Synthesis and biological evaluation of matrine derivatives as anti-hepatocellular cancer agents

Article abstract of DOI:10.1016/j.bmcl.2016.07.045

We delineate herein the synthesis and anti-cancer effects of 15 matrine derivatives. The in vitro growth inhibitory assays showed that most of the prepared compounds exhibited improved anti-proliferative activities towards cancer cells with IC₅₀

Full text of DOI:10.1016/j.bmcl.2016.07.045

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of matrine derivatives
as anti-hepatocellular cancer agents
Lichuan Wu a,y, Shuaibing Liu a,y, Jinrui Wei a,b, Dong Li a, Xu Liu a, Jianyi Wang a, Lisheng Wang a,
⇑
a School of Chemistry and Chemical Engineering, Guangxi University, Nanning, Guangxi 530004, PR China
b Guangxi Scientific Research Center of Traditional Chinese Medicine, Guangxi University of Chinese Medicine, Nanning, Guangxi 530200, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
We delineate herein the synthesis and anti-cancer effects of 15 matrine derivatives. The in vitro growth
inhibitory assays showed that most of the prepared compounds exhibited improved anti-proliferative
activities towards cancer cells with IC50 17–109 times lower than that of matrine. Compounds CH6
showed the most potent anti-proliferative activities in the four tested cancer cell lines. Moreover, com-
pound CH6 could induce G1 cell cycle arrest and inhibit cell migration in human hepatocellular cancer
cell lines Bel-7402 and HepG2 through up-regulation of P21, P27 and E-cadherin and down-regulation
of N-cadherin.
Received 16 May 2016
Revised 18 July 2016
Accepted 20 July 2016
Available online xxxx
Keywords:
Matrine derivatives
Hepatocarcinoma
Anticancer activity
Cell cycle
Ó 2016 Elsevier Ltd. All rights reserved.
Migration
Hepatocellular cancer (HCC) is the second cause of cancer-
related death and more than 50% of the HCC cases occur in China.1
Currently, chemotherapy is the main treatment for HCC.2–5
Although the survival of HCC patients has been improved with
the emergence of sorafenib6–14, a kinase inhibitor, there still comes
with new issues, such as drug resistance. It is still urgent to
develop novel drugs to improve patient outcomes.
Matrine is the main chemical ingredient of Fufang Kushen injec-
tion which was approved by Chinese FDA (CFDA) in 1995 as an
Adjuvant to treat liver cancer and non small cell lung cancer.15–20
Owing to its druggable advantages, such as flexibility structure
and favorable safety profiles, matrine has been considered as an
ideal lead compound for further modification.21–23
In the present study, we designed and synthesized 15 matrine
derivatives which displayed improved anti-cancer effects than
matrine in four tested cancer cell lines. The synthetic route is out-
lined in Scheme 1. First, treatment of matrine with lithium diiso-
propylamide (LDA) provided the lithium enolate intermediates.
Then lithium enolate intermediates reacted with compounds B
which were prepared via a reported medthod24 to give intermedi-
ate products D. Intermediate D could easily convert to product E
quantitatively via deprotection at 0 °C with trifluoroacetic acid
and methylene chloride (the volume ratio was 1:20). Subsequently,
as described25, cyclodehydration of product E in the presence of
poly-phosphonic ester (PPE) at 120 °C in an inert atmosphere of
nitrogen gave the desired products (CH1–CH10) with yields rang-
ing from 15% to 25%. While preparing compound CH11, two hydro-
xyl groups in methyl 2, 5-dihydroxybenzoate needed to be
protected. Briefly, the 2-position hydroxyl group was initially pro-
tected by methyl ether, following a protection of 5-position hydro-
xyl group by benzyl group. Interestingly, B11 was successfully
obtained via recrystallization with a yield of 60%. Compounds
CH12 and CH13 were prepared successfully by removing methyl
ether protection of CH7 and CH8 in the condition of 40% HBr/CH3-
COOH mixture, respectively. In order to promote the reaction rate,
acetic acid was replaced by acetic anhydride or a certain amount of
PBr3 was added to increase the HBr concentration. For the synthe-
sis of compounds CH14–15, prior obtained compounds CH4–5
were oxidized at the nitrogen atom with m-CPBA as an oxidant.
All the compounds are characterized (Supplementary information).
All the available matrine derivatives were evaluated for their
cytotoxic activities against four human cancer cell lines, including
A549 (lung cancer cell), MCF-7 (breast cancer cell), SGC-7901 (gas-
tric cancer cell) and Bel-7402 (hepatocellular cancer cell) using
MTT assay.26 The anti-proliferative activities of 15 matrine deriva-
tives were depicted in Table 1. Most of the derivatives exhibited
improved anti-cancer effects with IC50 17–109 times lower than
that of matrine. Especially, compounds CH6 showed the most
⇑
Corresponding author.
E-mail address: w_lsheng@163.com (L. Wang).
The authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmcl.2016.07.045
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wu, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.045

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L. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Scheme 1. Synthetic routes of matrine derivatives. Reaction conditions: (a) LDA, 0 °C to 25 °C, 3 h; (b) TFA/DCM (v:v = 1:20), 8 h; (c) PPE, N2, reflux, 8 h; (d) BnBr, K2CO3,
15 °C, 3 h; (e) H2 balloon, 3% Pd/C, 12 h; (f) CH12: 40% HBr/Acetic anhydride, reflux, 48 h; CH13: 40% HBr/CH3COOH, PBr3, reflux, 48 h; (g) m-CPBA, 0 °C, 3 h.
Table 1
To evaluate the anti-proliferative activity of compound CH6 on
hepatocellular cancer cells, MTT assay was performed in Bel-7402
The anti-proliferative activities of matrine derivatives CH1-15
and HepG2 cells. The results demonstrated that compound CH6
could inhibit the proliferation of hepatocellular cancer cells in a
time- and dose-dependent manner (Fig. 1).
Compd
IC50 (lM)
SGC-7901
MCF-7
>100
A549
Bel-7402
CH1
>100
>100
>100
To determine whether the cytotoxicity of compound CH6
against Bel-7402 and HepG2 cells involved cell cycle arrest, we
tested the effects of CH6 on cell cycle distribution for 24 h. The
results indicated that compound CH6 induced G1 cell cycle arrest
in Bel-7402 and HepG2 cells in a dose-dependent manner (Fig. 2A).
The percentage of G1 phase in Bel-7402 cells increased from 49% to
CH2
CH3
CH4
CH5
CH6
CH7
CH8
CH9
CH10
CH11
CH12
CH13
CH14
CH15
Matrine
45.25 4.83
40.45 3.97
46.75 3.99
34.90 4.36
28.94 3.76
67.15 4.13
81.71 9.02
81.59 4.33
51.46 8.20
>100
>100
>100
57.14 8.26
79.09 6.30
1380 116
48.22 1.66
57.15 5.13
48.56 5.33
30.73 0.71
28.06 2.90
77.43 3.61
74.13 5.42
75.43 7.79
52.85 30.52
>100
>100
>100
53.27 5.17
73.78 5.71
2497 208
56.99 6.73
97.24 9.24
52.91 6.90
41.2 4.24
36.03 2.07
97.63 7.55
93.55 10.17
96.47 7.97
73.25 6.63
>100
>100
>100
73.78 2.51
113.78 11.92
3923 243
36.17 2.35
36.96 2.04
30.29 1.29
31.45 2.64
25.23 2.46
47.98 4.37
69.45 4.59
71.87 6.72
40.74 2.86
>100
>100
>100
49.29 7.94
66.01 7.07
2057 192
67% (10
(Fig. 2B, left). Consistently, the percentage of G1 phase in HepG2
cells increased from 48% to 54% (10 M, p <0.05) and 65%
(20 M, p <0.01), respectively (Fig. 2B, right).
lM, p <0.05) and 79% (20 lM, p <0.05), respectively
l
l
Migration and invasion are the main manifestations of tumor
progression.27 To test whether compound CH6 could inhibit cancer
cell migration, wound healing assays were conducted in Bel-7402
and HepG2 cells for 48 h. The results indicated that under CH6
treatment, cancer cell migration was inhibited (Fig. 3A). CH6
decreased the percentage of cell migration from 60% (control) to
Note: IC50 values are taken as a mean from 3 experiments. Mean SD.
75% (10
and from 58% (control) to 80% (10
p <0.01) in HepG2 cells, respectively (Fig. 3B).
To further dissect the molecular mechanism of CH6 induced G1
cell cycle arrest and migration inhibition, western blot assays were
conducted as described28 to detect regulators involved in cell cycle
and migration regulation. The results demonstrated that upon
l
M, p <0.01) and 85% (20
l
l
M, p <0.01) in Bel-7402 cells
potent anti-cancer effects. The IC50 of CH6 were 25–36 lM which
M, p <0.01) and 83% (20 M,
l
was about 48–109 times lower than that of matrine, respectively.
Interestingly, most of the derivatives displayed greater cytotoxicity
against human hepatocellular cancer cell line Bel-7402 than the
other three human cancer cell lines. Thus, we subsequently studied
the anti-cancer mechanism of compound CH6 on hepatocellular
cancer cells.
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L. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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Figure 1. Compound CH6 inhibits hepatocellular cancer cells proliferation.
Figure 2. Compound CH6 induces G1 cell cycle arrest in hepatocellular cancer cells. (A) CH6 induces G1 cell cycle arrest in Bel-7402 and HepG2 cells in a dose-dependent
manner. (B) Distributions of G1, S and G2/M in Bel-7402 and HepG2 cells under CH6 treatment.
treatment with CH6 for 24 h, P21 and P27 were dramatically up-
regulated. Upon treatment with CH6 for 48 h, E-cadherin was sig-
nificantly up-regulated while N-cadherin was down-regulated in a
dose-dependent manner in both Bel-7402 and HepG2 cells (Fig. 4).
In summary, a group of matrine derivatives were synthesized,
and their anti-proliferative activities against four selected human
cancer cell lines were investigated. Most of the derivatives
exhibited improved anti-cancer effects with IC50 17–109 times
lower than that of matrine and displayed greater cytotoxicity
against human hepatocellular cancer cell line Bel-7402 than the
other three human cancer cell lines. Compound CH6 exhibited
the most significant growth inhibition of cancer cells. Further stud-
ies indicated that CH6 exerted its anti-cancer activity by inducing
G1 cell cycle arrest and inhibiting cell migration in Bel-7402 and
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L. Wu et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Figure 3. Compound CH6 suppresses cell migration in hepatocellular cancer cells. (A) Inhibition of cell migration by CH6 was confirmed by the wounding healing assay in
Bel-7402 and HepG2 cells. (B) CH6 decreased the percentage of cell migration from 60% (control) to 75% (10 M, p <0.01) and 85% (20 M, p <0.01) in Bel-7402 cells and from
58% (control) to 80% (10 M, p <0.01) and 83% (20 M, p <0.01) in HepG2 cells.
l
l
l
l
education (gui jiao ren [2014] 49 hao), and the Postdoctoral
Science Foundation of Guangxi province of China. We also thank
Professor Hu Tingjun of school of Zoology Science and Technology
of Guangxi University for the experiment support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07.
045. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21262005), the high level innovation team
and outstanding scholar project of Guangxi institutions of higher
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