NMR (CDCl3, 500 MHz):
d
7.45 (1H, d, J ¼ 6.84 Hz, H-
10), 6.67 (1H, d, J ¼ 10.2 Hz, H-4), 5.96 (1H, m, H-27), 5.54 (1H, d,
J ¼ 10.2 Hz, H-3), 3.84 (3H, s, 6-OCH3), 3.45 (3H, s, COOCH3), 3.43
(1H, m, H-11), 3.30 (1H, d, H-31a), 3.15 (1H, m, H-31b), 2.98 (2H, d,
J ¼ 6.99 Hz, H-26), 2.70 (2H, m, H-37, H-32), 2.51 (1H, d, J ¼ 7.4 Hz,
H-22), 2.31 (1H, m, H-21a), 1.85 (2H, m, H-36), 1.76 (3H, s, H-25),
1.69 (3H, s, H-29), 1.66 (3H, s, H-34), 1.63 (3H, s, H-35), 1.60 (3H, s,
H-39), 1.58 (3H, s, H-40), 1.44 (1H, m, H-21b), 1.47 (3H, m, H-24),
1.28 (3H, s, H-19). ESI-MS: 661 [M þ H]þ, 683 [M þ Na]þ, 659 [M-
H]ꢀ. HRMS (M þ Na) m/z 683.2821 (Calcd for C38H44O10Na
667.2883).
878 cmꢀ1; 1H NMR (CDCl3, 300 MHz):
d
7.45 (1H, d, J ¼ 6.84 Hz, H-
10), 6.67 (1H, d, J ¼ 10.2 Hz, H-4), 5.96 (1H, m, H-27), 5.54 (1H, d,
J ¼ 10.2 Hz, H-3), 5.07 (1H, m, H-32), 3.84 (3H, s, 6-OCH3), 3.45 (3H,
s, COO CH3), 3.43 (1H, m, H-11), 3.30 (1H, d, 31a), 3.15 (1H, m, H-
31b), 2.98 (2H, d, J ¼ 6.99 Hz, H-26), 2.68 (1H, t, H-37), 2.51 (1H, d,
J ¼ 7.4 Hz, H-22), 2.31 (1H, m, H-21a), 1.85 (2H, m, H-36), 1.76 (3H, s,
H-25), 1.69 (3H, s, H-29),1.66 (3H, s, H-34), 1.63 (3H, s, H-35), 1.60
(3H, s, H-39), 1.58 (3H, s, H-40), 1.44 (1H, m, H-21b), 1.47 (3H, m, H-
24), 1.28 (3H, s, H-19); ESI-MS: 673 [M þ H]þ, 695 [M þ Na]þ.
4.1.4. (9,10)-Dihydroxy-gambogic acid 4
To a stirred solution of GA (314 mg, 0.5 mmol) in 95% ethanol
(15 ml) at room temperature, was added dropwise KMnO4 in
Fig. 6. Fluorescence-activated cell sorter analysis for Annexin-V and PI staining. (A) Control, (B) 1.0
apoptotic cells labeled with PI and Annexin V–FITC. LL (lower left): fully viable cells. LR (lower right): early apoptotic cells labeled with Annexin V–FITC but not with PI. After
exposure to compound 10, the cells of LR had increased from 4.3% (Control) to 12.9% (6.0 M).
mM, (C) 2.0 mM, (D) 6.0 mM compound 10, UR (up right): necrotic cells and late
m

2618
J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
water (1.5 ml, 80 mg, 0.5 mmol). The brown suspension was stir-
red for 6 h, and then the whole mixture was filtered through
a silica gel pad, the solvent was removed in vacuo and the residue
was taken up in EtOAc, then the organic layer was washed with
aqueous saturated Na2CO3, H2O and brine, respectively, then dried
over Na2SO4 and concentrated. The brown residue was purified by
flash chromatography using (petroleum ether:EtOAc ¼ 1:2), and
yielded a brown powder 4 (56 mg, 38%). M.p. 238–240 ꢃC. IR (KBr):
3424, 2971, 2927,1742, 1694, 1629,1594, 1446, 1375, 1190, 1086,
105–107 ꢃC. IR (KBr): 3470, 3418, 2972, 2924, 2854, 1736, 1687, 1634,
1592, 1454, 1436, 1400, 1380, 1331, 1240, 1176, 1138, 1046, 1018 cmꢀ1
1H NMR (CDCl3, 300 MHz):
12.80 (1H, s, 6-OH), 9.32 (1H, s, CHO),
.
d
7.57 (1H, d, J ¼ 6.9 Hz, H-10), 7.32 (1H, m, H-32), 6.68 (1H, d,
J ¼ 10.1 Hz, H-4), 6.50 (1H, d, J ¼ 10.1 Hz, H-3), 6.3 (1H, m, H-27), 5.4
(1H, m, H-3), 3.95(2H, brd, J ¼ 12.7 Hz, H-34), 3.50(1H, m, H-11), 3.30
(2H, m, H-31), 2.98 (2H, m, H-26), 2.07 (1H, m, H-36a), 2.72 (1H, d,
J ¼ 9.3 Hz, H-22), 2.37 (1H, m, H-21a), 2.15 (1H, m, H-36b),1.45 (3H, s,
H-24),1.28 (3H, s, H-19),1.55 (3H, s, H-40),1.68 (3H, s, H-35),1.72 (2H,
m, H-20), 2.31 (1H, m,H-21a), 1.40 (1H, m, H-21b), 1.70 (3H, s, H-29).
ESI-MS: 657 [M-H]þ. HRMS (M þ Na) m/z 681.2643 (Calcd for
C38H42O10Na 681.2676). Compound 7: M.p. 85–87 ꢃC. IR (KBr): 3474,
3416, 2963, 2923, 1734, 1714,1635,1592,1435,1401, 1330, 1176,1260,
1140 cmꢀ1 1H NMR (CDCl3, 300 MHz):
. d 11.40 (1H, s, 6-OH), 6.74
(1H, m, H-3), 6.20 (1H, m, H-27), 5.29 (1H, m, H-4), 5.09 (1H, m, H-
32), 5.20 (1H, m, H-37), 4.55 (2H, br s, 9-OH, 10-OH), 3.59 (1H, m,
10-H), 3.35 (1H, m, H-11), 3.30 (1H, m, H-31a), 3.21 (1H, m, H-31b),
2.98 (2H, dd, J ¼ 9.7 Hz, 7.9 Hz, H-26), 2.51 (1H, m, H-22), 2.36 (1H,
dd, J ¼ 13.0 Hz, 4.6 Hz, H-21a), 2.10 (2H, m, H-36), 1.76 (3H, s, H-
25), 1.72 (2H, m, H-20), 1.68 (9H, br s, H-34, H-35, H-39), 1.60 (3H,
s, H-40), 1.29 (3H, s, H-19), 1.40 (3H, s, H-24), 1.32 (1H, m, H-21b).
1095,1022, 802 cmꢀ1.1H NMR (CDCl3, 300 MHz):
d 137.61 (1H, d,
J ¼ 6.9 Hz, H-10), 6.65 (1H, d, J ¼ 9.9 Hz, H-4), 5.9 (1H, m, H-27), 5.58
(1H, d, J ¼ 10.0 Hz, H-3), 5.39(2H, m,H-32,37), 3.9(4H,brd, J ¼ 9.6 Hz,
H-34, H-40), 3.52 (1H, m, H-11), 3.33 (2H, m, H-31a), 3.10 (1H, dd,
J ¼ 14.7 Hz, 7.6 Hz, H-31b), 2.93 (2H, d, J ¼ 7.23 Hz, H-26), 2.51 (1H, d,
J ¼ 9.0 Hz, H-22), 2.31 (1H, dd, J ¼ 4.5 Hz, 4.8 Hz, H-21a), 2.13 (2H, m,
H-36),1.46 (1H, m, H-24),1.45 (1H, m, H-21b),1.80 (3H, m, H-25),1.78
(3H, s, H-29), 1.72 (3H, m, H-20), 1.68 (3H, s, H-35), 1.69 (3H, s, H-39),
13C NMR (CDCl3, 75 MHz):
d 17.9 (C-40), 18.0 (C-34), 20.2 (C-29),
22.0 (C-31), 27.5 (C-21), 25.7 (C-36), 25.8 (C-35), 27.7 (C-19), 29.8
(C-24), 30.2 (C-25), 33.6 (C-26), 39.0 (C-20), 47.5 (C-11), 48.5 (C-
22), 46.5 (C-10), 70.0 (C-9), 82.4 (C-2), 85.2 (C-13), 86.3 (C-23),
92.0 (C-14), 107.43 (C-17), 100.1 (C-7), 105.1 (C-5), 109.0 (C-4),
122.3 (C-32), 126.8 (C-28), 123.5 (C-37), 126.94 (C-3), 131.0 (C-33),
135.82 (C-38), 138.9 (C-27), 155.5 (C-6), 160.6 (C-16), 161.6 (C-18),
171.5 (C-30), 192.5 (C-8), 208.8 (C-12). ESI-MS: 663 [M þ H]þ, 685
[M þ Na]þ.
1.56 (3H, s, H-19).13C NMR (CDCl3, 75 MHz):
d 21.2 (C-29), 22.0 (C-31),
23.1 (C-36), 25.3 (C-21), 25.7 (C-35), 27.8 (C-19), 29.2 (C-24), 29.7 (C-
26), 30.0 (C-25), 42.3 (C-20), 48.0 (C-11), 50.0 (C-22), 68.2 (C-34), 68.3
(C-40), 82.1 (C-2), 84.4 (C-13), 84.5 (C-23), 92.3 (C-14), 101.3 (C-7),
103.4 (C-5), 108.1 (C-17), 116.7 (C-4), 123.4 (C-32), 123.4 (C-37), 124.6
(C-3),129.0 (C-28),134.1 (C-9),136.0 (C-38),136.8 (C-33),135.1 (C-10),
136.5(C-27),158.5(C-6),158.8(C-16),162.0(C-18),168.4 (C-30),180. 5
(C-8), 203.9 (C-12). ESI-MS: 661 [M þ H]þ, 683 [M þ Na]þ. HRMS
(M þ Na) m/z 683.2820 (Calcd for C38H44O10Na 683.2832).
4.1.5. 34-Hydroxy-gambogic acid 5
GA (628 mg, 1 mmol)) was dissolved in CH2Cl2 (2 ml) and added
dropwise to a clear solution of SeO2 (6.0 mg, 0.05 mmol) and t-
BuOOH (75%, 270 mg, 3 mmol in CH2Cl2 (10 ml) at room tempera-
ture. After being stirred at room temperature for 5 h, the reaction
mixture was diluted with Et2O and sequentially with aqueous 10%
KOH, H2O and brine, then dried and concentrated. The resulting
yellowoil was purified by flash column chromatography on silica gel
to yield a yellow oil 5 (130 mg, 21%) M.p. 103–105 ꢃC. IR (KBr): 3457,
3420, 2970, 2923, 1736, 1689, 1623, 1592, 1454, 1436, 1400, 1381,
4.1.7. (37R,38)-Dihydroxy-6-methoxy-gambogic acid
methyl ester 8
To GA-methylester(1 g,1.59 mmol)wasaddedamixtureof t-BuOH
(5 ml), H2O (5 ml), AD-mix-a (2.67 g, 1.91 mmol), and meth-
anesulfonyl amide (182 mg,1.91 mmol). The solution was stirred for
48 h at 0 ꢃC. After the addition of saturated Na2SO3 (2.0 g,
1.38 mmol), the mixture was stirred for 40 min and diluted with
EtOAc. The organic layer was washed with H2O and brine, dried over
MgSO4 and concentrated to give the crude product purified by silica
gel column chromatography, yielded yellow solid 8 (90 mg, 11%).
Unreacted GA-methyl ester (220 mg). M.p.108–110 ꢃC. IR (KBr): 3315,
3034, 3012, 2934, 2927,1713,1641,1589,1435,1413,1316,1143,1086,
1330,1176,1138,1047 cmꢀ1.1H NMR (CDCl3, 300 MHz):
d 7.54 (1H, d,
J ¼ 6.5 Hz, H-10), 6.59 (1H, d, J ¼ 10 Hz, H-4), 5.75 (1H, m, H-27), 5.43
(1H, d, J ¼ 10.0 Hz, H-3), 5.38 (1H, m, H-37), 5.05 (1H, m, H-32), 3.96
(2H, d, J ¼ 5.5 Hz, H-34), 3.49 (1H, m, H-11), 3.31 (1H, m, H-31a), 3.13
(1H, dd, J ¼ 8.6 Hz, 8.6 Hz, H-31b), 2.93 (2H, dd, J ¼ 4.4 Hz, 4.3 Hz, H-
26), 2.50 (1H, d, J ¼ 9.3 Hz, H-22), 2.31 (1H, dd, J ¼ 4.5 Hz, 4.4 Hz, H-
21a), 2.03 (2H, m, H-36), 1.78 (3H, m, H-25), 1.72 (3H, s, H-29), 1.70
(3H, m, H-20),1.48 (3H, s, H-35),1.66 (3H, s, H-39),1.55 (3H, s, H-40),
1.41 (1H, m, H-24), 1.37 (1H, m, H-21b). 1.25 (3H, s, H-19). 13C NMR
1029 cmꢀ1. 1H NMR (CDCl3, 300 MHz):
d
7.46 (1H, d, J ¼ 6.9 Hz, H-
10), 6.65 (1H, d, J ¼ 10.1 Hz, H-4), 6.06 (1H, t, H-27), 5.56 (1H, d,
J ¼ 10.2 Hz, H-3), 5.07 (1H, br t, H-32), 4.74 (2H, br s, 37-OH, 38-OH),
3.82 (3H, s, 6-OCH3), 3.76 (1H, d, J ¼ 10.9 Hz, 37-H), 3.43 (1H, m, H-
11), 3.41 (3H, s, COOCH3), 3.34 (2H, m, H-31a), 3.10 (1H, dd,
J ¼ 14.7 Hz, 7.6 Hz, H-31b), 2.92 (2H, d, J ¼ 7.23 Hz, H-26), 2.51 (1H, d,
J ¼ 9.0 Hz, H-22), 2.31 (1H, dd, J ¼ 13.5 Hz, 4.8 Hz, H-21a), 1.46
(1H, m, H-24), 1.39 (1H, m, H-21b), 1.76 (5H, m, H-20, H-25), 1.65
(3H, s, H-35),1.69 (6H, s, H-29, H-34),1.67 (3H, s, H-39), 2.03 (2H, m,
H-36), 1.28 (3H, s, H-19). ESI-MS: 691 [M þ H]þ, 713 [M þ Na]þ, 729
[M þ K]þ. HRMS (M þ H) m/z 691.3483 (Calcd for C40H51O10
691.3482).
(CDCl3, 75 MHz): d 17.51 (C-40), 20.8 (C-29), 20.9 (C-21), 21.2 (C-31),
22.6 (C-36), 25.3 (C-35), 25.5 (C-39), 27.1 (C-19), 28.8 (C-24), 29.7 (C-
26), 30.0 (C-25), 41.7 (C-20), 46.9 (C-11), 49.0 (C-22), 68.3 (C-34), 81.2
(C-2), 83.6 (C-13), 83.8 (C-23), 90.7 (C-14), 100.4 (C-7), 102,8 (C-5),
106.9 (C-17),115.8 (C-4),123.7 (C-32),123.8 (C-37),124.6 (C-3),128.5
(C-28), 131.8 (C-38), 133.3 (C-9), 134.2 (C-33), 135.1 (C-10), 136.5 (C-
27), 157.4 (C-6), 157.6 (C-16), 161.2 (C-18), 170.3 (C-30), 178.8 (C-8),
203.0 (C-12). ESI-MS: 667 [M þ Na]þ: 683 [M þ K]þ. HRMS (M þ Na)
m/z 667.2888 (Calcd for C38H44O9Na 667.2883).
4.1.6. 34-Hydroxy-39-formyl-gambogic acid 6 and (34,39)-
dihydroxy-gambogic acid 7
4.1.8. (37S,38)-Dihydroxy-6-methoxy-gambogic acid methyl ester 9
To GA-methyl ester (250 mg, 0.38 mmol) was added a mixture of
GA (628 mg, 1 mmol) was dissolved in CH2Cl2 (2 ml) and added
dropwise to a clear solution of SeO2 (12 mg, 0.1 mmol and t-BuOOH
(75%, 270 mg, 3 mmol in CH2Cl2 (10 ml) at room temperature. After
being stirred at room temperature for 5 h, the reaction mixture was
diluted with Et2O and sequentially with aqueous 10% KOH, H2O and
brine, then dried and concentrated. The resulting yellow oil was
purified by flash column chromatography on silica gel to yield the
yellow powder 6 (40 mg, 6%) and 7 (100 mg,15%). Compound 6: M.p.
t-BuOH (3.5 ml), H2O (3.5 ml), AD-mix-b (590 mg, 0.42 mmol), and
methanesulfonyl amide (36 mg, 0.38 mmol). The solution was
stirred for 48 h at 0 ꢃC. After the addition of saturated Na2SO3
(72 mg, 0.57 mmol), the mixture was stirred for 40 min and diluted
with EtOAc. The organic layer was washed with H2O and brine,
dried over MgSO4 and concentrated to give the crude product
purified by silica gel column chromatography, yielded yellow
solid 9 (62 mg, 48%). Unreacted GA-methyl ester (120 mg). M.p.

J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
2619
110–113 ꢃC. IR (KBr): 3315, 3034, 3012, 2934, 2927, 1713, 1641, 1589,
1435, 1413, 1316, 1143, 1086, 1029 cmꢀ1. 1H NMR (CDCl3, 300 MHz):
40),1.28 (3H, s, H-19), 1.38 (1H, m, H-21b),1.44 (3H, s, H-24). ESI-MS:
715 [M þ H]þ 737 [M þ Na]þ, 753 [M þ K]þ.
d
7.45 (1H, d, J ¼ 6.9 Hz, H-10), 6.65 (1H, d, J ¼ 10.1 Hz, H-4), 6.06
(1H, t, H-27), 5.56 (1H,d, J ¼ 10.2 Hz, H-3), 5.07 (1H, br t, H-32), 4.76
(2H, br s, 37-OH, 38-OH), 3.82 (3H, s, 6-OCH3), 3.75 (1H, d,
J ¼ 10.9 Hz, 37-H), 3.43 (1H, m, H-11), 3.41 (3H, s, COOCH3), 3.33
(1H, m, H-31a), 3.10 (1H, dd, J ¼ 14.7 Hz, 7.6 Hz, H-31b), 2.93 (2H, d,
J ¼ 7.23 Hz, H-26), 2.51 (1H, d, J ¼ 9.0 Hz, H-22), 2.31 (1H, dd,
J ¼ 13.5 Hz, 4.8 Hz, H-21a), 1.46 (1H, m, H-24), 1.39 (1H, m, H-21b),
1.76 (5H, m, H-20, H-25), 1.65 (3H, s, H-35), 1.69 (6H, s, H-29, H-34),
1.67 (3H, s, H-39), 2.03 (2H, m, H-36), 1.28 (3H, s, H-19). 13C NMR
4.2. Biological materials and methods
4.2.1. Biology
Compounds 1–11 and gambogic acid (supplied by the School of
Pharmacy, China Pharmaceutical University) were dissolved in
DMSO (Sigma) (0.1 ml) to make a final concentration of 10ꢀ2 M and
was stored at ꢀ20 ꢃC. The solutions of compounds 1–11 and GA
were freshly diluted with RPMI-1640 (GIBCO, USA) to the appro-
priate final concentrations at time of use.
(CDCl3, 75 MHz):
d 18.14 (C-34), 20.9 (C-29), 22.0 (C-31), 23.6 (C-
36), 25.6 (C-21), 25.4 (C-35), 26.8 (C-19), 28.8 (C-24), 29.2 (C-25),
29.8 (C-26), 29.5 (C-40), 38.9 (C-39), 43.4 (C-20), 46.5 (C-11), 46.7
(C-37), 49.0 (C-22), 51.1 (30-COOCH3), 62.1 (6-OCH3), 72.9 (C-38),
80.4 (C-2), 83.6 (C-13), 83.7 (C-23), 91.1 (C-14), 107.4 (C-17), 109.9
(C-7), 113.0 (C-5), 116.8 (C-4), 121.7 (C-32), 127.2 (C-3), 127.6 (C-28),
131.8 (C-33), 133.9 (C-9), 135.7 (C-10), 136.4 (C-27), 155.2 (C-6),
158.5 (C-16), 159.6 (C-18), 167.6 (C-30), 174.6 (C-8), 203.8 (C-12),
68.76 (C-40). ESI-MS: 691[M þ H]þ, 713[M þ Na]þ, 729[M þ K]þ.
HRMS (M þ H) m/z 691.3483 (Calcd for C40H51O10 691.3482).
Tumor cells: HT-29, Bel-7402, BGC-823, A549 and SKOV3,
HepG2 cell lines were purchased from Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Science. Cells were grown in RPMI-1640 medium supplemented
with 10% heat-inactivated calf serum (Sijiqing, Hangzhou, China),
100 U/ml penicillin G, and 100 U/ml streptomycin, pH 7.4, in
a water jacketed CO2 incubator (Thermo Forma, USA) with
a humidified atmosphere of 5% CO2 at 37 ꢃC.
Reagents: MTT (Fluka, USA) was dissolved in 0.01 M PBS.
Primary antibody of actin was obtained from CalBiochem. Primary
antibodies of Bcl-2 and Bax were obtained from Santa Cruz
Biotechnology Inc., USA. Secondary antibodies were all obtained
from Rockland Inc., USA. CycleTestÔ Plus DNA reagent lit was
obtained from Becton-Dickinson, USA. Annexin V-EGFP Apoptosis
Detection kit was obtained from KeyGen, USA.
4.1.9. 39-Hydroxy-6-methoxy-gambogic acid methyl ester 10
GA-methyl ester (450 mg, 0.69 mmol) was dissolved in CH2Cl2
(2 ml) and added dropwise to a clear solution of SeO2 (7.6 mg,
0.068 mmol) and t-BuOOH (75%, 185 mg, 2.06 mmol in CH2Cl2
(10 ml) at room temperature. After being stirred at room temper-
ature for 19 h, the reaction mixture was diluted with Et2O and
sequentially with aqueous 10% NaOH, H2O and brine, then dried
and concentrated. The resulting yellow oil was purified by flash
column chromatography on silica gel to yield a yellow sheet solid
10 (280 mg, 61%) .IR (KBr): 3467, 3416, 2966, 2925, 1734, 1709,1655,
1607, 1586, 1463, 1427, 1384, 1225, 1143, 1046 cmꢀ1. 1H NMR (CDCl3,
4.2.2. Methods
4.2.2.1. MTT assay of growth inhibition [30]. Cells (5 ꢂ103 per well in
a 96-well plate) were treated with 1–11 (0.01, 0.1, 1.0, 10, 100 and
1000 mM) and GA (0.01, 0.1, 1.0, 10, 100 and 1000 mM) for 48 h,
respectively. Then, 0.5% MTT was added into each well and incubated
for 4 h at 37 ꢃC. The supernatant was discarded and 0.1 ml DMSO
was added to dissolve the precipitate. The mixture was shaken on
a micro-vibrator for 5 min and the absorbance was measured at
570 nm by an Automated Microplate Reader ELx800 (Bio Tek).
Inhibition ratio (IR%) was calculated by the following method:
300 MHz):
d
7.53 (1H, d, J ¼ 6.70 Hz, H-10), 6.68 (1H, d, J ¼ 2.3 Hz,
H-4), 5.95 (1H, m, H-27), 5.53 (1H, d, J ¼ 6.1 Hz, H-3), 5.32 (1H, m,
H-37), 5.16 (1H, m, H-32), 4.11 (2H, s, H-40), 3.81 (3H, s, 6-OCH3),
3.43 (3H, s, COOCH3), 3.41 (1H, m, H-11), 3.37 (1H, m, H-31a), 3.26
(1H, m, H-31b), 2.96 (2H, m, H-26), 2.51 (1H, d, J ¼ 9.31 Hz, H-22),
2.30 (1H, m, H-21a), 2.04 (2H, m, H-36), 1.75 (3H, s, H-25), 1.69 (3H,
s, H-34), 1.65 (3H, s, H-35), 1.68 (3H, s, H-40), 3.95 (2H, s, H-39), 1.28
(3H, s, H-19), 1.38 (1H, m, H-21b), 1.44 (3H, s, H-24). 13C NMR
Average absorbance of treated group
Average absorbance of control group
1 ꢀ
ꢂ 100
(CDCl3, 75 MHz): d 18.1 (C-34), 20.7 (C-29), 20.8 (C-40), 22.1 (C-31),
The IC50 was defined as the concentration that caused 50%
inhibition of cell proliferation, and was calculated by SAS statistical
software. The data are presented as the mean ꢁ standard deviation
of three observations.
22.3 (C-36), 25.5 (C-21), 25.7 (C-35), 27.9 (C-19), 28.9 (C-24), 29.4
(C-25), 29.9 (C-26), 41.7 (C-20), 46.8 (C-11), 49.0 (C-22), 50.9 (30-
COOCH3), 62.1 (6-OCH3), 68.8 (C-39), 80.5 (C-2), 83.6 (C-13), 83.8
(C-23), 90.9 (C-14), 107.4 (C-17), 100.7 (C-7), 112.6 (C-5), 116.9 (C-4),
121.9 (C-32), 125.3 (C-37), 126.9 (C-3), 12, 158.6 (C-16), 159.7 (C-18),
174.6 (C-8), 203.9 (C-12), 167.3 (C-30). ESI-MS: (M þ H)þ m/z
673.3369 (Calcd for C40H49O9 673.3377).
4.2.3. Cell morphological assessment
HepG2 cell line was cultured in RPMI-1640 until mid-log phase.
Compound 10 (1, 2, or 3 mM) was then added to the culture media
and the cells were further incubated for 20 h. At the end of incu-
bation, the morphology of the cells was monitored under an
inverted light microscope. All floating and attached cells were
harvested with 0.02% (w/v) EDTA and 0.25% (w/v) trypsinase. The
cell suspension was fixed with ice-cold 4% paraformaldehyde for
20 min and washed with ice-cold PBS. The cell suspension was
permeabilized with 0.3% Triton X-100 and washed with ice-cold
PBS, stained with fluorochrome dye DAPI (Santa Cruz, USA), and
observed under a fluorescence microscope (OlympusIX51, Japan)
with a peak excitation wavelength of 340 nm.
4.1.10. 40-Acetoxy-6-methoxy-gambogic acid methyl ester 11
Compound 10 (85 mg, 0.13 mmol) was dissolved in CH2Cl2 (5 ml),
to the resulting solution was added pyridine (0.25 ml), freshly
distilled Ac2O (0.29 ml) and DMAP (2 mg, 0.016 mmol) at room
temperature with vigorous stirring for 4 h. The reaction mixture was
diluted with Et2O and sequentially with 10% HCl aqueous solution,
H2O, and brine, then dried and concentrated. The resulting crude was
purified by flash column chromatography on silica gel to yield
a yellow oil characterized as 11 (30 mg, 33%). 1H NMR (CDCl3,
300 MHz):
d
7.53 (1H, d, J ¼ 6.70 Hz, H-10), 6.68 (1H, d, J ¼ 2.3 Hz, H-
4), 5.95 (1H, m, H-27), 5.53 (1H, d, J ¼ 6.1 Hz, H-3), 5.12 (1H, m, H-37),
5.09 (1H, m, H-32), 4.45 (2H, s, H-39), 4.11 (2H, s, H-40), 3.81 (3H, s, 6-
OCH3), 3.43 (3H, s, COOCH3), 3.41 (1H, m, H-11), 3.37 (1H, m, H-31a),
3.26 (1H, m, H-31b), 2.96 (2H, m, H-26), 2.51 (1H, d, J ¼ 9.31 Hz, H-
22), 2.30 (1H, m, H-21a), 2.04 (2H, m, H-36), 1.90 (3H, s, –CH3CO–),
1.75 (3H, s, H-25),1.69 (3H, s, H-34), 1.65 (3H, s, H-35), 1.69 (3H, s, H-
4.2.4. AOEB staining
Aliquots of compound 10 (1.0–3.0
cells and incubated for an additional 18 h. Paraformaldehyde (4%,
500 l) was then added and fixed for 20 min followed by addition of
0.1% Triton-100 (300 l), permeabilizing for 20 min, and addition of
mM) were added to HepG2
m
m

2620
J. Wang et al. / European Journal of Medicinal Chemistry 44 (2009) 2611–2620
300
m
l AOEB in PBS for staining. Five minutes later, the cells were
and the Projects of Natural Science Foundation of Jiangsu Province
(BK2006149) for financial support.
observed under a fluorescence microscope (Olympus, Japan) with
peak excitation wavelength of 490 nm.
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All results shown represent the mean ꢁ SD from triplicate
experiments performed in a parallel manner unless otherwise
indicated. Statistical analyses were performed using an unpaired,
two-tailed Student’s t-test. All comparisons are made relative to
untreated controls and significance of difference is indicated as
*P < 0.05 and **P < 0.01.
Acknowledgments
The authors thank the 863 High-Tech Project of China
(2002AA2Z3112) (2004AA2Z3A10), the Key Projects of Science and
Technology Research of Ministry of Education of PRC (2006-106094)