Synthesis and synergetic anti-tumor activity evaluation of dihydroartemisinin-organogermanium(IV) compound

Article abstract of DOI:10.1016/j.bmcl.2014.09.048

Dihydroartemisinin (DHA), a semi-synthetic derivative of the herb artemisinin, has shown commendable bioactivity. In this paper, a novel dihydroartemisinin-organogermanium (DHA-Ge) compound was synthesized, characterized and its potential anti-tumor activity was evaluated by various methods. MTT results demonstrated that DHA-Ge could effectively inhibit the proliferation of HepG2 cells and showed their dose-dependent properties. The IC₅₀ value of inhibition effect on HepG2 cells of DHA-Ge was 10.23 μg/ml which was lower than 39.44 μg/ml of DHA. Flow cytometric results suggested that DHA-Ge could induce apoptosis of HepG2 cells and the apoptosis rate was 20.26% after 24 h treatment with 56.8 μg/ml DHA-Ge concentration. Atomic force microscopy images showed that HepG2 cells were collapsed and the cell nucleus were fragmented after 24 h treatment. All these results together showed that the DHA-Ge possessed desirable synergetic enhanced anti-tumor effects and could be developed as a suitable tumor therapeutic agent.

Full text of DOI:10.1016/j.bmcl.2014.09.048

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and synergetic anti-tumor activity evaluation
of dihydroartemisinin-organogermanium(IV) compound
⇑
Peng Lu, Shuguang Yao, Jiye Cai, Pei-hui Yang
Department of Chemistry, Jinan University, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Dihydroartemisinin (DHA), a semi-synthetic derivative of the herb artemisinin, has shown commendable
bioactivity. In this paper, a novel dihydroartemisinin-organogermanium (DHA-Ge) compound was
synthesized, characterized and its potential anti-tumor activity was evaluated by various methods.
MTT results demonstrated that DHA-Ge could effectively inhibit the proliferation of HepG2 cells and
showed their dose-dependent properties. The IC50 value of inhibition effect on HepG2 cells of DHA-Ge
Received 19 May 2014
Revised 28 August 2014
Accepted 17 September 2014
Available online xxxx
was 10.23
DHA-Ge could induce apoptosis of HepG2 cells and the apoptosis rate was 20.26% after 24 h treatment
with 56.8 g/ml DHA-Ge concentration. Atomic force microscopy images showed that HepG2 cells were
collapsed and the cell nucleus were fragmented after 24 h treatment. All these results together showed
that the DHA-Ge possessed desirable synergetic enhanced anti-tumor effects and could be developed as a
suitable tumor therapeutic agent.
lg/ml which was lower than 39.44 lg/ml of DHA. Flow cytometric results suggested that
Keywords:
Dihydroartemisinin
Ge-132
Organogermanium
Synergetic anti-tumor activity
l
Ó 2014 Elsevier Ltd. All rights reserved.
Germanium (Ge) is an active ingredient of many Chinese med-
icines, such as ginseng, which plays a significant role in the phar-
macological effects of the plants.1,2 Germanium was used as
dietary supplements for cancer and AIDS, but it is reported that
many germanium compounds have potent toxicity.3,4 Studies
showed that organic germanium compounds not only had much
lower toxicity, but also possessed immunomodulatory activity,
anti-oxidation, antibacterial and anti-tumor activity.5–9 Since Ge-
132 (carboxyethylgermanium sesquioxide) was found.10 Great
efforts have been made to investigate organic germanium com-
pounds for low-toxic and effective antitumor drugs,11–17 but most
of these achievements fell short of the desired results or prema-
turely terminated research on clinical uses.18,19 Accordingly, drugs
with better antitumor effects are required to fight against cancer
cells. It was found that some of Ge-132 derivatives showed stron-
ger anticancer activities than Ge-132,20,21 therefore selecting an
appropriate modify molecule offers a thrill route to devise anti-
tumor reagent with enhanced activity.
artemisinin.24 Recently, it was proved that DHA inhibited cell
proliferation and induced apoptosis in various tumor cells.25–29
variety of classes of DHA derivatives had been made and their
effect towards diverse tumor cell lines was compared with that
of artemisinin and anti-tumor drugs.25,29
A
In order to develop an anti-tumor reagent with enhanced bioac-
tivity and take advantage of the synergetic effects of Ge-132 and
DHA,
a dihydroartemisinin-organogermanium (DHA-Ge) com-
pound was prepared. The scheme of synthesis route is showed in
Figure 1. ESI-MS, 1H NMR, Cyclic voltammetry, FT-IR and elemental
analysis were performed to characterize the synthesized com-
pound. The ESI-MS information in Supplementary data (Fig. S1)
showed the fragment ions in agreement with the expected struc-
ture of DHA-Ge compound. The disappearance of the signal at
3.70 ppm originated from HO- of DHA in the 1H NMR (Fig. S2) dem-
onstrated that the DHA and the Ge-132 were connected. In the FT-
IR spectra of DHA-Ge compound (Fig. S3), there is a peak at
1739 cmÀ1 attributed to the stretching vibration of C@O, which
further indicated the binding of DHA with Ge-132. While the peak
at 460 cmÀ1 corresponded to the stretching vibration of Ge-C30
indicated the existence of Ge-132. Furthermore, the cyclic
voltammogram (Fig. S4) showed the endoperoxide-bridge of
DHA in DHA-Ge molecule retained intact during the synthesis.
The elementary analysis data (Table S1) showed that the experi-
mental results of the DHA-Ge compound were in accord with the
theoretical ones. All of these results demonstrated the successful
preparation of DHA-Ge compound.
As traditional Chinese herbal, artemisinin has been applied to
treat fever for many centuries22 and used to synthesize some
modern anti-malarial drugs.23 But its application was limited due
to poor solubility. Dihydroartemisinin (DHA) is the main active
derivative of artemisinin which possess a better bioactivity than
⇑
Corresponding author. Tel./fax: +86 20 85223569.
E-mail address: typh@jnu.edu.cn (P.-h. Yang).
http://dx.doi.org/10.1016/j.bmcl.2014.09.048
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lu, P.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.09.048

2
P. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Schematic representation of the synthesis of DHA-Ge. 1-(3-Dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide
(NHS), 4-dimethylaminopyridine (DMAP), dihydroartemisinin (DHA).
Figure 2. Compared inhibitory effects of DHA, DHA-Ge and Ge-132 on HepG2 cells
by MTT assay.
The inhibitory effects on proliferation of HepG2 cells after expo-
sure of DHA-Ge were evaluated by the MTT assay. Ge-132 and DHA
were evaluated as compare subjects. The data (Fig. 2) showed that
the DHA-Ge could effectively inhibit the growth and proliferation
of HepG2 cells. The survival rate of HepG2 cells was decreased with
the increased concentration of DHA-Ge compound, which sug-
gested their concentration-dependent properties. The value of
IC50 of DHA-Ge compound for HepG2 cells was 10.23
lg/ml which
was lower than 39.44 g/ml of DHA, which indicated that the inhi-
l
bition of DHA-Ge compound was more obvious than that of DHA.
These results suggested that the DHA-Ge compound possessed
much higher antitumor activity compared with DHA and Ge-132.
The better antitumor activity derived from DHA-Ge compound
retained the basic structure of DHA and Ge-132 and they were well
functioned, DHA and Ge-132 played a synergetic enhanced effect
in DHA-Ge compound.
Figure 3. DHA-Ge stimulates morphological changes of HepG2 cells (A–E) by
inverted microscope and effects of DHA-Ge on cell apoptosis by flow cytometry
(A1–E1) after 24 h treatment. Concentration of DHA-Ge (A/A1) 0
lg/mL; (B/B1)
7.1 g/mL; (C/C1) 14.2 g/mL; (D/D1) 28.4 g/mL; (E/E1) 56.8 g/mL.
l
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with the increased concentration of DHA-Ge compound after
24 h treatment. HepG2 cells were in a state that these cells were
apoptosis at early phase. The apoptosis of HepG2 cells induced
by DHA-Ge compound after 24 h of exposure was determined by
flow cytometry. The result suggested that apoptosis of HepG2 cells
depended on the concentration of DHA-Ge compound and the rate
Inverted microscope was used to obtain morphology of HepG2
cells which were treated with different concentration of DHA-Ge
compound for 24 h. The cells in the control group showed normal
morphological characteristics and were full in shape and were
evenly distributed (Fig. 3). But cell morphology changed to round
from fusiform and the number of cells in round shape increased
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P. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Figure 4. AFM images of changes in morphology and ultrastructure of HepG2 cells after various concentrations of DHA-Ge treatment for 24 h. Concentration of DHA-Ge: (A)
g/mL; (B) 3.5 g/mL (C) 7.1 g/mL; (D) 14.2 g/mL; (E) 28.4 g/mL; (F) 56.8 g/mL) (A1–F1) 3D images of cells (50 m); (A2–F2) ultrastructure images
m  50
m  3 m) on corresponding cell region indicated by blue frame; (A3–F3) histograms of cell membrane particle size distributions of ultrastructure.
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of apoptosis was raised as the increased concentration of DHA-Ge
compound. When the concentration of DHA-Ge compound was
56.8
lg/ml, the apoptosis rate was 20.26% after 24 h treatment,
but in the control group, it was 2.77%.
Please cite this article in press as: Lu, P.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.09.048

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P. Lu et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
The atomic force microscope (AFM) is not only a tool to image
Supplementary data
the morphology and mechanical property changes of cells exposed
to drugs, it can also be used to provide valuable information about
ultrastructure.31–33 Ultrastructure analysis indicated changes of
roughness and size distribution of cell surface particle induced
by drugs.34,35 Therefore, AFM was employed to measure changes
of cell morphology, cell membrane ultra-structure and mechanical
property of HepG2 cells treated with different concentration of
DHA-Ge compound for 24 h. As shown in Figure 4, HepG2 cells in
control group were fusiform and cell surface was smooth and pseu-
dopodium grew around the cell (Fig. 4A1–A3). After co-cultured
with DHA-Ge compound for 24 h, morphology of HepG2 cells
changed into round gradually along with the increase of concentra-
tion of DHA-Ge compound. There were gradual shrinkage in cell
shape and fragmentation in cell nucleus for high concentration
group, which were typical characteristics of cell apoptosis. More-
over, ultra-structure analysis showed as the increase of concentra-
tion of DHA-Ge compound, cell surface became rougher and the
average size of surface particle on cells increased, which resulted
from the drug-induced cell apoptosis (Fig. 4A3–F3). Mechanical
properties of cells such as Young’s modulus and Adhesion force
are regulators of physiological processes.36–38 Here, AFM force
spectroscopy was employed to extract Young’s modulus from
mechanical properties. Figure S5 in Supplementary data showed
elasticity histograms recorded on top of the cell membrane and
the average Young’s modulus decreased as the increase of concen-
tration of DHA-Ge compound. After treatment with DHA, cell apop-
tosis was identified with differences in cell morphology, cell
membrane ultrastructure and mechanical property.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.
048.
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In summary, a novel DHA-Ge compound was synthesized by
coupling highly bioactive DHA and Ge-132. Our results showed
that the enhanced anti-tumor activity of DHA-Ge could be attrib-
uted to the synergetic effects of DHA and Ge-132. It may be devel-
oped to be a promising anti-tumor reagent.
Acknowledgments
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This work was supported by the National Natural Science
Foundation of China (No. 21375048), the Major State Basic
Research Development Program of China (973 Program) (No.
2010CB833603).
Please cite this article in press as: Lu, P.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.09.048