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