Spectacular modification of Gambogic acid on microwave irradiation in methanol: Isolation and structure identification of two products with potent anti-tumor activity

Article abstract of DOI:10.1016/j.bmcl.2010.03.021

Treatment of gambogic acid with methanol in acidic condition under microwave irradiation led to the formation of two new products bearing spectacular A ring systems which were different from reported Garcinia natural products. The chemical structures of these two compounds were elucidated by extensive NMR and MS spectroscopic analysis as well as crystallographic study. Both of the two products could significantly inhibit the growth of various tumor cell lines in vitro with IC₅₀ values at μM level through apoptotic pathway.

Full text of DOI:10.1016/j.bmcl.2010.03.021

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2438–2442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spectacular modification of Gambogic acid on microwave irradiation
in methanol: Isolation and structure identification of two products
with potent anti-tumor activity
Xiaojian Wang a, , Na Lu b, , Qian Yang a, Qinsheng Dai b, Lei Tao b, Xiaoke Guo a, Qinglong Guo b,
,
*
Qidong You a,b,
*
a Department of Medicinal Chemistry, 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
Article history:
Treatment of gambogic acid with methanol in acidic condition under microwave irradiation led to the
formation of two new products bearing spectacular A ring systems which were different from reported
Garcinia natural products. The chemical structures of these two compounds were elucidated by extensive
NMR and MS spectroscopic analysis as well as crystallographic study. Both of the two products could sig-
Received 12 January 2010
Revised 26 February 2010
Accepted 4 March 2010
Available online 7 March 2010
nificantly inhibit the growth of various tumor cell lines in vitro with IC50 values at
apoptotic pathway.
lM level through
Keywords:
Anti-tumor effect
Apoptosis
Ó 2010 Elsevier Ltd. All rights reserved.
Gambogic acid
Microwave
Gamboge resin from Garcinia hanburyi tree in Southeast Asia was
reported in traditional folk medicine records to possess many inter-
esting biological profiles, including anti-viral, anti-inflammatory,
and anti-infective activities. Gambogic acid (GA, CAS No. 2752-65-
0) is one of the representative ingredients secreted from the gam-
boge resin.1 The chemical structure of GA has been well established
by NMR spectroscopic analysis2,3 and crystallographic studies.4 GA
contains the same unique 4-oxa-tricyclo[4.3.1.03,7]dec-2-one scaf-
fold (the so called caged ring system) as other natural products iso-
lated from Garcinia genus.5 Biological evidences indicated that GA
could significantly inhibit the growth of human cancer cells and pre-
vent tumor angiogenesis and metastasis without affecting normal
cells both in vitro and in vivo.6–9 Now GA has been approved to enter
Phase II clinical trials in China.
so far since it does not possess any directly modifiable site. There-
fore, it is highly desirable to expand the molecular diversity of A
ring system to explore SAR information and develop novel GA
derivatives. In this Letter, two GA derivatives with neo-type A ring
systems that differed from traditional Garcinia natural products
were generated during microwave irradiation in acidic condition
(Scheme 1). Details of the structure resolution, mechanism elucida-
tion and biological evaluation for these new compounds were re-
ported herein.
The reaction was conducted under microwave irradiation
(200 W, 80 °C, 5 min) using small amount of H2SO4 as acid and
methanol as both the solvent and reactant. Two products, encoded
1 and 2, were obtained and purified by silica column chromatogra-
phy (eluent, PE:EA = 8:1). 1H NMR, 13C NMR and 2D NMR analysis
showed that both 1 and 2 maintained the same bridged ring sys-
tem as GA. Subsequently, the structure of 1 was confirmed by sin-
gle crystal X-ray analysis14 (Fig. 1). Then the structure of 2 was
identified by comparing NMR data with 1. In accordance with pre-
viously reported microwave assisted esterification methodology,15
the esterification of GA’s carboxylic acid group was achieved under
the microwave condition, whereas the isopentenyl group was cy-
clized with the alkene on the A ring forming a new bridged ring
skeleton (compound 1) or a new six-membered ring structure
(compound 2).
During recent years, total synthesis and SAR studies of GA as
well as other Garcinia natural products have been springing up vig-
orously due to their specific caged scaffold and striking bioactivi-
ties.7,10–13 Previous structure modifications of GA mainly focused
on the 6-hydroxyl group, 9,10-double carbon bond of a,b-unsatu-
rated ketone, 30-carboxyl group and the isopentenyl groups.7,10,11
However, the A ring modified GA derivatives were rarely reported
* Corresponding authors. Tel./fax: +86 25 83271351 (Q.-D.Y.); +86 25 83271055
(Q.-L.G.).
E-mail addresses: anticancer_drug@yahoo.com.cn (Q. Guo), youqd@163.com,
youqidong@gmail.com (Q. You).
Since this reaction was unprecedented and the A ring systems
1 and 2 were different from reported Garcinia natural
in
These two authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.021

X. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2438–2442
2439
O
O
O
39
35
40
38
34
33
29
35
34
31
28
30
OH
33
O
O
O
34
3
32
33
32
27
24
25
32
37
26 O
36
31
17
31
O
35
20
1
O1
A
O
C
17
36
37
38
40
20
MW, 80°C , 5 min
CH3OH, H+
O
O
O
O
O
O
O
O
1
2
22
2
3
12
11
B
O
2
21
3
39
9
8
4
6
6
6
4
4
10
40
20
OH
O
38OH
O
OH
37
40
36
Gambogic acid
1
39
2
Scheme 1. The formation of products 1 and 2.
Figure 1. X-ray structure of compound 1.
products,13 the reaction condition were investigated. As shown in
Table 1, compounds 1 and 2 were not formed without microwave
irradiation (entries 1 and 2). The product ratio of 1 and 2 was nei-
ther proportional to the reaction time nor related to the degree of
acidity (entries 3–5). Subsequently, pure 1 and 2, respectively,
were subjected to the same microwave conditions in the presence
of methanol and acid. The results showed that 1 and 2 existed in
equilibrium amount without the formation of other products after
microwave heating. All the evidences suggested the thermody-
namic control of the product distribution.
On the basis of the above investigations, a possible mechanism
of the reactions was proposed to explain the formation of 1 and 2
(Scheme 2). Presumably, in the presence of acid, the alkene of the A
ring was protonated, which led to the formation of a carbocation
on the 4-position. This reaction is probably facilitated by the fact
that the carbocation formed is benzylic and that there are two elec-
tron-donating groups on C-17, and C-6 of the same phenyl ring,
which helps to stabilize the positive charge. Then attack of this car-
bocation by the
p-electrons of the double bond between C37 and
C38 led to the formation of the new bridged ring of compound 1.
Table 1
The attempted reaction conditions and their corresponding yields of 1 and 2
Entry
Conditions
Yieldsa
GA recovery (mg)
Temperature (°C)
Acids/solvent
Time
Compd 1 (%)
Compd 2 (%)
1
2
3
rt
80
MW, 80
H2SO4/CH3OH
H2SO4/CH3OH
H2SO4/CH3OH
48 h
6 h
0
0
0
0
185
180
20
15
15
18
8
5 min
10 min
15 min
30 min
5 min
5 min
49
50
50
50
47
46
28
28
27
27
40
37
4
5
MW, 80
MW, 80
CH3SO2H/CH3OH
p-CH3-PhSO2H/CH3OH
10
a
In each entry, 200 mg GA was used.

2440
X. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2438–2442
38
37
O
O
O
O
O
CH3OH,H+
H
O
17
OH
O
O
O
O
OH
CH3OH
O
O
O
+
O
H
H
O
O
4
HO
O
HO
HO
O
O
Gambogic acid
CH3OH
O
O
O
O
HO
O
H
O
O
O
O
-H+
O
O
O
O
O
O
O
O
O
HO
HO
O
O
HO
O
1
3
H
H
+
O
O
-H+
O
O
HO
O
CH3OH,H+
O
O
O
O
O
O
O
O
HO
O
HO
O
2
Scheme 2. Proposed mechanism for the formation of 1 and 2.
The esterification of the carboxylic acid group can be independent
of the cyclization event. As for the step leading to the formation of
compound 2, the O atom of the 1 bridged ring can be protonated,
facilitating an SN2 attack by methanol, resulting in ring cleavage
and intermediate 3. Protonation of the isopentenyl group on 17-
position and the subsequent formation of a carbocation initiate
the second cyclization thus resulting for the formation of com-
pound 2.
Compounds 1 and 2 were evaluated for their in vitro anti-tumor
effects against four human cancer cell lines including human hepa-
tocellular liver carcinoma HepG2 cell line, human gastric carci-
noma BGC-803 cell line, human stomach carcinoma SGC-7901
cell line, human mammary carcinoma MCF-7 cell line using MTT
assay.16 Interestingly, after incubation for 48 h, both 1 and 2
showed comparable anti-proliferative activities against these tu-
mor cell lines to GA and methyl gambogate, the methyl-esterified
derivative of GA (Table 2).7 HepG2 cell line was chosen for further
investigation since both 1 and 2 showed better anti-proliferative
activities than GA and methyl gambogate in this cell line. Cell mor-
phological observations were subsequently performed under an in-
verted-microscope. Under the light microscope (Â200), incubation
of 1.25 M, 2.5 M and 5 M of compound 1 and 0.5 M, 1 M and
M of compound 2, respectively, in HepG2 cells for 24 h led to
l
l
l
l
l
2
l
severe distortion or elongation, membrane blebbing and cell
shrinkage. With the gradient enhancement of drug concentration,
a large proportion of cells became round in shape and necrosed,
while the untreated cells displayed normal shape and clear skele-
ton under the same conditions (Fig. 2A and B). Under the fluores-
cence microscope (Â400), HepG2 cells treated with indicated
concentration of 1 and 2 for 24 h displayed the morphological fea-
tures of early apoptotic cells, such as bright nuclear condensation
identified by DAPI staining, and the apoptotic body appeared sig-
nificantly with the increased concentration of 1 and 2 (Fig. 2C
and D).
The explorations of 1 and 2 on apoptosis induction were per-
formed using Annexin-V/propidium iodide (PI) double staining as-
say on flow cytometry instrument. For compound 1, the percentage
of apoptotic cells (Annexin V+/PIÀ staining) in the control group
was 5.3%. After incubation of 1.25 lM, 2.5 lM and 5 lM of 1 for
24 h, the early and median apoptotic cells (lower right section of
fluorocytogram) were increased to 5.7%, 13.6% and 20.7%, respec-
tively, while the late apoptotic and necrotic cells (upper right sec-
tion of fluorocytogram) were increased slightly (Fig. 3A). For
compound 2, the percentage of apoptotic cells in the control group
Table 2
The in vitro anti-tumor effects of compounds 1 and 2a
was 3.5%. After incubation of 0.5 lM, 1 lM and 2 lM of 2 for 24 h,
Cell lines
IC50
GA
(lM)
1
2
Methyl gambogateb
the early and median apoptotic cells were increased to 4.3%, 12.9%
and 35.0%, respectively, whereas the late apoptotic and necrotic
cells were increased slightly (Fig. 3B). These results suggested that
1 and 2 could induce cell death mainly via apoptosis rather than
necrosis. Further explorations of the apoptotic induction mecha-
nism of 1 and 2 on the expression level of apoptosis related pro-
HepG2
1.96
1.45
2.15
1.88
1.53
2.01
1.51
1.24
2.40
1.54
1.47
0.50
2.53
1.24
0.79
0.52
BGC-803
SGC-7901
MCF-7
a
The IC50 values were determined from eight different concentrations of com-
pounds at two fold dilutions, and were the means of two separate experiments.
teins were performed. After incubation with 1.25
lM, 2.5 lM and
b
Methyl gambogate was synthesized according to Ref. 7.
5
lM of compound 1 for 24 h, the expression level of bcl-2 and

X. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2438–2442
2441
Figure 2. Cell morphological assessment of compounds 1 and 2 under light microscope instrument (A, B) and under fluorescence microscope instrument (C, D) on HepG2
cells. (A) Compound 1: (a) control group; (b) 1.25 M of 1; (c) 2.5 M of 1; (d) 5 M of 1. (B) Compound 2: (a) control group; (b) 0.5 M of 2; (c) 1 M of 2; (d) 2 M of 2. (C)
Compound 1: (a) control group; (b) 1.25 M of 1; (c) 2.5 M of 1; (d) 5 M of 1. (D) Compound 2: (a) control group; (b) 0.5 M of 2; (c) 1 M of 2; (d) 2 M of 2.
l
l
l
l
l
l
l
l
l
l
l
l
Figure 3. Fluorescence-activated cell sorter analysis of compounds 1 and 2 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) Compound 1: (a) control group; (b) 1.25 M of 1; (c) 2.5 M of 1; (d) 5 M of 1. (B) Compound 2: (a) control
group; (b) 0.5 M of 2; (c) 1 M of 2; (d) 2 M of 2.
l
l
l
l
l
l
pro-caspase-3 were decreased significantly in HepG2 cells, while
the protein level of bax had no significant change compared with
activator protein bax was increased, and pro-caspases-3 was trans-
formed to its active caspase-3 form, all of which indicated the ini-
tiation of apoptosis. Therefore, it was concluded that 1 and 2 could
inhibit cancer cell growth through apoptotic pathway.
control (Fig. 4A). For compound 2, being treated with 0.5
lM,
1
l
M and 2 M concentration, the expression level of bax were in-
l
creased, while the protein level of bcl-2 had no obvious change.
Meanwhile, the expression level of pro-caspase-3 was decreased,
accompanied by the increasing expression of active form cas-
pase-3 (Fig. 4B). The results indicated that after treatment with 1
and 2, the general ratio of apoptosis suppressor protein bcl-2 and
In summary, a novel intramolecular cyclization reaction of gam-
bogic acid under microwave irradiation was reported. Two new
products named 1 and 2 with new types of ring systems which
could not be obtained without microwave heating were obtained.
A possible mechanism was proposed by deep investigation of the

2442
X. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2438–2442
Figure 4. Effects of compounds 1 and 2 on the expression of apoptotic related proteins of HepG2 cells. (A) Compound 1; (B) compound 2.
reaction condition and the structures of 1 and 2. This new reaction
indicated that the specific 4-oxa-tricyclo[4.3.1.03,7]dec-2-one ring
system can tolerate acidic conditions under microwave heating,
thus make it possible to extend the application of the microwave
reaction to other Garcinia natural products. The preliminary biolog-
ical studies identified that these two compounds with novel A ring
systems could inhibit tumor cell growth through apoptotic path-
way. Their disparities in the mechanistic studies also precipitated
further investigations.
References and notes
1. Auterhoff, H.; Frauendorf, H.; Liesenklas, W.; Schwandt, C. Arch. Pharm. 1962,
295/67, 833.
2. Ollis, W. D.; Ramsay, M. V. J.; Sutherland, I. O.; Mongkolsuk, S. Tetrahedron
1965, 21, 1453.
3. Cardillo, G.; Merlini, L. Tetrahedron Lett. 1967, 8, 2529.
4. Weakley, T. J. R.; Cai, S. X.; Zhang, H.-Z.; Keana, J. F. W. J. Chem. Crystallogr. 2002,
31, 501.
5. Xu, Y. J.; Yip, S. C.; Kosela, S.; Fitri, E.; Hana, M.; Goh, S. H.; Sim, K. Y. Org. Lett.
2000, 2, 3945.
6. Asano, J.; Chiba, K.; Tada, M.; Yoshii, T. Phytochemistry 1996, 41, 815.
7. Zhang, H. Z.; Kasibhatla, S.; Wang, Y.; Herich, J.; Guastella, J.; Tseng, B.; Drewe,
J.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 309.
Acknowledgments
8. Zhao, L.; Guo, Q. L.; You, Q. D.; Wu, Z. Q.; Gu, H. Y. Biol. Pharm. Bull. 2004, 27,
998.
9. Yang, Y.; Yang, L.; You, Q. D.; Nie, F. F.; Gu, H. Y.; Zhao, L.; Wang, X. T.; Guo, Q. L.
Cancer Lett. 2007, 256, 259.
10. Kuemmerle, J.; Jiang, S.; Tseng, B.; Kasibhatla, S.; Drewe, J.; Cai, S. X. Bioorg.
Med. Chem. 2008, 16, 4233.
11. Wang, J.; Zhao, L.; Hu, Y.; Guo, Q.; Zhang, L.; Wang, X.; Li, N.; You, Q. Eur. J. Med.
Chem. 2009, 44, 2611.
Financial support from National Natural Science Foundation of
China (No. 90713038) and National Key Program for New Drug
Development (No. 2009ZX09501-003). We thank Professor Bing-
he Wang, Department of Chemistry, Georgia State University;
and Professor James R. McCarthy, Department of Chemistry and
Chemical Biology, Indiana University Purdue University Indianapo-
lis, for their helpful suggestions on this Letter.
12. Pang, X.; Yi, T.; Yi, Z.; Cho, S. G.; Qu, W.; Pinkaew, D.; Fujise, K.; Liu, M. Cancer
Res. 2009, 69, 518.
13. Han, Q. B.; Xu, H. X. Curr. Med. Chem. 2009, 16, 3775.
14. Crystallographic data for compound 1 have been deposited with the Cambridge
Crystallographic Data Centre (deposition number CCDC: 733795).
15. Yang, Q.; Wang, X. J.; Li, Z. Y.; Sun, L.; You, Q. D. Synth. Commun. 2008, 38, 4107.
16. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.021.