Design, synthesis and anticancer activity of novel nopinone-based thiosemicarbazone derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.04.024

A series of new nopinone-based thiosemicarbazone derivatives were designed and synthesized as potent anticancer agents. All these compounds were identified by ¹H NMR, ¹³C NMR, HR-MS spectra analyses. In the in vitro anticancer activity, most derivatives showed considerable cytotoxic activity against three human cancer cell lines (MDA-MB-231, SMMC-7721 and Hela). Among them, compound 4i exhibited most potent antitumor activity against three cancer cell lines with the IC₅₀ values of 2.79?±?0.38, 2.64?±?0.17 and 3.64?±?0.13?μM, respectively. Furthermore, the cell cycle analysis indicated that compound 4i caused cell cycle arrest of MDA-MB-231 cells at G2/M phase. The Annexin V-FITC/7-AAD dual staining assay also revealed that compound 4i induced the early apoptosis of MDA-MB-231 cells.

Full text of DOI:10.1016/j.bmcl.2017.04.024

Accepted Manuscript
Design, synthesis and anticancer activity of novel nopinone-based thiosemicar-
bazone derivatives
Yunyun Wang, Wen Gu, Yu Shan, Fei Liu, Xu Xu, Yiqin Yang, Qiangjian
Zhang, Yan Zhang, Hongbo Kuang, Zhonglong Wang, Shifa Wang
PII:
S0960-894X(17)30400-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.024
BMCL 24877
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 January 2017
4 April 2017
7 April 2017
Please cite this article as: Wang, Y., Gu, W., Shan, Y., Liu, F., Xu, X., Yang, Y., Zhang, Q., Zhang, Y., Kuang, H.,
Wang, Z., Wang, S., Design, synthesis and anticancer activity of novel nopinone-based thiosemicarbazone
derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.024
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Design, synthesis and anticancer activity of novel nopinone-based
thiosemicarbazone derivatives
Yunyun Wanga, Wen Gua,d, Yu Shanb, Fei Liub, Xu Xua,d, Yiqin Yangc, Qiangjian Zhanga,
Yan Zhanga, Hongbo Kuanga, Zhonglong Wanga, Shifa Wanga,c
a College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, People’s Republic of
China
b Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem.Sun
Yat-Sen), 210014, People’s Republic of China
c
Institute of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037,
People’s Republic of China
d Jiangsu Key Lab of Biomass-based green Fuels and Chemicals, Nanjing 210037, People’s Republic ofChina
 Corresponding author. Tel.: +86 25 85427812; fax: +86 25 85427812.
E-mail: wangshifa65@163.com (S. Wang).

Abstract
A series of new nopinone-based thiosemicarbazone derivatives were designed and synthesized as
1
potent anticancer agents. All these compounds were identified by H-NMR, 13C-NMR, HR-MS
spectra analyses. In the in vitro anticancer activity, most derivatives showed considerable cytotoxic
activity against three human cancer cell lines (MDA-MB-231, SMMC-7721 and Hela). Among them,
compound 4i exhibited most potent antitumor activity against three cancer cell lines with the IC50
values of 2.79 ± 0.38, 2.64 ± 0.17 and 3.64 ± 0.13 M, respectively. Furthermore, the cell cycle
analysis indicated that compound 4i caused cell cycle arrest of MDA-MB-231 cells at G2/M phase.
The Annexin V-FITC/7-AAD dual staining assay also revealed that compound 4i induced the early
apoptosis of MDA-MB-231 cells.
Keywords: Nopinone; Thiosemicarbazone; Anticancer; Apoptosis

Malignant tumor is a kind of disease that causes tremendous threat to human life and health. There
is increasing number of people died because of cancer in recent years1. Nowadays, chemotherapy is
still the main method in the treatment of cancer. In spite of new anticancer agents developed,
accumulation of toxicity has limited its application as antitumor drugs2,3. Thus, the investigations of
new anticancer drugs with high efficacy and low side effects are still an urgent demand for medicinal
chemists.
Thiosemicarbazone, containing thiourea based functional nucleus, is a vital chelator of metal ions
and many of them are seriously significant with biological activities, especially in anticancer
activity4,5. Thus, thiosemicarbazone derivatives were often considered as a tumor inhibitor via their
capability of chelation with iron and copper ions in cancer cells6. Recently, a lot of
thiosemicarbazone derivatives have been synthesized and their antitumor activities were also
reported7-9. For example, 3-AP is a thiosemicarbazone derivative with potent anticancer activity and
ribonucleotide reductase inhibitory activity, which has been advanced into clinical trials10, 11. In
addition, some other thiosemicarbazones such as DpT, DpC, Dp44Mt also showed potent anticancer
activity12. Therefore, thiosemicarbazone have proved to be a promising resource for the discovery of
new anticancer agents.
As natural products have complex and diverse structures, it plays an important role in new drug
discovery. As a major component of turpentine, β-pinene is an important natural terpene resource
and is widely used for producing natural perfume. β-pinene possess an extensive spectrum of
biological activities such as anticancer, antibacterial, antiviral and antidepressant activities13-16, but
the application of β-pinene in pharmaceuticals is less than its application in spices. β-pinene is easily
oxidized to nopinone. From the literature, nopinone was converted to a variety of derivatives and it

has experienced considerable attention for its amazing applications as drugs, fluorescent materials,
spices, etc17,18, 21,22
.
In our previous study, many applications of nopinone have been investigated such as
anticancer19,20, antimicrobial20, insecticidal activities21 and fluorescent dyes22,23. Nevertheless, there
are few literatures reporting the synthesis of thiosemicarbazone derivatives with nopinone moiety.
The aforementioned findings give us an impetus to design and synthesize thiosemicarbazone
derivatives of nopinone and explore their potential values as anticancer agents.
Scheme 1. Synthetic route for the final compounds 4a-4i from β-pinene. Reagents and conditions: (a)
KMnO4, H2SO4, acetone, room temperature, 6h; (b) substituted benzaldehyde, sodium ethoxide,
ethyl alcohol, reflux, 8h; (c) hydrochloric acid, ethyl alcohol, reflux, 4-6h.
The target compounds were synthesized via a 3-step synthetic route from β-pinene (1) as shown in
Scheme 1. β-Pinene (1) was easily oxidized to nopinone (2) by potassium permanganate. The key
intermediates, α, β-unsaturated ketones (3a-3f) of nopinone were synthesized by reacting compound
2 with substituted benzaldehyde in the presence of absolute ethyl alcohol and sodium ethoxide
according to our earlier work24. At last, the ketones 3a-3f were further reacted with different
substituted thiosemicarbazides to obtain target products 4a-4r. All the synthesized compounds were

purified by recrystallization or silica gel column chromatography, and their structures were
characterized by HR-MS, 1H-NMR and 13C-NMR spectra analyses.
Table 1 The anticancer activities of compounds 4a-4r
IC50 (M)
compound
MBA-MD-231
4.57±0.21
SMMC-7721
9.53±0.10
Hela
Hlf-1
>40
>40
>40
4a
4b
4c
12.79±0.01
14.35±0.01
10.63±0.02
3.98±0.16
9.04±0.04
6.21±0.14
8.30±0.14
16.90±0.08
12.36±0.45
14.64±0.24
>40
4d
4e
4f
3.38±0.59
4.09±0.44
13.88±0.14
10.93±1.10
10.63±0.09
24.55±0.21
23.32±0.03
>40
>40
>40
>40
4g
＞40
4h
4i
8.66±0.17
2.79±0.38
3.94±0.18
2.64±0.17
>40
7.11±0.04
3.64±0.13
6.97±0.08
>40
>40
>40
4j
＞40
＞40
＞40
＞40
＞40
＞40
＞40
＞40
4k
>40
>40
20.42±0.03
18.98±0.10
24.84±0.09
21.94±0.11
>40
>40
>40
>40
>40
>40
>40
>40
>40
4l
4m
7.46±0.11
19.26±0.09
>40
4n
4o
31.15±0.10
4p
>40
>40
4q
16.99±0.16
>40
8.81±0.12
9.97±0.13
7.89±0.79
4r
＞40
Etoposide
29.27±0.45
40.44±0.17
The designed 18 compounds were evaluated for cytotoxicity against human breast cancer cell line

(MDA-MB-231), human cervical carcinoma cell line (Hela) and human hepatocarcinoma cell line
(SMMC-7721) and human normal embryonic lung fibroblast (Hlf-1) using MTT assay. Etoposide
was selected as positive control and the IC50 values for inhibition of proliferation were illustrated in
Table 1.
In cytotoxic assay, eight compounds (4a-4f, 4h, 4i) showed strong antineoplastic activity against
three tested cancer cell lines (MDA-MB-231, SMMC-7721 and Hela cells) in vitro，especially
compound 4i showed the most potent activity with IC50 values of 2.79 M (for MDA-MB-231 cells),
2.65 M (for SMMC-7721 cells) and 3.64 M (for Hela cells), stronger than those of positive
control etoposide. In addition, compounds 4m, 4n and 4q exhibited good antitumor activity against
two tested cancer cell lines (SMMC-7721 and Hela cells) and compounds 4g, 4j-4l, 4o, 4r were only
active to one tumor cell (HeLa cells). However, compound 4p had no significant anticancer activity
for all three tumor cell lines. It is important to note that this class of compounds did not show notable
cytotoxicity against human embryonic lung fibrobiast (HLF-1) with IC50 > 40 M.
From structure-activity relationships of all compounds, these nopinone-based thiosemicarbazide
derivatives 4a-4f with no substitution at terminal amino group (R2 = H) showed more potent
anticancer activities. When hydrogen atoms of the active amino group were replaced with a methyl
group (4m-4r, R2 = CH3), the activity of thiosemicarbazide derivatives decreased significantly. In
addition, it can be found that compounds (4a-4f, 4g-4l or 4m-4r) with the p-electron-donating group
(CH3, OCH3) on phenyl ring conferred good antitumor activity than their analogs. Whereas, the
electron-withdrawing (Cl, F, NO2) group have greatly reduced the activity of the corresponding
derivatives. Interestingly, when phenyl-substitution at amino position (R2
= Ph) and
p-electron-donating group (R1 = CH3, OCH3) appeared in the same molecule, the compounds (4h, 4i)

obtained prominent antitumor activity.
Then, to investigate whether compound 4i induced cell apoptosis, compound 4i-treated
MDA-MB-231 cells were stained with Annexin V-FITC/7-AAD and then analyzed by flow
cytometry. As shown in Fig. 1A, compound 4i induced cell apoptosis in a dose-dependent manner.
The percentage of early apoptotic cells (Fig. 1A) significantly increased from 1.61% to 42.9% after
treatment with different concentrations of compound 4i (0, 2.5, 5, 10 M). These results suggested
that compound 4i could efficiently induce apoptosis of MDA-MB-231 cells in a dose-dependent
manner.
Figure 1. (A)Annexin V-FITC/7-AAD dual staining assay. MDA-MB-231 cells were treated with
different concentrations of compound 4i (0, 2.5, 5, 10 M) and stained with Annexin
V-FITC/7-AAD and analyzed for apoptosis using flow cytometer. (B) Cell cycle assay.
MDA-MB-231 cells were treated with different concentrations of compound 4i (0, 2.5, 5, 10 M)
and stained with propidium iodide and analyzed for cell cycle using flow cytometer.
Finally, the cell cycle analysis was carried out in the study the mechanism of cytotoxicity toward
MDA-MB-231 cells. MDA-MB-231 cells were treated with 0, 2.5, 5 and 10 M of 4i for 48h. As
shown in Fig. 1B, compound 4i led to a dose-dependent induction of cell cycle arrest in the G2/M

phase. When compared to untreated cell group, the percentage of 4i-treated cells in G2/M phase
increased from 14.98% to 31.09% and the percentage of 4i-treated cells in G0/G1 phase decreased
from 61.73% to 38.03%. These phenomena proved that compound 4i induced MDA-MB-231 cell
cycle arrest in G2/M phase.
Thiosemicarbazones are a structurally diverse family of compounds that have been extensively
studied because of their broad spectrum of pharmacological applications25. Many compounds
belonging to the thiosemicarbazone family have been examined for cytotoxic activity against several
cancer types26, 27. Although thiosemicarbazones with antiproliferative activity exhibit a wide
structural diversity, most of them exhibit mechanisms of action including ribonucleotide reductase
and topoisomerase IIinhibition28, reactive oxygen species generation29, DNA damage30, transition
metal chelators31 and induce redox intracellular imbalance32. In case of our study, compound 4i can
significantly arrest the cell cycle at G2/M phase and induce the apoptosis of cancer cells, which are
important characteristics of numerous tubulin polymerization inhibitors previously reported33-36
.
Therefore, we deduce that this class of compounds may probably be antimitotic agents by inhibiting
tubulin polymerization and thus induce the apoptosis of cancer cells.
In conclusion, 18 new nopinone-based thiosemicarbazone derivatives (4a-4r) have been
synthesized. It is noteworthy that some compounds exhibited strong growth inhibitory activity
against MDA-MB-231, Hela and SMMC-7721 cancer cells compared with positive control
etoposide. Among these thiosemicarbazone derivatives, compound 4i exhibited obvious
anti-proliferative activity against all tested cancer cell lines and low cytotoxicity on human normal
embryo lung fibroblasts (Hlf-1). These findings highlighted the potential of this class of derivatives
as new anticancer agents. More research will be carried out to explore the in-depth structure-activity

relationships and the antitumor mechanisms, especially tubulin polymerization activity of these
derivatives.
Acknowledgements
This work was supported by the University Science Research Project of Jiangsu Province (No.
14KJ220001), the National Natural Science Foundation of China (No. 31470592), the Open Funding
of Jiangsu Key Laboratory of Biomass Energy and Materials (No. JSBEM2014010), the Key
Technology of Green Processing and Efficient Utilization on Oleoresin (No. 2016YFD0600804), and
Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Supplementary data
Supplementary data associated with this article can be found, in the online version, at

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Graphic Abstract