Design, synthesis, cytotoxicity and mechanism of novel dihydroartemisinin-coumarin hybrids as potential anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.04.005

To develop novel agents with anticancer activities, thirty-four new dihydroartemisinin-coumarin hybrids were designed and synthesized in this study. Those compounds were identified that had great anticancer activity against two cancer cell lines (MDA-MB-231 and HT-29). The structure-activity relationships of the derivatives were also discussed, and the results of docking analysis had shown that carbonic anhydrases IX (CA IX) was very likely to be one of the drug targets of the hybrids. Meanwhile, to clarify the mechanism of the anticancer activity of the hybrids molecule, we did further exploration in the bioactivity of the hybrids. The results had shown that these derivatives obviously inhibited proliferation of HT-29 cell lines, arrested G₀/G₁ phase of HT-29 cells, suppressed the migration of tumor cells, and induced a great decrease in mitochondrial membrane potential leading to apoptosis of cancer cells. Interestingly, the hybrids also induced the other cell death pathway-ferroptosis.

Full text of DOI:10.1016/j.ejmech.2018.04.005

Accepted Manuscript
Design, synthesis, cytotoxicity and mechanism of novel dihydroartemisinin-coumarin
hybrids as potential anti-cancer agents
Haonan Yu, Zhuang Hou, Ye Tian, Yanhua Mou, Chun Guo
PII:
S0223-5234(18)30332-5
10.1016/j.ejmech.2018.04.005
EJMECH 10354
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 January 2018
Revised Date: 1 April 2018
Accepted Date: 2 April 2018
Please cite this article as: H. Yu, Z. Hou, Y. Tian, Y. Mou, C. Guo, Design, synthesis, cytotoxicity and
mechanism of novel dihydroartemisinin-coumarin hybrids as potential anti-cancer agents, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.005.
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ACCEPTED MANUSCRIPT

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Design, synthesis, cytotoxicity and mechanism of novel dihydroartemisinin-coumarin hybrids as
potential anti-cancer agents
Haonan Yu1#, Zhuang Hou1#, Ye Tian1, Yanhua Mou2* and Chun Guo1*
1School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
China;
2 Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China;
#Haonan Yu and Zhuang Hou contributed equally to this work.
*Correspondence to
Chun Guo, Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education,
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, No. 103 Wenhua Road,
Shenyang, 110016, Liaoning Province, China. Tel: +86-24-4352-0226, Email: chunguo@syphu.edu.cn;
Yanhua Mou, Department of Pharmacology, Shenyang Pharmaceutical University, No. 103 Wenhua
Road, Shenyang 110016, Liaoning Province, China. Email: mu_hua_jj@sina.com
Authors’ emails
yhna380@hotmail.com (HY)
houzhuang8@sina.com (ZH)
tianye870918@126.com (YT);
mu_hua_jj@sina.com (YM)
chunguo@syphu.edu.cn (CG)
Abbreviations
DHA, Dihydroartemisinin; Carbonic anhydrases, CA; Acetazolamide, AAZ; doxorubicin, DOX.
Acknowledgment
We greatly appreciate the funding support for this research provided by the National Natural Science
Foundation of China (Grant No. 81573292).
Conflict of interest: None.

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Financial support
National Natural Science Foundation of China (Grant No. 81573292).
Total words: 8818
Tables: 1
Figures: 10
Scheme: 2
Author contributions
Haonan Yu performed experiments, analyzed data and drafted the manuscript. Zhuang Hou
performed experiments, analyzed data and revised the manuscript. Ye tian performed experiments.
Yanhua Mou and Chun Guo conceived the work, gave critical comments and revised the manuscript.

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Abstract
To develop novel agents with anticancer activities, thirty-four new dihydroartemisinin-coumarin
hybrids were designed and synthesized in this study. Those compounds were identified that had great
anticancer activity against two cancer cell lines (MDA-MB-231 and HT-29). The structure-activity
relationships of the derivatives were also discussed, and the results of docking analysis had shown that
carbonic anhydrases IX (CA IX) was very likely to be one of the drug targets of the hybrids.
Meanwhile, to clarify the mechanism of the anticancer activity of the hybrids molecule, we did further
exploration in the bioactivity of the hybrids. The results had shown that these derivatives obviously
inhibited proliferation of HT-29 cell lines, arrested G0/G1 phase of HT-29 cells, suppressed the
migration of tumor cells, and induced a great decrease in mitochondrial membrane potential leading to
apoptosis of cancer cells. Interestingly, the hybrids also induced the other cell death
pathway-ferroptosis.
Keywords: anticancer; artemisnin; coumarin; hybrid; apoptosis; ferroptosis.

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1. Introduction:
Artemisinin, a well-known anti-malarial agent, has been reported to possess a potent and broad
antitumor spectrum against human cancer cell lines. Numbers of the artemisinin derivatives had been
designed and synthesized and some of them have shown excellent anticancer activities and
pharmacokinetic properties [1-5]. Some novel artemisinin derivatives could even overcome multidrug
resistance in cancer therapy [6]. Meanwhile, the coumarin derivatives have already been extensively
studied as anticoagulant, anti-inflammatory [7, 8], antibacterial [9] and anticancer [10, 11] agents. As
the anticancer agents, it selectively target CA IX, by which it inhibited tumor cells [12-15]. Owing to
the rapid proliferation, the tumor cells had always been in hypoxia condition along with low pH values.
The main function of CA IX is to homeostasis an acidification of extracellular environment that its pH
of tumor tissues was about 6.0-6.5 while that of normal tissue was about 7.4. CA IX catalyzed the
-
-
reaction of carbon dioxide hydration to yield HCO3 and H+. HCO3 was transferred into the tumor cells
to neutralize H+ and maintained appropriate micro-environment of tumor tissue through the anion
-
transporter and Na+/HCO3 co-transporter [16-18].
Previously, four series of compounds were obtained referring to the principle of hybridization (Fig.
1). Each of them contained coumarin moiety, dihydroartemisinin (DHA) moiety and a triazole ring
linker. The cytotoxic activities of those compounds under anoxic condition were 1 to 10 times than that
under the normoxic condition. Some compounds had selectivity in inhibiting HT-29 cells
(overexpressed CA IX) and compound A (Fig. 1) was considered a better cytotoxic activity than others
via experiment [19]. In this article, we designed and synthesized of new molecular hybrids of DHA and
coumarin tethered directly or by the alkane chain other than triazole ring. The synthetic approaches
have been designed as follows (Fig. 2):
(i) no linker
(ii) ethyl and propyl as the linker
(ii) linkers containing triazole ring
Herein, a variety of novel dihydroartemisinin-coumarin molecules containing different linkers
(1b-1e, 2a-2e, 3a-3e, 4a-4d, 4f, 5a-5d, 5f, 6g-6k and 7g-7k) were synthesized and were also evaluated
for their cytotoxic and anti-tumor activities. The structure-activity relationships of
dihydroartemisinin-coumarin hydrids were discussed. In addition, a series of pharmacological activity

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of compound A was tested to reveal the mechanism of the dihydroartemisinin-coumarin.
Fig. 1. Structures of previous dihydroartemisinin-coumarin molecules
Fig. 2. Structures of new designed dihydroartemisinin-coumarin molecules

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2. Results and discussion
2.1. Chemistry
The synthesis of the intermediates a-f, I-VII was presented at Scheme 1. 7-hydroxylcoumarin
derivatives a-d were obtained by substitution of ethyl acetoacetate in 1, 3-dihydroxybenzene, while e
and f were synthesized by the cyclization between 2, 4-dihydroxybenzaldehyde and diethyl
malonate/dimethyl malonate. Azidation reaction was conducted to obtain intermediates Va-Vd, Vf,
VIa-VId and VIf after each coumarin derivative’s (a-d and f) reaction with 1, 2-dibromoethane or
1,3-dibromopropane. The synthesis of 7-aminocoumarin derivatives g-k was achieved by the process
starting from m-aminophenol and its corresponding substituted ethyl acetoacetate. Intermediates
VIIg-VIIk were synthesized by g-k via diazotization reaction and azidation reaction. Intermediates I, II,
III, IV were respectively obtained by 2-bromoethanol, 3-bromo-1-propanol, propargyl alcohol and
3-Butyn-1-ol in the substrate of dihydroartemisinin (DHA).

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R3
R3
R3
R4
(4)
R4
(5)
R4
z
+
z
Br
z
Br
O O
O
O O
a-d, f
OH
O O
Va-Vd, Vf (z=2)
O
Br
N3
VIa-VId, VIf (z=3)
Va, VIa: R3=CH3, R4=H
Vb, VIb: R3=CF3, R4=H
Vc, VIc: R3=CH3, R4=CH3
Vd, VId: R3=CH3, R4=Cl
Vf, VIf: R3=H, R4=COOMe
O
(7)
O
O
+
(6)
(8)
O
+
EtO
H2N
OH
N
R5
OEt
H2N
OH
EtO Cl
H
R6
R5
R6
R5
O
R5
O
(9)
O
R6
O
R6
O
EtO
O O
N
N3
H
g-k
g, VII
g: R5=CH3, R6=H
VIIg-VIIk
h, VIIh: R5=CF3, R6=H
i, VIIi: R5=CH3, R6=CH3
j, VII
j: R5=CH3, R6=Cl
k, VIIk: R5=CH3, R6=F
.
Reagents and conditions: (1) H2SO4, r.t.; (2) piperidine (cat.), EtOH, reflux; (3) BF3 Et2O, CH2Cl2, 0 ;
(4) K2CO3, DMF, 60 ; (5) NaN3, DMF, 60 ; (6) Et3N, CH2Cl2; (7) H2SO4, r.t.; (8) AcOH, H2SO4; (9)
①NaNO2, HCl; ②NaN3;
Scheme 1. Synthetic routes of intermediates.
The synthesis of the desired new compounds was presented at Scheme 2, target compounds 1b-1e
were prepared by the reaction between b-e and DHA. Target compounds 2a-2e and 3a-3e were
obtained by the reaction between each coumarin derivatives (a-e) with I and II. Target compounds
4a-4d, 4f, 5a-5d and 5f were synthesized by intermediates V and VI with intermediate IV via click
chemistry. Target compounds 6g-6k and 7g-7k were obtained by reaction of VIIg-VIIk with III and IV,
respectively via click chemistry.

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.
Reagents and conditions: (1) (CF3CO)2O, Et3N, CH2Cl2; (2) K2CO3, DMF, 60 ; (3) CuSO4 5H2O,
sodium ascorbate, DMF, r.t..
Scheme 2. Synthetic routes of target compounds 1b-1e, 2a-2e, 3a-3e, 4a-4d, 4f, 5a-5d, 5f, 6g-6k and
7g-7k.
2.2. Biological assay
2.2.1. In vitro cytotoxicity studies
The cytotoxicity of new compounds toward sensitive HT-29 (CA IX high express) and
MDA-MB-231 (CA IX normal express) cells were evaluated by the MTT assay. As the highly-relative

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expression of CA IX towards the growth and metastasis of tumor which could be more significant in
the anoxia environment, the influence of O2 was also considered in the experiment. Acetazolamide
(AAZ, standard carbonic anhydrase inhibitor), doxorubicin (DOX) and DHA were selected to be
positive reference drugs.
The inhibitory concentration 50% (IC50) of the target compounds were shown in Table 1. The
cytotoxicity of the most compounds on HT-29 cells were higher than that of MDA-MB-231 cells.
Meanwhile, there showed a general promotion under hypoxia conditions, indicating the cytotoxicity
effects of the coumarin moiety of the target compounds.
The structure-activity relationship of the target compounds was also concluded. i): in the same series
of compounds, the 3-chloro, 4-methyl substituent in coumarin moiety generally showed a better
exhibition than that of the others. ii): on the contrary, the 3-ethoxycarbonyl group or
3-methoxycarbonyl group in the 3-position of coumarin moiety showed poorer activity than others in
the same series of compouds. iii): in the series of compounds 1, 2 and 3，compounds 2 and 3 generally
had better cytotoxicity exhibition than that of the series of compounds 1 which indicated increasing the
distance between DHA and coumarin moiety could improve the activity. iv): although the distance
between DHA moiety and coumarin moiety were different, the cytotoxicity activity of the series of
compounds 4, 5, 6 and 7 which having 1, 2, 3- triazole linker showed no difference. v): the compounds
having 1, 2, 3- triazole linker generally had better cytotoxicity exhibition than the others.
Table 1. IC50 of tested samples in two cell lines.
IC50(µM)
Compd.
HT-29
MDA-MB-231
Hypoxia
>100
0.25
Normoxia
>100
Hypoxia
Normoxia
>100
>100
5.98
5.64
7.51
3.25
6.44
AAZ
DOX
DHA
A
0.24
1.07
18.52
0.17
>100
47.11
18.37
56.84
78.81
1.88
22.3
11.09
11.79
1b
30.28
1c

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0.41
7.17
3.69
5.59
57.22
7.02
69.96
8.71
1.19
3.54
8.05
8.69
10.69
4.01
14.56
2.37
75.68
0.11
4.29
0.11
1.68
-
1.28
3.45
2.35
54.82
12.82
23.92
5.52
13.82
4.57
8.71
2.55
13.82
5.72
3.58
3.47
5.28
67.19
6.87
5.88
5
1.4
2.85
0.1
50.54
21.93
4.29
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4f
6.14
12.85
5.36
6.02
22.94
8.86
7.92
2.38
22.96
18.25
14.25
52.28
7.24
58.44
14.4
6.58
33.19
6.07
-
15.2
57.21
18.03
17.02
29.96
13.57
17.13
0.6
42.89
17
17.88
56.71
7.53
60.97
72.25
32.5
5a
5b
5c
5d
5f*
6g
6h
6i
>100
30.98
-
1.19
-
5.09
5.33
0.02
1.15
7.39
10.93
10.27
10.84
5.22
2.67
4.27
3.35
8.95
6.07
49.82
49.6
4.85
24.68
2.6
21.49
>100
23.44
50.53
3.47
6j
6k
7g
7h
9.96
95.74
3.78
1.85

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0.08
0.01
5.66
4.71
1.5
3.58
1.03
3.36
3.75
5.19
3.84
7i
7j
7k
9.89
*5f was insoluble
2.2.2. Anti-tumor mechanisms of compound A.
Dihydroartemisinin-coumarin hybrids had exhibited a great value in anticancer activity, but the
specific mechanisms were still unclear. Compound A as one of the dihydroartemisinin-coumarin
hybrids was chosen to be further studied.
2.2.2.1 Docking analysis
Maresca et al. revealed that coumarin moiety were hydrolyzed within the CA active site[20].
Therefore, as shown in (d) of Fig. 3, the hydrolyzed compound A was used as the ligand of this
docking analysis. The molecular docking results showed the coumarin segment of compound A could
integrated closely with the CA IX active site cavity (Fig. 3) with minimum binding energy of (∆G)
−11.4 kcal/mol. At the active site of CA IX, compound A formed six H-bonds with His-96, His-119,
Thr-199, Thr-200, Asn-62 and Gln-67. The triazole substation was involved in the interactions, which
might explain why the compounds having 1, 2, 3- triazole linker generally had better cytotoxicity
exhibition than the others. Combined with the results of the cytotoxicity studies, CA IX was very likely
to be one of the drug targets of the dihydroartemisinin-coumarin hybrids.

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Fig. 3. Interaction diagrams of the selected docked conformations for hydrolyzed compound A inside
the active site of CA IX enzyme. (a) The surface representation of binding pocket has been shown at
the top of the figure. (b) 2D ligand interactions diagram. (c) 3D ligand interactions diagram. (d) The
ligand of this docking analysis.
2.2.2.2. Cell morphology
Different concentration of compound A were administered to HT-29 and then cultured for 96 h,
which showed a proliferation inhibition in a dose dependent manner (Fig. 4). As shown in F1 and F2 of
Fig. 4, the cell volume significantly enlarged, the cell amount, meanwhile, was less than others,
indicating the proliferate inhibition effect of compound A.

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Fig. 4. Inhibition of compound A on cell viability under normoxic condition (A1-F1); Inhibition of
compound A on cell viability under hypoxic condition (A2-F2). Cell were treated with (A1, A2) 0 µM,
(B1, B2) 1.25 µM, (C1, C2) 2.5 µM, (D1, D2) 5.0 µM, (E1, E2) 10.0 µM, (F1, F2) 20.0 µM compound
A for 96 h. Representative images were shown. 100×
2.2.2.3. Analysis of cell cycle progression by flow cytometry
The effect of compound A on cell cycle progression and apoptosis was examined by flow cytometry.
The distribution of HT-29 in cell cycle was tested after treated with different concentrations of
compound A (A1-A5 of Fig. 5). These results explained the possible correlation between growth
inhibition of compound A on HT-29 and G0/G1 phase arrest. We also took the time factor into the
consideration. HT-29 cells were treated with 10 µM compound A. As shown in B of Fig. 5, Sub-G1

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phase was not obviously evident before 48 h. The ratio increased during 48 h to 72 h in a time
dependent manner. The maximum ratio of Sub-G1 reached up to 60%. All these results revealed that
compound A could arrest the growth of HT-29 within a short time-frame (0 h-48 h). But when
prolonged treated period, there induced obviously apoptosis of HT-29 cells, indicating that the duration
of the treatment was at least 48 h enough to induce apoptosis.
Fig. 5. G0/G1-phase cell cycle arrest in HT-29 cell line induced by compound A at 48 h (A1-A5); Cell
cycle arrest in HT-29 cell line induced by 10 µM compound A at different time (B).
2.2.2.4. Evaluation of apoptosis by Annexin V-FITC assay
For further confirming the evidence of apoptosis, early apoptosis was measured by Annexin V-FITC
assay. As illustrated in Fig. 6, cells in the early apoptotic phase were positive after treatment of HT-29
cells with different compound A concentrations.

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Fig. 6. Compound A induced apoptosis in HT-29 at 48 h. Cells were treated with different
concentrations of compound A, and then treated with 0.25 % trypsin. Cells were collected and washed
with PBS, incubated with ANNEX-FITC and PI in binding buffer, and at final detected by flow
cytometry.
2.2.2.5. Mitochondrial membrane potential (∆Ψm) analysis
The effect of compound A on mitochondrial membrane potential (MMP) of HT-29 was investigated
by flow cytometry after treated with rhodamine-123 (Rh-123). As shown in Fig. 7, compound A
induced dramatically decrease of MMP at 3 h, but it was gradually recovered until 24 h. Interestingly,
after 24 h, we observed that the MMP of HT-29 gradually inclined again in time dependent manner.
Compound A was considered to induce cell cycle arrest within 24 h and showed apoptosis effect after
24 h according to the previous experiment. The dramatically decrease at 3 h was possibly related to
extra function of Rh-123 on cells. Other than MMP, Rh-123 could also evaluate the ATP level of cells.
We tend to think the possibility that compound A induced ATP metabolic disorder of HT-29.

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Fig. 7. A induced transmitochondria membrain potential decrease in HT-29 cell line. Cells were treated
with 20 µM A for different times. Stained with Rh123 for 40 min and then treated with 0.25% trypsin.
Cells were collected and washed with PBS for 3 times and detected by flow cytometry.
2.2.2.6. Intracellular pH analysis
Using BCECF-AM as the fluorescent pH indicator, we investigated the effect of compound A on the
intracellular pH. As shown in Fig. 8, after 3 h and 12 h administration (color green and pink), the
fluorescence intensity was obviously lower than 0 h (color red). After 24 h administration (color blue),
the fluorescence intensity recovered but still lower than 0 h. These results indicated the acidification
occurred in the cells, which showed the possible suppression of CA IX by compound A resulted in the
H+accumulated.

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Fig. 8. Compound A induced cellular pH decrese in HT-29 cells. Cells were treated with 10 µM A for
different times. Stained with BCECF-AM, a PH sensetive fluorodye, for 40 min and then treated with
0.25% trypsin. Cells were collected and washed with PBS and detected by flow cytometry.
2.2.2.7. Ferroptosis
Ferroptosis was a newly found cell death pathway. It was demonstrated that overwhelming,
iron-dependent accumulation of lethal lipid reactive oxygen species (ROS) could trigger cell death in
the form of ferroptosis [21]. Artesunate had already been identified as an activator of ferroptosis[22].
Our target compounds had a similar artesunate DHA group. Therefore, we supposed our target
compounds had the same effect as well. As shown in A1-A3 of Fig. 9, HT-29 cells were treated with
liproxstatin-1 which is ferroptosis inhibitor, which resulted in strong death suppression at 72 h after
A/Liproxstatin-1 treatment. As shown in B of Fig. 9, the death prevention of liproxstatin-1 showed a
dose dependent manner at 48 h after HT-29 cells were treated with compound A. These results
demonstrated that compound A induced ferroptosis in HT-29 Cells.

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Fig. 9. Compound A-induced ferroptosis in HT-29 cells (A1-A3). Cells were pretreated with 10 µM;
Liproxstatin-1 reversed compound A-induced growth inhibition in HT-29 cells (B). Cells were
pretreated with different concentrations of Liproxstatin-1 for 1 h, and then 10 µM A was added and
cultured for 48 h. At the end points MTT assay was measured.
2.2.2.8. Wound-Healing Assay
HT-29 cells were plated in 24-well plates for 48 h and then the wound was created. The tumor cells
were treated with 0 µM, 0.31 µM, 0.63 µM, 1.25 µM, 2.5 µM and 5 µM compound A respectively, then
cultured in normoxia condition. 0 h and 72 h were observed by inverted microscope. As shown in Fig.
10, there showed the migrate inhibition of compound A in a concentration dependent manner.
Fig. 10. Inhibition of compound A on cell migration in HT-29 cells by wounded healing test. (0 h

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above, 72 h below)
2.2.2.9. Transwell assay
The migrate inhibition of compound A was tested by Transwell assay. As shown in Fig. 11, 1.25 µM
and 2.5 µM compound A could obviously inhibit HT-29 cell migration.
Fig. 11. Inhibition of compound A on cell migration in HT-29 cells by transwell test.
3. Conclusion
We designed and synthesized new series of dihydroartemisinin-coumarin hybrids. These compounds
exhibited moderate cytotoxic activities against HT-29 and MDA-MB-231 cell lines especially under
hypoxia condition. Then we did further study in the compound A which was reported before. We found
that compound A could inhibited proliferation of HT-29 cell lines, arrested the cell cycle progression of
HT-29 cells, and induce both apoptosis and ferroptosis of HT-29 cells. It also exhibited the inhibition of
the migration of HT-29 cells. Therefore we preliminarily clarified the mechanism of
dihydroartemisinin-coumarin hybrids’ anticancer activity.
4. Experimental section
4.1 Chemistry
Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech

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1
Instrument Co., Ltd., Beijing, China) without calibration. The H-NMR and 13C-NMR spectra were
measured by a Bruker AV-400 spectrometer (Bruker Bioscience, Billerica, MA, USA), with
tetramethylsilane as an internal standard. High-resolution mass spectra (HRMS) were measured with an
Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) in ESI mode. Reaction progress
was monitored by TLC on silica gel precoated GF254 plates (Qingdao Haiyang Chemical Co. Ltd.,
Qingdao, Shandong, China). Preparative flash column chromatography was performed on the 200–300
mesh silica gel (Qingdao Haiyang Chemical Co. Ltd., Qingdao, Shandong, China). Unless otherwise
noted, all solvents and reagents were commercially available and used without further purification.
4.1.1. General Procedure for the Synthesis of Compounds 1b-1e
DHA (0.01 mol) and triethylamine (0.02 mol) were added in 40 mL dichloromethane and stirred for
10 min at -5~0 . Trifluoroacetic anhydride was added dropwise while stirring. After the completion of
addition, the mixture was stirred at 0~5 . After 10 h, coumarin (b-e) (0.02 mol) was added and the
reaction mixture kept stirring for 8 h. The mixture was washed with saturation sodium bicarbonate (100
mL×5), dried over anhydrous sodium sulfate and evaporated to dryness. The product was purified
through column chromatography.
4.1.1.1. 4-(trifluoromethyl)-7-((10S)-Dihydroartemisin-10-oxy)-2H-1-chromen-2-one (1b)
1
White solid; 12.1% yield; m.p. 186~187 ; H NMR (400 MHz, CDCl3) δ (ppm): 7.65-7.59 (m, 1H),
7.18 (d, J = 2.4 Hz, 1H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.62 (s, 1H), 5.60 (d, J = 3.5 Hz, 1H), 5.41 (s,
1H), 2.91-2.81 (m, 1H), 2.41-2.32 (m, 1H), 2.05-1.99 (m, 1H), 1.98-1.82 (m, 3H), 1.69-1.73 (m, 1H),
1.66-1.57 (m, 1H), 1.45-1.52 (m, 1H), 1.43 (s, 3H), 1.27-1.40 (m, 3H), 1.03 (d, J = 7.3 Hz, 3H), 0.96 (d,
J = 5.8 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 160.06, 159.28, 158.36, 155.01, 125.37, 121.45,
119.63, 113.44, 111.77, 106.86, 103.75, 99.41, 87.36, 79.73, 76.20, 75.99, 75.78, 51.37, 43.11, 36.43,
35.21, 33.49, 29.71, 24.94, 23.58, 23.37, 19.25, 11.75; ESI-HRMS [M+Na]+: (m/z) Calcd. for
C25H27F3O7Na: 519.1601. Found: 519.1585.
4.1.1.2. 3, 4-dimethyl-7-((10S)-Dihydroartemisin-10-oxy)-2H-1-chromen-2-one (1c)
1
White solid; 17.0% yield; m.p. 162~164 ; H NMR (400 MHz, CDCl3) δ (ppm): 7.48 (d, J = 8.8 Hz,
1H), 7.07 (d, J = 2.5 Hz, 1H), 7.01 (dd, J = 8.7, 2.4 Hz, 1H), 5.54 (d, J = 3.4 Hz, 1H), 5.42 (s, 1H),
2.87-2.77 (m, 1H), 2.40-2.37 (m, 1H), 2.35 (s, 3H), 2.17 (s, 3H), 2.03 (s, 1H), 1.93-1.84 (m, 3H), 1.70

ACCEPTED MANUSCRIPT
(dd, J = 13.2, 3.2 Hz, 1H), 1.61-1.55 (m, 2H), 1.42 (s, 3H), 1.33-1.22 (m, 3H), 1.01 (d, J = 7.3 Hz, 3H),
0.95 (d, J = 5.9 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 161.34, 158.13, 152.26, 145.00, 124.20,
118.45, 114.15, 112.21, 103.31, 103.23, 99.44, 87.25, 79.83, 76.22, 76.01, 75.80, 51.43, 43.23, 36.40,
35.26, 33.55, 29.84, 25.00, 23.60, 23.39, 19.27, 14.06, 12.20, 11.84; ESI-HRMS [M+Na]+: (m/z) Calcd.
for C26H32O7Na: 479.2040. Found: 479.2045.
4.1.1.3. 3-chloro-4-methyl-7-((10S)-Dihydroartemisin-10-oxy)-2H-1-chromen-2-one (1d)
White solid; 17.5% yield; m.p. 97~98 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.51 (d, J = 8.8 Hz, 1H),
7.11 (d, J = 2.4 Hz, 1H), 7.07 (dd, J = 8.8, 2.4 Hz, 1H), 5.56 (d, J = 3.4 Hz, 1H), 5.41 (s, 1H), 2.86-2.82
(m, 1H), 2.53 (s, 3H), 2.40-2.32 (m, 1H), 2.06-1.98 (m, 1H), 1.96-1.81 (m, 3H), 1.75-1.67 (m, 1H),
1.62-1.57 (m, 1H), 1.50-1.44 (m, 1H), 1.42 (s, 3H), 1.36-1.21 (m, 3H), 1.02 (d, J = 7.3 Hz, 3H), 0.95 (d,
J = 5.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 160.155, 157.308, 152.814, 147.791, 125.878,
118.207, 114.130, 104.389, 104.268, 100.451, 88.324, 80.786, 77.412, 77.094, 76.776, 52.404, 44.181,
37.411, 36.247, 34.533, 30.789, 25.966, 24.609, 24.381, 20.281, 16.180, 14.203, 12.823; ESI-HRMS
[M+Na]+: (m/z) Calcd. for C25H29ClO7Na: 499.1494. Found: 499.1501.
4.1.1.4. Ethyl 7-((10S)-Dihydroartemisin-10-oxy)-2H-1-chromen-2-one-3-carboxylate (1e)
White solid; 16.7% yield; m.p. 82~83 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.49 (s, 1H), 7.51 (d, J
= 8.6 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.6, 2.3 Hz, 1H), 5.60 (d, J = 3.4 Hz, 1H), 5.40 (s,
1H), 4.39 (q, J = 7.1 Hz, 2H), 2.88-2.84 (m, 1H), 2.43-2.32 (m, 1H), 2.06-2.00 (m, 1H), 1.98-1.78 (m,
3H), 1.76-1.68 (m, 1H), 1.64-1.58 (m, 1H), 1.43 (s, 3H), 1.40 (t, J = 7.1 Hz, 4H), 1.32-1.21 (m, 3H),
1.03 (d, J = 7.3 Hz, 3H), 0.96 (d, J = 5.7 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 163.43,
162.65, 157.18, 157.08, 148.83, 130.74, 114.71, 114.53, 112.46, 104.46, 103.86, 100.48, 88.37, 80.72,
77.24, 77.03, 76.81, 61.77, 52.39, 44.13, 37.44, 36.22, 34.51, 30.74, 25.97, 24.60, 24.36, 20.27, 14.28,
12.78; ESI-HRMS [M+Na]+: (m/z) Calcd. for C27H32O9Na: 523.1939. Found: 523.1939.
4.1.2. General Procedure for the Synthesis of Compounds 2a-2e and 3a-3e
Substituted coumarin (a-e) (0.001 mol), substituted DHA (I, II) (0.001 mol) and anhydrous
potassium carbonate (0.0011 mol) were added in 15 mL DMF and stirred for 5 h. After the completion
of reaction, the mixture was poured in the water and extracted with ethyl acetate (50 mL×3). The

ACCEPTED MANUSCRIPT
organic layer was dried over anhydrous sodium sulfate and evaporated to dryness. The product was
purified through column chromatography.
4.1.2.1. 4-methyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-ethyoxyl)-2H-1-chromen-2-one (2a)
White solid; 61.7% yield; m.p. 64~65 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.48 (d, J = 8.7 Hz, 1H),
6.85 (dd, J = 8.7, 2.4 Hz, 1H), 6.82 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 1.6 Hz, 1H), 5.44 (s, 1H), 4.88 (d, J
= 3.4 Hz, 1H), 4.25-4.13 (m, 3H), 3.85-3.76 (m, 1H), 2.68-2.59 (m, 1H), 2.39 (d, J = 1.4 Hz, 3H), 2.34
(dd, J = 13.9, 3.8 Hz, 1H), 2.06-2.00 (m, 1H), 1.91-1.82 (m, 1H), 1.78-1.52 (m, 3H), 1.51-1.45 (m, 2H),
1.43 (s, 3H), 1.41-1.14 (m, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 7.3 Hz, 3H); 13C NMR (151 MHz,
CDCl3) δ (ppm): 161.90, 161.21, 155.21, 152.51, 125.58, 113.68, 112.56, 112.00, 104.10, 102.24,
101.59, 87.89, 81.04, 67.86, 66.29, 52.51, 44.34, 37.47, 36.38, 34.57, 30.83, 26.17, 24.70, 24.40, 20.34,
18.69, 12.93; ESI-HRMS [M+Na]+: (m/z) Calcd. for C27H34O8Na: 509.2146. Found: 509.2151.
4.1.2.2. 4-(trifluoromethyl)-7-(2-((10S)-Dihydroartemisin-10-oxy)-ethyoxyl)-2H-1-chromen-2-one (2b)
1
White solid; 46.3% yield; m.p. 60~61 ; H NMR (400 MHz, CDCl3) δ (ppm): 7.61 (dd, J = 9.0, 1.9
Hz, 1H), 6.91 (dd, J = 8.9, 2.6 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.61 (s, 1H), 5.42 (s, 1H), 4.88 (d, J =
3.4 Hz, 1H), 4.27-4.16 (m, 3H), 3.85-3.78 (m, 1H), 2.68-2.59 (m, 1H), 2.39-2.31 (m, 1H), 2.06-1.99 (m,
1H), 1.89-1.84 (m, 1H), 1.72-1.69 (m 1H), 1.68-1.64 (m, 1H), 1.52 (dd, J = 13.3, 3.2 Hz, 1H), 1.45 (d,
J = 5.7 Hz, 1H), 1.43 (s, 3H), 1.42-1.40 (m, 1H), 1.30-1.16 (m, 3H), 0.91 (d, J = 5.5 Hz, 3H), 0.88 (d, J
= 7.4 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 162.80, 159.31, 156.29, 126.37, 126.36, 113.66,
112.32, 112.29, 107.11, 104.15, 102.31, 102.10, 87.90, 81.00, 77.28, 77.07, 76.85, 68.09, 66.22, 52.49,
44.31, 37.53, 36.37, 34.55, 30.80, 26.15, 24.69, 24.43, 20.31, 12.91; ESI-HRMS [M+Na]+: (m/z) Calcd.
for C27H31F3O8Na: 563.1863. Found: 563.1862.
4.1.2.3. 3,4-dimethyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-ethyoxyl)-2H-1-chromen-2-one (2c)
1
White solid; 58.0% yield; m.p. 146~148 ; H NMR (400 MHz, CDCl3) δ (ppm): 7.48 (d, J = 8.8 Hz,
1H), 6.83 (dd, J = 8.8, 2.6 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 5.44 (s, 1H), 5.28 (s, 1H), 4.87 (d, J = 3.4
Hz, 1H), 4.20-4.13 (m, 3H), 3.84-3.75 (m, 1H), 2.66-2.58 (m, 1H), 2.35 (s, 3H), 2.35-2.30 (m, 1H),
2.16 (s, 3H), 2.06-1.98 (m, 1H), 1.89-1.82 (m, 1H), 1.75-1.64 (m, 2H), 1.53-1.46 (m, 2H), 1.43 (s, 3H),
1.40 (d, J = 4.2 Hz, 1H), 1.25-1.16 (m, 2H), 0.90 (d, J = 5.3 Hz, 3H), 0.88 (d, J = 7.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3) δ (ppm): 162.377, 160.807, 153.503, 146.194, 125.278, 118.999, 114.291,

ACCEPTED MANUSCRIPT
112.375, 104.100, 102.237, 101.318, 87.906, 81.052, 77.398, 77.080, 76.763, 67.780, 66.354, 52.527,
44.367, 37.458, 36.399, 34.583, 30.841, 26.165, 24.698, 24.402, 20.327, 15.071, 13.148, 12.925;
ESI-HRMS [M+Na]+: (m/z) Calcd. for C28H36O8Na: 523.2302. Found: 523.2300.
4.1.2.4. 3-chloro-4-methyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-ethyoxyl)-2H-1-chromen-2-one (2d)
1
White solid; 38.4% yield; m.p. 124~126 ; H NMR (400 MHz, CDCl3) δ (ppm): 7.52 (d, J = 8.9 Hz,
1H), 6.90 (dd, J = 8.9, 2.5 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 5.44 (s, 1H), 4.88 (d, J = 3.4 Hz, 1H),
4.23-4.15 (m, 3H), 3.85-3.78 (m, 1H), 2.68-2.58 (m, 1H), 2.54 (s, 3H), 2.40-2.32 (m, 1H), 2.06-2.01 (m,
1H), 1.92-1.82 (m, 1H), 1.79-1.64 (m, 3H), 1.52 (dd, J = 13.2, 3.2 Hz, 3H), 1.43 (s, 3H), 1.27-1.19 (m,
2H), 0.91 (d, J = 5.4 Hz, 3H), 0.88 (d, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 161.836,
157.371, 153.071, 147.926, 125.926, 117.847, 113.393, 113.278, 104.142, 102.291, 101.507, 87.914,
81.030, 77.389, 77.071, 76.754, 67.991, 66.281, 52.508, 44.337, 37.510, 36.384, 34.578, 30.823,
26.163, 24.709, 24.424, 20.341, 16.184, 12.924; ESI-HRMS [M+Na]+: (m/z) Calcd. for C27H33ClO8Na:
543.1756. Found: 543.1763.
4.1.2.5. Ethyl 7-(2-((10S)-Dihydroartemisin-10-oxy)-ethyoxyl)-2H-1-chromen-2-one-3-carboxylate (2e)
White solid; 62.5% yield; m.p. 73~76 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.48 (s, 1H), 7.49 (d, J
= 8.7 Hz, 1H), 6.86 (dd, J = 8.7, 2.4 Hz, 1H), 6.80 (d, J = 2.3 Hz, 1H), 5.41 (s, 1H), 4.86 (d, J = 3.5 Hz,
1H), 4.36 (q, J = 7.1 Hz, 2H), 4.23-4.14 (m, 3H), 3.84-3.76 (m, 1H), 2.66-2.56 (m, 1H), 2.39-2.28 (m,
1H), 2.04-1.98 (m, 1H), 1.89-1.80 (m, 1H), 1.73-1.62 (m, 2H), 1.54-1.42 (m, 3H), 1.40 (d, J = 9.3 Hz,
4H), 1.36 (d, J = 7.1 Hz, 3H), 1.78-1.22 (m, 2H), 0.89 (d, J = 5.6 Hz, 3H), 0.86 (d, J = 7.3 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) δ (ppm): 164.382, 163.406, 157.483, 157.069, 148.884, 130.768, 114.197,
113.854, 111.727, 104.146, 102.317, 101.070, 87.905, 81.004, 77.400, 77.082, 76.764, 68.167, 66.180,
61.713, 52.492, 44.311, 37.523, 36.367, 34.553, 30.803, 26.148, 24.699, 24.435, 20.333, 14.280,
12.906; ESI-HRMS [M+Na]+: (m/z) Calcd. for C29H36O10Na: 567.2201. Found: 567.2202.
4.1.2.6. 4-methyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-propoxy)-2H-1-chromen-2-one (3a)
White solid; 65.9% yield; m.p. 66~68 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.48 (d, J = 8.7 Hz, 1H),
6.86 (dd, J = 8.8, 2.3 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.12 (d, J = 1.2 Hz, 1H), 5.25 (s, 1H), 4.79 (d, J
= 3.4 Hz, 1H), 4.10 (t, J = 6.1 Hz, 3H), 3.53-3.47 (m, 1H), 2.62-2.57 (m, 1H), 2.38 (s, 3H), 2.33-2.27
(m, 1H), 2.11-2.05 (m, 2H), 2.01-1.96 (m, 1H), 1.81-1.42 (m, 5H), 1.42 (s, 3H), 1.39-1.21 (m, 2H),

ACCEPTED MANUSCRIPT
1.16-1.09 (m, 1H), 0.86 (d, J = 7.2 Hz, 3H), 0.82-0.78 (m, 1H), 0.74 (d, J = 5.0 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 162.07, 161.21, 155.29, 152.52, 125.52, 113.53, 112.70, 111.90, 104.03,
101.87, 101.19, 87.73, 80.94, 77.40, 77.08, 76.76, 65.18, 63.89, 52.38, 44.28, 37.19, 36.37, 34.45,
30.85, 28.87, 26.18, 24.55, 24.50, 20.18, 18.68, 13.00; ESI-HRMS [M+Na]+: (m/z) Calcd. for
C28H36O8Na: 523.2302. Found: 523.2300.
4.1.2.7. 4-(trifluoromethyl)-7-(2-((10S)-Dihydroartemisin-10-oxy)-propoxy)-2H-1-chromen-2-one (3b)
White solid; 50.6% yield; m.p. 102~104 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.62 (dd, J = 9.0, 2.0
Hz, 1H), 6.93 (dd, J = 9.0, 2.5 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.61 (s, 1H), 5.24 (s, 1H), 4.80 (d, J =
3.5 Hz, 1H), 4.15-4.10 (m, 3H), 3.54-3.49 (m, 1H), 2.66-2.57 (m, 1H), 2.36-2.31 (m, 1H), 2.13-2.07 (m,
2H), 2.02-1.96 (m, 1H), 1.82-1.44 (m, 5H), 1.43 (s, 3H), 1.42-1.09 (m, 4H), 0.87 (d, J = 7.4 Hz, 3H),
0.76 (d, J = 5.1 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 162.96, 159.26, 156.36, 126.32, 126.31,
113.81, 112.18, 112.14, 106.95, 104.04, 101.86, 101.67, 87.70, 80.87, 77.31, 77.10, 76.89, 65.47, 63.73,
53.47, 52.35, 44.24, 37.26, 36.32, 34.44, 30.81, 28.74, 26.13, 24.51, 20.17, 12.98; ESI-HRMS
[M+Na]+: (m/z) Calcd. for C28H33F3O8Na: 577.2020. Found: 577.2029.
4.1.2.8. 3, 4-dimethyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-propoxy)-2H-1-chromen-2-one (3c)
White solid; 60.3% yield; m.p. 68~70 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.48 (d, J = 8.9 Hz, 1H),
6.85 (dd, J = 8.9, 2.5 Hz, 1H), 6.78 (t, J = 2.5 Hz, 1H), 5.26 (s, 1H), 4.79 (d, J = 3.4 Hz, 1H), 4.14-4.07
(m, 3H), 3.53-3.47 (m, 1H), 2.61-2.58 (m, 1H), 2.35 (s, 3H), 2.32-2.27 (m, 1H), 2.17 (d, J = 2.0 Hz,
3H), 2.07 (t, J = 6.2 Hz, 2H), 2.01-1.96 (m, 1H), 1.81-1.57 (m, 3H), 1.46-1.43 (m, 1H), 1.42 (s, 3H),
1.38-1.08 (m, 3H), 0.86 (d, J = 7.3 Hz, 3H), 0.85-0.76 (m, 2H), 0.74 (d, J = 5.9 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ (ppm): 162.36, 160.96, 153.56, 146.21, 125.24, 118.87, 114.13, 112.48, 104.02,
101.87, 100.89, 87.73, 80.95, 77.40, 77.08, 76.76, 65.08, 63.95, 52.40, 44.30, 37.16, 36.37, 34.46,
30.85, 28.92, 26.18, 24.55, 24.49, 20.18, 15.07, 13.14, 13.00; ESI-HRMS [M+Na]+: (m/z) Calcd. for
C29H38O8Na: 537.2459. Found: 537.2463.
4.1.2.9. 3-chloro-4-methyl-7-(2-((10S)-Dihydroartemisin-10-oxy)-propoxy)-2H-1-chromen-2-one (3d)
White solid; 64.2% yield; m.p. 85~87 ; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.52 (d, J = 8.9 Hz, 1H),
6.91 (d, J = 8.9 Hz, 1H), 6.81 (s, 1H), 5.27 (d, J = 1.9 Hz, 1H), 4.82-4.77 (m, 1H), 4.12-4.09 (m, 3H),
3.55-3.47 (m, 1H), 2.62-2.59 (m, 1H), 2.55-2.52 (m, 3H), 2.36-2.28 (m, 1H), 2.12-2.04 (m, 2H),

ACCEPTED MANUSCRIPT
2.03-1.94 (m, 1H), 1.86-1.56 (m, 4H), 1.49-1.44 (m, 1H), 1.42 (d, J = 2.0 Hz, 3H), 1.39-1.32 (m, 1H),
1.31-1.22 (m, 1H), 1.20-1.09 (m, 1H), 0.94 (dd, J = 6.0, 1.8 Hz, 1H), 0.86 (dd, J = 7.5, 1.8 Hz, 3H),
0.77 (dd, J = 6.1, 2.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 162.013, 157.374, 153.152,
147.956, 125.898, 117.742, 113.405, 113.252, 104.077, 101.950, 101.134, 87.774, 80.952, 77.405,
77.088, 76.770, 65.400, 64.011, 52.406, 44.295, 37.283, 36.376, 34.489, 30.858, 28.925, 26.185,
24.599, 24.524, 20.233, 16.182, 13.019; ESI-HRMS [M+Na]+: (m/z) Calcd. for C28H35ClO8Na:
557.1913. Found: 557.1917.
4.1.2.10. Ethyl 7-(2-((10S)-Dihydroartemisin-10-oxy)-propoxy)-2H-1-chromen-2-one-3-carboxylate
(3e)
White solid; 66.2% yield; m.p. 82~84 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.50 (s, 1H), 7.50 (d, J
= 8.7 Hz, 1H), 6.89 (dd, J = 8.7, 2.4 Hz, 1H), 6.80 (d, J = 2.3 Hz, 1H), 5.28 (d, J = 6.8 Hz, 1H), 4.80 (d,
J = 3.5 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 4.17-4.06 (m, 3H), 3.55-3.50 (m, 1H), 2.66-2.57 (m, 1H),
2.38-2.27 (m, 1H), 2.13-2.07 (m, 2H), 2.02-1.97 (m, 1H), 1.86-1.57 (m, 4H), 1.51-1.46 (m, 1H), 1.42 (s,
3H), 1.39 (t, J = 7.1 Hz, 4H), 1.36-1.26 (m, 1H), 1.19-1.12 (m, 1H), 0.99-0.90 (m, 1H), 0.87 (d, J = 7.4
Hz, 3H), 0.79 (d, J = 6.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 164.54, 163.45, 157.60,
157.09, 148.93, 130.69, 114.11, 114.08, 111.61, 104.10, 101.97, 100.65, 87.78, 80.94, 77.34, 77.02,
76.71, 65.64, 63.95, 61.74, 52.40, 44.28, 37.36, 36.36, 34.48, 30.85, 28.87, 26.18, 24.62, 24.55, 20.30,
14.29, 13.01; ESI-HRMS [M+Na]+: (m/z) Calcd. for C30H38O10Na: 581.2357. Found: 581.2366.
4.1.3. General Procedure for the Synthesis of Compounds 4a-4d, 4f, 5a-5d and 5f
Substituted coumarin (Va-Vd, Vf, VIa-VId, VIf) (10 mmol) and compound IV (10 mmol) were added
in 20 mL CH2Cl2. A mixture of CuSO4·5H2O (12.5 mg) and sodium ascorbate (30 mg) in water (10 mL)
was added and the reaction mixture was stirred at room temperature for 8 h. The reaction mixture was
filtered, washed with water, dried over anhydrous sodium sulfate and evaporated to dryness. The
product was purified through column chromatography.
4.1.3.1.
4-methyl-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-ethoxy)-2H-1-chr
omen-2-one (4a)
White solid; 37.4% yield; m.p. 73~75 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.53 (s, 1H), 7.50 (d, J

ACCEPTED MANUSCRIPT
= 8.7 Hz, 1H), 6.82 (dd, J = 8.7, 2.5 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.15 (t, J = 1.4 Hz, 1H), 5.27 (s,
1H), 4.85-4.71 (m, 3H), 4.41 (t, J = 5.0 Hz, 2H), 4.16-4.07 (m, 1H), 3.70-3.64 (m, 1H), 3.07-2.95 (m,
2H), 2.60-2.56 (m, 1H), 2.39 (s, 3H), 2.32 (dd, J = 14.1, 4.0 Hz, 1H), 2.03-1.97 (m, 1H), 1.87-1.82 (m,
1H), 1.73-1.63 (m, 2H), 1.60-1.54 (m, 1H), 1.47-1.38 (m, 5H), 1.31-1.13 (m, 3H), 0.92 (d, J = 6.0 Hz,
3H), 0.80 (d, J = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 160.92, 160.64, 155.11, 152.27,
145.60, 125.83, 122.38, 114.40, 112.59, 112.07, 104.07, 101.87, 101.81, 87.87, 81.02, 77.36, 77.04,
76.72, 67.22, 66.89, 52.49, 49.33, 44.30, 37.36, 36.39, 34.60, 30.80, 26.59, 26.17, 24.67, 24.34, 20.37,
18.68, 12.98; ESI-HRMS [M+Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2647.
4.1.3.2.
4-(trifluoromethyl)-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-ethoxy)-
2H-1-chromen-2-one (4b)
1
White solid; 39.1% yield; m.p. 71~73 ; H NMR (600 MHz, CDCl3) δ (ppm): 7.64 (dd, J = 9.0, 1.8
Hz, 1H), 7.52 (s, 1H), 6.89 (dd, J = 9.0, 2.5 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 6.65 (s, 1H), 5.28 (s, 1H),
4.82-4.76 (m, 3H), 4.45 (t, J = 5.1 Hz, 2H), 4.12-4.08 (m, 1H), 3.71-3.67 (m, 1H), 3.10-2.95 (m, 2H),
2.61-2.58 (m, 1H), 2.37-2.32 (m, 1H), 2.05-1.97 (m, 1H), 1.88-1.83 (m, 1H), 1.75-1.62 (m, 2H),
1.59-1.55 (m, 1H), 1.48-1.35 (m, 5H), 1.30-1.17 (m, 2H), 0.93 (d, J = 6.2 Hz, 3H), 0.91-0.84 (m, 1H),
0.82 (d, J = 7.3 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 161.51, 159.00, 156.13, 145.63, 126.66,
126.65, 122.38, 113.20, 112.99, 107.80, 104.07, 102.24, 101.86, 87.87, 81.00, 77.27, 77.06, 76.85,
67.20, 67.07, 53.46, 52.47, 49.20, 44.28, 37.37, 36.38, 34.59, 30.78, 26.56, 26.16, 24.66, 24.34, 20.36,
12.99; ESI-HRMS [M+Na]+: (m/z) Calcd. for C31H36F3N3O8Na: 658.2347. Found: 658.2374.
4.1.3.3.
3,4-dimethyl-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-ethoxy)-2H-1-
chromen-2-one (4c)
White solid; 33.2% yield; m.p. 77~79 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.53 (s, 1H), 7.49 (d, J
= 8.9 Hz, 1H), 6.80 (dd, J = 8.8, 2.5 Hz, 1H), 6.76 (d, J = 2.6 Hz, 1H), 5.27 (s, 1H), 4.79-4.72 (m, 3H),
4.39 (t, J = 5.0 Hz, 2H), 4.13-4.07 (m, 1H), 3.70-3.64 (m, 1H), 3.09-2.94 (m, 2H), 2.61-2.51 (m, 1H),
2.36 (s, 3H), 2.34-2.29 (m, 1H), 2.17 (s, 3H), 2.02-1.97 (m, 1H), 1.88-1.82 (m, 1H), 1.75-1.62 (m, 2H),
1.59-1.53 (m, 1H), 1.49-1.42 (m, 1H), 1.40 (s, 3H), 1.39-1.36 (m, 1H), 1.30-1.12 (m, 3H), 0.92 (d, J =
6.0 Hz, 3H), 0.79 (d, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 162.124, 159.562,

ACCEPTED MANUSCRIPT
153.412, 145.941, 145.576, 125.538, 122.411, 119.675, 115.022, 111.900, 104.063, 101.880, 101.524,
87.874, 81.024, 77.365, 77.047, 76.729, 67.228, 66.834, 52.503, 49.386, 44.310, 37.359, 36.401,
34.609, 30.799, 26.584, 26.155, 24.663, 24.332, 20.357, 15.085, 13.202, 12.965; ESI-HRMS [M+Na]+:
(m/z) Calcd. for C32H41N3O8Na: 618.2786. Found: 618.2801.
4.1.3.4.
3-chloro-4-methyl-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-ethoxy)-
2H-1-chromen-2-one (4d)
White solid; 35.8%; m.p. 85~88 ; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.53 (d, J = 5.2 Hz, 1H), 7.52
(s, 1H), 6.86 (dd, J = 8.9, 2.5 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 5.27 (s, 1H), 4.80-4.75 (m, 3H), 4.42 (t,
J = 5.1 Hz, 2H), 4.11-4.08 (m, 1H), 3.69-3.65 (m, 1H), 3.06-2.96 (m, 2H), 2.54 (s, 3H), 2.36-2.30 (m,
1H), 2.04-1.97 (m, 1H), 1.88-1.82 (m, 1H), 1.76-1.63 (m, 3H), 1.58-1.55 (m, 1H), 1.48-1.42 (m, 1H),
1.40 (s, 3H), 1.40-1.35 (m, 1H), 1.26-1.15 (m, 3H), 0.92 (d, J = 6.2 Hz, 3H), 0.80 (d, J = 7.4 Hz, 3H);
13C NMR (150 MHz, CDCl3) δ (ppm): 160.481, 157.035, 152.840, 147.629, 145.513, 126.092, 122.321,
118.351, 113.991, 112.693, 103.989, 101.786, 101.631, 87.787, 80.929, 77.184, 76.972, 76.759, 67.131,
66.899, 52.395, 49.197, 44.204, 37.276, 36.302, 34.517, 30.703, 26.490, 26.074, 24.578, 24.251,
20.282, 16.117, 12.897; ESI-HRMS [M+Na]+: (m/z) Calcd. for C31H38ClN3O8Na: 638.2240. Found:
638.2260.
4.1.3.5. Methyl
7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-ethoxy)-2H-1-chromen-2-o
ne-3-carboxylate (4f)
White solid; 39.3% yield; m.p. 81~84 ; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.51 (s, 1H), 7.52 (d, J
= 2.1 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 6.85 (dd, J = 8.7, 2.3 Hz, 1H), 6.78 (d, J = 2.3 Hz, 1H), 5.26 (s,
1H), 4.78 (t, J = 4.9 Hz, 3H), 4.45 (t, J = 5.0 Hz, 2H), 4.11-4.06 (m, 1H), 3.91 (s, 3H), 3.69-3.63 (m,
1H), 3.07-2.92 (m, 2H), 2.60-2.54 (m, 1H), 2.37-2.28 (m, 1H), 2.03-1.95 (m, 1H), 1.89-1.61 (m, 4H),
1.59-1.52 (m, 1H), 1.49-1.41 (m, 1H), 1.40 (s, 3H), 1.25-1.16 (m, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.79 (d,
J = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ (ppm): 163.84, 163.14, 157.30, 156.78, 149.10, 145.59,
131.04, 122.37, 114.49, 113.46, 112.28, 104.05, 101.84, 101.24, 87.85, 80.99, 77.39, 77.07, 76.76,
67.18, 67.15, 52.77, 52.47, 49.17, 44.28, 37.35, 36.38, 34.59, 30.78, 26.55, 26.14, 24.65, 24.33, 20.35,
12.97; ESI-HRMS [M+Na]+: (m/z) Calcd. for C32H39N3O10Na: 648.2528. Found: 648.2555.

ACCEPTED MANUSCRIPT
4.1.3.6.
4-methyl-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-propoxy)-2H-1-ch
romen-2-one (5a)
White solid; 33.2% yield; m.p. 65~68 ; 1H NMR (600 MHz, CDCl3) δ (ppm) 7.47 (d, J = 8.8 Hz, 1H),
7.35 (s, 1H), 6.81 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 6.10 (d, J = 1.4 Hz, 1H), 5.23 (s,
1H), 4.75 (d, J = 3.4 Hz, 1H), 4.53 (t, J = 6.8 Hz, 2H), 4.08-4.04 (m, 1H), 4.01 (t, J = 5.8 Hz, 2H),
3.66-3.62 (m, 1H), 3.02-2.92 (m, 2H), 2.59-2.52 (m, 1H), 2.42-2.38 (m, 2H), 2.37 (d, J = 1.2 Hz, 3H),
2.34-2.28 (m, 1H), 2.02-1.95 (m, 1H), 1.85-1.80 (m, 1H), 1.71-1.59 (m, 2H), 1.55-1.51 (m, 1H),
1.45-1.34 (m, 5H), 1.28-1.11 (m, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.79 (d, J = 7.3 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ (ppm): 161.277, 160.991, 155.058, 152.395, 145.299, 125.650, 121.662, 113.847,
112.078, 112.045, 103.935, 101.757, 101.535, 87.734, 80.900, 77.254, 77.042, 76.829, 67.175, 64.623,
52.369, 46.633, 44.187, 37.236, 36.280, 34.493, 30.695, 29.668, 26.469, 26.051, 24.554, 24.222,
20.251, 18.576, 12.879; ESI-HRMS [M+Na]+: (m/z) Calcd. for C32H41N3O8Na: 618.2786. Found:
618.2805.
4.1.3.7.
4-(trifluoromethyl)-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-propoxy
)-2H-1-chromen-2-one (5b)
White solid; 37.8% yield; m.p. 70~73 ; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.64 (d, J = 8.4 Hz, 1H),
7.35 (s, 1H), 6.90 (dd, J = 9.0, 2.6 Hz, 1H), 6.85 (s, 1H), 6.63 (s, 1H), 5.26 (s, 1H), 4.79 (d, J = 3.5 Hz,
1H), 4.55 (d, J = 6.8 Hz, 2H), 4.12-4.02 (m, 4H), 2.63-2.57 (m, 1H), 2.44 (t, J = 6.2 Hz, 2H), 2.37-2.32
(m, 1H), 2.04-1.99 (m, 1H), 1.89-1.83 (m, 1H), 1.74-1.62 (m, 3H), 1.56 (dd, J = 13.2, 3.3 Hz, 1H),
1.52-1.44 (m, 1H), 1.41 (s, 3H), 1.41 (s, 2H), 1.30-1.17 (m, 3H), 0.93 (d, J = 6.1 Hz, 3H), 0.83 (d, J =
7.4 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ (ppm): 162.23, 159.20, 156.23, 145.50, 126.54, 121.67,
113.29, 112.61, 107.42, 104.08, 102.11, 101.88, 100.11, 91.10, 87.86, 81.01, 77.25, 77.04, 76.83, 67.27,
64.93, 52.47, 46.62, 44.29, 37.37, 36.38, 34.60, 30.79, 29.68, 26.58, 26.17, 24.68, 24.35, 20.36, 13.00;
ESI-HRMS [M+Na]+: (m/z) Calcd. for C32H38F3N3O8Na: 672.2503. Found: 672.2523.
4.1.3.8.
3,4-dimethyl-7-((2-(4-(2-((10S)-Dihydroartemisin-10-oxy)-ethyl)-1H-1,2,3-triazol-1-yl))-propoxy)-2H-
1-chromen-2-one (5c)