Combating P-glycoprotein-mediated multidrug resistance with 10-O-phenyl dihydroartemisinin ethers in MCF-7 cells

Article abstract of DOI:10.1016/j.ejmech.2015.10.040

A series of 10-β-phenyl ethers of dihydroartemisinin (DHA) with piperazine substitutions were synthesized with the goal of overcoming multidrug resistance in cancer therapy. These novel compounds exerted significant antiproliferative activities in breast

Full text of DOI:10.1016/j.ejmech.2015.10.040

European Journal of Medicinal Chemistry 108 (2016) 720e729
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Combating P-glycoprotein-mediated multidrug resistance with
0-O-phenyl dihydroartemisinin ethers in MCF-7 cells
1
a
b
a
a
a
b
Hang Zhong , Xuan Zhao , Zhizhong Zuo , Jingwei Sun , Yao Yao , Tao Wang ,
Dan Liu
a
a,
**
a, *
, Linxiang Zhao
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
School of Life Sciences and Biopharmaceutis, Shenyang Pharmaceutical University, Shenyang 110016, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 April 2015
Received in revised form
A series of 10-b-phenyl ethers of dihydroartemisinin (DHA) with piperazine substitutions were syn-
thesized with the goal of overcoming multidrug resistance in cancer therapy. These novel compounds
exerted significant antiproliferative activities in breast cancer MCF-7 and MCF-7/Adr cell lines at the
submicromolar level and were shown to be approximately 100- to 300-times more potent than the lead
compound DHA. Remarkably, the P-gp-overexpressed MCF-7/Adr cell line showed collateral sensitivity
towards these derivatives. Furthermore, compounds 3d and 5c, with the highest selectivity for MCF-7/
Adr towards MCF-7 cells, were free from P-gp efflux in a MDCK-MDR1 assay. Flow cytometry and
western blot assays suggested that the antiproliferative effects of 5c were associated with cell cycle arrest
at G1 phase through the downregulation of Cyclin D1 and Cyclin B1.
25 September 2015
Accepted 22 October 2015
Available online 10 November 2015
Keywords:
10-O-phenyl dihydroartemisinin ethers
Multidrug-resistance
MCF-7/Adr
© 2015 Published by Elsevier Masson SAS.
P-glycoprotein
MDCK-MDR1
Antiproliferative effects
Collateral sensitivity
1. Introduction
required pharmacophore and displays improved pharmacokinetic
profiles. Artemisinins have demonstrated potential anticancer ac-
The overexpression of P-glycoprotein (P-gp) is one of multiple
mechanisms of multidrug resistance (MDR) in cancer, which is a
hindrance to successful chemotherapy [1]. Clinical chemothera-
peutic agents such as paclitaxel, adriamycin (Adr) [2], and even
highly selective kinase inhibitors erlotinib [3] and sorafenib [4]
often suffer reduced efficacy as a consequence of the efflux func-
tion of P-gp. Despite great efforts to inhibit P-gp directly to over-
come P-gp-mediated resistance, none of the current P-gp inhibitors
have shown improved clinical efficacy due to inherent side effects
tivity, especially in some P-gp-overexpressed MDR cancer cells
[6,7]. For instance, the growth inhibitory activities of artesunate
(ART) against 55 NCI cell lines were unaffected by the content of P-
gp in a microarray analysis, and no cross-resistance occurred with
P-gp-overexpressed multidrug resistance in an HL-60 cell line [8].
The preserved anti-cancer potency of ART was also observed in
drug resistant neuroblastoma cells [9]. Recently, a series of homo-
dimeric ARTs (e.g.1, Fig. 1) displayed great potency in P-gp-over-
expressed CEM/ADR5000 leukemia cells [10,11]. These studies
informed our hypothesis that P-gp-overexpressed MDR cancer cells
might exhibit collateral sensitivity towards DHA derivatives [12].
In a previous study, DHA derivatives with a substituted piper-
azine moiety exhibited superior antiproliferative activities
compared to DHA against mouse lymphoma P388 and adriamycin
resistant P388/Adr cells. Particularly, compound 2 (Fig. 2) showed
higher efficacy against P388/Adr than the sensitive P388 cells [13].
To improve the antiproliferative effect and sensitivity of DHA
against MDR cells, a series of novel 10-phenyl ethers with N-aryl
piperazines of DHA (3ae3e, 4ae4e and 5ae5e, Fig. 2) were syn-
thesized based on further modifications of compound 2. As re-
ported by O'Neill et al. [14], C-10-phenyl ethers of DHA contributed
[5]. The development of agents to evade the P-gp efflux effect
seems to be a practical approach to overcoming P-gp-mediated
multidrug resistance in cancer.
Artemisinins (Fig. 1) have been widely used in the treatment of
drug resistant malaria. Dihydroartemisinin (DHA), the simplest
derivative of artemisinin, retains the endoperoxide bridge as
*
Corresponding author.
** Corresponding author.
E-mail addresses: sammyld@163.com (D. Liu), linxiang.zhao@vip.sina.com
(
L. Zhao).
http://dx.doi.org/10.1016/j.ejmech.2015.10.040
223-5234/© 2015 Published by Elsevier Masson SAS.
0

H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
721
benzyl, phenylacetyl and cinnamoyl linkers were prepared from
DHA and corresponding side chains 6ae6e, 7ae7e and 8ae8e,
respectively (Scheme 1) [13]. 6ae6e were prepared through
Leuckart-Wallach reductive amination [16], and compounds 7ae7e
and 8ae8e were obtained by reacting p-hydroxyphenylacetyl and
p-hydroxycinnamoyl chlorides with N-substituted piperazines. The
activation of DHA with trifluoroacetic anhydride (TFAA) in the
presence of Et
the reaction of 9 with the corresponding side chains 6ae6e, 7ae7e,
and 8ae8e in CH Cl gave aryl ethers of DHA 3ae3e, 4ae4e and
ae5e as C-10 -diastereomers.
The chemical structures of the target compounds were
3
N at room temperature gave the active ester 9, and
2
2
5
b
1
13
confirmed by IR, H NMR, C NMR and MS spectroscopy data. The
stereochemistry was determined by analyzing the coupling con-
stant values between H-9 and H-10 (
d
¼ 5.64) in the scaffold of DHA,
and all target compounds were -isomers with similar coupling
b
constant values (JH9-10 ¼ 3.0e3.3 Hz) [17e19]. The stereoselective
results were similar to those described in Haynes’ study [19] as a
kinetic controlled
b
a
-ester 9 was exclusively formed and then gave a
-ether through a S 2 process. The cinnamoyl double bonds of
ae5e were designed as trans-configurations according to the two
¼ 7.50 and 7.20 ( ¼ 7.72 and 7.57 for 5e),
displaying a coupling constant value of 15.3 Hz.
Fig. 1. The chemical structures of reported artemisinin derivatives.
N
5
related H-atoms at about
d
d
to an improved half-life and metabolic stability and were therefore
retained in our design. For systematic SAR studies, the benzoyl
linker of 2 was reduced to benzyl or extended to phenylactetyl and
2
2
.2. Pharmacology
a,b-unsaturated cinnamoyl to optimize the linker; additionally,
various substituted aryls were introduced into the N-4 position of
piperazine, as a previous study had showed that a phenyl motif was
more preferable than other substitutions [13].
.2.1. Artemisinin-phenyl ethers inhibited cell growth in MCF-7 and
MCF-7/Adr cells
The antiproliferative activities of 3ae3e, 4ae4e and 5ae5e were
evaluated against the human breast tumor cell line MCF-7 and
adriamycin-resistant MCF-7/Adr by the MTT method [20,21]; the
results were summarized in Table 1.
All tested artemisinin ethers showed greater potency against
MCF-7 cells than DHA but were less potent than the positive control
adriamycin. 2 and 5a were the most potent artemisinin ethers
Breast cancer retains the highest morbidity rates of cancer in
females, and chemotherapy often fails due to the unavoidable
development of drug resistance [15]. Antiproliferative activities of
newly synthesized ethers were evaluated against human breast
cancer MCF-7 cells and P-gp-overexpressed MCF-7/Adr cells, the
latter exhibiting collateral sensitivity to DHA and newly synthe-
sized artemisinin-phenyl ethers. A further mechanism study was
also performed and found that the derivatives were unaffected by
P-gp efflux; therefore, G1 cell cycle arrest might play a role in their
growth inhibition abilities.
(
GI50 ¼ 0.10
m
M and 0.11
mM) and displayed 300-fold higher anti-
proliferative activities than DHA (33.22
m
M) against MCF-7 cells. In
MCF-7/Adr cells, all artemisinin ethers exhibited significantly
improved potency compared to DHA as well as adriamycin. 5c
proved to be the most effective compound against the MCF-7/Adr
2
. Results and discussion
cell line with a GI50 value of 0.021
fold much more potent than DHA (3.32
(0.52 M), respectively.
m
M, which was 150- and 25-
mM) and adriamycin
2.1. Chemistry
m
Intriguingly, MCF-7/Adr cells showed collateral sensitivity for all
novel artemisinin-phenyl ethers, which could be quantitatively
The target artemisinin ethers 3ae3e, 4ae4e and 5ae5e with
Compd.
X
Y
R
H
3a
CH
CH
CH
CH
N
3b
2-OCH
3
3
3
3c
3-CF
4-F
H
3
3d
3
e
a
4
CH
CH
CH
CH
N
H
4b
2-OCH
4c
3-CF
4-F
H
3
4d
4
5
e
a
CH
CH
CH
CH
N
H
5b
2-OCH
5c
3-CF
4-F
H
3
5d
5e
Fig. 2. Design of target compounds.

722
H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
Scheme 1. The synthetic routes of target compounds.
evaluated by selectivity ratio value (SR ¼ GI50 sensitive/GI50 MDR,
the ratio of a compound's cytotoxicity over parental cell line) [12].
d had the highest selectivity between MCF-7 (GI50 ¼ 0.53 M) and
MCF-7/Adr (GI50 ¼ 0.025 M) with an SR value of 21.20.
3
m
m
Table 1
A critical SAR analysis illustrated that the introduction of side
chains through phenyl ether into DHA was able to improve anti-
proliferative activity significantly. The alterations of linkers and
substituents on the N-4-position of piperazine did bring increased
selectivities against MCF-7/Adr cells compared with that of com-
pound 2, while little influence on inhibitory activities was
observed. For example, 3d with a benzyl linker and N-4-(p-fluo-
rophenyl) piperazine showed the maximum SR value of 21.20. It
should be noted that newly synthesized compounds were superior
to 2 with a benzoyl linker and N-4-phenyl piperazine in selectivity
The antiproliferative activities of artemisin ethers in MCF-7 and MCF-7/Adr cells.
Compd.
GI50
/
m
M
SR
MCF-7
MCF-7/Adr
2
0.10 ± 0.12
0.24 ± 0.19
0.43 ± 0.30
0.11 ± 0.03
0.58 ± 0.46
0.47 ± 0.03
0.14 ± 0.04
0.22 ± 0.03
0.26 ± 0.04
0.32 ± 0.03
0.53 ± 0.10
0.29 ± 0.16
0.27 ± 0.06
0.17 ± 0.11
0.96 ± 0.19
0.29 ± 0.07
33.22 ± 6.33
0.054 ± 0.020
0.075 ± 0.017
0.021 ± 0.015
0.090 ± 0.047
0.051 ± 0.038
0.15 ± 0.03
0.043 ± 0.020
0.031 ± 0.002
0.033 ± 0.020
0.041 ± 0.006
0.021 ± 0.007
0.025 ± 0.019
0.044 ± 0.028
0.038 ± 0.003
0.055 ± 0.051
0.080 ± 0.048
0.11 ± 0.06
1.33
11.43
4.78
2.16
3.87
10.93
4.52
6.67
3
4
5
3
4
5
3
4
5
3
4
5
3
4
5
a
a
a
b
b
b
c
(SR value of 1.33).
c
c
6.34
15.24
21.20
7.11
8.57
3.09
12.00
2.64
10.00
0.10
d
d
d
e
e
e
2.2.2. 5c and 3d were free from P-gp efflux determined by MDCK-
MDR1 and MDCK-WT cell transport assays
Artemisinin ether derivatives selectively inhibited the prolifer-
ation of MCF-7/Adr cells, which are MDR phenotypic cells induced
from MCF-7 by adriamycin to overexpress P-gp steadily. To clarify
the selectivity and sensitivity for MCF-7/Adr cells, it's necessary to
determine whether the novel ethers were transported substrates of
P-gp. MadineDarby canine kidney (MDCK) assays have been widely
DHA
Adr
3.32 ± 0.64
0.52 ± 0.14
SR: sensitivity ratio.

H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
723
utilized in the study of drug transport [22] and for MDCK-MDR1
transfected cells in the study of efflux effects by P-gp [23]. The
transcellular permeability of 5c and 3d was assessed in MDCK-
MDR1 and MDCK-WT cell monolayers, and the experimental re-
sults were summarized in Table 2. Apparent permeability co-
efficients (Papp) from A-to-B (apical to basolateral of the cell
monolayers, A-B) and B-to-A (basolateral to apical of the cell
monolayers, B-A) were obtained by measuring the content of the
compounds transported from the donor compartment to the
receiver after incubation, with quantification achieved by LC-MS/
MS analysis. The P-gp efflux effects were assessed by efflux ratio
cell cycle progression. Treatment of MCF-7 cells with 5c for 24 h
resulted in a higher number of cells in G1 phase at the indicated
concentrations [0.25
mM (70.0%), 0.50 mM (73.8%); Fig. 3A],
compared with the control (62.5%). A greater accumulation of cells
at G1 phase (83.4%) was observed when treated with 5c for 48 h at
0.50
mM G1-phase arrest also appeared when MCF-7/Adr cells were
treated with 5c (Fig. 3A). Treatment with 5c at 0.05
mM and 0.25
mM
for 24 h led to moderate increases in the number of cells at G1
phase (71.5% and 70.2%, respectively) compared to the untreated
cells (54.6%). Treatment with 5c for a prolonged period (48 h) at
0.25 mM resulted in a smaller increase of cells in G1 phase (62.6%)
(
the ratio of Papp in the direction of B-A over that of A-B). A com-
compared to that of 24 h and untreated cells (70.2%, 56.9%). How-
ever, slight apoptosis was observed as sub-G1 phase cells increased
from 2.3% to 8.7%, which might explain the decreasing cells in G1
phase during prolonged treatment.
pound with an efflux ratio value greater than 2.0 was generally
considered to be a potential substrate for P-gp [24]. Quinidine (an
efflux substrate), Metoprolol (a high permeability control), Atenolol
(
a low permeability control) and DHA were included as the parallel
These data demonstrated clearly that 5c induced a dose-
dependent G1 phase arrest in MCF-7 cells, while it caused G1
phase arrest and a slight apoptosis effect in an extended-time
manner in MCF-7/Adr cells, which might explain the 5c-induced
growth inhibition in both cell lines.
Cyclins facilitate cell cycle progression and are considered pos-
itive regulators, while kinase inhibitor proteins (KIP) are commonly
regarded as negative regulators of the cell cycle [26]. To further
investigate the potential mechanism for 5c-induced cell cycle ar-
rest, the effect of 5c on cell cycle regulatory proteins (Cyclin D1 and
B1) in both cell lines was studied. As illustrated in Fig. 3B, treatment
references in the same experiments.
According to the data in Table 2, DHA was highly permeable
ꢁ
6
(
P
app > 10.0 ꢀ 10
cm/s) and unaffected by P-gp (Efflux
ratio ¼ 0.89). 3d and 5c were similar (Efflux ratio < 1.0) in the price
of decreased permeabilities. Our results might surmount the pre-
vious research by Steglich et al. [25], in which an artemisinin de-
rivative SM616 was found to inhibit P-gp specifically for the reason
mentioned above [5]. Our data indicated that DHA, 3d and 5c could
inhibit cell growth of MCF-7/Adr selectively without the limitation
of P-gp, so it was reasonable to conclude that these novel molecules
could effectively overcome the P-gp mediated MDR through
bypassing the efflux effects of P-gp.
with 0.5
cyclin B1 while increasing the expression of p27 in MCF-7 cells.
While in MCF-7/Adr cells, treatment with 5c at 0.1 M for 48 h
mM 5c decreased the expression of both cyclin D1 and
m
sharply decreased the levels of cyclin D1 and cyclin B1 but not p27.
The proteolytic cleavage of Poly (ADP-ribose) polymerase (PARP)
2
.2.3. 5c induced G1-phase cell cycle arrest in human MCF-7 and
MCF-7/Adr cells
[27] was not observed under these conditions, indicating that
The growth-inhibitory effects of 5c towards MCF-7 and MCF-7/
Adr cells inspired us to investigate whether 5c had any effects on
apoptosis did not dominate in either cell line. These primary ex-
periments demonstrated that 5c-induced G1 phase arrest was
associated with the downregulation of Cyclin D1 and Cyclin B1 in
MCF-7 and MCF-7/Adr cells.
Table 2
AeB and BeA Papp of DHA, 3d and 5c across MDCK-WT and MDCK-MDR1
monolayers.
3
. Conclusion
Test Articale Cell Type
Direction
P
app(10-6 cm/
s)
P
app(B ꢁ A)/ Papp(A ꢁ B)
Fifteen novel 10-O-dihydroartemisinin derivatives were
designed and synthesized, and the structures were confirmed by IR,
MS, HR-MS and NMR data. All compounds displayed improved
antiproliferative effects in both sensitive MCF-7 and resistant MCF-
7/Adr cells compared with DHA. MCF-7/Adr cells revealed collateral
sensitivity towards all novel artemisinin-phenyl ethers. 5c, 3d and
DHA proved to be free from P-gp efflux by MDCK-MDR1 assay. Flow
cytometry and western blot assays suggested that the anti-
proliferative effects of 5c against both MCF-7 and MCF-7/Adr cells
were associated with cell cycle arrest at G1 phase. Our compounds
have the potential to combat P-gp-mediated MDR by evading the
efflux effect.
RSD
a
3
d
c
MDR1
AeB
BeA
<1.34 N/A N/A
<0.85 N/A
<1.33 N/A N/A
<0.83 N/A
<1.83 N/A N/A
<1.00 N/A
<1.82 N/A N/A
<0.93 N/A
a
a
Wild Type AeB
BeA
MDR1
a
a
5
AeB
BeA
a
a
Wild Type AeB
BeA
MDR1
a
DHA
AeB
BeA
27.97
24.87
31.37
25.10
4.37
0.06 0.89
0.08
0.08 0.80
0.01
0.05 7.81
0.01
0.01 2.34
0.03
0.04 1.07
0.00
0.01 0.76
0.05
0.27 0.76
0.14
Wild Type AeB
BeA
MDR1
Quinidine
Metoprolol
Atenolol
AeB
BeA
34.10
9.98
4. Experimental section
Wild Type AeB
BeA
MDR1
23.39
22.19
23.69
24.30
18.54
1.25
4.1. Chemistry
AeB
BeA
Melting points were determined on an X-4 melting point
Wild Type AeB
BeA
MDR1
1
apparatus and uncorrected. Proton nuclear magnetic resonance ( H
13
AeB
BeA
NMR) and C NMR spectra were measured on Bruker ARX-300, AV
00 MHz or ARX-600 spectrometers with tetramethylsilane (TMS)
as the internal standard. Chemical shifts are reported in (ppm).
0.95
4
a
Wild Type AeB
BeA
<0.23 N/A N/A
<0.12 N/A
a
d
Infrared (IR) spectra were recorded on BRUKER IR-27G or Spec-
trum100 (PerkinElmer) spectrometers. Mass spectra (MS) were
determined on Finnigan MAT/USA spectrometer (LCeMS). High-
resolution mass spectra were obtained on BRUKER microOTOF-Q
N/A: not acquired
a
P
app values were expressed as “<” than the values that were calculated using the
minimum concentration of the standards for receiver sides due to the fact that real
concentration in the receivers were below quantitation limit.

724
H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
Fig. 3. G1-phase cell cycle arrest in human MCF-7 and MCF-7/Adr induced by 5c. A: Cell cycle progressions were analyzed by Flow cytometry method. B: Levels of cycle related
proteins were tested by Western blot.
in the ESI mode (HR-ESI-MS). DHA was purchased from Jiangsu
swellxin bio-pharm. pty. Ltd. China, with purity over 91%. The other
reagents were obtained from commercial suppliers and used
without purification. TLC analysis was carried out on silica gel
plates GF254 (Qindao Haiyang Chemical, China). Column chroma-
tography was performed with silica gel 60 (200e300 mesh). All

H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
725
target compounds are ꢂ95% purity through HPLC analysis.
4.1.1.3.1. (E)-3-(4-hydroxyphenyl)-1-(4-phenylpiperazin-1-yl)
prop-2-en-1-one (8a). To a solution of p-hydroxycinnamic acid
(
1.64 g, 10 mmol) in anhydrous dichloromethane (30 mL), anhy-
drous Et N (1.01 g, 1.39 mL, 10 mmol) was added slowly, the com-
plex was stirred under reflux for 30 min. Then thionyl chloride
0.87 mL,12 mmol) was added drop-wise to the mixture and stirred
for another 2 h. Subsequently, the solution was cooled to room
temperature and concentrated in vacuum. The residue was soluted
in 30 mL anhydrous dichloromethane and N-phenylpiperazine
4
4
.1.1. General procedure for side chains
3
.1.1.1. Compounds 6ae6e
4
.1.1.1.1. 4-((4-phenylpiperazin-1-yl)methyl)phenol
(6a).
(
N-phenylpiperazine (1.78 g, 11 mmol) and p-hydroxybenzaldehyde
ꢃ
(
1.22 g, 10 mmol) were added into a flask, and heated to 60 C to
generate a eutectic body. Subsequently, formic acid (0.37 mL,
2 mmol) was added drop-wise to the mixture, which was heated
and stirred under refluxing for another 2 h. After the solution was
cooled to room temperature, about 30 mL ethyl acetate was added,
the generated solids were filtrated in vacuum, washed with a small
amount of ethyl acetate to obtain brown solid 1.06 g, yield: 39.5%.
1
(1.78 g, 11 mmol) was added and stirred under reflux for 2 h. The
solution was cooled to room temperature and a white solid was
precipitated from solvent, 0.97 g. Yield: 32.1%.
4.1.1.3.2. (E)-3-(4-hydroxyphenyl)-1-(4-(2-methoxyphenyl)piper-
azin-1-yl)prop-2-en-1-one (8b). The product was obtained by the
similar procedure of 8a, from N-(2-methoxyphenyl)piperazine and
p-hydroxycinnamic acid, as a light yellow powder 2.16 g, yield:
4
.1.1.1.2. 4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenol
6b). The product was obtained by the similar procedure of 6a,
from N-(2-methoxyphenyl)piperazine and p-hydrox-
ybenzaldehyde, as a white powder 2.04 g, yield: 68.3%.
.1.1.1.3. 4-((4-(3-trifluoromethylphenyl)piperazin-1-yl)methyl)
phenol (6c). The product was obtained by the similar procedure of
a, from N-(3-trifluoromethylphenyl)piperazine and p-hydrox-
ybenzaldehyde, as a white powder 2.40 g, yield: 71.4%.
.1.1.1.4. 4-((4-(4-fluorophenyl)piperazin-1-yl)methyl)phenol
6d). The product was obtained by the similar procedure of 6a,
(
63.8%.
4.1.1.3.3. (E)-3-(4-hydroxyphenyl)-1-(4-(3-trifluoromethylphenyl)
4
piperazin-1-yl)prop-2-en-1-one (8c). The product was obtained by
the similar procedure of 8a, from N-(3-trifluoromethylphenyl)
piperazine and p-hydroxycinnamic acid, as a light yellow powder
6
þ
1
2
9
.33 g, yield: 61.9%. MS: 377.1 [MþH] ; H NMR (DMSO,
d (ppm)):
4
.84 (1H, s, AreOH),7.57 (2H, d, J ¼ 8.4 Hz, AreH), 7.45 (1H, d,
(
J ¼ 15.3 Hz, -CH]CH-), 7.44(1H, s, AreH), 7.26 (1H, m, AreH),
.21(1H, s, AreH), 7.11 (1H, m, AreH),7.09 (1H, d, J ¼ 15.2 Hz, -CH]
CH-), 6.79 (2H, d, J ¼ 8.4 Hz, AreH) 3.78 (4H, d, -NCH -), 3.28 (4H, s,
NCH -).
.1.1.3.4. (E)-3-(4-hydroxyphenyl)-1-(4-(4-fluorophenyl)piper-
from N-(4-fluorophenyl)piperazine and p-hydroxybenzaldehyde,
as a yellow powder 2.11 g, yield: 73.5%.
7
2
4
.1.1.1.5. 4-((4-(pyridin-2-yl)piperazin-1-yl)methyl)phenol (6e).
The product was obtained by the similar procedure of 6a, from N-
pyridin-2-yl)piperazine and p-hydroxybenzaldehyde, as a white
powder 2.21 g, yield: 82.1%.
-
2
4
(
azin-1-yl)prop-2-en-1-one (8d). The product was obtained by the
similar procedure of 8a, from N-(4-fluorophenyl)piperazine and p-
hydroxycinnamic acid, as a light yellow powder 2.47 g, yield: 75.6%.
4
.1.1.2. Compounds 7ae7e
.1.1.2.1. 2-(4-hydroxyphenyl)-1-(4-phenylpiperazin-1-yl)etha-
none (7a). To a solution of p-hydroxyphenylacetic acid (1.52 g,
0 mmol) in anhydrous dichloromethane (30 mL), anhydrous Et
1.01 g, 1.39 mL, 10 mmol) was added slowly, the complex was
stirred under reflux for 30 min. Then thionyl chloride (0.87 mL,
2 mmol) was added drop-wise to the mixture and stirred for
4
.1.1.3.5. (E)-3-(4-hydroxyphenyl)-1-(4-(pyridin-2-yl)piperazin-
4
1-yl)prop-2-en-1-one (8e). The product was obtained by the similar
procedure of 8a, from N-(pyridin-2-yl)piperazine and p-hydrox-
ycinnamic acid, as a light yellow powder 2.21 g, yield: 71.5%.
1
(
3
N
4.1.2. General procedure for 3ae3e, 4ae4e and 5ae5e
1
A solution of DHA (0.568 g, 2 mmol) and Et N (0.56 g, 4 mmol) in
3
ꢃ
another 2 h. Subsequently, the solution was cooled to room tem-
perature and N-phenylpiperazine (1.78 g, 11 mmol) was added and
stirred at room temperature for 2 h. The solution was concentrated
in vacuum and the residue was purified by silica gel column
chromatography eluting with dichloromethaneeacetone system, as
a light yellow powder 1.06 g, yields: 41.3%.
3
3
0 mL anhydrous dichloromethane was stirred under 0 C for
0 min. Then, trifluoroacetic anhydride (0.57 g, 4 mmol) was added
drop-wise, the mixture was allowed to warm to room temperature
and stirred overnight, corresponding side chains (6ae6e, 7ae7e
and 8ae8e) (6 mmol) were added to the mixture and reacted for
1
2 h. Finally, the solution was washed by 5% aqueous sodium hy-
droxide (30 mL ꢀ 3) and saturated brine (30 mL ꢀ 3), the organic
layer was separated, dried over anhydrous Na SO and concen-
4
.1.1.2.2. 2-(4-hydroxyphenyl)-1-(4-(2-methoxyphenyl)piper-
azin-1-yl)ethanone (7b). The product was obtained by the similar
procedure of 7a, from N-(2-methoxyphenyl)piperazine and p-
hydroxyphenylacetic acid, as a yellow powder 0.84 g, yield: 25.7%.
2
4
trated in vacuum. The crude product was purified by silica gel
column chromatography eluting with petroleum ethereethyl ace-
tate system.
4
.1.1.2.3. 2-(4-hydroxyphenyl)-1-(4-(3-trifluoromethylphenyl)
piperazin-1-yl)ethanone (7c). The product was obtained by the
similar procedure of 7a, from N-(3-trifluoromethylphenyl)pipera-
zine and p-hydroxyphenylacetic acid, as a white powder 1.50 g,
yield: 41.2%.
4
.1.2.1. 10-O-(4-((4-phenylpiperazin-1-yl)methyl)phenyl)-(10S)-
dihydroartemisinin (3a). White solid, 0.129 g; yield: 12.1%, mp:
ꢃ
5
9
d
2e53 C; IR (KBr): 3439, 2921, 2873, 1600, 1507, 1384, 1227, 1035,
4.1.1.2.4. 2-(4-hydroxyphenyl)-1-(4-(4-fluorophenyl)piperazin-1-
ꢁ1
þ
1
59, 876, 837, 758 cm ; MS: 535.5 [MþH] ; H NMR (DMSO,
yl)ethanone (7d). The product was obtained by the similar proce-
dure of 7a, from N-(4-fluorophenyl)piperazine and p-hydrox-
yphenylacetic acid. The white solid was precipitated from solvent,
(ppm)): 7.27 (2H, d, J ¼ 8.7 Hz, AreH), 7.20 (2H, m, AreH), 7.04
(
2H, d, J ¼ 8.7 Hz, AreH), 6.91 (2H, d, J ¼ 8.1 Hz, AreH), 6.76 (1H, m,
AreH), 5.52 (1H, d, J ¼ 3.0 Hz, H-10), 5.44 (1H, s, H-12), 3.47 (2H, s,
ArCH -), 3.12 (4H, s, -NCH -), 2.59 (1H, m, H-9), 2.50 (4H, s, -NCH -),
.21 (1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81 (2H,
1.78 g. Yield: 56.7%.
2
2
2
4.1.1.2.5. 2-(4-hydroxyphenyl)-1-(4-(pyridin-2-yl)piperazin-1-yl)
2
ethanone (7e). The product was obtained by the similar procedure
of 7a, from N-(pyridin-2-yl)piperazine and p-hydroxyphenylacetic
acid. The white solid was precipitated from solvent, 2.32 g. Yield:
m, H-7 and H-8), 1.66e1.50 (2H, m, H-5 and H-8a), 1.40 (2H, m, H-6
and H-8),1.29 (3H, s, H-14), 1.25e1.07 (2H, m H-5a and H-7), 0.97
13
(
(
1
3H, d, J ¼ 7.2 Hz, H-16), 0.91 (3H, d, J ¼ 6.0 Hz, H-15). C NMR
7
8.1%.
DMSO,
03.9, 99.8, 87.8, 80.8, 61.9, 52.9, 52.4, 48.6, 44.1, 36.9, 36.4, 34.5,
30.8, 26.0, 24.6, 24.4, 20.6, 13.0. HRMS (ESI) m/z calcd for
d (ppm)): 156.4, 151.4, 131.8, 130.4, 129.2, 119.1, 117.1, 115.7,
4.1.1.3. Compounds 8ae8e

726
H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
C
H
32 43
N
2
O
5
[MþH]þ 535.3165, found 535.3166.
2.60 (1H, m, H-9), 2.43 (4H, m, -NCH
2
-), 2.19 (1H, m, H-4), 2.03e1.90
(2H, m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50
4
.1.2.2. 10-O-(4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)
(2H, m, H-5 and H-8a), 1.39 (2H, m, H-6 and H-8), 1.29 (3H, s, H-14),
phenyl)-(10S)-dihydroartemisinin (3b). White solid, 0.121 g; yield:
1.25e1.07 (2H, m H-5a and H-7), 0.97 (3H, d, J ¼ 7.2 Hz, H-16), 0.91
ꢃ
13
1
0.7%, mp: 135e137 C; IR (KBr): 2935, 2816, 1595, 1500, 1373,
(3H, d, J ¼ 7.5 Hz, H-15). C NMR (CDCl
3
, d (ppm)): 158.0, 151.7,
ꢁ
1
þ
1
242,1226, 1039, 981, 877, 740 cm ; MS: 565.1 [MþH] , 587.2
139.7, 129.6, 129.4, 123.1, 119.9, 116.3, 115.7, 104.5, 100.8, 88.6, 81.4,
63.2, 53.4, 52.9, 49.3, 44.8, 37.7, 36.7, 35.0, 31.3, 26.4, 25.0, 24.8, 20.7,
þ
þ 1
[
MþNa] , 1165.3 [2MþH] ; H NMR (DMSO,
d (ppm)): 7.26 (2H, d,
J ¼ 8.1 Hz, AreH), 7.03 (2H, d, J ¼ 8.4 Hz, AreH), 6.92 (2H, d,
13.3. HRMS (ESI) m/z calcd for C31
536.3115.
42
H N
3
O
5
[MþH]þ 536.3119, found
J ¼ 8.1 Hz, AreH), 6.87 (2H, d, J ¼ 8.4 Hz, AreH), 5.53 (1H, d,
J ¼ 3.0 Hz, H-10), 5.43 (1H, s, H-12), 3.76 (3H, s, -OCH
3
), 3.48 (2H, s,
ArCH
2
-), 2.95 (4H, s, -NCH
2
-), 2.58 (1H, m, H-9), 2.19 (1H, m, H-4),
4
.1.2.6. 10-O-(4-(2-oxo-2-(4-phenylpiperazin-1-yl)ethyl)phenyl)-
2
.09 (4H, s, -NCH
2
-), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81 (2H,
(
10S)- dihydroartemisinin (4a). White solid, 0.276 g; yield: 24.5%,
m, H-7 and H-8), 1.66e1.50 (2H, m, H-5 and H-8a), 1.39 (2H, m, H-6
and H-8), 1.29 (3H, s, H-14), 1.25e1.07 (2H, m H-5a and H-7), 0.97
ꢃ
mp: 122e124 C; IR (KBr): 3439, 2922, 2872,1645, 1599, 1509, 1450,
1382, 1342, 1280, 1231, 1095, 1066, 1035, 979, 959, 876 cm ; MS:
5
ꢁ1
1
3
(
(
3H, d, J ¼ 7.2 Hz, H-16), 0.91 (3H, d, J ¼ 6.0 Hz, H-15). C NMR
þ
þ 1
63.4 [MþH] , 585.4 [MþNa] ; H NMR (DMSO,
d (ppm)): 7.22 (2H,
DMSO, (ppm)): 156.4, 152.4, 141.6, 130.5, 122.7, 121.2, 118.3, 117.1,
d
d, J ¼ 8.4 Hz, AreH), 7.19 (2H, m, AreH), 7.01 (2H, d, J ¼ 8.4 Hz,
115.3, 112.3, 103.9, 99.8, 87.8, 80.8, 61.9, 55.7, 53.1, 52.4, 50.4, 44.1,
AreH), 6.93 (2H, d, J ¼ 8.1 Hz, AreH), 6.80(1H, m, AreH), 5.50 (1H,
3
6.9, 36.4, 34.5, 30.8, 26.0, 24.6, 24.4, 20.6, 13.0. HRMS (ESI) m/z
[MþH]þ 565.3272, found 565.3250.
d, J ¼ 3.0 Hz, H-10), 5.42 (1H, s, H-12), 3.70 (2H, s, ArCH
2
-), 3.62 (4H,
45 2 6
calcd for C33H N O
m, -NCH
), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8),
.64e1.49 (2H, m, H-5 and H-8a), 1.38 (2H, m, H-6 and H-8), 1.28
(3H, s, H-14),1.25e1.07 (2H, m H-5a and H-7), 0.96 (3H, d, J ¼ 7.2 Hz,
2 2
-), 3.07 (4H, m, -NCH -), 2.57 (1H, m, H-9), 2.19 (1H, m, H-
4
1
4.1.2.3. 10-O-(4-((4-(3-trifluoromethylphenyl)piperazin-1-yl)methyl)
phenyl)-(10S)- dihydroartemisinin (3c). White solid, 0.116 g; yield:
ꢃ
9
1
6
.6%, mp: 65e67 C; IR (KBr): 2950, 1610, 1509, 1450, 1351, 1319,
13
ꢁ
1
þ
H-16), 0.90 (3H, d, J ¼ 6.0 Hz, H-15); C NMR (DMSO,
d (ppm)):
228, 1165, 1122, 1035, 979, 957, 877 cm ; MS: 603.2 [MþH] ,
þ
þ 1
169.6,156.0,151.2,130.4,129.7,129.4,119.8,117.5,116.3,104.0,100.0,
25.2 [MþNa] , 641.1 [MþK] ; H NMR (DMSO,
d (ppm)): 7.40 (1H,
8
2
5
7.9, 80.9, 52.6, 49.2, 48.7, 44.2, 41.5, 37.0, 36.5, 34.6, 30.9, 26.1, 24.7,
t, AreH), 7.26 (2H, d, J ¼ 8.4 Hz, AreH), 7.19 (1H, d, J ¼ 8.4 Hz, AreH),
.13 (1H, s, AreH), 7.05 (1H, d, J ¼ 8.4 Hz, AreH), 7.03 (2H, d,
J ¼ 8.4 Hz, AreH), 5.53 (1H, d, J ¼ 3.0 Hz, H-10), 5.43 (1H, s, H-12),
.47 (2H, s, ArCH -), 3.21 (4H, s, -NCH -), 2.59 (1H, m, H-9), 2.50
4H, s, -NCH -), 2.19 (1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5),
.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50 (2H, m, H-5 and H-8a),
4.5, 20.6, 13.1. HRMS (ESI) m/z calcd for C33
H
43
N
2
O
6
[MþH]þ
7
36.3116, found 563.3117.
3
(
1
2
2
2
4
.1.2.7. 10-O-(4-(2-(4-(2-methoxyphenyl)piperazin-1-yl)-2-
oxoethyl)phenyl)-(10S)-dihydroartemisinin
(4b). White
solid,
1.39 (2H, m, H-6 and H-8), 1.28 (3H, s, H-14), 1.25e1.07 (2H, m H-5a
ꢃ
0.122 g; yield: 10.3%, mp: 99e102 C; IR (KBr): 2943, 1644, 1509,
and H-7), 0.97 (3H, d, J ¼ 7.2 Hz, H-16), 0.91 (3H, d, J ¼ 6.3 Hz, H-15).
ꢁ1
1
459, 1375, 1241, 1095, 1065, 1035, 979, 959, 877 cm ; MS: 593.3
13
C NMR (DMSO,
d
(ppm)): 156.4, 151.6, 131.8, 130.4, 130.3, 125.7,
þ
þ 1
[
MþH] , 615.3 [MþNa] ; H NMR (DMSO,
d (ppm)): 7.18 (2H, d,
123.9, 119.1, 117.1, 114.8, 111.2, 103.9, 99.8, 87.8, 80.8, 61.8, 52.6, 52.4,
J ¼ 8.7 Hz, AreH), 7.01 (2H, d, J ¼ 8.7 Hz, AreH), 6.95 (2H, m, AreH),
4
8.0, 44.1, 36.9, 36.4, 34.5, 30.8, 26.0, 24.6, 24.4, 20.5, 13.0. HRMS
6
3
.86(2H, m, AreH), 5.50 (1H, d, J ¼ 3.3 Hz, H-10), 5.43 (1H, s, H-12),
.78 (3H, s, -OCH ), 3.69 (2H, s, ArCH -), 3.61 (4H, m, -NCH -), 2.88
-), 2.58 (1H, m, H-9), 2.19 (1H, m, H-4), 2.03e1.90 (2H,
(
ESI) m/z calcd for
C H
33 42
3
F N
2
O
5
[MþH]þ 603.3040, found
3
2
2
6
03.3046.
(4H, m, -NCH
2
m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50 (2H, m,
H-5 and H-8a), 1.39 (2H, m, H-6 and H-8), 1.28 (3H, s, H-14),
4
.1.2.4. 10-O-(4-((4-(4-fluorophenyl)piperazin-1-yl)methyl)phenyl)-
(
10S)- dihydroartemisinin (3d). White solid, 0.120 g; yield: 10.9%,
1
.25e1.07 (2H, m H-5a and H-7), 0.96 (3H, d, J ¼ 7.2 Hz, H-16), 0.90
ꢃ
mp: 71e73 C; IR (KBr): 3429, 2972, 1610, 1511, 1453, 1384, 1229,
13
(
3H, d, J ¼ 6.3 Hz, H-15). C NMR (DMSO,
d (ppm)): 169.4, 155.9,
ꢁ1
þ
þ
1
094, 1037, 979, 958, 877 cm ; MS: 553.5 [MþH] , 575.4 [MþNa] ,
1
8
3
C
52.4, 141.1, 130.3, 129.7, 123.2, 121.2, 118.6, 117.4, 112.3, 103.9, 99.9,
þ
1
1
106.1 [2MþH] ; H NMR (DMSO,
d
(ppm)): 7.26 (2H, d, J ¼ 8.7 Hz,
7.8, 80.8, 55.7, 52.4, 50.9, 50.4, 46.1, 44.1, 41.8, 36.9, 36.4, 34.5, 30.8,
0.0, 26.0, 24.5, 24.4, 20.6, 13.0. HRMS (ESI) m/z calcd for
AreH), 7.04 (2H, d, J ¼ 8.4 Hz, AreH), 7.01 (2H, d, J ¼ 8.7 Hz, AreH),
6
.93 (2H, d, J ¼ 8.4 Hz, AreH), 5.53 (1H, d, J ¼ 3.3 Hz, H-10), 5.43
[MþH]þ 593.3221, found 593.3216.
34 45 2 7
H N O
(
1H, s, H-12), 3.46 (2H, s, ArCH -), 3.06 (4H, s, -NCH -), 2.60 (1H, m,
-), 2.19 (1H, m, H-4), 2.03e1.90 (2H, m, H-4
2
2
H-9), 2.50 (4H, s, -NCH
2
and H-5), 1.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50 (2H, m, H-5
and H-8a), 1.39 (2H, m, H-6 and H-8), 1.29 (3H, s, H-14), 1.25e1.07
4.1.2.8. 10-O-(4-(2-(4-(3-trifluoromethylphenyl)piperazin-1-yl)-2-
oxoethyl)phenyl)-(10S)- dihydroartemisinin (4c). White solid,
0.134 g; yield: 10.4%, mp: 96e97 C; IR (KBr): 2945, 1646, 1610,
ꢃ
(
2H, m H-5a and H-7), 0.97 (3H, d, J ¼ 7.2 Hz, H-16), 0.91 (3H, d,
13
J ¼ 6.3 Hz, H-15). C NMR (DMSO,
d
(ppm)): 156.7, 156.0, 155.1,
1510, 1450, 1348, 1309, 1231, 1121, 1095, 1075, 1035, 979, 958,
ꢁ
1
þ 1
1
47.9, 130.0, 117.1, 117.0, 116.7, 115.2, 115.1, 103.5, 99.4, 87.4, 80.4,
877 cm ; MS: 631.3 [MþH] ; H NMR (DMSO,
d (ppm)): 7.43 (1H,
6
1.4, 52.4, 52.0, 49.0, 43.7, 36.5, 35.9, 34.1, 30.4, 25.6, 24.1, 24.0, 20.1,
t, AreH), 7.20 (4H, m, AreH), 7.09 (1H, d, J ¼ 7.6 Hz, AreH), 7.01 (2H,
d, J ¼ 8.4 Hz, AreH), 5.51 (1H, d, J ¼ 3.2 Hz, H-10), 5.42 (1H, s, H-12),
12.6. HRMS (ESI) m/z calcd for C32
H42FN
2
O
5
[MþH]þ 553.3072,
found 553.3080.
2 2 2
3.71 (2H, s, ArCH -), 3.63 (4H, m, -NCH -), 3.19 (4H, m, -NCH -), 2.58
(1H, m, H-9), 2.18 (1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5),
4
.1.2.5. 10-O-(4-((4-(pyridin-2-yl)piperazin-1-yl)methyl)phenyl)-
1.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50 (2H, m, H-5 and H-8a),
1.39e1.36 (2H, m, H-6 and H-8), 1.28 (3H, s, H-14), 1.25e1.07 (2H, m
(
10S)- dihydroartemisinin (3e). White solid, 0.095 g; yield: 8.9%,
ꢃ
mp: 68e70 C; IR (KBr): 3437, 2944, 1596, 1509, 1481, 1440, 1384,
H-5a and H-7), 0.96 (3H, d, J ¼ 7.2 Hz, H-16), 0.90 (3H, d, J ¼ 6.3 Hz,
ꢁ
1
þ
1
13
1
(
7
(
224, 1094, 1034, 978, 941, 877 cm ; MS: 536.3 [MþH] ; H NMR
DMSO,
(ppm)): 8.08 (1H, d, J ¼ 1.5 Hz, AreH), 7.51 (1H, m, AreH),
.26 (2H, d, J ¼ 8.4 Hz, AreH), 7.03 (2H, d, J ¼ 8.4 Hz, AreH), 6.78
1H, d, J ¼ 8.4 Hz, AreH), 6.62 (1H, m, AreH), 5.53 (1H, d, J ¼ 3.3 Hz,
H-10), 5.43 (1H, s, H-12), 3.45 (2H, s, ArCH -), 3.41 (4H, s, -NCH -),
H-15). C NMR (DMSO,
d
(ppm)): 169.5, 155.9, 151.3, 130.4, 130.3,
d
129.6, 125.7, 123.9, 119.4, 117.4, 115.3, 111.6, 103.9, 99.9, 87.7, 80.8,
52.4, 48.3, 47.9, 44.1, 41.2, 36.9, 36.3, 34.5, 30.8, 26.0, 24.5, 24.4, 20.5,
þ
13.0. HRMS (ESI) m/z calcd for C34
found 653.2801.
H
41
F
3
N
2
O
6
Na [MþNa] 653.2809,
2
2

H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
727
4
.1.2.9. 10-O-(4-(2-(4-(4-fluorophenyl)piperazin-1-yl)-2-oxoethyl)
1.67e1.50 (2H, m, H-5 and H-8a), 1.39e1.35 (2H, m, H-6 and H-8),
1.29 (3H, s, H-14), 1.25e1.07 (2H, m H-5a and H-7), 0.96 (3H, d,
phenyl)-(10S)-dihydroartemisinin (4d). White solid, 0.172 g; yield:
ꢃ
13
14.8%, mp: 92e94 C; IR (KBr): 3441, 2922, 2873, 1645, 1510, 1452,
J ¼ 7.2 Hz, H-16), 0.90 (3H, d, J ¼ 6.0 Hz, H-15). C NMR (DMSO,
ꢁ1
1380, 1231, 1095, 1035, 979, 959, 939, 876, 827 cm ; MS: 581.8
d (ppm)): 165.0, 158.4, 152.4, 141.7, 141.2, 129.9, 129.4, 123.2, 121.2,
þ
þ
1
[
MþH] , 603.8[MþNa] ; H NMR (DMSO,
d
(ppm)): 7.17 (2H, d,
118.7, 117.4, 116.4, 112.3, 104.0, 99.5, 87.8, 80.7, 55.7, 52.4, 51.2, 50.6,
44.0, 36.9, 36.3, 34.5, 30.7, 26.0, 24.5, 24.3, 20.5,12.9. HRMS (ESI) m/
J ¼ 8.4 Hz, AreH), 7.00 (4H, m, AreH), 6.98 (2H, d, J ¼ 8.4 Hz, AreH),
5
.50 (1H, d, J ¼ 3.3 Hz, H-10), 5.42(1H, s, H-12), 3.70 (2H, s, ArCH
2
-),
z calcd for C35
H
45
N
2
O
7
[MþH]þ 605.3221, found 605.3218.
3
.61 (4H, m, -NCH -), 3.00 (4H, m, -NCH -), 2.57 (1H, m, H-9), 2.19
2
2
(
1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81 (2H, m, H-7
and H-8), 1.66e1.50 (2H, m, H-5 and H-8a), 1.38 (2H, m, H-6 and H-
), 1.28 (3H, s, H-14), 1.25e1.07 (2H, m H-5a and H-7), 0.96(3H, d,
4.1.2.13. 10-O-((E)-4-(3-oxo-3-(4-(3-trifluoromethylphenyl)piper-
8
azin-1-yl)prop-1-enyl)phenyl)-(10S)-dihydro-
White solid, 0.114 g; yield: 8.9%, mp: 100e102 C; IR (KBr): 2941,
artemisinin
(5c).
13
ꢃ
J ¼ 7.2 Hz, H-16), 0.90 (3H, d, J ¼ 6.3 Hz, H-15). C NMR (DMSO,
d
(ppm)): 169.4, 157.4, 155.9, 148.1, 148.0, 130.3, 129.6, 118.1, 118.0,
2872, 1649, 1602, 1509, 1448, 1349, 1231, 1122, 1097, 1035, 978, 956,
ꢁ
1
þ
þ
þ 1
117.4, 115.8, 115.6, 103.9, 99.9, 87.8, 80.8, 52.4, 49.9, 49.4, 44.1, 41.4,
876 cm ; MS: 643.4 [MþH] , 665.4 [MþNa] , 681.4 [MþK] ;
H
3
6.9, 36.4, 34.5, 30.8, 26.0, 24.5, 24.4, 20.5, 13.0. HRMS (ESI) m/z
NMR (DMSO,
d
(ppm)): 7.70 (2H, d, J ¼ 8.7 Hz, AreH), 7.49 (1H, d,
calcd for C33
H42FN
2
O
6
[MþH]þ 581.3021, found 581.3045.
J ¼ 15.3 Hz, -CH]CH-), 7.20 (1H, d, J ¼ 15.3 Hz, -CH]CH-), 7.08 (4H,
m, AreH), 7.00 (2H, m, AreH), 5.64 (1H, d, J ¼ 3.0 Hz, H-10), 5.41
4
.1.2.10. 10-O-(4-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)
2 2
(1H, s, H-12), 3.78 (4H, d, -NCH -), 3.10 (4H, s, -NCH -), 2.60 (1H, m,
phenyl)-(10S)-dihydroartemisinin (4e). White solid, 0.168 g; yield:
H-9), 2.20 (1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81
(2H, m, H-7 and H-8), 1.67e1.50 (2H, m, H-5 and H-8a), 1.39e1.35
ꢃ
1
4.9%, mp: 116e118 C; IR (KBr): 2922, 2872, 1646, 1593, 1510, 1480,
ꢁ1
1435, 1376, 1230, 1095, 1035, 979, 959, 877 cm ; MS: 564.8
(2H, m, H-6 and H-8), 1.29 (3H, s, H-14), 1.25e1.10 (2H, m H-5a and
þ
1
13
[
MþH] ; H NMR (DMSO,
d
(ppm)): 8.11 (1H, m, AreH), 7.56 (1H, m,
H-7), 0.98 (3H, d, J ¼ 7.5 Hz, H-16), 0.92 (3H, d, J ¼ 6.3 Hz, H-15).
C
AreH), 7.19 (2H, d, J ¼ 8.4 Hz, AreH), 6.99 (2H, d, J ¼ 8.4 Hz, AreH),
NMR (DMSO,
d
(ppm)): 165.2, 158.5, 151.5, 141.9, 130.5, 130.0, 129.4,
6
1
3
.80 (1H, m, AreH), 6.65 (1H, m, AreH), 5.50 (1H, d, J ¼ 3.0 Hz, H-
0), 5.42 (1H, s, H-12), 3.71 (2H, s, ArCH -), 3.58 (4H, m, -NCH -),
-), 2.57 (1H, m, H-9), 2.19 (1H, m, H-4), 2.03e1.90
126.2, 123.5, 119.5, 117.5, 116.4, 115.4, 111.7, 104.0, 99.6, 87.9, 80.8,
52.5, 48.7, 48.1, 44.1, 37.0, 36.4, 34.6, 30.8, 26.0, 24.6, 24.4, 20.6, 13.0.
2
2
.45 (4H, m, -NCH
2
HRMS (ESI) m/z calcd for C35
643.3001.
H F
42 3
N
2
O
6
[MþH]þ 643.2989, found
(
2H, m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8), 1.66e1.50
(
2H, m, H-5 and H-8a), 1.38 (2H, m, H-6 and H-8), 1.28 (3H, s, H-14),
1
.25e1.07 (2H, m H-5a and H-7), 0.96 (3H, d, J ¼ 7.2 Hz, H-16), 0.90
13
(
3H, d, J ¼ 6.0 Hz, H-15). C NMR (DMSO,
d
(ppm)): 169.6, 159.1,
4.1.2.14. 10-O-((E)-4-(3-oxo-3-(4-fluorophenyl)piperazin-1-yl)prop-
1-enyl)phenyl)-(10S)-dihydroartemisinin (5d). Yellow solid, 0.245 g;
1
8
2
5
55.9, 147.9, 138.0, 130.3, 129.6, 117.4, 113.6, 107.6, 103.9, 99.9, 87.8,
ꢃ
0.8, 52.4, 45.4, 44.8, 44.1, 41.2, 36.9, 36.4, 34.5, 30.8, 26.0, 24.5,
yield: 20.7%, mp: 88e89 C; IR (KBr): 2926, 1647, 1602, 1509, 1436,
[MþH]þ
1375, 1229,1095,1035, 978, 957, 877, 829 cm ; MS: 593.3 [MþH] ;
ꢁ1
þ
4.4, 20.6, 13.0. HRMS (ESI) m/z calcd for C32
H
42
N
3
O
6
1
64.3068, found 564.3066.
H NMR (DMSO,
d
(ppm)): 7.70 (2H, d, J ¼ 8.7 Hz, AreH), 7.49 (1H, d,
J ¼ 15.2 Hz, -CH]CH-), 7.20 (1H, d, J ¼ 15.2 Hz, -CH]CH-), 7.08 (4H,
4.1.2.11. 10-O-((E)-4-(3-oxo-3-(4-phenylpiperazin-1-yl)prop-1-enyl)
m, AreH), 7.01 (2H, m, AreH), 5.64 (1H, d, J ¼ 3.0 Hz, H-10), 5.41
phenyl)-(10S)-dihydro artemisinin (5a). White solid, 0.238 g; yield:
2 2
(1H, s, H-12), 3.78 (4H, d, -NCH -), 3.10 (4H, s, -NCH -), 2.60 (1H, m,
ꢃ
2
1
[
0.7%, mp: 88e89 C; IR (KBr): 3444, 2922, 2872, 1731, 1647, 1602,
H-9), 2.20 (1H, m, H-4), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81
(2H, m, H-7 and H-8), 1.67e1.50 (2H, m, H-5 and H-8a), 1.39e1.35
(2H, m, H-6 and H-8), 1.28 (3H, s, H-14), 1.25e1.07 (2H, m H-5a and
ꢁ1
508, 1384, 1210, 1035, 957, 876, 830, 742, 700 cm ; MS: 575.4
þ
þ 1
MþH] , 597.3 [MþNa] ; H NMR (DMSO,
d (ppm)): 7.71 (2H, d,
13
J ¼ 8.4 Hz, AreH), 7.50 (1H, d, J ¼ 15.3 Hz, -CH]CH-), 7.24 (2H, m,
AreH), 7.20 (1H, d, J ¼ 15.3 Hz, -CH]CH-),7.11 (2H, d, J ¼ 8.4 Hz,
AreH), 6.97 (2H, d, J ¼ 8.1 Hz, AreH), 6.81 (1H, t, AreH), 5.64 (1H, d,
H-7), 0.98 (3H, d, J ¼ 7.5 Hz, H-16), 0.92 (3H, d, J ¼ 6.3 Hz, H-15).
C
NMR (DMSO, (ppm)): 165.0, 158.4, 155.9, 148.1, 141.8, 130.0, 129.4,
d
118.2, 117.4, 116.4, 115.6, 104.0, 99.5, 87.8, 80.7, 52.4, 50.2, 49.6, 44.0,
36.9, 36.3, 34.5, 30.7, 26.0, 24.5, 24.3, 20.5, 12.9. HRMS (ESI) m/z
J ¼ 3.3 Hz, H-10), 5.41 (1H, s, H-12), 3.84e3.73 (4H, m, -NCH
2
-), 3.17
(
4H, s, -NCH
2
-), 2.62 (1H, m, H-9), 2.20 (1H, m, H-4), 2.03e1.90 (2H,
calcd for C34
H42FN
2
O
6
[MþH]þ 593.3021, found 593.3019.
m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8), 1.67e1.50 (2H, m,
H-5 and H-8a), 1.39e1.35 (2H, m, H-6 and H-8), 1.29 (3H, s, H-14),
1
.25e1.07 (2H, m H-5a and H-7), 0.97 (3H, d, J ¼ 6.9 Hz, H-16), 0.91
4.1.2.15. 10-O-((E)-4-(3-oxo-3-(4-(pyridin-2-yl)piperazin-1-yl)prop-
1-enyl)phenyl)-(10S)-dihydroartemisinin (5e). White solid, 0.094 g;
13
(
3H, d, J ¼ 5.7 Hz, H-15). C NMR (DMSO,
d
(ppm)): 165.0, 158.4,
ꢃ
1
8
2
51.2, 141.8, 130.0, 129.4, 119.7, 117.4, 116.4, 116.2, 104.0, 99.5, 87.8,
yield: 8.2%, mp: 67e69 C; IR (KBr): 2943, 2859, 1726, 1647, 1598,
ꢁ1
0.7, 52.4, 49.4, 48.8, 44.0, 36.9, 36.3, 34.5, 30.7, 26.0, 24.5, 24.3,
1509, 1481, 1435, 1375, 1229, 1207, 1097, 1037, 979, 957, 876 cm
;
þ
þ
þ 1
0.5, 12.9. HRMS (ESI) m/z calcd for C34
H
43
N
2
O
6
[MþH] 575.3116,
MS: 576.3 [MþH] ,1152.5 [2MþH] ; H NMR (CDCl
(1H, d, J ¼ 3.3 Hz, AreH), 7.72 (1H, d, J ¼ 15.3 Hz, -CH]CH-), 7.57
1H, d, J ¼ 15.3 Hz, -CH]CH-), 7.54 (3H, m, AreH), 7.14 (2H, d,
J ¼ 8.4 Hz, AreH), 6.69 (2H, d, J ¼ 8.4 Hz, AreH), 5.56 (1H, d,
J ¼ 3.0 Hz, H-10), 5.48 (1H, s, H-12), 3.85 (4H, m, -NCH -), 3.65 (4H,
m, -NCH -), 2.84 (1H, m, H-9), 2.40 (1H, m, H-4), 2.24e2.18 (1H, m,
3
, d (ppm)): 8.23
found 575.3133.
(
4.1.2.12. 10-O-((E)-4-(3-oxo-3-(4-(2-methoxyphenyl)piperazin-1-yl)
prop-1-enyl)phenyl)-(10S)-dihydro- artemisinin (5b). White solid,
0
2
ꢃ
.115 g; yield: 9.5%, mp: 144e146 C; IR (KBr): 2941, 2845, 1646,
2
ꢁ
1
1
6
d
599, 1508, 1435, 1374, 1227, 1097, 1036, 979, 955, 876 cm ; MS:
H-4), 2.07e1.80 (3H, m, H-5, H-7 and H-8), 1.76e1.70 (2H, m, H-5
and H-8a), 1.62 (2H, m, H-6 and H-8), 1.46 (3H, s, H-14), 1.34e1.27
(2H, m H-5a and H-7), 0.96 (3H, d, J ¼ 7.2 Hz, H-16), 0.91 (3H, d,
þ
þ
þ 1
05.3 [MþH] , 627.3 [MþNa] , 1029.7 [2MþH] ; H NMR (DMSO,
(ppm)): 7.69 (2H, d, J ¼ 8.4 Hz, AreH), 7.47 (1H, d, J ¼ 15.3 Hz,
1
3
-
CH]CH-), 7.18 (1H, d, J ¼ 15.3 Hz, -CH]CH-), 7.10 (2H, d,
J ¼ 6.0 Hz, H-15). C NMR (DMSO,
d (ppm)): 167.3, 159.1, 158.4,
J ¼ 8.4 Hz, AreH), 6.96 (2H, m, AreH), 6.90 (2H, m, AreH), 5.64 (1H,
d, J ¼ 2.4 Hz, H-10), 5.41 (1H, s, H-12), 3.80 (3H, s, OCH ), 3.71 (4H,
m, -NCH -), 2.97 (4H, s, -NCH -), 2.61 (1H, m, H-9), 2.19 (1H, m, H-
), 2.03e1.90 (2H, m, H-4 and H-5), 1.85e1.81 (2H, m, H-7 and H-8),
148.0, 141.8, 138.0, 130.0, 122.6, 117.4, 116.4, 113.6, 107.6, 104.0, 87.8,
80.7, 52.4, 45.4, 44.9, 44.0, 36.9, 36.3, 34.5, 30.7, 26.0, 24.5, 24.3,
20.5, 12.9. HRMS (ESI) m/z calcd for C33
3
H
42
N
3
O
6
[MþH]þ 576.3068,
2
2
4
found 576.3061.

728
H. Zhong et al. / European Journal of Medicinal Chemistry 108 (2016) 720e729
4.2. Biological assays
aprotinin (pH, 8.0)] were separated on 10% or 15% sodium dodecyl
sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose
membranes. After blocking with 5% nonfat milk, the membranes
loading protein were incubated with specific antibodies overnight
at 4 C. Immunocomplexes were visualized using enhanced
chemiluminescence Western blotting detection reagents (Amer-
sham Biosciences Inc., Piscataway, NJ) [29].
Reagents and chemicals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), Quinidine, Metoprolol and
Atenolol were obtained from Sigma and penicillin/streptomycin
was purchased from Invitrogen. Fetal bovine serum (FBS), phos-
phate buffered saline (PBS), DMEM and trypsin were purchased
from Biosera. Adriamycin and dimethyl sulfoxide were obtained
from EBEWE Pharma and Merck, respectively. Propidium iodide (PI)
was purchased from BD Biosciences (San Diego, CA). Antibodies to
ꢃ
Acknowledgments
Parp, p27, cyclin D1, cyclin B1 and
Cruz Biotechology, Inc. (Santa Cruz, CA).
b-actin were obtained from Santa
This work was supported by the National Major Scientific and
Technological Special Project for ‘Significant New Drugs Develop-
ment’ during the Twelve Five-year Plan Period (grant no.
4.2.1. The antiproliferative activity assay
2012ZX09103101-023) and National Science Foundation of China
Cell cultures. MCF-7 and MCF-7/Adr cell lines were maintained
(81072533). All authors would like to thank Tripos for offering
ꢃ
at 37 C in humidified air containing 5% CO
2
. Both cell lines were
software for free.
maintained in DMEM supplemented with 10% FBS, and 100 units/
ml penicillin-G and 100 g/ml streptomycin and 0.37% NaHCO
Method. MCF-7 and MCF-7/Adr cells were planted into 96-well
m
3
.
Appendix A. Supplementary data
4
micro-plates at a density of 1.5e4.0 ꢀ 10 cells/mL (100
mL per
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.040.
ꢃ
well). After overnight incubation at 37 C, 5e9 different concen-
trations of test compounds were added to the wells. Compounds
were dissolved in DMSO before being diluted in the growth me-
dium. The concentration of DMSO in the wells did not exceed 0.5%.
cells were further incubated for 96 h, at the end of the incubation
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