Study on the structure-activity relationship of dihydroartemisinin derivatives: Discovery, synthesis, and biological evaluation of dihydroartemisinin-bile acid conjugates as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2021.113754

A series of dihydroartemisinin derivatives was synthesized, and their anti-proliferation activity against cancer cells was evaluated. Structure-activity relationship studies led to the discovery of dihydroartemisinin-bile acid conjugates that exhibit broa

Full text of DOI:10.1016/j.ejmech.2021.113754

European Journal of Medicinal Chemistry 225 (2021) 113754
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Study on the structure-activity relationship of dihydroartemisinin
derivatives: Discovery, synthesis, and biological evaluation of
dihydroartemisinin-bile acid conjugates as potential anticancer agents
Xiaosu Zou 1, Chang Liu c 1, Congcong Li , Rong Fu , Wei Xu , Hongzhu Bian ,
b
,
,
b
,
1
a
b
d
Xun Dong , Xiaozhen Zhao , Zhenye Xu , Jinghua Zhang a , Zhengwu Shen a
School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing Road, Shanghai, 200025, China
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 CaiLun Road, Shanghai, 201203, China
Department of Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South WanPing Road, Shanghai, 200032, China
Yunnan Baiyao Group Co. Ltd, 3686 Yunnan Baiyao Street, Kunming, 650200, China
d
c
c
,
*
,
**
, b, ***
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2021
Received in revised form
A series of dihydroartemisinin derivatives was synthesized, and their anti-proliferation activity against
cancer cells was evaluated. Structure-activity relationship studies led to the discovery of
dihydroartemisinin-bile acid conjugates that exhibit broad-spectrum anti-proliferation activities. Among
them, the dihydroartemisinin-ursodeoxycholic acid conjugate (49) was the most potent, with IC50 values
5
August 2021
Accepted 5 August 2021
Available online 9 August 2021
between 0.04 and 0.96 mM when tested to determine its inhibitory properties against 15 various cancer
cell lines. In vivo experiments showed that compound 49 effectively suppressed tumor growth in an
A549 cell xenograft model at the dosage of 10 mg/kg body weight and in Lewis lung cancer cell trans-
plant model at the dosage of 12 mg/kg body weight.
Keywords:
Artemisinin
Bile acid
©
2021 Elsevier Masson SAS. All rights reserved.
Ursodeoxycholic acid
Conjugates
Anticancer agent
1
. Introduction
proliferation activities, with half-maximal inhibitory concentra-
tion (IC50) values at the nano-molar level. However, a lack of in vivo
activity was observed when these compounds were tested [16].
Because the anti-cancer mechanism of artemisinin derivatives is
still unclear, the research methods and cancer cell lines used by
different research groups have been different, and thus, it is difficult
to summarize a clear structure-activity relationship for artemisinin
derivatives. However, based on the large number of artemisinin
derivatives that have been reported to date, it was suggested that
the dihydroartemisinin (DHA) dimers or trimers may have higher
in vitro anti-cancer activity than that of monomers [16]. To validate
this preliminary assumption, we decided to initiate a structure-
activity relationship study of DHA dimers in order to discover a
more optimal and effective anticancer artemisinin derivative.
Since Deng et al. reported in the early nineties that artemisinin-
based derivatives selectively inhibit the proliferation of P388 leu-
kemia cells [1], a large number of studies have reported that many
different types of artemisinin derivatives exhibit anti-cancer ac-
tivity [2e24]. Despite their ambiguous mechanism of action, no
artemisinin-based anticancer drugs have been approved for the
market thus far [25e27]. However, many medicinal chemists
continue to study the anti-cancer properties of artemisinin-based
compounds due to their unique structure and low toxicity.
Among the numerous known artemisinin derivatives, there are
no shortages of compounds with strong anti-cancer activity. As
illustrated in Fig. 1, compounds 2e5 exhibited excellent anti-
*
Corresponding author.
*
*
* Corresponding author.
** Corresponding author.School of Medicine, Shanghai Jiao Tong University, 280 South Chongqing Road, Shanghai, 200025, China.
E-mail addresses: xuzhenye1947@126.com (Z. Xu), 183930@shsmu.edu.cn (J. Zhang), Jeff_shen_1999@yahoo.com, Jeff.shen@sjtu.edu.cn (Z. Shen).
These authors contributed equally.
1
https://doi.org/10.1016/j.ejmech.2021.113754
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Fig. 1. Artemisinin and anti-cancer artemisinin derivatives [10,28e30].
2
. Results and discussion
upon the different cancer cell lines being tested (6 vs 7, 8, and
; 12 vs 13).
9
2.1. Structure-activity relationship studies of dihydroartemisinin
dimers
In order to further evaluate the structure-activity relationship of
the linker of DHA dimers, as depicted in Fig. 3, fifteen DHA dimers
were synthesized. Among them, compounds 23 and 24 were
already known [10]. The linkers of compounds 24e29 are linear
and flexible, while the linkers of compounds 31e36 contain aro-
matic rings or steroid moieties that limit the conformational
change of the molecule and are considered as rigid linkers. Com-
pounds 30 and 37 were synthesized as comparators of the corre-
sponding dimers. The rigid structure of the aromatic ring or
steroids may allow us to identify the optimized spatial distance of
two DHA units in DHA dimers. In addition, it was reported that the
DHA-cholate conjugates (38e46) exhibited satisfactory anti-cancer
activity (Fig. 4) [35]. Among them, compound 43 is the most active,
with the IC50 at 19 nM against CCRF-CEM cells and at 340 nM
against CEM/ADR5000 cells. It was claimed that this compound was
more potent than clinically used doxorubicin against CEM/
ADR5000 cells. Hence, selecting the steroid as the linker may
enhance the anti-cancer activity of the DHA dimer.
The anti-cancer activity of compounds 23e37 was evaluated
with PC3, HeLa, Jurkat, 786-O, and MCF-7 cancer cell lines. The
results are depicted in Table 2. Compounds 23 and 35 were inactive,
but compounds 24 and 25 were considered to be active. From
compound 26 to compound 29, the activity gradually decreases
with the extension of the carbon chain of the linkers. Therefore, the
preferred length of the linear linkers of DHA dimers should be
between 3 and 5 atoms (the O of the hemiacetal of the DHA unit
was not counted). However, to our surprise, DHA monomer 30 was
more active than the corresponding dimers (31e33). This obser-
vation suggested that the rigid linker of DHA dimers may fix the
spatial distance of the DHA units at an unfavorable position, and
thus, these DHA dimers are less active than their corresponding
monomer. Hence, as expected, the anti-cancer activity of 37 was
significantly higher than that of the corresponding dimers (34e36).
In fact, compound 37 was the most active DHA derivative we tested
thus far. This finding suggests that conjugates consisting of bile
acids and DHA may be new lead anti-cancer compounds.
According to previous studies, DHA dimers or trimers may have
higher anti-cancer activity [9,16,31e34]. In order to validate this
assumption, a group of DHA dimers and trimers was designed and
synthesized, and their in vitro anti-proliferation activity was eval-
uated. As depicted in Fig. 2, compounds 6e9 represent DHA dimers
or trimer with linear and flexible linkers. Because excellent anti-
proliferation activity was reported for compound 6 [31,32], it was
likely that a change in the substitute on the linker could further
improve the activity of the dimers. Compounds 10e13 represent
DHA dimers or trimer with the linker containing a rigid structure.
DHA monomers (14e22) were synthesized as comparators to those
dimers and trimers.
The in vitro anti-cancer activity (IC50) of the above-mentioned
compounds against four cancer cell lines (HeLa, K562, HepG2,
H322) was measured. As depicted in Table 1, compound 6 exhibited
satisfactory anti-proliferation activities. However, an additional
methyl, hydroxyethylene, or a third DHA unit substituted on the
nitrogen atom of the linker of compound 6 did not improve the anti-
cancer activity (entries 7, 8, and 9), except that DHA trimer (9) only
showed higher activity against K562 cells. Compound 10 and com-
pound 11 exhibit similar anti-proliferation activities, although 10 is a
DHA dimer, while 11 is a DHA trimer. Compounds 12 and 13 are DHA
dimers with 3, 5-bis-(oxymethylene)benzene as the linker, and the
only difference between them is the 1-substituent (-COOH vs eOH)
on the benzene ring. The activity of compound 12 is 10-fold stronger
in inhibiting K562 cells than that of compound 13, while compound
13 is three-fold more active than that of compound 12 in inhibiting
HeLa cells. Based on the above discussion and a simple structure-
activity comparison between corresponding monomers and di-
mers, we came to the following initial conclusions:
(
1) DHA dimers are more effective in inhibiting the proliferation
of cancer cells than their corresponding artemisinin mono-
mers (10 vs 18; 12 vs 19; 13 vs 20). However, compared with
DHA dimers, DHA trimers did not exhibit a significant
improvement in activity (8 vs 9; 10 vs 11).
(
2) The structure of the linkers is the most important factor that
determines the activity of DHA dimers. A small change in the
substituent on the linkers will significantly affect the anti-
cancer activity, and there are different impacts depending
2.2. Synthesis of bile acid-DHA conjugates and their anticancer
activity
Based on the results discussed above, deoxycholic acid, cheno-
deoxycholic acid, ursodeoxycholic acid, lithocholic acid, and
2

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Fig. 2. DHA monomers, dimers, and trimers for activity evaluation.
hyodeoxycholic acid as well as other naturally existing steroids
were selected to form conjugates with dihydroartemisinin. The
condensation of DHA and bile acid was highly regio-selective and
highly stereo-selective under the reaction condition. Only 3-OH of
inactive. It was also found that the DHA unit directly condensate to
the 3-OH of bile acids delivered stronger activity than the DHA
units attached to the steroid core through linkers (49 vs 54, 51 vs
55, and 34 vs 35). In addition, the position and the conformation of
the hydroxyl group on the bile acid core affect the activity of the
conjugates.
the bile acid reacted with DHA, and the bile acid-10b-DHA conju-
gate was isolated as the sole product [36]. The absolute stereo-
chemistry of compound 49 was confirmed by X-ray single crystal
diffraction.
For example, if there is one free hydroxyl group on the steroid
skeleton, the anti-cancer activity of the conjugate will follow the
As depicted in Fig. 5, compounds 47e56 were synthesized, and
their in vitro anti-cancer activity against PC3, HeLa, and 786-O cells
was evaluated. The IC50 values of compounds 47e56 are depicted in
Table 3. Compared with DHA, the anti-cancer activity of DHA-bile
acid conjugates was significantly increased. Among these conju-
gates, compounds 47, 49, and 51 are isomers, and exhibit the
strongest anti-cancer activities. Compared with compound 37,
there is one free hydroxyl group on the steroid skeleton for these
three isomers, and the higher lipophilicity leads to increased anti-
cancer activity, but also a further increase in the lipophilicity of
the conjugates. For example, the more lipophilic compound 50 is
less active, while compounds 52, 53, and 56 are completely
order of 7
b
-OH ¼ 12
a-OH > 6a-OH > 7a-OH. However, additional
hydroxyl groups introduced on the steroid (37 vs 47) or further
increases in the lipophilicity of the conjugates (50 vs 48) will
decrease their activity. In order to further assess the conjugates
with stronger activity, namely, compounds 37, 47, 49, and 51, fifteen
cancer cell lines were selected to evaluate their in vitro anti-cancer
activities.
As depicted in Table 4, compounds 47 and 49 exhibited excellent
anti-cancer activity, with more activity than taxol against OV-90
and A549 cancer cell lines. In order to further optimize the struc-
ture, we decided to modify the side chain of the steroids and then
evaluate the activity of these new conjugates.
3

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Table 1
a
In vitro anti-cancer activity evaluation of DHA monomers, dimers, and trimers. .
Compounds
IC50b
HeLa
(mM)
K562
HepG2
H322
6
7
8
9
0.84
8.32
3.72
5.89
1.78
1.15
4.57
1.41
>10
1.70
>10
10.00
>10
>10
>10
>10
>10
0.72
0.79
0.59
0.01
0.58
0.29
<0.01
0.18
>10
3.24
>10
>10
>10
>10
>10
>10
>10
1.51
4.17
1.74
2.51
1.74
1.10
0.87
0.32
>10
7.52
>10
>10
>10
>10
>10
>10
>10
0.79
2.37
1.68
6.61
0.79
1.62
5.50
7.08
>10
6.22
>10
6.73
>10
>10
>10
>10
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
Fig. 4. Previously reported DHA-cholate conjugates [35].
2
.3. Pharmacokinetic study
a
The data shown are mean values.
Concentrations required to inhibit 50% of human tumor cell proliferation after
b
Compound 49 was chosen for in vivo pharmacokinetic studies
7
2 h, IC50 determined by the CCK-8 assay of three repetitions.
because of its excellent cellular activities. Preliminary pharmaco-
kinetic evaluations were conducted in male Sprague-Dawley (SD)
rats. The pharmacokinetic parameters are summarized in Table 6.
Compound 49 displayed acceptable maximum concentration and
oral exposure. In addition, a typical artemisinin-associated short
half-life of 0.8 h and good plasma clearance were observed for 49.
As shown in Fig. 6, we reduced or amidated the 24-carboxylate
of bile acids to obtain new conjugates of DHA with corresponding
4-hydroxyl steroids (57, 58) or 24-amide steroids (63, 64). Further
2
modification of the 24-hydroxyl either by esterification or ammo-
nization yielded 24-ester (59e62) or 24-amine steroid derivatives
2
.4. In vivo anti-cancer effects of DHA-ursodeoxycholic acid
(
65e68). As depicted in Table 5, 24-amine derivatives 65 and 67
conjugate (49)
exhibited a better overall anti-cancer activity against five cell-lines
tested. However, unfortunately, these two compounds showed
higher toxicity on Vero cell. Therefore, compound 49 was selected
for in vivo anti-cancer assessment.
Two tumor animal models (the A549 cell nude mouse xenograft
model and Lewis cell C57 mouse transplant model) were used to
study the in vivo antitumor effect of compound 49. The procedures
for animal use and care were approved by the Animal Care Com-
mittee of Shanghai Jiao Tong University School of Medicine
Fig. 3. DHA dimers with various linkers.
4

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Table 2
Table 3
a
a
In vitro anti-cancer activity of DHA dimers. .
In vitro anti-cancer activity of DHA-steroid conjugates .
Compounds
IC50b
PC3
(
m
M)
Compounds
IC50b(
PC3
mM)
HeLa
Jurkat
786-O
MCF7
HeLa
786-O
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
>10
3.12
1.38
0.97
1.16
0.89
>10
0.88
0.77
2.83
9.48
3.09
>10
>10
0.63
2.49
>10
2.07
2.41
0.72
0.99
0.54
7.17
0.41
0.25
0.64
3.97
7.17
>10
>10
0.39
2.66
<0.01
>10
3.64
0.78
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.15
5.21
<0.01
>10
0.77
0.68
0.13
0.13
0.06
7.30
0.09
0.48
4.8
3.08
>10
>10
1.65
0.20
0.63
<0.01
>10
>10
7.9
37
47
48
49
50
51
52
53
54
55
56
DHA
Taxol
0.63
0.01
1.56
0.26
0.97
0.53
>10
>10
1.05
0.80
>10
2.49
0.39
0.45
0.72
0.04
3.90
0.22
>10
>10
1.36
1.01
>10
2.66
<0.01
0.20
0.01
0.97
0.06
1.80
0.03
>10
>10
0.11
0.09
>10
0.63
<0.01
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.13
6.88
<0.01
<0.01
a
The data shown are mean values.
b
Concentrations required to inhibit 50% of human tumor cell proliferation after
2 h, IC50 determined by the CCK-8 assay of three repetitions.
DHA
Taxol
7
<0.01
a
The data shown are mean values.
b
Concentrations required to inhibit 50% of human tumor cell proliferation after
2 h, IC50 determined by the CCK-8 assay of three repetitions.
7
examined the concentration of the immune cell population and the
expression levels of immune checkpoint receptors. We collected
blood samples of 200 mL/mouse, and spleens from the above in vivo
(
approval number: A-2018-002). The A549 cell xenograft model
showed that compound 49 significantly reduced tumor growth,
with an inhibitory rate of 50.24% (n ¼ 6, 10 mg/kg body weight,
Fig. 7A). In the Lewis cell C57 mouse transplant model, when
compound 49 was administered at the dosage of 12 mg/kg, a
experiment (Section 2.4) were collected, labeled with antibodies,
and then subjected to flow cytometry analysis. The flow cytometry
analysis indicated that compound 49 significantly increased the
þ
concentration of CD3 T cells and decreased the concentration of
þ
4
4.90% inhibitory rate was observed, which was significantly
CD19 B cells. The FOXP3 experiment indicated that compound 49
þ
þ
higher than that of the taxol group (14.91%, 10 mg/kg, P < 0.05,
n ¼ 10, Fig. 7B).
increased the concentration of CD4 FOXP3 Treg cells in both
blood and spleen. In addition, there was a significant increase in
þ
þ
þ
þ
CD4 PD-1 and CD4 CD28 , which indicated that compound 49
may have synergistic effects with PD-1 inhibitors (Fig. 8). Com-
pound 49 affected the proportion of immune cells in mice, but
whether this effect indicates that compound 49 can exert strong
anti-tumor action requires further verification.
2.5. Flow cytometry analysis
To determine whether compound 49 can change the proportion
of immune cells in the Lewis cell C57 mouse transplant model, we
Fig. 5. DHA-steroid conjugates.
5

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
þ
Table 4
compound 49 significantly increased the concentration of CD3
(
a
The in vitro anti-cancer activity of DHA-bile acid conjugates .
þ
P < 0.05) and decreased the concentration of CD19 (P < 0.01). The
Cell lines
IC50b(
Taxol
m
M)
FOXP3 experiment showed that compound 49 significantly
increased the concentration of Tregs (P < 0.01). Immunotherapy is
an important means of tumor treatment in clinical practice, and
this finding indicates that the DHA-ursodeoxycholic acid conjugate
may potentially be applied for the treatment of autoimmune-
related diseases as well.
37
47
49
51
K562
NCleH446
OV-90
0.01
0.15
5.19
0.02
0.01
0.10
0.06
0.03
0.01
0.01
2.40
0.61
0.42
0.01
0.62
0.16
0.12
0.12
0.26
0.38
0.67
2.20
0.34
0.69
0.16
0.57
4.34
2.02
0.39
0.39
0.06
0.07
0.05
0.22
0.28
0.16
0.68
0.26
0.29
0.37
0.29
0.77
0.60
0.45
0.77
0.06
0.05
0.06
0.13
0.17
0.38
0.96
0.33
0.42
0.06
0.05
0.93
0.85
0.04
0.93
0.09
0.11
0.12
0.10
0.36
0.32
1.78
0.27
0.42
0.50
0.05
0.65
1.22
0.22
1.22
HCT116
HepG2
RCC-4
RCC4-VHL
Ue2OS
U-87MG
Kasumi-1
A549
4. Experimental Section
4.1. Material and methods
Jurkat
All reactions were performed in glassware containing a Teflon-
coated stirring bar. Solvents and chemical reagents were obtained
from commercial sources and used without further purification. H
786-O
HeLa
MCF
1
13
NMR and C NMR spectral data were recorded in CDCl
DMSO‑d on a Bruker Avance III 400 M spectrometer. Chemical
shifts ( ) are reported in ppm downfield from an internal tetra-
3
or
a
The data shown are mean values.
6
b
Concentrations required to inhibit 50% of human tumor cell proliferation after
2 h, IC50 determined by the CCK-8 assay of three repetitions.
d
7
methylsilane (TMS) standard. Coupling constants are reported in
units of hertz (J value, Hz; integration and splitting patterns: where
s ¼ singlet, d ¼ double, t ¼ triplet, q ¼ quartet, brs ¼ broad singlet,
and m ¼ multiple). Low- and high-resolution mass spectra were
obtained in electrospray ionization (ESI) mode. Flash column
chromatography on silica gel (200e300 mesh) was used for the
routine purification of reaction products. The column output was
monitored by thin-layer chromatography (TLC) on silica gel
(200e300 mesh) precoated on glass plates, and spots were visu-
alized by ultraviolet (UV) light at 254 nm or 5% phosphomolybdic
acid in EtOH with subsequent heating. The specific chemical syn-
thesis of reaction intermediates and compounds is detailed in the
Supporting Information.
3
. Conclusions
In the process of studying the structure-activity relationship of
DHA dimers, the DHA-bile acid conjugates were found to have
excellent anti-tumor activity. Among them, the DHA-
ursodeoxycholic acid conjugate (49) and DHA-deoxycholic acid
conjugate (47) were the most potent compounds. The IC50 values of
these two compounds against fifteen cancer cells were under 1 mM.
Compound 49 showed good in vivo anti-tumor activity in the
A549 lung cancer cell xenograft model and the Lewis lung cancer
cell transplant model. All of the results demonstrated that com-
pound 49 is a potential anticancer agent that warrants further
study.
Cells were cultured in Dulbecco's modified Eagle's medium
(DMEM) or Roswell Park Memorial Institute (RPMI)-1640 medium
(Hyclone) containing 10% fetal bovine serum (Gibco) and 100 U/mL
Flow cytometry analysis of blood samples indicated that
penicillin (Invitrogen) and 100
mg/mL streptomycin (Invitrogen).
Fig. 6. Conjugates derived from DHA-cholic acid and DHA-deoxycholic acid.
6

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Table 5
NMR (400 MHz, CDCl
)
d
5.39 (s, 2H), 4.80 (d, J ¼ 3.4 Hz, 2H), 3.97
3
In vitro anti-cancer activity of conjugates derived from DHA-cholic acid and DHA-
deoxycholic acid .
(
2
d, J ¼ 9.6 Hz, 2H), 3.73 (d, J ¼ 8.3 Hz, 4H), 3.36 (d, J ¼ 9.6 Hz, 2H),
a
.76-2.61 (m, 2H), 2.39 (td, J ¼ 14.0, 3.9 Hz, 2H), 2.22 (t, J ¼ 5.6 Hz,
Compounds
IC50b(
HeLa
mM)
2H), 2.07-2.00 (m, 2H), 1.90 (m, 2H), 1.85-1.75 (m, 2H), 1.74-1.62 (m,
13
K562
HepG2
H322
786-O
4H), 1.59-1.34 (m, 12H), 1.34-1.22 (m, 4H), 0.95 (m, 12H). C NMR
100 MHz, CDCl 104.18, 102.84, 87.91, 80.96, 68.97, 64.96, 52.54,
4.94, 44.24, 37.39, 36.46, 34.57, 30.89, 26.20, 24.56, 20.40, 13.09.
(
3
) d
37
47
49
57
58
59
60
61
62
63
64
65
66
67
68
0.39
0.45
0.38
0.16
1.26
7.94
0.93
>10
0.08
3.55
>10
0.33
0.19
0.58
0.98
0.16
0.07
<0.01
<0.01
0.16
<0.01
0.39
0.06
0.04
0.6
0.38
0.28
2.45
1.29
1.74
1.68
3.24
>10
1.51
4.57
>10
0.56
0.72
0.84
0.81
6.56
5.28
4.22
15.14
7.76
2.79
5.85
>10
>10
7.94
>10
1.05
1.00
1.20
0.94
2.02
0.60
0.32
0.04
1.86
0.17
2.00
0.32
1.12
2.40
>10
0.36
1.17
0.47
1.51
4
4.2.2. Synthesis of compounds 26e29 and 31e33
Under an argon atmosphere, DCC (513 mg, 2 mmol) and DMAP
(13 mg, 0.1 mmol) were added to a solution of dihydroartemisinin
(569 mg, 2 mmol) and dicarboxylic acid analogues (1 mmol) in
dichloromethane (15 mL) at 0 C. The mixture was stirred for 24 h
at room temperature before being filtered. The filtrate was
concentrated under reduced pressure, and the residue was purified
by silica gel column chromatography to obtain the corresponding
compound.
ꢁ
>10
0.13
0.56
0.20
0.58
ꢁ
1
a
Compound 26. White solid. Yield: 21.3%. Mp: 87.4e90.5 C. H
NMR (400 MHz, CDCl
5.79 (d, J ¼ 9.9 Hz, 2H), 5.44 (s, 2H), 2.64-
2.50 (m, 2H), 2.45-2.32 (m, 2H), 2.13 (s, 2H), 2.02 (m, 2H), 1.93-1.86
The data shown are mean values.
Concentrations required to inhibit 50% of human tumor cell proliferation after
b
3
) d
72 h, IC50 determined by the CCK-8 assay of three repetitions.
(m, 2H),1.74 (m, 3H),1.61 (m,1H),1.51-1.39 (m, 7H),1.37 (s,1H),1.32
(
0
m, 3H), 1.28 (s, 3H), 1.25 (s, 6H), 1.20 (m, 2H), 0.97-0.94 (m, 4H),
Cell Counting Kit-8 (Dojindo) was used to evaluate the anti-
proliferation activities of the compounds.
13
13
.85 (m, 4H). C NMR (100 MHz, CDCl
169.88, 104.51, 91.88, 91.52, 80.16, 51.58, 45.2, 44.63, 37.30,
6.23, 34.10, 31.78, 29.72, 26.00, 24.60, 22.02, 21.16, 20.24, 12.11.
3
)
d
C NMR (100 MHz,
3
CDCl ) d
3
ꢁ
1
4
4
.2. General synthesis procedure
Compound 27. White solid. Yield: 41.3%. Mp: 137.2e139.6 C. H
NMR (400 MHz, CDCl
3
)
d
5.80 (d, J ¼ 9.9 Hz, 2H), 5.45 (s, 2H), 2.90-
.2.1. Synthesis of compounds 24 and 25
2.66 (m, 4H), 2.65-2.52 (m, 2H), 2.39 (td, J ¼ 14.0, 4.0 Hz, 2H), 2.07-
2.01 (m, 2H), 1.91 (m, 2H), 1.84-1.69 (m, 4H), 1.68-1.62 (m, 2H), 1.53-
1.44 (m, 8H), 1.41-1.26 (m, 6H), 1.07-0.94 (m, 8H), 0.88 (d, J ¼ 7.1 Hz,
Under an argon atmosphere, BF.
3 2
Et O (1 mL) was added drop-
wise to a solution of dihydroartemisinin (569 mg, 2 mmol) and
pentaerythritol (136.15 mg, 1 mmol) or glycerol (92.1 mg, 1 mmol)
13
3
6H). C NMR (100 MHz, CDCl ) d 171.09, 104.48, 92.19, 91.50, 80.13,
ꢁ
in diethyl ether (50 mL) at ꢀ78 C. The temperature of this mixture
77.03, 76.72, 51.57, 45.25, 37.29, 36.23, 34.11, 31.80, 28.87, 25.98,
24.60, 22.02, 20.24, 12.10.
was slowly raised to room temperature, and stirring was continued
ꢁ
overnight. Saturated aqueous sodium bicarbonate (NaHCO
was added to the reaction mixture and extracted with ethyl acetate
30 mL ꢂ 3). The combined organic phase was dried over anhydrous
Na SO and concentrated under vacuum. The residue was purified
3
, 30 mL)
Compound 28. White solid. Yield: 42.5%. Mp: 142.8e145.3 C.
1
H NMR (400 MHz, CDCl
3
)
d
5.81 (d, J ¼ 9.9 Hz, 2H), 5.46 (s, 2H),
(
2.62-2.54 (m, 2H), 2.49 (t, J ¼ 7.4 Hz, 4H), 2.45-2.34 (m, 2H), 2.09-
1.98 (m, 4H), 1.94-1.87 (m, 2H), 1.83-1.70 (m, 4H), 1.68-1.62 (m, 2H),
1.56-1.50 (m, 1H), 1.49-1.43 (m, 8H), 1.39-1.28 (m, 5H), 1.09-1.00 (m,
2
4
by silica gel column chromatography to obtain the corresponding
compound.
13
2H), 0.98 (d, J ¼ 6.0 Hz, 6H), 0.86 (d, J ¼ 7.1 Hz, 6H). C NMR
Compound 24. [10] Colorless oil. Yield: 42.8%. 1H NMR
(100 MHz, CDCl 171.69, 104.48, 91.88, 91.50, 80.14, 51.58, 45.25,
3
) d
(
3
1
1
(
8
2
400 MHz, CDCl
.48 (m, 2H), 2.66 (m, 2H), 2.46-2.31 (m, 3H), 2.08-1.99 (m, 2H),
.93-1.84 (m, 2H), 1.82-1.72 (m, 3H), 1.68 (m, 3H), 1.54-1.51 (m, 1H),
.48 (m, 3H), 1.42 (m, 6H), 1.36 (m, 2H), 1.31-1.21 (m, 4H), 0.98-0.90
m, 12H). 13C NMR (100 MHz, CDCl
104.14, 102.78, 102.69, 87.93,
1.01, 69.91, 69.75, 69.69, 52.51, 44.32, 37.37, 36.44, 34.57, 30.88,
6.17, 24.58, 20.38, 13.09.
Compound 25. White solid. Yield: 24.3%. Mp: 94.8e96.9 C. H
3
)
d
5.38 (s, 2H), 4.82 (t, J ¼ 3.3 Hz, 2H), 3.90 (m, 3H),
37.28, 36.23, 34.10, 33.19, 31.77, 25.98, 24.59, 22.01, 20.24, 19.57,
12.17.
ꢁ
Compound 29. White solid. Yield: 36.2%. Mp: 164.9e167.7 C.
1
H NMR (400 MHz, CDCl
2.57 (ddd, J ¼ 9.9, 7.1, 4.6 Hz, 2H), 2.49 (t, J ¼ 7.4 Hz, 4H), 2.44-2.34
3
)
d
5.80 (d, J ¼ 9.9 Hz, 2H), 5.45 (s, 2H),
3
)
d
(m, 2H), 2.09-1.99 (m, 4H), 1.94-1.89 (m, 2H), 1.82-1.72 (m, 4H),
1.66-1.60 (m, 2H), 1.50-1.30 (m, 16H), 1.02-0.96 (m, 8H), 0.86 (d,
ꢁ
1
13
J ¼ 7.1 Hz, 6H). C NMR (100 MHz, CDCl
3
) d 172.01, 156.81, 104.46,
Table 6
a
Pharmacokinetic data for 49 in rats .
Plasma concentration-time data of 49 after an IV dose of 5 mg/kg in male
SD rats
Plasma concentration-time data of 49 after a PO dose of 10 mg/kg in male
SD rats
PK parameters
CL
Unit
Mean
PK parameters
Unit
Mean
L/h/kg
L/kg
h
h*ng/mL
h*ng/mL
h
0.492
0.346
0.773
10203
10209
0.704
T
C
T
max
max
1/2
h
1.667
1110
0.825
4190
4434
20.6
V
ss
ng/mL
h
h*ng/mL
h*ng/mL
%
T
1/2
AUClast
AUCINF
MRTINF
AUClast
AUCINF
b
F
a
Three animals were used for each dose group. The data shown are mean values.
The F value was determined by the following equation: If AUClast/AUCINF>80%: F¼(AUCINF-EX ꢂ DOSEIV)/(AUCINF-IV ꢂ DOSEEX)*100%; If AUClast/AUCINF<80%:
b
F¼(AUClast-EX ꢂ DOSEIV)/(AUClast-IV ꢂ DOSEEX)*100%.
7

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
Fig. 7. In vivo anti-tumor effect of compound 49
(
A) A549 cell BALB/c nude mouse xenograft model. Compound 49 was intraperitoneally injected at the dosage of 10 mg/kg. (B) Lewis cell C57 BL/6 mouse transplant model.
Compound 49 was intraperitoneally injected at the dosage of 12 mg/kg. The data are presented as the mean ± standard error. * Indicates P < 0.05, and ** indicates P < 0.01.
ꢁ
1
9
2
1.79, 91.50, 80.14, 51.59, 49.08, 45.27, 37.28, 36.24, 33.98, 31.80,
5.97, 25.65, 24.98, 24.59, 24.05, 22.01, 20.23, 12.16.
Compound 31. White solid. Yield: 57.6%. Mp: 135.9e138.4 C. H
NMR (400 MHz, CDCl
8.20 (s, 4H), 6.04 (d, J ¼ 9.8 Hz, 2H), 5.56 (s,
3
) d
Fig. 8. Flow cytometry analysis of a specimen from C57 BL/6 mice from the in vivo experiment
A) The proportion of change in immune cells in peripheral blood. (B) The proportion of change in immune cells in the spleen. The data are presented as the mean ± standard error. *
Indicates P < 0.05, and ** indicates P < 0.01 vs control.
(
8

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
2
2
2
0
1
3
H), 2.91-2.66 (m, 2H), 2.42 (td, J ¼ 14.2, 3.9 Hz, 2H), 2.15-1.99 (m,
H), 1.94 (m, 2H), 1.89-1.81 (m, 2H), 1.81-1.68 (m, 4H), 1.56-1.50 (m,
H), 1.45 (m, 8H), 1.35 (m, 3H), 1.14-1.04 (m, 2H), 1.03-0.98 (m, 6H),
1H NMR (400 MHz, CDCl
3.69-3.52 (m, 2H), 2.67-2.57 (m, 1H), 2.47-2.34 (m, 2H), 2.28 (m,
1H), 2.04 (m, 2H),1.94-1.72 (m, 9H),1.71-1.15 (m, 25H),1.15-1.02 (m,
3
)
d
5.46 (s, 1H), 4.92 (d, J ¼ 3.4 Hz, 1H),
.98-0.91 (m, 6H). 13C NMR (100 MHz, CDCl
)
d
164.54, 133.74,
30.02, 104.51, 92.95, 91.66, 80.17, 51.64, 45.33, 37.30, 36.25, 34.12,
1.96, 26.93, 25.97, 24.60, 22.08, 20.26, 12.29.
Compound 32. White solid. Yield: 60.3%. Mp: 95.0e97.2 C. H
NMR (400 MHz, CDCl
7.26 (s, 4H), 5.78 (d, J ¼ 9.9 Hz, 2H), 5.45 (s,
H), 3.80-3.62 (m, 4H), 2.56 (m, 2H), 2.39 (td, J ¼ 13.9, 3.9 Hz, 2H),
.08-2.02 (m, 2H), 1.95-1.86 (m, 2H), 1.77-1.70 (m, 3H), 1.61 (m, 2H),
2H), 0.97 (m, 9H), 0.90 (d, J ¼ 7.3 Hz, 3H), 0.70 (s, 3H). C NMR
13
3
3
(100 MHz, CDCl ) d 178.71, 104.07, 100.21, 88.08, 81.22, 71.41, 55.61,
54.92, 52.61, 44.51, 43.79, 42.37, 40.07, 39.19, 37.49, 36.99, 36.48,
35.21, 35.01, 34.71, 34.25, 33.60, 30.82, 30.75, 28.85, 28.63, 26.90,
26.27, 24.70, 24.56, 23.53, 21.24, 20.41, 18.42, 13.16, 12.15, 0.02.
ꢁ
1
3
) d
ꢁ
2
2
Compound 50. White solid. Yield: 65.5%. Mp: 126.1e128.7 C.
H NMR (400 MHz, CDCl
1
3
)
d
5.47 (s, 1H), 4.92 (d, J ¼ 3.3 Hz, 1H),
1.55-1.43 (m, 8H), 1.43-1.23 (m, 7H), 1.07-0.94 (m, 8H), 0.72 (d,
3.73-3.53 (m,1H), 2.66-2.56 (m,1H), 2.39 (m, 2H), 2.27 (m,1H), 2.05
(m, 1H), 1.99-1.94 (m, 1H), 1.93-1.70 (m, 8H), 1.68-1.55 (m, 3H), 1.51-
13
J ¼ 7.1 Hz, 6H). C NMR (100 MHz, CDCl
3
)
d
170.27, 132.50, 129.58,
13
104.48, 92.22, 91.55, 80.14, 51.56, 45.24, 40.93, 37.26, 36.22, 34.08,
1.21 (m, 21H), 1.08 (m, 4H), 1.00-0.89 (m, 13H), 0.66 (s, 3H). C NMR
(100 MHz, CDCl 180.33, 104.04,100.16, 88.07, 81.26, 56.42, 55.98,
31.87, 26.01, 24.59, 21.97, 20.24, 11.97.
3
) d
ꢁ
Compound 33. White solid. Yield: 66.1%. Mp: 129.8e132.3 C.
52.63, 44.57, 42.77, 41.97, 40.42, 40.12, 37.48, 36.50, 35.85, 35.32,
35.42, 34.74, 32.59, 31.00, 30.85, 30.78, 29.72, 29.01, 28.20, 27.33,
26.40, 26.27, 24.63 (d), 24.21, 23.51, 20.89, 20.42, 18.30, 13.18, 12.07.
1
H NMR (400 MHz, CDCl
3
)
d
8.24 (d, J ¼ 8.4 Hz, 4H), 7.72 (d,
J ¼ 8.5 Hz, 4H), 6.06 (d, J ¼ 9.8 Hz, 2H), 5.57 (s, 2H), 2.80 (m, 2H),
.42 (td, J ¼ 14.1, 3.9 Hz, 2H), 2.14-2.02 (m, 2H), 1.99-1.67 (m, 9H),
ꢁ
1
2
Compound 51. White solid. Yield: 83.7%. Mp: 134.5e137.3 C. H
NMR (400 MHz, CDCl
13
1
d
.56-1.30 (m, 14H), 1.12-0.94 (m, 14H). C NMR (100 MHz, CDCl
165.03, 144.71, 130.79, 129.20, 127.25, 104.49, 92.66, 91.67, 80.23,
1.67, 45.37, 37.31, 36.28, 34.14, 32.05, 26.00, 24.61, 22.10, 20.28,
3
)
3
)
d
5.41 (s, 1H), 4.96 (d, J ¼ 3.5 Hz, 1H), 4.14-
4.02 (m, 1H), 3.64 (dt, J ¼ 15.5, 5.5 Hz, 1H), 2.68-2.58 (m, 1H), 2.35
(m, 3H), 2.12-1.95 (m, 2H), 1.95-1.56 (m, 12H), 1.56-1.21 (m, 16H),
1.12 (m, 6H), 1.02-0.89 (m, 10H), 0.86 (d, J ¼ 7.3 Hz, 3H), 0.65 (s, 3H).
5
12.29.
13
3
C NMR (100 MHz, CDCl ) d 179.13, 104.09, 98.78, 88.20, 81.19,
4
.2.3. Synthesis of compounds 37, 47e53, and 56
72.49, 71.73, 56.21, 55.93, 52.64, 47.88, 44.45, 42.88, 40.04, 39.94,
37.55, 36.51, 36.00, 35.43, 35.34, 34.69, 34.51, 31.64, 30.24, 30.36,
30.44, 30.75, 30.77, 30.87, 29.67, 28.11, 26.26, 24.68, 24.60, 24.22,
23.77, 20.75, 20.41, 18.25, 13.16, 12.02.
Under an argon atmosphere, BF.
Et O (1 mL) was added drop-
3
2
wise to a solution of dihydroartemisinin (569 mg, 2 mmol) and
cholic acid or steroid analogues (2 mmol) in diethyl ether (50 mL)
ꢁ
ꢁ
1
at ꢀ78 C. The temperature of the resulting mixture was slowly
Compound 52. White solid. Yield: 67.5%. Mp: 167.1e168.9 C. H
NMR (400 MHz, CDCl 5.56 (m, 1H), 5.49 (s, 1H), 5.39 (m, 1H),
raised to room temperature and stirred overnight before being
3
) d
quenched with saturated aqueous sodium bicarbonate (NaHCO
0 mL) and extracted with ethyl acetate (30 mL ꢂ 3). The combined
organic phase was dried over Na SO and concentrated under
3
,
5.33-5.14 (m, 2H), 4.97 (d, J ¼ 3.4 Hz,1H), 3.70 (m,1H), 2.69-2.58 (m,
1H), 2.56-2.46 (m, 1H), 2.38 (m, 2H), 2.11-2.01 (m, 3H), 2.00-1.82
(m, 7H),1.79-1.65 (m, 4H),1.65-1.56 (m, 2H),1.53-1.45 (m, 5H),1.42-
1.21 (m, 10H), 1.04 (d, J ¼ 6.7 Hz, 3H), 0.89e0.98 (m, 12H), 0.85 (t,
3
2
4
vacuum. The residue was purified by silica gel column chroma-
13
tography to provide the corresponding compound.
J ¼ 6.4 Hz, 6H), 0.64 (s, 3H). C NMR (100 MHz, CDCl
3
) d 140.99,
ꢁ
1
Compound 37. White solid. Yield: 53.8%. Mp: 135.8e137.2 C. H
NMR (400 MHz, CDCl
5.47 (s, 1H), 4.93 (d, J ¼ 3.3 Hz, 1H), 4.00 (s,
H), 3.88 (s, 1H), 3.51 (m, 1H), 2.64-2.56 (m, 1H), 2.47-2.21 (m, 4H),
.10-2.02 (m, 2H), 1.99-1.85 (m, 5H), 1.72 (m, 8H), 1.55-1.43 (m,
0H), 1.39-1.30 (m, 4H), 1.31-1.24 (m, 4H), 1.16 (m, 1H), 1.06-0.95 (m,
139.97, 135.63, 131.93, 119.47, 116.47, 104.05, 99.60, 88.10, 81.24,
75.07, 55.69, 54.50, 52.63, 46.05, 44.52, 42.82, 42.80, 40.50, 39.07,
38.81, 37.97, 37.48, 37.26, 36.49, 34.72, 33.10, 30.77, 29.72, 28.35,
27.98, 26.25, 24.52, 24.70, 23.03, 21.13, 21.05, 20.40, 19.98, 19.67,
17.62, 16.21, 13.11, 12.07.
3
) d
1
2
1
7
1
13
ꢁ
H), 0.89 (m, 8H), 0.71 (s, 3H). C NMR (100 MHz, CDCl
04.03, 100.14, 88.08, 81.29, 68.67, 52.65, 46.86, 46.51, 44.56, 42.21,
1.33, 39.32, 37.47, 36.51, 35.58, 35.29, 35.22, 34.87, 30.84, 30.68,
3
)
d
178.40,
Compound 53. White solid. Yield: 64.8%. Mp: 146.8e149.1 C.
1
H NMR (400 MHz, CDCl
3
)
d
5.48 (s,1H), 5.36 (d, J ¼ 5.0 Hz,1H), 4.94
4
(d, J ¼ 3.4 Hz, 1H), 3.73-3.49 (m, 1H), 2.67-2.56 (m, 1H), 2.44-2.22
3
2
0.54, 30.31, 30.18 29.72, 29.04, 28.37, 27.58, 26.70, 26.30, 24.70,
3.22, 22.60, 20.41, 17.24, 13.08, 12.46.
(m, 3H), 2.12-1.79 (m, 8H), 1.74 (m, 1H), 1.69-1.62 (m, 1H), 1.56-1.09
13
(m, 25H), 1.03-0.99 (m, 4H), 0.99-0.77 (m, 18H), 0.68 (s, 3H).
C
ꢁ
1
Compound 47. White solid. Yield: 54.8%. Mp: 129.4e131.6 C. H
NMR (400 MHz, CDCl
5.47 (s, 1H), 4.91 (d, J ¼ 3.4 Hz, 1H), 4.02 (s,
H), 3.67-3.55 (m, 1H), 2.65-2.56 (m, 1H), 2.48-2.25 (m, 3H), 2.07-
.01 (m, 1H), 1.92-1.81 (m, 5H), 1.77-1.70 (m, 3H), 1.68-1.58 (m, 4H),
NMR (100 MHz, CDCl 140.71, 121.67, 104.03, 99.51, 88.08, 81.27,
3
) d
3
)
d
76.32, 56.72, 56.14, 52.64, 50.11, 44.55, 42.32, 40.32, 39.78, 39.53,
37.48, 36.99, 36.72, 36.50, 36.20, 35.82, 34.73, 31.93, 31.89, 31.64,
30.78, 29.66, 28.27, 28.03, 27.65, 26.93, 26.26, 24.70, 24.52, 24.31,
23.84, 22.85, 22.59, 21.09, 20.41, 19.49, 18.73, 13.11, 11.87.
1
2
1.54 (m, 2H), 1.50-1.34 (m, 12H), 1.27 (m, 8H), 1.18-1.06 (m, 2H), 1.01
ꢁ
1
(
d, J ¼ 6.2 Hz, 3H), 0.96 (d, J ¼ 6.3 Hz, 3H), 0.93-0.85 (m, 7H), 0.71 (s,
Compound 56. White solid. Yield: 72.1%. Mp: 164.6e167.0 C. H
NMR (400 MHz, CDCl
13
3
8
3
3
2
H). C NMR (100 MHz, CDCl
3
)
d
179.27, 104.03, 100.49, 88.10,
3
)
d
5.48 (s,1H), 5.36 (d, J ¼ 4.9 Hz,1H), 4.94 (d,
1.30, 73.32, 52.62, 48.36, 47.33, 46.46, 44.55, 42.00, 37.43, 36.51,
6.06, 35.26, 35.04, 34.66, 34.30, 33.79, 32.64, 31.95, 31.45, 30.91,
0.82, 30.68, 30.20, 29.72, 28.98, 27.43, 27.27, 26.29, 26.06, 24.69,
4.58, 23.62, 23.34, 20.38, 17.33, 13.20, 12.77.
J ¼ 3.4 Hz, 1H), 4.43 (dd, J ¼ 15.0, 7.5 Hz, 1H), 3.60 (m, 1H), 3.50 (m,
1H), 3.40 (t, J ¼ 10.9 Hz, 1H), 2.67-2.57 (m, 1H), 2.44-2.26 (m, 3H),
2.02 (m, 3H), 1.93-1.80 (m, 5H), 1.78-1.72 (m, 2H), 1.71-1.57 (m, 7H),
1.56-1.44 (m, 8H), 1.40-1.09 (m, 8H), 1.06-0.93 (m, 11H), 0.90 (d,
ꢁ
1
13
Compound 48. White solid. Yield: 57.8%. Mp: 138.1e140.5 C. H
NMR (400 MHz, CDCl
5.46 (s, 1H), 4.93 (d, J ¼ 3.1 Hz, 1H), 3.88 (s,
H), 3.48 (t, J ¼ 10.8 Hz, 1H), 2.65-2.55 (m, 1H), 2.46-2.33 (m, 2H),
.27 (m,1H), 1.87 (m,16H),1.52-1.13 (m, 20H), 0.95 (m, 14H), 0.66 (s,
J ¼ 7.3 Hz, 3H), 0.81 (m, 6H). C NMR (100 MHz, CDCl
3
) d 140.74,
3
)
d
121.39, 109.32, 104.05, 99.45, 88.09, 81.27, 80.87, 76.13, 66.87, 62.05,
56.46, 52.62, 50.01, 44.52, 41.61, 40.27, 39.78, 37.47, 36.94, 36.86,
36.49, 34.71, 32.07, 31.87, 31.42, 31.39, 30.76, 30.32, 28.81, 27.59,
26.28, 24.70, 24.52, 20.87, 20.42, 19.52, 17.18, 16.32, 14.57, 13.12.
1
2
3
8
3
3
13
H). C NMR (100 MHz, CDCl3)
d 179.74, 104.02, 100.40, 88.09,
1.30, 77.49, 68.70, 55.79, 52.63, 50.53, 44.59, 42.73, 41.33, 39.56,
9.33, 37.47, 36.51, 35.86, 35.37, 35.23, 34.89, 34.72, 32.88, 30.92,
0.79, 29.12, 28.18, 26.29, 24.70, 24.57, 23.70, 22.90, 20.64, 20.41,
4.3. In vivo anti-cancer experiments
1
8.27, 13.20, 11.78.
Compound 49. White solid. Yield: 76.2%. Mp: 148.7e150.2 C.
4.3.1. A549 cell-derived xenograft model
ꢁ
Thirty BALB/c nude mice (females; 7e9 weeks of age; 18e25 g
9

X. Zou, C. Liu, C. Li et al.
European Journal of Medicinal Chemistry 225 (2021) 113754
6
weight) were subcutaneously inoculated with A549 cells (2 ꢂ 10 ),
and 33 days after the inoculation, the tumor volume reached
Time (min)
Mobile Phase B (%)
3
approximately 1 cm . The 30 mice were randomly divided into 3
0
0
.20
.60
5
groups, namely the model group, the taxol group, and the admin-
istration group, with 10 mice in each group. The model group was
intraperitoneally injected with 0.1 mL solution (10% DMSO, 10%
propylene glycol, 10% castor oil, 70% normal saline) every day for
seven consecutive days with two days of rest. This comprised one
cycle and four consecutive cycles of administration. For the
administration group, intraperitoneal injection of 10 mg/kg of
compound 49 (2 mg/mL, in 10% DMSO, 10% propylene glycol, 10%
castor oil, 70% saline) was continuously administered for seven
days, with two days of rest. Nine days were considered as one cycle,
and four consecutive cycles were administered. For the taxol group,
60
80
80
5
1.40
2.00
2
2
.01
.40
Stop
The retention time of 49 is 1.32 min and Glipizide is 0.93 min.
4.4. Flow cytometry analysis
The spleen and 200
mL blood/mouse from the above in vivo
1
0 mg/kg taxol (2 mg/mL, in 10% DMSO, 10% propylene glycol, 10%
experiment (Section 2.4) were collected in each tube. The spleens
were milled, filtered with 350 mesh nylon membrane, and centri-
fuged at 1500 rpm for 5 min, and the supernatant was removed.
Themolysin was added, cells were lysed for 10 min and centrifuged
at 1500 rpm for 5 min, and the supernatant was removed. The cells
were rinsed with PBS and centrifuged at 1500 rpm for 5 min, and
then the supernatant was removed. The cells were subsequently
labeled with surface antibodies (LY6G-FITC, CD11b-BV421, Gr-1-
APC, LY6C-PECY7, CD3-FITC, CD8-BV510, CD4-APC, CD19-
PERCP5.5) (BD Biosciences), and incubated for 30 min in the dark
for flow cytometry analysis (BD FACSDiva 8.0.1).
castor oil, 70% saline) was injected intraperitoneally every three
days, with nine days per cycle, and four consecutive cycles of
administration. The tumor volume was measured every two days in
the A549 cell nude mouse model.
4.3.2. Lewis lung cancer cell transplant tumor model
Thirty C57 mice (females; 7e9 weeks of age; 18e25 g weight)
were subcutaneously inoculated with Lewis lung cancer cells
6
(
2 ꢂ 10 ). Ten days after the inoculation, the tumor volume reached
3
approximately 0.5 cm . The mice were randomly divided into 3
groups, namely the model group, the taxol group, and the admin-
istration group, with 10 mice in each group. The model group was
intraperitoneally injected with 0.1 mL solution (10% DMSO, 10%
propylene glycol, 10% castor oil, 70% normal saline) every day for
five consecutive days with two days of rest. Seven days was
considered to be one cycle, and three consecutive cycles were
administered. The administration group received intraperitoneal
injection of 12 mg/kg of compound 49 (2 mg/mL, in 10% DMSO, 10%
propylene glycol, 10% castor oil, 70% saline) with continuous
administration for five days, and two days of rest. Seven days were
considered as one cycle, and three consecutive cycles were
administered. For the taxol group, 12 MPK taxol (2.4 mg/mL, in 10%
DMSO, 10% propylene glycol, 10% castor oil, 70% saline) was injected
intraperitoneally every three days. The tumor weight was
measured after completion of the experiment.
4
4
.5. In vitro anti-cancer experiments
.5.1. Cell lines
K562, Jurkat and Kasumi-1: The human leukemia cell lines.
HepG2: The human hepatocellular carcinoma cell line. H322 and
NCIeH446: The human non-small cell lung cancer cell lines. PC3:
The human prostate cancer cell line. HeLa: The human cervical
cancer cell line. 786-O: The human renal clear cell carcinoma cell
line. MCF-7: The human breast cancer cell line. OV-90: The human
ovarian cancer cell lines. HCT116: The human colon cancer cell
lines. RCC-4, RCC4-VHL: The human renal cell carcinoma cell lines.
Ue2OS: human osteosarcoma cell line. U-87MG: The human gli-
oma cell line.
4
.5.2. Cell Counting Kit-8 assay
Cells were cultured in Dulbecco's modified Eagle's medium
4
.3.3. Pharmacokinetic studies of 49 in rats
Six male SD rats were divided into two groups. For Group
(N ¼ 3), 5 mg/kg (2 mL/kg) of 49 was injected via foot dorsal vein
(
(
DMEM) or Roswell Park Memorial Institute (RPMI)-1640 medium
Hyclone) containing 10% fetal bovine serum (Gibco) and 100 U/mL
1
injection. For group 2 (N ¼ 3), 10 mg/kg (5 mL/kg) of 49 was
penicillin (Invitrogen) and 100
m
g/mL streptomycin (Invitrogen) at
administrated via oral gavage. The animal was restrained manually
ꢁ
37 C with 5% CO
2
in humidified atmosphere. Cells were seeded in
at the designated time points, approximately 150
mL of blood
appropriate density (3000 cells/well) in 96-well plates and cultured
overnight. After 72 h of treatment with different concentrations of
compounds, 10 mL CCK-8 (Dojindo) was added to each well and
there was a further incubation of the plates for 2 h. The plates were
measured the absorbance at 450 nm using a multimode microplate
reader. The IC50 was calculated using GraphPad software.
sample was collected via tail vein for serial bleeding into K2EDTA
tubes. Sampling at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 h, 8 time points.
Serial bleeding for plasma generation. The blood samples were
maintained in wet ice first and centrifuged to obtain plasma (2000
g, 4 C, 5 min) within 15 min post sampling. Plasma samples were
stored at approximately ꢀ70 C until analysis.
ꢁ
ꢁ
An aliquot of 30 mL plasma sample was added with 200 mL IS
Author contributions
(
Glipizide, 150 ng/mL in ACN). The mixture was vortexed for 10 min
at 750 rpm and centrifuged at 6000 rpm for 10 min. An aliquot of
L supernatant was injected for LC-MS/MS analysis. The Instru-
ZWS designed the overall project and wrote the manuscript. JHZ
molecule design and synthesis, WX and HZB performed the syn-
thesis, XSZ, CCL, XD performed in-vitro proliferation test. ZYX and
XZZ designed and led the in-vivo experiments. CL performed the
in-vivo experiments. RF performed the flow cytometry analysis.
8
m
ment used for this analysis is UPLC/MS-MS-031 (API-6500), Glipi-
zide was used as internal standard.
MS conditions: Negetive ion, ESI, MRM detection.
HPLC conditions:
Column: ACQUITY UPLC BEH C18 (2.1 ꢂ 50 mm, 1.7
mm)
Flow rate: 0.60 mL/min.
Funding sources
Mobile Phase A: H
2
O-0.1%NH
3
.H
2
O
Mobile Phase B: MeOH-0.1%NH
3
.H
2
O
The National Natural Science Foundation of China.
10

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Declaration of competing interest
[
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
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15] Y. Tian, Z. Liang, H. Xu, Y. Mou, C. Guo, Design, synthesis and cytotoxicity of
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The work was supported by the National Natural Science
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Project of Innovation of Shanghai Education Committee, fund
approval number 2017-01-07-00-10-E00064. We thank LetPub
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[
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113754.
226e234.
[
20] J. Wang, J. Zhang, Y. Shi, C. Xu, C. Zhang, Y.K. Wong, Y.M. Lee, S. Krishna, Y. He,
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specific anticancer property of artemisinin and its combination with amino-
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Abbreviations
DHA
DMSO
dihydroartemisinin
dimethyl sulfoxide
phosphate buffered saline
boron trifluoride diethyl etherate
1,3-dicyclohexylcarbodiimide
4-dimethylaminopyridine.
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artemisinin dimers with potent anticancer activities, Eur. J. Med. Chem. 150
PBS
(2018) 829e840.
.
BF
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Et
2
O
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