Novel artemisinin derivatives with potent anticancer activities and the anti-colorectal cancer effect by the mitochondria-mediated pathway

Article abstract of DOI:10.1016/j.bioorg.2020.104496

Many artemisinin derivatives have good inhibitory effects on malignant tumors. In this work, a novel series of artemisinin derivatives containing piperazine and fluorine groups were designed and synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and HRMS technologies. The in vitro cytotoxicity against various cancer cell lines was evaluated. Among the derivatives, compound 12h was found to exhibit not only the best activity against HCT-116 cells (IC₅₀ = 0.12 ± 0.05 μM), but also low toxicity against normal cell line L02 (IC₅₀ = 12.46 ± 0.10 μM). The mechanisms study revealed that compound 12h caused the cell cycle arrest in G1 phase, induced apoptosis in a concentration-dependent manner, significantly reduced mitochondrial membrane potential, increased intracellular ROS and Ca²⁺ levels, up-regulated the expression of Bax, cleaved caspase-9, cleaved caspase-3, and down-regulated the expression of Bcl-2 protein. A series of analyses confirmed that 12h can inhibit HCT-116 cells migration and induce apoptosis by a mechanism of the mitochondria-mediated pathway in the HCT-116 cell line. The present work indicates that compound 12h may merit further investigation as a potential therapeutic agent for colorectal cancer.

Full text of DOI:10.1016/j.bioorg.2020.104496

Journal Pre-proofs
Novel artemisinin derivatives with potent anticancer activities and the anti-
colorectal cancer effect by the mitochondria-mediated pathway
Lan Lin, Wenyu Lu, Tianzhi Dai, Huan Chen, Tong Wang, Li Yang, Xuelian
Yang, Ying Liu, Dequn Sun
PII:
S0045-2068(20)31794-6
DOI:
https://doi.org/10.1016/j.bioorg.2020.104496
YBIOO 104496
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20 November 2020
Please cite this article as: L. Lin, W. Lu, T. Dai, H. Chen, T. Wang, L. Yang, X. Yang, Y. Liu, D. Sun, Novel
artemisinin derivatives with potent anticancer activities and the anti-colorectal cancer effect by the mitochondria-
mediated pathway, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104496
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Novel artemisinin derivatives with potent anticancer activities
and the anti-colorectal cancer effect by the
mitochondria-mediated pathway
Lan Lin 1, a, b, Wenyu Lu 1, a, c, Tianzhi Dai a, c, Huan Chen a, Tong Wang a, Li Yang a,
Xuelian Yang a, Ying Liu b, *, Dequn Sun a, *
a. School of Life Science and Engineering, Southwest University of Science and Technology, No. 59, Middle
Section of Qinglong Avenue , Mianyang 621010, China.
b. Institute of Materials, China Academy of Engineering Physics, Jiangyou 621907, China.
c. Marine College, Shandong University at Weihai, No.180, Wenhua West Road, Weihai 264209, P. R. China.
1. two persons contribute this work equally.
* Corresponding author: liuying2016@caep.cn
* Corresponding author: dqsun@swust.edu.cn
Corresponding author:
Ying Liu: liuying2016@caep.cn
Dequn Sun: dqsun@swust.edu.cn
Abstract
Many artemisinin derivatives have good inhibitory effects on malignant tumors.
In this work, a novel series of artemisinin derivatives containing piperazine and
fluorine groups were designed and synthesized and their structures were confirmed by
1H-NMR, 13C-NMR and HRMS technologies. The in vitro cytotoxicity against
various cancer cell lines was evaluated. Among the derivatives, compound 12h was
found to exhibit not only the best activity against HCT-116 cells (IC50= 0.12 ±
0.05μM), but also low toxicity against normal cell line L02 (IC50= 12.46±0.10 μM.
The mechanisms study revealed that compound 12h caused the cell cycle arrest in G1
phase, induced apoptosis in a concentration-dependent manner, significantly reduced
mitochondrial membrane potential, increased intracellular ROS and Ca2+ levels,
up-regulated the expression of Bax, cleaved caspase-9, cleaved caspase-3, and
down-regulated the expression of Bcl-2 protein. A series of analyses confirmed that
12h can inhibit HCT-116 cells migration and induce apoptosis by a mechanism of the
mitochondria-mediated pathway in the HCT-116 cell line. The present work indicates
that compound 12h may merit further investigation as a potential therapeutic agent for
colorectal cancer.
Keywords
Artemisinin derivatives; Piperazine and fluorine groups; Anti-cancer activity;
Colorectal cancer; Mitochondria-mediated pathway.
1

1. Introduction
Artemisinin (ART), a sesquiterpene lactone isolated from Artemisia annua, has
been widely used to treat malaria. In 2015, Madam Youyou Tu won the Nobel Prize
for her contribution to the development of novel antimalarial drugs, artemisinin and
dihydroartemisinin [1]. The bioactivity of artemisinin (Fig.1) is closely related to the
endoperoxide bridge, which is involved in the production of reactive oxygen species
(ROS) and peroxy free radicals, inducing oxidative stress, DNA damage, alkylation of
target proteins and cell apoptosis [2]. Besides the antimalarial effects, the artemisinin
derivatives exhibit a wide range of biological activities [3], such as antimicrobial
[4,5], antiviral [6] and anti-multiple tumor cell lines [7-9]. Artemisinin derivatives
have the advantages of low cross-resistance, synergistic effect with traditional
chemotherapy anticancer drugs, and low toxicity to normal tissues or cell lines [10].
Over the years, artemisinin has been used as a lead compound for the treatment of
cancer. Many studies retain the backbone of the endoperoxide bridge and modify
artemisinin at the 10-position carbon [11,12].
Piperazine is an important pharmaceutical intermediate. Piperazine derivatives
show broad spectrum of pharmacological activities such as antidepressant, anxiolytic,
and antipsychotic [13]. Previously, we found artemisinin derivatives with piperazine
groups have good antitumor activity [14]. On the other hand, fluorine is the most
abundant halogen in the earth's crust and ranks 13th among all elements [15]. Since
the fluorine atom has the strongest electronegativity, its introduction into the active
molecules can significantly change the physicochemical properties [16]. In 1957, the
anti-metabolite 5-fluorouracil was synthesized for the first time [17]. Since then, the
introduction of fluorine atoms or fluorine-containing groups into drug molecules has
become an important means of drug modification. Our previous work showed that the
introduction of fluorine atoms into sulfur heterocyclic derivatives caused good
antitumor activity [18]. Grellepois [19] introduced trifluoromethyl group at the C-10
position of artemisinin, confirming that the compound had good antimalarial activity.
We previously, introduced piperazine-containing groups at C-10 position to obtain
four new artemisinin derivatives, among which the most active compound had the
IC50 value of about 4.1 μM for HepG-2 cell line [14]. Furthermore, Li [20] in our
group introduced fluorine atoms into piperazine-containing groups to synthesize a
new kind of artemisinin derivatives, among which the IC50 value of the most active
compound was about 2.1 μM for MCF-7 cell line. Compared with the original
designed derivatives, the activity of the derivative was improved. Zhang [21]
synthesized five-membered cyclic artemisinin dimers with potent anticancer activity.
They retained the endoperoxide bridge and obtained a compound with good activity
on PC12 cell line, but it is uncertain whether the five-membered cyclic artemisinin
containing piperazine and fluorine groups have good antitumor activity.
Although artemisinin derivatives showed strong anti-tumor potential, its targets
are diverse and exact antitumor mechanisms have not yet been elucidated. It has been
found that artemisinin drugs can inhibit tumor cell proliferation, induce cell cycle
2

arrest [22], inhibit tumor cell invasion and metastasis [23], promote tumor cell
apoptosis, and induce cell death through the mitochondrial pathway [24]. In the latest
research on the anti-tumor activity of artemisinin derivatives, Wang’s group reported
a series of new compounds with good activity against colorectal cancer cell line
(HCT-116), and the IC50 value of the most active compound was about 0.110 μM
[25], although the toxicity and anti-tumor mechanisms were not explored. Krishna's
team reported an oral artesunate neoadjuvant therapy with good tolerance in a
randomized double-blind pilot clinical phase II trial to treat colorectal cancer (CRC),
indicating that ART derivatives could possibly to be developed as an anti-CRC drug
[26]. In addition, Yu’s group synthesized a series of dihydroartemisinin piperazine
dithiocarbamate derivatives with prominent inhibitory activity (its IC50 was about 25
nM) against human liver cancer cell line [7], but for normal liver cells, its selectivity
is unsatisfactory.
In this work, we attempted to design and synthesize a novel series of artemisinin
derivatives containing both piperazine and fluorine groups in order to find leading
compounds with strong bioactivity and low toxicity. We also exploited the
mechanisms so as to explore their potentials as the anti-cancer drug candidates for
clinical research.
H
5
6
4O
5a
7
8
O
2
3
1
13
8a
12a
12
O
H
H
O11
9
10
O
Fig.1. The structure of artemisinin.
2. Results and Discussion
2.1. Chemistry
Our investigation
commenced
with
the
synthesis
of
phenyl
piperazine-1-carboxylate 4 and N-phenylpiperazine-1-carbothioamide 7 (Scheme 1
and 2). Commercially available phenols were treated with triphosgene (BTC) and
trimethylamine to give the aryl chloroformate 2 [27,28], which was treated with
Boc-protected
piperazine
to
obtain
1-(tert-butyl)
4-phenyl
piperazine-1,4-dicarboxylate 3 [29]. Next, the deprotection of Boc group of 3 with
trifluoroacetic acid (TFA) provided desired intermediate 4 [30,31], which was used to
synthesize the target compounds 12a-12h and 18a-18h. Commercially available aryl
isothiocyanates were treated with Boc-protected piperazine to obtain tert-butyl
4-(phenylcarbamothioyl)piperazine-1-carboxylate 6 [32]. Next, the deprotection of
Boc group of 6 with trifluoroacetic acid (TFA) afforded desired intermediate 7
[30,31], which was used to synthesize the target compounds 13a-13e and 19a-19e.
3

Cl
Boc
N
H
N
OH
O
O
c
b
a
R1
N
Boc
N
R1
R1
N
O
R1
O
O
4
O
N
H
1
2
3
R1: a = p-CH3 , b = p-F , c = m-F , d = o-F , e = p-CF3 , f = m-CF3 , g = m-OCF3 , h = p-SCF3
Scheme 1. The synthetic routes of phenyl piperazine-1-carboxylates 4a-4h. Reagents and
conditions: (a) BTC (0.33 equiv), TEA (1 equiv), DCM, 0°C to r.t.; (b) TEA (1.1 equiv), DCM,
r.t., 47-64%; (c) TFA : DCM=1:4, 0°C to r.t.
Boc
H
N
NCS
N
b
a
N
Boc
N
N
R2
R2
R2
S
N
H
S
N
H
N
H
5
6
7
R2: a = p-F , b = m-F , c = o-F , d = m-CF3 , e = p-CF3 , f = p-OCF3
Scheme 2. The synthetic routes of N-phenylpiperazine-1-carbothioamides 7a-7f. Reagents and
conditions: (a) DCM, r.t., 85-99.6%; (b) TFA: DCM=1:4, 0°C to r.t.
The target compounds 12a-12h and 13a-13e were synthesized starting from
artemisinin according to the literature procedure (Scheme 3). Firstly, artemisinin was
subjected to NaBH4-mediated reduction to afford dihydroartemisinin (DHA). Then,
DHA was treated with trimethylchlorosilane and triethylamine in DCM at 0–5°C to
generate DHA α-trimethylsilyl ether 10 [20]. DHA α-trimethylsilyl ether 10 reacted
with trimethylsilyl bromide (TMSBr) to give intermediate bromide 11 which is then
treated with an amine nucleophile to form target compounds 12a-12h and 13a-13e
[33].
As previously reported [21], the synthesis of glycal 14 was prepared from DHA
in the presence of a Lewis acid, typically borontrifluoride-diethyl ether.
Hydrobromination of 14 gave the bromoacetal 15 in good yield as a mixture of two
diastereoisomers. Bromohydrin 15 underwent rearrangement with trimethylamine,
leading to aldehyde 16 as a single isomer in excellent yield. Oxidation of 16 with
sodium chlorite afforded the corresponding carboxylic acid 17. With compound 17 in
hand, compounds 18a-18h and 19a-19e were prepared (Scheme 4). The structures of
all the compounds were confirmed by 1H-NMR, 13C-NMR and HRMS
(Supplementary materials).
4

H
O
O
O
H
H
O
N
N
d
R3
H
O
O
H
H
H
O
O
O
O
O
O
O
O
12
c
a
b
O
O
O
O
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
Br
11
OH
9
OTMS
10
O
O
H
H
8
O
e
N
N
R4
R3: a = p-CH3 , b = p-F , c = m-F , d = o-F, e = p-CF3 , f = m-CF3 , g = m-OCF3 , h = p-SCF3
R4: a = p-F , b = m-F , c = o-F , d = p-CF3 , e = p-OCF3
S
N
H
13
Scheme 3. The synthetic routes of six-membered cyclic carbamate artemisinin derivatives
12a-12h, 13a-13e. Reagents and conditions: (a) NaBH4 (3.0 equiv), MeOH, 0-5°C, 89%; (b)
TMSCl (1.2 equiv), TEA (2.0 equiv), DCM, 0°C to r.t., 94%; (c) TMSBr (1.02 equiv), DCM,
0-5°C; (d) amine (0.95 equiv), TEA (2 equiv), DCM, 0°C to r.t., 15-30%; (e) amine (0.95 equiv),
TEA (2 equiv), DCM/THF, 0°C to r.t., 24-31%.
H
H
H
O
O
H
O
O
O
O
O
O
O
O
H
c
a
b
O
O
O
H
Br
H
H
H
H
H
H
O
H
O
O
OH
15
O
OH
14
9
16
H
O
O
O
H
O
R5
O
e
H
N
H
N
O
O
O
d
O
H
O
18
O
H
OH
f
O
H
O
O
17
O
H
HN
S
R6
O
H
N
N
O
19
R5: a = p-CH3, b = p-F, c = m-F, d = o-F, e = p-CF3, f = m-CF3, g = m-OCF3, h = p-SCF3
R6: a = p-F, b = m-F, c = o-F, d = m-CF3, e = p-CF3
Scheme 4. The synthetic routes of five-membered cyclic carbamate artemisinin derivatives
18a-18h, 19a-19e. Reagents and conditions: (a) BF3-Et2O (10.0 equiv), diethyl ether, 0°C to r.t.,
90%; (b) Br2 (1.5 equiv), CCl4, H2O, r.t., 96%; (c) TEA (2.5 equiv), DCM, r.t., 91%; (d)
aminosulfonic acid (4.0 equiv), sodium chlorite (4.0 equiv), water/dioxane, r.t., 80%; (e) HATU
(1.2 equiv), DIPEA, DCM, r.t., 50-74%; (f) HATU (1.2 equiv), DIPEA, DCM/THF, r.t., 27- 45%.
5

2.2. In vitro anticancer evaluations
Twenty-six novel artemisinin derivatives were evaluated for their
antiproliferative activities against human neuroblastoma cancer cells (SH-SY5Y),
human lung adenocarcinoma cell (A549), breast cancer cells (MCF-7),
pheochromocytoma cells (PC12), human glioblastoma (U87MG), human brain
astrocyte tumor (U-118MG), colorectal carcinoma cells (HCT-116) and human
normal liver cell line (L02) by MTT assay. The IC50 for each compound with respect
to these cell lines was calculated and the results were summarized in Table 1. These
values represent the concentration at which a 50% decrease in cell growth is observed
after 72 h incubation at the presence of the drug and compared with the control cells
treated with ART and DHA, nerve cells treated with Temozolomide (TMZ), as well
as positive control cells treated with doxorubicin (DOX) under the same conditions.
As shown in Table 1, the derivatives showed enhanced antitumor activity
compared to ART and DHA, and lower cytotoxicity against L02 than DOX. For the
first series, compounds 12a-12h exhibited broad-spectrum antitumor effects on
MCF-7, PC12 and HCT-116 cells, with IC50 values between 0.12-12.36 μM. What
was more noteworthy was that these compounds exhibited excellent inhibitory effects
on HCT-116 cells, with IC50 values between 0.12-2.16 μM. Compound 12h exhibited
the strongest inhibitory activity, with an IC50 value of 0.12 μM, which was far
superior to the positive control DOX (1.86 μM)，and less cytotoxic to L02. Therefore
compound 12h was considered for further study. For the second series, some
compounds among 13a-13e showed certain inhibitory effects on MCF-7 and PC12
cells, with the IC50 values between 2.59-20.54 μM. Compared to the first series, the
activity was slightly reduced. But it was noteworthy that all the compounds of the
second series still showed good effects on HCT-116, with the IC50 values between
1.08-6.57 μM. In the third and fourth series (18a-18h, 19a-19e), compound 18e
displayed moderate antiproliferation activity against PC12, SH-SY5Y and U-118MG
cell lines, with the IC50 values between 4.50-8.84 μM, while the IC50 values for the
other compounds were larger than 10 μM.
Almost all the compounds tested had no effects on the brain tumor-related cell
lines U-118MG and U87MG (the IC50>40 μM), except that 18e showed an IC50 value
of 8.14 μM for U-118MG. This indicated that artemisinin derivatives containing
fluorine and piperazine groups had no significant anti-brain tumor effects. Similarly,
all the compounds did not show good inhibitory activity on A549 cells. However, for
the SH-SY5Y cell line, we were pleasant to find that 12g exhibited prominent effects
with an IC50 value of 0.29 μM, which was equivalent to that of the positive control
DOX (0.30 μM), but the cytotoxicity to L02 is much less than DOX, indicating that
12g is also a compound worthy of further research.
The structure activity relationship (SAR) studies revealed that the compounds
containing both fluorine and piperazine groups have more remarkable activities.
Among compounds (12a, 12b, 12d, 12f, 12g, 12h, 13a, 13b and 18e) with excellent
activity (IC50<10 μM), 12a, 12b, 12d, 12f, 12g, 12h, 13a, 13b belong to
six-membered ring artemisinin derivatives, and only 18e is five-membered ring
6

artemisinin derivative. Table 1 shows that the inhibitory activity of the six-membered
ring compounds in each cell line is superior to that of the five-membered ring
compound, which may indicate that retaining the six-membered ring structure of
artemisinin is necessary to improve the antitumor activity of its derivatives. When we
analyzed the selectivity of these compounds for cell lines, we found that all
six-membered ring compounds exhibited strong inhibitory activity against HCT-116
cells, indicating that the six-membered ring artemisinin derivatives containing both
fluorine and piperazine groups is promising for the treatment of colon cancer cells.
Among these compounds, only 13a and 13b belong to thiourea derivatives, while the
rest are carbamate derivatives. Carbamate derivatives displayed better bioactivity than
thiourea derivatives. The derivatives (18e, IC50<10 μM) containing fluorine groups
exhibited better bioactivity than that without fluorine groups (18a, IC50>40 μM),
indicating that fluorine groups in same substituent position could improve the
bioactivity of compounds. Moreover, we explored the SAR regarding the substituents
on the phenyl ring in each series. For the first series (12a-12h), the substition of
fluorine atom
both on para (12b) and ortho (12d) positions exhibited good
anticancer activity, especially in HCT-116 cell line, and the substitent on para position
(12b) displayed low cytotoxicity for L02 cells. The fluorine substitent on meta
position (12c) decreased the water-solubility of the compound. The trifluoromethoxy
group (-CF3) exhibited certain activities in the meta position (12f), while poor water
solubility in the para position (12e). For the second series (13a-13e), similarly to
compound 12b, the substition of fluorine atom on para position (13a) showed better
activity. Yet, the five-membered ring artemisinin derivative (18a-18h) showed
opposite results. The substituent offluorine atom on meta position (18c) showed good
activity against PC12 cells, but the water-solubility is poor when it was substituted on
para position (18b). Opposite to 12e, -CF3 on the para position (18e) showed
prominent anticancer activity.
It is noted that 12h and 12g are two isosteres and similar in their chemical
structural formulas. Coincidentally, these two compounds are both candidate
compounds that exhibit excellent activity and worthy of further study. Compared with
our previous work [14, 20], the activity and selectivity of the trifluoromethylthio
(-SCF3) group at the same substituent position are higher than trifluoromethoxy
(-OCF3) group, and the compounds with both fluorine and piperazine rings have
better antitumor activity than those containing only fluorine or piperazine rings. The
results indicate that trifluoromethylthio (-SCF3) group, fluorine and piperazine rings
could be meaningful pharmacophores for antitumor activity. In addition, the
water-solubility of five-membered ring artemisinin derivatives is opposite to
six-membered ring artemisinin derivatives, under the same substituent.
7

Table 1. Inhibitory activities of 12a-12h, 13a-13e, 18a-18h and 19a-19e against
seven cancer cell lines and one human normal liver cell.
Compound
IC50 Values（μM）a
MCF-7
A549
>40
PC12b
SH-SY5Y
U87MG
>40
U-118MG
>40
HCT-116
2.16±0.50
0.64±0.12
NT
L02
>40
12a
12b
12c
12d
12e
12f
3.01±0.36
2.62±0.21
>40
>40
H
O
O
12.36±1.51 11.73±0.70 11.10±1.97
>40
>40
28.41±1.02
NT
O
NTc
6.39±0.66
NT
NT
>40
NT
NT
NT
NT
NT
H
H
O
4.79±1.94
NT
>40
>40
>40
0.65±0.12
NT
14.49±1.20
NT
N
N
NT
NT
NT
R3
1.81±0.64
6.08±0.21
1.96±0.91
4.06±0.94
>40
>40
>40
>40
>40
>40
>40
5.91±0.71
2.11±0.38
>40
>40
>40
>40
0.81±0.26
0.51±0.01
0.12±0.05
1.08±0.33
1.67±0.15
2.73±0.14
7.15±0.56
21.03±1.93
12.46±0.10
2.09±0.51
19.54±0.26
>40
O
O
12
12g
12h
13a
13b
13c
0.29±0.02
4.03±0.11
>40
33.12±1.17 24.96±1.19
>40
>40
>40
>40
2.59±0.12
3.61±0.78
H
O
O
>40
33.74±0.97
>40
>40
O
H
H
O
>40
20.54±0.29 32.31±1.02
33.35±0.54
N
N
13d
13e
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
3.69±0.25
6.57±0.31
15.41±1.41
>40
R4
S
N
H
13
18a
18b
18c
>40
NT
>40
NT
27.52±0.01
NT
>40
NT
>40
NT
>40
NT
>40
NT
>40
NT
H
O
O
O
>40
>40
11.08±0.71
>40
>40
>40
>40
>40
>40
>40
NT
>40
>40
>40
O
H
O
O
N
N
R5
18d
18e
>40
>40
>40
>40
>40
>40
H
14.43±0.87
>40
>40
4.51±0.24
35.11±0.14
13.54±0.38
NT
8.09±0.01
>40
8.84±0.04
>40
>40
>40
O
18
18f
>40
>40
>40
18g
>40
>40
>40
>40
>40
>40
18h
19a
NT
NT
NT
NT
NT
NT
H
>40
>40
17.97±0.47
30.95±1.97
>40
>40
>40
>40
>40
>40
>40
>40
>40
NT
>40
>40
>40
O
O
O
H
19b
19c
>40
>40
>40
>40
>40
>40
HN
N
N
R6
O
H
>40
>40
>40
>40
>40
>40
S
O
19d
19e
>40
>40
23.33±0.24
>40
>40
>40
32.26±2.13
>40
>40
19
>40
>40
>40
>40
>40
ART
DHA
DOX
TMZ
>40
>40
>40
>40
>40
>40
>40
>40
>40
8.36±0.11
0.08±0.01
>40
>40
>40
9.11±0.88
1.86±0.12
NT
9.38±0.13
0.98±0.04
NT
0.35±0.01
NT
0.31±0.13
NT
0.30±0.03
>40
NT
>40
>40
a Values are average of three biological replicates and deviation from the average is less than 5% of the average
value.
b NGF-differentiated PC12.
c NT: not tested. 12c, 12e, 18b and 18h were not tested because of poor solubility in DMSO.
Next, we carried out selectivity index (SI) analysis to test the selectivity of
compound 12h and 12g, dividing the IC50 value for L02 by that for cancer cell lines
SH-SY5Y and HCT-116. According to the data in Table 2, the selectivity of both
compounds was better than the positive control DOX. Since the maximum SI of 12h
was 103.83 against HCT-116 cells and was higher than that of 12g (72.52 against
SH-SY5Y cells), compound 12h was chosen for further mechanisms study. As shown
in Fig. 2, compound 12h caused a significant inhibitory effect on the growth of
HCT-116 cells. The effect occurred in a concentration- and time-dependent manner.
8

Table 2. The selectivity of compounds 12g and 12h for L02 over the SH-SY5Y and
HCT-116 cell lines.
Compound
IC50 Values（μM）
SI
SI
(IC50 L02/
IC50SH-SY5Y)
72.52
(IC50 L02/
IC50HCT-116)
41.24
SH-SY5Y
HCT116
L02
12g
12h
0.29±0.07
4.03±0.11
0.30±0.03
0.51±0.01
0.12±0.05
1.86±0.12
21.03±1.93
12.46±0.10
0.23±0.03
3.34
3.27
103.83
0.53
DOX
a Values are average of three biological replicates and deviation from the average is less than 5% of the average
value.
9

Fig.2. Quantification of inhibitory effects of compound 12h. Inhibition rate of
HCT-116 cells was tested at 24, 48 and 72 h after treatment with compound 12h
（0.1875, 0.375, 0.75, 1.25, 2.5 and 5 μM）. Data were expressed as mean ± SD (n=
3).
2.3. Cell cycle arrest induced by compound 12h
Cell-cycle arrest is one of anti-tumor mechanisms. To investigate whether the
compound 12h caused the cell-cycle arrest, the DNA content of cell nuclei was
detected by flow cytometry at the indicated time. As shown in Fig. 3, compound 12h
led to significant (P<0.05) accumulation of cells at the G1 phase from 49.6% (0 μM)
to 70.3% (1 μM), 75.9% (2 μM) and 81.00% (4 μM). The percentage of cells in S and
G2 phase were decreased from 28.9% (0 μM) to 6.6% (4 μM) and 21.2% (0 μM) to
11.9% (4 μM), respectively. It is confirmed that the cell DNA replication was
inhibited and the cell cycle was arrested in G1 phase by 12h.
A
B
10

Fig.3. Representative histograms and quantitative analysis showing cell cycle arrest
induced by compound 12h. (A) Cells were treated with 1, 2 and 4 μM of 12h for 24 h,
stained with PI before flow cytometry analysis. (B) The cell distribution rate in G1, S
and G2/M phase. Data were expressed as mean ± SD (n= 3), *P < 0.05, **P < 0.01
versus the controls (0 μM).
2.4. Apoptosis induced by compound 12h
The induction of cell apoptosis is one of the effective approaches in cancer
treatment [34]. Here, we explored whether the anti-colon cancer activity of compound
12h was associated with apoptosis. As shown in Fig. 4A, after treatment with various
concentrations of compound 12h, the percentages of apoptotic cells were 8.0%,
23.2%, 26.2%, 35.1%, 38.4% and 57.1% (Q1UR+Q1LR), respectively, indicating that
compound 12h can induce HCT-116 cells apoptosis and inhibit cells proliferation.
Furthermore, Hoechst 33258 was used to stain apoptotic cells immediately. In Fig.
4B, HCT-116 cells showed typical apoptosis features such as improved brightness of
apoptotic body, cell shrinkage, nuclear condensation (Fig. 4B enlarge part). These
nuclear morphological changes are different from ferroptosis [35], which mainly
damages the mitochondria instead of the nucleus. Our results indicated 12h probably
induced cell death in a different way. The quantitative analysis of apoptotic cells
indicated that 12h induced obvious cell apoptosis in a dose-dependent manner (Fig.
4A).
11

A
B
12

Fig.4. Pro-apoptotic effect of compound 12h on HCT-116 cell. (A) Representative
scatter diagram and quantitative analysis of apoptotic cells (the upper region).
HCT-116 cell were treated with compound 12h at 0, 0.25, 0.5, 1, 2 and 4 μM for 24
h and stained with Annexin V- FITC/PI before flow cytometry analysis. Data were
expressed as mean ± SD (n= 3), *P < 0.05, **P < 0.01 versus the controls (0 μM).
(B) Representative microscopy images of HCT-116 cells. Cells were treated with
compound 12h at 0, 0.25, 0.5, 1, 2 and 4 μM for 48 h, stained with Hoechst 33258
and visualized under a fluorescent microscope (×20). The blue color indicates cell
nuclei. Apoptotic bodies are pointed by the white arrows, and nomal cells are pointed
by the red arrows.
2.5. The change of mitochondrial membrane potential
Mitochondria are involved in a variety of cellular processes and functions,
playing a critical role in determining cell survival or death [36]. The change of
mitochondrial membrane potential (ΔΨm) is related to cell apoptosis. To verify
whether compound 12h acts on mitochondria, we performed ΔΨm staining with JC-1,
which formed orange fluorescent aggregates and accumulated in the mitochondria of
healthy cells. When the ΔΨm is low, JC-1 produces green fluorescence as a monomer.
Fig. 5A displays that 12h induces a decrease of cell population with intense orange
fluorescence in a concentration-dependent manner and an accompanying increase of
cell population with intense green fluorescence. These data indicate that 12h causes a
loss of ΔΨm, and notably, a rigorous mitochondrial damage response.
We also used the TMRE Mitochondrial Membrane Potential Assay Kit to
perform flow cytometry analysis and quantitatively measure ΔΨm. HCT-116 cells
treated with 12h and stained with TMRE. Fig. 5A shows that as the concentration
increases, the fluorescence intensity decreases, indicating that compound 12h reduces
ΔΨm in a concentration-dependent manner. All the results prove compound 12h has a
powerful influence on mitochondria, and the anti-tumor effect of compound 12h
maybe through the mitochondrial-mediated apoptosis pathway.
13

A
B
14

Fig.5. Effects of 12h on ΔΨm. (A) Representative microscopy images showing in
ΔΨm in HCT-116 cells. Cells were treated with compound 12h at 0, 0.5, 1 and 2 μM
for 48 h, stained with JC-1 and visualized under a fluorescent microscope (×20). The
red color indicates JC-1 gathered in the mitochondria of healthy cells. The green
indicates mitochondrial membrane is damaged and JC-1 is released as a monomer.
(B) Representative histograms and quantitative analysis showing the change of ΔΨm.
HCT-116 cell were treated with compound 12h at 0, 0.25, 0.5, 1, 2 and 4 μM for 24 h
and stained with TMRE before flow cytometry analysis. Data were expressed as mean
± SD (n= 3). **p <0.01 versus the controls (0 μM).
2.6. Effects on reactive oxygen species (ROS) and intracellular free calcium
The mitochondrial function disturbance is an important event in apoptosis via the
mitochondria-mediated pathway, which is commonly involved in the cell death
stimuli. It can cause downstream caspase cascade activation and changes of
mitochondrial permeability, intracellular Ca2+ concentration and ROS level [37].
Using DCFH fluorescence intensity as an index of ROS accumulation, we
demonstrated that the cells treated with compound 12h showed a prominent
accumulation of ROS compared to the control cells (0 μM). ROS can initiate
oxidative stress and ultimately cause cellular damages, which lead to destructive
actions on both DNA and proteins [34]. Therefore, compound 12h can increase the
ROS level and cause oxidative damage to HCT-116 cells.
The FLOU-3 fluorescence was used to test the intracellular free calcium levels.
Fig.6B showed that 12h induced a gradual increase in intracellular free Ca2+ levels in
a concentration-dependent manner. Compared with the ROS levels, the effect of
compound 12h on intracellular free Ca2+ levels is more prominent. Therefore, it is
most likely that compound 12h stimulates cells to cause mitochondrial membrane
damage. In turn, the ΔΨm decreases, meanwhile Ca2+ and reactive oxygen flow out.
15

A
B
Fig.6. Effects on ROS and intracellular calcium levels. (A) Representative histograms
and quantitative analysis indicating ROS levels by flow cytometry. HCT-116 cells
treated with 12h at the concentrations of 0, 0.5, 1, 2, 4 and 8 μM for 12 h and loaded
with DCFH-DA fluorescent probe before analysis. (B) Representative histograms and
quantitative analysis indicating intracellular free calcium levels by flow cytometry.
HCT-116 cells treated with 12h at the concentrations of 0, 0.5, 1, 2 and 4 μM for 24 h,
before the fluorescence of FLOU-3 was determined. ROS or Ca2+ levels were
represented as the flurescence intensity. Data were expressed as mean ± SD (n= 3). *p
<0 .05 and **p <0.01 versus the controls (0 μM).
2.7. Effects of 12h on apoptosis-related proteins
To further explore the potential molecular mechanisms of 12h-induced apoptosis,
the effects of 12h on the expression level of proteins related with mitochondria
mediated apoptosis were examined. It has been demonstrated that BH3-only family
proteins play a crucial role in regulating the intrinsic apoptotic pathway. When cells
are stimulated by apoptotic factors, BH3-only family in the cytoplasm can sense
various apoptotic signals transmitted upstream and activate corresponding protein
expressions, including pro-apoptotic proteins (Bax, Bad) and anti-apoptotic
proteins(Bcl-2). Bax opens the osmotic transport pores in the mitochondrial
membrane, releasing cytochrome C which combines with apoptosis protein activating
16

factor (Apaf-1) and ATP / dATP to form an apoptosis complex. This complex recruits
Pro-caspase-9, leading to the hydrolytic activation of Pro-caspase-9, causing the
downstream caspase cascade to activate and induce apoptosis [37]. The expression
levels of mitochondrial pathway related proteins in HCT-116 cells pretreated with
12h are shown in Fig. 7. The immunoblot results showed that treatment with
compound 12h decreased Bcl-2 expression and increased Bax in
a
concentration-dependent manner (Fig. 7A). As in Fig. 7B, the proportion of
Bax/Bcl-2 increased. Since the Bax/ Bcl-2 ratio is the "molecular switch" that initiates
apoptosis, we can infer that compound 12h can induce apoptosis by elevating the ratio
of Bax/Bcl-2. Compared with the control group, the expressions of cleaved caspase-9
and caspase-3 were also up-regulated, indicating that the mitochondrial pathway is
activated. All these molecular expression results indicate that compound 12h can
induce apoptosis of HCT-116 cells through a mitochondrial-mediated pathway.
A
12h (μM)
0
0.125 0.25
0
0.125
0.25
Caspase 9
Procaspase 3
Caspase 3
Bcl-2
Bax
17
β-acting

Fig.7. The expression of mitochondrial-mediated pathway related protein in HCT-116
cells. Cells were treated with 0, 0.125 and 0.25 μM of compound 12h for 48 h and
β-acting was used as the internal control. (A) The expression of caspase and Bcl-2
family proteins detected by western blot. (B) The protein levels of caspase-9,
procaspase-3, caspase-3, and the rate changes of Bax/Bcl-2 in treated HCT-116 cells.
The two panels are independent replicates of the same experiment.
2.8. Effects on tubule morphology and the HCT-116 migration
The establishment of the vascular system is an important process for the growth
and survival of tumor cells. The growth of primary tumors and secondary tumor cells
requires a continuous supply of nutrients from blood vessels. Most artemisinin
derivatives possess vascular disrupting activity, which is thought to disrupt
microtubule dynamics to induce the changes in endothelial cell morphology. To
evaluate the anti-vascular activity of compound 12h, we used primary human
umbilical vein endothelial cells (HUVEC) culture assay to assess the ability of 12h to
inhibit vascular activity. As shown in Fig.8A, after treatment with 12h for 24 h, we
18

did not find the number of microtubules reduced (Fig.8S) or microtubule disrupted
compared with the control group, indicating that compound 12h did not show
anti-vascular activity. Because colorectal cancer is a malignant tumor, easy to
metastasize to lymph, liver, lung, bone and other tissues, endangering life safety, we
tested the effect of compound 12h on cell migration. According to the results of the
scratch experiment (Fig. 8B), 12h can obviously inhibit the migration of HCT-116
cells, and in turn inhibit the metastasis and spread of colon cancer cells to other
tissues. It is promising for clinical treatment of early colorectal cancer.
A
19

B
Fig.8. Effects on tube formation and the HCT-116 migration. (A) Images depicting
the formation of HUVEC capillary-like tubular network by treatment with 12h (0, 0.5,
1, 2 and 4 μM) for 24 h. (B) Scratches were created with a sterile 20 μL pipette tip
and images were captured at 0 and 24 h after treatment with 12h (0, 0.25, 0.5, 1 and 2
μM).
3. Conclusion
In summary, a series of novel artemisinin derivatives containing fluorine atoms
and piperazine group were synthesized and their cytotoxic activity against seven
tumor cell lines and one human normal liver cell line were determined. Compared to
ART and DHA, most of derivatives showed improved bioeffects against tumor cell
lines and yet less cytotoxicity towards normal cells. The SAR study revealed that
six-membered cyclic carbamate derivatives containing both fluorine and piperazine
groups tended to have better inhibitory activities. However, the change of
artemisinin's six-membered lactone into a five-membered lactone did not increase its
activity, and the water-solubility of five-membered ring artemisinin derivatives is
opposite to six-membered ring artemisinin derivatives, under the same substituent. In
particular, the compound 12h was the most active derivative and effectively
suppressed the tumor growth in vitro with an IC50 of 0.12 μM with low cytotoxicity to
L02. It also displayed prominent selectivity compared with the control DOX. Its
mechanisms of action against HCT-116 cell line was studied and revealed. Compound
12h could inhibit DNA replication and arrest cell cycle in G1 phase and induce cell
apoptosis in a dose-dependent manner. The change of mitochondrial membrane
potential proved that 12h could damage mitochondria and increase the levels of
intracellular ROS and Ca2+. Furthermore, 12h could up-regulate the expression levels
of Bax, cleaved caspase-9, cleaved caspase-3 and down-regulate Bcl-2. Compound
12h did not show the rupture of microtubule, but inhibited the migration of HCT-116
cells. From all above, compound 12h has a selective and significant anti-cancer
activity, inhibiting HCT-116 cells migrate and inducing apoptosis by a mechanism of
the mitochondria-mediated pathway. The compound 12h may merit further
20

investigation as a potential therapeutic agent for colorectal cancer.
4. Experimental section
4.1. Materials and methods
All chemicals were purchased from commercial source and used without further
purification unless otherwise stated. The reactions were monitored by TLC using
ultraviolet (UV) light at 254 nm; analytical thin-layer chromatography was carried out
on silica gel GF254. The products were purified by column chromatography by using
silica gel (200-300 mesh). Reagents were all analytically or chemically pure. All the
solvents and liquid reagents were dried by standard methods in advance or distilled
1
before use. H NMR spectra were obtained on an Agilent 400MR, Varian 500MR or
Bruker Avance 600MR spectrometer at ambient temperature. Data were reported as
follows: chemical shift on the δ scale using residual proton solvent as internal
standard [δ 0.00 (TMS)], multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q
13
= quartet, m = multiplet), integration, and coupling constant (s) in hertz. C NMR
spectra were obtained with proton decoupling on an Agilent 400MR (100 MHz) or
Varian 500MR (125 MHz) spectrometer and were reported in ppm with residual
solvent for internal standard [δ 77.16 (CDCl3)]. The NMR data was processed by
software Mest Re-Nova (Ver. 9.0.0.12821, mestrelab research S.L.). High resolution
mass spectra were obtained on an Agilent spectrometer. Melting point was determined
by WRS-2 Digital Melting Point Apparatus and was uncorrected.
4.2. Chemical synthesis
4.2.1. General procedure for the preparation of aryl chloroformates (2a-2h)
1.0 g (3.37 mmol) BTC was dissolved in 30 mL dry CH2Cl2, and cooled in ice
bath. 10.11 mmol substituted phenol was slowly added with stirring, then the mixed
solution of 10.11mmol triethylamine and 10 mL dry CH2Cl2 was dropped at 0-5°C.
The solution was stirred for 1 h at 0-5°C, then stirred for 2 h at room temperature. The
crude product was diluted with CH2Cl2 and washed with HCl (1 N), saturated
NaHCO3, and finally with saturated NaCl solution. The collected organic layer was
dried with Na2SO4 and the solvent was removed under vacuum to give the product
which was used in the next step without further treatment [27,28].
4.2.2. General procedure for the preparation of 1-(tert-butyl) 4-phenyl
piperazine-1,4-dicarboxylates (3a-3h)
To the solution of 1-BOC-piperazine (1 g, 5.36 mmol) in dichloromethane (5
mL) and triethylamine (1.1 equiv, 817 µL) was added compound 2 (1 equiv). The
reaction mixture was stirred at room temperature until TLC showed the completion of
reaction, the reaction mixture was diluted with CH2Cl2, then washed with brine, the
organic layer was dried with Na2SO4, filtered and evaporated in vacuo to afford crude
product which was purified through column chromatography to give compounds
3a-3h [29].
4.2.2.1. 1-(tert-butyl) 4-(p-tolyl)piperazine-1,4-dicarboxylate (3a)
21

The product was obtained as white solid (1.08 g, 63%), m.p.: 145.5-146.4°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.15 (d, J = 8.2 Hz, 2H, Ph-3 and Ph-5), 6.98
(d, J = 8.3 Hz, 2H, Ph-2 and Ph-6), 3.57 (d, J = 54.0 Hz, 8H, piperazine-H ), 2.33 (s,
3H, CH3), 1.49 (s, 9H, Boc-H) [38].
4.2.2.2. 1-(tert-butyl) 4-(4-fluorophenyl)piperazine-1,4-dicarboxylate (3b)
The product was obtained as white solid (0.94 g, 54%), m.p.: 127.7-128.7°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.09-7.00 (m, 4H, Ph-H), 3.57 (d, J = 48.1
Hz, 8H, piperazine-H), 1.49 (s, 9H, Boc-H) [39].
4.2.2.3. 1-(tert-butyl) 4-(3-fluorophenyl)piperazine-1,4-dicarboxylate (3c)
1
The product was obtained as white solid (0.82 g, 47%), m.p.: 65.3-67.5°C. H
NMR (400 MHz, Chloroform-d) δ ppm: 7.32 (dd, J = 15.0, 8.0 Hz, 1H, Ph-5),
6.96-6.86 (m, 3H, Ph-2 Ph-4 and Ph-6), 3.47 (d, J = 48.3 Hz, 8H, piperazine-H), 1.49
(s, 9H, Boc-H) [38].
4.2.2.4. 1-(tert-butyl) 4-(2-fluorophenyl)piperazine-1,4-dicarboxylate (3d)
1
The product was obtained as white solid (0.87 g, 50%), m.p.: 69.7-74.3°C. H
NMR (500 MHz, Chloroform-d) δ ppm: 7.19-7.10 (m, 4H, Ph-H), 3.59 (d, J = 75.8
13
Hz, 8H, piperazine-H), 1.48 (s, 9H, Boc-H); C NMR (125 MHz, Chloroform-d) δ
ppm: 154.57 (C=O), 154.47 (d, J = 249.0 Hz, Ph-2), 152.71 (C=O), 138.74 (d, J =
12.6 Hz, Ph-1), 126.69 (d, J = 7.1 Hz, Ph-4), 124.34 (d, J = 3.7 Hz, Ph-5), 124.08
(Ph-6), 116.59 (d, J = 18.4 Hz, Ph-3), 80.31 (Boc-C), 44.62 (piperazine-C), 44.01
(piperazine-C), 42.98 (piperazine-C), 28.39 (CH3). HRMS (ESI) Calcd. for
[C16H21FN2O4Na+]: 347.1378, Found, 347.1344.
4.2.2.5. 1-(tert-butyl) 4-(4-(trifluoromethyl)phenyl)piperazine-1,4-dicarboxylate
(3e)
The product was obtained as white solid (1.10 g, 55%), m.p.: 161.5-168.4°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.60 (d, J = 8.5 Hz, 2H, Ph-3 and Ph-5), 7.21
(d, J = 8.4 Hz, 2H, Ph-2 and Ph-6), 3.56 (d, J = 52.4 Hz, 8H, piperazine-H), 1.46 (s,
9H, Boc-H); 13C NMR (100 MHz, Chloroform-d) δ ppm: 154.49 (C=O), 153.66
(Ph-1), 152.81 (C=O), 127.49 (q, J = 32.5 Hz, Ph-C), 126.60 (q, J = 3.5 Hz, Ph-C),
123.89 (q, J = 270.3 Hz, CF3), 122.05 (Ph-C), 80.35 (Boc-C), 44.39 (piperazine-C),
43.76 (piperazine-C), 42.79 (piperazine-C), 28.29 (CH3). HRMS (ESI) Calcd. for
[C17H21F3N2O4Na+]: 397.1346, Found, 397.1305.
4.2.2.6. 1-(tert-butyl) 4-(3-(trifluoromethyl)phenyl)piperazine-1,4-dicarboxylate
(3f)
1
The product was obtained as white solid (1.20 g, 61%), m.p.: 75.2-79.6°C. H
NMR (500 MHz, Chloroform-d) δ ppm: 7.49 (d, J = 6.8 Hz, 2H, Ph-4 and Ph-6), 7.40
(s, 1H, Ph-2), 7.33 (d, J = 6.6 Hz, 1H, Ph-5), 3.59 (d, J = 65.9 Hz, 8H, piperazine-H),
1.49 (s, 9H, Boc-H); 13C NMR (125 MHz, Chloroform-d) δ ppm: 154.57 (C=O),
153.04 (Ph-1), 151.29 (C=O), 131.84 (q, J = 32.9 Hz, Ph-3), 129.87 (Ph-5), 125.29
(Ph-6), 123.57 (q, J = 270.6 Hz, CF3),122.25 (q, J = 3.5 Hz, Ph-C), 119.00 (q, J = 3.8
Hz, Ph-C), 80.43 (Boc-C), 44.46 (piperazine-C), 43.86 (piperazine-C), 43.14
(piperazine-C), 28.39 (CH3). HRMS (ESI) Calcd. for [C17H21F3N2O4Na+]: 397.1346,
Found, 397.1295.
4.2.2.7. 1-(tert-butyl) 4-(3-(trifluoromethoxy)phenyl)piperazine-1,4-dicarboxylate
22

(3g)
The product was obtained as white solid (1.30 g, 64%), m.p.: 132.6-136.2°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.38 (t, J = 8.3 Hz, 1H, Ph-5), 7.08 (d, J =
8.2 Hz, 2H, Ph-4 and Ph-6), 7.04 (s, 1H, Ph-2), 3.58 (d, J = 62.1 Hz, 8H,
piperazine-H), 1.49 (s, 9H, Boc-H); 13C NMR (125 MHz, Chloroform-d) δ ppm:
154.58 (Ph-C), 152.97 (C=O), 151.89 (Ph-C), 149.50 (C=O), 130.00 (Ph-C), 120.40
(q, J = 255.9 Hz, OCF3), 120.21 (Ph-C), 117.80 (Ph-C), 115.00 (Ph-C), 80.42
(Boc-C), 44.46 (piperazine-C), 43.85 (piperazine-C), 43.03 (piperazine-C), 28.39
(CH3). HRMS (ESI) Calcd. for [C17H21F3N2O5Na+]: 413.1295, Found, 413.1285.
4.2.2.8. 1-(tert-butyl) 4-(4-((trifluoromethyl)thio)phenyl)piperazine-1,4-
dicarboxylate (3h)
The product was obtained as white solid (1.30 g, 60%), m.p.: 110.1-113.1°C.1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.66 (d, J = 8.5 Hz, 2H, Ph-3 and Ph-5), 7.19
(d, J = 8.6 Hz, 2H, Ph-2 and Ph-6), 3.58 (d, J = 63.4 Hz, 8H, piperazine-H), 1.49 (s,
9H, Boc-H); 13C NMR (125 MHz, Chloroform-d) δ ppm: 154.57 (C=O), 153.45
(C=O), 152.85 (Ph-1), 137.68 (Ph-C), 129.45 (q, J = 306.5 Hz, SCF3), 122.78 (Ph-C),
120.86 (Ph-C), 116.54 (Ph-C), 80.46 (Boc-C), 44.49 (piperazine-C), 43.85
(piperazine-C), 42.98 (piperazine-C), 28.39 (CH3). HRMS (ESI) Calcd. for
[C17H21F3N2O4SNa+]: 429.1066, Found, 429.1008.
4.2.3. General procedure for the preparation of phenyl piperazine-1-carboxylates
(4a-4h)
Trifluoroacetic acid (2.5 mL) was added dropwise into a solution of 3a-3h (2
mmol) in 10 mL of dichloromethane. The reaction was stirred at room temperature for
1 h. The solvent was removed under vacuum to give residual, then the saturated
potassium carbonate solution was added dropwise in an ice bath, and the solution was
extracted with dichloromethane (3×20 mL), the organic layer was dried over
anhydrous Na2SO4, and concentrated under vacuum to obtain the product 4a-4h
[30,31].
4.2.4.
General
procedure
for
the
preparation
of
tert-butyl
4-(phenylcarbamothioyl) piperazine-1-carboxylate (6a-6f)
To the solution of 1-BOC-piperazine (0.5 g, 2.68 mmol) in dry dichloromethane
(20 mL) was added the corresponding isothiocyanate (1.2 equiv). The reaction
mixture was stirred at room temperature until TLC showed the completion of
reaction, the reaction was diluted with CH2Cl2 and then washed with HCl (1 N),
saturated NaHCO3, and finally with saturated NaCl solution. The collected organic
layer was dried with Na2SO4 and the solvent was removed under vacuum. The
compounds were purified by flash chromatography on silica gel using the appropriate
eluent [32].
4.2.4.1. tert-butyl 4-((4-fluorophenyl)carbamothioyl)piperazine-1-carboxylate
(6a)
The product was obtained as white solid (0.90 g, 99.6%), m.p.: 147.4-149.3°C.
23

1H NMR (400 MHz, Chloroform-d) δ ppm: 7.15 (dd, J = 8.8, 4.8 Hz, 2H, Ph-H), 7.03
(m, 3H, Ph-H, NH), 3.86 (s, 4H, piperazine-H ), 3.55 (m, 4H, piperazine-H ), 1.46 (s,
9H, Boc-H) [40].
4.2.4.2. tert-butyl 4-((3-fluorophenyl)carbamothioyl)piperazine-1-carboxylate
(6b)
The product was obtained as white solid (0.84 g, 92%), m.p.: 155.0-158.8°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.94 (s, 1H, NH), 7.14 (dd, J = 14.7, 7.6 Hz,
1H, Ph-H), 6.84 (m, 2H, Ph-H), 6.74 (t, J = 7.9 Hz, 1H, Ph-H), 3.69 (s, 4H,
piperazine-H), 3.36 (m, 4H, piperazine-H), 1.37 (s, 9H, Boc-H) [40].
4.2.4.3. tert-butyl 4-((2-fluorophenyl)carbamothioyl)piperazine-1-carboxylate
(6c)
The product was obtained as white solid (0.77 g, 85%), m.p.: 187.3-189.3°C.1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.57 (t, J = 6.9 Hz, 1H, Ph-6), 7.20-7.06 (m,
4H, Ph-H and NH), 3.91 (s, 4H, piperazine-H), 3.56 (m, 4H, piperazine-H), 1.47 (s,
13
9H, Boc-H); C NMR (125 MHz, Chloroform-d) δ ppm: 182.86 (C=S), 155.15 (d, J
= 247.8 Hz, Ph-2), 154.55 (C=O), 127.60 (d, J = 11.1 Hz, Ph-5), 126.64 (Ph-4),
126.19 (Ph-6), 124.15 (d, J = 3.6 Hz, Ph-1), 115.80 (d, J = 19.7 Hz, Ph-3), 80.55
(Boc-C), 48.33 (piperazine-C), 42.94 (piperazine-C), 41.95 (piperazine-C), 28.36
(CH3). HRMS (ESI) Calcd. for [C16H23FN3O2SH+]: 340.1490, Found, 340.1487.
4.2.4.4. tert-butyl 4-((3-(trifluoromethyl)phenyl)carbamothioyl)piperazine-1-
carboxylate (6d)
The product was obtained as white solid (1.00 g, 96%), m.p.: 162.5-163.7°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.47-7.39 (m, 5H, Ph-H, NH), 3.86 (s, 4H,
piperazine-H), 3.54 (m, 4H, piperazine-H), 1.47 (s, 9H, Boc-H); 13C NMR (125 MHz,
Chloroform-d) δ ppm: 183.10 (C=S), 154.54 (C=O), 140.28 (Ph-1), 131.45 (q, J =
32.8 Hz, Ph-C), 129.54 (Ph-C), 126.85 (Ph-C), 123.70 (q, J = 270.8 Hz, CF3),121.90
(q, J = 3.6 Hz, Ph-C), 120.09 (q, J = 3.6 Hz, Ph-C), 80.67 (Boc-C), 48.69
(piperazine-C), 43.09 (piperazine-C), 42.21 (piperazine-C), 28.36 (CH3). HRMS
(ESI) Calcd. for [C17H22F3N3O2S2H+]: 390.1458, Found, 390.1438.
4.2.4.5. tert-butyl 4-((4-(trifluoromethyl)phenyl)carbamothioyl)piperazine-1-
carboxylate (6e)
The product was obtained as white solid (1.04 g, 99.2%), m.p.: 174.5-175.4°C.
1H NMR (400 MHz, Chloroform-d) δ ppm: 7.70 (s, 1H, NH), 7.55 (d, J = 8.4 Hz, 2H,
Ph-3 and Ph-5), 7.24 (d, J = 8.3 Hz, 2H, Ph-2 and Ph-6), 3.83 (s, 4H, piperazine-H),
3.84 (m, 4H, piperazine-H), 1.47 (s, 9H, Boc-H) [40].
4.2.4.6. tert-butyl 4-((4-(trifluoromethoxy)phenyl)carbamothioyl)piperazine-1-
carboxylate (6f)
The product was obtained as white solid (1.08 g, 99%), m.p.: 185.9-194.8°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.30 (s, 1H, NH), 7.23-7.17 (m, 4H, Ph-H),
3.86 (s, 4H, piperazine-H), 3.54 (m, 4H, piperazine-H), 1.47 (s, 9H, Boc-H); 13C
NMR (125 MHz, Chloroform-d) δ ppm: 183.23 (C=S), 154.54 (C=O), 146.32 (Ph-C),
138.27 (Ph-C), 124.98 (Ph-C), 121.66 (Ph-C), 120.43 (q, J = 255.8 Hz, OCF3), 80.63
(Boc-C), 48.61(piperazine-C), 43.05(piperazine-C), 41.91(piperazine-C), 28.36(CH3).
HRMS (ESI) m/z calcd. for [C17H22F3N3O3SH+]: 406.1407, Found, 406.1402.
24

4.2.5. General procedure for the preparation of N-phenylpiperazine-1-
carbothioamides (7a-7f)
Trifluoroacetic acid (2.5 mL) was added dropwise into a solution of 6a-6f (2
mmol) in 10 mL of dichloromethane. The reaction was stirred at room temperature for
1 h. The solvent was removed under vacuum to give residual, then the saturated
potassium carbonate solution was added dropwise in an ice bath, and the solution was
extracted with dichloromethane (3×20 mL), the organic layer was dried over
anhydrous Na2SO4, and concentrated under vacuum to afford the product 7a-7f
[30,31].
4.2.6. General procedure for the preparation of dihydroartemisinin 9
Artemisinin (5 g, 17.73 mmol) was dissolved in anhydrous methanol (120 mL)
and cooled in an ice bath to 0–5°C. To the solution was added sodium borohydride (3
equiv, 2.02 g) in portion within 30 minutes. The mixture was kept stirring for
approximately 30 minutes at 0–5°C, neutralized to PH 7.0 using acetic acid, The
reaction solution was concentrated to remove most of methanol, diluted with cold
water (60 mL) and stirred for 15 min at room temperature. The precipitate was
collected, washed with water (3×20 mL) and dried to afford product as white solid,
1
4.51 g, m.p.: 146.7-148.5°C. yield: 89%. H NMR (500 MHz, Chloroform-d) δ 5.61
(s, 1H, 12a-H), 5.29 (t, J = 3.3 Hz, 1H, 10-H), 2.70-2.60 (m, 2H, 6-H and 9-H), 2.38
(td, J = 13.6, 4.0 Hz,1H, 8a-H), 2.07-2.02 (m, 1H, 5a-H), 1.93-1.79 (m, 3H),
1.68-1.62 (m, 1H), 1.56-1.46 (m, 2H), 1.43 (s, 3H, 3-CH3), 1.41-1.22 (m, 2H), 0.96
(d, J = 7.9 Hz, 6H, 6-CH3 and 9-CH3) [20].
4.2.7. General procedure for the preparation of trimethyl(((3R,5aS,6R,8aS,
9R,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isoc
hromen-10-yl)oxy)silane 10
To the solution of dihydroartemisinin 9 (1 g, 3.52 mmol) and triethylamine (2
equiv, 0.36 mg) in anhydrous dichloromethane (15 mL) at 0–5°C was added dropwise
a cold solution of trimethylchlorosilane (2 equiv, 1.14 g) in anhydrous
dichloromethane (30 mL). After 15 minutes, the reaction was kept stirring for
approximately 0.5 h at room temperature. Then solvent was removed under vacuum.
The white residue was diluted by petroleum ether (30 mL), then the organic layers
was washed with H2O (3×20 mL), dried over anhydrous Na2SO4 and concentrated to
1
afford product as white solid, 1.18 g, yield: 94%. m.p.: 85.6-87.7°C. H NMR (500
MHz, Chloroform-d) δ 5.32 (s, 1H, 12a-H), 4.76 (d, J = 9.0 Hz, 1H, 10-H), 2.41-2.28
(m, 2H, 6-H and 9-H), 2.01 (td, J = 14.5, 3.0 Hz,1H, 8a-H), 1.91-1.84 (m, 1H, 5a-H),
1.77-1.65 (m, 2H), 1.56-1.43 (m, 2H), 1.41 (s, 3H,3-CH3 ),1.34-1.21 (m, 3H),0.95 (d,
J = 6.2 Hz, 3H, 6-CH3 ), 0.86 (d, J = 7.2 Hz, 3H, 9-CH3), 0.19 (s, 9H) [20].
4.2.8. General procedure for the preparation of (3R,5aS,6R,8aS,9R,10R,12S,
12aR)-10-bromo-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]
isochromene 11
25

A cold stirred solution of compound 10 (0.2 g, 0.56 mmol) indichloromethane (5
mL) was treated drop wise with a cold solution of bromotrimethylsilane (1.02 equiv)
in dichloromethane (2 mL) at 0°C. After completion (confirmed by TLC), the reaction
solution was used in the next step directly without further purification.
4.2.9. General procedure for the preparation of compounds (12a-12h)
A cold stirred solution of amines 4a-4h (0.95 equiv) in anhydrous
dichloromethane (5 mL) was treated drop wise with a cold solution of triethylamine
(2 equiv, 0.12 g). The above solution (containing 11) was transferred to a stirred
solution of amines 4a-4h. The reaction mixture was stirred overnight at room
temperature, then diluted by dichloromethane (20 mL), washed with saturated sodium
bicarbonate solution (3×30 mL) and water (3×30 mL), dried over anhydrous Na2SO4,
concentrated under reduced pressure to yield the crude product, which was further
purified by chromatography on silica gel to obtain the products 12a-12h.
4.2.9.1. p-tolyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-trimethyldecahydro-12H-
3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)piperazine-1-carboxylate (12a)
The product was obtained as white solid (60 mg, 23%), m.p.: 139.0-145.8°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.14 (d, J = 8.2 Hz, 2H, Ph-3 and Ph-5), 6.98
(d, J = 8.4 Hz, 2H, Ph-2 and Ph-4), 5.29 (s, 1H, 12a-H), 4.06 (d, J = 10.2 Hz, 1H,
10-H), 3.70-3.48 (m, 4H, piperazine-H), 3.04 (s, 2H, piperazine-H), 2.75-2.69 (m, 2H,
piperazine-H), 2.65-2.56 (m, 1H, 9-H), 2.37 (dd, J = 14.0, 3.9 Hz, 1H, 6-H), 2.33 (s,
3H, Ph-CH3), 2.05-1.97 (m, 1H, 8a-H), 1.90-1.85 (m, 1H, 5a-H), 1.75-1.68 (m, 2H),
1.59-1.43 (m, 3H), 1.41 (s, 3H, 3-CH3), 1.38-1.20 (m, 3H), 0.95 (d, J = 6.3 Hz, 3H,
6-CH3), 0.84 (d, J = 7.1 Hz, 3H, 9-CH3); 13C NMR (125 MHz, Chloroform-d) δ ppm:
153.92 (C=O), 149.21 (Ph-1), 134.71 (Ph-4), 129.73 (Ph-2 and Ph-6), 121.48 (Ph-3
and Ph-5), 104.00 (3-C), 91.67 (12a-C), 90.84 (10-C), 80.30 (12-C), 51.74 (5a-C),
47.32 (piperazine-C), 47.17 (piperazine-C), 45.83 (piperazine-C), 44.85
(piperazine-C), 44.29 (8a-C), 37.40 (6-C), 36.33 (4-C), 34.29 (7-C), 28.48 (9-C),
26.03 (3-CH3), 24.76 (5-C), 21.65 (8-C), 20.83 (Ph-CH3), 20.29 (6-CH3), 13.50
(9-CH3). HRMS (ESI) m/z calcd. for [C27H38N2O6H+]: 487.2803, Found, 487.2830.
4.2.9.2. 4-fluorophenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12b)
1
The product was obtained as white solid (74 mg, 28%), m.p.: 120.9-125.1°C. H
NMR (400 MHz, Chloroform-d) δ ppm: 7.09-6.98 (m, 4H, Ph-H), 5.30 (s, 1H,
12a-H), 4.07 (d, J = 10.2 Hz, 1H, 10-H), 3.70-3.49 (m, 4H, piperazine-H), 3.04 (s,
2H, piperazine-H), 2.76-2.67 (m, 2H, piperazine-H), 2.64-2.56 (m, 1H, 9-H), 2.35 (td,
J = 13.9, 3.9 Hz, 1H, 6-H), 2.06-1.98 (m, 1H, 8a-H), 1.91-1.83 (m, 1H, 5a-H),
1.77-1.67 (m, 2H), 1.59-1.45 (m, 2H), 1.41 (s, 3H, 3-CH3), 1.37-1.22 (m, 4H), 0.96
13
(d, J = 6.1 Hz, 3H, 6-CH3), 0.84 (d, J = 7.1 Hz, 3H, 9-CH3); C NMR (100 MHz,
Chloroform-d) δ ppm: 159.86 (d, J = 242.0 Hz,Ph-4), 153.52 (C=O), 147.21 (Ph-1),
123.15 (d, J = 9.0 Hz,Ph-2 and Ph-6), 115.79 (d, J = 23.0 Hz, Ph-3 and Ph-5), 103.96
(3-C), 91.63 (12a-C), 90.74 (10-C), 80.26 (12-C), 51.66 (5a-C), 47.34 (piperazine-C),
47.06 (piperazine-C), 45.75 (piperazine-C), 44.82 (piperazine-C), 44.30 (8a-C), 37.36
26

(6-C), 36.26 (4-C), 34.22 (7-C), 28.43 (9-C), 26.03 (3-CH3), 24.71 (5-C), 21.61 (8-C),
20.30 (6-CH3), 13.51 (9-CH3). HRMS (ESI) m/z calcd. for [C26H35FN2O6H+]:
491.2552, Found, 491.2547.
4.2.9.3. 3-fluorophenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12c)
1
The product was obtained as white solid (53 mg, 21%), m.p.:118.3-121.1°C. H
NMR (500 MHz, Chloroform-d) δ ppm: 7.30 (dd, J = 14.8, 8.0 Hz, 1H, Ph-5),
6.93-6.86 (m, 3H, Ph-2, Ph-4 and Ph-6), 5.29 (s, 1H, 12a-H), 4.07 (d, J = 10.2 Hz, 1H,
10-H), 3.70-3.49 (m, 4H, piperazine-H), 3.05 (s, 2H, piperazine-H), 2.76-2.69 (m, 2H,
piperazine-H), 2.65-2.56 (m, 1H, 9-H), 2.36 (td, J = 14.0, 3.9 Hz, 1H, 6-H), 2.06-1.99
(m, 1H, 8a-H), 1.90-1.85 (m, 1H, 5a-H), 1.76-1.67 (m, 2H), 1.59-1.44 (m, 2H), 1.41
(s, 3H, 3-CH3), 1.38-1.20 (m, 4H), 0.96 (d, J = 6.3 Hz, 3H, 6-CH3), 0.84 (d, J = 7.2
Hz, 3H, 9-CH3); 13C NMR (125 MHz, Chloroform-d) δ ppm:162.84 (d, J = 245.0 Hz,
Ph-3), 153.03 (Ph-1), 152.34 (C=O), 129.87 (d, J = 9.6 Hz,Ph-5), 117.53 (d, J = 2.5
Hz,Ph-6), 112.16 (d, J = 21.3 Hz,Ph-4), 109.84 (d, J = 24.4 Hz,Ph-2), 104.00 (3-C),
91.66 (12a-C), 90.80 (10-C), 80.29 (12-C), 51.73 (5a-C), 47.36 (piperazine-C), 47.13
(piperazine-C), 45.81 (piperazine-C), 44.93 (piperazine-C), 44.37 (8a-C), 37.41 (6-C),
36.31 (4-C), 34.28 (7-C), 28.47 (9-C), 26.03 (3-CH3), 24.76 (5-C), 21.64 (8-C), 20.29
(6-CH3), 13.50 (9-CH3). HRMS (ESI) m/z calcd. for [C26H35FN2O6H+]: 491.2552,
Found, 491.2544.
4.2.9.4. 2-fluorophenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12d)
The product was obtained as white solid (66 mg, 25%), m.p.: 130.1-131.4°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.22-7.05 (m, 4H, Ph-H), 5.30 (s, 1H,
12a-H), 4.07 (d, J = 10.2 Hz, 1H, 10-H), 3.70-3.49 (m, 4H, piperazine-H), 3.05 (s,
2H, piperazine-H), 2.76-2.70 (m, 2H, piperazine-H), 2.65-2.56 (m, 1H, 9-H), 2.35 (td,
J = 14.0, 3.9 Hz, 1H, 6-H), 2.04-1.97 (m, 8a-H), 1.91-1.84 (m, 1H, 5a-H), 1.76-1.66
(m, 2H), 1.61-1.44 (m, 3H), 1.41 (s, 3H, 3-CH3), 1.38-1.19 (m, 3H), 0.96 (d, J = 6.3
Hz, 3H, 6-CH3), 0.84 (d, J = 7.1 Hz, 3H, 9-CH3); 13C NMR (125 MHz, Chloroform-d)
δ ppm: 154.59 (d, J = 246.3 Hz, Ph-2), 152.65 (C=O), 138.93 (Ph-1), 126.38 (d, J =
7.0 Hz, Ph-C), 124.23 (Ph-C), 116.50 (d, J = 14.2 Hz, Ph-C), 103.99 (3-C), 91.65
(12a-C), 90.83 (10-C), 80.28 (12-C), 51.73 (5a-C), 47.34 (piperazine-C), 47.20
(piperazine-C), 45.82 (piperazine-C), 45.05 (piperazine-C), 44.53 (8a-C), 37.40 (6-C),
36.32 (4-C), 34.29 (7-C), 28.48 (9-C), 26.02 (3-CH3), 24.76 (5-C), 21.64 (8-C), 20.29
(6-CH3), 13.51 (9-CH3). HRMS (ESI) m/z calcd. for [C26H35FN2O6H+]: 491.2552,
Found, 491.2558.
4.2.9.5. 4-(trifluoromethyl)phenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12e)
The product was obtained as white solid (74 mg, 26%), m.p.: 156.6-162.7°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.62 (d, J = 8.5 Hz, 2H, Ph-3 and Ph-5), 7.24
(d, J = 8.4 Hz, 2H, Ph-2 and Ph-6), 5.30 (s, 1H, 12a-H), 4.08 (d, J = 10.3 Hz, 1H,
27

10-H), 3.68-3.46 (m, 4H, piperazine-H), 3.06 (s, 2H, piperazine-H), 2.75-2.67 (m, 2H,
piperazine-H), 2.62-2.55 (m, 1H, 9-H), 2.32 (td, J = 13.9, 3.9 Hz, 1H, 6-H), 2.01-1.95
(m, 1H, 8a-H), 1.89-1.81 (m, 1H, 5a-H), 1.74-1.65 (m, 2H), 1.57-1.42 (m, 2H), 1.38
(s, 3H, 3-CH3), 1.36-1.30 (m, 2H), 1.26-1.20 (m, 2H), 0.96 (d, J = 6.1 Hz, 3H, 6-CH3),
13
0.84 (d, J = 7.1 Hz, 3H, 9-CH3); C NMR (100 MHz, Chloroform-d) δ ppm: 153.95
(Ph-1), 152.74 (C=O), 127.24 (q, J = 32.5 Hz, Ph-C), 126.53 (q, J = 3.9 Hz, Ph-C),
123.97 (q, J = 270.2 Hz, CF3), 122.11(Ph-C), 103.97 (3-C), 91.63 (12a-C), 90.73
(10-C), 80.26 (12-C), 51.65 (5a-C), 47.30 (piperazine-C), 47.04 (piperazine-C), 45.74
(piperazine-C), 44.93 (piperazine-C), 44.36 (8a-C), 37.36 (6-C), 36.25 (4-C), 34.21
(7-C), 28.42 (9-C), 26.02 (3-CH3), 24.70 (5-C), 21.60 (8-C), 20.28 (6-CH3), 13.49
(9-CH3). HRMS (ESI) m/z calcd. for [C27H35F3N2O6H+]: 541.2520, Found, 541.2511.
4.2.9.6. 3-(trifluoromethyl)phenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12f)
1
The product was obtained as white solid (91 mg, 30%), m.p.: 80.0-81.1°C. H
NMR (400 MHz, Chloroform-d) δ ppm: 7.50-7.40 (m, 2H, Ph-4 and Ph-6), 7.40 (s,
1H, Ph-2), 7.32 (d, J = 7.2 Hz, 1H, Ph-5), 5.30 (s, 1H, 12a-H), 4.07 (d, J = 10.2 Hz,
1H, 10-H), 3.72-3.49 (m, 4H, piperazine-H), 3.06 (s, 2H, piperazine-H), 2.78-2.69 (m,
2H, piperazine-H), 2.67-2.55 (m, 1H, 9-H), 2.35 (td, J = 13.9, 3.9 Hz, 1H, 6-H),
2.05-1.98 (m, 1H, 8a-H), 1.91-1.83 (m, 1H, 5a-H), 1.76-1.67 (m, 2H), 1.60-1.45 (m,
2H), 1.40 (s, 3H, 3-CH3), 1.38-1.31 (m, 2H), 1.29-1.22 (m, 2H), 0.96 (d, J = 6.1 Hz,
13
3H, 6-CH3), 0.84 (d, J = 7.2 Hz, 3H, 9-CH3); C NMR (100 MHz, Chloroform-d) δ
ppm: 152.90 (Ph-1), 151.47 (C=O), 131.61 (q, J = 32.8 Hz, Ph-C), 129.73 (Ph-C),
125.40 (Ph-C), 123.59 (q, J = 270.7 Hz, CF3), 121.93 (q, J = 3.6 Hz, Ph-C), 119.04 (q,
J = 3.6 Hz, Ph-C), 103.97 (3-C), 91.62 (12a-C), 90.73 (10-C), 80.25 (12-C), 51.65
(5a-C), 47.30 (piperazine-C), 47.04 (piperazine-C), 45.74 (piperazine-C), 44.91
(piperazine-C), 44.36 (8a-C), 37.36 (6-C), 36.25 (4-C), 34.21 (7-C), 28.43 (9-C),
26.01 (3-CH3), 24.71 (5-C), 21.60 (8-C), 20.28 (6-CH3), 13.49 (9-CH3). HRMS (ESI)
m/z calcd. for [C27H35F3N2O6H+]: 541.2520, Found, 541.2546.
4.2.9.7. 3-(trifluoromethoxy)phenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12g)
1
The product was obtained as white solid (45 mg, 15%), m.p.: 91.9-93.8°C. H
NMR (500 MHz, Chloroform-d) δ ppm: 7.36 (t, J = 8.2 Hz, 1H, Ph-5), 7.10-7.02 (m,
3H, Ph-2, Ph-4 and Ph-6), 5.30 (s, 1H, 12a-H), 4.07 (d, J = 10.2 Hz, 1H, 10-H),
3.70-3.50 (m, 4H, piperazine-H), 3.05 (s, 2H, piperazine-H), 2.76-2.69 (m, 2H,
piperazine-H), 2.65-2.57 (m, 1H, 9-H), 2.34 (td, J = 14.0, 3.9 Hz, 1H, 6-H), 2.04-1.99
(m, 1H, 8a-H), 1.91-1.84 (m, 1H, 5a-H), 1.76-1.69 (m, 2H), 1.59-1.46 (m, 2H), 1.41
(s, 3H, 3-CH3), 1.38-1.30 (m, 2H), 1.21-1.27 (m, 2H), 0.96 (d, J = 6.3 Hz, 3H,
6-CH3), 0.84 (d, J = 7.2 Hz, 3H, 9-CH3); 13C NMR (125 MHz, Chloroform-d) δ ppm:
152.88 (Ph-C), 152.18 (Ph-C), 149.45 (C=O), 129.86 (Ph-C), 120.40 (q, J = 255.9 Hz,
OCF3), 120.32 (Ph-C), 117.49 (Ph-C), 115.06 (Ph-C), 104.01 (3-C), 91.66 (12a-C),
90.81 (10-C), 80.29 (12-C), 51.73 (5a-C), 47.35
(piperazine-C), 47.10
(piperazine-C), 45.81 (piperazine-C), 44.95 (piperazine-C), 44.40 (8a-C), 37.41
28

(6-C), 36.32 (4-C), 34.28 (7-C), 28.47 (9-C), 26.03 (3-CH3), 24.76 (5-C), 21.65 (8-C),
20.29 (6-CH3), 13.49 (9-CH3). HRMS (ESI) m/z calcd. for [C27H35F3N2O7H+]:
557.2469, Found, 557.2458.
4.2.9.8. 4-((trifluoromethyl)thio)phenyl 4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carboxylate (12h)
The product was obtained as white solid (70 mg, 25%), m.p.: 149.2-153.5°C. 1H
NMR (500 MHz, Chloroform-d) δ ppm: 7.64 (d, J = 8.5 Hz, 2H, Ph-3 and Ph-5), 7.19
(d, J = 8.6 Hz, 2H, Ph-2 and Ph-6), 5.30 (s, 1H, 12a-H), 4.07 (d, J = 10.2 Hz, 1H,
10-H), 3.72-3.50 (m, 4H, piperazine-H), 3.05 (s, 2H, piperazine-H), 2.77-2.69 (m, 2H,
piperazine-H), 2.65-2.57 (m, 1H, 9-H), 2.35 (td, J = 14.0, 3.9 Hz, 1H, 6-H), 2.04-1.98
(m, 1H, 8a-H), 1.91-1.84 (m, 1H, 5a-H), 1.76-1.67 (m, 2H), 1.58-1.43 (m, 3H), 1.40
(s, 3H, 3-CH3), 1.38-1.19 (m, 3H), 0.96 (d, J = 6.3 Hz, 3H, 6-CH3), 0.84 (d, J = 7.1
13
Hz, 3H, 9-CH3); C NMR (125 MHz, Chloroform-d) δ ppm: 153.76 (C=O), 152.74
(Ph-1), 137.62 (Ph-C), 133.11 (Ph-C), 129.47 (q, J = 306.3 Hz, SCF3), 122.86 (Ph-C),
120.40 (Ph-C), 104.01 (3-C), 91.67 (12a-C), 90.81 (10-C), 80.29 (12-C), 51.73
(5a-C), 47.35 (piperazine-C), 47.12 (piperazine-C), 45.81 (piperazine-C), 44.99
(piperazine-C), 44.41 (8a-C), 37.41 (6-C), 36.31 (4-C), 34.28 (7-C), 28.47 (9-C),
26.04 (3-CH3), 24.75 (5-C), 21.65 (8-C), 20.29 (6-CH3), 13.49 (9-CH3). HRMS (ESI)
Calcd. for [C27H35F3N2O6SH+]: 573.2241, Found, 573.2216.
4.2.10. General procedure for the preparation of compounds (13a-13e)
A cold stirred solution of amines 7a-7c, 7e-7f (0.95 equiv) in anhydrous
tetrahydrofuran (5 mL) was treated drop wise with a cold triethylamine (2 equiv, 0.12
g). The above solution (containing 11) was transferred to a stirred solution of amines
7a-7c, 7e-7f. The reaction mixture was stirred overnight at room temperature, then
concentrated under reduced pressure and the residual was diluted by dichloromethane
(30 mL), then washed with saturated sodium bicarbonate solution (3×30 mL) and
water (3×30 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure
to yield the crude product, which was purified by chromatography on silica gel to
obtain the products 13a-13e.
4.2.10.1. N-(4-fluorophenyl)-4-((3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-
trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl)
piperazine-1-carbothioamide (13a)
The product was obtained as white solid (67 mg, 31%), m.p.: 204.4-207.2°C. 1H
NMR (400 MHz, Chloroform-d) δ ppm: 7.15 (t, J = 8.6 Hz, 2H, Ph-2 and Ph-6), 7.02
(t, J = 8.6 Hz, 2H, Ph-3 and Ph-5), 5.28 (s, 1H, 12a-H), 4.07 (d, J = 10.6 Hz, 1H,
10-H), 3.86 (s, 4H, piperazine-H), 3.04 (dt, J = 11.0, 5.0 Hz, 2H, piperazine-H), 2.76
(dt, J = 11.0, 5.0 Hz, 2H, piperazine-H), 2.58-2.51 (m, 1H, 9-H), 2.33 (td, J = 14.0,
3.8 Hz, 1H, 6-H), 2.04-1.98 (m, 1H, 8a-H), 1.92-1.83 (m, 1H, 5a-H), 1.75-1.68 (m,
2H), 1.57-1.41 (m, 2H), 1.39 (s, 3H, 3-CH3), 1.34-1.27 (m, 2H), 1.26-1.18 (m, 2H),
0.95 (d, J = 6.1 Hz, 3H, 6-CH3), 0.82 (d, J = 7.1 Hz, 3H, 9-CH3); 13C NMR (100 MHz,
Chloroform-d) δ ppm: 182.51(C=S), 160.23 (d, J = 245.0 Hz, Ph-4), 135.96 (d, J =
3.0 Hz, Ph-1), 126.44 (d, J = 8.3 Hz, Ph-2 and Ph-6), 115.64 (d, J = 22.7 Hz, Ph-3 and
29