Synthesis and anti-glioblastoma effects of artemisinin-isothiocyanate derivatives

Article abstract of DOI:10.1039/C8RA08162J

A series of novel artemisinin (ART) derivatives containing an isothiocyanate (ITC) group were synthesized. All the compounds showed more potent anti-tumor effects than those of parent dihydroartemisinin (DHA) towards glioblastoma multiforme U87 in vitro. Among them, 5b had the strongest cytotoxic activity which exerted its effects in a concentration-dependent but not time-dependent manner (IC₅₀ 7.41 μM for 24 h, 7.35 μM for 72 h). Pyknosis was observed in 5b-treated U87 cells. Multiple intrinsic apoptotic pathways were induced by 5b including the upregulation of caspase 9, the release of cytochrome c, an increase of the proapoptotic protein Bax, a decrease of the anti-apoptotic protein Bcl 2, and the activation of execution pathways by the upregulation of caspase 3. In addition to apoptosis, an autophagic mechanism was also involved in 5b-induced cytotoxicity to human GBM U87 cells by upregulating the expression of LC3-II and downregulating p62. Furthermore, 5b also significantly attenuated the migration of U87 cells. Therefore, our results suggest that 5b may be a promising molecule for the further development of a novel drug for the treatment of glioblastoma.

Full text of DOI:10.1039/C8RA08162J

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PAPER
Synthesis and anti-glioblastoma effects of
artemisinin-isothiocyanate derivatives†
Cite this: RSC Adv., 2018, 8, 40974
Chan Myae Nyein,‡abc Xiaolin Zhong,‡a Junfeng Lu,b Huijuan Luo,a Jiamin Wang,a
ef
d
g
a
Simona Rapposelli,
Mingtao Li, Ying Ou-yang, Rongbiao Pi bd and Xixin He
*
*
A series of novel artemisinin (ART) derivatives containing an isothiocyanate (ITC) group were synthesized. All
the compounds showed more potent anti-tumor effects than those of parent dihydroartemisinin (DHA)
towards glioblastoma multiforme U87 in vitro. Among them, 5b had the strongest cytotoxic activity
which exerted its effects in a concentration-dependent but not time-dependent manner (IC50 7.41 mM
for 24 h, 7.35 mM for 72 h). Pyknosis was observed in 5b-treated U87 cells. Multiple intrinsic apoptotic
pathways were induced by 5b including the upregulation of caspase 9, the release of cytochrome c, an
increase of the proapoptotic protein Bax, a decrease of the anti-apoptotic protein Bcl 2, and the
activation of execution pathways by the upregulation of caspase 3. In addition to apoptosis, an
autophagic mechanism was also involved in 5b-induced cytotoxicity to human GBM U87 cells by
upregulating the expression of LC3-II and downregulating p62. Furthermore, 5b also significantly
attenuated the migration of U87 cells. Therefore, our results suggest that 5b may be a promising
molecule for the further development of a novel drug for the treatment of glioblastoma.
Received 2nd October 2018
Accepted 19th November 2018
DOI: 10.1039/c8ra08162j
rsc.li/rsc-advances
activities; they are still the rst-line drug for malarial treatment.
ARTs have not only antimalarial property but also anticancer
effects.4,5 It has been conrmed that dihydroartemisinin (DHA),
Introduction
Glioblastoma multiforme (GBM) is the most prevalent and
severe primary central nervous system tumor. Even when using
conventional treatments such as surgery, radiation therapy and
chemical treatment, the average survival time is less than 15
months.1 The average occurrence rate of GBM is 3.19/100 000
population, and the median age of diagnosis is 64 years old. The
occurrence is higher in men than in women.2 Therefore, the
discovery of effective therapy for GBM is an area of interest.
Artemisinin (ART) and its derivatives (collectively termed as
artemisinins (ARTs)) are sesquiterpene lactones isolated from
sweet wormwood (Artemisia annua), which are used as a remedy
for fevers and chills in Chinese Traditional Medicine.3 The
endoperoxide bridge of ARTs is very important for their
the active metabolite of ARTs, showed cytotoxic activity on ten
human glioma cell lines through the induction of autophagy.6
DHA also exerted an anti-tumor role through induced ROS-
mediated mitochondrion and ER stress apoptotic pathways as
well as autophagic cell death in human GBM cells.7 Although
ART and its derivatives are extremely effective in their antima-
larial role, they are less potent as anticancer agents, especially
as monotherapy drugs because of their mild activity and short
elimination half-life.8 Therefore, to improve the clinical
outcome of ARTs, future efforts should be focused on the
combination of longer-acting drugs for maximum efficacy or the
development of potent derivatives.9–11
Naturally occurring isothiocyanates (ITCs), which are
generated by the enzymatic hydrolysis of glucosinolates, are
abundant in cruciferous vegetables (Fig. 1).12–14 It has been
proven that some natural and synthetic ITCs have anticancer
activities with multiple targets and mechanisms. ITCs can
enhance the antitumor efficacy of other drugs, or act as covalent
inhibitors.15 ITCs alone are able to suppress the growth of
various cancer cell lines such as non–small-cell lung cancer
cells, human malignant astrocytoma cells, and HCT 116 human
colon cancer cells.16–20 They exert their effects mainly by
increasing the production of reactive oxygen species (ROS),
induction of apoptosis, induction of cell cycle arrest and so
on.18–23 However, the exact mechanisms of ITCs against tumors
are still not clear. Some research uncovered that aryl
aSchool of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine,
Guangzhou 510006, China. E-mail: mark07@gzucm.edu.cn
bSchool of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006,
China. E-mail: pirb@mail.sysu.edu.cn
cBiotechnology Research Department, Ministry of Education, Kyauk-se, Myanmar
dGuangdong Provincial Key Laboratory of Brain Function and Disease, Zhongshan
School of Medicine, Sun Yat-Sen University, Guangzhou, China
eDepartment of Pharmacy, University of Pisa, Via Bonanno, 6, 56126 Pisa, Italy
fInterdepartmental Research Center for Biology and Pathology of Aging, University of
Pisa, Via Bonanno, 6, 56126 Pisa, Italy
gDepartment of Pediatrics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University,
Guangzhou 510120, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra08162j
‡ These authors contributed equally to this work.
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Fig. 1 The representatives of naturally occurring isothiocyanates.
isothiocyanates (e.g., benzyl isothiocyanate (BITC), phenyl iso- bromo-substituted alkanol in CH2Cl2 by using boron triuoride
thiocyanate (PITC)) and allyl isothiocyanates (AITC) (Fig. 1) act ethyl ether (BF3$Et2O) as a catalyst afforded substituted ART
as H2S donor agents.24,25 H2S as the third most important enantiomers. 1a–c were obtained by silica gel column chroma-
gasotransmitter is involved in the development of cancer by tography elution with petroleum ether/ethyl acetate (97 : 3) as
shortening the cell cycle and inducing proliferation.26,27
the b conguration (indicated the coupling constants between
Based on those studies, we hypothesized that artemisinin- 9-H and 10-H in 1H NMR, J9,10 ¼ 3–4 Hz).30 1a–c were then
based isothiocyanates might be more efficacious compounds converted to 4a–c via an azidation and reduction reaction.
towards human GBM cells with multiple mechanisms. In this Finally, the intermediates 4a–c bearing an amino group reacted
study, the synthesis, bio-evaluation and mechanism of with carbon disulde (CS2) in dried THF31 to afford the target
artemisinin-based isothiocyanates against GBM in vitro have compounds 5a–c. All the synthesized compounds were puried
been carried out, and we found a novel DHA-ITC derivative 5b to by silica gel column chromatography.
have potent GBM cell toxicity.
The structures of compounds 5a–c were elucidated on the
basis of 1H NMR, 13C NMR and HR-ESI-MS spectra. In a typical
example, the HR-ESI-MS of compound 5b displayed a peak at m/
z 384.1802, corresponding to the quasimolecular ions [M + H]+,
conrming the molecular formula of compound 5b as
Results and discussion
Chemistry
C
19H29NO5S. In the 1H-NMR spectrum of 5b, the signals of one
The synthetic strategy employed for the synthesis of the target
compounds is depicted in Scheme 1. A series of novel hybrids
were afforded by linking DHA at C-10 and an isothiocyanate
group. The detailed synthetic route was described in Scheme 2.
The DHA derivatives 4a–c were synthesized according to the
literature.28,29 The reaction of DHA with the corresponding
singlet at d 1.42 and two doublets at d 0.89 and 0.94 could be
attributed to Me-14, Me-16 and Me-15, respectively. The doublet
peak appearing at d 4.78 (d, J ¼ 3.2 Hz) indicated H-10 in the
a conguration.30 In the 13C-NMR spectrum, there were 19
peaks appearing in the range from d 12 to 130 ppm. Among
them, a weak peak appearing at d 129.5 could be ascribed to the
carbon of the isothiocyanate (–N]C]S). The other NMR
characterizations have been assigned through the comparison
of relative data in literature.32,33
Biological evaluation
Artemisinin-based isothiocyanate derivatives induced cyto-
toxicity in glioblastoma U87 cells. The cytotoxicity of
artemisinin-based isothiocyanate derivatives on human glio-
blastoma U87 cells was rstly tested by MTT assay. U87 cells
were exposed to various concentrations of compounds for 24 h,
and all compounds could induce cell death by MTT assay (Table
1). Cells shrunk and fragmented when exposed to compounds
under the phase-contrast microscope (Fig. 2). Among all the
compounds, 5b showed the most cytotoxic activity with an
IC50value of 7.41 mM and was thus selected for further experi-
ments. As shown in Fig. 3, 5b reduced the viability of GBM cells
in a concentration-dependent manner. To evaluate the effects of
5b on the proliferative cancer cell line, U87 glioblastoma cells
were exposed to various concentrations of 5b for different
durations. 5b signicantly suppressed the survival of U87 cells
Scheme 1 The design strategy.
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Scheme 2 The synthetic route of artemisinin-isothiocyanate derivatives 5a–c. Reagents and conditions:aNaBH4/MeOH, 0 ꢀC, 2 h, 94%;
bBF3$Et2O/CH2Cl2, bromo-substituted alkanol; cNaN3/NaI/DMF, 60 ꢀC, 4 h, 85–98%; d(1) (Ph)3P/THF, 60 ꢀC, 2 h; (2) H2O, 70 ꢀC, 2 h, 65–91%;
eCS2/THF/Et3N, 0 ꢀC-r.t, 30 min, then AcCl, 0 ꢀC-r.t, 30 min, 49–76%.
and the inhibitory efficiency was stronger than that of DHA (see a well-established test of cancer cell migration. To investigate
Table 1 and Fig. 3).
the effects of 5b on wound-healing phenomena, cells were
Artemisinin-based isothiocyanate derivative 5b inhibits cell exposed to 4 mM (around half of the IC50) of 5b for 0–72 h.35 The
migration. Cell migration is a landmark of cancer invasion and migration of U87 cells was reduced signicantly by 5b (Fig. 4).
metastasis, immune responses, angiogenesis, wound repair,
Artemisinin-based isothiocyanate derivative 5b induces
and embryonic morphogenesis.34 Wound-healing assay is apoptosis in U87 cells. To determine the cell death pathway, the
U87 cells were treated with two doses (8 and 16 mM) of 5b, which
were selected to determine the apoptotic effects of 5b in
Table 1 IC50 values (mM) against human U87 cells
a concentration-dependent manner. Aer 16 h of incubation,
apoptotic markers of treated cells were analyzed with uores-
cein isothiocyanate (FITC)-conjugated annexin V and PI.
Annexin
V detects phosphatidylserine (PS) on the cell
membrane during apoptosis. Propidium iodide (PI) stains the
DNA of apoptotic and necrotic cells. As shown in Fig. 5A, 5b
induced a higher proportion of early apoptotic and necrotic
cells. The nuclear morphological changes were examined by
DAPI staining. Pyknosis and nuclear shrinkage were observed in
5b-treated cells (Fig. 5B).
To support the fact that apoptosis was involved in the cyto-
toxic effects of 5b on GBM cells, we further investigated the
caspase family apoptotic proteins and the Bcl2 family apoptotic
proteins. As there are three classical apoptotic pathways, we
investigated intrinsic and execution pathways. As shown in
Fig. 6, the caspase family apoptotic proteins were induced in
U87 cells treated with various concentrations of 5b (4–16 mM)
and DHA (120 mM) for 16 h. Caspase 9 and cytochrome c were
induced in U87 cells treated with 5b. It was assumed that
a
Compounds
n
R
IC50
4b
4c
5a
5b
1
4
0
1
4
–NH2
–NH2
–N]C]S
–N]C]S
–N]C]S
>120
118.26 ꢁ 15.12
27.32 ꢁ 3.65
7.41 ꢁ 1.56
20.29 ꢁ 4.17
118.95 ꢁ 12.39
5c
Dihydroartemisinin (DHA)
a
Aer 24 h incubation.
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Fig. 2 Morphological examination of U87 cells treated with artemisinin-isothiocyanates (5a–c) and the key intermediates (4b, 4c). The cells were
treated with 30 mM of the compounds for 24 h and the morphological changes were examined with phase contrast microscope. Scale bar ¼ 50
mm.
Artemisinin-based isothiocyanate derivative 5b induces
autophagy in U87 cells. Autophagy assures cellular homeostasis
and is gaining increasing importance in cancer treatment,
where it impacts carcinogenesis as well as the propagation of
the malignant phenotype and the development of resistance.38
To observe whether autophagy was also involved in 5b-induced
cytotoxicity, autophagic markers, such as LC3-II, microtubule-
associated proteins 1A/1B light chain 3B, and p62/SQSTM1,
were investigated in U87 cells treated with 5b for 16 h. LC3-II
expression increased in the 5b-treated group compared to the
control groups. Moreover, the decrease of p62 was observed in
the 5b-treated group (Fig. 7).
Fig. 3 5b possesses the cytotoxic activity on human GBM cell lines,
Conclusion
U87. The cells were treated with indicated concentrations of 5b for
24 h. Cell viability was tested via MTT assay and IC50 value was
calculated. 5b reduced the cell viability of U87 cells in a concentration
In conclusion, novel ART derivatives containing an ITC group
have been synthesized. All the compounds showed more potent
dependent manner. All data were represented as mean ꢁ SD from
activity than that of the parent DHA towards glioblastoma
multiforme U87 in vitro. The data in the present study demon-
strated that 5b signicantly induced cytotoxicity (IC50 7.41 mM)
towards human GBM cells through inducing both apoptotic
and autophagic cell death and also inhibited the migration of
human GBM cells. It was a stronger inducer in GBM cells
compared to DHA (about twenty times stronger). Therefore, it
suggested that 5b may be a potential multifunctional small
molecule for developing new drugs for the treatment of GBM.
three independent experiments; ***P < 0.001 versus control.
release of cytochrome c activated the caspase-dependent mito-
chondrial pathway. Cytochrome c binds and activates Apaf1 as
well as procaspase-9, forming an “apoptosome”. The clustering
of procaspase-9 leads to caspase-9 activation.36,37 The anti-
apoptotic protein Bcl-2 was downregulated and pro-apoptotic
protein BAX was upregulated in 5b-treated cells. The Bcl-2
family of proteins governs mitochondrial membrane perme-
ability via the regulation of cytochrome c released from the
mitochondria.36 Caspase 3 is considered to be the most
important of the executioner caspases in apoptotic cell death.
Cleaved (active) caspase 3 was increased in 5b-treated cells
(Fig. 6).
Experimental
Chemistry
Melting points were measured on an XT-4 apparatus (Taike-
1
Corp., Beijing, China) and were uncorrected. H and 13C NMR
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Fig. 4 5b inhibits the migration of human glioblastoma cells, U87. Data were expressed as means ꢁ SD. ***P < 0.001 versus control. All
experiments were performed in triplicates.
spectra were recorded on Bruker 400 MHz, and the data were chromatography elution with petroleum ether/ethyl acetate
recorded using CDCl3 as the solvent. Chemical shis (d) are (97 : 3) (indicated are the coupling constants between 9-H and
1
reported in ppm downeld from an internal TMS standard. HR- 10-H in H NMR, J9,10 ¼ 3–4 Hz).30
ESI-MS spectra were determined on an AB SciexTripleTOF 5600+
The solution of DHA (3.0 mmol) and 3-bromo-1-propanol, or
apparatus. Flash column chromatography on silica gel (200–300 2-bromo-1-ethanol, or 6-bromo-hexanol (4.6 mmol) in CH2Cl2
mesh) was used for the routine purication of the reaction (20 mL) below 0 ꢀC was added to BF3$Et2O (0.63 mL, 5.0 mmol).
products. Analytical TLC was performed on silica gel 60 F254 The mixture was stirred for about 50 min at the same temper-
plates (Merck KGaA, Darmstadt, Germany). All solvents and ature under nitrogen protection and monitored by TLC. At the
chemical reagents were obtained from commercial sources and end of the reaction, the organic layer was collected and washed
used without further purications.
with saturated NaHCO3 solution (1 ꢂ 20 mL), water (1 ꢂ 20 mL)
and brine (1 ꢂ 20 mL), dried over anhydrous Na2SO4 and then
concentrated in vacuo to give the crude product, which was
puried by silica gel column chromatography (ether/ethyl
acetate, 97 : 3,v/v) to afford compounds 1a–c.
1-Bromo-2-(10b-dihydroartemisinoxy)ethane (1a). Colorless
solid; yield: 36%; mp 73–74 ꢀC; 1H NMR (refer to literature 28);
13C NMR (100 MHz, CDCl3) d 104.3(C-3), 102.2(C-10), 88.3(C-12),
General procedure for the synthesis of compounds 1a–c
The DHA derivatives 1a–c were synthesized via the reaction of
DHA with the corresponding bromo-substituted alkanol in
CH2Cl2 using boron triuoride ethyl ether (BF3$Et2O)28,29 and
isolation in the
b conguration by silica gel column
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Fig. 5 Apoptotic analysis of 5b on U87 glioblastoma cells. (A) U87 cells were treated with DMSO or 8 and 16 mM concentrations of 5b for 16 h, and
apoptotic rates were analyzed by flow cytometry after annexin V/PI staining. (B) U87 cells were treated with DMSO or 8 mM of 5b for 16 h and the
nuclear morphological changes were observed using DAPI staining and fluorescence microscope. (C) Data are expressed as mean ꢁ SD of three
independent experiments presented in (A). *P < 0.05 versus control.
81.3(C-12a), 68.4(–OCH2–), 52.8(C-5a), 44.5(C-8a), 37.6(C-6),
36.6(C-4), 34.9(C-7), 31.6(C-9), 31.1(BrCH2–), 26.4(C-14),
24.8(C-5), 24.6(C-8), 20.6(C-15), 13.2(C-16).
1-Bromo-3-(10b-dihydroartemisinoxy)propane (1b). Color-
less solid; yield: 39%; mp: 69–71 ꢀC;1H NMR (refer to literature
29);;13C NMR (100 MHz, CDCl3) d 104.3(C-3), 102.3(C-10),
88.1(C-12), 81.2(C-12a), 65.8(–OCH2–), 52.7(C-5a), 44.5(C-8a),
37.6(C-6), 36.6(C-4), 34.8(C-7), 32.7(C-9), 31.1(BrCH2–),
1-Bromo-6-(10b-dihydroartemisinoxy)hexane (1c). Colorless
oil; yield: 40%; 1H NMR (400 MHz, CDCl3) d 5.35 (s, 1H, H-12),
4.76 (d, J ¼ 3.0 Hz, 1H, H-10), 3.98–3.85 (m, 1H, –OCH2–), 3.46–
3.37 (m, 2H, –CH2Br), 3.34 (t, J ¼ 6.7 Hz, 1H,–OCH2–), 2.68–2.50
(m, 1H, H-9), 2.34 (td, J ¼ 14.1, 3.7 Hz, 1H, H-4), 2.01 (d, J ¼
14.3 Hz, 1H, H-4), 1.66–1.56 (m, 2H), 1.53–1.42 (m, 2H), 1.40 (s,
3H, CH3-14), 1.36–1.26 (m, 1H), 1.25–1.18 (m, 1H), 0.92 (d, J ¼
6.2 Hz, 3H, CH3-15), 0.87 (d, J ¼ 7.2 Hz, 3H, CH3-16), 0.87 (m,
1H, H-7); 13C NMR (100 MHz, CDCl3) d 104.3(C-3), 102.2(C-10),
88.1(C-12), 81.2(C-12a), 68.4(–OCH2–), 52.8(C-5a), 44.7(C-8a),
37.7(C-6), 36.7(C-4), 34.9(C-7), 34.0, 32.9(C-9), 31.1, 29.7, 28.1,
26.4(C-14), 25.7, 24.9(C-5), 24.7(C-8), 20.6(C-15), 13.1(C-16).
30:8ðBrCH2 CH2 ꢃ Þ; 26.4(C-14), 24.8(C-5), 24.7(C-8), 20.5(C-
15), 13.2(C-16).
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Fig. 6 5b induces apoptosis by regulating caspase family proteins and Bcl2 family proteins in human GBM cells. Cells were treated with the
indicated concentrations of 5b and DHA for 16 h. The proteins were collected for western blot assay and b-actin was used as protein loading
control. The result was obtained by three different independent experiments.
General procedure for the synthesis of compounds 3a–c
The intermediate 3a–c was synthesized from 1a–c according to
the procedure previously reported.28,29 To the solution of 1a–c
(0.89 mmol) and NaI (10.0 mg) in DMF (5.0 mL), NaN3 (2.7
mmol) was added. The reaction mixture was heated to 60 ꢀC for
4 h. Aer the disappearance of the starting material, the solu-
tion was poured into ice water (20 mL), stirred for 1 h, and then
extracted with CH2Cl2 (3 ꢂ 20 mL), dried over anhydrous
Na2SO4 and concentrated to give a light yellow oil. The crude
product was further puried by silica gel column chromatog-
raphy (petroleum ether/ethyl acetate, 9 : 1, v/v) to provide 3a–c.
Table 2 Information of antibodies for western blot
Antibody name Manufacturer
Catalog
Dilutions
Primary antibody
Bax
BCL2
Caspase 3
Caspase 9
Cytochrome C
LC3B
ABclonal
ABclonal
Cell signaling technology 9665
Cell signaling technology 9508
Cell signaling technology 11 940
Cell signaling technology 3868
A2211
A0208
1 : 1000
1 : 1000
1 : 1000
1 : 1000
1 : 1000
1 : 1000
1 : 1000
P62
MBL
PM045
Fig. 7 5b induces autophagy in human GBM cells. Cells were treated
with the indicated concentrations of 5b and DHA for 16 h. The proteins
were collected for western blot assay with LC3B, p62 and b-actin
antibodies. The result was obtained by three different independent
experiments.
b-Actin
Thermo Fisher
MA5-11869 1 : 2500
Secondary antibody
Anti-rabbit
Anti-mouse
Beyotime
Beyotime
A0208
A0216
1 : 1000
1 : 1000
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2-(10b-Dihydroartemisinoxy)ethyl azide (3a). Colorless solid;
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6.9 Hz, 2H, –CH2NH2), 2.66–2.52 (m, 1H, H-9), 2.34 (td, J ¼ 14.0,
3.7 Hz, 1H, H-4), 2.01 (m, 1H, H-4), 1.86 (m, 1H),1.79–1.66 (m,
4H), 1.66–1.60 (m, 1H), 1.53–1.42 (m, 2H),1.41 (s, 3H, CH3-14),
1.30 (m, 1H), 1.26–1.16 (m, 2H),0.92 (d, J ¼ 6.1 Hz, 3H, CH3-15),
0.86 (d, J ¼ 7.4 Hz, 3H, CH3-16), 0.86 (m, 1H, H-7); 13C NMR (100
MHz, CDCl3) d 104.3(C-3), 102.3(C-10), 88.1(C-12), 81.3(C-12a),
66.6(–OCH2–), 52.8(C-5a), 44.6(C-8a), 39.9(–CH2NH2), 37.7(C-
yield: 85%; mp: 86–87 C; H NMR (refer to literature 28); 13C
NMR (100 MHz, CDCl3) d 104.34(C-3), 102.59(C-10), 88.14(C-12),
81.22(C-12a), 67.47(–OCH2–), 52.70(C-5a), 51.33(–CH2N3),
44.54(C-8a), 37.57(C-6), 36.56(C-4), 34.75(C-7), 30.93(C-9),
26.32(C-14), 24.84(C-5), 24.56(C-8), 20.53(C-15), 13.10(C-16).
3-(10b-Dihydroartemisinoxy)propyl azide (3b). Colorless
solid; yield: 94%; mp: 80–82 ꢀC; 1H NMR (400 MHz, CDCl3)
d 5.35 (s, 1H, H-12), 4.76 (d, J ¼ 3.1 Hz, 1H, H-10), 3.97–3.84 (m,
1H, –OCH2–), 3.49–3.38 (m, 1H, –OCH2–), 3.34 (t, J ¼ 6.7 Hz, 2H,
–CH2N3), 2.68–2.53 (m, 1H, H-9), 2.34 (td, J ¼ 14.0, 3.8 Hz, 1H,
H-4), 2.00 (m, 1H, H-4), 1.83 (m, 3H), 1.77–1.68 (m, 2H), 1.66–
1.56 (m, 2H), 1.54–1.42 (m, 2H), 1.40 (s, 3H, CH3-14), 1.35–1.24
(m, 1H), 1.24–1.12 (m, 1H), 0.92 (d, J ¼ 6.2 Hz, 3H, CH3-15), 0.87
(d, J ¼ 7.3 Hz, 3H, CH3-16), 0.87 (m, 1H, H-7); 13C NMR (100
MHz, CDCl3) d 104.3(C-3), 102.3(C-10), 88.1(C-12), 81.2(C-12a),
65.2(–OCH2–), 52.7(C-5a), 48.8(–CH2N3), 44.5(C-8a), 37.6(C-6),
1
ꢀ
6), 36.6(C-4), 34.8(C-7), 33:8ð ꢃ CH2 CH2NH2Þ; 31.1(C-9),
26.4(C-14), 24.9(C-5), 24.7(C-8), 20.6(C-15), 13.2(C-16).
6-(10b-Dihydroartemisinoxy)hexylamine (4c). Colorless oil;
yield: 86%; HR-ESI-MS (m/z) [M + H]+ calcd for: 384.2750, found:
1
384.2711; H NMR (400 MHz, CDCl3) d 5.34 (s, 1H, H-12), 4.73
(d, J ¼ 3.0 Hz, 1H, H-10), 3.78 (dt, J ¼ 15.9, 6.6 Hz, 1H, –OCH2–),
3.41–3.24 (dt, J ¼ 15.9, 6.6 Hz, 1H, –OCH2–), 2.64 (t, J ¼ 7.0 Hz,
2H, –CH2NH2), 2.59–2.51 (m, 1H, H-9), 2.32 (td, J ¼ 14.0, 3.7 Hz,
1H, H-4), 1.99 (m, 1H, H-4), 1.91–1.64 (m, 7H), 1.64–1.41 (m,
7H),1.40 (s, 3H,CH3-14), 1.38 (m, 1H), 1.29 (m, 6H), 1.24–1.11
(m, 2H), 0.91 (d, J ¼ 6.2 Hz, 3H, CH3-15), 0.88 (m, 1H, H-7),0.85
(d, J ¼ 7.2 Hz, 3H, CH3-16); 13C NMR (100 MHz, CDCl3)
d 104.2(C-3), 102.1(C-10), 88.1(C-12), 81.3(C-12a), 68.5(–OCH2–),
52.7(C-5a), 44.7(C-8a), 41.8(–CH2NH2), 37.7(C-6), 36.6(C-4),
36.6(C-4), 34.8(C-7), 31.1(C-9), 29:3ðN3CH2 CH2 ꢃ Þ; 26.8(C-
14), 24.8(C-5), 24.7(C-8), 20.5(C-15), 13.2(C-16).
6-(10b-Dihydroartemisinoxy)hexyl azide (3c). Colorless oil;
yield: 98%; 1H NMR (400 MHz, CDCl3) d 5.36 (s, 1H, H-12), 4.75
(d, J ¼ 3.0 Hz, 1H, H-10), 3.89–3.74 (m, 1H, –OCH2–), 3.40–3.30
(m, 1H, –OCH2–), 3.23 (t, J ¼ 6.5 Hz, 2H, –CH2N3), 2.59 (m,
1H,H-9), 2.34 (t, J ¼ 13.9 Hz, 1H, H-4), 2.00 (m, 1H, H-4), 1.80 (m,
3H), 1.51 (m, 5H), 1.41 (s, 3H, CH3-14), 1.38 (m, 7H), 1.26–1.12
34.8(C-7), 32:9ð ꢃ CH2 CH2NH2Þ; 31.1(C-9), 29.8, 26.8, 26.4(C-
14), 26.3, 24.9(C-5), 24.6(C-8), 20.6(C-15), 13.2(C-16).
(m, 3H),0.93 (d, J ¼ 6.0 Hz, 3H, CH3-15), 0.87 (d, J ¼ 6.6 Hz, 3H, General procedure for the synthesis of compounds 5a–c
CH3-16), 0.85–0.78 (m, 1H, H-7); 13C NMR (100 MHz, CDCl3)
The desired compounds 5a–c were prepared according to the
d 104.3(C-3), 102.2(C-10), 88.1(C-12), 81.3(C-12a), 68.4(–OCH2–),
literature. CS2 (47 mL, 0.77 mmol) was added dropwise to
52.8(C-5a), 51.6(–CH2N3), 44.7(C-8a), 37.7(C-6), 36.7(C-4),
a solution of compounds 4a–c (0.64 mmol) and Et3N (0.24 mL,
34.9(C-7), 31.1(C-9), 29.7, 29.0, 26.7, 26.4(C-14), 26.1, 24.9(C-5),
1.9 mmol) in anhydrous THF (5.0 mL) in an ice bath. Then, the
24.7(C-8), 20.6(C-15), 13.2(C-16).
solution was stirred for 30 min at r.t. and AcCl (54 mL, 0.77
mmol) was added into the solution at 0 C. Aer 5 min at the
same temperature, it was warmed to r.t and stirred for 15–
ꢀ
General procedure for the synthesis of compounds 4a–c
The key intermediates 4a–c were prepared by the reduction of 30 min. Then, the reaction was quenched with 2.0 mL of 5%
3a–c according to literature.28,29 Triphenylphosphine (0.85 HCl (aq.) and extracted with ethyl acetate three times (3 ꢂ 10
mmol) was added to the solution of 3a–c (0.71 mmol) in THF mL). The organic layers were combined, dried over anhydrous
(5.0 mL). The solution was stirred at 55 ꢀC for 2 h. Then, 1.0 mL Na2SO4, and concentrated to afford the crude product, which
of water was added, and stirred at room temperature for about was puried by silica gel column chromatography (petroleum
5 h. Aer removal of the solvent, 20 mL of water was added and ether/ethyl acetate 97 : 3, v/v) to provide 5a–c as a colorless oil.
then extracted with dichloromethane (3 ꢂ 20 mL). The organic
2-(10b-Dihydroartemisinoxy)ethyl
isothiocyanate
(5a).
layers were combined, dried over anhydrous Na2SO4, and Colorless oil; yield: 49%; HR-ESI-MS (m/z) [M + H]+ calcd for:
concentrated to afford the crude product, which was further 370.1688, found: 370.1643; 1H NMR (400 MHz, CDCl3) d 5.43 (s,
puried by silica gel column chromatography (methanol/ 1H, H-12), 4.81 (d, J ¼ 2.9 Hz, 1H, H-10), 4.00 (dt, J ¼ 10.3,
dichloromethane, 5 : 95, v/v) to provide a colorless oil.
5.1 Hz, 1H,–OCH2–), 3.65 (t, J ¼ 5.1 Hz, 2H, –CH2NCS), 3.56 (dt, J
2-(10b-Dihydroartemisinoxy)ethylamine (4a). Colorless oil; ¼ 9.7, 4.7 Hz, 1H, –OCH2–), 2.69–2.57 (m, 1H, H-9), 2.33 (td, J ¼
yield: 65%; HR-ESI-MS (m/z) [M + H]+ calcd for: 328.2124, found: 14.1, 3.7 Hz, 1H, H-4), 2.00 (d, J ¼ 14.5 Hz, 1H, H-4), 1.90–1.79
328.2094;1H NMR (refer to literature 28); 13C NMR (100 MHz, (m, 1H), 1.75 (m, 2H), 1.64 (m, 1H), 1.45 (m, 3H), 1.40 (s, 3H,
CDCl3) d 104.34(C-3), 102.39(C-10), 88.11(C-12), 81.27(C-12a), CH3-14), 1.28–1.14 (m, 2H), 0.91 (d, J ¼ 6.4 Hz, 3H,CH3-15), 0.91
71.08(–OCH2–), 52.76(C-5a), 44.60(C-8a), 42.26(–CH2NH2), (d, J ¼ 6.4 Hz, 3H,CH3-16), 0.88–0.79 (m, 1H, H-7); 13C NMR (100
37.69(C-6), 36.62(C-4), 34.81(C-7), 31.15(C-9), 26.40(C-14), MHz, CDCl3) d 128.99(–N]C]S), 104.35(C-3), 102.57(C-10),
24.88(C-5), 24.82(C-8), 20.55(C-15), 13.27(C-16).
88.24(C-12), 81.21(C-12a), 66.65(–OCH2–), 52.68(C-5a), 45.65(–
3-(10b-Dihydroartemisinoxy)propylamine (4b). Colorless oil; CH2NCS), 44.45(C-8a), 37.42(C-6), 36.55(C-4), 34.74(C-7),
yield: 91%; HR-ESI-MS (m/z) [M + H]+ calcd for: 342.2280, found: 30.88(C-9), 26.30(C-14), 24.82(C-5), 24.64(C-8), 20.50(C-15),
1
342.2245; H NMR (400 MHz, CDCl3) d 5.36 (s, 1H, H-12), 4.76 13.16(C-16).
(d, J ¼ 3.1 Hz, 1H, H-10), 3.96–3.84 (dt, J ¼ 10.9, 6.0 Hz, 1H,
3-(10b-Dihydroartemisinoxy)propyl isothiocyanate (5b).
–OCH2–), 3.42 (dt, J ¼ 10.9, 6.0 Hz, 1H, –OCH2–), 2.77 (t, J ¼ Colorless oil; yield: 53%; HR-ESI-MS (m/z) [M + H]+ calcd for:
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384.1845, found: 384.1802; 1H NMR (400 MHz, CDCl3) d 5.36 (s, conuence, a scratch in the cell monolayer was made using
1H, H-12), 4.78 (d, J ¼ 3.2 Hz, 1H, H-10), 4.02–3.88 (dt, J ¼ 9.5, a sterilized pipette tip (10 mL). Thereaer, all wound-healing
6.4 Hz, 1H, –OCH2–), 3.60 (t, J ¼ 6.6 Hz, 2H, –CH2NCS), 3.53– processes were performed in serum-free conditions. Aer
3.41 (dt, J ¼ 9.5, 6.4 Hz, 1H,–OCH2–), 2.70–2.57 (m, 1H,H-9), washing with 0.01 M phosphate-buffered saline (PBS, pH 7.4),
2.35 (td, J ¼ 14.1, 3.8 Hz, 1H, H-4), 2.01 (m, 1H, H-4), 1.97– the plates were incubated with 5b or with DMSO, and images
1.91 (m, 2H), 1.87 (m, 1H), 1.81–1.67 (m, 2H), 1.63 (m, 2H), 1.49 were captured at 0, 24, 48 and 72 h using an EVOS® Digital
(m, 2H), 1.42 (s, 3H, CH3-14),0.94 (d, J ¼ 6.3 Hz, 3H,CH3-15), microscope. Cell death would affect the wound healing results.
0.89 (d, J ¼ 7.3 Hz, 3H, CH3-16), 0.87–0.80 (m, 1H, H-7); 13C Thus, to avoid too much cell death, half of the IC50 (4 mM) of 5b
NMR (100 MHz, CDCl3) d 129.5(–N]C]S), 104.4(C-3), 102.4(C- was used. The distance of wound healing was calculated with
10), 88.1(C-12), 81.2(C-12a), 64.8(–OCH2–), 52.8(C-5a), 44.5(C- the MRI wound healing tool of the Image J soware (National
8a), 42.5(–CH2NCS), 37.7(C-6), 36.6(C-4), 34.8(C-7), 31.0(C-9), Institutes of Health, MD, USA).
30:3ð ꢃ CH2 CH2NCSÞ 26.4(C-14), 24.9(C-5), 24.8(C-8), 20.5(C-
DAPI staining
15), 13.2(C-16).
Morphological changes of the nuclear chromatin of apoptotic
6-(10b-Dihydroartemisinoxy)hexyl isothiocyanate (5c). Color-
cells were identied by staining with the DNA binding dye 40,6-
diamidino-2-phenylindole (DAPI, cat. no. D8417, Sigma
Aldrich). Cells were grown in 12-well plates at a density of 1 ꢂ
105 cells per well followed by the desired treatment. Aer 16 h of
incubation, the cells were washed with cold PBS, xed with 4%
paraformaldehyde for 30 min at room temperature, rewashed
and then stained with DAPI solution (1 : 1000in PBS) at 37 ꢀC for
5 min. Aer removing the staining solution, apoptotic cells were
visualized using a uorescence microscope.
less oil; yield: 76%; HR-ESI-MS (m/z) [M + H]+ calcd for: 426.2314,
found: 426.2302; 1H NMR (400 MHz, CDCl3) d 5.35 (s, 1H, H-12),
4.74 (d, J ¼ 3.2 Hz, 1H, H-10), 3.80 (dt, J ¼ 9.5, 6.4 Hz, 1H, –OCH2–
), 3.48 (t, J ¼ 6.6 Hz, 2H, –CH2NCS), 3.33 (dt, J ¼ 9.5, 6.4 Hz, 1H,
–OCH2–), 2.65–2.47 (m, 1H, H-9), 2.33 (td, J ¼ 14.0, 3.8 Hz, 1H, H-
4), 2.06–1.94 (m, 1H, H-4), 1.92–1.80 (m, 1H), 1.80–1.70 (m, 2H),
1.70–1.61 (m, 3H), 1.53 (m, 4H), 1.41 (s, 3H, CH3-14), 1.35 (m,
3H), 1.20 (m, 2H), 0.92 (d, J ¼ 6.2 Hz, 3H, CH3-15), 0.89 (m, 1H, H-
7), 0.86 (d, J ¼ 7.2 Hz, 3H, CH3-16); 13C NMR (100 MHz, CDCl3)
d 129.8(–N]C]S), 104.2(C-3), 102.2(C-10), 88.1(C-12), 81.3(C-
12a), 68.4(–OCH2–), 52.7(C-5a), 45.2(–CH2NCS), 44.6(C-8a),
37.7(C-6), 36.6(C-4), 34.8(C-7), 31.1(C-9), 30.1, 29.6, 26.5, 26.4(C-
14), 25.7, 24.9(C-5), 24.7(C-8), 20.6(C-15), 13.2(C-16).
Apoptotic analysis by ow cytometry
For this assay, U87 cancer cells were seeded at a density of 2 ꢂ
106 cells per mL and then incubated with 0, 8 and 16 mM of 5b
for 16 h. The cells were harvested using trypsinization and then
washed with ice-cold PBS and resuspended in binding buffer.
Aer adding Annexin V-FITC and PI, the cells were incubated at
Cell lines and reagents
In the present study, a U87 cell line (from Prof. Wenbo Zhu,
Zhongshan Medical School, Sun Yat-sen University) was used.
The cells were maintained in Dulbecco's modied Eagle's
medium (Gibo; Thermo Scientic, China) supplemented with
10% fetal bovine serum (PAN BIOTECH) in a humidied incu-
ꢀ
4 C in the dark for 15 minutes according to the manufacturer
instructions. The samples were analyzed by ow cytometer
(Beckman Coulter, Epics XL) for the percentage of apoptotic and
necrotic cells.
ꢀ
bator at 37 C with 5% CO2. Artemisinin-based isothiocyanate
Western blot assay
derivatives were dissolved in dimethyl sulfoxide and stored at
ꢃ20 ꢀC in the dark. Annexin V-FITC apoptosis detection kits
were from Best Bio(China). The antibodies used are listed in
Table 2.
Treated cells were washed with cold PBS and lysed in a radio-
immunoprecipitation assay (RIPA) buffer supplemented with
a proteinase inhibitor for extracting total proteins. Protein
concentrations were determined by the bicinchoninic acid
(BCA) protein assay. Aer denaturation, the proteins were
separated in SDS polyacrylamide gel electrophoresis and
transferred onto PVDF membranes. Nonspecic binding was
blocked with 5% milk in TBST buffer for 2 h, followed by
incubation with primary antibodies at 4 ꢀC overnight and
secondary antibodies at room temperature for 2 h. Blots were
visualized using ECL detection reagents.
Cytotoxic assay
The cells were seeded in 96-well plates at a density of 1 ꢂ 104
cells/100 mL and treated with different concentrations of the
compounds ranging from 1 to 250 mM. Aer 24 hours of incu-
bation, MTT (Sigma-Aldrich) was added to cells following the
treatment and incubated for 4 h at 37 ꢀC. DMSO was subse-
quently added, and the absorbance was measured with
a microplate reader aer shaking for ꢄ30 s. The half maximal
inhibitory concentration (IC50) was calculated. The cytotoxicity
of 5b and DHA were investigated for the 24 and 72 h
incubations.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Wound healing assay
The wound healing assay was performed in 6-well tissue culture This study was supported by the Guangdong Provincial Inter-
plates. Aer the cells were allowed to attach and reach 80% national Cooperation Project of Science & Technology (No.
40982 | RSC Adv., 2018, 8, 40974–40983
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RSC Advances
2013B051000038), the National Natural Science Foundation of 16 Y. J. Jeong, H. J. Cho, F. L. Chung, X. Wang, H. S. Hoe,
China (No. 31371070, No. 81671264), Grant (No. 201704020222,
No. 201807010094) from Guangzhou Science, Technology and
K. K. Park, C. H. Kim, H. W. Chang, S. R. Lee and
Y. C. Chang, Oncotarget, 2017, 8, 63949–63962.
Innovation Commission and the Fundamental Research Funds 17 Y. T. Yang, Y. Shi, M. Jay and A. J. Di Pasqua, Chem. Res.
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He, (2014Y2-00500) to M. Li. The authors also thank the and E. Mazzon, Fitoterapia, 2016, 110, 1–7.
Talented Young Scientist Program supported by the China 19 K. C. Liu, T. Y. Shih, C. L. Kuo, Y. S. Ma, J. L. Yang, P. P. Wu,
Science and Technology Exchange Center for the fellowship.
Y. P. Huang, K. C. Lai and J. G. Chung, Am. J. Chin. Med.,
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RSC Adv., 2018, 8, 40974–40983 | 40983