Design, synthesis and biological evaluation of artemisinin derivatives containing fluorine atoms as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2018.05.035

Ten novel artemisinin derivatives containing fluorine atoms were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and HRMS technologies in this study. The in vitro cytotoxicity against U87MG, SH-SY5Y, MCF-7, MDA-MB-231, A549 and A375 cancer cell lines was evaluated by MTT assay. Compound 9j was the most potent anti-proliferative agent against the human breast cancer MCF-7 cells (IC₅₀ = 2.1 μM). The mechanism of action of compound 9j was further investigated by analysis of cell apoptosis and cell cycle. Compound 9j induced cell apoptosis and arrested cell cycle at G1 phase in MCF-7 cells. Our promising findings indicated that the compound 9j could stand as potential lead compound for further investigation.

Full text of DOI:10.1016/j.bmcl.2018.05.035

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of artemisinin derivatives
containing fluorine atoms as anticancer agents
Shu Lia, Gongming Lia, Xiaohong Yangb, Qian Menga, Shuo Yuana, Yun Heb, Dequn Suna,⁎
a
Marine College, Shandong University at Weihai, No. 180, Wenhua West Road, Weihai 264209, PR China
School of Pharmaceutical Sciences, Chongqing University, 55 Daxuecheng South Road, Chongqing 401331, PR China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fluorine
Artemisinin derivatives
Synthesis
Cytotoxic
Ten novel artemisinin derivatives containing fluorine atoms were synthesized and their structures were con-
firmed by 1H NMR, 13C NMR and HRMS technologies in this study. The in vitro cytotoxicity against U87MG, SH-
SY5Y, MCF-7, MDA-MB-231, A549 and A375 cancer cell lines was evaluated by MTT assay. Compound 9j was
the most potent anti-proliferative agent against the human breast cancer MCF-7 cells (IC50 = 2.1 μM). The
mechanism of action of compound 9j was further investigated by analysis of cell apoptosis and cell cycle.
Compound 9j induced cell apoptosis and arrested cell cycle at G1 phase in MCF-7 cells. Our promising findings
indicated that the compound 9j could stand as potential lead compound for further investigation.
Apoptosis
Cell cycle
Artemisinin (ART) is the first natural endoperoxide containing ses-
quiterpene lactone isolated from Chinese herb Artemisia annua.
Artemisinin and its derivatives (Fig. 1) have been widely used in the
treatment of malaria due to their high anti-malarial activity and low
toxicity.1 With the established safety record in millions of malarial
patients,2,3 other activities in addition to antimalaria of artemisinins
were also being investigated, such as anti-viral,4 anti-parasitic,5 anti-
fungal,6 anti-inflammatory7 and anti-cancer.8 In 1993, antitumor ac-
tivity of artemisinins was firstly reported.9 Subsequently, many arte-
misinin derivatives, such as ester,10 ether,11 dimer, trimer and tet-
ramer,12 were synthesized as antitumor drug candidates. Despite the
fact that hemiacetal structure provides a chance for the preparation of
artemisinin derivatives, current synthetic strategies have only focused
on the C-9 and C-10 atoms mainly due to the difficulty to introduce
functionalities on the ring systems by conventional chemical methods.
Soomro13 found that C-10 derivatives were more cytotoxic towards
cancer cells than C-9 derivatives. Introduction of additional heteroa-
toms to the skeleton of artemisinin may afford derivatives with novel
biological properties. In our previous work, we found artemisinin de-
rivatives with piperazine group at C-10 site displayed potent antitumor
activity.14
synthesis artemisinin-like compounds which present excellent anti-
malarial activity in oral treatment of infected mice. In our previous
research, we found that introduction of fluorine atoms to thiazole ex-
hibited enhanced antitumor activities.19
With this background, we attempted to design and synthesize a
series of artemisinin derivatives containing fluorine atoms. The in vitro
cytotoxicity against cancer cell lines (U87MG, SH-SY5Y, MCF-7, MDA-
MB-231, A549 and A375) was evaluated. Furthermore, the mechanism
of action of compound 9j against breast cancer cell (MCF-7) was in-
vestigated.
Our investigation commenced with the synthesis of key inter-
mediate 4 (Scheme 1). Commercially available aromatic amines were
treated with triphosgene (BTC) and trimethylamine to give the aryl
isocyanates 2,20 which was treated with Boc-protected piperazine to
obtain intermediate 3. Then following deprotection of Boc group of 3
with trifluoroacetic acid (TFA) to provide desired intermediate 4,21
which was used to synthesize the target compounds 9h, 9i and 9j.
The target compounds 9a–j were synthesized starting from artemi-
sinin according to the literature procedure (Scheme 2). Firstly, arte-
misinin was subjected to NaBH4-mediated reduction to afford dihy-
droartemisinin
(DHA).22
Then,
DHA
was
treated
with
Fluorine has been exploited extensively in drug design and devel-
opment.15 An increasing number of fluorinated antimitotic/antitumor
agents have now becoming available for cancer treatment.16 In the
design of analogues of bioactivity compounds, replacement of a CeH
bond or CeO bond with fluorine has special advantages.17 Grellepois18
introduced a trifluoromethyl substituent at C-10 of artemisinin to
trimethylchlorosilane and triethylamine in DCM at 0–5 °C to give DHA
a-trimethylsilyl ether 7.23 The target compounds 9a–j were obtain from
DHA a-trimethylsilyl ether 7 with trimethylsilyl bromide (TMSBr),
followed by treatment of the intermediate bromide 8 formed in suit with
the amine nucleophile.24 The structures of all compounds were con-
firmed by 1H NMR, 13C NMR and HRMS (Supporting Information).
⁎
Corresponding author.
E-mail address: dequn.sun@sdu.edu.cn (D. Sun).
https://doi.org/10.1016/j.bmcl.2018.05.035
Received 19 March 2018; Received in revised form 10 May 2018; Accepted 16 May 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Li,S.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.05.035

S. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
The in vitro cytotoxicity of artemisinin derivatives were determined
against U87MG, SH-SY5Y, MCF-7, MDA-MB-231, A549, A375 by using
MTT assay. Artemisinin, dihydroartemisinin, doxorubicin (DOX) and
temozolomide (TMZ) were used as positive controls. The IC50 for each
compound with respect to these cell lines were calculated and the re-
sults were summarized in Table 1. Artemisinin derivatives 9a–j were
generally more effective inhibiting cancer cells growth than the parent
compound artemisinin, but presented lower cytotoxicity than doxor-
ubicin against human normal liver cell line L02. While regarding to
human neuroblastoma cell lines U87, compounds 9a, 9f, 9g, 9h, 9i and
9j have moderate antiproliferation activity (IC50 values from 15.2 μM to
28.7 μM), while 9b, 9c, 9d and 9e have no pronounced inhibition ac-
tivity (IC50 > 50 μM). For SH-SY5Y cells, compounds 9f, 9h, 9i and 9j
showed potent cytotoxic activities with IC50 values of 5.0, 5.0, 3.2 and
3.6 µM, respectively. Among human breast cancer cell MCF-7, com-
pounds 9a–j exhibited significantly higher cytotoxicity compared with
artemisinin (IC50 values from 2.1 μM to 28.8 μM). It is remarkable that
compound 9j presented the strongest cytotoxic activities
(IC50 = 2.1 μM) against MCF-7 cells. For MDA-MB-231cells, compounds
9i and 9j showed significantly higher cytotoxicity compared with ar-
temisinin (the IC50 values were 5.8 μM and 8.4 μM, respectively).
Fig. 1. The structures of artemisinin and its analogues.
Scheme 1. The synthetic routes of compound 4a–c.
Scheme 2. The synthetic routes of artemisinin derivatives containing fluorine atoms.
Table 1
Cytotoxic activity of 9a–j and control drugs against cell lines.
Compound
IC50 Values(μM)a
U87
SH-SY5Y
MCF-7
MDA-MB-231
A549
A375
L02
9a
9b
9c
9d
9e
9f
9g
9h
25.5
> 50
> 50
> 50
> 50
17.7
28.0
28.7
15.2
19.7
> 50
> 50
> 50
NTc
2.7
> 50
> 50
11.2
15.0
> 50
5.0
20.3
5.0
3.2
28.8
27.0
7.5
3.5
6.8
10.2
5.3
5.5
2.7
2.1
0.1
5.7
0.5
0.2
1.4
0.8
> 50
> 50
> 50
> 50
12.9
35.9
44.4
17.9
41.8
17.6
21.1
19.9
19.9
> 50
46.8
2.3
> 50
40.9
20.0
12.8
46.5
10.9
26.5
9.1
11.2
20.5
9.1
17.1
23.2
35.7
9.3
15.6
4.0
6.7
1.6
0.5
1.0
1.6
4.0
2.8
2.1
2.2
0.2
0.2
3.1
4.6
0.1
3.0
0.1
1.5
0.9
2.7
12.5
12.0
32.9
21.8
22.9
10.9
5.8
0.3
0.7
2.9
3.0
3.4
2.5
0.1
0.3
4.0
0.7
2.4
3.2
3.3
2.6
1.3
0.5
1.1
0.3
2.4
2.6
0.4
1.9
0.2
03
0.1
0.1
0.2d
1.6
0.5
9i
1.7
0.7
15.0
3.4
1.1
9j
3.6
2.3d
8.4
1.6d
0.7d
9kb
> 50
> 50
> 50
NTc
15.2
> 50
> 50
1.0
19.2
> 50
45.7
2.8
0.7
NTc
> 50
> 50
34.0
0.9
artemisinin
dihydroartemisinin
doxorubicin
temozolomide
> 50
38.5
0.4
2.7
0.5
0.7
0.2
2.6
0.1
1.6
0.1
0.1
> 50
> 50
NTc
NTc
NTc
NTc
NTc
a
IC50 values are indicated as the mean
Reported in Ref. 14.
NT means not tested.
SD (standard error) of at least three independent experiments. The cells were continuously treated with compounds for 72 h.
b
c
d
The IC50 values of compound 9j against SH-SY5Y, MCF-7, MDA-MB-231 and A375 cells (IC50 < 10 μM).
2

S. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
cell lines. Our results showed that the structure modification of arte-
misinin, i.e., introducing the group of piperazine with attached amide
bond into C10 position of artemisinin, led to a dramatic increase of the
inhibitory effect on certain cancer cells in some degree.
Above results showed that compound 9j presented the strongest
cytotoxic activities (IC50 = 2.1 μM) against MCF-7 cells and showed
lower cytotoxicity (IC50 = 6.7 μM) than doxorubicin (IC50 = 0.9 μM)
against L02. Therefore, we decided to further investigate the pre-
liminary mechanism of compound 9j on MCF-7 cells. As shown in
Fig. 2, compound 9j caused significant inhibitory effect on the growth
of MCF-7 cells. The effect occurred in a concentration- and time- de-
pendent manner.
Compound 9j was further tested to evaluate whether the cytotoxic
effect is related to apoptosis. MCF-7 cells were treated with 1 μM–20 μM
of 9j for 48 h, and then, the cells were harvested and stained with
Annexin V-FITC and propidium (PI) and analyzed by flow cytometry. As
shown in Fig. 3a, the percentages of apoptosis cells were 7.49%,
19.83%, 35.00%, 54.16% and 73.58%, respectively, being elevated
fromthe control group (6.72%). As shown in Fig. 3b, 9j induced cell
apoptosis in a concentration-dependent manner. These phenomena
were also verified by fluorescence microscopy assay. As shown in
Fig. 3c, compound 9j induced prominent apoptotic morphological al-
terations, including cell shrinkage and granular apoptotic body for-
mation. Therefore, the anti-proliferative effect of compound 9j might
be involved in the induction of apoptosis. These results were similar to
that of 9k reported in our previous work.14
In our previous work, artemisinin derivative 9k induced significant
G2/M phase arrest.14 Hence, a flow cytometry analysis was performed
to determine the arrest effect of 9j on cell cycle distribution. MCF-7
cells were treated with increased concentrations of 9j (0–10 μM) for
48 h. As a result, compound 9j led to significant accumulation of cells at
G1 phase from 45.21% to 47.95% (0.5 μM), 56.57% (1 μM), 64.46%
(2.5 μM), 76.68% (5 μM) and 83.50% (10 μM), along with concomitant
losses in the S phase from 41.55% to 9.20% (Fig. 4a). Compound 9j
effectively arrested cells at the G1 phase in a concentration-dependent
manner (Fig. 4b). These results illustrated the basic mechanism of 9j on
Fig. 2. Quantification of inhibitory effect compound 9j.
Regarding to human lung cancer cell line A549, compounds 9c–j ex-
erted moderate cytotoxicity compared with artemisinin (IC50 values
from 12.9 μM to 44.4 μM). Among human melanoma cell A375, com-
pounds 9h and 9j have significantly higher cytotoxicity compared with
artemisinin (the IC50 values were 9.1 μM and 3.4 μM, respectively).
From the antitumor activity results, the activity of the screened com-
pounds could be correlated with structure modification and alteration.
As compared with compounds 9d–e, compounds 9h–j exhibited higher
inhibitory activities against U87, SH-SY5Y, MDA-MB-231, A549 and
A375 cells, suggesting that the introduction of amide group could en-
hance inhibitory effect toward these cancer cells. It is noticeably that
compound 9j presented higher cytotoxicity against SH-SY5Y, MCF-7,
MDA-MB-231 and U87 cells (the IC50 values were 3.6, 2.1, 8.4 and
19.7 µM, respectively) than 9k14 reported in our previous work. Com-
pound 9j have potent to be broad antitumor reagent for human tumor
Fig. 3. 9j-induced apoptosis in MCF-7 cells. (a) Result of MCF-7 treated with or without 9j by flow cytometric analysis. (b) The total apoptosis ratio in treated cells
and control cells (c) Hoechst33258 staining of MCF-7 cells treated or untreated with 9j. *P < 0.05, **P < 0.01 versus control group (0 µM).
3

S. Li et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Fig. 4. Cell cycle analysis of 9j on MCF-7 Cells. *P < 0.05, **P < 0.01 versus control group (0 µM).
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In summary, a series of novel artemisinin derivatives containing
fluorine atoms were synthesized. The in vitro cytotoxicity of compounds
9a–j were evaluated against six human cancer cell lines by MTT assay.
Among all artemisinin derivatives, the optimal compound 9j displayed
excellent activity against MCF-7 cells with a concentration- and time-
dependent manner. The mechanism of action of compound 9j was
further investigated by analysis of cell apoptosis and cell cycle.
Compound 9j induced cell apoptosis and arrested cell cycle at G1 phase
in MCF-7 cells. These results suggested that compound 9j have potential
for further development as anticancer agent.
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This work was funded by National Natural Science Foundation of
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