Synthesis and antitumor activities of piperazine- and cyclen-conjugated dehydroabietylamine derivatives

Article abstract of DOI:10.1515/hc-2015-0025

A series of piperazine- and cyclen-conjugated dehydroabietylamine derivatives were synthesized and characterized by ¹H NMR, ¹³C NMR, and HRMS. The in vitro antitumor activities of conjugates 10-13 against MCF-7 and HepG-2 tumor cell

Full text of DOI:10.1515/hc-2015-0025

Heterocycl. Commun. 2015; 21(4): 233–237
Xinbin Yang*, Xiaolin Qin, Qin Wang and Yu Huang*
Synthesis and antitumor activities of piperazine-
and cyclen-conjugated dehydroabietylamine
derivatives
DOI 10.1515/hc-2015-0025
Received March 7, 2015; accepted June 29, 2015; previously published
online July 25, 2015
antioxidative, and antitumor activities [3–12]. These
results arouse our interest in screening for new potential
antitumor drugs by the introduction of various functional
groups to the dehydroabietylamine skeleton.
A number of piperazine and 1,4,7,10-tetraazacyclodo-
decane (cyclen) heterocyclic derivatives as chemothera-
peutic drugs have attracted considerable attention during
the past decade. Studies have shown that the introduction
of piperazine and cyclen moieties can modulate the phys-
icochemical properties and enhance the bioactivity of the
compounds [13–21]. However, the studies on the synthesis
and antitumor activities of heterocyclic derivatives derived
from dehydroabietylamine have not been reported. Our
present work is to design and synthesize a series of pipera-
zine- and cyclen-conjugated dehydroabietylamine deriva-
tives and to evaluate in vitro antitumor activities of these
conjugates against HepG-2 and MCF-7 cells. Furthermore,
the apoptotic effect induced by the representative conju-
gates is also investigated by flow cytometry.
Abstract: A series of piperazine- and cyclen-conjugated
dehydroabietylamine derivatives were synthesized and
1
13
characterized by H NMR, C NMR, and HRMS. The in vitro
antitumor activities of conjugates 10–13 against MCF-7
and HepG-2 tumor cell lines were evaluated using CCK-8
assay. The results show that the synthesized compounds
cause a dose-dependent inhibition of cell proliferation
and display different antitumor activities with the IC50
values ranging from 23.56 to 78.92 μꢀ. Moreover, the anti-
tumor activity of conjugate 10 against the MCF-7 cell line
is superior to that of the positive control 5-fluorouracil.
In addition, flow cytometric assay revealed that the rep-
resentative conjugate 10 could induce apoptosis in MCF-7
tumor cells in a dose-dependent manner.
Keywords: antitumor activity; conjugate; cyclen; dehydro-
abietylamine; piperazine.
Results and discussion
Introduction
The synthetic route to the target conjugates 10–13
is shown in Scheme 1. The reaction between tri-Boc-
Cancer remains the primary cause of death due to the lack protected cyclen 1 or Boc-protected piperazine 2 and chlo-
of effective drugs [1]. Natural compounds have played racetyl chloride afforded the respective products 3 and 4.
an important role in anticancer drug discovery, where Compound 6 was obtained by the reaction between 3 and
the fraction of the drugs derived from natural products 5a in the presence of KOH. Compounds 7–9 were synthe-
amounts to 60% [2]. Dehydroabietylamine is an abietane sized using a similar methodology. The target compounds
diterpenic amine that is obtained as a part of a mixture of 10–13 were obtained by deprotection of Boc group in HCl-
amines derived from rosin. Recent studies have found that ethanol solution. All these products exhibit good water
dehydroabietylamine derivatives demonstrate broad bio- solubility. The structures of all synthesized conjugates
1
13
logical activities, such as antibacterial, antiinflammatory, were confirmed by H NMR, C NMR, and HRMS.
The in vitro antitumor activities of the conjugates 10–13
*
Corresponding authors: Xinbin Yang, Rongchang Campus,
were evaluated by means of CCK-8 assay against MCF-7 and
HepG-2 tumor cell lines. The inhibition rates of cell viability
with different concentrations of conjugates are shown in
Southwest University, Chongqing 402460, P. R. China,
e-mail: yangxbqq@126.com; and Yu Huang, Pharmacy College,
Ningxia Medical University, Yinchuan 750004, P. R. China,
e-mail: huangyuxmu@163.com
Figure1,andtheIC valuesaregiveninTable1.Itcanbeseen
50
that the treatment with increasing doses of all conjugates
causes a dose-dependent inhibition of cell proliferation. As
Xiaolin Qin and Qin Wang: Rongchang Campus, Southwest
University, Chongqing 402460, P. R. China
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34ꢀꢀꢀꢀꢁꢀX. Yang et al.: Piperazine- and cyclen-conjugated dehydroabietylamine derivatives
Boc
Boc
N
N
HN
N
Boc
1
ClCH2COCl
Et3N
O
R
O
O
Boc
Cl 5a: R = H2NCH2
Boc
Boc
R
R
N
N
5
b: R= 4-HO-C6H4CH2HNCH2
N
N
N
N
HCl
NH
N
4HCl
N
N
EtOH
NHHN
Boc
Boc
KOH/THF
Boc
3
6
7
: R = HNCH2
: R = 4-O-C6H4PhCH2HNCH2
1
1
0: R = HNCH2
1: R = 4-O-C6H4PhCH2HNCH2
Boc
N
NH
2
ClCH2COCl
Et3N
R
5
a: R = H2NCH2
O
O
O
5b: R= 4-HO-C6H4CH2HNCH2
HCl
Boc
N
N
Boc
N
N
HN
N
KOH/THF
R
EtOH
R
Cl
2
HCl
4
8
9
: R = HNCH2
: R = 4-O-C6H4PhCH2HNCH2
12: R = HNCH2
13: R = 4-O-C6H4PhCH2HNCH2
Scheme 1ꢀ
(
i) 1
(ii) 100
10
00
10
12
1
1
1
2
11
80
60
40
20
0
80
60
40
20
0
13
1
3
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
-6
-6
(
Compound)/10 mol/l
(Compound)/10 mol/l
Figure 1ꢀAntiproliferation effect of conjugates 10, 11, 12, and 13 against MCF-7 (i) and HepG-2 (ii) tumor cells.
Cells were plated and incubated with the indicated concentrations of 10, 11, 12, and 13 (5, 10, 20, 40, and 80 μꢂ). After 24 h of treatment,
cell proliferation was measured by the CCK-8 assay. Data represent the means±SD of triplicate experiments.
Table 1ꢀThe IC50 values of the conjugates 10–13 and 5-FU against tumor cell lines.
Cell lineꢁꢁ
ICꢂꢃ value (μꢄ)
ꢅꢃꢁ
ꢅꢅꢁ
ꢅꢆꢁ
ꢅꢇꢁ
ꢂ-FU
MCF-ꢃ
ꢄ
ꢅꢆ.ꢇꢅ±ꢅ.ꢈꢇꢄ
ꢅꢃ.ꢈꢆ±ꢋ.ꢍꢍꢄ
ꢇꢉ.ꢊꢋ±ꢌ.ꢅꢊꢄ
ꢆꢅ.ꢊꢌ±ꢅ.ꢉꢇꢄ
ꢊꢅ.ꢆꢆ±ꢇ.ꢈꢋꢄ
ꢅꢌ.ꢆꢊ±ꢋ.ꢇꢆꢄ
ꢃꢍ.ꢉꢅ±ꢅ.ꢊꢍꢄ
ꢃꢋ.ꢉꢊ±ꢅ.ꢌꢌꢄ
ꢅꢃ.ꢋꢅ±ꢋ.ꢉꢆ
ꢊ.ꢃꢍ±ꢈ.ꢊꢆ
HepG-ꢅ ꢄ
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X. Yang et al.: Piperazine- and cyclen-conjugated dehydroabietylamine derivativesꢎꢁꢀꢀꢀꢎ235
Control
10 µM
20 µM
40 µM
04
03
02
01
00
104
103
102
101
100
104
103
102
101
100
104
103
102
101
100
1
1
1
1
1
Q1
Q2
.68%
Q1
Q2
8.97%
Q1
Q2
36.73%
Q1
Q2
31.73%
3
Q4
.42%
Q4
1.49%
Q4
8.92%
Q4
22.02%
0
1
00
101
102
103
104
100
101
102
APC
103
104
100
101
102
APC
103
104
100
101
102
APC
103
104
APC
Figure 2ꢀApoptosis ratio of conjugate 10 as detected by Annexin V-APC/PI double-staining assay of MCF-7 cells.
The MCF-7 cells were treated with 10, 20, and 40 μꢂ of conjugate 10 for 24 h. Four quadrant images were observed by flow cytometric analy-
sis: the Q1 area represents damaged cells appearing in the process of cell collection, the Q2 region shows necrotic cells and later-period
apoptotic cells, the normal cells are located in the Q3 area, and the Q4 area shows early apoptotic cells.
evident from the obtained results, conjugate 10 with cyclen conjugate 10 against the MCF-7 cells was slightly superior
moiety is the most potent cytotoxic agent against MCF-7 to that of the positive control 5-FU. In addition, flow cyto-
and HepG-2 cells among the tested conjugates. Compound metric assay indicated that the representative conjugate
1
0 displays a prominent inhibitory activity by almost 100% 10 induces apoptosis in MCF-7 cells in a dose-dependent
at 40 μꢀ, with the IC value of 25.42 μꢀ for MCF-7 cell and manner. These results encourage us to synthesize addi-
50
2
7.0 5 μꢀ for HepG-2 cell. Furthermore, the effect of conju- tional new dehydroabietylamine derivatives with the
gate 10 against the MCF-7 tumor cell line is slightly supe- expected more potent antitumor activity.
rior to that of the positive control 5-fluorouracil (5-FU), as
indicated by the IC50 values. In addition, conjugate 12 with
piperazine moiety shows selectivity for HepG-2 over MCF-7
Experimental
All reagents were purchased from commercial sources and used with-
with the corresponding IC50 values of 23.56 and 62.55 μꢀ.
Meanwhile, conjugates 11 and 13 containing benzene ring
as part of the R group exhibit only mild cytotoxic activities out further purification. The HepG-2 (liver hepatocellular carcinoma
against MCF-7 and HepG-2 cells.
cell) and MCF-7 (human breast adeno-carcinoma cell) were obtained
from ATCC. High resolution mass spectrometry (HRMS) data
The effect of conjugate 10 on apoptosis in MCF-7 cell
was investigated. Apoptosis assay may provide prelimi-
nary information about the mechanism of growth inhi-
bition of tumor cells. The apoptosis ratios (including the
early and late apoptosis rates) induced by conjugate 10
in MCF-7 cell lines were determined by flow cytometry.
The results are given in Figure 2. The apoptotic rate was
1
were recorded on a Bruker Daltonics Bio TOF instrument. H NMR
1
3
(
400 MHz) and C NMR (100 MHz) spectra were measured on a Var-
ian INOVA-400 spectrometer. Flow cytometry was performed using a
BD FASAria Cell Sorter.
Preparation of compound 5b
4
.10% of the total number of cells in the control group.
The percentage of apoptotic cells were increased to 10.46,
5.65, and 53.75% by the treatment with 10, 20, and 40 μꢀ
The ethanol solution (30 mL) of dehydroabietylamine (5a, 1.45 g,
5
mmol) and p-hydroxybenzaldehyde (0.61 g, 5 mmol) was heated
4
under reꢁux for 4 h. Af er cooling, sodium borohydride (0.185 g,
conjugate 10, respectively. The results indicate that apo- 5 mmol) was added and the mixture was stirred at room temperature
for 12 h. Then, the mixture was concentrated under reduced pressure
ptosis induction of the conjugate 10 in MCF-7 tumor cells
and quenched with water (10 mL). The aqueous phase was extracted
changes in a dose-dependent manner.
with ethyl acetate (3ꢂ×ꢂ30 mL). The solvent was removed under
reduced pressure and the residue was purified by column chromatog-
raphy eluting with petroleum ether/ethyl acetate, 3:1, to give product
1
5b as a white solid: yield 72%; mp 143–145°C; H NMR (CDCl ): δ 0.95
3
Conclusion
A series of cyclic polyamine-dehydroabietylamine con-
jugates 10–13 were synthesized and characterized. The
in vitro antitumor activities of these compounds against
(s, 3H), 1.21 (s, 3H), 1.26 (d, 6H, J ꢂ=ꢂ 12 Hz), 1.35–1.79 (m, 8H), 2.24–2.31
m, 2H), 2.49 (d, H, J ꢂ=ꢂ 10.8 Hz), 2.80–2.84 (m, 3H), 3.63–3.72 (m, 2H),
.72 (d, 2H, J ꢂ=ꢂ 8.0 Hz), 6.88 (s, 1H), 6.98 (d, 1H, J ꢂ=ꢂ 8.0 Hz), 7.14 (d, 2H,
(
6
1
3
J ꢂ=ꢂ 7.0 Hz), 7.17 (d, H, J ꢂ=ꢂ 7.0 Hz); C NMR (CDCl ): δ 154.8, 147.5, 145.4,
3
1
34.8, 132.3, 129.4, 126.8, 124.3, 123.8, 115.3, 60.8, 54.1, 45.5, 38.4, 37.4,
3
6.9, 36.2, 33.4, 30.3, 25.4, 24.0, 19.3, 18.8, 18.7. ESI-HRMS. Anal. Calcd
+
HepG-2 and MCF-7 cells were evaluated. The effect of for C H NO ([M+H] ): m/z 392.2953. Found: m/z 392.2956.
2
7
38
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36ꢀꢀꢀꢀꢁꢀX. Yang et al.: Piperazine- and cyclen-conjugated dehydroabietylamine derivatives
Preparation of compounds 3 and 4
156.5, 154.5, 147.5, 145.4, 134.8, 132.2, 129.3, 126.7, 124.2, 123.7, 114.3,
0.3, 68.0, 60.8, 54.0, 45.3, 45.2, 42.0, 38.5, 37.4, 37.0, 36.2, 33.4, 30.3,
8.3, 25.3, 23.9, 19.3, 18.8, 18.7. ESI-HRMS. Anal. Calcd for C H N O
8
2
38
56
3
4
Compound 3 was prepared according to the literature [22] by the
+
(
[M+H] ): m/z 618.4271. Found: m/z 618.4274.
reaction of 1 with chloracetyl chloride in the presence of Et N at 0°C.
3
Purification by silica gel column chromatography eluting with petro-
leum ether/ethyl acetate, 1:1, gave product 3 as a white solid: yield
1
6
5%; H NMR (CDCl ): δ 1.46–1.49 (s, 27H, Boc-H), 3.38–3.56 (m, 16H, General procedure for the preparation of compounds 10–13
3
+
CH ), 4.06 (s, 2H); MS-ESI: m/z 548 (M ).
2
Compound 4 was synthesized using a similar procedure: yield
To a stirred solution of 6, 7, 8, or 9 (0.3 mmol) in ethanol (10 mL) at
room temperature was slowly added 5 mL of 3 ꢀ HCl in ethanol solu-
tion. Af er stirring overnight, the reaction mixture was concentrated
under reduced pressure. The residue was washed by anhydrous ether
to furnish a hydrochloride salt of 10, 11, 12, or 13 as a white powder.
1
7
7%; H NMR (CDCl ): δ 1.47 (s, 9H, Boc-H), 3.43–3.61 (m, 8H, CH ),
3
2
+
4
.05 (s, 2H); ESI-MS: m/z 262 (M ).
General procedure for the preparation of compounds 6–9
Compound 10ꢃYield 87%; mp 224–226°C; 1H NMR (DMSO-d ):
6
δ 0.99 (s, 3H), 1.14 (s, 3H), 1.21 (d, 6H, J ꢂ=ꢂ 6.8 Hz), 1.50–1.72 (m, 8H),
The tetrahydrofuran (THF) solution (30 mL) of 3 or 4 (0.5 mmol), 5a or
2
.17–2.31 (m, 2H), 2.50 (d, H, J ꢂ=ꢂ 10.8 Hz), 2.79–2.87 (m, 3H), 3.02–3.19
5
b (0.5 mmol), and KOH (0.084 g, 1.5 mmol) was stirred at 60°C for 10
(
(
m, 16H), 3.44–3.51 (m, 2H), 6.86 (s, 1H), 6.98 (d, 1H, J ꢂ=ꢂ 7.6 Hz), 7.17
h. Af er cooling, the mixture was filtered and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel
column chromatography eluting with petroleum ether/ethyl acetate,
1
3
d, H, J ꢂ=ꢂ 8.0 Hz); C NMR (DMSO-d ): δ 166.9, 146.7, 145.1, 134.2, 126.3,
23.8, 123.5, 58.7, 50.8, 48.2, 46.2, 45.3, 44.4, 43.1, 37.3, 36.9, 36.0, 34.6,
6
1
3
2.8, 28.7, 24.9, 23.9, 18.2, 18.0. ESI-HRMS. Anal. Calcd for C H N O
30 52 5
+
1:1, to give desired product 6–9 as a white solid.
(
[M+H] ): m/z 498.4172. Found: m/z 498.4174.
Compound 6ꢃThis compound was synthesized from 3 and 5a: yield
1
Compound 11ꢃYield 73%; mp 244–246°C; H NMR (DMSO-d ): δ 0.89
6
1
7
5%; mp 108–110°C; H NMR (CDCl ): δ 0.95 (s, 3H), 1.21 (s, 3H), 1.26
3
(s, 3H), 1.13 (s, 3H), 1.20 (d, 6H, J ꢂ=ꢂ 12 Hz), 1.38–1.61 (m, 8H), 2.24–2.28
(
d, 6H, J ꢂ=ꢂ 12 Hz), 1.35–1.75 (m, 35H), 2.17–2.31 (m, 2H), 2.50 (d, H,
(
m, 2H), 2.51 (d, H, J ꢂ=ꢂ 12 Hz), 2.73–2.80 (m, 3H), 3.05–3.16 (m, 16H),
J ꢂ=ꢂ 10.8 Hz), 2.79–2.87 (m, 3H), 3.38–3.47 (m, 18H), 6.86 (s, 1H), 6.98
3
.43–3.58 (m, 2H), 4.91 (s, 2H), 6.85 (s, 1H), 6.96 (d, 1H, J ꢂ=ꢂ 4.0 Hz), 7.03
3
(
d, 1H, J ꢂ=ꢂ 8.0 Hz), 7.17 (d, H, J ꢂ=ꢂ 8.0 Hz); 1 C NMR (CDCl ): δ 170.0,
13
3
(d, 2H, J ꢂ=ꢂ 8.0 Hz), 7.13 (d, H, J ꢂ=ꢂ 8.0 Hz), 7.46 (d, 2H, J ꢂ=ꢂ 8.0 Hz);
C
1
55.4, 147.4, 145.3, 134.8, 126.7, 124.3, 123.7, 80.4, 62.1, 51.7, 51.5, 49.7,
NMR (DMSO-d ): δ 168.8, 158.8, 146.6, 145.2, 134.1, 131.8, 129.0, 126.2,
6
4
9.6, 45.5, 38.4, 37.4, 37.0, 36.1, 33.4, 30.3, 28.5, 25.3, 24.0, 19.3, 19.1,
1
23.8, 123.4, 115.0, 65.7, 56.1, 50.4, 45.6, 44.6, 44.0, 42.9, 42.5, 37.4, 36.9,
+
1
8.8. ESI-HRMS. Calcd for C H N O ([M+H] ): m/z 798.5745. Found:
45
76
5
7
35.7, 34.7, 32.8, 28.6, 24.7, 23.9, 18.1, 18.0. ESI-HRMS. Anal. Calcd for
m/z 798.5736.
+
C H N O ([M+H] ): m/z 604.4591. Found: m/z 604.4593.
37
58
5
2
Compound 7ꢃThis compound was synthesized from 3 and 5b: yield
1
Compound 12ꢃYield 75%; mp 194–196°C; H NMR (DMSO-d ): δ 0.99
6
1
6
8%; mp 99–101°C; H NMR (CDCl ): δ 0.95 (s, 3H), 1.21 (s, 3H), 1.26
3
(s, 3H), 1.14 (s, 3H), 1.21 (d, 6H, J ꢂ=ꢂ 12 Hz), 1.47–1.72 (m, 8H), 2.27–2.31
(
d, 6H, J ꢂ=ꢂ 12 Hz), 1.34–1.73 (m, 35H), 2.17–2.27 (m, 2H), 2.50 (d, H, J ꢂ=ꢂ
0.8 Hz), 2.80–2.85 (m, 3H), 3.34–3.57 (m, 16H), 3.71–3.73 (m, 2H), 4.64
s, 2H), 6.87 (d, 2H, J ꢂ=ꢂ 4.4 Hz), 6.90 (s, 1H), 6.98 (d, 1H, J ꢂ=ꢂ 8.0 Hz),
(
m, 2H), 2.76 (d, H, J ꢂ=ꢂ 8.0 Hz), 2.81–2.90 (m, 3H), 3.05–3.18 (m, 8H),
1
3
8
.55–3.70 (m, 2H), 6.87 (s, 1H), 6.95 (d, 1H, J ꢂ=ꢂ 7.2 Hz), 7.17 (d, H, J ꢂ=ꢂ
(
13
.0 Hz); C NMR (DMSO-d ): δ 163.7, 146.7, 145.1, 134.4, 126.3, 123.8,
6
13
7
.14 (d, H, J ꢂ=ꢂ 7.2 Hz), 7.21 (d, 2H, J ꢂ=ꢂ 6.0 Hz); C NMR (CDCl ): δ 170.1,
3
123.5, 58.7, 47.5, 44.2, 42.1, 41.1, 38.1, 37.3, 37.0, 36.1, 34.8, 32.9, 28.8,
1
57.0, 155.6, 147.5, 145.4, 134.8, 132.3, 129.2, 126.7, 124.3, 123.7, 114.6,
+
2
5.3, 23.9, 18.6, 18.2. ESI-HRMS. Anal. Calcd for C H N O ([M+H] ):
2
6
42
3
8
0.5, 67.0, 60.6, 51.4, 50.4, 49.9, 49.6, 45.3, 38.5, 37.4, 36.9, 36.2, 33.4,
m/z 412.3328. Found: m/z 412.3329.
3
0.9, 28.5, 25.4, 24.0, 19.3, 18.8,18.7. ESI-HRMS. Calcd for C H N O
52 82 5 8
+
(
[M+H] ): m/z 904.6163. Found: m/z 904.6166.
1
Compound 13ꢃYield 70%; mp 249–251°C; H NMR (DMSO-d ): δ 0.90
6
(
(
s, 3H), 1.13 (s, 3H), 1.26 (d, 6H, J ꢂ=ꢂ 12 Hz), 1.39–1.68 (m, 8H), 2.24–2.28
m, 2H), 2.51 (d, H, J ꢂ=ꢂ 12 Hz), 2.73–2.80 (m, 3H), 3.10–3.32 (m, 8H),
Compound 8ꢃThis compound was synthesized from 4 and 5a: yield
1
8
0%; mp 117–119°C; HNMR (CDCl ): δ 0.93 (s, 3H), 1.18 (s, 3H), 1.26
3
3.65–3.70 (m, 2H), 4.92 (s, 2H), 6.85 (s, 1H), 6.94 (d, 1H, J ꢂ=ꢂ 4.0 Hz),
.01 (d, 2H, J ꢂ=ꢂ 8.4 Hz), 7.13 (d, H, J ꢂ=ꢂ 8.4 Hz), 7.45 (d, 2H, J ꢂ=ꢂ 8.4 Hz);
C NMR (DMSO-d ): δ 165.9, 158.5, 146.7, 145.2, 134.2, 131.8, 129.0,
126.3, 123.8, 123.5, 115.3, 65.4, 56.2, 50.5, 44.0, 42.5, 41.1, 38.1, 37.4, 36.9,
5.7, 34.7, 32.9, 28.6, 24.8, 23.9, 18.2, 18.0. ESI-HRMS. Anal. Calcd for
C H N O ([M+H] ): m/z 518.3747, Found: m/z 518.3750.
(
d, 6H, J ꢂ=ꢂ 12 Hz), 1.38–1.77 (m, 17H), 2.27–2.31 (m, 2H), 2.60 (d, H, J ꢂ=ꢂ
0.8 Hz), 2.81–2.87 (m, 3H), 3.02–3.20 (m, 8H), 3.45–3.51 (m, 2H), 6.86
7
1
(
13
6
13
s, 1H), 6.95 (d, 1H, J ꢂ=ꢂ 8.0 Hz), 7.17 (d, H, J ꢂ=ꢂ 8.0 Hz); C NMR (CDCl ):
3
δ 170.0, 154.5, 147.4, 145.4, 134.7, 126.7, 124.2, 123.7, 80.3, 62.1, 51.9, 45.5,
3
4
1
4.4, 41.6, 39.5, 38.4, 37.4, 36.2, 33.4, 30.2, 28.3, 25.6, 24.0, 19.1, 18.8,
+
33
48
3
2
+
8.4. ESI-HRMS. Anal. Calcd for C H N O ([M+H] ): m/z 512.3852.
3
1
50
3
3
Found: m/z 512.3851.
In vitro cytotoxicity assay
Compound 9ꢃThis compound was synthesized from 4 and 5b: yield
1
7
8%; mp 112–114°C; HNMR (CDCl ): δ 0.90 (s, 3H), 1.21 (s, 3H), 1.26
3
(
1
d, 6H, J ꢂ=ꢂ 12 Hz), 1.37–1.65 (m, 17H), 2.17–2.28 (m, 2H), 2.51 (d, H, J ꢂ=ꢂ Cytotoxicities of all compounds against MCF-7 and HepG-2 cell lines
2 Hz), 2.82–2.86 (m, 3H), 3.40–3.57 (m, 8H), 3.68–3.70 (m, 2H), 4.68 were determined using a cell counting kit-8 (CCK-8) assay. The cells
4
(
7
s, 2H), 6.87 (d, 2H, J ꢂ=ꢂ 8.4 Hz), 6.90 (s, 1H), 6.98 (d, 1H, J ꢂ=ꢂ 8.0 Hz), were plated in 96-well culture plates at density of 1ꢂ×ꢂ10 cells per well
13
.17 (d, H, J ꢂ=ꢂ 8.0 Hz), 7.24 (d, 2H, J ꢂ=ꢂ 8.4 Hz); C NMR (CDCl ): δ 166.8, and incubated for 24 h at 37°C in a wet atmosphere containing 5% CO2.
3
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X. Yang et al.: Piperazine- and cyclen-conjugated dehydroabietylamine derivativesꢎꢁꢀꢀꢀꢎ237
The tested compound was dissolved in PBS and then the diluted solu- [10] Xing, Y. H.; Zhang, W.; Song, J. J.; Zhang, Y. X.; Jiang, X. X.;
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Wang, R. Anticancer effects of a novel class rosin-derivatives
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2
5
-FU was used as a positive control. Then, 10 μL of a freshly diluted
CCK-8 solution [5 mg/mL in phosphate buffer saline (PBS)] was added
to each well for 2 h. The cell survival was evaluated by measuring the [11] Huang, X. C.; Wang, M.; Wang, H. S.; Chen, Z. F.; Zhang, Y.;
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estimation. All experiments were carried out in triplicate.
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Assessment of cell apoptosis
[
The MCF-7 cells were plated in 6-well culture plates at density of 3ꢂ×ꢂ105
cells per well. The cells were incubated with different concentrations
of compound 10 for 24 h. The cultured MCF-7 cells were washed twice
with PBS (pH 7.4) and then resuspended gently in 400 μL of bind-
ing buffer. The cell solution was then stained with Annexin V-APC/PI
apoptosis Kit according to the protocol of the company.
[
Acknowledgments: This study was financially supported
by the National Science Foundation of China (No. 21172182
and 21362026).
[
[
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[
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