Synthesis and cytotoxicity of novel artemisinin derivatives containing sulfur atoms

Article abstract of DOI:10.1016/j.ejmech.2016.08.015

Ten novel artemisinin derivatives containing sulfur atoms were designed and synthesized and their structures were confirmed by¹H NMR,¹³C NMR and HRMS technologies in this study. All compounds were reported for the first time. The in?

Full text of DOI:10.1016/j.ejmech.2016.08.015

European Journal of Medicinal Chemistry 123 (2016) 763e768
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and cytotoxicity of novel artemisinin derivatives containing
sulfur atoms
,
b
,
, **
Cang-Cang Xu a, Juan-Juan Wu a, Tao Xu a, Chun-Hua Yao a, Bo-Yang Yu a *, Ji-Hua Liu a
a Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, Jiangsu 211198, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten novel artemisinin derivatives containing sulfur atoms were designed and synthesized and their
structures were confirmed by 1H NMR, 13C NMR and HRMS technologies in this study. All compounds
were reported for the first time. The in vitro cytotoxicity against PC-3, SGC-7901, A549 and MDA-MB-
435s cancer cell lines was evaluated by MTT assay. Compounds 4a and 4f displayed potent antitumor
Received 29 March 2016
Received in revised form
8 August 2016
Accepted 9 August 2016
Available online 10 August 2016
activity against PC-3, SGC-7901 and A549 cells with IC50 ranging from 1.6 to 30.5
mM, which values are
compared to that of 5-FU (IC50 from 6.8 to 42.5 M). Compounds 4a and 4f showed high specificity
m
towards human lung cancer A549 cells compared to normal human hepatic L-02 cells with selectivity
index of 16.1 and 50.1 respectively. Our promising findings indicated that the compounds 4a and 4f could
stand as potential lead compounds for further investigation.
Keywords:
Synthesis
Novel artemisinin derivatives
Cytotoxicity
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
used element beyond C, H, O and N. The appearance of sulfur atom
even enhance the cellular uptake percentage and the level of
Artemisinin is extracted from the Chinese herb qinghaosu
(Artemisia annua or annual wormwood) containing a 1, 2, 4-
tiroxane ring system [1], which is a powerful antimalarial drug
and has also been proved to possess antiviral [2e6], immunosup-
pressive [7e9] and anticancer [10e14] activities. Its anticancer ac-
tivity with strong selectivity [15,16], reversing multidrug resistance
[17] and sensitization radiation and chemotherapy [16,18] attracts
extensive attention. Many artemisinin derivatives have been syn-
thesized as anticancer agents [2,10,14,19e27]. However, current
modification strategies have only focused on the hemiacetal
structure of artemisinin due to the difficulty to introduce func-
tionalities on the ring systems by conventional chemical methods.
Biotransformation technology successfully solved the above prob-
reactive oxygen species (ROS) [30], which is a crucial factor for the
antitumor activity of artemisinins [31,32]. Herein, ten novel arte-
misinin ester derivatives containing sulfur atoms with alkyl or ar-
omatic side chains were reported and in vitro cytotoxicity against
four cancer cell lines (PC-3, SGC-7901, A549 and MDA-MB-435s)
was evaluated.
2. Results and disscussion
2.1. Chemistry
9a-OH DHA was obtained according to our previous work
[28,33]. The synthesis of compounds 3ae3e began with m-bro-
mobenzoic acid (1) and different thiols (2ae2e) in the presence of
Pd2(dba)3 and Xantphos in 1,4-dioxane, with heating reflux for 6 h
under nitrogen. The reaction mixture was acidified with acetic acid
to reach pH 3e4 then filtered and concentrated. The crude product
was purified by silica gel chromatography to give target compounds
(3ae3e) (Scheme 1). The synthesis of compounds 4ae4j was the
lem. 9a-OH-dihydroartemisinin (9a-OH DHA) with double hy-
droxyl modification sites was obtained in our previous work [28],
which make chemical modification on the ring system come true.
According to the analysis of the elemental composition of U.S.
FDA approved drug architectures [29], the sulfur is the fifth most
esterification of 9a-OH-DHA and compounds 3ae3e respectively
* Corresponding author. Jiangsu Key Laboratory of TCM Evaluation and Trans-
lational Research, China Pharmaceutical University, Nanjing, Jiangsu 211198, China.
** Corresponding author.
E-mail addresses: boyangyu59@163.com (B.-Y. Yu), liujihua@cpu.edu.cn
(J.-H. Liu).
using 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydro-
chloride (EDCI) as coupling agent and 4-dimethylaminopyridine
(DMAP) as catalyst in dichloromethane (Scheme 2).
The structures of ten novel artemisinin derivatives were
http://dx.doi.org/10.1016/j.ejmech.2016.08.015
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

764
C.-C. Xu et al. / European Journal of Medicinal Chemistry 123 (2016) 763e768
Scheme 1. The synthesis route of compounds 3ae3e. Reagents and conditions: 2.5 mol% Pd2(dba)3, 5 mol% Xantphos, 2 eq. i-Pr2NEt, 1,4-dioxane, reflux, 6 h.
Scheme 2. The synthesis route of compounds 4ae4j. Reagents and conditions: EDCI/DMAP, dry DCM, 0 ꢀC-rt, 12 h.
confirmed by 1H NMR, 13C NMR and HRESIMS. For compounds
4ae4j, the 12 -isomer was exclusively attained, which was evident
from the large coupling constants (J > 9 Hz) of H-11 and H-12 in 1H
NMR (the
-isomers, J z 3e4 Hz) [34]. Additionally, the 13C NMR
spectrum chemical shift of C-4 at circa 104 ppm in all compounds
suggested the presence of the intact peroxide [35]. The di-
esterification or mono-esterification products were determinated
by the numbers of carbonyl signals in 13C NMR spectrum. The
HMBC correlations of H-12 or H-9 with carbonyl group indicated
that acetoxy group were attached to C-12 or C-9.
compounds 4ae4j were showed in Table 1. The compounds 4a and
a
b
Table 1
Cytotoxicity of compounds 4ae4j against PC-3, SGC-7901, A549 and MDA-MB-435s
cell lines.
Compounds Cytotoxicity, IC50 (mM)
PC-3
SGC-7901
A549
MDA-MB-435s
4a
4b
4c
4d
4e
30.5 4.7
30.0 13.6
114.9 12.6 85.0 13.4
NA
6.0 1.7
74.2 13.4
4.3 1.7
44.4 2.6
106.6 42.3
223.0 24.0
NA
107.6 24.8
111.5 25.4
NA
288.9 46.6
NA
NA
NA
2.2. Biological evaluation
NA
4f
4g
13.0 5.0
NA
7.1 1.5
NA
1.6 0.6
1435.0 485.3 119.9 14.3
123.7 42.8
The biological activities of all compounds were evaluated
against selected four cancer cell lines (human prostate cancer PC-
3 cells, human gastric cancer SGC-7901 cells, non-small-cell-lung
cancer A549 cells and human breast cancer MDA-MB-435s cells)
using the MTT assay. DHA and 5-fluorouracil (5-FU) were used as
the positive controls. Compounds 3ae3e showed no activity to any
cancer cell lines (the data is not shown). The IC50 values of
4h
4i
4j
DHA
5-FU
464.4 27.9 172.8 18.7 96.1 14.6
146.2 20.2 NA 95.0 11.1
204.4 38.1 553.3 62.2 33.3 11.6
NA
223.0 37.1
29.60 5.4
21.9 1.1
15.2 0.9
NA
NA
80.4 5.8
6.8 0.8
42.5 3.7
11.9 1.3
NA: no activity.
The bold values idicate a relatively good antitumor activity.

C.-C. Xu et al. / European Journal of Medicinal Chemistry 123 (2016) 763e768
765
4f displayed potent cytotoxicity against PC-3, SGC-7901 and
A549 cell lines with IC50 ranging from 1.6 to 30.5 M. Other de-
4.2. Preparation of the compounds
m
rivatives showed poor or no cytotoxic activities against the selected
four cancer cell lines. To further investigate the specificity between
the cancer cell and the normal cell lines, the cytotoxicity of com-
pounds 4a and 4f against human normal hepatic L-02 cells were
examined. In A549 cells, compounds 4a and 4f were 4.2 fold and
13.2 fold less cytotoxic than that of 5-FU respectively. In SGC-
7901 cells, compounds 4a and 4f were 5.2 fold and 5.1 fold less
cytotoxic than that of 5-FU respectively (Table 2).
The structure activity relationship analysis revealed no clear
trend for the effects of the side chains on the cytotoxicity of com-
pounds 4ae4j. Compounds 4a and 4f with the p-methox-
ythiophenyl substituent group on benzoic acid showed potent
cytotoxicity implied that the different substituents of benzoic acid
have a significant impact on cytotoxicity. The cytotoxicity of com-
pound 4b with ethylthio substituent group was better than that of
compound 4c with butylthio substituent group, which manifested
the length of alkyl carbon chain might have a negative effect on the
antitumor activity.
4.2.1. General procedure of synthesis of compounds 3ae3e
The compounds 3ae3e were obtained by palladium-catalyzed
coupling according to reference [36]. To a three-necked flask
(100 mL) were added 3-bromobenzoic acid (2 mmol), i-Pr2NEt
(4 mmol), dry 1,4-dioxane (8.0 mL). The mixture was evacuated and
backfilled with nitrogen (3 cycles). Catalyst Pd2(dba)3 (0.05 mmol),
Xantphos (0.1 mmol) and corresponding thiols (2 mmol) were
added and then the mixture was degassed twice more. The mixture
was heated to reflux for 6 h. Then the reaction mixture was allowed
to reach ambient temperature and acidified with acetic acid to
reach pH 3e4, filtered and concentrated. The crude product was
purified by column chromatography on silica gel to afford the target
compounds.
4.2.1.1. 3-(40-Methoxyphenyl)sulfanylbenzoic acid (3a). Yield: 60%.
White solid. mp. 120e121 ꢀC. 1H NMR (400 MHz, CDCl3)
d
¼ 7.89e7.86 (m, 1H), 7.84 (ddd, J ¼ 7.9, 5.6, 3.7 Hz, 1H), 7.49e7.43
(m, 2H), 7.37e7.29 (m, 2H), 6.99e6.88 (m, 2H), 3.85 (s, 3H). The 1H
NMR data were consistent with the reported data [36]. HR-ESI-MS
m/z 259.0437 [M ꢁ H]ꢁ, (calcd for C14H11O3S, 259.0434).
3. Conclusions
4.2.1.2. 3-(Ethylthio)benzoic acid (3b). Yield: 66%. White solid. mp.
In conclusion, ten novel artemisinin derivatives containing sul-
fur atoms were synthesized and evaluated for their cytotoxicity
against selected four human cancer cell lines. Compounds 4a and 4f
with the p-methoxythiophenyl substituent group on benzoic acid
were potent cytotoxicity and high specificity. The structure-activity
relationship study revealed that the substitutes on benzoic acid and
the length of alkyl carbon chain had an impact on their cytotoxicity.
Our results indicated compounds 4a and 4f could be further
investigated as antitumor drug candidates.
107e110 ꢀC. 1H NMR (400 MHz, CDCl3)
d
¼ 8.26 (t, J ¼ 1.7 Hz, 1H),
7.93e7.88 (m, 1H), 7.55 (ddd, J ¼ 7.8, 1.9, 1.1 Hz, 1H), 7.40 (t,
J ¼ 9.4 Hz, 1H), 3.01 (q, J ¼ 7.4 Hz, 2H), 1.35 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d
¼ 171.68, 137.98, 133.87, 131.21, 129.90,
128.81, 128.91, 27.38, 14.18. HR-ESI-MS m/z 181.0321 [M ꢁ H]ꢁ,
(calcd for C9H9O2S, 181.0329).
4.2.1.3. 3-(Butylthio)benzoic acid (3c). Yield: 50%. White solid. mp.
135e138 ꢀC. 1H NMR (400 MHz, CDCl3)
d
¼ 8.04 (t, J ¼ 1.7 Hz, 1H),
7.92e7.88 (m, 1H), 7.54 (ddd, J ¼ 7.8, 1.8, 1.1 Hz, 1H), 7.38 (t,
J ¼ 7.8 Hz, 1H), 2.99 (t, J ¼ 7.3 Hz, 2H), 1.66 (ddd, J ¼ 12.6, 8.4, 6.4 Hz,
2H), 1.48 (dq, J ¼ 14.4, 7.3 Hz, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H). 13C NMR
4. Experimental section
(100 MHz, CDCl3)
d
¼ 171.99, 138.38, 133.69, 129.94, 129.63, 128.87,
4.1. Materials and measurements
127.23, 32.96, 31.01, 21.95, 13.62. HR-ESI-MS m/z 209.0660
[M ꢁ H]ꢁ, (calcd for C11H13O2S, 209.0642).
Melting points (mp) were determined in duplicate on an X-4
digital display microscope melting point apparatus. 1H NMR and
13C NMR spectra was obtained in CDCl3 solution on a Brucker
Avance-400 or Brucker 300 spectrophotometer using TMS as the
internal standard. High resolution ESI mass spectra were measured
on Aglient LC-Q-TOF-MS 6520. Column chromatography was done
using silica gel (Qingdao Marine Chemical Co., Ltd., China), or
Sephadex LH-20 (GE healthcare, Sweden).
All commercially available reagents were used without further
purification unless otherwise stated. The progress of the reactions
was monitored by analytical thin layer chromatography (TLC) per-
formed on homemade HSGF254 precoated silica gel plates. Visu-
alization was performed by UV or development using vanillin
solution in sulfuric acid and ethanol (4/1 v/v). Yields were reported
after chromatographic purification.
4.2.1.4. 3-(benzylthio)benzoic acid (3d). Yield: 52%. White solid.
mp. 128e130 ꢀC. 1H NMR (400 MHz, CDCl3)
d
¼ 8.07 (s, 1H), 7.91 (d,
J ¼ 7.7 Hz, 1H), 7.50 (d, J ¼ 7.9 Hz, 1H), 7.39e7.27 (m, 6H), 4.18 (s,
2H). 13C NMR (100 MHz, CDCl3)
d
¼ 171.17, 137.47, 136.81, 134.67,
130.81, 129.89, 128.90, 128.86 (ꢂ2), 128.59 (ꢂ2), 127.93, 127.39,
38.70. HR-ESI-MS m/z 243.0495 [M ꢁ H]ꢁ, (calcd for C14H11O2S,
243.0485).
4.2.1.5. 3-(cyclohexylthio)benzoic acid (3e). Yield: 79%. White solid.
mp. 84e86 ꢀC. 1H NMR (400 MHz, CDCl3)
d
¼ 8.13 (t, J ¼ 1.6 Hz, 1H),
7.97e7.92 (m, 1H), 7.62 (ddd, J ¼ 7.8, 1.9, 1.2 Hz, 1H), 7.40 (q,
J ¼ 7.7 Hz, 1H), 3.24e3.15 (m, 1H), 2.05e1.96 (m, 2H), 1.83e1.72 (m,
2H), 1.63 (dd, J ¼ 10.5, 4.3 Hz, 1H), 1.47e1.20 (m, 5H). 13C NMR
Table 2
Cytotoxicity of compounds 4a and 4f on human normal hepatic L-02 cells.
Lꢁ02
A549
Lꢁ02
SGCꢁ7901
/IC )
50
Compounds
IC50
(
m
M)
SI (IC
/IC
)
SI (IC
50
50
50
A549
SGC-7901
L-02
4a
4f
DHA
5-FU
4.3 1.7
1.6 0.6
80.4 5.8
6.8 0.8
6.0 1.7
7.1 1.5
NA
69.2 19.6
80.2 5.9
145.9 3.6
25.9 2.3
16.1
50.1
1.8
11.5
11.3
11.9 1.3
3.8
2.2
NA: no activity.
The bold values idicate a relatively good antitumor activity.

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C.-C. Xu et al. / European Journal of Medicinal Chemistry 123 (2016) 763e768
(100 MHz, CDCl3)
d
¼ 171.51, 136.69, 136.48, 132.80, 129.87, 128.86,
d
¼ 8.00 (dt, J ¼ 33.1, 1.7 Hz, 2H), 7.93e7.89 (m, 1H), 7.86e7.82 (m,
128.18, 46.52, 33.25 (ꢂ2), 25.97 (ꢂ2), 25.72. HR-ESI-MS m/z
1H), 7.47 (tdd, J ¼ 8.8, 1.9, 1.1 Hz, 2H), 7.36e7.21 (m, 12H), 6.00 (d,
J ¼ 9.8 Hz,1H), 5.65 (s,1H), 4.73 (td, J ¼ 10.9, 4.4 Hz,1H), 4.18 (s, 2H),
4.15 (s, 2H), 2.82e2.72 (m, 1H), 2.50e2.40 (m, 1H), 2.20 (dt, J ¼ 12.9,
4.4 Hz, 1H), 2.11 (dt, J ¼ 7.2, 6.2 Hz, 1H), 2.02e1.92 (m, 2H),
1.79e1.54 (m, 4H), 1.46 (s, 3H), 1.01 (d, J ¼ 6.3 Hz, 3H), 0.90 (d,
235.0814 [M ꢁ H]ꢁ, (calcd for C13H15O2S, 235.0798).
4.2.2. General procedure of synthesis of compounds 4ae4j
To a three-necked flask (100 mL) that contained a solution of 9a-
OH DHA (1 equiv), 4-dimethyaminopyridine (DMAP, 2.6 equiv) and
1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride
(EDCI, 3 equiv) in dry CH2Cl2 (20 mL) was added the corresponding
3ae3e (2.6 equiv) at ice bath. The resulting mixture was stirred at
0 ꢀC-rt for 12 h, the solvent was moved. Then the residue was taken
up with EtOAc, washed with water (20 mL) and saturated NaCl
solution (20 mL), dried over anhydrous MgSO4, and purified using
column chromatography to obtain the target compounds (Scheme
2).
J ¼ 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
¼ 165.58,164.67,137.35,
d
137.16, 136.91, 134.49, 134.16, 130.90, 130.66, 130.36, 130.11, 128.84
(ꢂ6), 128.73, 128.57 (ꢂ4), 127.97, 127.40, 127.35 (ꢂ2), 104.61, 92.34,
91.26, 79.07, 76.18, 49.09, 42.78, 41.36, 38.81 (ꢂ2), 36.09, 31.66,
27.52, 25.88, 24.57, 15.51, 12.12. HR-ESI-MS m/z 775.2388 [MþNa]þ,
(calcd for C43H44NaO8S2, 775.237).
4.2.2.5. Dihydroartemisinin
(4e). Yield: 16.7%. White powder. mp. 95e97 ꢀC. 1H NMR (300 MHz,
CDCl3)
9a,12a-di-(3-cyclohexylthio)-benzoate
d
¼ 8.08 (dt, J ¼ 18.4, 1.7 Hz, 2H), 7.91 (dd, J ¼ 20.2, 7.7 Hz,
4.2.2.1. Dihydroartemisinin
benzoate (4a). Yield: 31.0%. White powder. mp. 75 ꢀC. 1H NMR
(400 MHz, CDCl3)
9
a,12
a-di-[3-(p-methoxyphenyl)thio]-
2H), 7.63e7.53 (m, 2H), 7.36 (q, J ¼ 7.7 Hz, 2H), 6.01 (d, J ¼ 9.8 Hz,
1H), 5.65 (s, 1H), 4.74 (td, J ¼ 10.7, 4.3 Hz, 1H), 3.23e3.11 (m, 2H),
2.86e2.72 (m, 1H), 2.52e2.35 (m, 1H), 2.24 (dt, J ¼ 12.7, 4.4 Hz, 1H),
2.06e1.93 (m, 8H), 1.78 (d, J ¼ 4.6 Hz, 7H), 1.62 (d, J ¼ 10.4 Hz, 6H),
1.45 (s, 3H), 1.43e1.22 (m, 6H), 1.04 (d, J ¼ 6.2 Hz, 3H), 0.93 (d,
d
¼ 7.89e7.82 (m, 3H), 7.80e7.74 (m, 1H),
7.47e7.41 (m, 4H), 7.30 (td, J ¼ 5.3, 1.9 Hz, 4H), 6.95e6.89 (m, 4H),
5.92 (d, J ¼ 9.8 Hz, 1H), 5.61 (s, 1H), 4.68 (td, J ¼ 10.8, 4.4 Hz, 1H),
3.83 (s, 3H), 3.82 (s, 3H), 2.79e2.66 (m, 1H), 2.41 (dd, J ¼ 18.5,
8.8 Hz, 1H), 2.18 (ddd, J ¼ 15.0, 9.7, 5.3 Hz, 1H), 2.14e2.06 (m, 1H),
2.00e1.88 (m, 2H),1.78e1.51 (m, 4H),1.44 (s, 3H), 0.99 (d, J ¼ 6.2 Hz,
J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3)
¼ 165.67, 164.75, 136.43,
d
136.34,136.26,136.16,132.92,132.46, 130.63,130.09,128.79,128.65,
128.17, 127.60, 104.56, 92.35, 91.25, 79.07, 76.24, 49.12, 46.61, 42.81,
41.36, 36.08, 33.24(ꢂ3), 33.20(ꢂ3), 31.66, 27.52, 25.91 (ꢂ3), 25.85,
25.69 (ꢂ2), 24.54, 15.45, 12.09. HR-ESI-MS m/z 759.3000 [MþNa]þ,
(calcd for C41H52NaO8S2, 759.2996).
3H), 0.86 (d, J ¼ 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d
¼ 165.64,
164.71, 160.26, 160.23, 140.08, 139.89, 135.92 (ꢂ2), 135.90 (ꢂ2),
132.46, 132.24, 130.72, 130.19, 129.00, 128.87, 128.74, 128.71, 127.25,
126.68, 123.18, 123.10, 115.40 (ꢂ4), 104.58, 92.34, 91.22, 79.08,
76.26, 55.40 (ꢂ2), 49.09, 42.77, 41.33, 36.08, 31.63, 27.48, 25.88,
24.54, 15.44, 12.05. HR-ESI-MS m/z 807.2262 [MþNa]þ, (calcd for
4.2.2.6. 9
thio]-benzoate (4f). Yield: 44.8%. White powder. mp. 64 ꢀC. 1H NMR
(400 MHz, CDCl3)
¼ 7.90 (d, J ¼ 0.9 Hz, 1H), 7.89e7.85 (m, 1H),
a-Hydroxy-dihydroartemisinin 12a-[3-(p-methoxyphenyl)
C
43H44NaO10S2, 807.2268).
d
7.47e7.40 (m, 2H), 7.33e7.27 (m, 2H), 6.97e6.88 (m, 2H), 5.95 (d,
J ¼ 9.8 Hz, 1H), 5.57 (s, 1H), 3.84 (s, 3H), 3.25 (td, J ¼ 10.6, 4.3 Hz,
1H), 2.77e2.58 (m, 1H), 2.40 (td, J ¼ 14.4, 3.9 Hz, 1H), 2.09 (ddd,
J ¼ 8.9, 4.5, 1.7 Hz, 1H), 1.90 (ddd, J ¼ 16.4, 8.6, 5.1 Hz, 1H), 1.83 (dd,
J ¼ 9.3, 4.9 Hz, 1H), 1.64e1.47 (m, 3H), 1.44 (s, 3H), 1.40e1.29 (m,
2H), 1.08 (d, J ¼ 6.1 Hz, 3H), 0.88 (d, J ¼ 7.1 Hz, 3H). 13C NMR
4.2.2.2. Dihydroartemisinin
Yield: 40.3%. White powder. mp. 69e72 ꢀC. 1H NMR (400 MHz,
CDCl3)
9a,12a-di-(3-ethylthio)-benzoate (4b).
d
¼ 8.21 (dt, J ¼ 26.8, 1.7 Hz, 2H), 8.06e7.96 (m, 2H),
7.76e7.68 (m, 2H), 7.40e7.30 (m, 2H), 6.01 (d, J ¼ 9.7 Hz, 1H), 5.65
(s, 1H), 4.76 (td, J ¼ 10.8, 4.3 Hz, 1H), 3.01 (q, J ¼ 7.4 Hz, 2H), 2.99 (q,
J ¼ 7.5 Hz, 2H), 2.85e2.74 (m, 1H), 2.43 (dt, J ¼ 14.4, 3.8 Hz, 1H),
2.26e2.17 (m, 1H), 2.12 (t, J ¼ 12.4 Hz, 1H), 2.00e1.91 (m, 2H),
1.83e1.51 (m, 4H), 1.46 (s, 3H), 1.34 (t, J ¼ 11.4 Hz, 3H), 1.32 (t,
(100 MHz, CDCl3)
d
¼ 164.75, 160.21, 139.85, 135.91, 135.88, 132.44,
130.24, 129.18, 128.87, 127.29, 123.18, 115.22 (ꢂ2), 104.54, 92.50,
91.31, 79.45, 73.81, 55.40, 49.10, 44.15, 42.99, 36.12, 31.60, 31.07,
25.90, 24.49, 15.43, 12.15. HR-ESI-MS m/z 565.1858 [MþNa]þ, (calcd
for C29H34NaO8S, 565.1867).
J ¼ 11.4 Hz, 3H), 1.03 (d, J ¼ 6.3 Hz, 3H), 0.93 (d, J ¼ 7.1 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d
¼ 165.73, 164.81, 136.68, 136.38, 136.16,
133.18, 132.60, 130.07 (ꢂ2), 128.73, 128.18, 127.41, 122.60, 122.46,
104.65, 92.37, 91.27, 79.02, 76.53, 49.07, 42.79, 41.33, 36.08, 31.68,
29.69, 27.55 (ꢂ2), 25.86, 24.55, 15.50, 14.20 (ꢂ2), 12.11. HR-ESI-MS
m/z 651.2061 [MþNa]þ, (calcd for C33H40NaO8S2, 651.2057).
4.2.2.7. 9
(4g). Yield: 42.5%. White powder. 75e77 ꢀC. 1H NMR (400 MHz,
CDCl3)
a-Hydroxy-dihydroartemisinin 12a-(3-ethylthio)-benzoate
d
¼ 8.26e7.88 (m, 1H), 8.08e8.01 (m, 1H), 7.72e7.48 (m, 1H),
7.42e7.30 (m, 1H), 6.01 (d, J ¼ 9.7 Hz, 1H), 5.65 (s, 1H), 4.76 (td,
J ¼ 10.8, 4.3 Hz, 1H), 3.00 (q, J ¼ 7.4 Hz, 2H), 2.84e2.72 (m, 1H), 2.39
(dt, J ¼ 14.4, 3.7 Hz, 1H), 2.26e2.16 (m, 1H), 2.10 (d, J ¼ 14.4 Hz, 1H),
1.97 (dd, J ¼ 21.4, 11.6 Hz, 2H), 1.85e1.53 (m, 4H), 1.45 (s, 3H), 1.32 (t,
4.2.2.3. Dihydroartemisinin
Yield: 30.3%. White powder. mp. 115e118 ꢀC. 1H NMR (400 MHz,
CDCl3)
¼ 8.00 (dt, J ¼ 24.0, 1.6 Hz, 2H), 7.92e7.86 (m, 1H),
9a,12a-di-(3-butylthio)-benzoate (4c).
d
7.84e7.80 (m, 1H), 7.54e7.48 (m, 2H), 7.36 (dd, J ¼ 17.1, 7.8 Hz, 2H),
6.00 (d, J ¼ 9.8 Hz, 1H), 5.65 (s, 1H), 4.74 (td, J ¼ 10.9, 4.4 Hz, 1H),
2.97 (q, J ¼ 5.6 Hz, 4H), 2.83e2.73 (m, 1H), 2.49e2.38 (m, 1H), 2.22
(dt, J ¼ 12.8, 4.4 Hz, 1H), 2.10 (dd, J ¼ 15.0, 3.8 Hz, 1H), 2.03e1.91 (m,
2H), 1.77 (td, J ¼ 10.8, 6.1 Hz, 1H), 1.72e1.55 (m, 9H), 1.51e1.46 (m,
2H), 1.45 (s, 3H), 1.04 (d, J ¼ 6.3 Hz, 3H), 0.92 (d, J ¼ 7.2 Hz, 3H), 0.93
(t, J ¼ 7.3 Hz, 3H), 0.94 (t, J ¼ 7.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
J ¼ 7.8 Hz, 3H), 1.03 (d, J ¼ 6.3 Hz, 3H), 0.91 (t, J ¼ 8.3 Hz, 3H). 13
C
NMR (100 MHz, CDCl3)
d
¼ 164.83, 136.35, 133.59, 133.02, 129.93,
127.40, 122.45, 104.58, 92.71, 91.37, 79.46, 73.80, 49.11, 44.16, 42.98,
36.12, 31.65, 31.09, 27.55, 25.89, 24.50, 15.43, 14.21, 12.21. HR-ESI-
MS m/z 487.1712 [MþNa]þ, (calcd for C24H32NaO7S, 487.1761).
4.2.2.8. 9
(4h). Yield: 33.7%. White powder. mp. 72e75 ꢀC. 1H NMR
(300 MHz, CDCl3)
a-Hydroxy-dihydroartemisinin 12a-(3-butylthio)-benzoate
d
¼ 165.74, 164.82, 138.25, 138.05, 133.45, 133.12, 130.67, 130.12,
129.86, 129.32, 128.86, 128.73, 127.27, 126.67, 104.60, 92.36, 91.27,
79.10, 76.22, 49.12, 42.82, 41.38, 36.09, 33.13, 33.09, 31.68, 31.08,
31.05, 27.55, 25.88, 24.56, 21.93 (ꢂ2), 15.49, 13.63, 13.61, 12.12. HR-
ESI-MS m/z 707.2700 [MþNa]þ, (calcd for C37H48NaO8S2, 707.2683).
d
¼ 8.04 (t, J ¼ 1.6 Hz, 1H), 7.94e7.87 (m, 1H), 7.51
(ddd, J ¼ 7.8,1.7,1.1 Hz,1H), 7.35 (t, J ¼ 7.8 Hz,1H), 6.01 (d, J ¼ 9.8 Hz,
1H), 5.57 (s, 1H), 3.25 (td, J ¼ 10.7, 4.3 Hz, 1H), 3.02e2.92 (m, 2H),
2.83e2.67 (m, 1H), 2.48e2.31 (m, 1H), 2.07 (dt, J ¼ 13.7, 4.2 Hz, 2H),
1.86 (m, 2H), 1.71e1.45 (m, 6H), 1.43 (s, 3H), 1.40e1.24 (m, 2H), 1.10
4.2.2.4. Dihydroartemisinin 9
a,12
a
-di-(3-benzylthio)-benzoate (4d).
(d, J ¼ 5.9 Hz, 3H), 0.94 (d, J ¼ 7.1 Hz, 3H), 0.95 (t, J ¼ 7.2 Hz, 3H). 13
C
Yield: 17.2%. White powder. mp. 72 ꢀC. 1H NMR (400 MHz, CDCl3)
NMR (100 MHz, CDCl3)
d
¼ 164.84, 138.03, 133.42, 130.19, 129.85,

C.-C. Xu et al. / European Journal of Medicinal Chemistry 123 (2016) 763e768
767
128.73, 127.26, 104.54, 92.50, 91.36, 79.46, 73.81, 49.11, 44.17, 43.01,
References
36.14, 33.13, 31.65, 31.12, 31.07, 25.89, 24.50, 21.93, 15.43, 13.61,
12.21. HR-ESI-MS m/z 515.2078 [MþNa]þ, (calcd for C26H36NaO7S,
515.2074).
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ate (4i). Yield: 47.4%. White powder. mp. 83 ꢀC. 1H NMR (300 MHz,
CDCl3)
a-Hydroxy-dihydroartemisinin 12a-(3-benzylthio)-benzo-
d
¼ 8.05 (s,1H), 7.91 (d, J ¼ 7.8 Hz,1H), 7.46 (d, J ¼ 7.8 Hz,1H),
7.31 (dd, J ¼ 8.8, 5.6 Hz, 6H), 5.99 (d, J ¼ 9.8 Hz, 1H), 5.57 (s, 1H), 4.16
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d
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136.93, 134.45, 130.89, 130.77, 130.19, 128.82, 128.69, 128.55, 127.95,
127.84, 127.31, 104.51, 92.48, 91.33, 79.41, 73.82, 49.11, 44.16, 43.00,
38.82, 36.13, 31.61, 31.10, 25.85, 24.48, 15.39, 12.16. HR-ESI-MS m/z
549.1925 [MþNa]þ, (calcd for C29H34NaO7S, 549.1917).
4.2.2.10. 9
benzoate (4j). Yield: 26.8%. White powder. mp. 90e92 ꢀC. 1H NMR
(300 MHz, CDCl3)
a-Hydroxy-dihydroartemisinin
12a-(3-cyclohexylthio)-
d
¼ 8.11 (dd, J ¼ 4.5, 2.9 Hz, 1H), 7.98e7.91 (m,
1H), 7.61e7.55 (m, 1H), 7.37 (q, J ¼ 7.4 Hz, 1H), 6.00 (d, J ¼ 9.8 Hz,
1H), 5.57 (s, 1H), 3.32e3.10 (m, 2H), 2.83e2.68 (m, 1H), 2.40 (dt,
J ¼ 14.4, 4.0 Hz, 1H), 2.11e1.95 (m, 5H), 1.92e1.72 (m, 4H), 1.67e1.47
(m, 4H), 1.43 (s, 3H), 1.41e1.24 (m, 5H), 1.10 (d, J ¼ 5.9 Hz, 3H), 0.94
(d, J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3)
d
¼ 164.77, 136.40,
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Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
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This work was supported by the Specialized Research Fund for
the Doctoral Program of Higher Education Priority Development
Field (No. 20110096130002), and the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD). Au-
thors are also thankful to the Professor Zhou Jia-hong (Nanjing
Normal University) for NMR data.
Appendix A. Supplementary data
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