Novel steroidal 5α,8α-endoperoxide derivatives with semicarbazone/thiosemicarbazone side-chain as apoptotic inducers through an intrinsic apoptosis pathway: Design, synthesis and biological studies

Article abstract of DOI:10.3390/molecules25051209

A series of novel steroidal 5α,8α-endoperoxide derivatives bearing semicarbazone (7a-g) or thiosemicarbazone (7h-k) side chain were designed, synthesized and evaluated for their cytotoxicities in four human cancer cell lines (HepG2, HCT-116, MCF-7, and A549) using the MTT assay in vitro. The results showed that compound 7j exhibited significant cytotoxic activity against HepG2 cells (IC50 = 3.52 μM), being more potent than ergosterol peroxide. Further cellular mechanism studies in HepG2 cells indicated that compound 7j triggered the mitochondrial-mediated apoptosis by decreasing mitochondrial membrane potential (MMP), which was associated with up-regulation of Bax, down-regulation of Bcl-2, activation levels of the caspase cascade, and formation of reactive oxygen species (ROS). The above findings indicated that compound 7j may be used as a promising skeleton for antitumor agents with improved efficacy.

Full text of DOI:10.3390/molecules25051209

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
molecules
Article
Novel Steroidal 5α,8α-Endoperoxide Derivatives
with Semicarbazone/Thiosemicarbazone Side-chain
as Apoptotic Inducers through an Intrinsic Apoptosis
Pathway: Design, Synthesis and Biological Studies
Liwei Ma 1, Haijun Wang 2, Jing Wang 2, Lei Liu 2, Song Zhang 3 and Ming Bu 2,
*
1
Research Institute of Medicine & Pharmacy, Qiqihar Medical University, Qiqihar 161006, China;
maliwei@qmu.edu.cn
College of Pharmacy, Qiqihar Medical University, Qiqihar 161006, China; wanghaijun@qmu.edu.cn (H.W.);
qyyx305@qmu.edu.cn (J.W.); liuleiqiyi@sina.com (L.L.)
Basic Medical Science College, Qiqihar Medical University, Qiqihar 161006, China; zhangsong@qmu.edu.cn
Correspondence: buming@qmu.edu.cn; Tel.: +86-0452-266-3881
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 February 2020; Accepted: 6 March 2020; Published: 7 March 2020
Abstract: A series of novel steroidal 5
thiosemicarbazone (7h ) side chain were designed, synthesized and evaluated for their cytotoxicities
in four human cancer cell lines (HepG2, HCT-116, MCF-7, and A549) using the MTT assay in vitro
The results showed that compound 7j exhibited significant cytotoxic activity against HepG2 cells
(IC50 = 3.52 M), being more potent than ergosterol peroxide. Further cellular mechanism studies
α,8α-endoperoxide derivatives bearing semicarbazone (7a–g) or
–
k
.
µ
in HepG2 cells indicated that compound 7j triggered the mitochondrial-mediated apoptosis by
decreasing mitochondrial membrane potential (MMP), which was associated with up-regulation of
Bax, down-regulation of Bcl-2, activation levels of the caspase cascade, and formation of reactive
oxygen species (ROS). The above findings indicated that compound 7j may be used as a promising
skeleton for antitumor agents with improved efficacy.
Keywords: ergosterol peroxide; semicarbazone; thiosemicarbazone; anti-tumor activity; apoptosis;
HepG2 cells
1. Introduction
Cancer is a serious disease that poses a risk people’s health. The discovery of potent anticancer
drugs from natural products is one of the important directions in drug research. Nowadays, it’s still
more than 50% of drugs used in the areas of cancer and infectious diseases are based on natural
origins [
bond (-O-O-) in their structures as cyclic organic compounds, such as artemisinin, schinalactone A,
talaperoxides B and gracilioethers A, yingzaosu, etc. (Figure 1) [ ]. Most of natural endoperoxides
have been proved with antiviral, anticancer or antifungal activity [ ]. The most representative
discovery of endoperoxide from natural products is artemisinin by Youyou Tu (2015 Nobel Award
winner) [ ]. Artemisinin was extracted from a traditional Chinese medicine (Artemisia annua L.), and
1,2]. Natural endoperoxides are widely found in nature, which all have a distinct peroxide
3–5
6–8
9
many derivatives of it have been synthesized and used for treatment of malaria [10,11].
Steroidal compounds have drawn attention not only due to their unusual and interesting chemical
structures, but also due to their widespread application as anti-inflammatory, diuretic, anabolic,
contraceptive, and anticancer agents [12]. Most steroidal drugs in use today are semisynthetic
compounds widely used in traditional medicines by modification of the steroid ring system and side
chains [13]. The interesting structural and stereochemical features of the steroid nucleus provide
Molecules 2020, 25, 1209; doi:10.3390/molecules25051209
www.mdpi.com/journal/molecules

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Molecules 2020, 25, 1209
2 of 16
additional fascination for researchers, and the introduction of heteroatoms, heterocycles, or amides,
or replacement of one or more carbon atoms in the steroidal skeleton has been envisaged to discover
new chemical entities with the potential to become promising future drugs, resulting in notable
modifications in biological activity [14–16].
Figure 1. Representative compounds of natural endoperoxides with bioactivities.
Ergosterol peroxide (5
fungal secondary metaboliters of sterol 5
biological activities have been attributed to EP, including antitumor, anti-inflammatory, antiviral
and immunomodulatory activities and antioxidant activities [16 21]. As an important active lead
compound in drug discovery, EP is well known for its 5 ,8 -peroxy moiety. During the last years,
α
,8
α
-epidioxiergosta-6,22-dien-3
β-ol, EP) is a member of a class of
α
,8α-endoperoxidederivatives (Figure 2). A number of
–
α
α
our research group has been working on the modifications of steroids to obtained more active
compounds as potential antitumor agents. In our previous studies, we synthesized a series of steroidal
5α,8α-endoperoxide derivatives with aliphatic side-chain. We found that most of these derivatives
exhibited significant anticancer activities compared to ergosterol, which has no peroxidic bridge at C-5
and C-8 positions with barely inhibition activity against all the tested cells. The studies showed that
the 5
several series of steroidal 5
and hydrazone hybrid side-chain. The MTT assay indicated that some derivatives possessing the
,8 -peroxy group and different side chains displayed distinct cytotoxic activity against some cancer
cell lines [23 27]. Based on the above results, we speculate that the difference between side chains at
α
,8α
-peroxygroup is requisite pharmacophore for these derivatives [22]. Then we synthesized
α
,8α-endoperoxide derivatives with 17-(O-carboxymethyl)oxime side-chain
5
α α
–
C-17 position and 5α,8α-peroxy group would provide a synergistic effect for the bioactivity.
Figure 2. Structure of ergosterol peroxide (EP).
Semicarbazones and thiosemicarbazones are a class of Schiff bases that have been gaining
considerable attention due to their wide array of pharmacological effects or biological activities [28,29]
.
In a literature review, we found that semicarbazone based derivatives have shown anticancer,
anti-protozoal, antimicrobial, pesticidal activity, etc. [30–33]. In addition, thiosemicarbazones (TSCs)
have a wide clinical antitumor spectrum with efficacy in various tumor types such as bladder cancer,
pancreatic cancer, cervical cancer, non-small cell lung cancer, breast cancer, prostate cancer and
leukemia [34–37]. Figure 3 shows some representative compounds with thiosemicarbazones structure
in the clinical trial. In addition, it is reported that combining steroids (progesterone and testosterone)
with semicarbazide or thiosemicarbazide moieties could be a good anticancer drug [38].

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Molecules 2020, 25, 1209
3 of 16
Figure 3. Representative compounds with thiosemicarbazones structure in the clinical trial.
Inspired by the good biological properties of semicarbazones and thiosemicarbazones, and in
continuation with our previous work on the modifications of steroids, we here report the design and
synthesis of a series of novel steroidal endoperoxide derivatives with semicarbazone/thiosemicarbazone
side-chain (Figure 4) and their underlying action mechanisms of anticancer activity, wishing to find
new effective antitumor agents.
Figure 4. Design strategy of novel steroidal derivatives.
2. Results and Discussion
2.1. Chemistry
The general procedure for the synthesis of steroidal derivatives is shown in Scheme 1.
Dehydroepiandrosterone (DHEA,
and dichloromethane to give compound
N-Bromosuccinimide (NBS) and debromination with 2,4,6-collidineto afford compound
was reacted with potassium hydroxide for deacetylation to give compound . Subsequently, compound
, using phloxine B as photosensitizer. Then, compound
was reacted with hydrazine hydrate to obtain the corresponding steroidal hydrazine . At last, the
was reacted with different phenyl isocyanate or isothiocyanate substituents to
,8 -endoperoxide steroidal conjugation semicarbazone/thiosemicarbazone
The structures of all compounds were characterized by MS, 1H-NMR and
1
) was reacted with acetic anhydride in the presence of pyridine
. Compound further underwent bromination with
. Compound
2
2
3
3
4
4
5
was reacted with oxygen to give endoperoxide 5
6
resulting hydrazine
obtain target novel 5
derivatives 7a
13C-NMR spectrum.
6
α
α
–k.
2.2. Biological Evaluation
2.2.1. In Vitro Cytotoxic Activity
All of the newly synthesized steroidal endoperoxide derivatives 7a–k were investigated for
their anti-proliferative activities against human hepatocellular carcinomacells (HepG2), human breast
carcinoma cells (MCF-7), human colorectal cells (HCT-116), and human lung carcinoma cells (A549) by
the MTT assay in vitro. Mitomycin was used as positive reference drug. The results of the MTT assay
were summarized as IC50 values in Table 1.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Molecules 2020, 25, 1209
4 of 16
O
O
O
ii
i
AcO
HO
AcO
DHEA, 1
3
2
O
O
iii
v
iv
O
O
HO
HO
4
5
X
R
N NH2
N
N
N
vi
H
H
O
O
O
O
HO
6
HO
7a-k
7a R = 4-Cl
7d R = 3-CH3
7k R = 4-CH3
7b R = 3-CF3
7c R = 4-OCH3
7g R = 4-CN
X = O
7e R = 4-CF3
7h R = 4-Cl
7f R = 3-CF3-4-Cl
X = S
7i R = 4-OCH3
7j R = 3-Cl
Scheme 1. Synthesis of compounds 7a–k. Reagents and conditions: (i) Ac2O, pyridine, DCM, rt, 97%;
(ii) Cyclohexane, NBS, reflux, 1 h; 2,4,6-collidine, xylene, reflux, 3 h, 33%; (iii) KOH, MeOH, petroleum
ether, reflux, 1 h, 93%; (iv) MeOH, O2, phloxine B, light, 0◦C, 0.5 h, 44%; (v) NH2NH2-H2O, EtOH,
45◦C,1 h, 82%; (vi) EtOH, AcOH, different isocyanates and thioisocyanates for 7h
–k, rt, 2~5 h, 85%~93%.
Table 1. In vitro anti-proliferativeactivities of compounds7a–k in human cancer cells.
IC50 (µM) a
Compound
X
R
HepG2
HCT-116
MCF-7
A549
7a
7b
7c
7d
7e
7f
7g
7h
O
O
O
O
O
O
O
S
S
S
S
-
4-Cl
3-CF3
4-OCH3
4-CH3
4-CF3
3-CF3-4-Cl
4-CN
4-Cl
4-OCH3
3-Cl
4-CH3
-
8.65 ±0.72
5.73 ± 0.78
>60
32.59 ± 2.18
10.48 ± 0.59
>60
19.31 ± 1.08
8.62 ± 0.71
52.32 ± 2.14
>60
14.90 ± 0.90
7.37 ± 0.42
8.98 ± 0.70
7.66 ± 0.49
26.73 ± 1.75
4.88 ± 0.42
25.73 ± 1.52
16.04 ± 0.81
16.56± 1.09
21.43 ± 1.12
18.04 ± 0.93
>60
47.33 ± 2.17
21.88 ± 1.21
6.03 ± 0.81
16.93 ± 1.77
11.33 ± 0.69
>60
37.50 ± 2.26
7.51 ± 0.77
4.34 ± 0.41
6.06 ± 0.69
5.44 ± 0.42
14.35± 0.82
3.52± 0.35
19.07 ± 1.09
21.35± 1.13
29.36 ± 1.14
>60
26.44 ± 1.43
12.78 ± 1.42
14.07 ± 1.58
15.41 ± 1.63
>60
9.79 ± 0.55
48.36 ± 4.43
26.42± 1.32
10.52± 0.87
7i
7j
7k
7.70 ± 0.62
>60
EP
19.48 ± 1.16
12.47 ± 1.04
Mitomycin
-
-
a
IC50: Concentration of the tested compound that inhibits 50% of cell growth. All data are presented as means
of three independent experiments.
±
SD