Synthesis, cytotoxicity, and in vivo antitumor activity study of parthenolide semicarbazones and thiosemicarbazones

Article abstract of DOI:10.1016/j.bmc.2020.115557

Parthenolide is an important sesquiterpene lactone with potent anticancer activities. In order to further improve its biological activity, a series of parthenolide semicarbazone or thiosemicarbazone derivatives was synthesized and evaluated for their anti

Full text of DOI:10.1016/j.bmc.2020.115557

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Synthesis, cytotoxicity, and in vivo antitumor activity study of parthenolide
semicarbazones and thiosemicarbazones
Xinxin Jia, Qi Liu, Shiyi Wang, Binglin Zeng, Guohua Du, Chen Zhang, Yan
Li
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DOI:
Reference:
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https://doi.org/10.1016/j.bmc.2020.115557
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29 April 2020
5 May 2020
Please cite this article as: Jia, X., Liu, Q., Wang, S., Zeng, B., Du, G., Zhang, C., Li, Y., Synthesis, cytotoxicity,
and in vivo antitumor activity study of parthenolide semicarbazones and thiosemicarbazones, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115557
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Synthesis, cytotoxicity, and in vivo antitumor activity study of
parthenolide semicarbazones and thiosemicarbazones
Xinxin Jia†, Qi Liu†, Shiyi Wang†, Binglin Zeng§, Guohua Du§, Chen Zhang*†‡, Yan Li*§
†
School of Chemical Engineering, Beijing Institute of Petrochemical Technology, Beijing,
02617, P. R. China;
1
§
State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Beijing
Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, P.R. China;
‡
*
Beijing Key Laboratory of Fuels Cleaning and Advanced Catalytic Emission Reduction
Technology, Beijing Institute of Petrochemical Technology, Beijing 102617, P. R. China;
To whom correspondence should be addressed
zhangc@bipt.edu.cn
liyanxiao@imm.ac.cn

Abstract:
Parthenolide is an important sesquiterpene lactone with potent anticancer activities. In order to
further improve its biological activity, a series of parthenolide semicarbazone or
thiosemicarbazone derivatives was synthesized and evaluated for their anticancer activity.
Derivatives were tested in vitro against 5 human tumor cell lines, and many of these showed
higher cytotoxicity than parthenolide. Five compounds were further studied for their antitumor
activity in mice. The in vivo result indicated that compound 4d showed both promising
antitumor activity against mice colon tumor and small side effects on immune systems. The
cell apoptosis and cell cycle distribution of compound 4d were also studied. Molecular docking
studies revealed multiple interactions between 4d and NF-κB. Our findings demonstrate the
potential of semicarbazones as a promising type of compounds with anticancer activity.
Keywords:
Anticancer
Parthenolide
Semicarbazone
In vivo

1
. Introduction
The lethality of cancer has encouraged numerous efforts to develop new anticancer drugs.
Parthenolide (PTL, 1a), a sesquiterpene lactone isolated from feverfew, has shown biological
1
activity in many illnesses, including inflammatory diseases, kidney stones, infant colic, etc. In
recent years, parthenolide attracted particular attention due to its inhibiting activity on acute
myeloid leukemia (AML) and glioblastoma (GBM). It is proposed that the antitumor activity
2
of PTL resulted from its ability to inhibit NF-κB and induce redox stress. Moreover, PTL was
identified as the first small molecule to selectively eliminate cancer stem cells.1, 3 Structure-
activity relationship studies revealed that the α-methylene-γ-lactone structure and the epoxide
group of PTL, which could react with biological nucleophiles of proteins, are the most
important moieties for its biological activities.4, 5
Despite of its exceptional efficacy in eradicating cancer stem cells, it is reported that PTL were
1
still not as effective in killing bulk tumor cells as in killing cancer stem cells. Therefore,
modifying the structure of PTL to enhance its interaction with target molecules while retaining
the crucial α,β-unsaturated carbonyl structure could be an effective strategy to design PTL
derivatives and reveal structure-activity relationships. Semicarbazones and thiosemicarbazones
have been extensively studied for their broad bioactivities in antiviral, antibacterial,
antimalarial, and anticancer applications.6-8 Previous research showed that the oxygen/sulfur
atom and nitrogen atoms of semicarbazone/thiosemicarbazone may interact with target kinases
9
through the formation of hydrogen bonds, resulting in an inhibition of kinases. In addition,
structure-activity relationship studies on diaryl urea suggested that the aromatic ring attached
to amide nitrogen could insert into pockets of some kinases and resulting in an inhibition effect
1
0
on tumors. Thiosemicarbazones were also widely studied as transition metal chelators with
effective antitumor activity.11 Therefore, inspired by these researches, the incorporation of
semicarbazones or thiosemicarbazones scaffold into the PTL structure is expected to enhance
the interaction between synthesized compounds and target molecules such as NF-κB, to
increase their anticancer activity.
The goal of this research is to combine PTL structure with substituted semicarbazones or
thiosemicarbazones to obtain PTL derivatives with higher anticancer activity and explore the
structure-activity relationship between substituent groups of semicarbazone/thiosemicarbazone
and the anticancer activity. The comparison between semicarbazones and thiosemicarbazones
could also reveal valuable information about the importance of hydrogen bonds and metal
chelation in anticancer activity. In order to attach semicarbazones or thiosemicarbazones to
PTL, a two-step oxidation of PTL was conducted to yield an aldehyde (PTL-CHO, 1c), which
1
2
displayed slightly lower in vitro activity relative to PTL. PTL-CHO was then condensed with
semicarbazones or thiosemicarbazones to form a series of novel PTL derivatives, which were
tested for their in vitro and in vivo anticancer activities.

2
2
. Results and discussion
.1 Chemistry
The synthesis procedures of PTL semicarbazone or thiosemicarbazone derivatives are shown
in Scheme 1. Initially, PTL (1a) was oxidized with SeO /t-BuOOH to give melampomagnolide
2
B (1b), followed by the reaction with Dess-Martin periodinane to furnish the aldehyde PTL-
CHO (1c).12 Semicarbazones (2a-i) were synthesized by the condensation of amines with
phenyl chloroformate to yield the carbamate, followed by the reaction with hydrazine hydrate.13,
1
4
Substituted thiosemicarbazones (3a-l) were either purchased from chemical companies or
synthesized by reacting functionalized amines with carbon disulphide, followed by the reaction
1
5
with hydrazine hydrate. The synthesized semicarbazones/thiosemicarbazones were refluxed
1
5
with PTL-CHO (1c) catalyzed by acetic acid to yield the target compounds (4a-i, 5a-l). The
1
13
chemical structures of all compounds (4a-i, 5a-l) were confirmed by H NMR, C NMR, and
high-resolution mass spectroscopy. Although both E and Z isomeric forms are possible for the
imine double bond (-CH=N-), acidic synthesis environment as in this study generally yields E
isomers.8, 13 This is corroborated by the single peak observed in H NMR of synthesized
compounds for the hydrogen attached to carbon of imine, which is usually located at around δ
1
7
.8 ppm for most compounds.9 15
clogP values of synthesized derivatives were calculated by the ALOGPS 2.1 software from the
Virtual Computational Chemistry Laboratory (http://www.vcclab.org).16, 17 It could be seen
from the result in Table 1 that synthesized PTL derivatives were generally more lipophilic
compared to PTL or PTLCHO, despite the introduction of hydrogen bond donors/receptors as
the =N-NH-CO-NH- or =N-NH-CS-NH- structure. Also, thiosemicarbazones were more
lipophilic than semicarbazones, perhaps due to the lower polarity of the sulfur atom compared
to that of the oxygen atom.
Scheme 1. General synthesis of PTL semicarbazone/thiosemicarbazone derivatives (4a-i, 5a-
l). Reagents and conditions: (a) phenyl chloroformate, pyridine, 50 °C, 4 h; (b) hydrazine
hydrate, dimethoxyethane, 80 °C, 12 h; (c) ethanol, acetic acid, 50 °C, 4 h; (d) SeO /t-BuOOH,
2
CH Cl , 45 °C, 4 h; (e) Dess-Martin periodinane, CH Cl , NaHCO , room temperature, 2 h; (f)
2
2
2
2
3
CS , DMF, NaOH, room temperature, 4 h; (g) hydrazine hydrate, 65 °C, 2 h.
2

2
.2
In vitro cytotoxic studies
The cytotoxic activities of target compounds (4a-i, 5a-l) were evaluated in HCT116 (human
colorectal carcinoma), U87-MG (human glioblastoma), HepG2 (human liver carcinoma),
BGC823 (human gastric cancer), and PC9 (human lung cancer) using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Both Taxol (paclitaxel)
and PTL (1a) were used as positive control, while PTL was also a well-known NF-κB inhibitor.2,
1
8, 19
PTL-CHO (1c) were also included for comparison. The IC values were reported in Table
5
0
1
. PTL was more cytotoxic than PTL-CHO, which is consistent with a previous study.20 All
synthesized derivatives displayed moderate to prominent anticancer activities against 5 cancer
cell lines. All semicarbazones except 4b displayed stronger in vitro inhibitory activities against
5
cell lines compared to PTL, with most of IC values lower than PTL.
50

Table 1. In vitro antitumor activity of target compounds 4a-4i, 5a-5l against 5 human cancer
cell lines.
H
N
H
N
N
R
X
O
O
O
Compound
X
R
clogP a
IC50 (μmol/L)
HCT116
U87-MG
HepG2
5.88
BGC823
14.40
PC9
8.67
4
4
4
4
4
4
4
4
4
5
5
5
a
b
c
d
e
f
O
O
O
O
O
O
O
O
O
S
2.95
3.02
3.65
3.61
3.50
4.08
2.96
2.75
3.71
2.58
3.42
3.91
2.72
4.77
14.02
4.58
2.81
3.16
2.49
3.05
2.72
1.47
2.27
5.44
17.29
14.47
3.48
7.43
2.77
2.83
2.86
1.74
3.23
6.54
3.66
8.99
17.44
6.15
15.60
12.83
2.02
6.44
5.02
F
5.35
3.90
OCH3
CF3
4.24
2.90
13.60
4.66
4.16
1.44
g
h
i
8.09
1.08
4.18
N
6.91
3.77
16.74
3.12
N
3.31
1.77
N
a
b
c
16.09
19.11
11.98
16.48
34.17
19.69
11.47
34.20
22.36
H
S
S

Compound
X
S
R
clogP a
IC50 (μmol/L)
5
5
5
5
5
5
5
5
5
1
1
d
3.84
15.21
18.74
5.99
18.31
3.33
25.63
15.35
15.26
13.52
18.70
15.17
24.21
34.36
9.30
35.08
15.55
5.10
e
S
S
S
S
S
S
S
S
-
F
4.23
4.26
4.50
4.76
4.40
4.96
4.97
4.86
3.03
2.01
14.01
7.77
F
f
3.24
14.86
4.75
g
4.48
2.43
4.27
h
Cl
17.92
15.60
17.92
18.78
8.64
14.74
4.38
17.25
3.77
18.05
7.31
i
OCH3
j
CF3
17.16
9.75
17.15
16.82
7.33
9.22
CF3
k
7.73
l
14.55
5.75
12.40
6.61
-
-
-
a (PTL)
3.13
5.71
15.77
16.39
c (PTL-CHO)
-
14.46
7.37
4.77
10.69
Taxol
-
0.000206 0.000380 0.000762 0.000077 0.000056
a
clogP: calculated by the ALOGPS 2.1 software from Virtual Computational Chemistry Laboratory
(VCCLAB).
In general, semicarbazone derivatives were more cytotoxic than thiosemicarbazones, as can be
seen in the comparison between 4a and 5c, 4d and 5g, etc. This is indicative of the importance
of the oxygen atom in the carbonyl group. Thiosemicarbazones are an important type of
anticancer agents, which could inhibit the DNA synthesis by chelating with metal ions.7
However, the inferior activity of PTL thiosemicarbazones compared to PTL as can be seen in

Table 1 suggested that the ability to chelate did not contributed to the cytotoxicity in this study.
In addition, the lower electronegativity of the thiosemicarbazone sulfur atom than the
semicarbazone oxygen atom may also hamper the ability to form hydrogen bonds and weaken
the interaction between synthesized compounds and target proteins.
One noticeable trend for both semicarbazone and thiosemicarbazone derivatives is that the
alkyl-substituted compounds were generally less potent than aryl-substituted ones, as can be
seen for 4a, 4b, 5b, 5c and 5d. Previous docking study on diaryl urea indicated that an aromatic
ring attached to the urea moiety may insert into the hydrophobic pockets of various kinases and
1
0
inhibit proliferation-related kinases. Due to the structure similarity, this mechanism could also
be a possible explanation for the higher potency of aryl-substituted compounds, implying that
an aromatic ring attached to the urea moiety may be beneficial for increasing cytotoxicity.
Among aryl-substituted derivatives, for both semicarbazones and thiosemicarbazones, the p-
methylphenyl-substituted compound (4d and 5g) stood out in terms of their cytotoxicity against
all 5 cell lines.
2
.3
In vivo anticancer activity studies
Based on the promising results of in vitro assay, five synthesized compounds, including 4d, 4f,
g, 4i, and 5g, were selected for further evaluation of in vivo antitumor activity in mice bearing
4
murine colon adenocarcinoma cell MC38. The mice were divided into one control group and
six experimental groups (6 mice per group) treated with 5-fluorouracil (5-FU), 4d, 4f, 4g, 4i,
and 5g. As shown in Table 2 and Figure 1, 5-FU showed the highest tumor inhibition rate
among all tested compounds, while its toxicity was also severe according to the decreased body
weight, spleen index, and thymus index compared to the control group. Five synthesized
compounds showed distinct in vivo antitumor activities, with compound 4d and 4f displayed
the highest inhibition activity against MC38. However, 4f showed considerable toxicity during
the treatment, with the mice body weight much lower than the control or other groups. 4i, which
showed prominent activity in MTT assay, showed the lowest tumor inhibition rate among all
compounds as well as pronounced toxicity. Interestingly, although previous studies have
suggested that the low water solubility of PTL inhibited its activity,21 no direct correlation
between clogP values and tumor inhibition rates was observed in this study. For example,
compound 4h with 2-pyridyl substituent showed the lowest clogP value among all
semicarbazone derivatives, while its in vivo antitumor activity was moderate compared to other
compounds.
Spleen and thymus are important immune organs, which are also the major sites for NF-κB to
2
2
function. Mice treated with 5-FU displayed much lower thymus indices than the control group
P < 0.001), demonstrating the suppression effect of 5-FU on immune system as reported
(

2
3
previously. In contrast, all 5 PTL derivatives showed higher spleen indices and much higher
thymus indices than 5-FU, suggesting that synthesized compounds induced beneficial
influences on immune systems compared to 5-FU during treatments in tumor-bearing mice. In
addition, synthesized compounds exerted less side effects than 5-FU as the body weights of 4d,
4
g, and 4i treated mice showed increased body weights compared to the control group (Table
2
).
Table 2. In vivo antitumor effects in MC38-bearing mice (mean ± SD).
Groups
N
Dose
Mice body weight (g)
Tumor weight (g) Inhibition rate (%)
(mg/kg)
Begin
End
Control
6
6
6
6
6
6
6
-
14.5 ± 0.5
14.2 ± 0.6
14.0 ± 0.9
14.5 ± 0.6
14.2 ± 0.7
14.0 ± 0.5
14.0 ± 0.6
16.6 ± 1
2.67 ± 0.59
1.76 ± 0.32**
1.97 ± 0.36*
1.96 ± 0.24*
2.03 ± 0.76
2.29 ± 0.49
2.11 ± 0.33
-
5
4
4
4
4
5
-FU
40.0
40.0
40.0
40.0
40.0
40.0
16.0 ± 1.5
17.4 ± 0.9
14.5 ± 2.5
17.8 ± 1.5
18.0 ± 1.4
16.1 ± 1.1
34.27
26.22
26.78
24.09
14.23
20.85
d
f
g
i
g
N, number of mice in each group.
*
*
P < 0.05, compared with model group.
* P < 0.01, compared with model group.
Figure 1. Evaluation of spleen indices and thymus indices of tumor-bearing mice.
1
1
20
00
Spleen index
Thymus index
8
6
4
2
0
0
0
0
0
Control 5-FU
4d
4f
4g
4i
5g

Previous studies have reported that PTL alone was not able to significantly inhibit tumor
growth.1, 24, 25 According to the in vivo experiment in the present study, PTL semicarbazone 4d
showed promising antitumor activity, with slightly lower tumor inhibition rate but less overall
side effects compared to 5-FU. Therefore, it is deduced that an increased dose of 4d might be
more effective against tumor in vivo, and this will be studied in future research.
Considering the possibility of semicarbazone decomposition, the chemical stability of the most
active compound (4d) over 6 h was studied in phosphate buffer solution of pH = 7.4 at 37 °C,
to simulate its stability in serum. The relative change of 4d concentration versus time is shown
in Figure 2. It could be seen in the figure that 4d is relatively stable, with no obvious decrease
in concentration for 6 h. This is consistent with a previous study, reporting that semicarbazone
scaffold is stable in both buffer solutions and rat plasma.13
Figure 2. Chemical stability of compound 4d in phosphate buffer solution (pH = 7.4) at 37 °C
in 6 h.
1
00%
8
6
4
2
0%
0%
0%
0%
0
%
0
2
Time (h)4
6
2
.4
Cell apoptosis and cell cycle studies
Based on the in vitro and in vivo result, PTL semicarbazone derivative 4d displayed the best
biological activity among synthesized compounds. In order to provide further insights on the
antitumor activity of 4d, effects of 4d on cell apoptosis and cell cycle of human glioblastoma
U87-MG were studied by flow cytometry. It could be seen in Figure 3 that 4d induced
concentration-dependent apoptosis in U87-MG tumor cell, demonstrating the antitumor activity
of 4d, in agreement with the MTT assay in this study. Also, it is observed that the cell apoptosis
induced by 4d is mainly the early stage of apoptosis (Q2), while the percentage of late apoptosis
and necrosis cells (Q3) is generally unchanged.
The effect of compound 4d on U87-MG cell cycle was also studied and the result is shown in
Figure 4. The cell population in G0/G1 phase of the cell cycle was slightly decreased in 4d-
treated cells compared to the control group, suggesting a cancer cell cycle arrest in G0/G1 phase.

Although NF-κB has been reported previously to induce G0/G1 phase arrest,26, 27 the role of
NF-κB on cell cycle distribution is still controversial and has yet reached an agreement.28
Therefore, further experiments will be necessary to reveal the actual influence of 4d on cell
cycle.
Figure 3. Compound 4d induced apoptosis of U87-MG cells incubated at various
concentrations (0, 4, and 16 μM).
Figure 4. The effect of compound 4d on cell cycle in U87-MG cancer cells. (A) U87-MG
cells were incubated at various concentrations of 4d (0, 4, and 16 μM). (B) Histograms
display the percentage of cell cycle distribution.
2
.5
Molecular docking studies:
1
In literature, PTL has been widely regarded as a strong NF-κB inhibitor. In order to reveal the
binding modes of compound 4d with NF-κB, molecular docking simulations were conducted
using Discovery Studio (Accelrys Inc., USA). Compound 4d was docked into the active site of
NF-κB. As shown in Figure 5, multiple interactions were observed between compound 4d and
the NF-κB residues. For the PTL moiety of compound 4d, a hydrogen bond was formed
between residue SER476 and the epoxide group, consistent with other studies proposing that
the epoxide group is crucial for the anticancer activity of PTL.29 Three H-bond interactions

were noticed for the semicarbazone structure: one between CYS533 and the imine nitrogen
atom, one between CYS533 and the carbonyl oxygen atom, and one between GLU440 and the
amide hydrogen atom. Previous research proposed that the -NH-CO-NH- structure could bind
various enzymes and receptors through hydrogen bond interaction, with the amide hydrogen
1
0
being the proton donors and the carbonyl oxygen as the proton receptor. The p-methylphenyl
group of compound 4d also interacts with various residues of NF-κB. Three π-alkyl interactions
were formed between the benzene ring and residue ILE467, CYS444, and VAL453. The methyl
group is also interacting with residue LEU455, VAL453, and CYS444. In addition, this
compound would insert into the pocket formed by multiple protein residues via van der Waals
interaction, which further stabilized the ligand-receptor conformation. Previous research
suggested the formation of hydrogen bond between amide hydrogen of the urea group and the
carbamoyl moieties of IκB kinase, an important kinase complex in the NF-κB pathway,
resulting in a significant increase in anticancer activity.30 In this study, direct interactions
between the synthesized semicarbazone 4d and NF-κB were also discovered and could account
for the anticancer activity of 4d by inhibiting NF-κB activity.
Figure 5. Docking models of representative semicarbazone 4d. (A) 2D molecular docking
model of compound 4d and surrounding residues of NF-κB. (B) 3D molecular docking model
of compound 4d with NF-κB.
A
B

3
. Conclusion
In this study, 21 novel parthenolide semicarbazone or thiosemicarbazone derivatives were
synthesized. Most semicarbazones exhibited higher cytotoxicity against human cancer cell lines
than parthenolide. Preliminary analysis indicated that aryl-substituted semicarbazones were
generally more cytotoxic. Five synthesized compounds, including 4 semicarbazones and 1
thiosemicarbazone were further tested in MC38-bearing mice. The in vivo results showed that
4
d possessed relatively high tumor inhibitory effect, as well as nearly unchanged spleen and
thymus indices compared to the control group. Cell apoptosis experiments indicated a
concentration-dependent apoptosis induced by 4d in U87-MG cells, with a G0/G1 phase arrest
observed in cell cycle analysis. Molecular studies demonstrated the formation of various
interactions between NF-κB and semicarbazone moiety of 4d. This indicated that PTL
semicarbazones showed promising antitumor activity and are worth further investigation in
anticancer experiments.
4
4
. Experimental section
.1 Chemistry
Reagents, solvents and starting compounds were purchased from Innochem, Alladin, Adamas,
Alfa, or other chemical companies. Thin-layer chromatography (TLC) with Qingdao Haiyang
GF254 were used to monitor reaction. Column chromatograph were performed with silica gel
(100-200 mesh) from Qingdao Haiyang. The chromatograms were viewed under ultraviolet
light (254-265 nm). NMR analysis on Bruker AV 400 (Bruker Company, USA) were performed
to determine the structure of target compounds. CDCl , or DMSO-d were used as solvents and
3
6
1
13
tetramethylsilane was used as reference. H NMR were recorded at 400 or 500 MHz, and C
NMR were recorded at 100 or 126 MHz. Chemical shifts (δ) were reported in ppm and coupling
constants (J) were reported in Hz. High-resolution mass spectroscopy of target compounds was
conducted on Agilent Technologies 6224 TOF LC/MS equipped with an ESI source.
4
.1.1 General method for semicarbazones preparation
Typically, 10 mmol of substituted amines were dissolved in 10 mL of pyridine and allowed to
stir. 11 mmol of phenyl chloroformate were added dropwise into the pyridine solution. The
reaction was proceeded at 50 °C for 4 hours. After the total conversion of amines monitored by
TLC, 20 mL of dichloromethane were added, and the mixture was then washed with water (20
mL × 3). The organic phase was concentrated in a rotary evaporator at 50 °C to afford crude
carbamates. These carbamates could be directed used in the next step without further
purification.

All crude carbamates from the previous step were dissolved in 20 mL dimethoxyethane,
followed by the addition of 2.5 mL of 80% hydrazine hydrate. The reaction was stirred and
refluxed at 80 °C for 12 h. Afterwards, the mixture was cooled and concentrated to give
unpurified semicarbazones 2a-2i in the form of oily liquid. This liquid was either recrystallized
by 80% ethanol in water or purified by silica gel chromatography using a mixture of petroleum
ether and ethyl acetate (2:1 ~ 1:2) as the eluent, to yield semicarbazones (2a - 2i).
4
.1.2 General method for thiosemicarbazones preparation
Thiosemicarbazone 3a, 3j and 3k were purchased from chemical companies. For other
compounds, the following synthesis protocol was used. To 15 mL of N, N-dimethylformamide
(DMF), 10 mmol of substituted amines, 10 mmol of carbon disulphide, and 11 mmol of sodium
hydroxide were added. The reaction was stirred at room temperature for 4 h. 2.5 mL of 80% of
hydrazine hydrate was then added and the temperature was increased to 65 °C. The reaction
was continued with stirring for 2 hours. After the reaction is completed checked by TLC, 30
mL water was added, and the mixture was then cooled in the fridge overnight. In some cases, a
solid separated out which could be filtrated to yield the crude thiosemicarbazones. Otherwise,
the mixture was extracted by CH Cl (30 mL × 3). The organic phases were combined and
2
2
purified by column chromatography. The solids obtained by filtration or column
chromatography were then recrystallized by 90% ethanol in water to yield thiosemicarbazones
3
b-3i, and 3l.
4
.1.3 General method for PTL-CHO preparation
2
0
The synthesis method was adapted from a previous research. 1 mmol of parthenolide (1a) in
1
4
5 mL of CH Cl were oxidized with 0.4 mmol of SeO and 10 mmol of 70% t-BuOOH at
2 2 2
5 °C for 4 h. After the reaction was completed checked by TLC, the mixture was filtered
through kieselguhr. The liquid was concentrated under reduced pressure and purified by column
chromatography to give melampomagnolide B (1b) in 50% yield and PTL-CHO (1c) in yield
of 24%. 1 mmol of 1b could be further oxidized by 1.5 mmol of Dess-Martin periodinane in 10
mL CH Cl and 10 mmol NaHCO at room temperature for 2 h. To the mixture saturated
2
2
3
Na S O (10 mL) aqueous solution, 10 mL of water, and 20 mL of CH Cl were added and
2
2
3
2
2
stirred. After the liquid phases turned clear, the organic phase was washed with brine, dried
over anhydrous Na SO overnight, and then concentrated to afford the residue. The residue was
2
4
then purified by column chromatography using a mixture of petroleum ether and ethyl acetate
2:1 ~ 1:2) as the eluent, to give PTL-CHO (1c) in the yield of 87%.
(

4
.1.4 General synthesis method for PTL semicarbazone/thiosemicarbazone derivatives
To 5 mL of ethanol, 0.25 mmol of PTL-CHO (1c), 0.3 mmol of semicarbazone (2a-2i) or
thiosemicarbazone (3a-3l), and 0.1 mL of acetic acid were added. The reaction mixture was
stirred at 50 °C for 4 h, monitored by TLC. After the complete disappearance of PTL-CHO, the
liquid was cooled in the fridge overnight. In some cases, solids were separated out and direct
filtration will yield target compounds. Otherwise, 10 mL of saturated sodium bicarbonate
solution were added to the liquid, and the mixture was extracted by 30 mL of CH Cl 3 times.
2
2
The organic phases were combined, concentrated, and the solid residue was purified by column
chromatography to yield the target compounds.
4
.1.4.1:
(E)-N-butyl-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carboxamide (4a). 40% yield; H NMR (400 MHz, CDCl ) δ: 8.82
3
(
s, 1H), 7.40(s, 1H), 6.20 (d, J = 3.5 Hz, 1H), 5.93 (t, J = 8.3 Hz, 1H), 5.85 (t, J = 6.0 Hz, 1H),
.38 (d, J = 3.2 Hz, 1H), 3.82 (t, J = 9.4 Hz, 1H), 3.42-3.24 (m, 2H), 2.91-2.82 (m, 1H), 2.80
d, J = 9.6 Hz, 1H), 2.69-2.63 (m, 2H), 2.58-2.39 (m, 2H), 2.38-2.29 (m, 1H), 2.27-2.20 (m,
H), 1.71-1.63 (m, 1H), 1.59-1.50 (m, 5H), 1.45-1.34 (m, 2H), 1.18 (t, J = 13.2 Hz, 1H), 0.96
5
(
1
1
3
(
t, J = 7.43 Hz, 3H); C NMR (126MHz, CDCl ) δ: 169.27, 156.08, 144.42, 138.73, 138.50,
3
1
1
37.12, 119.70, 81.77, 63.05, 59.66, 41.62, 39.48, 36.79, 32.52, 25.87, 24.66, 23.19, 20.07,
+
8.00, 13.82; HRMS (ESI) calcd for C H N O [M + H ] 376.2236; found 376.2236.
2
0
29
3
4
4
.1.4.2:
(E)-N-(tert-butyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carboxamide (4b). 16% yield; H NMR (400MHz, CDCl ) δ: 8.39
3
(s, 1H), 7.34 (s, 1H), 6.23 (d, J = 3.4 Hz, 1H), 5.90 (t, J = 8.4 Hz, 1H), 5.80 (s, 1H), 5.42 (d, J
=
3.0 Hz, 1H), 3.82 (t, J = 9.4 Hz, 1H), 2.91-2.76 (m, 2H), 2.71-2.60 (m, 2H), 2.60-2.30 (m,
3
H), 2.29-2.19 (m, 1H), 1.72-1.61 (m, 1H), 1.56 (s, 3H), 1.42 (s, 9H), 1.18 (t, J = 13.3 Hz, 1H);
1
3
C NMR (126MHz, CDCl ) δ: 169.26, 154.83, 143.61, 138.84, 138.33, 137.11, 119.53, 81.82,
3
6
3.04, 59.64, 50.46, 41.60, 36.82, 29.71, 29.45, 29.32, 25.80, 24.66, 23.24, 18.01; HRMS (ESI)
+
calcd for C H N O [M + H ] 376.2236; found 376.2246.
2
0
29
3
4
4
.1.4.3:
(E)-N-(2-fluorophenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carboxamide (4c). 31% yield; H NMR (400MHz, CDCl ) δ: 9.00(s,
3
1
H), 8.29-8.25 (m, 2H), 7.50 (s, 1H), 7.18-7.11 (m, 2H), 7.07-7.01 (m, 1H), 6.16 (d, J = 3.5
Hz, 1H), 6.02 (t, J = 8.5 Hz, 1H), 5.49 (d, J = 3.2 Hz, 1H), 3.83 (t, J = 9.5 Hz, 1H), 3.05-2.95
(m, 1H), 2.82 (d, J = 9.5 Hz, 1H), 2.77-2.56 (m, 3H), 2.53-2.44 (m, 1H), 2.43-2.34 (m, 1H),
1
3
2
.29-2.23 (m, 1H), 1.79-1.69 (m, 1H), 1.58 (s, 3H), 1.21 (t, J = 13.2 Hz, 1H); C NMR
(
126MHz, CDCl ) δ: 169.57, 153.54, 152.94, 151.62, 145.87, 140.39, 138.53, 137.29, 126.61,
3

1
25.04, 123.67, 120.76, 115.10, 81.97, 63.22, 59.85, 41.99, 36.95, 25.66, 24.99, 23.44, 18.20;
+
HRMS (ESI) calcd for C H FN O [M + H ] 414.1829; found 414.1839.
2
2
24
3
4
4
.1.4.4:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
yl)methylene)-N-(p-tolyl)hydrazine-1-carboxamide (4d). 63% yield; 1H NMR (400MHz,
CDCl ) δ: 8.83 (s, 1H), 7.77 (s, 1H), 7.45 (s, 1H), 7.37 (s, 1H), 7.35 (s, 1H), 7.17 (s, 1H), 7.14
3
(s, 1H), 6.15 (d, J = 3.5 Hz, 1H), 5.98 (t, J = 8.8 Hz, 1H), 5.37 (d, J = 3.2 Hz, 1H), 3.83 (t, J =
9
2
1
1
2
4
.5 Hz, 1H), 2.96-2.87 (m, 1H), 2.81 (d, J = 9.5 Hz, 1H), 2.74-2.65 (m, 2H), 2.64-2.51 (m, 1H),
.51-2.42 (m, 1H), 2.41-2.30 (m, 4H), 2.29-2.23 (m, 1H), 1.77-1.67 (m, 1H), 1.59-1.54 (s, 3H),
1
3
.20 (m, J = 13.3 Hz, 1H); C NMR (126MHz, CDCl ) δ: 169.25, 153.35, 145.17, 139.54,
3
38.42, 136.92, 134.86, 133.57, 129.69, 120.07, 119.87, 81.72, 63.03, 59.64, 41.61, 36.73,
+
5.88, 24.74, 23.23, 20.84, 18.00; HRMS (ESI) calcd for C H N O [M + H ] 410.2084; found
2
3
27
3
4
10.2080.
.1.4.5: (E)-N-(4-methoxyphenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
4
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carboxamide (4e). 38% yield; H NMR (500MHz, CDCl ) δ: 8.98 (s,
3
1
5
H), 7.69 (s, 1H), 7.46 (s, 1H), 7.39-7.35 (m, 2H), 6.92-6.88 (m, 2H), 6.17 (d, J = 3.5 Hz, 1H),
.97 (t, J = 8.5 Hz, 1H), 5.38 (d, J = 3.2 Hz, 1H), 3.85-3.80 (m, 4H), 2.97-2.86 (m, 1H), 2.81
(d, J = 9.5Hz, 1H), 2.75-2.65 (m, 2H), 2.63-2.42 (m, 2H), 2.40-2.32 (m, 1H), 2.28-2.23 (m,
1
3
1
1
6
H), 1.67-1.61 (m, 1H), 1.57 (s, 3H), 1.20 (t, J = 13.5 Hz, 1H); C NMR (126MHz, CDCl ) δ:
3
69.23, 156.36, 153.66, 145.16, 139.44, 138.47, 136.94, 130.34, 122.13, 119.99, 114.35, 81.71,
3.03, 59.63, 55.53, 41.63, 36.74, 25.88, 24.73, 23.22, 18.00; HRMS (ESI) calcd for
+
C H N O [M + H ] 426.2024; found 426.2029.
2
3
27
3
5
4
.1.4.6:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
yl)methylene)-N-(4-(trifluoromethyl)phenyl)hydrazine-1-carboxamide (4f). 34% yield; 1H
NMR (400MHz, CDCl ) δ: 9.00 (s, 1H), 8.05 (s, 1H), 7.67-7.58 (m, 4H), 7.51 (s, 1H), 6.15 (d,
3
J = 3.5 Hz, 1H), 6.04 (t, J = 8.4 Hz, 1H), 5.34 (d, J = 3.1 Hz, 1H), 3.84 (t, J = 9.4 Hz, 1H),
2
.95-2.85 (m, 1H), 2.80 (d, J = 9.5 Hz, 1H), 2.76-2.57 (m, 3H), 2.55-2.35 (m, 2H), 2.30-2.24
1
3
(
m, 1H), 1.80-1.70 (m, 1H), 1.58 (s, 3H), 1.21 (t, J = 13.3 Hz, 1H); C NMR (126MHz, CDCl )
3
δ: 169.15, 166.85, 152.85, 145.99, 140.79, 140.32, 138.46, 136.74, 126.45, 126.42, 119.93,
1
18.79, 81.66, 62.98, 59.65, 41.62, 36.66, 25.79, 24.78, 23.20, 17.97; HRMS (ESI) calcd for
+
C H F N O [M + H ] 464.1801; found 464.1797.
2
3
24
3
3
4
4
.1.4.7:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(pyridin-2-yl)hydrazine-1-carboxamide (4g). 33% yield; H NMR (400MHz,
CDCl ) δ: 8.88-8.38 (m, 2H), 8.06 (s, 1H), 8.28 (d, J = 4.5 Hz, 1H), 7.74-7.68 (m, 1H), 7.55-
3

7
3
2
.48 (s, 1H), 7.04-7.00 (m, 1H), 6.14 (d, J = 3.5 Hz, 1H), 6.01 (t, J = 8.2 Hz, 1H), 5.68 (d, J =
.1 Hz, 1H), 3.83 (t, J = 9.4 Hz, 1H), 3.10 (s, 1H),2.82-2.45 (m, 5H), 2.42-2.33 (m, 1H), 2.30-
1
3
.23 (m, 1H), 1.78-1.67 (m, 1H), 1.58 (s, 3H), 1.20 (t, J = 13.2 Hz, 1H); C NMR (101 MHz,
CDCl ) δ: 169.47, 152.92, 151.45, 147.63, 146.59, 140.12, 138.37, 138.16, 137.25, 120.68,
3
1
18.93, 112.76, 81.86, 63.05, 59.59, 41.89, 36.80, 25.86, 24.71, 23.21, 17.99; HRMS (ESI)
+
calcd for C H N O [M + H ] 397.1881; found 397.1876.
2
1
24
4
4
4
.1.4.8:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(pyridin-3-yl)hydrazine-1-carboxamide (4h). 45% yield; H NMR (400MHz,
CDCl ) δ: 9.16 (s, 1H), 8.63 (d, J = 2.3 Hz, 1H), 8.36 (d, J = 4.6 Hz, 1H), 8.08 (d, J = 8.5 Hz,
3
1
H), 7.92 (s, 1H), 7.52 (s, 1H), 7.32 (dd, J = 3.6, 4.7 Hz, 1H), 6.16 (d, J = 3.6 Hz, 1H), 6.04 (t,
J = 8.3 Hz, 1H), 5.36 (d, J = 3.1 Hz, 1H), 3.84 (t, J = 9.4 Hz, 1H), 2.95-2.86 (m, 1H), 2.82 (d,
J = 9.6 Hz, 1H), 2.78-2.44 (m, 4H), 2.42-2.33 (m, 1H), 2.30-2.23 (m, 1H), 1.78-1.71 (m, 1H),
1
3
1
1
4
3
.58 (s, 3H), 1.21 (t, J = 13.7 Hz, 1H); C NMR (126MHz, CDCl ) δ: 169.22, 153.15, 146.13,
3
44.67, 140.77, 140.37, 138.51, 136.78, 134.64, 126.94, 123.92, 119.96, 81.68, 63.00, 59.67,
+
1.66, 36.67, 25.75, 24.79, 23.20, 17.99; HRMS (ESI) calcd for C H N O [M + H ]
2
1
24
4
4
97.1881; found 397.1877.
4
.1.4.9:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(quinolin-5-yl)hydrazine-1-carboxamide (4i). 35% yield; H NMR (500 MHz,
CDCl ) δ: 9.27 (s, 1H), 8.97 (d, J = 3.2 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.20 (s, 1H), 8.02 (d,
3
J = 8.5 Hz, 1H), 7.88 (d, J = 7.4 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.51-7.43 (m, 2H), 6.11 (d,
J = 3.1Hz, 1H), 5.96 (t, J = 8.3 Hz, 1H), 5.31 (s, 1H), 3.82 (t, J = 9.4 Hz, 1H), 3.00-2.91 (m,
1
H), 2.81-2.67 (m, 3H), 2.65-2.57 (m, 1H), 2.53-2.43 (m, 1H), 2.40-2.33 (m, 1H), 2.29-2.22
1
3
(
m, 1H), 1.74-1.68 (m, 1H), 1.57 (s, 3H), 1.20 (t, J = 13.2 Hz, 1H); C NMR (126MHz, CDCl )
3
δ: 169.21, 154.33, 150.50, 148.68, 146.07, 140.01, 138.66, 136.80, 132.38, 129.87, 129.49,
1
1
27.27, 123.47, 121.76, 121.12, 119.83, 81.67, 62.95, 59.68, 41.69, 36.66, 25.73, 24.75, 23.21,
+
7.95; HRMS (ESI) calcd for C H N O [M + H ] 447.2040; found 447.2032.
2
5
26
4
4
4
.1.4.10:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5a). 43% yield; H NMR (500 MHz, DMSO-d ) δ:
6
1
5
1.12 (s, 1H), 8.07 (s, 1H), 7.70 (s, 1H), 7.53 (s, 1H), 6.01 (s, 1H), 5.92 (t, J = 7.7 Hz, 1H),
.52 (s, 1H), 4.04 (t, J = 9.2 Hz, 1H), 3.01-2.92 (m, 1H), 2.84 (d, J = 9.5 Hz, 1H), 2.65-2.57
(m, 1H), 2.55-2.39 (m, 3H), 2.29-2.21 (m, 1H), 2.16-2.09 (m, 1H), 1.60-1.45 (m, 4H), 1.09-
1
3
1
1
.00 (m, 1H); C NMR (101 MHz, DMSO-d ) δ: 177.41, 169.19, 147.26, 140.05, 139.70,
6
37.18, 118.65, 81.55, 62.20, 59.52, 40.88, 36.19, 24.52, 24.14, 22.14, 17.26; HRMS calcd for
+
C H N O S [M + H ] 336.1382; found 336.1371.
1
6
21
3
3

4
.1.4.11:
(E)-N-isopropyl-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5b). 32% yield; H NMR (400MHz, CDCl ) δ: 9.27
3
(s, 1H), 7.49 (s, 1H), 6.92 (d, J = 8.51 Hz, 1H), 6.20 (d, J = 3.5 Hz, 1H), 6.06 (t, J = 8.50 Hz,
1
2
H), 4.65-4.55 (m, 1H), 3.82 (t, J = 9.2 Hz, 1H), 2.86-2.75 (m, 2H), 2.70-2.47 (m, 4H), 2.44-
.32 (m, 1H), 2.30-2.22 (m, 1H), 1.73-1.60 (m, 2H), 1.58-1.55 (s, 3H), 1.24-1.20 (m, 6H), 1.21
1
3
(
t, J = 13.0 Hz, 1H) ; C NMR (126MHz, CDCl ) δ: 176.18, 169.32, 146.28, 141.71, 138.89,
3
1
1
36.94, 119.83, 81.83, 63.12, 59.88, 46.49, 41.85, 36.83, 25.91, 25.11, 23.36, 23.07, 22.85,
+
8.21; HRMS (ESI) calcd for C H N O S [M + H ] 378.1851; found 378.1844.
1
9
27
3
3
4
.1.4.12:
(E)-N-butyl-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5c). 38% yield; H NMR (400MHz, CDCl ) δ: 9.02
3
(s, 1H), 7.45 (s, 1H), 7.13 (t, J = 5.5 Hz, 1H), 6.21 (d, J = 3.6 Hz, 1H), 6.05 (t, J = 8.4 Hz, 1H),
5
2
1
1
2
.34 (d, J = 3.2 Hz, 1H), 3.82 (m, 2H), 3.63 (m, 1H); 2.87-2.76 (m, 2H), 2.69-2.46 (m, 4H),
.41-2.34 (m, 1H), 2.29-2.24 (m, 1H), 1.70-1.63 (m, 3H), 1.59-1.55 (m, 5H), 1.48-1.18 (m,
1
3
H), 0.99 (t, J=7.2 Hz, 3H); C NMR (126MHz, CDCl ) δ: 177.45, 169.34, 146.20, 141.53,
3
38.83, 136.99, 119.97, 81.80, 63.15, 59.89, 44.43, 41.84, 36.83, 31.68, 25.94, 25.12, 23.37,
+
0.35, 18.20, 14.04; HRMS (ESI) calcd for C H N O S [M + H ] 392.2008; found 392.2004.
2
0
29
3
3
4
.1.4.13:
(E)-N-(tert-butyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5d). 15% yield; H NMR (400MHz, CDCl ) δ: 8.91
3
(s, 1H), 7.43 (s, 1H), 7.23 (s, 1H), 6.22 (d, J = 3.6 Hz, 1H), 6.04 (t, J = 8.6 Hz, 1H), 5.36 (d, J
=
3.1 Hz, 1H), 3.82 (t, J = 9.3 Hz, 1H), 2.87-2.76 (m, 2H), 2.69-2.33 (m, 5H), 2.29-2.22 (m,
1
3
1
H), 1.73-1.66 (m, 1H), 1.63-1.59 (m, 9H), 1.58-1.54 (m, 3H), 1.21 (t, J = 14.0 Hz, 1H); C
NMR (126MHz, CDCl ) δ: 175.93, 169.29, 144.84, 141.30, 138.93, 137.02, 119.81, 81.84,
3
6
3.15, 59.85, 53.84, 41.80, 36.86, 29.29, 25.89, 25.11, 23.40, 18.20; HRMS (ESI) calcd for
+
C H N O S [M + H ] 392.2008; found 392.2006.
2
0
29
3
3
4
.1.4.14: (E)-N-(4-fluorophenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5e). 32% yield; H NMR (500MHz, CDCl ) δ: 9.47
3
(s, 1H), 8.74 (s, 1H), 7.57 (s, 1H), 7.52-7.47 (m, 2H), 7.12 (t, J = 7.8 Hz, 2H), 6.18 (s, 1H),
6
9
1
.12 (t, J = 8.2 Hz, 1H), 5.33 (s, 1H), 3.83 (t, J = 9.4 Hz, 1H), 2.90-2.81 (m, 1H), 2.80 (d, J =
.8 Hz, 1H), 2.73-2.56 (m, 3H), 2.54-2.45 (m, 1H), 2.43-2.34 (m, 1H), 2.30-2.24 (m, 1H), 1.75-
1
3
.55 (m, 4H), 1,27-1.19 (m, 1H); C NMR (126MHz, CDCl ) δ: 176.55, 169.08, 160.01 (d, J
3
=
245.4 Hz), 146.81, 142.53, 138.54, 136.61, 133.46 (d, J = 3.4 Hz), 127.12 (d, J = 8.4 Hz),
1
19.85, 115.99 (d, J = 22.6 Hz), 81.56, 62.92, 59.62, 41.71, 36.58, 25.72, 24.99, 23.14, 17.98;
+
HRMS (ESI) calcd for C H FN O S [M + H ] 430.1601; found 430.1597.
2
2
24
3
3

4
.1.4.15: (E)-N-(2-fluorophenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5f). 23% yield; H NMR (400MHz, CDCl ) δ: 9.40
3
(s, 1H), 9.20 (s, 1H), 8.64-8.57 (m, 1H), 7.57 (s, 1H), 7.22-7.15 (m, 3H), 6.18 (d, J = 3.4 Hz,
1
1
2
H), 6.13 (t, J = 8.2 Hz, 1H), 5.46 (d, J = 3.1 Hz, 1H), 3.83 (t, J = 9.3 Hz, 1H), 3.02-2.91 (m,
H), 2.81 (d, J = 9.5 Hz, 1H), 2.76-2.59 (m, 3H), 2.53-2.45 (m, 1H), 2.44-2.35 (m, 1H), 2.30-
1
3
.24 (m, 1H), 1.77-1.67 (m, 1H), 1.58 (s, 3H), 1.23 (t, J = 11.20 Hz, 1H); C NMR (126MHz,
CDCl ) δ: 174.96, 169.32, 153.46 (d, J = 245.5 Hz), 146.84, 142.74, 138.21, 136.86, 126.37 (d,
3
J = 8.4 Hz), 126.16 (d, J = 10.1 Hz), 124.59, 124.20 (d, J = 3.4 Hz), 120.41 (d, J = 3.6 Hz),
1
15.13 (d, J = 20.4 Hz), 81.62, 62.93, 59.74, 41.73, 36.57, 25.41, 25.02, 23.12, 17.98; HRMS
+
(
ESI) calcd for C H FN O S [M + H ] 430.1601; found 430.1586.
2
2
24
3
3
4
.1.4.16:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(p-tolyl)hydrazine-1-carbothioamide (5g). 42% yield; H NMR (500MHz,
DMSO-d ) δ: 11.46 (s, 1H), 9.59 (s, 1H), 7.79 (s, 1H), 7.34 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.2
6
Hz, 2H), 5.98 (m, 2H), 5.40 (s, 1H), 4.05 (t, J = 9.2 Hz, 1H), 3.05-2.97 (m, 1H), 2.87 (d, J =
9
1
1
5
+
.5 Hz, 1H), 2.70-2.62 (m, 2H), 2.53-2.41 (m, 2H), 2.33-2.24 (m, 4H), 2.17-2.11 (m, 1H), 1.62-
1
3
.54 (m, 1H), 1.49 (s, 3H), 1.07 (t, J = 13.4 Hz, 1H); C NMR (126MHz, CDCl ) δ: 176.57,
3
69.30, 153.84, 146.45, 142.40, 138.63, 136.93, 135.10, 129.86, 125.13, 120.23, 81.77, 63.18,
9.79, 41.91, 36.84, 25.97, 25.19, 23.40, 21.32, 18.20; HRMS (ESI) calcd for C H N O S [M
2
3
27
3
3
+
H ] 426.1851; found 426.1839.
.1.4.17: (E)-N-(4-chlorophenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
4
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5h). 27% yield; H NMR (400MHz, CDCl ) δ: 9.53
3
(
s, 1H), 8.80 (s, 1H), 7.59-7.52 (m, 3H), 7.41-7.36 (m, 2H), 6.16 (d, J = 3.3 Hz, 1H), 6.13 (t, J
8.7 Hz, 1H), 5.31 (d, J = 3.1 Hz, 1H), 3.82 (t, J = 9.2 Hz, 1H), 2.90-2.77 (m, 2H), 2.74-2.58
m, 3H), 2.52-2.35 (m, 2H), 2.30-2.24 (m, 1H), 1.76-1.66 (m, 1H), 1.57 (s, 3H), 1.23 (t, J =
=
(
1
3
1
1
1
3.7 Hz, 1H); C NMR (126MHz, CDCl ) δ: 175.96, 169.08, 146.84, 142.67, 138.48, 136.58,
3
36.08, 131.95, 129.11, 125.86, 119.91, 81.55, 62.91, 59.63, 41.69, 36.57, 25.71, 25.00, 23.15,
+
7.98; HRMS (ESI) calcd for C H ClN O S [M + H ] 446.1305; found 446.1291.
2
2
24
3
3
4
.1.4.18: (E)-N-(4-methoxyphenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5i). 27% yield; H NMR (500 MHz, DMSO-d ) δ:
6
1
5
1.42 (s, 1H), 9.55 (s, 1H), 7.78 (s, 1H), 7.20 (d, J = 7.5 Hz, 2H), 6.94 (d, J = 7.0 Hz, 2H), 6.02-
.94 (m, 2H), 5.44 (s, 1H), 4.05 (d, J = 9.2 Hz, 1H), 3.77 (s, 3H), 3.08-2.98 (m, 1H), 2.86 (d, J
=
9.3 Hz, 1H), 2.70-2.62 (m, 2H), 2.52-2.41 (m, 2H), 2.31-2.23 (m, 1H), 2.16-2.11 (m, 1H),
1
3
1
.63-1.53 (m, 1H), 1.49 (s, 3H), 1.07 (t, J = 13.7 Hz, 1H); C NMR (126MHz, CDCl ) δ:
3

1
6
76.88, 169.09, 158.30, 146.28, 142.15, 138.48, 136.72, 130.30, 126.96, 119.96, 114.24, 81.57,
2.96, 59.58, 55.51, 41.72, 36.63, 25.76, 24.97, 23.17, 17.99; HRMS (ESI) calcd for
+
C H N O S [M + H ] 442.1801; found 442.1794.
2
3
27
3
4
4
.1.4.19:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(4-(trifluoromethyl)phenyl)hydrazine-1-carbothioamide (5j). 36% yield; H
NMR (400MHz, DMSO-d ) δ: 11.72 (s, 1H), 9.88 (s, 1H), 7.84-7.80 (m, 3H), 7.76-7.72 (s, 2H),
6
6
1
2
.02 (t, J = 8.4 Hz, 1H), 5.86 (d, J = 3.9 Hz, 1H), 5.37 (d, J = 2.6 Hz, 1H), 4.06 (t, J = 8.5 Hz,
H), 3.05-2.94 (m, 1H), 2.87 (d, J = 9.5 Hz, 1H), 2.73-2.64 (m, 2H), 2.54-2.42 (m, 2H), 2.34-
1
3
.25 (m, 1H), 2.18-2.12 (m, 1H), 1.65-1.55 (m, 1H), 1.50 (s, 3H), 1.08 (t, J = 12.5 Hz, 1H); C
NMR (126MHz, DMSO-d ) δ: 175.51, 169.23, 148.49, 142.78, 141.34, 139.92, 137.16, 125.60,
6
1
5
25.28 (q, J = 30.8 Hz), 125.15 (q, J = 31.0 Hz), 123.12 (q, J = 271 Hz), 118.31, 81.60, 62.22,
9.63, 40.91, 36.20, 24.57, 24.28, 22.16, 17.33; HRMS (ESI) calcd for C H F N O S [M +
2
3
24
3
3
3
+
H ] 480.1569; found 480.1555.
4
.1.4.20:
(E)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1
a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)-N-(3-(trifluoromethyl)phenyl)hydrazine-1-carbothioamide (5k). 18% yield; H
NMR (400MHz, CDCl ) δ: 9.40 (s, 1H), 8.91 (s, 1H), 7.90-7.84 (m, 2H), 7.60-7.51 (m, 3H),
3
6
2
.20-6.12 (m, 2H), 5.33 (d, J = 3.2 Hz, 1H), 3.83 (t, J = 9.1 Hz, 1H), 2.91-2.78 (m, 2H), 2.76-
1
3
.36 (m, 5H), 2.31-2.24 (m, 1H), 1.78-1.68 (m, 1H), 1.58 (s, 3H), 1.23 (t, J = 13.4 Hz, 1H); C
NMR (126MHz, CDCl ) δ: 175.85, 169.08, 147.19, 142.94, 138.51, 138.10, 136.52, 131.49 (q,
3
J = 33.0 Hz), 129.51, 127.77, 124.72 (q, J = 272 Hz), 123.02 (q, J = 4.0 Hz), 121.03 (q, J = 4.0
Hz), 119.88, 81.54, 62.89, 59.66, 41.72, 36.54, 25.69, 25.03, 23.14, 17.98; HRMS (ESI) calcd
+
for C H F N O S [M + H ] 480.1569; found 480.1570.
2
3
24
3
3
3
4
.1.4.21:
(E)-N-(4-ethylphenyl)-2-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-
oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[2',3':9,10]cyclodeca[1,2-b]furan-5-
1
yl)methylene)hydrazine-1-carbothioamide (5l). 51% yield; H NMR (500MHz, DMSO-d ) δ:
6
1
5
1.47 (s, 1H), 9.59 (s, 1H), 7.79 (s, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 7.8 Hz, 2H), 6.02-
.94 (m, 2H), 5.75 (s, 2H), 5.41(s, 1H), 4.05 (t, J = 9.4 Hz, 1H), 3.06-2.96 (m, 1H), 2.87 (d, J
=
9.6 Hz, 1H), 2.71-2.57 (m, 4H), 2.31-2.21 (m, 1H), 2.18-2.10 (m, 1H), 1.63-1.54 (m, 1H),
1
3
1
1
5
.49 (s, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.07 (t, 13.1 Hz, 1H); C NMR (126 MHz, DMSO-d ) δ:
6
69.24, 147.62, 140.91, 140.63, 139.96, 137.26, 136.63, 127.34, 126.00, 118.35, 81.63, 62.24,
9.63, 54.81, 40.92, 36.23, 27.62, 24.63, 24.24, 22.14, 17.33, 15.64; HRMS (ESI) calcd for
+
C H N O S [M + H ] 440.2008; found 440.2011.
2
4
29
3
3
4
.2 Cell proliferation assay

The growth inhibitory effect of compounds toward five cancer cell lines was evaluated by the
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were
3
plated at a density of 1500-2000 cells/well in 96-well plates and incubated for 24 h. Compounds
were added to the wells with various concentrations. After 96 h, a total of 50 μL MTT stock
solutions (2 mg/mL, BIOFROXX, Germany) were added to each well. The 96-well plates were
subsequently incubated for a further 2 h at 37˚C. The medium was replaced with DMSO (200
μL/well, Biosharp, Inc., Hefei, China;). Following gentle agitation, an ELISA reader (WD-
2
102A, Beijing Liuyi Biotech Co., Ltd., China) was used to measure the absorbance at 570 nm.
Three parallel samples were measured in each cell line. The absorbance values were normalized
to the values obtained for vehicle-treated cells to determine the percentage of surviving cells.
The median inhibitory concentration (IC ) was assessed from the dose response curve.
5
0
4
.3 In vivo antitumor experiments
The MC38 xenograft model was established in male C57BL/6 mice (6-8 weeks old). The
animals were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China) and housed in
controlled environment at 25 °C on a 12 h light/dark cycle. All animal protocols were
conformed to the Guidelines for the Care and Use of Laboratory Animals approved by the
Animal Care and Use Committee of Chinese Academy of Medical Sciences and Peking Union
Medical College. C57BL/6 mice were subcutaneously implanted with 0.2 mL MC38 cell
7
dilution (5×10 cells/mL) on the flank. 24 h after inoculation, mice were randomly divided into
7
groups (n = 6). One group received gavage (p.o.) of 25% PEG400 as a model control; one
group received an intraperitoneal injection of 40.0 mg/kg 5-FU (once every three days) used as
the positive control. 5 groups involved 40.0 mg/kg of 4d, 4f, 4g, 4i, and 5g (dissolved in 25%
PEG400) were continuously administrated p.o. for 12 days, once a day. The weight of tumor,
body, thymus and spleen at the end were measured.
4
.4 Chemical stability studies:
The chemical stability assay was adapted from a previous research.13 Compound 4d was
dissolved in DMSO to obtain a solution of 1.6 mM. 1 mL of this solution was diluted with pH
7
.4 phosphate buffer solution to a final concentration of 0.08 mM. 1 mL of the 0.08 mM solution
mixture were placed in water bath at 37 °C for different periods of time. At the end of each time
period, the sample was mixed with 5 mL of acetonitrile and filtered (Jinteng, 0.45 μm pore
size). Three parallel experiments were conducted for each time period. The liquid sample was
analyzed by a high-performance liquid chromatography (Wufeng LC100) equipped with a
Kromasil C18 column (4.6 mm * 250 mm, 5 μm) and an ultraviolet detector at the wavelength

of 220 nm. The mobile phase was 60% acetonitrile and 40% water at the flow rate of 1 mL/min.
Each sample was analyzed 3 times, and average values were used for plotting.
4
.5 Flow cytometric study of cell apoptosis and cell cycle
Cell apoptosis of 4d was studied using AnnexinV-FITC/PI labeled Apoptosis Assay Kit
(BestBio, Shanghai, China) according to the manufacturer’s instructions. Briefly, U87-MG was
6
cultured into 6-well plates and incubated for 48 h. Then, cells (10 cells/mL) were treated with
0
μM, 4 μM and 16 μM of 4d in DMSO for 12 hours. After the cells were collected and washed
twice with cold PBS and once with binding buffer, cells were stained by 5 μL of AnnexinV-
FITC and 5 μL of PI. Apoptic cells were analyze by a flow cytometry ACEA Novocyte.
For cell cycle analysis, incubated U87-MG were treated with 0 μM, 4 μM and 16 μM of 4d in
DMSO for 12 hours. Cells were then harvested, washed with PBS, and then fixed with ice-cold
7
0% ethanol at -20 °C overnight. PI/RNase A (BestBio, Shanghai, China) solution was used to
stain cells for 30 min in the dark. The samples were determined by a flow cytometry ACEA
Novocyte.
4
.6 Molecular docking studies:
Molecular docking was carried out using the Discovery Studio as implemented through the
graphical user interface DS-CDOCKER protocol. The 3D structures of compound 4d were
constructed using Chem3D software (Chemical Structure Drawing Standard; Cambridge Soft
corporation). The molecular docking in this study was performed by inserting compound 4d
into the binding pocket of NF-κB (PDB code: 1NFK). Types of interactions of the docked
protein with ligand-based pharmacophore model were analyzed after the end of molecular
docking.
Acknowledgements
This work was supported by the National Drug Innovation Major Project (2018ZX09711-001-
0
05).
Supplementary Information
Supplementary material is available on the publisher's website along with the published article.

Reference
1
2
3
.
.
.
Ghantous, A.; Sinjab, A.; Herceg, Z.; Darwiche, N. Drug Discov Today 2013, 18, 894.
Mathema, V. B.; Koh, Y.-S.; Thakuri, B. C.; Sillanpää, M. Inflammation 2012, 35, 560.
Guzman, M. L.; Rossi, R. M.; Karnischky, L.; Li, X.; Peterson, D. R.; Howard, D. S.; Jordan,
C. T. Blood 2005, 105, 4163.
Ghantous, A.; Gali-Muhtasib, H.; Vuorela, H.; Saliba, N. A.; Darwiche, N. Drug Discov
Today 2010, 15, 668.
Siedle, B.; García-Piñeres, A. J.; Murillo, R.; Schulte-Mönting, J.; Castro, V.; Rüngeler, P.;
Klaas, C. A.; Da Costa, F. B.; Kisiel, W.; Merfort, I. J Med Chem 2004, 47, 6042.
Tu, Y.; Wang, C.; Xu, S.; Lan, Z.; Li, W.; Han, J.; Zhou, Y.; Zheng, P.; Zhu, W. Bioorg. Med.
Chem. 2017, 25, 3148.
4
.
5
.
6
.
7
8
.
.
Kalinowski, D. S.; Quach, P.; Richardson, D. R. Future Med. Chem. 2009, 1, 1143.
Elsayed, H. E.; Ebrahim, H. Y.; Haggag, E. G.; Kamal, A. M.; El Sayed, K. A. Bioorg. Med.
Chem. 2017, 25, 6297.
9
2
1
1
.
Liu, Z.; Wu, S.; Wang, Y.; Li, R.; Wang, J.; Wang, L.; Zhao, Y.; Gong, P. Eur. J. Med. Chem.
014, 87, 782.
0. Laura, G.; Marinella, R.; Giovanni, B.; Mariarosaria, F. Curr. Med. Chem. 2016, 23, 1528.
1. Richardson, D. R.; Kalinowski, D. S.; Richardson, V.; Sharpe, P. C.; Lovejoy, D. B.; Islam, M.;
Bernhardt, P. V. J Med Chem 2009, 52, 1459.
2. Yang, Z. J.; Kuang, B. J.; Kang, N.; Ding, Y. H.; Ge, W. Z.; Lian, L. H.; Gao, Y.; Wei, Y. Q.;
Chen, Y.; Zhang, Q. Eur. J. Med. Chem. 2017, 127, 296.
3. Alves, M. A.; de Queiroz, A. C.; Alexandre-Moreira, M. S.; Varela, J.; Cerecetto, H.;
González, M.; Doriguetto, A. C.; Landre, I. M.; Barreiro, E. J.; Lima, L. M. Eur. J. Med. Chem.
1
1
2
1
1
1
015, 100, 24.
4. He, Z.-Y.; Huang, C.-F.; Tian, S.-K. Org Lett 2017, 19, 4850.
5. Tripathi, L.; Kumar, P.; Singh, R.; Stables, J. P. Eur. J. Med. Chem. 2012, 47, 153.
6. Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V. A.;
Radchenko, E. V.; Zefirov, N. S.; Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. Journal of
Computer-Aided Molecular Design 2005, 19, 453.
1
1
7. VCCLAB. 2005.
8. Patel, N. M.; Nozaki, S.; Shortle, N. H.; Bhat-Nakshatri, P.; Newton, T. R.; Rice, S.; Gelfanov,
V.; Boswell, S. H.; Goulet, R. J.; Sledge, G. W.; Nakshatri, H. Oncogene 2000, 19, 4159.
9. D'Anneo, A.; Carlisi, D.; Lauricella, M.; Puleio, R.; Martinez, R.; Di Bella, S.; Di Marco, P.;
1
Emanuele, S.; Di Fiore, R.; Guercio, A.; Vento, R.; Tesoriere, G. Cell Death & Disease 2013, 4,
e891.
2
0. Yang, Z.-J.; Ge, W.-Z.; Li, Q.-Y.; Lu, Y.; Gong, J.-M.; Kuang, B.-J.; Xi, X.; Wu, H.; Zhang, Q.;
Chen, Y. J Med Chem 2015, 58, 7007.
1. Kreuger, M. R. O.; Grootjans, S.; Biavatti, M. W.; Vandenabeele, P.; D’Herde, K. Anti-
cancer drugs 2012, 23, 883.
2
2
2
2
2. Hayden, M. S.; West, A. P.; Ghosh, S. Oncogene 2006, 25, 6758.
3. Shi, F.; Zhao, J.-H.; Liu, Y.; Wang, Z.; Zhang, Y.-T.; Feng, N.-P. Int J Nanomed 2012, 7, 2033.
4. Liu, Y.; Lu, W.-L.; Guo, J.; Du, J.; Li, T.; Wu, J.-W.; Wang, G.-L.; Wang, J.-C.; Zhang, X.; Zhang,
Q. J. Controlled Release 2008, 129, 18.
5. Zhang, D.; Qiu, L.; Jin, X.; Guo, Z.; Guo, C. Mol. Cancer Ther. 2009, 7, 1139.
2

2
6. Ralstin, M. C.; Gage, E. A.; Yip-Schneider, M. T.; Klein, P. J.; Wiebke, E. A.; Schmidt, C. M.
Mol. Cancer Ther. 2006, 4, 387.
7. Wyrębska, A.; Szymański, J.; Gach, K.; Piekielna, J.; Koszuk, J.; Janecki, T.; Janecka, A. Mol
Biol Rep 2013, 40, 1655.
2
2
2
8. Chen, F.; Castranova, V.; Shi, X. Am J Pathol 2001, 159, 387.
9. Fonrose, X.; Ausseil, F.; Soleilhac, E.; Masson, V.; David, B.; Pouny, I.; Cintrat, J.-C.;
Rousseau, B.; Barette, C.; Massiot, G.; Lafanechère, L. Cancer Research 2007, 67, 3371.
0. Sugiyama, H.; Yoshida, M.; Mori, K.; Kawamoto, T.; Sogabe, S.; Takagi, T.; Oki, H.; Tanaka,
3
T.; Kimura, H.; Ikeura, Y. Chem. Pharm. Bull. 2007, 55, 613.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: