Design, synthesis, and mechanism of dihydroartemisinin-coumarin hybrids as potential anti-neuroinflammatory agents

Article abstract of DOI:10.3390/molecules24091672

Cancer patients frequently suffer from cancer-related fatigue (CRF), which is a complex syndrome associated with weakness and depressed mood. Neuroinflammation is one of the major inducers of CRF. The aim of this study is to find a potential agent not only on the treatment of cancer, but also for reducing CRF level of cancer patients. In this study, total-thirty new Dihydroartemisinin-Coumarin hybrids (DCH) were designed and synthesized. The in vitro cytotoxicity against cancer cell lines (HT-29, MDA-MB-231, HCT-116, and A549) was evaluated. Simultaneously, we also tested the anti-neuroinflammatory activity of DCH. DCH could inhibit the activated microglia N9 release of NO, TNF-α, and IL-6. The docking analysis was shown that MD-2, the coreceptor of TLR4, might be one of the targets of DCH.

Full text of DOI:10.3390/molecules24091672

molecules
Article
Design, Synthesis, and Mechanism of
Dihydroartemisinin–Coumarin Hybrids as
Potential Anti-Neuroinflammatory Agents
Haonan Yu 1,† , Zhuang Hou 1,†, Xiaoguang Yang 1, Yanhua Mou 2,* and Chun Guo 1,
*
1
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China;
yhna380@hotmail.com (H.Y.); houzhuang8@sina.com (Z.H.); Xiaog_yang@163.com (X.Y.)
School of Life Sciences and Biological Pharmacy, Shenyang Pharmaceutical University,
Shenyang 110016, China
2
*
†
Correspondence: mu_hua_jj@sina.com (Y.M.); chunguo@syphu.edu.cn (C.G.); Tel.: +86-24-4352-0226 (C.G.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 April 2019; Accepted: 24 April 2019; Published: 28 April 2019
Abstract: Cancer patients frequently suffer from cancer-related fatigue (CRF), which is a complex
syndrome associated with weakness and depressed mood. Neuroinflammation is one of the major
inducers of CRF. The aim of this study is to find a potential agent not only on the treatment
of cancer, but also for reducing CRF level of cancer patients. In this study, total-thirty new
Dihydroartemisinin–Coumarin hybrids (DCH) were designed and synthesized. The in vitro
cytotoxicity against cancer cell lines (HT-29, MDA-MB-231, HCT-116, and A549) was evaluated.
Simultaneously, we also tested the anti-neuroinflammatory activity of DCH. DCH could inhibit the
activated microglia N9 release of NO, TNF-α, and IL-6. The docking analysis was shown that MD-2,
the coreceptor of TLR4, might be one of the targets of DCH.
Keywords: anticancer; anti-neuroinflammation; coumarin; dihydroartemisinin
1. Introduction
Chemotherapy is an important method in the therapy of cancer, but the adverse side effects of
anticancer agents should also be focused on. Gastrointestinal injury induced by chemotherapeutic
agents could result in bacterial/endotoxin translocation from the gut to the systemic blood
circulation [
neuroinflammation [
a complex syndrome associated with weakness and depressed mood. Diana M., Norden et al.
demonstrated that the neuroinflammation is one of the major reasons which induce CRF [ ]. Currently,
there were no effective method to reduce CRF [ ]. The aim of this study is to find a potential
1,
2]. The released endotoxin would cause a systemic inflammatory response including
3
]. Cancer patients frequently suffer from cancer-related fatigue (CRF), which is
4
5
anti-neuroinflammatory agents which might reduce CRF of cancer patients.
Central nervous system (CNS) consists of two major types of cells, nerve cells and glial cells.
Microglia is a major glial cell and plays an important role as the resident macrophage population
in the CNS [
neurotoxic. They release reactive oxygen species, nitric oxide (NO), tumor necrosis factor-alpha
(TNF- ), and other potentially cytotoxic molecules [ ]. It means that activation of microglia could
induce neuroinflammation.
6,7]. There has been wide recognition that the fully activated microglial cells are
α
8,9
Lipopolysaccharide (LPS), also known as endotoxin, is a natural Toll-like receptor 4 (TLR4)
ligand and it is also a potent microglial activator that can trigger the production of inflammatory
mediators. TLR-4, a transmembrane receptor, plays a vital role in the initiation and acceleration of
the inflammatory response induced by LPS [10
,
11]. TLR4 alone does not directly bind LPS and needs
Molecules 2019, 24, 1672; doi:10.3390/molecules24091672
www.mdpi.com/journal/molecules

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the coreceptor MD-2 [12]. MD-2 is linked with the extracellular domain of TLR4 and is indispensable
for LPS recognition [13]. MD-2 is a member of the MD-2-related lipid-recognition protein family [14].
MD-2 directly binds to LPS in its hydrophobic cavity with high affinity [15].
Artemisinin and its derivatives have been the first-line antimalarial drug since the late 1990s.
Dihydroartemisinin (DHA) is the primary bioactive metabolite of artemisinin and its derivatives,
but not artemisinin per se [16]. Additionally, DHA has higher bioavailability and less side effects
than artemisinin [17]. Recent research showed that besides antimalarial activity, DHA also exhibits
anticancer and anti-inflammatory activity [18–21]. As the anti-inflammatory agent, DHA could
decrease LPS-induced protein expression of TLR4 [22].
Coumarins are widely distributed in different parts of plants. There are various pharmacological
activities of coumarins, which includes anti-inflammatory [23
As the anticancer agents, coumarins were reported to exhibit negligible or mild side effects [27
,
24] and anticancer activity [25
,
26].
28].
,
Substitution at the C-3 or C-4 position of coumarins exhibit good cytotoxic activity in various cancer
cell lines [26 29 31]. Additionally, as anti-inflammatory agents, some kinds of coumarins could also
,
–
target TLR4/MD-2 complex [32,33].
In our previous study, Dihydroartemisinin–Coumarin hybrids (DCH) were designed and
synthesized referring to the principle of hybridization [34,35]. Each of them contains coumarin
moiety, DHA moiety and a linker. DCH could inhibit HT-29 cells proliferation and arrest the G0/G1
phase of HT-29 cells [35]. They could also suppress cancer cell migration, and induce apoptosis and
ferroptosis of HT-29 cancer cells [35]. It also has been confirmed that the DCH having 1,2,3-triazole
linker generally exhibit better cytotoxicity exhibition than the others [35].
With this background, total-thirty new DCH were designed and synthesized, and the synthetic
approaches were shown in Figure 1. All of them contained 1,2,3-triazole linker. We focused on
the modifications of positions 3 and 4 of the coumarin moiety. Due to the new synthesized DCH
were for the cancer patients, we evaluated the in vitro cytotoxicity against cancer cell lines (HT-29,
MDA-MB-231, HCT-116, and A549). In addition, DCH could inhibit the activated N9 cell release of NO,
TNF-α and IL-6. Furthermore, to investigate the mechanism of the anti-neuroinflammatory activity,
docking analysis was applied. The results might support that the anti-neuroinflammatory activity was
related to inhibiting TLR4/MD-2 complex.
Figure 1. Structures of new designed Dihydroartemisinin–Coumarin hybrids.
2. Results and Discussion
2.1. Chemistry
The synthesis of the intermediates a–p and I–IV is presented in Scheme 1. The intermediates I and II
were obtained by 2-bromoethanol or 3-bromo-1-propanol, with dihydroartemisinin (DHA), respectively,
followed by treating the product with sodium azide. The intermediates III and IV were synthesized
by propargyl alcohol or 3-butyn-1-ol with DHA, respectively. The required 3-methylcoumarin
derivatives were obtained by substitution of salicylaldehyde with propionic anhydride in the
presence of sodium propionate and triethylamine. The substituted-3-bromomethylcoumarins were
prepared by the radical bromination of 3-methylcoumarin derivatives through the reaction with

Molecules 2019, 24, 1672
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N-Bromosuccinimide (NBS) in the presence of dibenzoylperoxide [36]. The intermediates a–e
were synthesized by substituted-3-bromomethylcoumarins via azidation reaction. The required
substituted-4-chloromethylcoumarins were prepared by the Pechmann cyclisation [37] of substituted
phenols with 4-chloroethylacetoacetate using sulfuric acid as the condensing agent. The intermediates
f
–
i
were synthesized by substituted-4-chloromethylcoumarin via azidation reaction. To synthesize
the intermediates , several substituted-4-hydroxycoumarin were prepared by condensing the
j–p
substituted phenol with malonic acid [38]. Then the products were treated with 3-bromo-1-propyne in
the presence of potassium carbonate.
.
Scheme 1. Synthetic routes of intermediates. Reagents and conditions: (1) BF3 Et2O, CH2Cl2, 0 ◦C;
(2) NaN3, DMF, 60 ◦C; (3) CH3CH2COONa, Et3N, reflux; (4) NBS, BPO, CCl4, reflux; (5) 70% H2SO4,
r.t.; (6) ZnCl2, POCl3, 65 ◦C; and (7) K2CO3, DMF, 60 ◦C.

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The synthesis of the desired new compounds was presented in Scheme 2. The target compounds
1e and 2a 2e were obtained by the reaction of the intermediates III or IV with via click chemistry,
respectively. The target compounds 3f 3i and 4f 4i were synthesized by intermediates III or IV with
via click chemistry, respectively. The target compounds 5j 5l 5o, and 6j 6p were prepared by the
reaction of the intermediates I or II with j–p via click chemistry, respectively.
1a
–
–
a–e
–
–
f
–i
,
–
–
H
H
O
O
1a, 2a: R1=H, R2=H
R1
O
O
1b, 2b: R1=Br, R2=H
1c, 2c: R1=Cl, R2=H
1d, 2d: R1=Cl, R2=Cl
1e, 2e: R1=Br, R2=Br
N3
(1)
O
O
H
H
+
O
O
O
O
R1
O
R2
O
O
N
a-e
x
x
III, IV
R2
N
N
1a-1e(x=1)
2a-2e(x=2)
O
............................................................................................................................................................................................
H
H
O
O
O
O
3f, 4f: R3=OH, R4=H, R5=H
: R =CH , R =H, R =CH
3h, 4h: R3=CH3, R4=H, R5=H
3i, 4i: R3=H, R4=CH3, R5=H
N3
R5
O
,
3g 4g
O
3
3
4
5
3
O
H
H
R4
R3
(1)
O
+
O
O
N
O
O
x
x
III, IV
f-i
R4
R5
O
N
N
R3
3f-3i(x=1)
4f-4i(x=2)
O
...........................................................................................................................................................................................
H
H
5j, 6j: R6=H, R7=H, R8=H, R9=H
6k: R6=H, R7=H, R8=CH3, R9=H
5l, 6l: R6=CH3, R7=H, R8=CH3, R9=H
5m, 6m: R6=H, R7=CH3, R8=H, R9=CH3
5n, 6n: R6=H, R7=H, R8=CH3, R9=CH3
5o, 6o: R6=H, R7=H, R8=H, R9=CH3
6p: R6=H, R7=CH3, R8=H, R9=H
O
O
R6
O
O
O
O
O
O
H
(1)
H
R7
R8
+
O
O
O
O
O
N3
y
R7
O
R9
j p
-
y
N
N
I, II
R6
R8
R9
N
O
5j, 5l-5o(y=2)
6j-6p(y=3)
O
Scheme 2. Synthetic routes of target compounds. Reagents and conditions: (1) CuI, triethylamine,
dichloromethane, r.t.
2.2. Biological Assay
2.2.1. In Vitro Cytotoxic Activity of the Compounds
As the anti-neuroinflammatory agents for cancer patients, all newly synthesized compounds
were evaluated in vitro for antitumor activity against human breast cancer cell line MDA-MB-231,
colorectal cancer cells HT-29 and HCT-116, and lung cancer cell line A549 by using the standard MTT
method, respectively. Doxorubicin (DOX) and DHA were used as the reference standard. Additionally,
owing to the rapid proliferation, the tumor cells actually was always in hypoxia condition. Therefore,
the in vitro cytotoxicity was evaluated in two different conditions (hypoxia and normoxia). All data
are summarized in Table 1. Most of the target compounds exhibited moderate activities against the
tested cell lines and they showed a general promotion under hypoxia conditions.

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The structure–activity relationship was evaluated: (i) the series of compounds
exhibited better cytotoxicity activity than that of the series of compounds and
the 4-oxygen group in the coumarin moiety as a part of linker could improve the antitumor activity;
(ii) although compounds and did not exhibit good activity against three cell lines (MDA-MB-231,
5
and
6
generally
3
4, which indicated that
3
4
HCT-116, and A549), they showed good activity against HT-29 like the other derivatives. Therefore,
4-methyl group in the coumarin moiety could improve the selectivity of DCH against HT-29.
In addition, almost all of the target compounds exhibited good cytotoxicity activity against HT-29.
Therefore, HT-29 was selected for the next explorations.
Table 1. IC50 (µM) of tested samples in MDA-MB-231, HT-29, HCT-116, and A549 cell lines.
HT-29
MDA-MB-231
HCT-116
A549
Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia Hypoxia Normoxia
DOX
DHA
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
<0.1
11.74
14.29
13.14
26.37
13.34
26.95
19.42
12.09
19.61
30.81
13.92
15.46
15.86
32.04
8.57
11.30
9.33
56.35
39.02
12.25
3.76
5.89
8.90
21.25
7.42
5.32
0.11
51.10
18.40
21.35
35.41
14.94
15.32
>100
9.75
17.46
19.54
38.52
37.83
23.89
>100
16.36
32.30
13.85
>100
>100
30.03
2.78
0.69
1.37
>100
64.97
>100
>100
68.85
77.89
>100
37.74
43.98
88.46
>100
>100
>100
>100
>100
>100
>100
>100
>100
100.00
>100
14.17
11.04
37.41
67.42
38.56
1.17
>2
1.29
>2
>2
45.93
30.47
>100
32.26
33.93
45.46
>100
36.86
32.47
47.92
44.14
63.07
>100
>100
22.81
78.79
22.71
>100
>100
69.41
>100
13.04
11.68
16.54
33.40
14.46
6.85
25.89
49.79
>100
40.38
48.61
>100
>100
>100
64.37
46.51
86.49
67.74
>100
>100
>100
>100
25.65
>100
>100
>100
>100
44.81
32.92
38.49
32.84
33.70
27.92
93.53
33.74
42.35
38.05
10.33
48.90
>100
60.30
60.01
54.45
>100
55.99
49.48
53.19
94.16
>100
>100
>100
64.99
>100
34.50
>100
>100
77.20
>100
62.82
25.64
29.69
25.90
28.13
26.90
42.05
22.51
12.09
35.33
>100
53.87
>100
48.70
45.42
59.75
>100
60.16
66.87
69.67
79.37
98.00
>100
>100
62.27
>100
>100
>100
>100
>100
>100
1.282
2.443
45.06
49.03
9.49
>100
83.48
>100
73.49
53.34
>100
>100
>100
87.15
96.48
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.670
2.107
50.37
53.37
14.92
6.88
3f
3g
3h
3i
4f
4g
4h
4i
5j
5l
5m
5n
5o
6j
6k
6l
6m
6n
6o
6p
12.88
10.19
16.09
10.49
6.85
8.41
22.43
9.16
3.40
5.90
4.19
29.02
12.48
9.78
18.74
10.84
18.57
10.33
24.64
13.35
47.65
22.08
20.14
0.039
39.92
18.82
27.87
30.40
78.07
18.85
81.57
7.49
2.2.2. Morphological Changes and Effect of Compound 5n on Cell Viability
As shown in A–F of Figure 2, there showed a concentration-dependent cell death after treatment
with compound 5n for 48 h. As the concentration up to 100 M, the compound 5n could obviously
µ
inhibit the proliferation of HT-29. As shown in the (G) of Figure 2, after HT-29 cells were treated with
different concentrations of 5n for 48 h, the cell viability decreased in a dose-dependent manner.

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Figure 2. Inhibition of compound 5n on cell viability in HT-29 cells by MTT staining. Cells were
treated with ( ) 0 M, ( ) 6.25 M, ( ) 12.5 M, ( ) 25.0 M, ( ) 50.0 M, and ( ) 100.0 M compound
5n for 48 h. Representative images were shown; 200 . ( ) Cells were treated with 6.25, 12.5, 25,
50, and 100 M compound 5n for 48 h. Data were shown as mean SD. * p < 0.05, ** p < 0.01 and
A
µ
B
µ
C
µ
D
µ
E
µ
F
µ
×
G
µ
±
*** p < 0.001 compared with group 0 µM.
2.2.3. Anti-Neuroinflammatory Activity
The anti-neuroinflammatory activity of DCH was evaluated and then the representative
compounds were selected for the further study. As shown in Table 2, murine microglial N9 cells were
treated with 1
screening. Most of the tested compounds exhibited good anti-inflammatory activity, and especially the
IC50 of some compounds were even less than 1 M. Among them, compound 5n had the best activity
and the IC50 reached 0.22 M. In addition, in order to exclude the death influence, MTT-IC20 of all the
µg/mL LPS for 48 h and the NO level in the medium was measured as the preliminary
µ
µ
compounds were measured. The results showed that DCH did not exhibit cytotoxic activity in N9 cells.
The exact reason was still unclear, but we supposed that it might be related to the homeostasis of cells.
The tumor cells actually were always in hypoxia condition, in which DCH could promote the cytotoxic
activity against tumor cells. In brief, DCH could reduce the release of NO caused by LPS-induced
microglial cells.

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Table 2. IC50 of tested samples in LPS-induced N9 cells for the inhibition of NO; IC20 of tested samples
in N9 cells for cytotoxic activity.
Compound.
NO-IC50 (µM)
MTT-IC20 (µM)
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3f
3g
3h
3i
4f
4g
4h
4i
5j
5l
5m
5n
5o
6j
6k
6l
6m
6n
6o
1.46
>10
5.33
1.60
0.48
0.97
0.64
2.66
1.35
0.94
0.37
>10
>10
0.72
2.03
1.36
5.69
>10
1.59
3.36
1.08
0.22
0.57
0.29
1.91
0.52
5.80
1.13
1.43
1.42
1.58
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
7.13
1.04
2.44
>10
>10
>10
>10
>10
6.53
>10
>10
>10
>10
6p
DHA
Minocycline
NO Capture Capability
Sodium nitroprusside (SNP) is a NO donor. In this experiment, the reason of the DCH reducing
NO level was preliminary explored. As shown in Figure 3, the results showed that the compounds
only partially reduced NO levels by direct NO capture. That means the main reason of the NO level
reduction is due to species-specific effect.
Figure 3. Compound 6l and 5n had NO radical scavenging activity. 6l (0.01
and 10 M) or 5n (0.01 M, 0.1 M, 1 M, and 10 M) was added to a concentration of 0.5 mM SNP
solution. After reacted for 1 h under normal sun light at room temperature, Griess reagent was added
µM, 0.1 µM, 1 µM,
µ
µ
µ
µ
µ
and the optical density was observed at 540 nm. Data were shown as mean
with control group.
±
SD. *** p < 0.001 compared

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Effect of Representative Compounds on TNF-α and IL-6 Release
As shown in Table 3, four kinds of DCH were involved to measure the effect on the release of
TNF- and IL-6 from the microglia cells which induced by LPS for 48 h. The results showed that
compounds 2b 2c 5n, and 6p could inhibit the release of TNF- with the inhibition rate of 22% to 32%
α
,
,
α
and the release of IL-6 with the inhibition rate of 26% to 43%. The results indicated that the inhibition
effect of DCH on NO release was not only by nonspecific effects, but also by slightly influencing the
synthesis and release of upstream inflammatory pathway.
Table 3. The inhibition of compounds (1
µM) on cytokines release (48 h) of the murine microglial cell
line N9 induced by lipopolysaccharide (LPS) (1 µg/mL).
TNF-α (pg/mL)
Inhibition (%)
IL-6 (pg/mL)
Inhibition (%)
Group
155.54 ± 79.61
4455.87 ± 518.24 #
3285.99 ± 1062.40
3077.03 ± 375.48
3508.08 ± 368.72
3451.29 ± 646.50
–
–
Con
LPS
2b
2c
5n
6p
#
0
±
–
–
1154.44
148.48 #
27.20
32.06
22.04
23.36
797.52 ± 140.74 *
850.24 ± 112.47
656.75 ± 79.49 **
747.80 ± 125.56 *
30.92
26.35
43.11
35.22
p < 0.001 compared with control group, * p < 0.05 ** p < 0.01 compared with LPS group.
Docking Analysis
Toll-like receptor 4 (TLR4) and its coreceptor—MD-2—are the target of LPS, which could influence
innate immune response to release potentially cytotoxic molecules. MD-2 is linked with the extracellular
domain of TLR4 and is indispensable for LPS recognition [13]. Therefore, this docking analysis was
applied to explore whether or not the target compounds inhibit MD-2 to reduce the release of
inflammatory mediators. Umeharu Ohto et al. [39] had revealed the crystal structure of mouse
TLR4/MD-2/LPS complex, which was shown in Figure 4. In their study, MD-2 directly bound to LPS
in its hydrophobic cavity with high affinity. With this background, compound 5n was used as the
ligand to explore the relationship between DCH and TLR4/MD-2/LPS complex. As shown in Figure 5,
compound 5n could integrate closely with the hydrophobic cavity of MD-2, which is the important
TLR4/MD-2/LPS binding position. That means the DCH might disturb the interactions between LPS
and TLR4/MD-2 complex. The minimum binding energy of (
result, we supposed TLR4/MD-2 complex is likely to be one of the drug targets of DCH.
∆
G) is 10.08 kcal/mol. Given to this
−
Figure 4. The surface representation of LPS (red) and Toll-like receptor 4 (TLR4) (green)/MD-2 (cyan)
binding pocket (PDB: 3VQ2).

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Figure 5. Interaction diagrams of the selected docked conformations of compound 5n to TLR4/MD-2
complex. ( ) The surface representation of compound 5n and TLR4 (limon)/MD-2 (yellow) binding
) The magnified representation of compound 5n (red) and TLR4 (limon)/MD-2 (cyan) binding
) The interface of compound 5n and TLR4/MD-2 complex; ( ) 3D ligand interactions diagram.
A
pocket. (
pocket. (
B
C
D
Dotted line; pink: alkyl hydrophobic; blue: pi hydrophobic; green: hydrogen bond.
3. Conclusions
Total-thirty new DCH were designed and synthesized. Most of the target compounds exhibited
moderate activities against the tested cell lines and they showed a general promotion under hypoxia
conditions. Meanwhile, the new potential activity of DCH was found that DCH could exhibit
anti-neuroinflammatory activity. DCH could inhibit the release of NO, TNF-α, and IL-6, which were
induced by LPS. The docking analysis was shown that MD-2, the coreceptor of TLR4, might be one of
the targets of DCH. Additionally, DCH could also reduce NO levels by direct NO capture. In brief,
DCH were the potential anti-neuroinflammatory agents which might reduce CRF of cancer patients.
4. Experimental Section
4.1. Chemistry
Unless otherwise specified, all solvents and reagents were commercially available and used
without further purification. Analytical TLC was performed using silica gel precoated GF254 plates.
The 1H-NMR and 13C-NMR spectra were recorded at 600 MHz and 150 MHZ on a Bruker AV-400
spectrometer (Bruker Bioscience, Billerica, MA, USA), using CDCl3 as solvent with tetramethylsilane as
the internal standard. NMR spectra were analyzed using MestReNova. High-resolution mass spectra
(HRMS) were measured using an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA)
instrument in ESI mode. The 1H-NMR and 13C-NMR spectra of synthesized compounds, please see
the supplementary materials.

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4.1.1. General Procedure for the Synthesis of Compounds 1a–1e and 2a–2e
Substituted coumarin (a–e) (10 mmol), substituted DHA (III or IV) (10 mmol), and triethylamine
(10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction
mixture was stirred at room temperature for 8 h under the protection of nitrogen. The mixture was
filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was
purified through column chromatography.
3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (1a) White
solid; m.p. 73–75 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.77 (s, 1H), 7.68 (s, 1H), 7.56 (t,
J = 7.9 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 5.47 (s, 2H), 5.39
(s, 1H), 4.94 (d, J = 12.6 Hz, 1H), 4.92 (d, J = 3.5 Hz, 1H), 4.68 (d, J = 12.6 Hz, 1H), 2.67–2.60 (m, 1H),
2.38–2.31 (m, 1H), 2.01–1.97 (m, 1H), 1.85–1.81 (m, 1H), 1.77–1.68 (m, 3H), 1.59 (dd, J = 13.2, 3.4 Hz, 1H),
1.46–1.43 (m, 1H), 1.41 (s, 3H), 1.31–1.18 (m, 1H), 1.26–1.16 (m, 2H), 0.91 (d, J = 6.3 Hz, 3H), 0.88 (d,
J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 159.64, 152.65, 144.62, 141.31, 131.46, 127.30,
123.92, 122.67, 121.84, 117.53, 115.71, 103.10, 100.77, 86.93, 80.06, 76.22, 76.01, 75.80, 60.70, 51.47, 47.97,
43.33, 36.33, 35.36, 33.52, 29.78, 25.09, 23.59, 23.41, 19.30, 11.99; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C28H33N3O7Na: 546.2216. Found: 546.2181.
6-bromo-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (1b
)
White solid; m.p. 78–80 ◦C; yield 48%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.75 (s, 1H), 7.64 (dd,
J = 8.8, 2.3 Hz, 1H), 7.61 (d, J = 2.3 Hz, 1H), 7.55 (s, 1H), 7.24 (d, J = 8.8 Hz, 1H), 5.47 (s, 2H), 5.39
(s, 1H), 4.94 (d, J = 12.7 Hz, 1H), 4.93 (d, J = 3.5 Hz, 1H), 4.69 (d, J = 12.8 Hz, 1H), 2.67–2.63 (m, 1H),
2.38–2.33 (m, 1H), 2.03–1.99 (m, 1H), 1.86–1.82 (m, 1H), 1.75–1.70 (m, 2H), 1.62–1.58 (m, 2H), 1.42 (s,
3H), 1.31–1.19 (m, 4H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ
(ppm): 158.90, 151.42, 144.74, 139.67, 134.18, 129.51, 123.17, 122.67, 119.00, 117.43, 116.53, 103.12, 100.87,
86.95, 80.06, 76.20, 75.99, 75.78, 60.76, 51.46, 47.82, 43.32, 36.36, 35.36, 33.52, 29.79, 25.10, 23.62, 23.42,
19.30, 12.00; ESI-HRMS [M + Na]+: (m/z) Calcd. for C28H32BrN3O7Na: 624.1321. Found: 624.1264.
6-chloro-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (1c
)
White solid; m.p. 93–95 ◦C; yield 41%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.75 (s, 1H), 7.56 (s, 1H),
7.51 (dd, J = 8.8, 2.5 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 8.9 Hz, 1H), 5.47 (s, 2H), 5.39 (s, 1H),
4.95 (d, J = 12.7 Hz, 1H), 4.93 (d, J = 3.6 Hz, 1H), 4.69 (d, J = 12.6 Hz, 1H), 2.68–2.62 (m, 1H), 2.35
(td, J = 14.0, 4.0 Hz, 1H), 2.03–1.99 (m, 1H), 1.87–1.82 (m, 1H), 1.75–1.71 (m, 2H), 1.62–1.59 (m, 1H),
1.48–1.43 (m, 2H), 1.42 (s, 3H), 1.31–1.19 (m, 3H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 7.4 Hz, 3H); 13
C
NMR (150 MHz, CDCl3) δ (ppm): 158.96, 150.95, 144.73, 139.79, 131.37, 129.27, 126.46, 123.19, 122.67,
118.51, 117.16, 103.12, 100.86, 86.95, 80.06, 76.21, 75.99, 75.78, 60.75, 51.47, 47.84, 43.33, 36.36, 35.36,
33.52, 29.79, 25.10, 23.62, 23.42, 19.30, 12.00; ESI-HRMS [M + Na]+: (m/z) Calcd. for C28H32ClN3O7Na:
580.1826. Found: 580.1788.
6,8-dichloro-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
1d) White solid; m.p. 113–115 ◦C; yield 60%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.75 (s, 1H), 7.61
(d, J = 2.3 Hz, 1H), 7.55 (s, 1H), 7.37 (d, J = 2.4 Hz, 1H), 5.48 (s, 2H), 5.37 (s, 1H), 4.96–4.92 (m, 2H),
4.70 (d, J = 12.7 Hz, 1H), 2.69–2.62 (m, 1H), 2.38–2.33 (m, 1H), 2.04–1.99 (m, 1H), 1.87–1.82 (m, 1H),
1.76–1.72 (m, 3H), 1.62–1.59 (m, 1H), 1.42 (s, 3H), 1.32–1.18 (m, 4H), 0.92 (d, J = 6.3 Hz, 3H), 0.89 (d,
J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 157.92, 146.92, 144.86, 139.45, 131.36, 129.12,
125.03, 124.04, 122.68, 121.71, 119.30, 103.13, 100.95, 86.94, 80.04, 76.20, 75.99, 75.78, 60.80, 51.44, 47.63,
43.31, 36.37, 35.35, 33.51, 29.79, 25.09, 23.61, 23.42, 19.28, 12.00; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C28H31Cl2N3O7Na: 614.1437. Found: 614.1428.
6,8-dibromo-3-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
1e) White solid; m.p. 103–105 ◦C; yield 61%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.90 (d, J = 2.2
Hz, 1H), 7.75 (s, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.52 (s, 1H), 5.48 (s, 2H), 5.37 (s, 1H), 4.96–4.92 (m, 2H),
4.70 (d, J = 12.7 Hz, 1H), 2.67–2.63 (m, 1H), 2.38–2.33 (m, 1H), 2.03–1.99 (m, 1H), 1.87–1.82 (m, 1H),

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1.76–1.72 (m, 2H), 1.62–1.58 (m, 4H), 1.42 (s, 3H), 1.25–1.19 (m, 2H), 0.92 (d, J = 6.3 Hz, 3H), 0.90 (d,
J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
(ppm): 158.01, 148.45, 144.87, 139.36, 136.89, 128.75,
δ
123.97, 122.67, 119.74, 116.48, 110.29, 103.13, 100.97, 86.95, 80.04, 76.20, 75.99, 75.78, 60.82, 51.45, 47.56,
43.31, 36.37, 35.35, 33.51, 29.79, 25.09, 23.62, 23.42, 19.30, 12.00; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C28H31Br2N3O7Na: 702.0426. Found: 702.0421.
3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (2a) White
solid; m.p. 143–145 ◦C; yield 66%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.65 (s, 1H), 7.63 (s, 1H), 7.56
(ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.47 (dd, J = 7.8, 1.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.30 (td, J = 7.5, 1.1
Hz, 1H), 5.44 (s, 2H), 5.29 (d, J = 9.7 Hz, 1H), 4.78 (d, J = 3.6 Hz, 1H), 4.13–4.09 (m, 1H), 3.69–3.65 (m,
1H), 3.07–2.97 (m, 2H), 2.61–2.55 (m, 1H), 2.33 (td, J = 14.0, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.85–1.81
(m, 1H), 1.71–1.67 (m, 2H), 1.59–1.56 (m, 1H), 1.41 (s, 3H), 1.40–1.38 (m, 1H), 1.28–1.16 (m, 3H), 0.91
(d, J = 6.3 Hz, 3H), 0.89–0.83 (m, 1H), 0.80 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm):
159.65, 152.64, 144.77, 141.15, 131.43, 127.30, 123.90, 121.98, 121.78, 117.52, 115.70, 103.03, 100.92, 86.84,
80.02, 76.21, 76.00, 75.78, 66.18, 51.47, 47.86, 43.28, 36.34, 35.36, 33.55, 29.79, 25.57, 25.14, 23.62, 23.34,
19.33, 11.93; ESI-HRMS [M + Na]+: (m/z) Calcd. for C29H35N3O7Na: 560.2373. Found: 560.2417.
6-bromo-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (2b
)
White solid; m.p. 138–140 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.64 (dd, J = 8.8, 2.3 Hz,
1H), 7.61 (d, J = 2.3 Hz, 1H), 7.60 (s, 1H), 7.52 (s, 1H), 7.23 (d, J = 8.8 Hz, 1H), 5.43 (s, 2H), 5.28 (s, 1H),
4.79 (d, J = 3.5 Hz, 1H), 4.13–4.09 (m, 1H), 3.70–3.66 (m, 1H), 3.07–2.98 (m, 2H), 2.62–2.57 (m, 1H), 2.34
(td, J = 14.0, 4.0 Hz, 1H), 2.01–1.98 (m, 1H), 1.86–1.81 (m, 1H), 1.71–1.67 (m, 2H), 1.60–1.56 (m, 1H), 1.42
(s, 3H), 1.40–1.37 (m, 1H), 1.27–1.17 (m, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.89–0.85 (m, 1H), 0.82 (d, J = 7.3
Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.90, 151.40, 144.86, 139.50, 134.15, 129.51, 123.31,
121.76, 118.99, 117.41, 116.51, 103.06, 100.92, 86.85, 80.00, 76.20, 75.99, 75.78, 66.18, 51.45, 47.72, 43.26,
36.37, 35.35, 33.55, 29.78, 25.56, 25.14, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C29H34BrN3O7Na: 638.1478. Found: 638.1508.
6-chloro-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (2c
)
White solid; m.p. 138–140 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.60 (s, 1H), 7.53 (s,
1H), 7.50 (dd, J = 8.8, 2.5 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 5.43 (d, J = 1.1
Hz, 2H), 5.28 (s, 1H), 4.79 (d, J = 3.6 Hz, 1H), 4.13–4.09 (m, 1H), 3.70–3.66 (m, 1H), 3.05–2.99 (m, 2H),
2.61–2.58 (m, 1H), 2.37–2.31 (m, 1H), 2.01–1.98 (m, 1H), 1.86–1.81 (m, 1H), 1.70–1.68 (m, 2H), 1.60–1.56
(m, 1H), 1.42 (s, 3H), 1.41–1.36 (m, 1H), 1.26–1.17 (m, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.89–0.85 (m, 1H),
0.82 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.97, 150.93, 144.87, 139.62, 131.34,
129.25, 126.47, 123.35, 121.76, 118.51, 117.14, 103.06, 100.93, 86.85, 80.00, 76.20, 75.99, 75.78, 66.19, 51.45,
47.72, 43.27, 36.37, 35.35, 33.56, 29.79, 25.57, 25.14, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (m/z)
Calcd. for C29H34ClN3O7Na: 594.1983. Found: 594.2002.
6,8-dichloro-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
2d) White solid; m.p. 101–103 ◦C; yield 50%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.60 (d, J = 2.2 Hz,
2H), 7.52 (s, 1H), 7.37 (d, J = 2.3 Hz, 1H), 5.45 (s, 2H), 5.27 (s, 1H), 4.79 (d, J = 3.5 Hz, 1H), 4.13–4.08 (m,
1H), 3.71–3.67 (m, 1H), 3.04–3.01 (m, 2H), 2.61–2.59 (m, 1H), 2.34 (td, J = 14.0, 4.0 Hz, 1H), 2.02–1.98 (m,
1H), 1.85–1.82 (m, 1H), 1.71–1.68 (m, 2H), 1.60–1.58 (m, 1H), 1.42 (s, 3H), 1.41–1.36 (m, 1H), 1.26–1.17
(m, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.90–0.85 (m, 1H), 0.82 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz,
CDCl3)
δ (ppm): 157.93, 146.90, 144.92, 139.29, 131.34, 129.12, 125.04, 124.16, 121.82, 121.70, 119.30,
103.07, 100.94, 86.86, 79.99, 76.20, 75.99, 75.78, 66.17, 51.44, 47.57, 43.26, 36.39, 35.35, 33.55, 29.78, 25.53,
25.13, 23.63, 23.35, 19.32, 11.95; ESI-HRMS [M + Na]+: (m/z) Calcd. for C29H33Cl2N3O7Na: 628.1593.
Found: 628.1360.
6,8-dibromo-3-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
2e) White solid; m.p. 93–95 ◦C; yield 44%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.83 (d, J = 2.1 Hz,
1H), 7.53 (s, 1H), 7.49 (d, J = 2.2 Hz, 1H), 7.42 (s, 1H), 5.38 (s, 2H), 5.21 (s, 1H), 4.73 (d, J = 3.4 Hz, 1H),

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4.06–4.02 (m, 1H), 3.64–3.60 (m, 1H), 2.97–2.94 (m, 2H), 2.56–2.51 (m, 1H), 2.30–2.25 (m, 1H), 1.95–1.91
(m, 1H), 1.79–1.75 (m, 1H), 1.64–1.60 (m, 2H), 1.52 (dd, J = 13.2, 3.3 Hz, 1H), 1.35 (s, 3H), 1.34–1.29 (m,
1H), 1.19–1.11 (m, 3H), 0.85 (d, J = 6.1 Hz, 3H), 0.83–0.78 (m, 1H), 0.75 (d, J = 7.4 Hz, 3H); 13C NMR
(150 MHz, CDCl3)
δ (ppm): 158.01, 148.41, 144.96, 139.16, 136.85, 128.75, 124.12, 121.77, 119.74, 116.47,
110.26, 103.07, 100.93, 86.86, 80.00, 76.21, 76.00, 75.79, 66.19, 51.44, 47.48, 43.26, 36.39, 35.35, 33.55, 29.78,
25.56, 25.13, 23.63, 23.35, 19.33, 11.96; ESI-HRMS [M + Na]+: (m/z) Calcd. for C29H33Br2N3O7Na:
716.0583. Found: 716.0381.
4.1.2. General Procedure for the Synthesis of Compounds 3f–3i and 4f–4i
Substituted coumarin (f–i) (10 mmol), substituted DHA (III or IV) (10 mmol) and triethylamine
(10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction
mixture was stirred at room temperature for 8 h under the protection of nitrogen. The mixture was
filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was
purified through column chromatography.
7-hydroxy-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (3f
)
White solid; m.p. 165–167 ◦C; yield 54%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 9.61 (s, 1H), 7.68 (d,
J = 8.8 Hz, 1H), 7.67 (s, 1H), 6.99 (dd, J = 8.8, 2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.15 (s, 1H), 5.76 (d, J
= 15.4 Hz, 1H), 5.57 (d, J = 15.3 Hz, 1H), 5.20 (s, 1H), 4.87 (d, J = 1.9 Hz, 1H), 4.86 (d, J = 7.4 Hz, 1H),
4.68 (d, J = 12.9 Hz, 1H), 2.62–2.60 (m, 1H), 2.34–2.29 (m, 1H), 2.02
1.66 1.61 (m, 3H), 1.61 1.57 (m, 1H), 1.45–1.42 (m, 1H), 1.40 (s, 3H), 1.20
Hz, 3H), 0.82 (d, J = 6.1 Hz, 3H); 13C NMR (150 MHz, CDCl3)
(ppm): 160.88, 159.75, 154.96, 146.45,
−
1.98 (m, 1H), 1.82–1.78 (m, 1H),
−
−
−
1.08 (m, 3H), 0.84 (d, J = 7.4
δ
145.19, 124.19, 122.29, 112.78, 111.57, 108.80, 103.21, 103.04, 101.40, 86.91, 79.92, 76.20, 75.99, 75.78, 60.44,
51.32, 50.16, 43.18, 36.28, 35.26, 33.42, 29.74, 25.00, 23.53, 23.32, 19.26, 11.89; ESI-HRMS [M + Na]+: (m/z)
Calcd. for C28H33N3O8Na: 562.2165. Found: 562.2172.
5,7-dimethyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
3g) White solid; m.p. 133~135 ◦C; yield 57%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.56 (s, 1H), 7.07 (s,
1H), 6.95 (s, 1H), 5.90 (d, J = 17.0 Hz, 1H), 5.86 (d, J = 17.0 Hz, 1H), 5.51 (s, 1H), 5.41 (s, 1H), 4.98 (d,
J = 12.8 Hz, 1H), 4.93 (d, J = 3.5 Hz, 1H), 4.73 (d, J = 12.8 Hz, 1H), 2.68 (s, 3H), 2.40 (s, 3H), 2.35 (dd,
J = 14.0, 4.0 Hz, 1H), 2.05
1H), 1.48–1.46 (m, 1H), 1.43 (s, 3H), 1.32
3H); 13C NMR (150 MHz, CDCl3)
(ppm): 158.67, 154.26, 149.33, 145.35, 142.10, 133.93, 129.26, 122.10,
−
2.01 (m, 1H), 1.88
−
1.85 (m, 1H), 1.75–1.70 (m, 2H), 1.60 (dd, J = 13.2, 3.4 Hz,
−
1.20 (m, 5H), 0.93 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 7.4 Hz,
δ
115.63, 113.36, 112.80, 103.16, 100.93, 86.98, 80.05, 76.21, 76.00, 75.79, 60.80, 52.05, 51.48, 43.31, 36.34,
35.36, 33.50, 29.78, 25.11, 23.61, 23.44, 23.28, 20.24, 19.31, 12.01; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C30H37N3O7Na: 574.2539. Found: 574.2553.
7-methyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (3h
)
White solid; m.p. 107–109 ◦C; yield 50%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.58 (s, 1H), 7.50 (d,
J = 8.1 Hz, 1H), 7.19 (s, 1H), 7.11 (d, J = 8.1 Hz, 1H), 6.01 (s, 1H), 5.74 (d, J = 16.3 Hz, 1H), 5.64 (d,
J = 16.0 Hz, 1H), 5.30 (s, 1H), 4.92–4.88 (m, 2H), 4.72 (d, J = 12.9 Hz, 1H), 2.65–2.61 (m, 1H), 2.45 (s,
3H), 2.34 (td, J = 14.0, 4.0 Hz, 1H), 2.04–2.00 (m, 1H), 1.86–1.81 (m, 1H), 1.71–1.66 (m, 2H), 1.58–1.55
(m, 1H), 1.42 (s, 3H), 1.26–1.18 (m, 5H), 0.91 (d, J = 5.7 Hz, 3H), 0.86 (d, J = 7.3 Hz, 3H); 13C NMR
(150 MHz, CDCl3)
δ (ppm): 159.01, 152.86, 146.75, 145.42, 143.16, 125.03, 122.23, 121.95, 116.71, 113.56,
113.41, 103.15, 101.10, 86.93, 80.00, 76.21, 75.99, 75.78, 60.90, 51.41, 49.29, 43.27, 36.34, 35.31, 33.47, 29.77,
25.06, 23.58, 23.38, 20.70, 19.30, 11.96; ESI-HRMS [M + Na]+: (m/z) Calcd. for C29H35N3O7Na: 560.2373.
Found: 560.2375.
6-methyl-4-((4-((10S)-dihydroartemisininoxy)methyl-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (3i
)
1
White solid; m.p. 88–90◦C; yield 60%; H NMR (600 MHz, CDCl3)
δ (ppm): 7.58 (s, 1H), 7.39 (d,
J = 8.8 Hz, 2H), 7.28 (d, J = 8.3 Hz, 1H), 6.01 (s, 1H), 5.75 (d, J = 16.3 Hz, 1H), 5.66 (d, J = 16.3 Hz, 1H),
5.32 (s, 1H), 4.92 (d, J = 12.8 Hz, 1H), 4.90 (d, J = 3.5 Hz, 1H), 4.73 (d, J = 12.8 Hz, 1H), 2.67–2.60 (m,

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1H), 2.41 (s, 3H), 2.34 (td, J = 14.0, 4.0 Hz, 1H), 2.04–1.99 (m, 1H), 1.86–1.81 (m, 1H), 1.72–1.68 (m, 2H),
1.57 (dd, J = 13.2, 3.1 Hz, 1H), 1.42 (s, 3H), 1.26–1.19 (m, 5H), 0.91 (d, J = 5.7 Hz, 3H), 0.86 (d, J = 7.3
Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.91, 150.87, 146.72, 145.41, 133.69, 132.71, 122.26,
121.95, 116.32, 115.68, 114.29, 103.15, 101.05, 86.94, 80.00, 76.21, 76.00, 75.78, 60.86, 51.42, 49.17, 43.27,
36.34, 35.31, 33.47, 29.76, 25.07, 23.59, 23.39, 20.01, 19.30, 11.96; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C29H35N3O7Na: 560.2373. Found: 560.2403.
7-hydroxy-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
4f) White solid; m.p. 121–123 ◦C; yield 54%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 9.98 (s, 1H), 7.68 (d,
J = 8.8 Hz, 1H), 7.49 (s, 1H), 7.01 (dd, J = 8.8, 2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.16 (s, 1H), 5.68–5.59
(m, 2H), 5.20 (s, 1H), 4.71 (d, J = 3.5 Hz, 1H), 4.08–4.04 (m, 1H), 3.67–3.63 (m, 1H), 3.03–2.94 (m, 2H),
2.56–2.53 (m, 1H), 2.33 (td, J = 14.1, 4.0 Hz, 1H), 2.03–1.99 (m, 1H), 1.86–1.82 (m, 1H), 1.58–1.54 (m, 2H),
1.51–1.48 (m, 1H), 1.40 (s, 3H), 1.40–1.35 (m, 2H), 1.27–1.25 (m, 1H), 1.20–1.16 (m, 2H), 0.88 (d, J = 5.6
Hz, 3H), 0.68 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 161.02, 159.72, 155.05, 146.56,
145.44, 124.28, 121.23, 112.79, 111.51, 108.74, 103.16, 103.05, 100.88, 86.87, 79.91, 76.20, 75.99, 75.78, 65.75,
51.37, 50.15, 43.11, 36.37, 35.30, 33.45, 29.66, 25.24, 25.09, 23.62, 23.29, 19.29, 11.73; ESI-HRMS [M +
Na]+: (m/z) Calcd. for C29H35N3O8Na: 576.2322. Found: 576.2352.
5,7-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
4g) White solid; m.p. 135–137 ◦C; yield 52%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.41 (s, 1H), 7.06
(s, 1H), 6.94 (s, 1H), 5.84 (s, 2H), 5.54 (s, 1H), 5.28 (s, 1H), 4.80 (d, J = 3.4 Hz, 1H), 4.15–4.10 (m, 1H),
3.73–3.69 (m, 1H), 3.11–3.00 (m, 2H), 2.66 (s, 3H), 2.61–2.58 (m, 1H), 2.40 (s, 3H), 2.35 (td, J = 14.1, 4.0
Hz, 1H), 2.03–1.99 (m, 1H), 1.89–1.84 (m, 1H), 1.70–1.63 (m, 2H), 1.59–1.55 (m, 1H), 1.49–1.43 (m, 1H),
1.42 (s, 3H), 1.29–1.18 (m, 3H), 0.93 (d, J = 6.3 Hz, 3H), 0.87 (dd, J = 12.4, 4.6 Hz, 1H), 0.80 (d, J = 7.3 Hz,
3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.63, 154.30, 149.34, 145.42, 142.09, 133.98, 129.26, 121.15,
115.62, 113.33, 112.98, 103.07, 100.86, 86.87, 80.00, 76.21, 76.00, 75.79, 65.93, 52.03, 51.47, 43.25, 36.36,
35.37, 33.55, 29.76, 25.58, 25.15, 23.66, 23.32, 23.21, 20.24, 19.35, 11.98; ESI-HRMS [M + Na]+: (m/z)
Calcd. for C31H39N3O7Na: 588.2686. Found: 588.2680.
7-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one
(
4h) White solid; m.p. 100–102 ◦C; yield 53%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.48 (d, J = 8.1 Hz,
1H), 7.40 (s, 1H), 7.18 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.00 (s, 1H), 5.68–5.62 (m, 2H), 5.29 (d, J = 12.6 Hz,
1H), 4.76 (d, J = 3.5 Hz, 1H), 4.11–4.08 (m, 1H), 3.69–3.65 (m, 1H), 3.06–2.96 (m, 2H), 2.60–2.54 (m, 1H),
2.45 (s, 3H), 2.34 (td, J = 14.0, 4.0 Hz, 1H), 2.05–1.99 (m, 1H), 1.88–1.83 (m, 1H), 1.66–1.63 (m, 2H),
1.60–1.58 (m, 1H), 1.56–1.53 (m, 1H), 1.41 (s, 3H), 1.27–1.17 (m, 3H), 0.93 (d, J = 6.1 Hz, 3H), 0.89–0.84
(m, 1H), 0.74 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.98, 152.88, 146.91, 145.50,
143.18, 125.06, 122.27, 120.82, 116.69, 113.56, 113.35, 103.08, 100.86, 86.86, 79.97, 76.21, 76.00, 75.78, 65.94,
51.44, 49.21, 43.21, 36.36, 35.35, 33.54, 29.72, 25.55, 25.14, 23.65, 23.31, 20.69, 19.34, 11.86; ESI-HRMS [M
+ Na]+: (m/z) Calcd. for C30H37N3O7Na: 574.2529. Found: 574.2567.
6-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methyl)-2H-1-chromen-2-one (4i
)
White solid; m.p. 100–102 ◦C; yield 43%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.40 (s, 1H), 7.40 (s, 1H),
7.38 (d, J = 1.9 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.00 (d, J = 1.2 Hz, 1H), 5.67 (s, 2H), 5.29 (s, 1H), 4.77
(d, J = 3.4 Hz, 1H), 4.13–4.09 (m, 1H), 3.71–3.67 (m, 1H), 3.05–2.99 (m, 2H), 2.61–2.56 (m, 1H), 2.41 (s,
3H), 2.36–2.32 (m, 1H), 2.03–2.00 (m, 1H), 1.88–1.84 (m, 1H), 1.66 (dd, J = 13.7, 3.5 Hz, 1H), 1.61 (d,
J = 4.2 Hz, 1H), 1.58–1.53 (m, 2H), 1.42 (s, 3H), 1.24–1.16 (m, 3H), 0.93 (d, J = 6.2 Hz, 3H), 0.88–0.85
(m, 1H), 0.75 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 158.88, 150.89, 146.89, 145.51,
133.70, 132.73, 122.29, 120.86, 116.31, 115.69, 114.22, 103.09, 100.87, 86.86, 79.98, 76.20, 75.99, 75.78, 65.94,
51.45, 49.06, 43.22, 36.37, 35.35, 33.54, 29.73, 25.57, 25.14, 23.65, 23.31, 20.03, 19.34, 11.86; ESI-HRMS [M
+ Na]+: (m/z) Calcd. for C30H37N3O7Na: 574.2529. Found: 574.2558.

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4.1.3. General Procedure for the Synthesis of Compounds 5j,5l–5o, and 6j–6p
Substituted coumarin (j–p) (10 mmol), substituted DHA (I or II) (10 mmol) and triethylamine
(10 mmol) were dissolved in 20 mL CH2Cl2. In addition, CuI (100 mg) was added and the reaction
mixture was stirred at room temperature for 8h under the protection of nitrogen. The mixture was
filtered, washed with water, dried over Na2SO4 and evaporated to dryness. The crude product was
purified through column chromatography.
4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one (5j) White
solid; m.p. 91–93 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.79–7.75 (m, 2H), 7.55 (ddd,
J = 8.7, 7.3, 1.6 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.25–7.22 (m, 1H), 5.87 (s, 1H), 5.34 (d, J = 2.7 Hz, 2H),
5.16 (s, 1H), 4.77 (d, J = 3.6 Hz, 1H), 4.72–4.68 (m, 1H), 4.58–4.54 (m, 1H), 4.32–4.29 (m, 1H), 3.87–3.83
(m, 1H), 2.63–2.60 (m, 1H), 2.33 (td, J = 14.0, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.87–1.81 (m, 1H), 1.68–1.64
(m, 1H), 1.58–1.55 (m, 1H), 1.53–1.51 (m, 1H), 1.41 (s, 3H), 1.38 (d, J = 11.2 Hz, 1H), 1.26–1.18 (m, 3H),
0.91 (d, J = 5.9 Hz, 3H), 0.88–0.83 (m, 1H), 0.78 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm):
163.90, 161.51, 152.34, 140.45, 131.58, 122.86, 122.85, 122.04, 115.80, 114.40, 103.21, 101.15, 90.15, 86.86,
79.73, 76.20, 75.99, 75.78, 65.36, 61.67, 51.32, 49.57, 42.97, 36.35, 35.25, 33.41, 29.53, 25.06, 23.58, 23.32,
19.29, 11.80; ESI-HRMS [M + Na]+: (m/z) Calcd. for C29H35N3O8Na: 576.2322. Found: 576.2336.
5,7-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (5l) White solid; m.p. 111–113 ◦C; yield 49%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.74 (s, 1H),
6.98 (s, 1H), 6.83 (s, 1H), 5.75 (s, 1H), 5.28 (s, 2H), 5.14 (s, 1H), 4.76 (d, J = 3.6 Hz, 1H), 4.70–4.66 (m,
1H), 4.58–4.54 (m, 1H), 4.31–4.27 (m, 1H), 3.87–3.84 (m, 1H), 2.84 (s, 1H), 2.61–2.59 (m, 1H), 2.53 (s,
3H), 2.36 (s, 3H), 2.35–2.30 (m, 1H), 2.04–1.97 (m, 2H), 1.87–1.82 (m, 1H), 1.58–1.54 (m, 1H), 1.52–1.50
(m, 1H), 1.41 (s, 3H), 1.25–1.17 (m, 3H), 0.91 (d, J = 5.9 Hz, 3H), 0.88–0.85 (m, 1H), 0.76 (d, J = 7.4 Hz,
3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 166.97, 161.69, 153.91, 141.76, 140.42, 135.57, 127.92, 122.68,
114.43, 110.62, 103.21, 101.15, 89.15, 86.85, 79.74, 76.21, 76.00, 75.79, 65.38, 61.48, 51.33, 49.59, 42.97,
36.36, 35.25, 33.42, 29.53, 25.05, 23.58, 23.30, 22.49, 20.35, 19.30, 11.80; ESI-HRMS [M + Na]+: (m/z)
Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2645.
6,8-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (5m) White solid; m.p. 111–113 ◦C; yield 49%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.77 (s, 1H),
7.39 (s, 1H), 7.22 (s, 1H), 5.83 (s, 1H), 5.32 (d, J = 2.5 Hz, 2H), 5.17 (s, 1H), 4.78 (d, J = 3.0 Hz, 1H),
4.72–4.68 (m, 1H), 4.59–4.55 (m, 1H), 4.33–4.29 (m, 1H), 3.87–3.83 (m, 1H), 2.64–2.58 (m, 1H), 2.41 (s,
3H), 2.37–2.33 (m, 1H), 2.33 (s, 3H), 2.02–1.98 (m, 1H), 1.88–1.82 (m, 1H), 1.68–1.63 (m, 1H), 1.58–1.53
(m, 1H), 1.53–1.48 (m, 1H), 1.41 (s, 3H), 1.41–1.38 (m, 1H), 1.28–1.16 (m, 3H), 0.91 (d, J = 5.9 Hz, 3H),
0.88–0.84 (m, 1H), 0.78 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 165.31, 162.94, 149.91,
141.55, 134.98, 133.03, 125.90, 123.94, 120.25, 114.82, 104.23, 102.18, 90.77, 87.87, 80.76, 77.24, 77.02, 76.81,
66.39, 62.54, 52.35, 50.58, 44.01, 37.37, 36.28, 34.44, 30.57, 26.07, 24.61, 24.35, 20.82, 20.31, 15.65, 12.84;
ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2639.
7,8-dimethyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (5n) White solid; m.p. 91–93 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.76 (s, 1H),
7.50 (d, J = 8.1 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 5.79 (s, 1H), 5.32 (d, J = 3.0 Hz, 2H), 5.15 (s, 1H), 4.77 (d,
J = 3.5 Hz, 1H), 4.71–4.67 (m, 1H), 4.58–4.53 (m, 1H), 4.33–4.27 (m, 1H), 3.86–3.82 (m, 1H), 2.63–2.58 (m,
1H), 2.37 (s, 3H), 2.36 (s, 3H), 2.34–2.30 (m, 1H), 2.01–1.97 (m, 1H), 1.86–1.81 (m, 1H), 1.68–1.62 (m,
1H), 1.58–1.53 (m, 1H), 1.51 (dd, J = 13.4, 3.5 Hz, 1H), 1.41 (s, 3H), 1.39–1.36 (m, 1H), 1.27–1.15 (m,
3H), 0.91 (d, J = 5.8 Hz, 3H), 0.88–0.82 (m, 1H), 0.77 (d, J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ
(ppm): 165.56, 163.09, 151.58, 142.30, 141.69, 125.34, 124.53, 123.85, 119.71, 113.06, 104.24, 102.18, 89.91,
87.88, 80.78, 77.25, 77.04, 76.82, 66.39, 62.61, 52.37, 50.60, 44.02, 37.37, 36.29, 34.45, 30.58, 26.09, 24.61,
24.35, 20.46, 20.33, 12.85, 11.67; ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2635.
Found: 604.2646.

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8-methyl-4-((4-(2-((10S)-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen- 2-one
5o) White solid; m.p. 84–86 ◦C; yield 43%; 1H NMR (600 MHz, CDCl3)
(ppm): 7.77 (s, 1H), 7.61 (d,
(
δ
J = 7.9 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 5.85 (s, 1H), 5.33 (d, J = 2.8 Hz, 2H), 5.15
(s, 1H), 4.77 (d, J = 3.5 Hz, 1H), 4.71–4.67 (m, 1H), 4.58–4.54 (m, 1H), 4.33–4.27 (m, 1H), 3.86–3.82 (m,
1H), 2.62–2.59 (m, 1H), 2.45 (s, 3H), 2.33 (td, J = 14.1, 4.0 Hz, 1H), 2.01–1.97 (m, 1H), 1.86–1.81 (m, 1H),
1.65 (dd, J = 14.0, 3.6 Hz, 1H), 1.58–1.54 (m, 1H), 1.51 (dd, J = 13.4, 3.5 Hz, 1H), 1.41 (s, 3H), 1.40–1.35
(m, 1H), 1.27–1.17 (m, 3H), 0.90 (d, J = 5.8 Hz, 3H), 0.88–0.84 (m, 1H), 0.77 (d, J = 7.3 Hz, 3H); 13C NMR
(150 MHz, CDCl3)
δ (ppm): 165.30, 162.70, 151.74, 141.60, 133.84, 126.28, 123.84, 123.42, 120.66, 115.15,
104.24, 102.18, 90.88, 87.89, 80.77, 77.25, 77.03, 76.82, 66.39, 62.71, 52.36, 50.60, 44.01, 37.38, 36.28, 34.45,
30.57, 26.09, 24.61, 24.35, 20.33, 15.76, 12.84; ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H37N3O8Na:
590.2478. Found: 590.2477.
4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one
(6j)
White solid; m.p. 74–76 ◦C; yield 47%; 1H NMR (600 MHz, CDCl3)
δ
(ppm): 7.79 (dd, J = 8.0, 1.6 Hz,
1H), 7.72 (s, 1H), 7.55 (ddd, J = 8.7, 7.4, 1.6 Hz, 1H), 7.32 (dd, J = 8.4, 1.1 Hz, 1H), 7.26–7.23 (m, 1H), 5.86
(s, 1H), 5.41 (s, 1H), 5.34 (s, 2H), 4.79 (d, J = 3.6 Hz, 1H), 4.56–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.41
(m, 1H), 2.69–2.63 (m, 1H), 2.39–2.34 (m, 1H), 2.27–2.20 (m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H),
1.79–1.75 (m, 1H), 1.67–1.64 (m, 1H), 1.51–1.46 (m, 2H), 1.42 (s, 3H), 1.39–1.33 (m, 1H), 1.28–1.22 (m,
2H), 0.96 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 163.96,
161.59, 152.33, 140.41, 131.55, 122.89, 122.40, 122.13, 115.78, 114.42, 103.18, 101.19, 90.16, 86.93, 79.93,
76.21, 76.00, 75.79, 63.62, 61.66, 51.47, 46.73, 43.23, 36.45, 35.33, 33.53, 29.78, 29.43, 25.12, 23.64, 23.54,
19.33, 12.07; ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H37N3O8Na: 590.2478. Found: 590.2463.
7-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one
(
6k) White solid; m.p. 80–82 ◦C; yield 51%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.71 (s, 1H), 7.65 (d,
J = 8.1 Hz, 1H), 7.12 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 5.80 (s, 1H), 5.41 (s, 1H), 5.32 (s, 2H), 4.79 (d, J = 3.6
Hz, 1H), 4.53–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.64 (m, 1H), 2.44 (s, 3H), 2.37
(td, J = 14.0, 4.0 Hz, 1H), 2.27–2.21 (m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.80–1.75 (m, 2H),
1.68–1.64 (m, 1H), 1.51–1.46 (m, 2H), 1.42 (s, 3H), 1.28–1.24 (m, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.94 (d, J
= 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): δ 164.21, 161.95, 152.44, 142.81, 140.51, 124.11,
122.39, 121.82, 115.88, 111.91, 103.19, 101.18, 89.25, 86.93, 79.94, 76.20, 75.99, 75.78, 63.62, 61.55, 51.47,
46.73, 43.24, 36.44, 35.33, 33.53, 29.78, 29.42, 25.12, 23.63, 23.53, 20.72, 19.33, 12.07; ESI-HRMS [M +
Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2617.
5,7-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (6l) White solid; m.p. 86–88 ◦C; yield 42%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.68 (s, 1H),
6.98 (s, 1H), 6.83 (s, 1H), 5.74 (s, 1H), 5.40 (s, 1H), 5.28 (s, 2H), 4.78 (d, J = 3.7 Hz, 1H), 4.53–4.48 (m,
2H), 3.91–3.87 (m, 1H), 3.42–3.38 (m, 1H), 2.69–2.64 (m, 1H), 2.53 (s, 3H), 2.40–2.33 (m, 4H), 2.25–2.19
(m, 2H), 2.05–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.80–1.77 (m, 1H), 1.76–1.73 (m, 1H), 1.67–1.64 (m, 1H),
1.51–1.47 (m, 2H), 1.42 (s, 3H), 1.30–1.21 (m, 3H), 0.96 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H); 13
C
NMR (150 MHz, CDCl3) δ (ppm): 166.99, 161.73, 153.90, 141.72, 140.46, 135.57, 127.92, 122.26, 114.43,
110.63, 103.18, 101.15, 89.14, 86.91, 79.93, 76.21, 76.00, 75.78, 63.49, 61.58, 51.46, 46.66, 43.23, 36.46, 35.33,
33.53, 29.78, 29.42, 25.11, 23.64, 23.54, 22.46, 20.35, 19.33, 12.08; ESI-HRMS [M + Na]+: (m/z) Calcd. for
C32H41N3O8Na: 618.2791. Found: 618.2827.
6,8-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (6m) White solid; m.p. 90–92 ◦C; yield 55%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.71 (s, 1H),
7.40 (s, 1H), 7.21 (s, 1H), 5.82 (s, 1H), 5.41 (s, 1H), 5.32 (s, 2H), 4.79 (d, J = 3.6 Hz, 1H), 4.55–4.46 (m,
2H), 3.94–3.90 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.63 (m, 1H), 2.40 (s, 3H), 2.39–2.34 (m, 1H), 2.33 (s,
3H), 2.27–2.21 (m, 2H), 2.06–2.01 (m, 1H), 1.91–1.86 (m, 1H), 1.79–1.77 (m, 1H), 1.76–1.73 (m, 1H),
1.67–1.63 (m, 1H), 1.51–1.45 (m, 2H), 1.42 (s, 3H), 1.37–1.35 (m, 1H), 1.28–1.22 (m, 2H), 0.95 (d, J = 6.4
Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 165.37, 163.02, 149.90, 141.55,

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134.97, 133.07, 125.88, 123.45, 120.35, 114.83, 104.21, 102.21, 90.79, 87.96, 80.97, 77.25, 77.04, 76.83, 64.68,
62.56, 52.51, 47.77, 44.27, 37.47, 36.37, 34.56, 30.82, 30.47, 26.15, 24.67, 24.57, 20.83, 20.36, 15.66, 13.11;
ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H41N3O8Na: 618.2791. Found: 618.2808.
7,8-dimethyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-
2-one (6n) White solid; m.p. 115–117 ◦C; yield 44%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.71 (s, 1H),
7.52 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 5.80 (s, 1H), 5.40 (s, 1H), 5.32 (s, 2H), 4.79 (d, J = 3.5
Hz, 1H), 4.56–4.45 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.69–2.64 (m, 1H), 2.39–2.34 (m, 7H),
2.27–2.19 (m, 2H), 2.06–2.01 (m, 1H), 1.92–1.86 (m, 1H), 1.81–1.77 (m, 1H), 1.74 (dd, J = 13.4, 3.5 Hz, 1H),
1.67–1.63 (m, 1H), 1.52–1.45 (m, 2H), 1.42 (s, 3H), 1.39–1.34 (m, 1H), 1.27–1.24 (m, 2H), 0.96 (d, J = 6.4
Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 165.61, 163.15, 151.58, 142.28,
141.66, 125.37, 124.51, 123.38, 119.78, 113.07, 104.21, 102.21, 89.92, 87.96, 80.97, 77.25, 77.04, 76.82, 64.66,
62.60, 52.51, 47.76, 44.27, 37.47, 36.37, 34.56, 30.82, 30.46, 26.15, 24.67, 24.57, 20.46, 20.37, 13.11, 11.67;
ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H41N3O8Na: 618.2791. Found: 618.2793.
8-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one
(
6o) White solid; m.p. 143–145◦C; yield 38%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.71 (s, 1H), 7.63 (d,
J = 8.0 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.14 (t, J = 7.7 Hz, 1H), 5.85 (s, 1H), 5.40 (s, 1H), 5.33 (s, 2H),
4.79 (d, J = 3.7 Hz, 1H), 4.55–4.46 (m, 2H), 3.93–3.89 (m, 1H), 3.44–3.40 (m, 1H), 2.70–2.63 (m, 1H), 2.45
(s, 3H), 2.37 (td, J = 14.0, 3.9 Hz, 1H), 2.28–2.19 (m, 2H), 2.05–2.01 (m, 1H), 1.92–1.86 (m, 1H), 1.81–1.72
(m, 2H), 1.67–1.64 (m, 1H), 1.53–1.46 (m, 2H), 1.42 (s, 3H), 1.39–1.33 (m, 1H), 1.30–1.22 (m, 2H), 0.96 (d,
J = 6.4 Hz, 3H), 0.94 (d, J = 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 165.35, 162.77, 151.73,
141.56, 133.82, 126.25, 123.45, 123.40, 120.73, 115.17, 104.22, 102.21, 90.89, 87.96, 80.97, 77.24, 77.03, 76.82,
64.65, 62.69, 52.51, 47.76, 44.27, 37.48, 36.37, 34.56, 30.82, 30.46, 26.15, 24.67, 24.57, 20.37, 15.76, 13.11;
ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2646.
6-methyl-4-((4-(3-((10S)-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-1-yl)-methoxy)-2H-1-chromen-2-one
(
6p) White solid; m.p. 80–82 ◦C; yield 45%; 1H NMR (600 MHz, CDCl3)
δ (ppm): 7.72 (s, 1H), 7.56 (s,
1H), 7.35 (dd, J = 8.5, 2.0 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 5.84 (s, 1H), 5.41 (s, 1H), 5.33 (s, 2H), 4.80 (d,
J = 3.6 Hz, 1H), 4.55–4.47 (m, 2H), 3.94–3.90 (m, 1H), 3.45–3.41 (m, 1H), 2.70–2.64 (m, 1H), 2.40–2.33 (m,
4H), 2.27–2.21 (m, 2H), 2.06–2.01 (m, 1H), 1.91–1.87 (m, 1H), 1.80–1.73 (m, 2H), 1.65 (dd, J = 13.3, 3.4 Hz,
1H), 1.53–1.46 (m, 2H), 1.42 (s, 3H), 1.38–1.34 (m, 1H), 1.28–1.22 (m, 2H), 0.96 (d, J = 6.4 Hz, 3H), 0.94 (d,
J = 7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ (ppm): 165.02, 162.88, 151.52, 141.46, 133.69, 133.59,
123.52, 122.80, 116.56, 115.08, 104.22, 102.22, 91.11, 87.97, 80.97, 77.24, 77.03, 76.82, 64.68, 62.60, 52.51,
47.80, 44.27, 37.48, 36.37, 34.57, 30.82, 30.48, 26.15, 24.67, 24.58, 20.90, 20.37, 13.12; ESI-HRMS [M +
Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2635. Found: 604.2621.
4.2. Biology
4.2.1. Storage and Preparation of Samples
All the target compounds were dissolved in dimethylsulfoxide and storage under
20 ◦C. They get
−
to be diluted to different concentration when they will be used.
4.2.2. Cell Culture
After recovery, the cells were cultured in 96-well plates for 24 h at 37 ◦C in a humidified atmosphere
with 95% air and 5% CO2. After treated with different tested compounds, the hypoxia groups were
placed in a sealed hypoxia incubator chamber (Stemcell Technologies, Inc., Vancouver, BC, Canada)
filled with 5% CO2 and 95% N2 for 24 h, and then transferred to the incubator chamber filled with air
for 72 h. On the contrary, the normoxia groups were cultured under air condition for 96h.
Cancer cells HT-29 (Human Colorectal Adenocarcinoma cell line), MDA-MB-231 (Human
Breast Cancer cell line), HCT-116 (Human Colorectal Carcinoma cell line), and A549 (Human Lung
Adenocarcinomic cell line) were from ATCC.

Molecules 2019, 24, 1672
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4.2.3. MTT Assay
When the adherent cells reached 80% confluence, the culture medium was discarded. The cells
were digested by trypsin and collected after centrifugation. The fresh culture medium was gently blown
into it to form single-cell suspension. Cell suspension will be diluted to 1.5–3
of 96-well plates was added 100
L cell suspension and incubated for 24 h (5% CO2; 37 ◦C). Then the
holes were added different concentrations of tested compounds. The early screening concentration is
set to 100 M, 50 M, 25 M, 12.5 M, and 6.25 M. Three replicates were made for each concentration
of the tested compounds. After the cells were grown for 96h, 20 L MTT (5 mg/mL) was added
to each well and incubated for 4 h. The medium was discarded and each well was added 100
×
104 cells/mL. Each hole
µ
µ
µ
µ
µ
µ
µ
µL
DMSO to dissolve the formazan blue. Absorbance was measured at 570 nm with a microplate reader
(Synergy-HT, BioTek Instruments, Winooski, VT, USA).
Hypoxia condition: The cells were treated with different concentration of tested compounds,
placed in hypoxia chamber (Catalog Number 27310, ◦Stemcell Technologies, Inc., Vancouver, BC,
Canada), and incubated for 24 h (5% air; 95% N2; 37 C). Then the cells were placed in normoxia
condition (5% CO2; 37 ◦C) and incubated for 72 h.
4.2.4. Preparation of N9 Cells
The murine microglial cell line N9 was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita
Degli Studi di Milano-Bicocca, Milan, Italy).
N9 cells, which were in the logarithmic phase, were incubated for 24 h by adherent culture.
Subsequently, the medium was changed to new one without serum. The cells were treated with
different concentration of tested compounds. After specific time in the LPS condition, the supernatants
were collected and analyzed.
4.2.5. Nitrite Measurement
The nitrite levels in medium were determined by Griess reaction. The absorbance was measured
at 540 nm using a microplate reader.
4.2.6. NO Capture Analysis
SNP was dissolved in PBS to prepare the 100 mM stock solution. In this experiment, SNP solution
of 25 µL was added to 975 µL PBS solution which included different concentration of tested compounds.
After 60 min under r.t. condition, the concentration of NO2− was tested using Griess assay.
4.2.7. Evaluation of Inflammatory Mediator
The secreted level of TNF-α and IL-6 was measured using mouse Th1/Th2/Th17 Cytokine Kit,
purchased from BD Pharningen. The data was analyzed by FCAP Array v3.0. IL-2, IL-4, IL-10, IL-17A,
and IFN-γ were not detected in the experiment.
4.2.8. Molecular Docking Simulations
The crystallographic structure of TLR4/MD-2 complex was retrieved from the Protein DataBank
(PDB ID: 3VQ2), which was optimized by removing water molecules, cofactors and heteroatoms.
Receptor grid was generally around the MD-2 active site. Docking calculations were accomplished
using AutoDock4. The docking results were further analyzed and visually optimized by Discovery
Studio 4.0 and PyMOL.
Supplementary Materials: The H and 13 C NMR spectra of synthesized compounds are available online.
Author Contributions: H.Y. performed experiments, analyzed data, and drafted the manuscript. Z.H. performed
experiments, analyzed data, and revised the manuscript. X.Y. performed experiments. Y.M. and C.G. conceived
the work, gave critical comments, and revised the manuscript.

Molecules 2019, 24, 1672
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Funding: We greatly appreciate the funding support for this research provided by the National Natural Science
Foundation of China (Grant No. 81573292).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
DHA
DCH
CRF
DOX
CNS
SNP
LPS
Dihydroartemisinin
Dihydroartemisinin–Coumarin hydrids
Cancer-related fatigue
Doxorubicin
Central nervous system
Sodium nitroprusside
Lipopolysaccharide
TLR4
Toll-like receptor 4
References
1.
Tsuji, E.; Hiki, N.; Nomura, S.; Fukushima, R.; Kojima, J.; Ogawa, T.; Mafune, K.; Mimura, Y.; Kaminishi, M.
Simultaneous onset of acute inflammatory response, sepsis-like symptoms and intestinal mucosal injury
after cancer chemotherapy. Int. J. Cancer 2003, 107, 303–308. [CrossRef] [PubMed]
Cerci, C.; Ergin, C.; Eroglu, E.; Agalar, C.; Agalar, F.; Cerci, S.; Bulbul, M. Effects of granulocyte-colony
stimulating factor on peritoneal defense mechanisms and bacterial translocation after administration of
systemic chemotherapy in rats. World J. Gastroenterol. 2007, 13, 2596–2599. [CrossRef] [PubMed]
Rivest, S. Molecular insights on the cerebral innate immune system. Brain Behav. Immun. 2003, 17, 13–19.
[CrossRef]
Norden, D.M.; Bicer, S.; Clark, Y.; Jing, R.; Henry, C.J.; Wold, L.E.; Reiser, P.J.; Godbout, J.P.; McCarthy, D.O.
Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in
muscle function. Brain Behav. Immun. 2015, 43, 76–85. [CrossRef] [PubMed]
Bower, J.E.; Lamkin, D.M. Inflammation and cancer-related fatigue: Mechanisms, contributing factors,
and treatment implications. Brain Behav. Immun. 2013, 30, S48–S57. [CrossRef]
Perry, V.H.; Gordon, S. Macrophages and microglia in the nervous system. Trends Neurosci. 1988, 11, 273–277.
[CrossRef]
Kreutzberg, G.W. Microglia: A sensor for pathological events in the CNS. Trends Neurosci. 1996, 19, 312–318.
[CrossRef]
Colton, C.A.; Gilbert, D.L. Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett.
1987, 223, 284–288. [CrossRef]
Banati, R.B.; Gehrmann, J.; Schubert, P.; Kreutzberg, G.W. Cytotoxicity of microglia. Glia 1993, 7, 111–118.
[CrossRef]
2.
3.
4.
5.
6.
7.
8.
9.
10. Lissauer, M.E.; Johnson, S.B.; Bochicchio, G.V.; Feild, C.J.; Cross, A.S.; Hasday, J.D.; Whiteford, C.C.;
Nussbaumer, W.A.; Towns, M.; Scalea, T.M. Differential expression of toll-like receptor genes: Sepsis
compared with sterile inflammation 1 day before sepsis diagnosis. Shock 2009, 31, 238–244. [CrossRef]
11. Steiner, A.A.; Chakravarty, S.; Rudaya, A.Y.; Herkenham, M.; Romanovsky, A.A. Bacterial lipopolysaccharide
fever is initiated via Toll-like receptor 4 on hematopoietic cells. Blood 2006, 107, 4000–4002. [CrossRef]
12. Shimazu, R.; Akashi, S.; Ogata, H.; Nagai, Y.; Fukudome, K.; Miyake, K.; Kimoto, M. MD-2, a Molecule
that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4. J. Exp. Med. 1999, 189, 1777–1782.
[CrossRef] [PubMed]
13. Nagai, Y.; Akashi, S.; Nagafuku, M.; Ogata, M.; Iwakura, Y.; Akira, S.; Kitamura, T.; Kosugi, A.; Kimoto, M.;
Miyake, K. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat. Immunol. 2002, 3,
667–672. [CrossRef] [PubMed]
14. Inohara, N.; Nuñez, G. ML—A conserved domain involved in innate immunity and lipid metabolism. Trends
Biochem. Sci. 2002, 27, 219–221. [CrossRef]
15. Viriyakosol, S.; Tobias, P.S.; Kitchens, R.L.; Kirkland, T.N. MD-2 binds to bacterial lipopolysaccharide. J. Biol.
Chem. 2001, 276, 38044–38051. [PubMed]

Molecules 2019, 24, 1672
19 of 20
16. Navaratnam, V.; Mansor, S.M.; Sit, N.W.; Grace, J.; Li, Q.; Olliaro, P. Pharmacokinetics of artemisinin-type
compounds. Clin. Pharmacokinet. 2000, 39, 255–270. [CrossRef] [PubMed]
17. Balint, G.A. Artemisinin and its derivatives: An important new class of antimalarial agents. Pharmacol. Ther.
2001, 90, 261–265. [CrossRef]
18. Chen, H.; Gu, S.; Dai, H.; Li, X.; Zhang, Z. Dihydroartemisinin Sensitizes Human Lung Adenocarcinoma
A549 Cells to Arsenic Trioxide via Apoptosis. Biol. Trace Elem. Res. 2017, 179, 203–212. [CrossRef]
19. Cao, L.; Duanmu, W.; Yin, Y.; Zhou, Z.; Ge, H.; Chen, T.; Tan, L.; Yu, A.; Hu, R.; Fei, L.; et al. Dihydroartemisinin
exhibits anti-glioma stem cell activity through inhibiting p-AKT and activating caspase-3. Die Pharm. 2014
69, 752–758.
,
20. Zhang, Z.; Guo, M.; Zhao, S.; Shao, J.; Zheng, S. ROS-JNK1/2-dependent activation of autophagy is required
for the induction of anti-inflammatory effect of dihydroartemisinin in liver fibrosis. Free Radic. Biol. Med.
2016, 101, 272–283. [CrossRef]
21. Jiang, L.B.; Meng, D.H.; Lee, S.M.; Liu, S.H.; Xu, Q.T.; Wang, Y.; Zhang, J. Dihydroartemisinin inhibits
catabolism in rat chondrocytes by activating autophagy via inhibition of the NF-kappaB pathway. Sci. Rep.
2016, 6, 38979. [CrossRef]
22. Huang, X.; Xie, Z.; Liu, F.; Han, C.; Zhang, D.; Wang, D.; Bao, X.; Sun, J.; Wen, C.; Fan, Y. Dihydroartemisinin
inhibits activation of the Toll-like receptor 4 signaling pathway and production of type I interferon in spleen
cells from lupus-prone MRL/lpr mice. Int. Immunopharmacol. 2014, 22, 266–272. [CrossRef]
23. Witaicenis, A.; de Oliveira, E.C.S.; Tanimoto, A.; Zorzella-Pezavento, S.F.G.; de Oliveira, S.L.; Sartori, A.;
Di Stasi, L.C. 4-methylesculetin, a coumarin derivative, ameliorates dextran sulfate sodium-induced intestinal
inflammation. Chem. Biol. Interact. 2018, 280, 59–63. [CrossRef] [PubMed]
24. Chougala, B.M.; Samundeeswari, S.; Holiyachi, M.; Naik, N.S.; Shastri, L.A.; Dodamani, S.; Jalalpure, S.;
Dixit, S.R.; Joshi, S.D.; Sunagar, V.A. Green, unexpected synthesis of bis-coumarin derivatives as potent
anti-bacterial and anti-inflammatory agents. Eur. J. Med. Chem. 2018, 143, 1744–1756. [CrossRef] [PubMed]
25. Emami, S.; Dadashpour, S. Current developments of coumarin-based anti-cancer agents in medicinal
chemistry. Eur. J. Med. Chem. 2015, 102, 611–630. [CrossRef] [PubMed]
26. Basanagouda, M.; Jambagi, V.B.; Barigidad, N.N.; Laxmeshwar, S.S.; Devaru, V.; Narayanachar. Synthesis,
structure-activity relationship of iodinated-4-aryloxymethyl-coumarins as potential anti-cancer and
anti-mycobacterial agents. Eur. J. Med. Chem. 2014, 74, 225–233. [CrossRef]
27. Devji, T.; Reddy, C.; Woo, C.; Awale, S.; Kadota, S.; Carrico-Moniz, D. Pancreatic anticancer activity of a novel
geranylgeranylated coumarin derivative. Bioorg. Med. Chem. Lett. 2011, 21, 5770–5773. [CrossRef] [PubMed]
28. Bronikowska, J.; Szliszka, E.; Jaworska, D.; Czuba, Z.P.; Krol, W. The coumarin psoralidin enhances anticancer
effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Molecules 2012, 17, 6449–6464.
[CrossRef] [PubMed]
29. Reddy, N.S.; Gumireddy, K.; Mallireddigari, M.R.; Cosenza, S.C.; Venkatapuram, P.; Bell, S.C.; Reddy, E.P.;
Reddy, M.V. Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer cell growth by inhibiting ErbB-2
and ERK1. Bioorg. Med. Chem. 2005, 13, 3141–3147. [CrossRef]
30. Harada, K.; Kubo, H.; Tomigahara, Y.; Nishioka, K.; Takahashi, J.; Momose, M.; Inoue, S.; Kojima, A.
Coumarins as novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors for potential treatment of
prostate cancer. Bioorg. Med. Chem. Lett. 2010, 20, 272–275. [CrossRef]
31. Vijay Avin, B.R.; Thirusangu, P.; Lakshmi Ranganatha, V.; Firdouse, A.; Prabhakar, B.T.; Khanum, S.A.
Synthesis and tumor inhibitory activity of novel coumarin analogs targeting angiogenesis and apoptosis.
Eur. J. Med. Chem. 2014, 75, 211–221. [CrossRef]
32. Jin, J.; Yu, X.; Hu, Z.; Tang, S.; Zhong, X.; Xu, J.; Shang, P.; Huang, Y.; Liu, H. Isofraxidin targets the TLR4/MD-2
axis to prevent osteoarthritis development. Food Funct. 2018, 9, 5641–5652. [CrossRef]
33. Liu, J.; Chen, Q.; Jian, Z.; Xiong, X.; Shao, L.; Jin, T.; Zhu, X.; Wang, L. Daphnetin Protects against Cerebral
Ischemia/Reperfusion Injury in Mice via Inhibition of TLR4/NF-kappaB Signaling Pathway. Biomed. Res. Int.
2016, 2016, 2816056. [CrossRef]
34. Tian, Y.; Liang, Z.; Xu, H.; Mou, Y.; Guo, C. Design, Synthesis and Cytotoxicity of Novel
Dihydroartemisinin-Coumarin Hybrids via Click Chemistry. Molecules 2016, 21, 758. [CrossRef]
35. Yu, H.; Hou, Z.; Tian, Y.; Mou, Y.; Guo, C. Design, synthesis, cytotoxicity and mechanism of novel
dihydroartemisinin-coumarin hybrids as potential anti-cancer agents. Eur. J. Med. Chem. 2018, 151, 434–449.
[CrossRef]

Molecules 2019, 24, 1672
20 of 20
36. Tan, Y.; Yang, X.-D.; Liu, W.-J.; Sun, X.-W. Novel one-pot asymmetric cascade approach toward densely
substituted enantioenriched α-methylene-γ-lactams. Tetrahedron Lett. 2014, 55, 6105–6108. [CrossRef]
37. Pechmann, H.V. Berichte der deutschen chemischen. Gesellschaft 1884, 17, 929.
38. Ji, Q.; Ge, Z.; Ge, Z.; Chen, K.; Wu, H.; Liu, X.; Huang, Y.; Yuan, L.; Yang, X.; Liao, F. Synthesis and biological
evaluation of novel phosphoramidate derivatives of coumarin as chitin synthase inhibitors and antifungal
agents. Eur. J. Med. Chem. 2016, 108, 166–176. [CrossRef]
39. Ohto, U.; Fukase, K.; Miyake, K.; Shimizu, T. Structural basis of species-specific endotoxin sensing by innate
immune receptor TLR4/MD-2. Proc. Natl. Acad. Sci. USA 2012, 109, 7421–7426. [CrossRef]
Sample Availability: Not available.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).