Isolation, characterization and cytotoxic activity of benzophenone glucopyranosides from Mahkota Dewa (Phaleria macrocarpa (Scheff.) Boerl)

Article abstract of DOI:10.1016/j.bmcl.2012.09.038

Two benzophenone glucopyranosides have been isolated from the nut shell part of Mahkota Dewa. The structures were identified as 2,4′,6-trihydroxy- 4-methoxy-benzophenone-2-O-β-d-glucoside (Mahkoside A) and 2,4′,6-trihydroxy-4-methoxy-6″-acetyl-benzophenone-2-O-β-d- glucoside (Mahkoside B). Mahkoside B was recognized as a novel compound. Furthermore, a series of benzophenone glucopyranoside derivatives (compounds 3-18) were synthesized and their bioactivities were characterized. Our results demonstrated that compound 18 has significant cytotoxicity against two esophageal cancer cell lines, stomach cancer cell line and prostate cancer cell line, with IC₅₀ less than 10 μM, indicating its potential activity against cancer cells.

Full text of DOI:10.1016/j.bmcl.2012.09.038

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6862–6866
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isolation, characterization and cytotoxic activity of benzophenone
glucopyranosides from Mahkota Dewa (Phaleria macrocarpa (Scheff.) Boerl)
Sai-Yang Zhang a, Quan-Hai Zhang a, Wen Zhao a,b, Xiao Zhang a, Qi Zhang a,b, Yue-Feng Bi a,b
,
Yan-Bing Zhang a,b,
⇑
a School of Pharmaceutical Science, Zhengzhou University, Zhengzhou, Henan, China
b New Drug Research and Development Center, Zhengzhou University, Zhengzhou, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2012
Revised 31 August 2012
Accepted 12 September 2012
Available online 20 September 2012
Two benzophenone glucopyranosides have been isolated from the nut shell part of Mahkota Dewa. The
structures were identified as 2,40,6-trihydroxy-4-methoxy-benzophenone-2-O-b-
-glucoside (Mahkoside
A) and 2,40,6-trihydroxy-4-methoxy-600-acetyl-benzophenone-2-O-b-
-glucoside (Mahkoside B).
Mahkoside B was recognized as a novel compound. Furthermore, a series of benzophenone glucopyran-
oside derivatives (compounds 3–18) were synthesized and their bioactivities were characterized. Our
results demonstrated that compound 18 has significant cytotoxicity against two esophageal cancer cell
D
D
Keywords:
lines, stomach cancer cell line and prostate cancer cell line, with IC50 less than 10
potential activity against cancer cells.
lM, indicating its
Mahkota Dewa
Benzophenone glucopyranoside
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
Bioactivity
Mahkota Dewa [Phaleria macrocarpa (Scheff.) Boerl] is mainly
distributed in Southeast Asia region, especially in Indonesia. Due
to its antihistamine, anti-oxidation and antitumor effects, it has
been used to treat many diseases, including cancer, impotency,
haemorrhoids and diabetes mellitus, et al.1 Mahkota Dewa is also
used as a major ingredient in some healthy drink or tea in Indone-
sia. Mahkoside A (2,40,6-trihydroxy-4-methoxy-benzophenone-2-
were also synthesized and their chemical structures were charac-
terized by NMR, HR-MS and UV. The current study is designed to
increase the diversity of the compounds and determine the bioac-
tivity of those compounds with different structures. Our goal is to
discover a safe and effective new drug with anti-cancer activity
and low toxicity from the title plant, through our in vitro screening
of the isolated compounds and the related derivatives synthesized.
Our results indicate that 14 of the 16 derivatives were synthesized,
for the first time, and have been recognized as novel compounds.
Their cytotoxicities against four different kinds of cancer cells were
also evaluated. Interestingly, we found that compound 18 has sig-
nificant cytotoxicity against four cancer cell lines with IC50 less
O-b-D-glucoside) was previously identified by our laboratory as a
component in Mahkota Dewa.2,3 Some of its derivatives were syn-
thesized and their in vitro cytotoxic activities were proved against
human esophageal cancer cell line EC9706.3 Due to their multiple
effects, the types of compound have been paid more and more
attention recently. They have been reported to exhibit notable
cytotoxicity against ovarian cancer cell line,4 colon cancer cell
line,5 human submandibular gland adenocarcinoma cell line and
primary human gingival fibroblast.6 They could also combat hu-
man parasite Trypanosoma cruzi (Chagas0s disease)7 and displayed
significant antioxidant activity.8 To further understand and devel-
op Mahkota Dewa for health and disease treatment applications, in
the current study, the known Mahkoside A and a new benzophe-
none glucopyranoside, 2,40,6-trihydroxy-4-methoxy-600-acetyl-
than 10 lM, indicating it may be a new candidate in our continue
searching for anti-cancer drugs.
The mature nut shells of Phaleria macrocarpa (Thymelaeaceae)
were collected at Pasuruan, East Java, Indonesia in 2007. The
botanical identification was made by Professor Zhang Wenhui of
the College of Forestry, Northwest Science-Technology, University
of Agriculture and Forestry, China. A voucher specimen of the plant
was deposited in the ‘New Drug Research and Development Center
of Zhengzhou University’. The dry nut shell of fruit of Phaleria
macrocarpa (1900 g) was crushed and extracted by 3 h refluxing
for three times with 95% ethanol (2000 ml). The mixture was then
evaporated under reduced pressure (50 °C) to obtain the total ex-
tract. Then, petroleum ether (1000 ml), chloroform (2000 ml) and
ethyl acetate (2000 ml) were added, respectively, for the total
products to be further extracted. The acquired ethyl acetate extract
was chromatographed on silica gel, using (3:1) or (4:1)
benzophenone-2-O-b-D-glucoside (Mahkoside B) were isolated
from the nut shell of Mahkota Dewa. 16 additional derivatives
⇑
Corresponding author. Address: School of Pharmaceutical Sciences, New Drug
Research and Development Center, Zhengzhou University, Zhengzhou 450001,
China.
E-mail address: zhangyb@zzu.edu.cn (Y.-B. Zhang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.038

S.-Y. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6862–6866
6863
MeO
OH
O
OH
at d (4.97, J = 7.7 Hz). Its linkage position at C-2 was ascertained
by HMBC. Thus, in the current study, compound 2, 2,40,6-tri-hydro-
xy-4-methoxy-600-acetylbenzophenone-2-O-glucoside, was iso-
lated from the title plant, for the first time, and recognized as a
novel compound, which was named as Mahkoside B.
OR
O
O
HO
HO
OH
In order to find benzophenone glucopyranoside derivatives
with potential anti-cancer bioactivity from the title plan, a variety
of structurally modified compounds (3–18) from three series,
including alkylation, acylation and etherification were synthesized,
using a nucleophilic substitutional reaction (Fig. 2 and Fig. 3).
For the synthesis of compounds 3, 4, 5, 7, 8, 9, 10 and 11, 206,
102, 136, 122, 126,115,105 and 122 mg Mahkoside A were com-
pletely dissolved in 4, 5, 3, 3, 3, 3, 4 and 4 ml DMF, followed by
adding 206, 121, 116, 136, 126, 126, 122 and 122 mg anhydrous
K2CO3. Then, 0.2 ml CH3I, 0.1 ml CH3CH2Br, 0.2 ml CH2@CHCH2Br,
0.1 ml Br(CH2)3CH3, 0.2 ml epoxy chlorpropane, 0.2 ml benzyl bro-
mide, 0.2 ml 4-methoxybenzyl chloride and 0.2 ml 3-methoxyben-
zyl chloride were added dropwise and stirred at 44 °C for 6, 6, 7, 7,
8, 7, 7 and 6 h, respectively. The whole reaction solution was fil-
tered to remove K2CO3 and concentrated in vacuum. The following
reaction procedures are shown in each compound’s synthesis.
For compound 6, 80 mg Makhoside A was completely dissolved
in 3 ml absolute ethyl alcohol, followed by adding 106 mg anhy-
drous K2CO3. Then, 0.1 ml BrCH2CH2CH2Br was added dropwise
and stirred at 44 °C for 10 h. The reaction solution was filtered to
remove K2CO3 and concentrated in vacuum.
1. Mahkoside A R= -H
2. Mahkoside B* R= -COMe
Figure 1. Benzophenone glucopyranosides isolated from the nut shell of Mahkota
Dewa. Note: ⁄Indicate that the compound was reported for the first time.
CHCl3/MeOH to give compound 1 (or Mahkoside A, 206 mg), and
compound 2 (or Mahkoside B, 17 mg), respectively.
The molecular formula C20H22O10 of compound 1 was deter-
mined on the basis of EI-MS data together with 1H and 13C NMR
spectra. The UV spectrum suggested that it has a polyphenolic
structure. Compound 1 has an identical NMR chemical shift pattern
compared with that of the known benzophenone glucopyranoside,
Mahkoside A, which had been previously isolated from Phaleria
macrocarpa Gnidia involucrate.2 Through detailed analysis of HSQC
and HMBC correlations, the identity of compound 1 was identified
as Mahkoside A.
The molecular formula C22H24O11 of compound 2 was deter-
mined on the basis of EI-MS data together with 1H and 13C NMR
spectra. Its UV spectrum is similar to that of compound 1, suggest-
ing a benzophenone structure again. The 1H NMR spectrum dis-
played similar signals besides an additional signal at d 2.05,
integrating for three protons. Two additional corresponding signals
at d 19.91 and d 170.39 was detected in the 13C NMR spectrum. A
HSQC correlation experiment suggested the presence of an acetyl
group (1H: d 2.05, 13C: d 19.91 and 170.39). Its position was as-
signed in C-600 thanks to an HMBC correlation experiment.
Multiplicities were revealed by a DEPT experiment. Gradient HSQC,
HMBC and COSY experiments (Fig. 1), allowed complete assign-
ment and identification of the glycone of 2 as 2,40,6-trihydroxy-
For the synthesis of compounds 12–15, 100–140 mg Mahkoside
A were completely dissolved in 4 ml pyridine, followed by adding
1.0–2.0 ml acetic anhydride, n-butyryl chloride, benzoyl chloride
or parachlorobenzoyl chloride dropwise, respectively. The solu-
tions were stirred at room temperature for 0.5 h. Then, 10 ml chlo-
roform was added, stirred and washed with 10 ml distilled H2O for
3 times. The chloroform solution was concentrated under vacuum
to afford compounds 12–15.
For the synthesis of compound 16, in 25 ml round bottomed
flask, 386 mg compound 9 was added to 6.0 ml absolute ethyl
alcohol, stirring at 25 °C to fully dissolve the compound. 2.0 ml
4-methoxybenzophenone. The presence of an O-b-
D-glucose was
confirmed by the characteristic 1H signal for the anomeric proton
MeO
OR1
OR2
MeO
OH
O
OH
MeO
OR
O
OR
(RCO)2O
or RCOCl
Pyd.
RX, K2CO3
DMF
OR
OH
OR
O
O
O
O
HO
O
O
HO
O
RO
RO
HO
HO
OH
OH
OR
3*. R1=-Me, R2=H
4*. R1=R2= -Et
12. R= -COCH3
1. Mahkoside A R= -H
13*. R= -COCH2CH2CH3
14*. R= -CO-C6H5
2*. Mahkoside B R= -COMe
5*. R1=R2= -CH2CH=CH2
6*. R1=R2= -(CH2)3Br
7*. R1=R2= -(CH2)3CH3
15*. R= -CO-C6H4 -p -Cl
8*. R1=R2= -CH2
O
9*. R1=R2= -CH2C6H5
10*.R1=R2= -CH2C6H4-p-OMe
11*.R1=R2= -CH2C6H4-m-OMe
Figure 2. Route of synthesis for compound 3–15. Note: ⁄Indicates that the compounds are reported for the first time.
MeO
OBn
O
OBn
MeO
OBn
O
OBn
Br(CH2)3Br
K2CO3, MeOH
EtOH,HCl
reflux
reflux
OH
O
9
HO
HO
O
16
OH
OH
MeO
OBn
O
OBn
MeO
OBn
OBn
piperidine, K2CO3
DMF
N
O
Br
O
O
*
17*
18
Figure 3. Route of synthesis for compound 16–18. Note: ⁄indicates that the compounds are reported for the first time.

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S.-Y. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6862–6866
Table 1
Structure of compounds (1–18) and their effects on cell viability
Samples
Yield
(%)
Mp (°C)
IC50
(
l
M/L, 72 h)
Cell lines
MGC-803
EC109
>100
EC9706
>100
PC-3
>100
MeO
OH
OH
1
2
3
105
>100
>100
>100
OH
O
O
O
O
O
HO
HO
OH
MeO
OH
OH
OH
Oil
matter
>100
>100
>100
>100
>100
>100
OAc
O
O
HO
HO
OH
MeO
OMe
47.9
96.9
74
61
OH
O
O
HO
HO
OH
MeO
OEt
OEt
4
5
>100
>100
>100
>100
>100
>100
>100
>100
OH
O
O
O
HO
HO
OH
MeO
OCH2CH=CH2
OCH2CH=CH2
87.8
80.6
93.9
67
55
52
OH
O
O
O
O
O
HO
HO
OH
MeO
O(CH2)3Br
O(CH2)3Br
6
7
23.057 1.363 31.260 1.495 53.767 1.731 40.000 1.602
OH
O
O
HO
HO
OH
MeO
O(CH2)3CH3
O(CH2)3CH3
29.022 1.463 41.763 1.621 49.155 1.692 32.042 1.506
OH
O
O
HO
HO
OH
O
O
OCH2
OCH2
MeO
8
9
80.3
79.8
69
62
>100
>100
>100
>100
OH
O
O
O
HO
HO
OH
MeO
OCH2Ph
OCH2Ph
22.680 1.356 41.062 1.613 44.265 1.646 28.933 1.461
OH
O
O
O
HO
HO
OH
OCH2
OCH3
OCH2
OCH3
MeO
10
75.4
58
17.079 1.232 20.516 1.312 28.180 1.450 11.747 1.070
OH
O
O
O
HO
HO
OH

S.-Y. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6862–6866
6865
Table 1 (continued)
Samples
Yield
(%)
Mp (°C)
IC50
(
l
M/L, 72 h)
Cell lines
MGC-803
EC109
EC9706
PC-3
OMe
OCH2
OCH2
MeO
11
76.5
59
74
12.323 1.091 20.852 1.319 18.385 1.264 6.922 0.840
OMe
OH
O
O
O
OAc
HO
HO
OH
MeO
OAc
12
91
84
50.662 1.705 86.061 1.935 47.313 1.675 34.063 1.532
OAc
O
O
O
AcO
AcO
OAc
OCO(CH2)2CH3
OCO(CH2)2CH3
MeO
Oil
matter
13
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
CH3(CH2)2COO
O
CH3(CH2)2COO
CH3(CH2)2COO
O
O
OCO(CH2)2CH3
OCOPh
MeO
OCOPh
14
15
72
77
72
PhCOO
O
O
O
PhCOO
PhCOO
OCOPh
OCOPh-Cl-p
MeO
OCOPh-Cl-p
124
p-Cl-PhCOO
O
O
O
p-Cl-PhCOO
p-Cl-PhCOO
OCOPh
OBn
MeO
OBn
16
17
78
96
119
105
>100
>100
>100
>100
>100
>100
>100
>100
OH
O
MeO
OBn
OBn
Br
O
O
MeO
OBn
OBn
18
58
193
5.139 0.711
4.971 0.696
3.121 0.494
3.474 0.541
N
O
O
Yield (%) and melting points (Mp°C) are indicated for each compound. Inhibition of four different cancer cell growth was expressed as IC50 (lM/L). EC109 and EC9706:
esophageal cancer cell lines; MGC-803: stomach cancer cell lines; PC-3: prostate cancer cell lines. Three independent experiments were performed in EC109, EC9706,
MGC-803 and PC-3 cell lines. Data are Mean SEM.
hydrochloric acid (9.5 M) was then added. The temperature was
raised to 105 °C and the solution was stirred for another 3 h and
ice-bath filtered to obtain the crystals. Absolute ethyl alcohol
was used to recrystallize the crystals and afford faint yellow needle
crystals of compound 16 (220 mg, 78% yield).
For the synthesis of compound 17, in 25 ml round bottomed
flask, 206 mg compound 9 was fully dissolved in 10.0 ml absolute
ethyl alcohol by stirring at 60 °C, followed by adding 220 mg anhy-
drous K2CO3 and stirred 20 min. 0.5 ml 1,3-dibromopropane was
then added dropwise and temperature was raised to 80 °C and stir-
red for another 2 h. It was further extracted by 10 ml chloroform
twice and white needle crystal (252 mg, 96% yield) was obtained.
For the synthesis of compound 18, in 10 ml round bottomed
flask, 96 mg compound 17 was fully dissolved in 4.0 ml DMF by
stirring at room temperature. 102 mg anhydrous K2CO3 was added
and stirred for 20 min. 0.3 ml piperidine was then added dropwise.
The solution was stirred 7 h at room temperature and further fil-
tered to remove K2CO3. Then, the reduced pressure distillation
was performed to remove DMF from the solution. The acquired
residue was purified by flash chromatography on silica gel (chloro-
form/methanol = 20:1) and vacuumized to afford compound 18
(56 mg, 58% yield) as a yellow oil matter.
In order to determine the relationship of structure and activity
of the compounds above, several cancer cell lines, including esoph-
ageal cancer cell lines: EC109 and EC9706, stomach cancer cell
line: MGC-803 and prostate cancer cell line: PC-3, were randomly
selected and cytotoxic activity was evaluated according to the
standard SRB (sulforhodamine B) assay. Briefly, EC109 and

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S.-Y. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6862–6866
and 7), the phenmethyl derivatives (compounds 9, 10 and 11)
and acylated derivatives 12. The derivatives with ꢀOMe replacing
groups (compounds 10 and 11) showed better activity without re-
spect to the position of the replacement. More importantly, we
found that compound 18 showed the best cytotoxic activity in four
cell lines with IC50 less than 10 lM (Table 1). From the structure of
compound 18, it is possible that the substitution of glucoside by
azotic alkyl group and the piperidine circle may play an important
role in this effect, when compared to compound 17. To further
determine the cytotoxic mechanism of compound 18, we chose
reactive oxygen species (ROS) inhibitor: L-NAC to pre-treat the
MGC-803 cells before compound 18 was added, based on our
hypothesis of its oxidative cell death effect. To our surprise, L-
NAC could not significant impair the effect of compound 18, indi-
cating that ROS may not play a role in this compound’s cytotoxicity
(Fig. 4).
In summary, in the current study, two benzophenone glucopyr-
anosides, Mahkosides A and B were isolated from the nut shell of
Mahkota Dewa fruit. Using Mahkoside A as a leading compound,
16 derivatives, including alkylation, acylation and etherification
substitutions, were synthesized. Among them, 14 were reported,
for the first time. The involved cytotoxic activities were also eval-
uated. Importantly, we found that compound 18 demonstrates
significant cytotoxicity against four different kinds of cancer cells.
Future studies will be performed to generate and characterize
more derivatives with azotic alkyl group and analyze their struc-
ture-effect relationships, based on the structure of compound 18.
Figure 4. Reactive oxygen species (ROS) inhibitor L-NAC could not block the
cytotoxic effect of compound 18. MGC-803 cells were cultured and treated with or
without 5 mM ROS scavenger L-NAC for 2 h before adding compound 18. SPSS16.0
software was used to analyze the data and calculate the index of 50% cell death
(IC50).
EC9706, MGC-803 and PC-3 cell lines, purchased from ‘Cell Bank,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences’, were cultured in DMEM, containing 10% FBS and 1% pen-
icillin and streptomycin. At the stage of logarithm, cells were
washed by PBS and digested with trypsin, then inoculated in 96-
well plates by 5000 cells/well. All the compounds used in this
study were dissolved in DMSO and made to 105
lM/L stock solu-
tions, according to their molecular weights. The final working solu-
tions were acquired by 1000 times dilution of the stock with
culture medium, less than 0.1% DMSO was included in each well.
The control cells were treated with the same conditions without
Acknowledgment
This work was supported by National Natural and Science
Foundation of China (81072539 and 81273393 by Zhang Yan-Bing)
and (81270270 by Wen Zhao).
compounds. After the cells were plated, 200
tions with final concentrations of 100, 50, 25, 12.5, 6.25, 3.125
and 1.5625 M for each compound were added to each well and
ll compound solu-
l
Supplementary data
the cells were incubated for another 72 h. 50% trichloroacetic acid
(TCA) were then added to each well to fix the cells for 10 min at
room temperature (RT) and 1 h at 4 °C. The plates were then
washed five times in distilled water and the cells were allowed
to dry in the air. After 0.4% (m/v) SRB (Sulforhodamine B) was
added, the cells were incubated at RT for 10 min. Then the SRB
solution was changed by quickly washing the plates with 1% v/v
acetic acid five times to remove unbound dye. The plates were then
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.038.
References and notes
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2. Yan-Bing, Zhang; Xiang-Jun, Xua; Hong-Min, Liu J. Asian Nat. Prod. Res. 2006, 8,
119.
dried in the air. To solubilize the bound SRB, 150 ll of 10 mM/L
unbuffered Tris Base (pH 10.5) was added to each well and the
plates were shaked for 5 min. The optical density (OD) of SRB in
each well is directly proportional to the cell number so the OD val-
ues can be plotted against concentration, with the working wave-
length 515 nm. SPSS16.0 software was used to analyze the data
and calculate the index of 50% cell death (IC50).
Interestingly, we found that some of the derivatives showed
good inhibition, especially the alkylation derivatives with more
than three saturated hydrocarbon lateral chains (compounds 6
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