Bioorganic Chemistry 111 (2021) 104903

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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Discovery of novel dihydroartemisinin-cinnamic hybrids inducing lung

cancer cells apoptosis via inhibition of Akt/Bad signal pathway
Yanping Hu 1, Yujin Wang 1, Na Li, Li Chen *, Jianbo Sun *
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of Traditional Pharmacy, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: A series of dihydroartemisinin-cinnamic acid hybrids were designed, synthesized and evaluated. Most of the
Dihydroartemisinin tested compounds showed enhanced anti-proliferative activities than artemisinin and dihydroartemisinin, among
Cinnamic acid which 16 g had the superior potency with IC50 values ranging from 5.07 μM to 7.88 μM against four tested cancer
Cytotoxicity
cell lines. The cell cycle arrest revealed that 16 g induced A549 cell cycle arrest at G0/G1 phase via regulation of
Apoptosis
PI3K/Akt/Bad pathway
G1-related protein expression (Cdk4). Further mechanism studies reveal that 16 g induced A549 cells apoptosis
via inhibiting Akt/Bad pathway. Moreover, 16 g depolarized the mitochondria membrane potentials and induced
ROS generation in A549. Additionally, 16 g blocked migration of A549 cells in a concentration-dependent
manner. What’s more, 16 g is barely nontoxic to zebrafish embryos. Overall, the cell cycle arrest, inhibition
of Akt/Bad signal pathway, ROS generation and migration blocked might explain the potent anti-proliferative
activities of these compounds.

1. Introduction
Drug repurposing is described as applying known drugs or com­
pounds with proven clinical safety to new diseases or indications which
result in significant time and cost savings [1,2]. Thus, drug repurposing
has become one of the important strategies for drug research. As a
natural product extracted and separated from Artemisia annua, artemi­
sinin is well known as an antimalarial reagent [3,4]. Recently, artemi­
sinin analogues were reported to possess antitumor activities, wherein
they could inhibit cell proliferation and migration, induce apoptosis
[5–9]. Dihydroartemisinin (DHA) is a semi-synthetic analogue of arte­
misinin, and showed a potent anti-angiogenetic function [10–12], thus
inhibiting proliferation, migration and invasion of cancer cells [13,14].
The underlying mechanisms research demonstrated that DHA could
downregulate the expression of PI3K/Akt/bad signal pathway through
inhibiting the binding activity of Akt [15,16]. As an ideal lead for the
design of antitumor drugs, several derivatives with perfect activities
against cancer cell lines were obtained by introducing different sub­
stituents into the 10 hydroxyl group of DHA. For example, compound 1
(Fig. 1) exhibited significantly antiproliferative activity with IC50
ranging from 0.025 μM to 0.093 μM against six cancer cell lines (HL-60,
U937, K562, NB-4, LNCaP, MCF-7/Adr) [17]. Compound 2 showed su­
perior activity against HeLa with IC50 valued 0.03 μM [18], and com­
pound 3 with IC50 7.4 nM against MV4-11 [19] (Fig. 1).
Recently, FDA approved of afatanib (2013), ibrutinib (2013), and
osimertinib (2015) that were designed to undergo Michael addition
reaction by trapping thiols in a biological media [20]. These drugs
contain the same structure of α, β-unsaturated carbonyl which could
react with sulfhydryl groups in vivo to form covalent bond and generates
important biological activity [21]. Therefore, due to the existence of α,
β-unsaturated carbonyl, cinnamic acid scaffolds presented potential
activities in glioblastoma, melanoma, prostate and lung carcinoma cells
[22]. Further research demonstrated that cinnamic acid scaffolds could
activate Akt-dependent pathways, thus decreased the expression of the
Bcl-2 family of proteins [23,24]. These data imply that cinnamic acid
scaffolds could serve as privilege fragments in the design of Akt
inhibitors.
Based on the above findings, cinnamic acid scaffolds were chosen as
the donation of α, β-unsaturated carbonyl to produce twenty-five new
DHA derivatives (14b-h, 16a-h, 19a-e and 22a-e) in this study. The
cytotoxic activities of these derivatives on the proliferation of human
breast cancer cell lines (MCF-7 and MDA-MB-231), human

* Corresponding authors.
E-mail addresses: chenli627@cpu.edu.cn (L. Chen), sunjianbo@cpu.edu.cn (J. Sun).
1
These authors (Yanping Hu and Yujin Wang) contributed equally to this work.

https://doi.org/10.1016/j.bioorg.2021.104903
Received 15 February 2021; Received in revised form 6 April 2021; Accepted 7 April 2021
Available online 20 April 2021
0045-2068/© 2021 Published by Elsevier Inc.
Y. Hu et al.
hepatocellular carcinoma cell line (HepG2), human non-small-cell lung
cancer cell line (A549) and human normal hepatic cell line (L02) were
determined by MTT method, respectively. Subsequently, the regulation
of Akt pathway and the simulation of Akt binding were studied. More­
over, cell cycle, apoptosis and migration of 16 g were biologically
evaluated to reveal the mechanisms of action of these compounds.
2. Results and discussion
2.1. Chemistry
The synthetic routes of target compounds 14a-h, 16a-h, 19a-e and
22a-e were illustrated in Scheme 1. Target compounds 14a-h were
synthesized by etherification reaction using 1-ethyl-3-(3-d-im-ethylami­
nopropyl) carbodiimide (EDCl) and 4-dimethyl-aminopyridine (DMAP)
as catalyst. The intermediates 15a-h were obtained through an amide
condensation reaction from 13a-h. Then, 15a-h stirred with DHA and
BF3⋅Et2O at 0 ◦ C for 2 h to produce 16a-h. The intermediates 18a-e were
prepared from the corresponding benzaldehyde by heating with cya­
noacetic acid at 100 ◦ C for 7 h, ammonium acetate was used as catalyst,
simultaneously [25]. Substituted benzeneacetonitrile reacted with 50%
glyoxylic acid in the presence of K2CO3 in methanol at 55–60 ◦ C for 5 h
to obtain compounds 21a-e [26]. Finally, the target compounds 19a-e,
22a-e were synthesized under the condition of EDCl and DMAP.
2.2. In vitro anticancer evaluations
All of the synthesized compounds were screened to their anticancer
activities against four human cancer cell lines, including human breast
cancer cell lines MCF-7 and MDA-MB-231, human non-small-cell lung
cancer cell line A549 and human hepatocellular carcinoma cell line
HepG-2 in vitro. Doxorubicin was used as the positive control. Notably,
as shown in Table 1, most of the synthesized compounds showed much
more potent anti-proliferative activities than artemisinin and dihy­
droartemisinin. Particularly, 16 g exhibited the strongest anticancer
activities against four tested cancer cell lines with IC50 values of 7.47,
5.07, 6.96 and 7.88 μM, respectively. It was reported that artemisinin
was resistant in the highly metastatic MDA-MB-231 breast cancer cell
line [27]. Surprisingly, all of the tested compounds displayed superior
activities against MDA-MB-231 cells. Especially 14d and 16 h with IC50
5.50 μM and 5.19 μM were comparable to doxorubicin (IC50 = 3.18 μM).
Moreover, further assay exhibited that all the active compounds showed
weak cytotoxicity in normal hepatic L02 cells with IC50 ranging from
31.73 μM to 83.07 μM while doxorubicin displayed high toxicity with
IC50 0.79 μM. Besides, the structure activity relationship (SAR) studies
revealed that the CN substitutes at α or β position of α, β-unsaturated
carbonyls resulted in decrease in anticancer efficacy.
Overall, 16 g displayed superior activities among the tested com­
pounds, exhibiting IC50 values from 5.07 to 7.88 μM against four cancer
cell lines. Thus, compound 16 g was selected for subsequent anticancer
mechanism.
Fig. 1. Artemisinin derivatives with potential anticancer activities.
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Bioorganic Chemistry 111 (2021) 104903
2.3. Effect of 16 g on cell cycle
To investigate the mechanism of cytotoxicity, propidium iodide (PI)
staining was used to examine the effect on cell cycle. A549 cells were
treated with different concentrations of 16 g for 48 h. As a result, 16 g
led to a significant increase of cells at G1 phase from 69.50% to 83.86%.
Meanwhile, cells at S phase reduced from 15.41% to 9.04% and cells at
G2 phase from 15.09% to 7.10% (Fig. 2). The result revealed that 16 g
induced A549 cell cycle arrest at G0/G1 phase.
Cdk4 (cyclin-dependent kinase), the major catalytic subunit of G1
cyclin, is a core part of the cell cycle regulatory machinery [28]. The
over expression of Cdk4 is closely related to the occurrence of tumors
[29]. Therefore, the expression levels of Cdk4 protein were detected
through western blot assay. As depicted in Fig. 5B, the level of Cdk4
protein was decreased in a dose-dependent manner which indicated that
16 g induced G0/G1 arrest may be correlated with a change of expres­
sion of Cdk4.
2.4. Effect of 16 g on Akt/Bad signal pathway
The Akt protein (also known as protein kinase B) plays an important
role in a diversity of cellular functions, such as cell proliferation, tran­
scription, glucose metabolism, cell cycle and apoptosis [30,31]. It was
reported that cell apoptosis might be inhibited due to the inappropri­
ately activated of the Akt/Bad pathway in many cancers [32–34]. Also of
note, p-Akt was overexpressed in none-small cell lung cancer and the
Akt/Bad cascade suppresses apoptosis from varies stimuli [35–37]. The
Bcl-2 protein families are the essential initiator of the mitochondrial
apoptotic pathway which contains both pro-apoptotic proteins Bad, Bax
and anti-apoptotic proteins Bcl-2 and Bcl-xL [38]. The activated Akt
phosphorylates the Bad and result in the release of Bcl-2, which
contribute to the cell survival. Bax, an inactive cytosolic protein, form
heterodimers with Bcl-2 and thereby neutralizing the anti-apoptotic
activity of Bcl-2 [39]. However, during apoptosis, Bax would trans­
locate into the mitochondria and form holes in the mitochondria
membrane through which cytochrome c is released into cytoplasm and
activates caspase-3 [40]. Caspase-3 acts as the final executor in
apoptosis and PARP is one of its substrates. The activated caspase-3
could cleave PARP and eliminating PARP’s ability to repair DNA and
led to apoptosis [41,42].
As shown in Fig. 3, 16 g significantly inhibited the activation of Akt
in concentration-dependent manners. Obviously, the expression of Bad
and Bax were markedly increased in the presence of 16 g while Bcl-2 was
decreased. What’s more, the level of cytochrome c in cytoplasm was
elevated which indicated that 16 g initiates mitochondrial apoptosis
signaling. More importantly, a dose-dependent activation of caspase-3
and accumulation of the cleavage products of PARP were observed. It
was suggested that 16 g induced apoptosis of A549 cells possibly
through inhibiting Akt/Bad signal pathway (Fig. 4).
2.5. Molecular docking
In order to describe the interaction mode of 16 g with target Akt, we
Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Scheme 1. Reagents and conditions: (i) EDCl/DMAP, dry DCM, r.t. 8 h; (ii) Ethanolamine, EDCl/HOBt, dry DCM, r.t. 2 h; (iii) BF3⋅Et2O, dry DCM, N2, 2 h, 0–5 ◦ C;
(iv) Cyanoacetic acid, NH4OAc, toluene, reflux; (v) 50% glyoxylic acid, K2CO3, methanol, reflux.

used Akt catalytic subunit (PDB 3O96) as a template to simulate the
molecular docking of 16 g and DHT (Fig. 5). Docking analysis revealed
that the oxygen on the DHA peroxy bridge forms a 3.2 Å hydrogen bond
with the carboxyl hydrogen of THR-82, while the introduction of the
oxygen substituent on the parent nucleus of 16 g made the hydrogen
bond distance with THR-82 shortened to 3.1 Å. In addition, the amino
hydrogen on the amide linker and the carbonyl oxygen of GLN-79
formed a 2.5 Å hydrogen bond and the introduced para-substituted
benzene ring formed a π-π stacking with the benzene ring of TRP-80. In
the 3D model diagram, DHA was well embedded in the cavity of the
active site of the AKT, while 16 g was better penetrated into the cavity
and interacted with the outer periphery due to the introduction of
substituents. The above results indicate that, compared with the parent
nucleus DHA, 16 g has an enhanced interaction with AKT due to the
introduction of substituents.
2.6. Effect of 16 g on cell apoptosis
To make further investigation of 16 g, the morphological apoptosis
was evaluated by Hoechst 33,342 staining and visualized under a fluo­
rescent microscope (×20). As shown in Fig. 6, A549 cells presented
typical features of apoptosis like the cells became brighter and the nu­
clear shrinkage. Then annexin V-FITC and PI staining were used and
performed on flow cytometry to explore the apoptotic cells mediated by

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Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Table 1 2.7. Effects of 16 g on mitochondrial depolarization and reactive oxygen

Anti-proliferative effects of new artemalogues against human cancer cell lines. species generation
Compd. IC50(μM)a
Increasing evidence showed that mitochondrial dysfunction could
MCF-7 A549 HepG-2 MDA-MB- L02
231 induce apoptosis and is considered to be the core of the apoptotic
pathway [43,44]. Early in the process, mitochondrial transmembrane
14a 12.05 ± 12.46 ± 19.43 14.82 ± 45.95 ±
potential (ΔΨ mt) is depolarized. To explore whether 16 g could induce
±
1.53 0.52 2.82 1.37 1.37
14b 11.32 ± 8.35 ± 14.52 ± 6.83 ± 61.93 ± mitochondrial dysfunction, mitochondrial membrane potential assay
0.22 0.41 3.40 0.58 2.96 was performed by JC-1 staining of mitochondria in A549 cells. As shown
14c 13.43 ± 10.23 ± 16.82 ± 7.55 ± 52.65 ± in Fig. 7A, cells treated with different concentrations (0, 2.5, 5 and 10
0.34 0.05 1.27 0.14 1.11
μM) of 16 g showed a concentration-dependent increase in the per­
14d 9.77 ± 11.82 ± 13.29 ± 5.50 ± 44.10 ±
1.89 0.87 2.25 0.31 0.08 centage of cells with low ΔΨ mt increased from 7.3% to 38.1%. The result
14e 10.08 ± 12.38 ± 19.33 ± 12.21 ± 34.45 ± suggested that 16 g induced mitochondrial depolarization and eventu­
1.16 0.35 3.12 0.86 2.63 ally triggers apoptosis.
14f 10.97 ± 18.32 ± 27.40 ± 20.61 ± 51.28 ± Increased reactive oxygen species (ROS) generation has been
0.29 2.14 5.47 1.34 3.10
14g 11.70 ± 10.41 ± 17.27 ± 8.77 ± 50.74 ±
detected in various cancers and is produced as a byproduct intracellu­
2.91 1.20 7.63 0.60 1.62 larly by mitochondria [45]. Mitochondrial membrane depolarization is
14h 11.43 ± 9.21 ± 15.32 ± 6.23 ± 45.73 ± related to mitochondrial production of ROS [46]. Therefore, the fluo­
0.83 0.15 1.65 0.17 0.57 rescent probe 2′ ,7′ -dichlorofluorescein diacetate (DCFH-DA) was used to
16a 32.39 ± 36.01 ± 42.00 13.3 ± 62.28 ±
±
detect the intracellular ROS levels. As shown in Fig. 7B, 16 g induced
1.13 3.12 2.02 0.53 3.21
16b 31.12 ± 49.82 ± >50 15.33 ± 73.89 ± intracellular ROS generation with a dose-dependent manner. However,
3.22 4.85 2.06 4.26 the increased ROS level could be inhibited by pre-incubation with 10
16c 17.43 ± 36.45 ± 31.05 ± 9.38 ± 40.05 ± mM of ROS scavenger (N-acetyl cysteine).
1.34 7.05 3.45 0.17 1.41
16d 23.76 ± 44.64 ± 15.21 ± 12.39 ± 69.59 ±
2.29 6.87 1.23 0.28 1.92
2.8. Effect of 16 g on cell migration
16e 26.23 ± 45.27 ± >50 14.53 ± 39.51 ±
1.26 2.14 1.87 2.25 Cell migration, an essential component of metastatic dissemination
16f 20.33 ± 42.25 ± >50 14.42 ± 54.57 ± of tumor cells, is the leading cause of cancer morbidity [47–49].
1.59 3.65 1.09 1.54
Therefore, a cell-based wound healing assay was performed according to
16g 7.47 ± 5.07 ± 6.96 ± 7.88 ± 31.73 ±
0.36 0.22 0.36 0.10 2.29 the standard method. As shown in Fig. 8, 16 g could block migration of
16h 14.52 ± 16.67 ± 16.09 ± 5.19 ± 32.18 ± A549 cells in a concentration-dependent manner.
0.09 0.45 2.16 0.07 0.54
19a 13.75 ± 17.94 ± 33.47 ± 9.12 ± 75.34 ± 2.9. Safety profile of 16 g in zebrafish embryo
1.37 3.32 3.90 0.22 0.98
19b 12.42 ± 15.42 ± 27.41 ± 10.04 ± 53.26 ±
1.63 2.66 2.58 0.54 2.92 To evaluate the safety of 16 g, zebrafish embryo toxicity experiment
19c 16.38 ± 22.46 ± 28.9 ± 9.187 ± 76.22 ± was carried out. The normal embryos were collected 2 h post fertiliza­
4.41 3.72 2.23 0.23 2.45 tion (hpf) and placed in 24-well plates (20 embyos/well), and at 2 hpf
19d 20.66 ± 17.41 ± 34.48 ± 23.00 ± 83.07 ±
exposed to different concentrations of 16 g and Doxorubicin in 0.1%
3.52 4.94 6.28 0.04 6.30
19e 16.19 ± 22.21 ± 24.61 ± 14.61 ± 64.12 ± DMSO incubated in E3 medium (0.3 g NaCl, 0.013 g KCl, 0.05 g CaCl2,
2.61 3.06 3.77 0.56 3.26 0.08 g MgSO4, and 0.05% methylene blue) at 28.5 ℃. The doses of
22a 12.64 ± 21.26 ± 29.80 ± 16.53 ± 42.83 ± compounds were based on our preliminary studies. Concentration
2.15 1.17 1.68 1.11 1.82 response curves were plotted for lethality from which LC50 values were
22b 11.17 ± 14.20 ± 30.51 ± 15.46 ± 53.72 ±
2.42 3.38 1.83 1.06 3.75
calculated for 72 h post-fertilization. As shown in Table 2, 16 g exhibited
22c 18.33 ± 17.32 ± 40.38 ± 15.84 ± 42.85 ± lower toxicity against zebrafish embryos compared with Doxorubicin. It
4.83 4.32 1.09 2.38 2.61 is noticeable that 16 g is barely nontoxic to zebrafish embryos.
22d 15.62 ± 23.56 ± 28.10 ± 13.80 ± 50.63 ±
2.31 6.25 3.26 0.53 4.62
2.10. Solubility and lipophilicity of 16 g
22e 14.18 ± 14.93 ± 28.28 ± 16.89 ± 36.18 ±
3.35 1.13 5.58 1.98 5.73
ART >50 >50 >50 >100 >100 Artemisinin was known with poor solubility both in oil and water
DHA 34.29 ± 25.04 ± 31.71 ± 58.69 ± 40.21 ± which limited its applications. To evaluate the antitumor candidate of
2.85 1.27 8.41 5.20 1.02 16 g, the lipophilicity and solubility were predicted. The partition co­
Doxorubicinb 1.13 ± 1.44 ± 0.65 ± 3.18 ± 0.79 ±
0.11 0.28 0.18 0.13 0.02
efficient (clog P) is used to estimate the lipophilicity and solubility of
chemical compounds. 16 g showed greater lipophilicity with clog P
a
MTT methods, cells were incubated with corresponding compounds for 48 h. values 2.03. The aqueous solubility of 16 g in phosphate buffered saline
IC50 values (means ± SD, n = 3).
b (PBS) was determined by high-performance liquid chromatography
Doxorubicin as the positive control.
(HPLC). As shown in Table 3, an improved solubility was achieved by
introducing of amide group.
16 g. A549 cells were treated with 16 g at different concentrations of 0,
2.5 μM, 5 μM, 10 μM, respectively. As can be seen from Fig. 9B, while the 3. Conclusion
concentrations of 16 g increased, the percentage of apoptosis was
significantly increased from 7.2% to 22.2%. The result indicated that the In conclusion, twenty-five new dihydroartemisinin-cinnamic acid
anti-proliferative of 16 g might be related to the induction of apoptosis. derivatives were synthesized and their anti-proliferative activities were
evaluated against MCF-7, A549, HepG-2, MDA-MB-231 and L02 cells.
Most of the hybrids exhibited enhanced anticancer activities compared
to artemisinin and dihydroartemisinin, and less cytotoxicity towards to
the normal liver cells than doxorubicin. Among them, 16 g exhibited

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Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Fig. 2. Cell cycle analysis of 16 g in A549. (A) Compound 16 g induced cell cycle arrest in A549. The cells were treated with 16 g at 0, 2.5 μM, 5 μM and 7.5 μM for
48 h. Then harvested, stained with PI, and analyzed by flow cytometry. (B) The expression level of Cdk4. A549 cells were treated with varying concentrations of 16 g
for 48 h. The expression of Cdk4 was determined by western blotting using specific antibody. β-actin was used as an internal control. Figures are representative of
three independent experiments. *P < 0.05, **P < 0.01, related to control.

Fig. 3. 16 g induced apoptosis-related proteins expression in A549 cells. Cells were treated with 16 g for 48 h. (A) The expression of p-Akt, PARP, Bcl-2, Bad, Bax,
cleaved-caspase 3 and cytochrome c (cytoplasm) were evaluated by Western Blotting using specific antibodies. β-actin was used as an internal control; (B) The density
ratios of proteins to β-actin are shown as relative expression. Figures are representative of three independent experiments. **P < 0.01, related to control.

stronger antiproliferative activities against four tested cancer cell lines. toxicity experiment indicated that 16 g exhibited lower toxicity against
Further mechanism studies revealed that 16 g induced A549 cell cycle zebrafish embryos compared with Doxorubicin. Overall, the current
arrest at G0/G1 phase via regulation of G1-related protein expression findings are valuable in understanding the anticancer activity of these
(Cdk4). Furthermore, the Hoechst 33324, annexin V-FITC and PI compounds as well as providing a new insight for the design of arte­
staining and western blot analysis indicated that 16 g induced cell misinin derivatives to enhance the efficacy of candidates.
apoptosis via inhibiting PI3K/Akt/Bad pathway. In addition, 16 g
depolarized the mitochondria membrane potentials and induced ROS
generation in A549 cells. What’s more, 16 g blocked migration of A549
cells in a concentration-dependent manner. Besides, the aqueous solu­
bility of 16 g was improved compared with artemisinin. In addition,

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Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Fig. 4. Schematic representation of the inhibition of Akt/Bad signaling pathway triggered by 16 g.

Fig. 5. Docking pose illustrates the binding simulations of 16 g at Akt catalytic subunit (PDB 3O96). The hydrogen bonds are shown by yellow dotted lines. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

6
Y. Hu et al.
Fig. 6. The apoptotic of 16 g on A549 cell. (A) Apoptotic assay by Hoechst 33342. A549 cell was treated with 16 g at 0, 2.5 μM, 5 μM, 10 μM for 48 h. Then cells were
stained with Hoechst 33,342 and visualized under a fluorescent microscope (×20). (B) Apoptotic assay by flow cytometry. A549 cell was treated with 16 g at 0, 2.5
μM, 5 μM, 10 μM for 48 h. Then annexin V-FITC and PI staining were used and performed on flow cytometry. Figures are representative of three independent
experiments. *P < 0.05, **P < 0.01, related to control.
4. Experimental section
4.1. Material and methods
All chemicals were purchased from commercial sources and used
without further purification, except for dichloromethane (DCM) that
was kept on molecular sieves (4 Å) prior to use in reactions. The re­
actions were monitored by TLC, visualized under UV light at 254 nm and
colored by vanillin - concentrated sulfuric acid. The column chroma­
tography was performed on 200–300 mesh silica gel. The 1H and 13C
NMR spectra were recorded on a Bruker ACF-300. Chemical shifts are
reported in parts per million δ (ppm), with the residual protons of the
solvent as reference. Data were reported as followed: s (singlet),
d (doublet), dd (doublet of doublet), t (triplet), q (quartet), p (pentet),
and m (multiplet). The NMR data were analyzed by Mest ReNova.
4.2. Chemical synthesis
4.2.1. General procedure for synthesis of hybrids 14a-h
Cinnamic acid derivatives (0.42 mmol, 1.2 eq) and DMAP (0.42
mmol, 1.2 eq) were dissolved in dry CH2Cl2 (10 ml). Then added EDCl
(0.42 mmol, 1.2 eq) and cooled to 0 ◦ C. After addition of DHA (0.35
mmol, 1 eq) the reaction mixture was slowly warmed to room temper­
ature and stirred overnight. The organic layer was washed by saturated
sodium chloride three times. The combined organic layers dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude
products were purified by column chromatography to obtain the target
7
Bioorganic Chemistry 111 (2021) 104903
compounds.
4.2.1.1. Compound 14a. The product was obtained as white solid (120
mg, 83%). ESI-MS: m/z 437 [M + Na]+. 1H NMR (500 MHz, CDCl3) δ
7.83 (d, J = 16.0 Hz, 1H), 7.58 (d, J = 3.1 Hz, 2H), 7.44 (s, 3H), 6.54 (d,
J = 16.0 Hz, 1H), 5.98 (d, J = 9.8 Hz, 1H), 5.54 (s, 1H), 2.74–0.94 (m,
21H) including three typical-CH3 of DHA such as 1.49 (s, 3H), 1.03 (d, J
= 6.1 Hz, 3H), 0.95 (d, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
165.5, 146.0, 134.3, 130.5, 128.9 (C × 2), 128.2 (C × 2), 117.6, 104.4,
92.1, 91.5, 80.1, 51.6, 45.3, 37.2, 36.2, 34.1, 32.0, 26.0, 24.6, 22.0,
20.2, 12.2. HRMS (ESI) calculated for C24H30O6Na [M + Na]+ 437.1940,
found 437.1932.
4.2.1.2. Compound 14b. The product was obtained as white solid (135
mg, 87%). ESI-MS: m/z 467 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.75 (d, J = 15.9 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz,
2H), 6.38 (d, J = 15.9 Hz, 1H), 5.95 (d, J = 9.8 Hz, 1H), 5.51 (s, 1H),
3.86 (s, 3H), 2.69–0.90 (m, 21H) including three typical-CH3 of DHA
such as 1.46 (s, 3H), 0.99 (d, J = 5.7 Hz, 3H), 0.91 (d, J = 7.1 Hz, 3H).
13
C NMR (75 MHz, CDCl3) δ 165.8, 161.6, 145.7, 129.9 (C × 2), 127.0,
115.0, 114.4 (C × 2), 104.4, 91.9, 91.5, 80.2, 55.4, 51.6, 45.3, 37.3,
36.3, 34.1, 32.0, 26.0, 24.6, 22.0, 20.2, 12.2. HRMS (ESI) calculated for
C25H32O7Na [M + Na]+ 467.2046, found 467.2033.
4.2.1.3. Compound 14c. The product was obtained as white solid (109
mg, 73%). ESI-MS: m/z 451 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Fig. 7. Effects of 16 g on mitochondrial membrane potential and ROS generation of A549. (A) After incubation with different concentrations (0, 2.5, 5 and 10 μM) of
16 g in A549 cells for 48 h prior to staining with JC-1 dye, the result was determined by flow cytometry analysis; (B) Generation of ROS was determined using ROS-
detecting fluorescent dye DCFH-DA. Figures are representative of three independent experiments. *P < 0.05, **P < 0.01, related to control.

7.77 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.9 Hz,
2H), 6.47 (d, J = 16.0 Hz, 1H), 5.95 (d, J = 9.8 Hz, 1H), 5.51 (s, 1H),
2.40 (s, 3H), 2.68–0.90 (m, 21H) including three typical-CH3 of DHA
such as1.46 (s, 3H), 0.99 (d, J = 5.8 Hz, 3H), 0.91 (d, J = 7.1 Hz, 3H).
13
C NMR (75 MHz, CDCl3) δ 165.0, 146.0, 140.9, 131.6, 129.6 (C × 2),
128.2 (C × 2), 116.5, 104.4, 92.0, 91.5, 80.1, 51.6, 45.3, 37.2, 36.2,
34.1, 32.0, 26.0, 22.0, 21.5, 20.2, 12.2. HRMS (ESI) calculated for
C25H32O6Na [M + Na]+ 451.2097, found 451.2083.
4.2.1.4. Compound 14d. The product was obtained as white solid (131
mg, 79%). ESI-MS: m/z 497 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.74 (d, J = 15.9 Hz, 1H), 7.13 (m, 1H), 7.08 (s, 1H), 6.89 (d, J = 8.3 Hz,
1H), 6.38 (d, J = 15.9 Hz, 1H), 5.94 (d, J = 9.8 Hz, 1H), 5.51 (s, 1H),
3.93 (s, 6H), 2.71–0.90 (m, 21H) including three typical-CH3 of DHA

8
Y. Hu et al. Bioorganic Chemistry 111 (2021) 104903

Fig. 8. Reduced migration of A549 cells by 16 g. (A) Solid lines indicate the borders of the wound edge closure at 0 h. Dash lines indicate the borders of the migrating
cells reached at 12 h and 24 h. (B and C) Quantitation of the areas of migrating cells and the width of the wound edge closure by migrating cells using Image J
software. Figures are representative of three independent experiments. **P < 0.01, related to control.

165.4, 162.3, 144.7, 130.5, 130.2, 130.0, 117.4, 116.2, 116.0, 104.5,
Table 2
92.1, 91.6, 80.2, 51.6, 45.3, 37.3, 36.2, 34.1, 32.0, 26.0, 24.6, 22.0,
Zebrafish embryo toxicity of 16 g.
20.2, 12.2. HRMS (ESI) calculated for C24H29FO6Na [M + Na]+
Compd. LC50 (μМ)a 455.1846, found 455.1833.
16 g 93.6 ± 8.79
Doxorubicin 7.3 ± 2.94 4.2.1.6. Compound 14f. The product was obtained as white solid (112
a
The embryos were placed in 24-well plates (20 mg, 70%). ESI-MS: m/z 482 [M + Na]+. 1H NMR (300 MHz, DMSO‑d6) δ
embryos/well) and at 2 hpf exposed to different 8.27 (d, J = 7.7 Hz, 2H), 8.07 (d, J = 8.2 Hz, 2H), 7.88 (d, J = 16.2 Hz,
concentrations of tested compounds (0.625–10.0 μM) 1H), 6.97 (d, J = 15.9 Hz, 1H), 5.82 (d, J = 9.4 Hz, 1H), 5.64 (s, 1H),
in 0.1% DMSO incubated in E3 medium (0.3 g NaCl, 2.73–0.83 (m, 21H) including three typical-CH3 of DHA such as 1.29 (s,
0.013 g KCl, 0.05 g CaCl2, 0.08 g MgSO4, and 0.05% 3H), 0.90 (d, J = 5.6 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H). 13C NMR (75
methylene blue) at 28.5 ◦ C. As a negative control MHz, CDCl3) δ 164.6, 142.9, 140.3, 135.0, 128.8 (C × 2), 124.2 (C × 2),
0.1% DMSO treated embryos were used. Digital im­
122.0, 104.5, 92.5, 91.6, 80.1, 51.6, 45.3, 37.3, 36.2, 34.1, 31.9, 25.9,
ages of embryos were taken under a stereomicroscope
24.6, 22.0, 20.2, 12.2. HRMS (ESI) calculated for C24H29NO8Na [M +
at 10× magnification (Nikon Eclipse SMZ745T with
ACT-1 imaging software) and LC50 values were Na]+ 482.1791, found 482.1775.
calculated for 72 hpf. The experiments were per­
formed in triplicate. 4.2.1.7. Compound 14g. The product was obtained as white solid (91
mg, 54%). ESI-MS: m/z 505 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.81 (d, J = 16.1 Hz, 1H), 7.66 (m, 4H), 6.58 (d, J = 16.0 Hz, 1H), 5.95
Table 3 (d, J = 9.8 Hz, 1H), 5.52 (s, 1H), 2.70–0.91 (m, 21H) including three
Solubility and Lipophilicity of artemisinin and 16 g. typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.8 Hz, 3H), 0.92
Compd. clog Pa Solubilityb (μg/ml) (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 165.0, 144.8, 137.6,
Artemisinin 1.09 [50] 82c [51] 134.7, 128.3 (C × 2), 125.9 (C × 2), 120.3, 104.5, 92.3, 91.6, 87.5, 80.2,
16 g 2.03 154 51.6, 45.3, 37.3, 36.2, 34.1, 32.0, 26.0, 24.6, 22.0, 20.2, 12.2. HRMS
a (ESI) calculated for C25H29F3O6Na [M + Na]+ 505.1814, found
clog P: calculated logarithm of the octanol-water partition coefficient.
b
Solubility in PBS (pH 7.4). 505.1797.
c
82 in water.
4.2.1.8. Compound 14h. The product was obtained as yellow solid (91
such as 1.45 (s, 3H), 0.99 (d, J = 5.8 Hz, 3H), 0.91 (d, J = 7.1 Hz, 3H). mg, 58%). ESI-MS: m/z 471 [M + Na]+. 1H NMR (500 MHz, CDCl3) δ
13
C NMR (75 MHz, CDCl3) δ 165.2, 150.8, 148.8, 145.4, 126.9, 122.3, 7.77 (d, J = 16.0 Hz,1H), 7.51 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz,
114.9, 110.6, 109.3, 103.9, 91.5, 91.0, 79.7, 55.5, 55.4, 51.1, 44.9, 36.8, 1H), 6.51 (d, J = 16.0 Hz, 1H), 5.97 (d, J = 9.8 Hz, 1H), 5.83 (s, 1H),
35.8, 33.7, 31.5, 25.4, 24.1, 21.5, 19.7, 11.6. HRMS (ESI) calculated for 2.71–0.93 (m, 21H) including three typical-CH3 of DHA such as 1.48 (s,
C26H34O8Na [M + Na]+ 497.2151, found 497.2141. 3H), 1.03 (d, J = 5.8 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 165.3, 144.5, 136.4, 132.8, 129.4 (C × 2), 129.2 (C × 2),
4.2.1.5. Compound 14e. The product was obtained as white solid (94 118.2, 104.5, 91.2, 91.5, 80.2, 51.6, 45.3, 37.3, 36.2, 34.1, 32.0, 26.0,
mg, 62%). ESI-MS: m/z 455 [M + Na]+. 1H NMR (300 MHz, DMSO‑d6) δ 24.6, 22.0, 20.2, 12.2. HRMS (ESI) calculated for C24H29ClO6Na [M +
7.87 (m, 2H), 7.77 (d, J = 16.2 Hz, 1H), 7.29 (t, J = 8.6 Hz, 2H), 6.71 (d, Na]+ 471.1550, found 471.1541.
J = 16.2 Hz, 1H), 5.81 (d, J = 9.6 Hz, 1H), 5.62 (s, 1H), 2.74–0.86 (m,
21H) including three typical-CH3 of DHA such as 1.29 (s, 3H), 0.90 (d, J 4.2.2. General procedure for synthesis of hybrids 15a-h
= 6.1 Hz, 3H), 0.83 (d, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ Cinnamic acid derivatives (1 mmol, 1 eq) and HOBt (1.2 mmol, 1.2

9
Y. Hu et al.
eq) were dissolved in dry CH2Cl2 (20 ml). Then added EDCl (1.2 mmol,
1.2 eq) and cooled to 0 ◦ C. After addition of ethanolamine (1.2 mmol,
1.2 eq) the reaction mixture was slowly warmed to room temperature
and stirred overnight. The organic layer was washed by saturated so­
dium chloride three times. The combined organic layers dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude
products were purified by column chromatography to obtain the target
compounds.
4.2.3. General procedure for synthesis of hybrids 16a-h
Artemisinin and compound 15a-h were dissolved in dry CH2Cl2 in
three-necked flask. While cooling to 0 ◦ C, the boron trifluoride etherate
was added under the protection of N2. The reaction was stirred for two
hours at the same condition. The reaction mixture was washed with
saturated NaHCO3 solution and brine. The combined organic layers
dried over Na2SO4, filtered and concentrated under reduced pressure.
The crude products were purified by column chromatography to obtain
the target compounds.
4.2.3.1. Compound 16a. The product was obtained as white solid (86
mg, 54%). ESI-MS: m/z 458 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 7.63
(d, J = 15.0 Hz, 1H), 7.51 (s, 2H), 7.35 (s, 3H), 6.89 (s, 1H), 6.52 (d, J =
15.5 Hz, 1H), 5.41 (s, 1H), 4.49 (d, J = 9.1 Hz, 1H), 3.90 (s, 2H), 3.68 (s,
1H), 3.55 (s, 1H), 2.67–0.91 (m, 21H) including three typical-CH3 of
DHA such as 1.43 (s, 3H), 0.97 (d, J = 5.2 Hz, 3H), 0.92 (d, J = 7.2 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 158.0, 149.1, 140.5, 129.5, 128.6 (C ×
2), 127.8 (C × 2), 121.1, 104.4, 100.9, 91.3, 84.3, 68.9, 51.4, 45.1, 40.1,
37.4, 36.1, 34.1, 32.4, 26.1, 24.6, 22.2, 20.2, 12.4. HRMS (ESI) calcu­
lated for C26H35NO6Na [M + Na]+ 480.2362, found 480.2349.
4.2.3.2. Compound 16b. The product was obtained as yellow solid (102
mg, 60%). ESI-MS: m/z 510 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.60 (d, J = 15.8 Hz, 1H), 7.48 (m, 2H), 6.90 (d, J = 8.6 Hz, 2H), 6.79 (s,
1H), 6.39 (d, J = 15.8 Hz, 1H), 5.41 (s, 1H), 4.49 (d, J = 9.4 Hz, 1H),
3.91 (s, 2H), 3.85 (s, 3H), 3.70 (s, 1H), 3.57 (s, 1H), 2.68–0.92(m, 21H)
including three typical-CH3 of DHA such as 1.44 (s, 3H), 0.98 (d, J = 5.7
Hz, 3H), 0.94 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 166.4, 160.7, 140.1,
129.3 (C × 2), 127.7, 118.9, 114.1 (C × 2), 102.6, 100.8, 91.2, 80.4,
68.9, 55.3, 51.5, 45.3, 39.9, 37.4, 36.3, 34.1, 32.5, 26.0, 24.7, 22.1,
22.1, 20.2, 12.6. HRMS (ESI) calculated for C27H37NO7Na [M + Na]+
510.2468, found 510.2459.
4.2.3.3. Compound 16c. The product was obtained as yellow solid (86
mg, 52%). ESI-MS: m/z 494 [M + Na]+. 1H NMR (300 MHz, DMSO‑d6) δ
8.05 (s, 1H), 7.43 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 16.1 Hz, 1H), 7.20 (d,
J = 7.8 Hz, 2H), 6.59 (d, J = 15.8 Hz, 1H), 5.41 (s, 1H), 4.49 (d, J = 9.2
Hz, 1H), 3.76 (dd, J = 10.0, 5.3 Hz, 1H), 3.56–3.48 (m, 1H), 3.35 (d, J =
5.6 Hz, 2H), 2.30 (s, 3H), 2.30–0.78 (m, 21H) including three typical-
CH3 of DHA such as 1.27 (s, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.79 (d, J =
7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.2, 140.4, 139.6, 132.2 (C
× 2), 129.4, 127.8, 120.1 (C × 2), 104.2, 100.8, 91.2, 80.3, 69.0, 51.5,
45.3, 40.0, 37.4, 36.3, 34.1, 32.5, 26.0, 24.7, 22.1, 22.1, 20.2, 12.6.
HRMS (ESI) calculated for C27H37NO6Na [M + Na]+ 494.2519, found
494.2508.
4.2.3.4. Compound 16d. The product was obtained as yellow solid (89
mg, 49%). ESI-MS: m/z 540 [M + Na]+. 1H NMR (500 MHz, CDCl3) δ
7.59 (d, J = 15.9 Hz, 1H), 7.13 – 7.09 (m, 2H), 6.89 (d, J = 8.2 Hz, 1H),
6.79 (s, 1H), 6.43 (d, J = 15.5 Hz, 1H), 5.44 (s, 1H), 4.52 (d, J = 9.3 Hz,
1H), 3.94 (d, J = 3.4 Hz, 6H), 3.90 (dd, J = 11.6, 8.8 Hz, 2H), 3.74 (dd, J
= 15.3, 8.5 Hz, 1H), 3.56 (s, 1H), 2.51–0.95 (m, 21H) including three
typical-CH3 of DHA such as 1.45 (s, 3H), 1.00 (d, J = 5.9 Hz, 3H), 0.95
(d, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 166.3, 149.3 (C × 2),
140.1, 130.2 (C × 2), 128.2, 124.7, 121.9, 110.7, 100.8, 91.3, 80.4,
68.9, 55.9, 55.8, 51.5, 45.3, 40.0, 37.4, 36.3, 34.2, 32.6, 26.0, 24.7,
10
Bioorganic Chemistry 111 (2021) 104903
22.1, 20.2, 12.6. HRMS (ESI) calculated for C28H39NO8Na [M + Na]+
540.2573, found 540.2564.
4.2.3.5. Compound 16e. The product was obtained as yellow solid (103
mg, 62%). ESI-MS: m/z 498 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.59 (d, J = 15.8 Hz, 1H), 7.52–7.48 (m, 2H), 7.04 (d, J = 8.6 Hz, 2H),
6.88 (s, 1H), 6.44 (d, J = 15.7 Hz, 1H), 5.41 (s, 1H), 4.49 (d, J = 9.2 Hz,
1H), 3.93–3.86 (m, 2H), 3.70 (d, J = 11.6 Hz, 1H), 3.57–3.52 (m, 1H),
2.45–0.91(m, 21H) including three typical-CH3 of DHA such as 1.42 (s,
3H), 0.98 (d, J = 5.6 Hz, 3H), 0.93 (d, J = 7.1 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ 165.0, 161.6, 139.3, 129.6, 129.5, 121.2, 116.0, 115.9,
115.7, 104.3, 102.8, 91.3, 80.2, 68.3, 51.5, 45.3, 40.0, 37.4, 36.2, 34.1,
32.5, 26.0, 24.7, 22.1, 20.2, 12.6. HRMS (ESI) calculated for
C26H34FNO6Na [M + Na]+ 498.2268, found 498.2258.
4.2.3.6. Compound 16f. The product was obtained as yellow solid (72
mg, 41%). ESI-MS: m/z 525 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.59 (dd, J = 15.6, 2.5 Hz, 1H), 7.49 (dd, J = 7.3, 4.3 Hz, 2H), 7.05 (t, J
= 8.5 Hz, 2H), 6.87 (s, 1H), 6.40 (dd, J = 24.9, 15.6 Hz, 1H), 5.41 (d, J =
4.4 Hz, 1H), 4.45 (t, J = 16.4 Hz, 1H), 3.86 (m, 2H), 3.79–3.50 (m, 2H),
2.67–0.91(m, 21H) including three typical-CH3 of DHA such as 1.43 (d,
J = 10.7 Hz, 3H), 0.97–0.92 (m, 3H), 0.92 (d, J = 6.3 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ 165.0, 161.6, 139.3, 129.6, 129.5, 121.2, 116.0,
115.9, 115.7, 104.3, 102.8, 91.3, 80.2, 68.3, 51.5, 45.3, 40.0, 37.4, 36.2,
34.1, 32.5, 26.0, 24.7, 22.1, 20.2, 12.6. HRMS (ESI) calculated for
C26H34N2O8Na [M + Na]+ 525.2213, found 525.2227.
4.2.3.7. Compound 16g. The product was obtained as yellow solid (103
mg, 56%). ESI-MS: m/z 548 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.59 (s, 5H), 7.09 (s, 1H), 6.58 (d, J = 15.6 Hz, 1H), 5.39 (s, 1H), 4.45 (d,
J = 12.6 Hz, 1H), 3.94 (m, 2H), 3.70–3.50 (m, 2H), 2.67–0.91(m, 21H)
including three typical-CH3 of DHA such as 1.43 (d, J = 10.7 Hz, 3H),
0.97–0.92 (m, 3H), 0.92 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
165.4, 139.3, 138.7, 127.9, 125.7, 125.6, 123.9, 104.5, 104.3, 102.9,
100.9, 91.3, 88.0, 80.3, 68.8, 51.5, 45.3, 40.1, 37.4, 36.2, 34.1, 32.5,
26.0, 25.0, 22.1, 20.2, 12.6. HRMS (ESI) calculated for C27H34F3NO6Na
[M + Na]+ 548.2236, found 548.2242.
4.2.3.8. Compound 16h. The product was obtained as yellow solid (89
mg, 52%). ESI-MS: m/z 514 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.57 (d, J = 15.7 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz,
2H), 6.97 (s, 1H), 6.50 (d, J = 15.7 Hz, 1H), 5.40 (s, 1H), 4.48 (d, J = 9.3
Hz, 1H), 3.96–3.83 (m, 2H), 3.69 (d, J = 13.9 Hz, 1H), 3.52 (d, J = 4.1
Hz, 1H), 2.44–0.85 (m, 21H) including three typical-CH3 of DHA such as
1.27 (s, 3H), 0.88 (s, 3H), 0.85 (s, 3H). 13C NMR (75 cMHz, CDCl3) δ
165.7, 139.1, 133.6, 129.0 (C × 4), 121.9, 104.5, 102.8, 100.9, 91.3,
80.4, 68.8, 51.5, 45.3, 40.0, 37.4, 36.2, 34.1, 32.5, 26.0, 24.7, 22.1,
20.2, 12.6. HRMS (ESI) calculated for C26H34ClNO6Na [M + Na]+
514.1972, found 514.1963.
4.2.4. General procedure for synthesis of hybrids 18a-e
The corresponding benzaldehyde (1 mmol, 1 eq) and Cyanoacetic
acid (1.11 mmol, 1.11 eq) were dissolved in toluene (10 ml). Then
ammonium acetate (0.17 mmol, 0.17 eq) was added as catalyst. The
reaction mixture was stirred at 100 ◦ C for 7 h. Filtered, the filter cake
was washed three times with the filtrate and dried under vacuum to
obtain the target compounds.
4.2.5. General procedure for synthesis of hybrids 18a-e
To a solution of 50% glyoxylic acid (0.95 mmol, 0.95 eq), the cor­
responding benzeneacetonitrile (1 mmol, 1 eq) and K2CO3 (1.5 mmol,
1.5 eq) in methanol (10 ml) was stirred at 60 ◦ C for 5–8 h until TLC
showed the reaction was completed. Then filtered, the filter cake was
washed three times with CH2Cl2。 Afterwards the filter cake was dis­
solved in water and using hydrochloric acid to adjust pH to 4. The
Y. Hu et al.
aqueous phase was extracted with ethyl acetate three times. The com­
bined organic layers dried over Na2SO4, filtered and concentrated under
reduced pressure to obtain hybrids 17a-e.
4.2.6. General procedure for synthesis of hybrids 19a-e, 22a-e
Cinnamic acid derivatives (0.42 mmol, 1.2 eq) and DMAP (0.42
mmol, 1.2 eq) were dissolved in dry CH2Cl2 (10 ml). Then added EDCl
(0.42 mmol, 1.2 eq) and cooled to 0 ◦ C. After addition of DHA (0.35
mmol, 1 eq) the reaction mixture was slowly warmed to room temper­
ature and stirred overnight. The organic layer was washed by saturated
sodium chloride three times. The combined organic layers dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude
products were purified by column chromatography to obtain the target
compounds.
4.2.6.1. Compound 19a. The product was obtained as white solid (88
mg, 57%). ESI-MS: m/z 462 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
8.33 (s, 1H), 8.04 (d, J = 7.0 Hz, 2H), 7.57 (m, 3H), 5.90 (d, J = 9.9 Hz,
1H), 5.53 (s, 1H),2.77–0.95 (m, 21H) including three typical-CH3 of
DHA such as 1.46 (s, 3H), 1.00 (d, J = 6.0 Hz, 3H), 0.96 (d, J = 7.0 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 163.5, 161.5, 154.5, 133.3 (C × 2),
123.8, 115.2, 114.3 (C × 2), 104.0, 93.3, 91.1, 87.2, 79.5, 55.1, 51.2,
44.7, 36.8, 35.7, 33.6, 31.3, 25.4, 24.1, 21.5, 19.7, 11.6. HRMS (ESI)
calculated for C25H29NO6Na [M + Na]+ 462.1893, found 462.1977.
4.2.6.2. Compound 19b. The product was obtained as white solid (107
mg, 65%). ESI-MS: m/z 492 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
8.25 (s, 1H), 8.05 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 8.9 Hz, 2H), 5.89 (d, J
= 9.8 Hz, 1H), 5.52 (s, 1H), 3.92 (s, 3H), 2.80–0.94 (m, 21H) including
three typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.8 Hz, 3H),
0.95 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 161.4, 156.0,
153.8, 133.6, 131.2 (C × 2), 129.3 (C × 2), 115.2, 104.5, 96.1, 94.1,
92.0, 79.8, 51.6, 45.3, 37.3, 36.2, 34.1, 32.0, 26.0, 24.6, 22.0, 20.2,
12.2. HRMS (ESI) calculated for C26H31NO7Na [M + Na]+ 492.1998,
found 492.2089.
4.2.6.3. Compound 19c. The product was obtained as white solid (86
mg, 54%). ESI-MS: m/z 476 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
8.29 (s, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 5.89 (d, J
= 10.0 Hz, 1H), 5.52 (s, 1H), 2.46 (s, 3H), 2.80–0.94 (m, 21H) including
three typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.8 Hz, 1H),
0.96 (d, J = 7.1 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 161.7, 155.8,
177.76, 131.5 (C × 2), 130.1 (C × 2), 128.8, 104.6, 101.7, 93.9, 91.7,
87.9, 80.0, 63.8, 51.6, 45.2, 37.2, 36.2, 34.1, 31.7, 25.9, 24.7, 22.0,
20.4, 12.2. HRMS (ESI) calculated for C26H31NO6Na [M + Na]+
476.2049, found 476.2134.
4.2.6.4. Compound 19d. The product was obtained as yellow solid (80
mg, 46%). ESI-MS: m/z 522 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
8.22 (s, 1H), 7.82 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 7.9 Hz,
1H), 5.88 (d, J = 9.8 Hz, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 2.73–0.85 (m,
21H) including three typical-CH3 of DHA such as 1.44 (s, 3H), 0.98 (d, J
= 4.5 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
162.1, 155.2 (C × 2), 153.7, 127.8, 124.2, 115.8, 111.8, 110.7, 104.6,
99.1, 93.6, 91.5, 80.0, 56.2, 56.1, 51.6, 45.2, 37.2, 36.2, 31.8, 31.3,
25.9, 24.6, 22.0, 20.2, 12.2. HRMS (ESI) calculated for C27H33NO8Na
[M + Na]+ 522.2104, found 522.2109.
4.2.6.5. Compound 19e. The product was obtained as white solid (82
mg, 51%). ESI-MS: m/z 480 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
8.28 (s, 1H), 8.09 (d, J = 5.3 Hz, 1H), 8.06 (d, J = 5.2 Hz, 1H), 7.23 (t, J
= 8.6 Hz, 2H), 5.90 (d, J = 9.9 Hz, 1H), 5.52 (s, 1H), 2.80–0.94 (m, 21H)
including three typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.8
Hz, 3H), 0.96 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 171.1,
154.5, 140.9, 135.0, 133.9, 133.8, 117.0, 116.7, 101.7, 96.4, 94.8, 91.3,
11
Bioorganic Chemistry 111 (2021) 104903
89.7, 87.9, 51.4, 44.5, 37.5, 36.2, 34.7, 30.8, 26.2, 24.7, 22.1, 20.4,
13.0. HRMS (ESI) calculated for C25H28FNO6Na [M + Na]+ 480.1798,
found 480.1882.
4.2.6.6. Compound 22a. The product was obtained as white solid (83
mg, 54%). ESI-MS: m/z 462 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.75 (d, J = 6.4 Hz, 2H), 7.51 (d, J = 7.5 Hz, 3H), 7.00 (s, 1H), 6.00 (d, J
= 9.8 Hz, 1H), 5.51 (s, 1H), 2.80–0.94 (m, 21H) including three typical-
CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.8 Hz, 3H), 0.94 (d, J =
7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 161.9, 131.8 (C × 2), 129.4,
128.6, 127.1, 114.9, 110.2, 104.6, 96.3, 93.1, 91.6, 80.1, 51.5, 45.3,
37.3, 36.2, 34.1, 31.8, 25.9, 24.6, 22.0, 20.2, 12.2. HRMS (ESI) calcu­
lated for C25H29NO6Na [M + Na]+ 462.1893, found 462.1889.
4.2.6.7. Compound 22b. The product was obtained as yellow solid (79
mg, 48%). ESI-MS: m/z 508 [M + K]+. 1H NMR (300 MHz, CDCl3) δ 7.71
(d, J = 8.9 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 6.87 (s, 1H), 5.99 (d, J =
10.0 Hz, 1H), 5.50 (s, 1H), 3.89 (s, 3H), 2.67–0.92 (m, 21H) including
three typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.7 Hz, 3H),
0.93 (d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 162.7, 162.3,
131.1, 128.9 (C × 2), 127.0, 125.5, 124.2, 114.8 (C × 2), 104.5, 92.9,
91.6, 80.1, 55.6, 51.5, 45.3, 37.3, 36.2, 31.8, 25.9, 24.6, 22.0, 20.2,
12.2. HRMS (ESI) calculated for C26H31NO7Na [M + Na]+ 492.1998,
found 492.1982.
4.2.6.8. Compound 22c. The product was obtained as white solid (71
mg, 45%). ESI-MS: m/z 476 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.64 (d, J = 8.1 Hz, 2H), 7.28 (s, 2H), 6.95 (s, 1H), 6.00 (d, J = 9.9 Hz,
1H), 5.51 (s, 1H), 2.43 (s, 3H), 2.70–0.92 (m, 21H) including three
typical-CH3 of DHA such as 1.46 (s, 3H), 1.00 (d, J = 5.7 Hz, 3H), 0.94
(d, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 162.2, 142.7, 130.1 (C
× 2), 129.0, 127.5, 127.2, 127.1 (C × 2), 115, 104.5, 93.0, 91.6, 80.1,
51.5, 45.3, 37.3, 36.2, 34.1, 31.8, 25.9, 22.0, 20.2, 12.2. HRMS (ESI)
calculated for C26H31NO6Na [M + Na]+ 476.2049, found 476.2036.
4.2.6.9. Compound 22d. The product was obtained as white solid (85
mg, 53%). ESI-MS: m/z 480 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.76 (s, 2H), 7.21 (d, J = 7.2 Hz, 2H), 6.93 (s, 1H), 5.99 (d, J = 9.5 Hz,
1H), 5.51 (s, 1H), 2.68–0.92 (m, 21H) including three typical-CH3 of
DHA such as 1.46 (s, 3H), 1.00 (d, J = 4.0 Hz, 3H), 0.93 (d, J = 6.3 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 166.4, 161.2, 129.4, 129.3, 128.4,
116.8 (C × 2), 116.5 (C × 2), 114.8, 104.5, 104.4, 93.2, 91.6, 80.1, 51.5,
45.3, 37.2, 36.2, 34.0, 31.8, 25.9, 24.6, 21.9, 20.2, 12.1. HRMS (ESI)
calculated for C25H28FNO6Cl [M + Cl]+ 492.1589, found 492.2038.
4.2.6.10. Compound 22e. The product was obtained as white solid (75
mg, 42%). ESI-MS: m/z 530 [M + Na]+. 1H NMR (300 MHz, CDCl3) δ
7.87 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.06 (s, 1H), 6.00 (d, J
= 9.9 Hz, 1H), 5.51 (s, 1H), 2.69–0.93 (m, 21H) including three typical-
CH3 of DHA such as 1.45 (s, 3H), 1.00 (d, J = 5.6 Hz, 3H), 0.94 (d, J =
7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 167.1, 158.8, 130.9, 127.6 (C
× 2), 126.5, 126.4, 126.3, 115.0, 113.2, 104.6, 93.4, 91.6, 80.1, 51.5,
45.3, 37.3, 36.2, 34.1, 31.7, 25.9, 24.6, 22.0, 20.2, 12.1. HRMS (ESI)
calculated for C26H28F3NO6Na [M + Na]+ 530.1766, found 530.1844.
4.3. Cell culture
The human breast cancer cell line MCF-7 and MDA-MB-231, human
non-small-cell lung cancer cell line A549, human hepatocellular carci­
noma cell line HepG-2 and human normal liver cell line L02 were grown
in DMED (for MCF-7, MDA-MB-231, HepG2 and L02) or 1640 (for
A549). All the media were supplemented with 10% fetal bovine serum.
The cells are cultured in an incubator (5% CO2 in air in 37 ◦ C).
Y. Hu et al.
4.4. Cell viability assay
Cell viability assay was measured using 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The cytotoxicity of
compounds was tested in MCF-7, MDA-MB-231, HepG2, A549 and L02
cells. Cells were plated in 24-well plates at a density of 15,000 cells per
well. After incubated overnight, cells were treated with artemisinin
derivatives at 0, 3, 6, 12, 24, 48 μM and incubated for 48 h. Then, each
well was added with 0.5 mg/ml of MTT 50 μL and incubated for 4 h. The
solution was removed and 0.5 ml dimethyl sulfoxide was added to each
well. Finally, we measured the absorbance at 570 nm.
4.5. Cell cycle assay
A549 cells were plated (1.5 × 105 cells/well) in six-well plates and
incubated for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 7.5
μM of 16 g for 48 h. Then the cells were digested, washed with PBS and
resuspended in PBS. The cooled 75% ethanol at 4 ◦ C was added to fix the
cells overnight. The cells were concentrated by centrifugation and
resuspended in PBS. Next, RNase A was added and incubated at 37 ◦ C for
30 min. Finally, propidium iodide was added and the mixture was
incubated for 10 min. Cell cycle analysis was evaluated via PI fluores­
cence and flow cytometry (Accuri C6, BD Biosciences). For each mea­
surement, at least 10,000 cells were counted.
4.6. Cell apoptosis assay
4.6.1. Hoechst 33,342 staining
A549 cells were plated (5 × 104 cells/well) in twelve-well plates and
incubated for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 10
μM of 16 g for 48 h. Then the cells were washed with PBS and incubated
with 10 μL Hoechst 33,342 for 5 min at 37 ◦ C under 5% CO2. The
morphological apoptosis was visualized under a fluorescent microscope
(Nikon, Ts2R, Japan).
4.6.2. Annexin V/PI detection
A549 cells were plated (1.5 × 105 cells/well) in six-well plates and
incubated for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 10
μM of 16 g for 48 h. Then the cells were digested, washed with PBS and
resuspended in PBS. Then, Annexin V-FITC and PI were added for 5 min
at room temperature protected from light. Cell apoptosis was measured
by flow cytometry (Accuri C6, BD Biosciences). For each measurement,
at least 10,000 cells were counted.
4.7. Western blotting analysis
A549 cells were seeded (5 × 105 cells) in 6 cm dishes and incubated
for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 10 μM of 16 g
for another 48 h. The cells were washed and lysed using lysis buffer. The
lysates were centrifuged at 10,000g for 20 min at 4 ◦ C. Then the proteins
were diluted to 5 mg/ml (BCA method). Each sample (10 μL) was
analyzed by SDS-PAGE (12% gel). Then the proteins were detected by
the conventional method. The monoclonal antibodies were purchased
(Abcam, Cambridge, UK).
4.8. MMP analysis
A549 cells were plated (1.5 × 105 cells/well) in six-well plates and
incubated for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 10
μM of 16 g for 48 h. Then the cells were digested, washed with PBS and
resuspended in 500 μL JC-1 incubation buffer at 37 ◦ C for 15 min. The
result was monitored by flow cytometry analysis (Accuri C6, BD
Biosciences).
12
Bioorganic Chemistry 111 (2021) 104903
4.9. Measurement of intracellular ROS generation
A549 cells were plated (1.5 × 105 cells/well) in six-well plates and
incubated for 24 h. The cells were treated with 0, 2.5 μM, 5 μM and 10
μM of 16 g for 48 h. The cells were incubated with 10 μM DCFH-DA at
37 ◦ C for 20 min. Then, cells were harvested and suspended in 1 ml PBS.
Samples were analyzed by flow cytometry (Accuri C6, BD Biosciences).
4.10. Wound healing assay
A549 cells were plated (4 × 105 cells/well) in six-well plates and
incubated for 24 h. Once the cells attached properly, the monolayer was
disrupted by a sterile pipette tip scraping the bottom of each well. Then
the cells were washed with PBS for three times and the remaining cells
were cultured in serum-free 1640. Different concentrations of 16 g (0,
2.5 μM, 5 μM and 10 μM) was added. Representative images of cells
migrating into the wounds were captured at 0 h, 12 h, and 24 h time
points at the same wounded region using a fluorescent microscope
(Nikon, Ts2R, Japan). The width of the wound edge closure was
measured by Image J.
4.11. Acute toxicity in zebrafish Embryo
The acute toxicities were investigated by the zebrafish embryo
experiment. After 2 h of fertilization, the embryos were placed in 24-
well plates (20 embryos/well) and added with or without different
concentrations of tested compounds (0.625–10.0 μM) in 0.1% DMSO
incubated in E3 medium (0.3 g NaCl, 0.013 g KCl, 0.05 g CaCl2, 0.08 g
MgSO4, and 0.05% methylene blue) at 28.5 ◦ C. Digital images of em­
bryos were taken under a stereomicroscope at 10 × magnification
(Nikon Eclipse SMZ745T with ACT-1 imaging software) and LC50 values
were calculated for 72 hpf (hours post-fertilization). The experiments
were performed in triplicate.
4.12. Solubility evaluation
The solubility of compound 16 g was determined via HPLC (Shi­
madzu LC-2030C). A linear calibration curve was determined in CH3CN
from seven different concentrations (0.96 mg/ml, 0.48 mg/ml, 0.38 mg/
ml, 0.29 mg/ml, 0.19 mg/ml, 0.096 mg/ml, 0.048 mg/ml) and the
calibration curve was set up via linear regression. Compound 16 g was
added to phosphate buffer (50 mM, pH 7.4) and stirred overnight at
ambient temperature. The sample was centrifuged and analyzed by
HPLC.
4.13. Statistical analyses
Statistical analyses were performed using GraphPad Prism5. Cell
cycle arrest was analyzed by C Flow Plus and Flowjo 7.6. Annexin V/ PI
detection was analyzed by C Flow Plus. Wound healing assay and
western blotting were measured by Image J.
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.
Acknowledgements
This work was financially supported by “Double First Class” Subject
Innovation Team Construction Project of China Pharmaceutical Uni­
versity (CPU2018GY12).
Y. Hu et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104903.
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