Design, synthesis and anticancer activity of matrine-1H-1,2,3-triazole-chalcone conjugates

Article abstract of DOI:10.1016/j.bmcl.2015.04.051

Abstract A series of novel matrine-1H-1,2,3-triazole-chalcone conjugates was synthesized and their anticancer activity against A549, Bel-7402, Hela, and MCF-7 cancer cells was evaluated. Most of the conjugates displayed higher potency than their component

Full text of DOI:10.1016/j.bmcl.2015.04.051

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2540–2544
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and anticancer activity
of matrine–1H-1,2,3-triazole–chalcone conjugates
⇑
Lihui Zhao, Lina Mao, Ge Hong, Xiaojiao Yang, Tianjun Liu
Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Tianjin 300192, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 November 2014
Revised 5 April 2015
Accepted 17 April 2015
Available online 22 April 2015
A series of novel matrine–1H-1,2,3-triazole–chalcone conjugates was synthesized and their anticancer
activity against A549, Bel-7402, Hela, and MCF-7 cancer cells was evaluated. Most of the conjugates dis-
played higher potency than their components. Compounds 6h and 6i exhibited more potent anticancer
activity than 5-fluorouracil against the four tested human cancer cell lines and lower cytotoxicity to
NIH3T3 normal cells. Flow cytometry tests demonstrated that compound 6h could induce apoptosis of
A549 cells in a concentration-dependent manner. Moreover, 6h could efficiently suppress human tumor
growth in mouse xenograft model without causing obvious toxicities.
Keywords:
Matrine
Chalcone
Ó 2015 Elsevier Ltd. All rights reserved.
1
,2,3-Triazole
Conjugate
Anticancer
Synthesis
Apoptosis
8
Chemotherapy is one of the most effective approaches used for
hybridization, we combined matrine, chalcones with 1H-1,2,3-tri-
azole to generate a series of novel matrine–1H-1,2,3-triazole–chal-
cone conjugates (Fig. 2). In the present study, we reported their
synthesis and preliminary anticancer activity studies in vitro and
in vivo, and some of them showed potent anticancer activity.
The matrine–1H-1,2,3-triazole–chalcone conjugates were
synthesized efficiently by Cu(I)-catalyzed azide/alkyne ‘click’ reac-
treating cancers in clinic. However, the lack of selectivity and
development of drug-resistance diminish the efficacy of cancer
chemotherapy. This dilemma makes it urgent to develop new
1
anticancer agents with high potency and less toxicity.
Matrine, a quinolizidine alkaloid, is an important active com-
pound in the root of Sophora flavescens Ait (also known as Kushen)
which was used as traditional Chinese herb for thousands of years
0
tion between 13-azido matrine and corresponding 4 -propargyloxy
2
3
4
9
in the treatment of liver diseases, cardiovascular diseases, asthma
chalcones. The 13-azido matrine was prepared in excellent
5
as well as tumors. While chalcones, with a scaffold of 1,3-diphenyl-
-propenone, possessing a variety of biological activities, are
yield by Michael addition reaction between sophocarpine and
excessive trimethylsilyl azide in the presence of acetic acid at
6
2
1
0
0
widely spreadin most natural fruits and vegetables as biologicalpre-
cursor of flavonoids. Notably, anticancer activityof both matrine and
chalcones has been confirmed, but efficacy of their conjugates in
anticancer activity has not been reported. The 1H-1,2,3-triazole is
an important structure contained in a huge amount of active
compounds mainly because of its metabolic stability, bioactivity
and convenient synthesis.7 Clinically used drugs containing a
ambient temperature. 4 -Propargyloxychalcones were prepared
by Claisen–Schmidt condensation reaction between substituted
1
1
acetophenones and various benzaldehydes.
Substituted ace-
tophenones (1a and 1b) was prepared via Williamson ether synthesis.
The synthetic route was depicted in Scheme 1. For activity
comparison, compounds 7a and 7b were also prepared starting
from 1a and 1b, respectively. All target compounds were
1
13
1
,2,3-triazole moiety include b-lactam antibiotics Tazobactam
characterized by IR, HRMS, H NMR, and C NMR (experimental
1
2
and Cefatrizine, as well as calcium channel blocker
Carboxyamidotriazole (CAI) (Fig. 1).
In our pursuit of natural product derivatives with high anti-
cancer potency and inspired by the idea of molecular
details and spectra were in Supplementary data).
Anti-proliferative activity against four cancer cell lines (human
lung cancer cells A549, human cervical epithelial carcinoma cells
Hela, human hepatocellular carcinoma cells Bel-7402 and human
breast cancer cells MCF-7) and one normal cell line (mouse embryo
fibroblasts NIH3T3) was evaluated based on a CCK-8 assay
methodology (see Supplementary data). The IC50 values of matrine,
⇑
Corresponding author. Tel./fax: +86 22 87893236.
E-mail address: liutianjun@hotmail.com (T. Liu).
http://dx.doi.org/10.1016/j.bmcl.2015.04.051
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

L. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2540–2544
2541
Figure 1. Chemical structures of matrine, chalcone and clinically used drugs contained 1,2,3-triazole moiety.
Figure 2. Design of matrine–1H-1,2,3-triazole–chalcones conjugates.
R2
H
O
R1
O
O
R1
O
O
R2
b
CH3
R2
1
a-b
N
N
N
R1
2
3
a-l
a-k
O
d
O
N
O
Br
H
H
a
H
O
O
N3
R1
N H
HO
5a-l
6a-k
N
N
c
H
H
H
H
H
CH3
H
O
N H
N H
N
N
N
R1
4
d
O
CH3
O
N
O
O
R1
H
H
H
CH3
N H
O
1
a-b
7a-b
Scheme 1. Synthetic route of the conjugates 5, 6 and 7. Reagent and reaction conditions: (a) propargyl bromide, K
2
CO
3
, Me
2
CO, rt ꢀ60 °C, 0.5–2.0 h; (b) KOH, EtOH, N
2
, 25–
6
0 °C, 6–24 h; (c) Me SiN , AcOH, DBU, toluene, N , rt, 36 h; (d) CuSO ꢁ5H O, sodium ascorbate, THF/H O (1:1), rt, 4–48 h.
3
3
2
4
2
2
some representative chalcones and their conjugates were listed in
Table 1.
Table 1 shows that matrine, chalcones or a simple mixture of
them displayed little cytotoxicity against normal cell NIH3T3, or
cancer cell like A549, Hela and MCF-7 (IC50 >50 lM), while
matrine–1H-1,2,3-triazole–chalcone conjugates showed moderate
toxicity toward NIH3T3 cells. The synergetic anticancer effect
toward cells could be interpreted from the following points: in
one case, the conjugate has a larger molecular skeleton with more
binding sites than the components, which cause a strong affinity to
the target sites; in another case, the conjugate contains both
matrine and chalcone species, so the multi-target binding sites
not only for matrine, chalcone alone, but also novel binding site
to potent anticancer activity to tested cell lines and relatively weak

2
542
L. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2540–2544
Table 1
Results of cell growth inhibition caused by conjugates and selected intermediates
Compd
R1
R2
IC50a
(lM)
NIH3T3
A549
Bel-7402
Hela
MCF-7
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
7
7
2
2
3
3
3
a
b
c
d
e
f
g
h
i
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
4-Cl
4-NO
4-OCHF
4-Me
4-Br
4-F
4-MeO
2-Cl
19.38 ± 1.17
31.30 ± 2.31
35.72 ± 2.94
27.33 ± 4.01
14.85 ± 2.53
30.91 ± 3.83
39.80 ± 4.02
23.12 ± 3.10
88.02 ± 7.68
38.15 ± 4.78
19.38 ± 1.33
41.06 ± 5.05
>100
75.24 ± 9.52
36.12 ± 4.74
29.58 ± 1.24
20.95 ± 2.30
20.43 ± 2.03
34.84 ± 4.11
39.21 ± 4.31
48.28 ± 5.82
13.34 ± 1.54
15.21 ± 2.21
42.88 ± 5.34
61.80 ± 7.20
72.88 ± 8.34
61.80 ± 7.20
66.56 ± 7.30
79.51 ± 8.19
94.67 ± 9.03
ND
32.18 ± 3.89
18.89 ± 1.30
14.46 ± 1.16
>50
32.42 ± 3.27
24.51 ± 2.75
18.74 ± 1.94
>50
14.25 ± 1.21
11.87 ± 1.07
8.72 ± 0.62
>50
16.82 ± 1.19
15.64 ± 1.28
18.30 ± 1.95
>50
20.73 ± 3.02
21.25 ± 2.32
16.22 ± 1.52
25.39 ± 2.62
19.30 ± 1.73
6.06 ± 0.63
>50
19.91 ± 1.81
17.14 ± 3.21
18.74 ± 1.43
>50
13.52 ± 1.54
15.23 ± 1.29
11.42 ± 1.01
>50
17.27 ± 1.29
10.72 ± 0.67
19.79 ± 1.84
>50
17.24 ± 1.34
14.32 ± 2.07
11.52 ± 0.98
ND
26.75 ± 2.90
22.73 ± 2.92
26.85 ± 3.49
ND
23.75 ± 2.84
10.25 ± 1.05
13.30 ± 0.91
ND
11.97 ± 1.40
12.67 ± 1.16
5.71 ± 0.72
>50
ND
ND
8.87 ± 0.63
6.25 ± 0.53
7.03 ± 0.63
ND
ND
>50
>50
>50
2
b
2
ND
35.55 ± 3.29
22.67 ± 1.73
22.18 ± 3.12
ND
21.79 ± 2.56
13.72 ± 1.03
11.93 ± 1.04
ND
17.33 ± 1.48
14.71 ± 0.99
9.02 ± 0.82
>50
ND
ND
12.14 ± 1.01
5.60 ± 0.37
12.44 ± 1.28
ND
ND
>50
>50
ND
ND
ND
ND
ND
j
k
l
3-Cl
3,4-Di-Cl
3,4-Di-MeO
H
4-Cl
4-F
4-MeO
4-Me
4-t-Butyl
3-CF
3-Cl
3,4-Di-Cl
a
b
c
d
e
f
g
h
i
>50
>50
36.46 ± 3.21
8.18 ± 0.93
5.01 ± 0.59
8.08 ± 0.94
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
36.27 ± 3.12
12.72 ± 1.08
7.31 ± 0.64
6.63 ± 0.17
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3
j
3,4-Di-MeO
3,4,5-Tri-MeO
—
—
k
a
b
a
k
a
h
i
OH
>50
>50
>50
>50
>50
>50
Matrine
>50
>50
Combination of 3a and matrinec
ND
Paclitaxel
20.01 ± 2.38
22.36 ± 2.09
2.82 ± 0.31
40.38 ± 4.61
ND
19.91 ± 2.68
0.38 ± 0.03
8.93 ± 0.79
ND
9.56 ± 1.01
5
-Fluorouracil
a
b
c
Data were shown as mean ± SD.
Not tested.
Molar ratio of 3a and matrine is 1:1.
with new action mode would occur, while each component has no
this advantage; thirdly, the conjugate is not physical addition in
physical properties like hydrophilicity, so the transmembrane
behavior for the conjugates will be different from that of each com-
ponent. The ensemble effect above would account for the differ-
ence in cytotoxicity for the conjugate and its corresponding
components. In the series of conjugates, 13-substituted matrine
with poor anticancer activity, would provide a geometry to help
the location of chalcone moiety, and the cytotoxicity difference is
from the contribution of the chalcone moiety. Both the position
and types of substituent on chalcone moiety have dominant influ-
ence on potency, as well as tumor-selectivity. Some structure–ac-
tivity relationships (SARs) could be extracted. First of all, that
structural integrity of chalcone moiety was essential for the anti-
cancer activity of the conjugate (7a, 7b), reveals the double bond
well as a,b-unsaturated moiety, thus decreasing reactivity of a,b-
unsaturated double bond as a Michael reaction acceptors in
organisms. On the contrary, conjugates (5d, 5h, 5l, 6d, 6e, 6j, 6k)
with EDGs (like 4-Me, 4-MeO, 4-OCHF
MeO) in B ring, have poor activity with IC50 more than 50
toward tested cancer cells. Finally, conjugates substituted with 4-
NO or 4-F in B ring showed selectivity among cancer cells, for
2
, 3,4-di-MeO and 3,4,5-tri-
l
M
2
compound 5c and 6c were the most potent than other
conjugates against Hela and MCF-7, respectively. Whether
matrine or triazole is essential to activity needs further study.
Table 1 shows that compound 6h is 8.0-fold more potent (IC50
5.01 ± 0.59
comparable to paclitaxel (IC50 2.82 ± 0.31
Besides, compound 6h, with a relatively broad anticancer spec-
trum, has less cytotoxicity (IC50 39.21 ± 4.31 M) than 5-fluo-
rouracil (IC50 22.36 ± 2.09 M) and paclitaxel (IC50
20.01 ± 2.38 M) against NIH3T3 cells. Thus compound 6h was
lM) than 5-fluorouracil (IC50 40.38 ± 4.61
lM) and is
l
M) against A549 cells.
l
of
a
,b-unsaturated moiety is of great importance to the anticancer
l
1
1b
activity, which was consistent with the reported literature.
l
0
Secondly, conjugates with a 2 -OH in A ring of chalcone moiety
showed more potent anticancer activity than those without –OH
substitution (5a vs 6a, 5b vs 6b, 5g vs 6c, 5j vs 6h), probably
chosen for further studies.
Flow cytometry assay with Annexin V-FITC/propidium iodide
double staining in A549 cells (results in Fig. 3) suggested that 6h
was capable to induce apoptosis in a concentration-dependent
manner, and percentage of both early and late apoptosis reached
to more than 30% in the high concentration treatment group
(two-fold IC50 of compound 6h, incubation for 24 h). This was sim-
ilar to that of the building blocks, matrine and chalcone. Matrine
could significantly increase rate of apoptosis in A549 cells from
0
because 2 -OH in A ring could induce intramolecular hydrogen
bond with chalcone carbonyl group which may change electron
density distribution of
a,b-unsaturated moiety or change
conformer of the conjugate. Thirdly, conjugates with electron-
withdrawing groups (EWGs) in B ring of chalcone moiety showed
better anticancer activity than those substituted with electron-
donating groups (EDGs) (5c vs 5h, 5k vs 5l, 6c vs 6d, 6i vs 6j),
mainly because EWGs could reduce electron density of B ring as
1
3
28.4% to 43.3% at 15–50
lM, while chalcone and their synthetic
derivatives, could also induce apoptosis in various cancer cell

L. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2540–2544
2543
Figure 3. Apoptosis of A549 cells induced by compound 6h after 24 h incubation. Experiments were done in triplicate. Left: representative graph of flow cytometry. (A)
Control (0.1% DMSO); (B) half IC50 of 6h; (C) IC50 of 6h; (D) two-fold IC50 of 6h. For each scatter diagram, lower right corner represents early apoptosis; upper right corner
⁄
represents late apoptosis. Right: statistical data of apoptotic cells at different concentrations. P <0.01, compared to control.
Figure 4. Tumor volume and mouse body weight change in xenograft model of A549 cells after treatment with compound 6h. Values were expressed by mean ± SE. Left:
tumor volume of different groups. Right: body weight change in each group. Tail vein injection every 3 days; n = 8 in treatment groups (6h and paclitaxel group), n = 6 in the
⁄
q
vehicle group (saline plus 0.2% Tween 80). P <0.01, compared to the vehicle; P >0.05, compared to paclitaxel group.
lines.14 The conjugates can strongly induce apoptosis of A549 cells,
which could be a reason for their anti-proliferative activity.
The antitumor efficacy of 6h in vivo was studied in the xeno-
graft nude mouse model of A549 cells (details in Supplementary
data). The results in Figure 4 indicated that treatment with
suggested that compound 6h could inhibit tumor growth in vivo
without causing obvious toxicity.
In summary, conjugation of matrine, 1H-1,2,3-triazol and chal-
cones could form novel anticancer agents with synergistic effect, in
which the double bond of
a,b-unsaturated moiety plays a domi-
0
5
.0 mg/kg and 10.0 mg/kg of compound 6h markedly suppressed
nant role; adding 2 -OH in A ring or substituting B ring of chalcone
with EWGs could increase anticancer activity of matrine–triazole–
chalcone conjugates. Among the conjugates compound 6h could
inhibit proliferation and induce apoptosis of human cancer cells,
and exhibited promising anticancer efficacy in vitro and in vivo,
which would be a potential scaffold for further development of
new anticancer agent.
tumor growth, while increase ratio of the body weight of tumor
baring mice was relatively higher than that of the paclitaxel group
or the vehicle group. This demonstrated that 6h probably have a
favorable safety profile in vivo. No significant difference was
observed in tumor volume between the 10.0 mg/kg group and
paclitaxel group after 8 days of treatment (P >0.05). Here percent-
age of tumor growth inhibition (% TGI, calculated by [1 ꢀ change of
tumor volume in treated group/change of tumor volume in vehicle
group] ꢂ 100) after 20 days treatment was dose-dependent (see
Supplementary data). The values of TGI were 43.9%, 64.8% and
Acknowledgment
This work was supported by the key technologies R & D
program of Tianjin (12ZCDZSY11900) and Peking Union
Medical College Graduate Innovation Fund Grant to Lihui Zhao
(NO. 20131007021).
8
9.6% for the dosage 2.5 mg/kg, 5 mg/kg and 10 mg/kg, respec-
tively. Particularly, after 16 days of treatment TGI in the group of
0.0 mg/kg 6h increased to as high as 85.4%. These results
1

2
544
L. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2540–2544
1. (a) Liaras, K.; Geronikaki, A.; Glamoclija, J.; Ciric, A.; Sokovic, M. Bioorg. Med.
1
Supplementary data
Chem. 2011, 19, 3135; (b) Srinivasan, B.; Johnson, T. E.; Lad, R.; Xing, C. J. Med.
Chem. 2009, 52, 7228.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.04.
12. General procedure for synthesis compound 5, 6 and 7: to a stirred solution of
compound 2, 3 or 1 (0.30 mmol) and compound 4 (0.3 mmol in 1.5 mL THF) in
4
1.5 mL water, freshly prepared 5% CuSO (1 mol %) and sodium ascorbate
0
51.
(
5 mol %) were added and this mixture was stirred for 4–48 h at room
temperature. After completion of the reaction monitored by TLC, THF was
removed under reduced pressure. The residue was purified by silica gel column
chromatography (300–400 mesh) using ethyl acetate–ethanol–ammonia
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