Design, synthesis and cytotoxicity of novel dihydroartemisinin-coumarin hybrids via click chemistry

Article abstract of DOI:10.3390/molecules21060758

In order to develop novel chemotherapeutic agents with potent anticancer activities, we designed four series of novel compounds employing hybridization strategy. Twenty novel dihydroartemisinin-coumarin hybrids, 10a-e, 11a-e, 12a-e, 13a-e, were synthesized via click chemistry in this study and their structures were characterized by HRMS and NMR. The cytotoxic activities were measured by MTT assay against three cancer cell lines (HCT-116, MDA-MB-231, and HT-29) under normoxic or anoxic conditions, respectively. The target compounds exhibited moderate activity with IC₅₀ values in the 0.05-125.40 μM range, and these compounds exhibited better activity against HT-29 cell line under anoxic condition. The cytotoxic activities of most compounds under anoxic condition displayed one- to 10-fold greater activity than under normoxic condition. Compounds 10a - e showed better selectivity against the HT-29 cell line than the other two cell lines. These results indicated that our design of CA IX inhibitors does correspond with its action mode to some degree and deserves further investigation.

Full text of DOI:10.3390/molecules21060758

molecules
Article
Design, Synthesis and Cytotoxicity of Novel
Dihydroartemisinin-Coumarin Hybrids via
Click Chemistry
Ye Tian 1, Zhen Liang 1, Hang Xu 1, Yanhua Mou 2 and Chun Guo 1,
*
1
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education,
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China;
tianye870918@126.com (Y.T.); llzz_666@126.com (Z.L.); xuhanglg2@126.com (H.X.)
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China;
mu_hua_jj@sina.com
2
*
Correspondence: chunguo@syphu.edu.cn; Tel.: +86-24-2398-6425
Academic Editor: Derek J. McPhee
Received: 16 May 2016; Accepted: 7 June 2016; Published: 10 June 2016
Abstract: In order to develop novel chemotherapeutic agents with potent anticancer activities,
we designed four series of novel compounds employing hybridization strategy. Twenty novel
dihydroartemisinin-coumarin hybrids, 10a–e, 11a–e, 12a–e, 13a–e, were synthesized via click
chemistry in this study and their structures were characterized by HRMS and NMR. The cytotoxic
activities were measured by MTT assay against three cancer cell lines (HCT-116, MDA-MB-231,
and HT-29) under normoxic or anoxic conditions, respectively. The target compounds exhibited
moderate activity with IC50 values in the 0.05–125.40 µM range, and these compounds exhibited better
activity against HT-29 cell line under anoxic condition. The cytotoxic activities of most compounds
under anoxic condition displayed one- to 10-fold greater activity than under normoxic condition.
Compounds 10a–e showed better selectivity against the HT-29 cell line than the other two cell lines.
These results indicated that our design of CA IX inhibitors does correspond with its action mode to
some degree and deserves further investigation.
Keywords: anticancer; artemisinin; coumarin; hybrid; carbonic anhydrase IX; cytotoxicity; anoxic
1. Introduction
Currently, the mortality rate associated with cancer shows an increasing tendency worldwide.
Chemotherapy is the most important therapeutic strategy against all types of cancer; however, the
available anticancer drugs usually cause adverse effects and lead to the development of resistance.
Therefore, there is a pressing need for novel chemotherapeutic agents with higher selectivity and more
potent anticancer activities [1,2].
Many natural products have been found to possess antineoplastic activity with minimal side
effects [
3
]. Artemisinin, a well-known anti-malarial agent, has been reported to possess a potent
]. However, the drawback, such as
and broad antitumor spectrum against human cancer cell lines [
4
drug resistance or unsatisfactory metabolism, constrains artemisinin to be employed as an antitumor
drug clinically. Numbers of artemisinin derivatives have been designed and synthesized to overcome
the imperfection mentioned above and some of them have shown excellent anticancer activities and
pharmacokinetic properties [5].
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc metalloenzymes [
6] that catalyze
a very simple, but important, reaction: the reversible hydration of carbon dioxide to bicarbonate
ion and proton, following a two-step catalytic mechanism [
7]. Out of the sixteen human CA (hCA)
Molecules 2016, 21, 758; doi:10.3390/molecules21060758
www.mdpi.com/journal/molecules

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isozymes that have been identified, CA IX is a tumor-associated isoform which over-expressed in a
broad range of solid tumor types, while in normal tissues, the expression was much more limited,
mainly in the glandular mucosa region in stomach [
IX is important in tumor growth and metastasis by increasing the tumor cell survival and invasion,
consequently, CA IX is considered to be a promising anti-cancer drug target [10 12] and developing
8,9]. In anoxia environments, the expression of CA
–
high selective inhibitors of CA IX may reduce side effects compared to classical anticancer agents [13].
In the last years, several approaches have been reported that the coumarins are truly CA IX-selective
inhibitors [14–17] which exhibit a very different binding mode [17].
Using the principle of hybridization, we designed and synthesized four new series of artemisinin
derivatives by means of click chemistry (Scheme 1), in which the coumarin moiety was attached to C-10
position of dihydroartemisinin to afford new hybrids. The details of the synthesis of target compounds
and evaluation of their activities against cancer cell lines are presented herein.
Scheme 1. The design of target compounds.
2. Results and Discussion
2.1. Chemistry
The synthetic approaches toward the target compounds 10a–e, 11a–e, 12a–e, and 13a–e were
illustrated in Scheme 2, proceeded in five steps, commencing from 1,3-dihydroxybenzene or
2,4-dihydroxybenzaldehyde, and DHA.
The coumarin derivatives 1a
1,3-dihydroxybenzene with corresponding substituted ethyl acetoacetate in H2SO4 afforded substituted
coumarins (1a ) [18], while 1e was synthesized by 2,4-dihydroxybenzaldehyde cyclized with
diethyl malonate [19]. The substituted coumarins (1a ) were then reacted with 1,2-dibromoethane,
1,3-dibromopropane or 3-bromo-1-propyne, respectively, to give 2a 3a 4a 7a 7b, and 9a
were obtained by dihydroartemisinin(DHA) reacted with 2-bromoethanol, 3-bromo-1-propanol, or
–e were synthesized according to the literature [18,19]. Reaction of
–
d
–e
–
e,
–
e
,
–
e,
,
3-bromo-1-propyne, respectively, as
β configuration (indicated by the small coupling constants between
9-H and 10-H in 1H NMR, J9,10 = 3–4 Hz [20]) by using boron trifluoride ethyl ether as a catalyst [21].
Further azidation of 2a–e, 3a–e, 7a, and 7b gave compounds 5a–e, 6a–e, 8a, and 8b [22].
Finally, the intermediate bearing azido group (5a
possessing terminal alkyne group (4a 9a), respectively, (5a
click chemistry [23] to get the target compounds.
–
e
,
6a
–
e
–
,
8a
,
8b) reacted with the intermediate
with 9a 8a 8b, with 4a ) via
–
e,
e,
6a
–
e
;
,
–e

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Scheme 2. Synthetic routes to target compounds 10a–e, 11a–e, 12a–e and 13a–e. Reagents and
conditions: (1) H2SO4/r.t. 12 h; (2) piperidine/EtOH/reflux 8–10 h; (3) 1,2-dibromoethane or
1,3-dibromoproane/Me2CO/K2CO3/56 ˝C 10 h; (4) 3-bromo-1-propyne/DMF/K2CO3/60 ˝C 4–5 h;
(5) NaN3/ DMF/70 ˝C 4–5 h; (6) 2-bromoethanol or 3-bromo-1-propanol/BF3
¨
Et2O/CH2Cl2/0 ˝C
5H2O/sodium ascorbate/DMF/r.t. 8–10 h. (9)
Et2O/CH2Cl2/0 ˝C 6–8 h; (10) CuSO4
5H2O/sodium ascorbate/DMF/r.t. 8–10 h.
6–8 h; (7) NaN3/DMF/70 ˝C 4–5 h; (8) CuSO4
2-propyn-1-ol/BF3
¨
¨
¨
2.2. Cytotoxicity
We focused our attention on the cytotoxicity of the four series of newly-synthesized compounds
11a 12a , and 13a ) under normoxic or anoxic conditions. The cytotoxic activities of target
(10a
–e
,
–
e,
–
e
–e
compounds were measured by MTT assay, working with MRC-5 (human fetal lung fibroblast cells,
normal lung cell), HCT-116 (human colorectal cancer cell line, do not express CA IX in response to
anoxia), MDA-MB-231 (human breast carcinoma cell line, CA IX negative), and HT-29 (human colon
carcinoma cell line, overexpresses high amounts of CA IX) [24,25], using doxorubicin as a positive
control, and acetazolamide (AAZ, standard carbonic anhydrase inhibitor) as reference drug.
The data was calculated by the Logit method and presented as IC50 values in Table 1. In 96 h MTT
assay results, cytotoxic activities for the twenty target compounds against four cell lines were found to
shown moderate activity compared to AAZ, which shows poor cytotoxicity (IC50 > 100
µM) with IC50
values in the 0.05–125.40 M range, but the cytotoxic activities were less than the positive control DOX.
µ

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Table 1. IC50 values of tested compounds against MRC-5, HCT-116, MDA-MB-231, and HT-29.
IC50 (µM)
MDA-MB-231
Anoxia Normoxia Value1
MRC-5
HCT-116
Normoxia Value 1
HT-29
Anoxia Normoxia Value 1
Compd.
Normoxia Anoxia
AAZ
DOX
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
13b
13c
>100
0.34
>100
0.15
11.94
8.31
6.89
5.30
28.80
6.02
9.33
16.56
6.00
28.14
18.27
24.35
12.07
5.86
35.67
9.65
2.75
>100
0.06
10.31
6.46
6.51
4.18
51.51
8.21
12.86
20.97
7.68
44.14
27.44
8.47
16.01
15.89
57.87
28.49
20.44
19.07
17.96
64.28
-
>100
0.37
>100
0.27
-
>100
0.05
1.47
2.18
1.82
0.05
8.69
1.22
5.06
23.20
1.34
12.03
10.64
2.46
2.87
3.63
20.75
6.40
5.31
2.80
4.68
7.09
>100
0.09
-
0.39
0.86
0.78
0.95
0.79
1.79
1.36
1.38
1.27
1.28
1.57
1.50
0.35
1.33
2.71
1.62
2.95
7.43
8.95
41.38
3.65
0.73
1.42
1.13
0.97
2.10
1.10
2.87
1.97
1.41
2.29
2.38
2.63
2.73
3.02
2.30
1.54
2.77
3.76
14.29
20.03
2.07
1.74
8.35
4.99
11.50
333.96
3.66
7.17
1.63
0.83
2.90
4.25
3.11
4.62
6.04
6.16
1.88
4.30
3.74
4.23
1.87
2.22
27.98
33.56
22.31
50.72
52.47
15.49
43.79
60.86
>100
38.74
37.40
18.51
33.84
62.33
69.55
26.17
21.40
27.78
31.47
27.24
36.74
24.68
33.19
29.19
91.21
17.82
36.90
39.75
22.51
50.67
47.74
14.67
33.16
43.51
65.06
36.06
26.57
4.17
52.02
28.01
32.34
61.41
>100
51.18
72.62
56.00
51.60
120.72
125.40
40.05
>100
>100
>100
>100
>100
59.60
72.50
53.68
12.29
10.88
20.97
17.70
31.83
8.78
8.23
19.28
3.90
51.09
33.05
11.34
17.32
22.37
38.96
27.51
19.83
11.86
8.76
2.13
0.43
17.60
13d
13e
3.62
25.94
15.74
1
The values stand for the activity promotion for each cancer cell line. Value = IC50(normoxia)/IC50(anoxia).
The line chart that depicted the IC50 values was shown in Figures 1–3. In order to gain a clearer
understanding of the activity data, we set the data “>100” in Table 1 as 100, for it would not affect the
result. Then we set out to evaluate the structure-activity relationship (SAR) against three cancer cell
lines (HCT-116, MDA-MB-231, and HT-29).
First, we found that for the four series of compounds: (i) in the coumarin ring, the 3-chloro,
4-methyl substituent exhibited better activity; (ii) on the contrary, the 3-ethoxycarbonyl group in the
3-position of coumarin showed poorer activity than others; and (iii) the 4-methyl or 4-trifluoromethyl
substituent showed no significant differences in the activity.
Then we analyzed the activity data against three cancer cell lines under normoxic or anoxic
condition separately compared with MRC-5 (normal cell line) under normoxic conditions (Figures 1–3).
In general, the target compounds exhibited better activity against HT-29 cell line under anoxic
conditions. The cytotoxicity of these compounds against HT-29 and MDA-MB-231 (CA IX negative [25])
had a general promotion (Figures 1 and 2) under anoxic condition, but the cytotoxicity to MDA-MB-231
exhibited a comparable activity to MRC-5 which mean poor selectivity to normal cells; while the
activity for HT-29 under anoxic conditions exhibited better selectivity and appeared less toxic to
normal cell MRC-5. For the HCT-116 cell line, which does not express CA IX in response to anoxia [24],
there was no significant change in cytotoxicity (Figure 3).
Moreover, the cytotoxic activities of most compounds under anoxic condition displayed one-
to 10-fold greater activity than under normoxic condition (Figure 4). For HT-29 cell line which over
expresses high amounts of CA IX, the first series 10a–e exhibited better selectivity compared to other
two cell lines.
Above all, these results indicated that our design of CA IX inhibitors does correspond with its
action mode, and deserved further investigation.

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Figure 1. The line chart of the IC50 values of tested compounds against MRC-5 and HT-29.
Figure 2. The line chart of the IC50 values of tested compounds against MRC-5 and MDA-MB-231.
Figure 3. The line chart of the IC50 values of tested compounds against MRC-5 and HCT-116.

Molecules 2016, 21, 758
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Figure 4. The cytotoxic activities of anoxia condition compared with normoxia codition.
3. Materials and Methods
3.1. Chemistry
Melting points were recorded on an X-4 microscope melting point apparatus (Beijing Tech
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.
3.1.1. General Procedure for the Synthesis of Compounds 1a–d
A solution of 1,3-dihydroxybenzene (11.0 g, 100 mmol) in substituted ethyl acetoacetate (100 mmol)
was added dropwise to stirring H2SO4 kept at 0 ˝C. After completion of the addition, the reaction
mixture was kept stirring for 12 h at room temperature and then poured onto ice-water. The crude
products were filtrated and recrystallized from ethanol [18].
7-Hydroxy-4-methyl-2H-chromen-2-one (1a): White solid; yield 73%. ESI-HRMS [M + H]+: (m/z) Calcd.
for C10H9O3: 177.0546. Found: 177.0556.
7-Hydroxy-4-(trifluoromethyl)-2H-chromen-2-one (1b): White solid; yield 52%. ESI-HRMS [M
(m/z) Calcd. for C10H6F3O3: 231.0275. Found: 231.0162.
´
H]´:
7-Hydroxy-3,4-dimethyl-2H-chromen-2-one (1c) White solid; yield 76%. ESI-HRMS [M + H]+: (m/z) Calcd.
for C11H11O3: 191.0703. Found: 191.0712.
3-Chloro-7-hydroxy-4-methyl-2H-chromen-2-one (1d): White solid; yield 74%. ESI-HRMS [M + H]+: (m/z)
Calcd. for C10H8ClO3: 211.0156. Found: 211.0166.

Molecules 2016, 21, 758
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3.1.2. The Synthesis of Compound 1e
2, 4-dihydroxybenzaldehyde (6.9 g, 0.05 mol), diethyl malonate (9.6 g, 0.06 mol) and piperidine
(0.2 g, 2.5 mmol) were refluxing in 150 mL ethanol for 10 h. After cooled to room temperature, the
crude products were filtrated and recrystallized from methanol [19].
Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (1e): Light yellow solid; yield 81%. ESI-HRMS [M +
Na]+: (m/z) Calcd. for C12H10O5Na: 257.0420. Found: 257.0434.
3.1.3. General procedure for the synthesis of compounds 2a–e, 3a–e
The compounds 2a
–e
and 3a
–
e
were synthesized according to the method from literature [26–28]
.
To a solution of compound 1a
–
e
(10.0 mmol) in acetone (30 mL), 1,2–dibromoethane or
1,3-dibromopropane (30.0 mmol) and potassium carbonate (12.3 mmol) were added. The reaction
˝
mixture was stirred at 56 C for 10 h, then poured into water, and extracted with ethyl acetate (30 mL ˆ 3)
.
The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
7-(2-Bromoethoxy)-4-methyl-2H-chromen-2-one (2a): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z)
Calcd. for C12H12BrO3: 282.9964. Found: 282.9981.
7-(2-Bromoethoxy)-4-(trifluoromethyl)-2H-chromen-2-one (2b): White solid; yield 53%. ESI-HRMS
[M + H]+: (m/z) Calcd. for C12H9BrF3O3: 336.9682. Found: 336.9712.
7-(2-Bromoethoxy)-3,4-dimethyl-2H-chromen-2-one (2c): White solid; yield 67%. ESI-HRMS [M + H]+:
(m/z) Calcd. for C13H14BrO3: 297.0121. Found: 297.0129.
7-(2-Bromoethoxy)-3-chloro-4-methyl-2H-chromen-2-one (2d): White solid; yield 70%. ESI-HRMS [M + H]+
(m/z) Calcd. for C12H11BrClO3: 316.9575. Found: 316.9589.
:
Ethyl 7-(2-bromoethoxy)-2-oxo-2H-chromene-3-carboxylate (2e): Yellow solid; yield 65%. ESI-HRMS
[M + H]+: (m/z) Calcd. for C14H14BrO5: 341.0019. Found: 341.0036.
7-(3-Bromopropoxy)-4-methyl-2H-chromen-2-one (3a): White solid; yield 62%. ESI-HRMS [M + H]+: (m/z)
Calcd. for C13H14BrO3: 297.0121. Found: 297.0132.
7-(3-Bromopropoxy)-4-(trifluoromethyl)-2H-chromen-2-one (3b): White solid; yield 62%. ESI-HRMS
[M + H]+: (m/z) Calcd. for C13H11BrF3O3: 350.9838. Found: 350.9851.
7-(3-Bromopropoxy)-3,4-dimethyl-2H-chromen-2-one (3c): White solid; yield 62%. ESI-HRMS [M + H]+:
(m/z) Calcd. for C14H16BrO3: 311.0277. Found: 311.0284.
7-(3-Bromopropoxy)-3-chloro-4-methyl-2H-chromen-2-one (3d): White solid; yield 62%. ESI-HRMS
[M + H]+: (m/z) Calcd. for C13H13BrClO3: 330.9731. Found: 330.9748.
Ethyl 7-(3-bromopropoxy)-2-oxo-2H-chromene-3-carboxylate (3e): Yellow solid; yield 62%. ESI-HRMS
[M + H]+: (m/z) Calcd. for C15H16BrO5: 355.0176. Found: 355.0183.
3.1.4. General Procedure for the Synthesis of Compounds 4a–e
To a solution of compound 1a (10.0 mmol) in DMF (20 mL), propargyl bromide (1.30 g, 10.9 mmol)
˝
and potassium carbonate (1.70 g, 12.3 mmol) were added. The reaction mixture was stirred at 60 C for
5 h, then was diluted with ethyl acetate (80 mL) and washed with water (100 mL 2). The organic layer
ˆ
was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude products 4a
were used without purification.

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The compounds 4b–e were synthesized by the same operation procedure of compound 4a.
4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4a): White solid; yield 91%; m.p. 129–132 ˝C.
ESI-HRMS [M + H]+: (m/z) Calcd. for C13H10O3: 215.0703. Found: 215.0715.
7-(Prop-2-yn-1-yloxy)-4-(trifluoromethyl)-2H-chromen-2-one (4b): White solid; yield 78%; m.p. 82–84 ˝C.
ESI-HRMS [M + H]+: (m/z) Calcd. for C13H8F3O3: 269.0420. Found: 269.0437.
˝
3,4-Dimethyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4c): White solid; yield 85%; m.p. 118–122 C.
ESI-HRMS [M + H]+: (m/z) Calcd. for C14H13O3: 229.0859. Found: 229.0875.
˝
3-Chloro-4-methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (4d):White solid; yield 82%; m.p. 131–134 C.
ESI-HRMS [M + H]+: (m/z) Calcd. for C13H10ClO3: 249.0313. Found: 249.0323.
˝
Ethyl 2-oxo-7-(prop-2-yn-1-yloxy)-2H-chromene-3-carboxylate (4e): White solid; yield 79%; m.p. 122–124 C.
ESI-HRMS [M + H]+: (m/z) Calcd. for C15H13O5: 273.0757. Found: 273.0764.
3.1.5. General Procedure for the Synthesis of Compounds 7a, 7b, 9a
To a solution of DHA (10.0 mmol) and 2-bromoethanol (10.0 mmol) in CH2Cl2 (30 mL), boron
fluoride ethyl ether (0.5 mL) was added dropwise at 0 ˝C. The reaction mixture was stirred at 0 ˝C
for 8 h, then washed with saturated NaHCO3 solution (20 mL
ˆ
3). The organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude products were purified by
silica gel column chromatography (PET/EtOAc = 10:1, v/v) to get the target compound 7a.
The compounds 7b and 9a were synthesized by the same operation procedure of compound 7a.
˝
1
1-Bromo-2-(10
(400 MHz, CDCl3)
β
-dihydroartemisinoxy)ethane (7a): White solid; yield 87%; m.p. 72–74 C. H-NMR
(ppm): 0.94 (d, J = 7.2 Hz, 3H), 0.96 (d, J = 6.4 Hz), 1.21–1.40 (m, 3H), 1.44
δ
(s, 3H), 1.45–1.56 (m, 2H), 1.63–1.68 (m, 1H), 1.73–1.79 (m, 1H), 1.84–1.96 (m, 2H), 2.01–2.07 (m, 1H),
2.33–2.41 (m, 1H), 2.61–2.69 (m, 1H), 3.52 (t, J = 5.2 Hz, 2H), 3.77–3.82 (m, 1H), 4.09–4.15 (m, 1H),
4.85 (d, J = 3.2 Hz, 1H), 5.49 (s, 1H). ESI-HRMS [M + Na]+: (m/z) Calcd. for C17H27BrO5Na: 413.0934.
Found: 413.0942.
˝
1
1-Bromo-3-(10
(400 MHz, CDCl3)
β
-dihydroartemisinoxy)propane (7b): White solid; yield 89%; m.p. 68–71 C. H-NMR
(ppm): 0.92 (d, J = 7.6 Hz, 3H), 0.96 (d, J = 6.0 Hz, 3H), 1.19–1.28 (m, 1H), 1.29–1.39
δ
(m, 1H), 1.44 (s, 3H), 1.45–1.56 (m, 2H), 1.60–1.69 (m, 1H), 1.72–1.78 (m, 2H), 1.85–1.92 (m, 1H), 2.01–2.14
(m, 3H), 2.37 (dt, J = 4.0 Hz, J = 13.6 Hz, 1H), 2.60–2.68 (m, 1H), 3.47–3.52 (m, 3H), 3.98–4.03 (m, 1H),
4.81 (d, J = 3.6 Hz, 1H), 5.43 (s, 1H). ESI-HRMS [M + Na]+: (m/z) Calcd. for C18H29BrO5Na: 427.1091.
Found: 427.1099.
1
3-(10
β
-Dihydroartemisinoxy)prop-1-yne (9a): White solid; yield 87%; m.p. 105–108 ˝C. H-NMR
(400 MHz, CDCl3)
δ
(ppm): 0.93 (d, J = 7.6 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 1.20–1.40 (m, 3H),
1.44 (s, 3H), 1.47–1.55 (m, 2H), 1.62–1.66 (m, 1H), 1.74–1.79 (m, 2H), 1.85–1.92 (m, 1H), 2.01–2.07 (m, 1H),
2.33–2.41 (m, 2H), 2.64–2.71 (m, 1H), 4.31 (d, J = 1.2 Hz, 2H), 4.98 (d, J = 3.2 Hz), 5.42 (s, 1H). ESI-HRMS
[M + Na]+: (m/z) Calcd. for C18H26O5Na: 345.1672. Found: 345.1683.
3.1.6. General Procedure for the Synthesis of Compounds 5a–e, 6a–e, 8a, 8b
These compounds were synthesized by the same operation in literature [20]. To a solution of
compound 2a (10˝mmol) in DMF (20 mL), sodium azide (30 mmol) was added, the reaction mixture
was stirred at 70 C for 4 h. the reaction mixture was poured into water and extracted with ethyl
acetate (40 mL
ˆ
3). The organic layer was dried over anhydrous Na2SO4, concentrated under reduced
pressure, and the crude products were used in next step immediately without further purification.

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3.1.7. General Procedure for the Synthesis of Compounds 10a–e, 11a–e, 12a–e, 13a–e
To a solution of compound 8a (10 mmol) and 4a (10 mmol) in CH2Cl2 (20 mL), 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 and the organic
layer was washed with water, then dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography (PET/EtOAc = 2:1, v/v)
to get the target compound 10a.
The compounds 10b–e, 11a–e, 12a–e, 13a–e were synthesized by the same operation procedure of
compound 10a (5a–e, 6a–e with 9a; 8a, 8b with 4a–e).
7-(2-(4-(10
β
-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-methyl-2H-chromen-2-one (10a):
(ppm): 0.87 (d, J = 7.6 Hz, 3H),
White solid; yield 31%; m.p. 83–85 ˝C. 1H-NMR (400 MHz, CDCl3)
δ
0.92 (d, J = 6.4 Hz, 3H), 1.19–1.28 (m, 3H), 1.44 (s, 3H), 1.45–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.70–1.75
(m, 1H), 1.80–1.90 (m, 1H), 1.99–2.04 (m, 1H), 2.33–2.37 (m, 1H), 2.40 (s, 3H), 2.60–2.68 (m, 1H), 4.43–4.46
(m, 2H), 4.76–4.71 (d, J = 12.4 Hz, 2H), 4.80–4.83 (m, 2H), 4.91 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 12.4 Hz,
1H), 5.42 (s, 1H), 6.16 (s, 1H), 6.79 (d, J = 2.4 Hz, 1H), 6.80–6.83 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.50
(d, J = 8.8 Hz, 1H), 7.69 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 160.90, 160.65, 155.12, 152.29,
145.40, 125.85, 123.49, 114.39, 112.56, 112.07, 104.13, 101.77, 101.58, 87.98, 81.06, 66.80, 61.61, 52.51, 49.41,
44.35, 37.36, 36.40, 34.56, 30.78, 26.14, 24.64, 24.42, 20.31, 18.65, 12.97. ESI-HRMS [M + Na]+: (m/z)
Calcd. for C30H37N3O8Na: 590.2473. Found: 590.2462.
7-(2-(4-(10
(
β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-trifluoromethyl-2H-chromen-2-one
˝
10b): White solid; yield 29%; m.p. 69–71 C. 1H-NMR (400 MHz, CDCl3)
δ (ppm): 0.88 (d, J = 7.2 Hz,
3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.30 (m, 3H), 1.44 (s, 3H), 1.55–1.59 (m, 1H), 1.62–1.80 (m, 3H),
1.84–2.05 (m, 3H), 2.33–2.41 (m, 1H), 2.61–2.68 (m, 1H), 4.48 (t, J = 4.8 Hz, 2H), 4.69 (d, J = 12.4 Hz,
1H), 4.84 (m, 2H), 4.91 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.66 (s, 1H), 6.86
(d, J = 2.8 Hz, 1H), 6.89 (dd, J = 2.4 Hz, J = 9.2 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.69 (s, H). 13C-NMR
(100 MHz, CDCl3)
δ (ppm): 161.42, 158.89, 156.07, 145.40, 130.83, 128.74, 126.57, 123.37, 113.10, 112.97,
112.93, 112.90, 112.86, 107.73, 104.06, 102.12, 101.52, 87.90, 80.97, 66.88, 65.47, 61.53, 52.40, 49.16,
44.24, 36.29, 34.46, 30.68, 26.05, 24.55, 24.33, 20.21, 12.88. ESI-HRMS [M + Na]+: (m/z) Calcd. for
C30H34F3N3O8Na: 644.2190. Found: 644.2183.
7-(2-(4-(10
(
β
-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-3,4-dimethyl-2H-chromen-2-one
(ppm): 0.87 (d, J = 7.2 Hz,
10c): White solid; yield 37%; m.p. 82–84 ˝C. 1H-NMR (400 MHz, CDCl3)
δ
3H), 0.92 (d, J = 6.0 Hz, 3H), 1.20–1.31 (m, 3H), 1.44 (s, 3H), 1.47–1.50 (m, 1H), 1.55–1.59 (m, 1H),
1.71–1.78 (m, 2H), 1.83–1.89 (m, 1H), 2.00–2.13 (m, 2H), 2.19 (s, 3H), 2.32–2.40 (m, 1H), 2.37 (s, 3H),
2.6–2.68 (m, 1H), 4.43 (t, J = 4.8 Hz, 2H), 4.68 (d, J = 12.8 Hz, 1H), 4.80–4.83 (m, 2H), 4.91 (d, J = 3.6 Hz,
1H), 4.95 (d, J = 12.8 Hz, 1H), 5.42 (s, 1H), 6.78 (s, 1H), 6.80 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.50
(d, J = 8.8 Hz, 1H), 7.70 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 162.13, 159.52, 153.41, 145.96,
145.41, 125.56, 123.49, 119.67, 115.01, 111.87, 104.14, 101.60, 101.43, 87.99, 81.09, 66.71, 61.65, 52.51, 49.45,
44.34, 37.37, 36.40, 34.56, 30.79, 26.18, 24.64, 24.43, 20.34, 15.18, 13.23, 13.00. ESI-HRMS [M + Na]+:
(m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2636.
3-Chloro-7-(2-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-4-methyl-2H-chromen-2-one
˝
10d): White solid; yield 39%; m.p. 75–77 C. 1H-NMR (400 MHz, CDCl3)
δ (ppm): 0.87 (d, J = 7.2 Hz,
(
3H), 0.92 (d, J = 6.4 Hz, 3H), 1.21–1.28 (m, 3H), 1.44 (s, 3H), 1.47–1.50 (m, 1H), 1.55–1.59 (m, 1H),
1.71–1.75 (m, 2H), 1.84–1.89 (m, 1H), 2.00–2.05 (m, 2H), 2.32–2.40 (m, 1H), 2.54 (s, 3H), 2.61–2.65
(m, 1H), 4.46 (m, 2H), 4.68–4.70 (d, J = 12.4 Hz, 2H), 4.81–4.84 (m, 2H), 4.90–4.91 (d, J = 3.6 Hz, 1H),
4.93–4.96 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.87 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H)
,
7.53 (d, J = 8.8 Hz, 1H), 7.70 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ
(ppm): 160.55, 157.08, 152.92, 147.70,

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145.39, 126.18, 123.51, 118.42, 114.07, 112.76, 104.14, 101.67, 101.55, 87.99, 81.06, 66.88, 61.57, 52.50, 49.36,
44.34, 37.37, 36.39, 34.56, 30.77, 26.16, 24.65, 24.43, 20.33, 16.19, 12.98. ESI-HRMS [M + Na]+: (m/z)
Calcd. for C30H36ClN3O8Na: 624.2083. Found: 624.2088.
Ethyl 2-oxo-7-(2-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy-2H-chromene-3-carboxylate
(
10e): White solid; yield 23%; m.p. 153–155 ˝C. 1H-NMR (400 MHz, CDCl3)
δ (ppm): 0.87
(d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 1.19–1.30 (m, 3H), 1.40 (t, J = 6.8 Hz, 3H), 1.44 (s, 3H),
1.46–1.60 (m, 3H), 1.70–1.78 (m, 2H), 1.83–1.90 (m, 1H), 2.00–2.06 (m, 1H), 2.33–2.41 (m, 1H), 2.61–2.68
(m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.48 (t, J = 4.8 Hz, 2H), 4.69 (d, J = 12.8 Hz, 1H), 4.84 (m, 2H), 4.91
(d, J = 3.2 Hz, 1H), 4.95 (d, J = 12.4 Hz, 1H), 5.42 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 2.4 Hz,
J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm):
163.21, 162.98, 157.26, 156.79, 148.62, 145.44, 130.98, 123.49, 114.93, 113.35, 112.33, 104.15, 101.58, 101.21,
87.99, 81.07, 67.01, 61.82, 61.58, 52.49, 49.25, 44.32, 37.38, 36.38, 34.55, 30.77, 26.17, 24.65, 24.43, 20.34,
14.27, 12.99. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H39N3O10Na: 648.2528. Found: 648.2525.
7-(3-(4-(10
White solid; yield 32%; m.p. 69–70 C. 1H-NMR (400 MHz, CDCl3)
(d, J = 6.0 Hz, 3H), 1.21–1.28 (m, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 1H), 1.55–1.59 (m, 1H), 1.65–1.76 (m, 3H),
1.84–1.89 (m, 1H), 1.99–2.05 (m, 2H), 2.41 (s, 3H), 2.46 (m, 2H), 2.61–2.64 (m, 1H), 4.04 (t, J = 5.6 Hz, 2H)
β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-methyl-2H-chromen-2-one (11a):
˝
δ
(ppm): 0.84 (d, J = 7.6 Hz, 3H), 0.92
,
4.60–4.63 (m, 2H), 4.67 (d, J = 12.8 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H), 4.93 (d, J = 12.4 Hz, 1H), 5.39 (s, 1H),
6.16 (s, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 3.2 Hz, J = 8.8 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H), 7.51
(d, J = 5.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.35, 161.11, 155.21, 152.43, 145.23, 125.75,
122.83, 114.02, 112.29, 112.11, 104.13, 101.71, 101.66, 87.96, 81.08, 64.69, 61.72, 52.49, 46.87, 44.34, 37.38,
36.40, 34.55, 30.79, 29.70, 26.16, 24.67, 24.42, 20.32, 18.69, 12.96. ESI-HRMS [M + Na]+: (m/z) Calcd. for
C31H39N3O8Na: 604.2629. Found: 604.2618.
7-(3-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-trifluoromethyl-2H-chromen-2-one
˝
11b): White solid; yield 19%; m.p. 67–69 C. 1H-NMR (400 MHz, CDCl3)
δ (ppm): 0.85 (d, J = 7.2 Hz,
(
3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.29 (m, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 2H), 1.55–1.59 (m, 1H),
1.66–1.76 (m, 2H), 1.84–1.89 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.48 (m, 2H), 2.59–2.67
(m, 1H), 4.07 (t, J = 6.0 Hz, 2H), 4.60–4.64 (m, 2H), 4.68 (d, J = 12.4 Hz, 1H), 4.90 (d, J = 3.2 Hz, 1H), 4.93
(d, J = 12.4 Hz, 1H), 5.39 (s, 1H), 6.64 (s, 1H), 6.85 (d, J = 2.8 Hz, 1H), 6.91 (dd, J = 2.4 Hz, J = 9.2 Hz,
1H), 7.52 (s, 1H), 7.64 (dd, J = 1.6 Hz, J = 8.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 162.23,
159.19, 156.25, 145.29, 126.57, 122.807, 120.19, 113.27, 112.66, 112.60, 107.44, 104.14, 102.10, 101.71, 87.96,
81.07, 64.95, 61.70, 52.48, 46.76, 44.33, 37.38, 36.39, 34.54, 30.78, 29.60, 26.15, 24.67, 24.41, 20.31, 12.95.
ESI-HRMS [M + Na]+: (m/z) Calcd. for C31H36F3N3O8Na: 658.2347. Found: 658.2350.
7-(3-(4-(10β-Dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-3,4-dimethyl-2H-chromen-2-one
(
11c): White solid; yield 35%; m.p. 83–85 ˝C. 1H-NMR (400 MHz, CDCl3)
δ (ppm): 0.85 (d, J = 5.2 Hz,
3H), 0.87 (d, J = 6.0 Hz, 3H), 0.90–0.99 (m, 1H), 1.15–1.27 (m, 2H), 1.30–1.31 (m, 1H), 1.55 (s, 3H),
1.58–1.72 (m, 3H), 1.74–1.79 (m, 1H), 1.82–1.89 (m, 1H), 2.19 (s, 3H), 2.38 (s, 3H), 2.40–2.48 (m, 3H),
3.57 (m, 1H), 4.00–4.03 (m, 2H), 4.59–4.65 (m, 3H), 4.87 (d, J = 4.4 Hz, 1H), 4.90 (d, J = 8.8 Hz, 1H),
5.26 (s, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 2.8 Hz, J = 8.8 Hz, 1H), 7.49 (s, 1H), 7.51 (d, J = 9.2
Hz, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 162.30, 160.24, 153.48, 146.11, 145.32, 125.44, 122.75,
119.31, 114.61, 111.96, 107.99, 101.32, 99.46, 93.80, 84.08, 77.26, 69.56, 64.53, 61.81, 46.90, 42.41, 40.63,
34.82, 34.70, 30.32, 29.72, 24.98, 20.99, 18.82, 15.10, 13.19, 12.26. ESI-HRMS [M + Na]+: (m/z) Calcd. for
C32H41N3O8Na: 618.2786. Found: 618.2784.
3-Chloro-7-(3-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-4-methyl-2H-chromen-2-one
1
(
11d): White solid; yield 34%; m.p. 150–152 ˝C. H-NMR (400 MHz, CDCl3)
δ
(ppm): 0.85
(d, J = 7.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 1.19–1.28 (m, 3H), 1.44 (s, 3H), 1.45–1.49 (m, 1H), 1.55–1.59

Molecules 2016, 21, 758
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(m, 1H), 1.65–1.81 (m, 3H), 1.83–1.90 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.47 (m, 2H), 2.56
(s, 3H), 2.59–2.66 (m, 1H), 4.05 (m, 2H), 4.61 (m, 2H), 4.67 (d, J = 12.8 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H),
4.92 (d, J = 12.8 Hz, 1H), 5.39 (s, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.88 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52
(s, 1H), 7.54 (d, J = 8.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.26, 157.26, 153.01, 147.84,
145.22, 126.09, 122.84, 118.10, 113.68, 112.81, 104.13, 101.67, 101.56, 87.96, 81.07, 64.84, 61.67, 52.48, 46.86,
44.33, 37.38, 36.39, 34.55, 30.78, 29.68, 26.16, 24.67, 24.42, 20.33, 16.21, 12.97. ESI-HRMS [M + Na]+:
(m/z) Calcd. for C31H38ClN3O8Na: 638.2240. Found: 638.2243.
Ethyl 2-oxo-7-(3-(4-(10β-dihydroartemisininoxy)methyl)-1H-1,2,3-triazol-1-yl)propoxy-2H-chromene-3-carboxylate
˝
(11e): White solid; yield 29%; m.p. 124–126 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.85 (d, J = 7.2 Hz,
3H), 0.92 (d, J = 6.0 Hz, 3H), 1.20–1.32 (m, 3H), 1.41 (t, J = 7.2 Hz, 3H), 1.44 (s, 3H), 1.46–1.50 (m, 1H),
1.55–1.59 (m, 1H), 1.64–1.81 (m, 3H), 1.83–1.90 (m, 1H), 1.99–2.05 (m, 1H), 2.32–2.40 (m, 1H), 2.48
(m, 2H), 2.59–2.67 (m, 1H), 4.08 (t, J = 6.0 Hz, 2H), 4.41 (q, J = 7.2 Hz, 2H), 4.61 (td, J = 2.0 Hz, J = 6.8 Hz
,
2H), 4.68 (d, J = 12.4 Hz, 1H), 4.90 (d, J = 3.2 Hz, 1H), 4.93 (d, J = 12.8 Hz, 1H), 5.39 (s, 1H), 6.79
(d, J = 2.0 Hz, 1H), 6.87 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 6.4 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H),
8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 163.78, 163.31, 157.37, 156.96, 148.80, 145.26, 130.91,
122.83, 114.48, 113.45, 111.97, 104.13, 101.69, 101.12, 87.95, 81.07, 65.11, 61.76, 61.68, 52.47, 46.82, 44.32,
37.37, 36.381, 34.54, 30.78, 29.62, 26.16, 24.66, 24.41, 20.32, 14.29, 12.97. ESI-HRMS [M + Na]+: (m/z)
Calcd. for C33H41N3O10Na: 662.2684. Found: 662.2679.
7-(1-(2-(10
β
-Dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (12a):
(ppm): 0.78 (d, J = 7.2 Hz, 3H),
White solid; yield 32%; m.p. 79–81 ˝C. 1H-NMR (400 MHz, CDCl3)
δ
0.92 (d, J = 5.6 Hz, 3H), 1.16–1.30 (m, 3H), 1.36–1.39 (m, 1H), 1.42 (s, 3H), 1.45–1.52 (m, 2H), 1.56–1.60
(m, 1H), 1.64–1.74 (m, 1H), 1.84–1.88 (m, 1H), 1.99–2.05 (m, 1H), 2.31–2.39 (m, 1H), 2.41 (s, 3H), 2.57–2.64
(m, 1H), 3.79–3.84 (m, 1H), 4.27–4.32 (m, 1H), 4.51–4.57 (m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz,
1H), 5.15 (s, 1H), 5.27 (s, 2H), 6.16 (s, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.95 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H),
7.52 (d, J = 8.8 Hz, 1H), 7.72 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.13, 155.15, 152.42, 143.10,
125.73, 123.49, 114.09, 112.33, 112.31, 104.21, 102.18, 102.13, 87.87, 80.80, 77.24, 66.40, 62.33, 52.38, 50.50,
44.04, 37.34, 36.31, 34.45, 30.59, 26.10, 24.61, 24.34, 20.36, 18.69, 12.81. ESI-HRMS [M + Na]+: (m/z)
Calcd. for C30H37N3O8Na: 590.2473. Found: 590.2471.
7-(1-(2-(10β-Dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-trifluoromethyl-2H-chromen-2-one
˝
(12b): White solid; yield 24%; m.p. 77–79 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H),
0.92 (d, J = 5.6 Hz, 3H), 1.16–1.31 (m, 3H), 1.42 (s, 3H), 1.48–1.76 (m, 4H), 1.84–1.88 (m, 1H), 2.00–2.05
(m, 1H), 2.31–2.34 (m, 1H), 2.59–2.62 (m, 1H), 3.80–3.85 (m, 1H), 4.25–4.32 (m, 1H), 4.51–4.70 (m, 2H),
4.76 (d, J = 2.7 Hz, 1H), 5.16 (s, 1H), 5.29 (s, 1H), 6.64 (s, 1H), 7.01–7.03 (m, 2H), 7.64 (d, J = 9.2 Hz, 1H),
7.73 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.928, 159.095, 156.088, 142.555, 126.430, 124.988,
123.535, 113.398, 112.573, 107.385, 104.107, 102.503, 102.073, 87.770, 80.677, 77.191, 76.979, 76.767, 66.282,
62.356, 52.277, 50.429, 43.935, 37.235, 36.202, 34.358, 30.480, 25.974, 24.517, 24.229, 20.241, 19.083, 12.691.
ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H34F3N3O8Na: 644.2190. Found: 644.2187.
7-(1-(2-(10
β
-Dihydroartemisininoxy)ethy˝l)-1H-1,2,3-triazol-4-yl)methoxy-3,4-dimethyl-2H-chromen-2-one
(12c): White solid; yield 36%; m.p. 84–86 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H),
0.91 (d, J = 4.4 Hz, 3H), 0.97–1.29 (m, 3H), 1.42 (s, 3H), 1.45–1.55 (m, 2H), 1.55–2.05 (m, 5H), 2.18
(s, 3H), 2.30–2.34 (m, 1H), 2.37 (s, 3H), 2.57–2.61 (m, 1H), 3.79–3.84 (m, 1H), 4.27–4.32 (m, 1H), 4.51–4.57
(m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz, 1H), 5.15 (m, 1H), 5.25 (m, 2H), 6.90–6.95 (m, 2H), 7.52
(dd, J = 3.6 Hz, J = 8.8 Hz, 1H), 7.72 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 162.26, 160.01, 153.39,
146.12, 143.21, 125.44, 123.52, 119.29, 114.66, 112.10, 104.16, 102.13, 101.76, 87.84, 80.78, 66.38, 62.25,
52.37, 50.47, 44.03, 37.29, 36.30, 34.43, 30.58, 26.07, 24.58, 24.31, 20.34, 15.08, 13.17, 12.80. ESI-HRMS
[M + Na]+: (m/z) Calcd. for C31H39N3O8Na: 604.2629. Found: 604.2645.

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3-Chloro-7-(1-(2-(10β-dihydroartemisininox˝y)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one
(12d): White solid; yield 33%; m.p. 76–78 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.2 Hz, 3H),
0.92 (d, J = 5.6 Hz, 3H), 1.16–1.32 (m, 3H), 1.32–1.42 (m, 2H), 1.42 (s, 3H), 1.44–1.69 (m, 5H), 1.84–1.88
(m, 1H), 1.99–2.15 (m, 2H), 2.31–2.39 (m, 1H), 2.58–2.62 (m, 1H), 3.79–3.85 (m, 1H), 4.27–4.32
(m, 1H), 4.51–4.58 (m, 1H), 4.63–4.69 (m, 1H), 4.76 (d, J = 3.2 Hz, 1H), 5.15 (s, 1H), 5.27 (s, 2H), 6.94
(d, J = 2.4 Hz, 1H), 7.00 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.73 (s, 1H). 13C-NMR
(100 MHz, CDCl3)
δ (ppm): 161.04, 157.29, 152.97, 147.83, 142.90, 126.07, 123.56, 118.17, 113.78, 113.02,
104.21, 102.18, 102.06, 87.87, 80.79, 66.40, 62.38, 52.38, 50.51, 44.03, 37.33, 36.31, 34.46, 30.59, 26.09,
24.61, 24.34, 20.36, 16.20, 12.82. ESI-HRMS [M + Na]+: (m/z) Calcd. for C30H36ClN3O8Na: 624.2083.
Found: 624.2111.
Ethyl 2-oxo-7-(1-(2-(10β-dihydroartemisininoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy-2H-chromene-3-carboxylate
˝
(12e): White solid; yield 26%; m.p. 71–73 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.78 (d, J = 7.6 Hz, 3H),
0.92 (d, J = 5.6 Hz, 3H), 1.16–1.28 (m, 3H), 1.39–1.42 (m, 8H), 1.47–1.52 (m, 1H), 1.55–1.60 (m, 1H),
1.65–1.69 (m, 1H), 1.84–1.89 (m, 1H), 1.99–2.05 (m, 1H), 2.31–2.39 (m, 1H), 2.57–2.64 (m, 1H), 3.80–3.85
(m, 1H), 4.27–4.32 (m, 1H), 4.40 (q, J = 3.2 Hz, 2H), 4.52–4.58 (m, 1H), 4.64–4.70 (m, 1H), 4.76
(d, J = 3.6 Hz, 1H), 5.15 (s, 1H), 5.29 (s, 2H), 6.94 (d, J = 2.0 Hz, 1H), 6.98 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H),
7.52 (d, J = 8.8 Hz, 1H), 7.74 (s, 1H), 8.51 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 163.55, 163.36,
157.34, 156.98, 148.79, 142.60, 130.88, 123.64, 114.63, 113.63, 112.08, 104.23, 102.19, 101.62, 87.88, 80.78,
66.40, 62.52, 61.79, 52.37, 50.54, 44.02, 37.35, 36.31, 34.46, 30.58, 26.10, 24.62, 24.34, 20.36, 14.29, 12.82.
ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H39N3O10Na: 648.2528. Found: 648.2530.
7-(1-(3-(10
β
-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one (13a):
(ppm): 0.93 (d, J = 7.2 Hz, 3H),
White solid; yield 39%; m.p. 75–77 ˝C. 1H-NMR (400 MHz, CDCl3)
δ
0.96 (d, J = 6.4 Hz, 3H), 1.22–1.33 (m, 3H), 1.43 (s, 3H), 1.46–1.55 (m, 2H), 1.63–1.69 (m, 1H), 1.76–1.78
(m, 1H), 1.86–1.93 (m, 1H), 2.02–2.13 (m, 2H), 2.17–2.25 (m, 2H), 2.33–2.38 (m, 1H), 2.41 (s, 3H), 2.62–2.69
(m, 1H), 3.36–3.42 (m, 1H), 3.87–3.93 (m, 1H), 4.44–4.52 (m, 2H),4.77 (d, J = 3.6 Hz, 1H), 5.27 (s, 2H),
5.40 (s, 1H), 6.15 (s, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz,
1H), 7.66 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.18, 161.13, 155.12, 152.50, 143.05, 125.74,
123.05, 114.07, 112.39, 112.29, 104.19, 102.19, 102.13, 87.94, 80.97, 64.65, 62.311, 52.50, 47.65, 44.27, 37.46,
36.37, 34.56, 30.81, 30.44, 26.15, 24.66, 24.55, 20.36, 18.70, 13.09. ESI-HRMS [M + Na]+: (m/z) Calcd. for
C31H39N3O8Na: 604.2629. Found: 604.2674.
7-(1-(3-(10β-Dihydroartemisininoxy)propyl)˝-1H-1,2,3-triazol-4-yl)methoxy-4-trifluoromethyl-2H-chromen-2-one
(13b): White solid; yield 25%; m.p. 69–71 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.94 (d, J = 7.2 Hz, 3H),
0.97 (d, J = 6.0 Hz, 3H), 1.22–1.39 (m, 3H), 1.43 (s, 3H), 1.46–1.55 (m, 2H), 1.64–1.78 (m, 3H), 1.87–1.92
(m, 1H), 2.02–2.06 (m, 1H), 2.16–2.28 (m, 2H), 2.34–2.41 (m, 1H), 2.63–2.70 (m, 1H), 3.37–3.43 (m, 1H),
3.87–3.93 (m, 1H), 4.45–4.54 (m, 2H), 4.78 (d, J = 3.2 Hz, 1H), 5.29 (s, 2H), 5.40 (s, 1H), 6.64 (s, 1H),
7.02–7.03 (m, 2H), 7.64–7.67 (m, 2H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 162.03, 159.26, 156.18,
142.64, 141.33, 126.52, 123.13, 113.55, 112.67, 112.62, 107.50, 104.21, 102.61, 102.21, 87.95, 80.96, 64.66,
62.45, 52.50, 47.68, 44.27, 37.47, 36.37, 34.56, 30.81, 30.44, 26.14, 24.66, 24.55, 20.36, 13.09. ESI-HRMS
[M + Na]+: (m/z) Calcd. for C31H36F3N3O8Na: 658.2347. Found: 658.2345.
7-(1-(3-(10
β
-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-3,4-dimethyl-2H-chromen-2-one
(13c): White solid; yield 37%; m.p. 68–70 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H),
0.96 (d, J = 6.4 Hz, 3H), 1.22–1.40 (m, 3H), 1.43 (s, 3H), 1.46–1.52 (m, 2H), 1.54–1.78 (m, 3H), 1.86–1.95
(m, 1H), 2.01–2.17 (m, 2H), 2.19 (s, 3H), 2.22–2.35 (m, 2H), 2.38 (s, 3H), 2.60–2.69 (m, 1H), 3.36–3.41
(m, 1H), 3.87–3.93 (m, 1H), 4.42–4.53 (m, 2H), 4.77 (d, J = 3.6 Hz, 1H), 5.26 (s, 2H), 5.40 (s, 1H), 6.91
(d, J = 2.4 Hz, 1H), 6.94 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H). 13C-NMR
˝
(100 MHz, CDCl3)
δ (ppm): 162.33, 160.02, 153.40, 146.16, 143.22, 125.44, 122.99, 119.31, 114.68, 112.18,
104.18, 102.18, 101.82, 87.93, 80.97, 64.65, 62.28, 52.51, 47.62, 44.27, 37.45, 36.37, 34.56, 30.82, 30.44,

Molecules 2016, 21, 758
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26.15, 24.66, 24.55, 20.36, 15.10, 13.19, 13.09. ESI-HRMS [M + Na]+: (m/z) Calcd. for C32H41N3O8Na:
618.2786. Found: 618.2785.
3-Chloro-7-(1-(3-(10β-Dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-4-methyl-2H-chromen-2-one
˝
(13d): White solid; yield 38%; m.p. 71–73 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz, 3H),
0.97 (d, J = 6.4 Hz, 3H), 1.22–1.29 (m, 2H), 1.32–1.38 (m, 1H), 1.43 (s, 3H), 1.46–1.51 (m, 2H), 1.58–1.82
(m, 3H), 1.87–1.92 (m, 1H), 2.02–2.07 (m, 1H), 2.17–2.25 (m, 2H), 2.33–2.41 (m, 1H), 2.56 (s, 3H),
2.62–2.70 (m, 1H), 3.36–3.42 (m, 1H), 3.87–3.92 (m, 1H), 4.41–4.56 (m, 2H), 4.77 (d, J = 3.2 Hz, 1H), 5.27
(s, 2H), 5.40 (s, 1H), 6.95 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz,
1H), 7.66 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ (ppm): 161.04, 157.34, 152.98, 147.86, 142.90, 126.07,
123.06, 118.17, 113.80, 113.08, 104.21, 102.21, 102.09, 87.95, 80.97, 64.65, 62.40, 52.51, 47.65, 44.28, 37.47,
36.38, 34.57, 30.82, 30.45, 26.16, 24.67, 24.56, 20.37, 16.21, 13.10. ESI-HRMS [M + Na]+: (m/z) Calcd. for
C31H38ClN3O8Na: 638.2240. Found: 638.2240.
Ethyl 2-oxo-7-(1-(3-(10β-dihydroartemisininoxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy-2H-chromene-3-carboxylate
˝
(13e): White solid; yield 30%; m.p. 79–81 C. 1H-NMR (400 MHz, CDCl3) δ (ppm): 0.93 (d, J = 7.6 Hz,
3H), 0.96 (d, J = 5.6 Hz, 3H), 1.22–1.32 (m, 2H), 1.39–1.43 (m, 6H), 1.47–1.56 (m, 2H), 1.61–1.73 (m, 2H),
1.76–1.78 (m, 1H), 1.86–1.93 (m, 1H), 1.97–2.07 (m, 2H), 2.18–2.26 (m, 2H), 2.33–2.41 (m, 1H), 2.62–2.68
(m, 1H), 3.37–3.43 (m, 1H), 3.87–3.92 (m, 1H), 4.40 (q, 2H), 4.45–4.55 (m, 2H), 4.78 (d, J = 3.2 Hz, 1H),
5.29 (s, 2H), 5.40 (s, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.99 (dd, J = 2.4 Hz, J = 8.8 Hz, 1H), 7.53 (d, J = 8.8 Hz
,
1H), 7.68 (s, 1H), 8.50 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δ
(ppm): 163.59, 163.40, 157.32, 157.03,
148.84, 146.18, 130.84, 114.59, 113.84, 112.13, 104.19, 102.13, 101.78, 87.97, 81.01, 80.63, 64.64, 62.26, 61.77,
52.54, 48.49, 44.34, 37.47, 36.39, 34.63, 30.85, 30.14, 29.71, 26.17, 24.68, 20.38, 14.29, 13.16. ESI-HRMS
[M + Na]+: (m/z) Calcd. for C33H41N3O10Na: 662.2684. Found: 662.2680.
3.2. In Vitro Cytotoxicity Study (MTT Assay)
All of the newly-synthesized compounds were screened for in vitro cytotoxicity by MTT assay.
The exponentially-growing MRC-5, HCT-116, MDA-MB-231, and HT-29 cells were allowed to grow
until 80% confluency, the culture medium was discarded, digested with trypsin, separated centrifugally,
then fresh culture medium was added and blown gently into single-cell suspension. Then, the
cell suspension was seeded into each well of 96-well microculture plates at a concentration of
1.5 ˆ 103–3 ˆ 103 cells/well.
˝
After incubation for 24 h at 37 C, the four lines of cells were treated with varying concentrations
(100, 50, 25, 12.5, and 6.25
with 50 L of 2 mg/mL MTT solution for 4 h in a humidified incubator. The supernatant was
discarded, and the media were then replaced with 200 L of dimethyl sulfoxide to dissolve the purple
colored formazan crystals formed in the wells, and their absorbances were measured at 492 nm with a
µM) of DOX, AAZ, or the derivatives for 96 h. The cells were incubated
µ
µ
microplate reader (Synergy-HT, BioTek Instruments, Winooski, VT, USA); 200
blank control.
µL DMSO was set as the
The anoxia condition was achieved by placing cells in a sealed hypoxia incubator chamber
(Catalog Number 27310, Stemcell Technologies, Inc., Vancouver, BC, Canada) filled with 5% CO2 and
95% N2. For the hypoxia group, the cells were treated with varying concentrations (100, 50, 25, 12.5,
and 6.25 µM) of DOX, AAZ, or the derivatives under anoxic condition for 24 h [29], and then moved
into nomoxic condition and cultured for additional 72 h.
4. Conclusions
In this study, four series of dihydroartemisinin–coumarin hybrids were designed and synthesized,
the target compounds were characterized by HRMS, 1H-, and 13C-NMR. The cytotoxic activities were
evaluated by MTT assay against HCT-116, MDA-MB-231, and HT-29 cell lines under normoxic or
anoxic conditions, respectively. The target compounds exhibited moderate activity with IC50 values

Molecules 2016, 21, 758
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in the 0.05–125.40
µM range, and these compounds exhibited better activity against HT-29 cell line
under anoxic condition. Under anoxic conditions, cytotoxic activities of most compounds under
anoxic conditions displayed one- to 10-fold greater activity than under normoxic condition. We were
delighted to find that the cytotoxic activities of compounds 10a–e exhibited better activity against the
HT-29 cell line under anoxic conditions compared with the other two cancer cell lines, which indicated
that our design of CA IX inhibitors do correspond with its action mode in some degree. On the basis
of the preliminary SARs we summarized, further investigations are currently in process.
Acknowledgments: This research was funded by the National Natural Science Foundation of China
(Grant No. 81573292).
Author Contributions: Y.T. designed and carried out the experimental and wrote the paper; Z.L. and H.X. assisted
in experiment; Y.M. supervised and directed the biological assay; C.G. supervised the whole experiment and
provided technical guidance. All authors have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 10a–e, 11a–e, 12a–e, 13a–e are available from the authors.
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