Synthesis and biological evaluation of 2-aryl-benzimidazole derivatives of dehydroabietic acid as novel tubulin polymerization inhibitors

Article abstract of DOI:10.1039/c8ra02078g

A series of novel 2-aryl-benzimidazole derivatives of dehydroabietic acid were synthesized and characterized by IR, ¹H NMR, ¹³C NMR, MS and elemental analyses. All the target compounds were evaluated for their in vitro cytotoxic activity against SMMC-7721, MDA-MB-231, HeLa and CT-26 cancer cell lines and the normal hepatocyte cell line QSG-7701 through MTT assays. Among them, compound 6j displayed the most potent cytotoxic activity with IC₅₀ values of 0.08 ± 0.01, 0.19 ± 0.04, 0.23 ± 0.05 and 0.42 ± 0.07 μM, respectively, and substantially reduced cytotoxicity against QSG-7701 cells (5.82 ± 0.38 μM). The treatment of SMMC-7721 cells with compound 6j led to considerable inhibition of cell migration ability. The influence of compound 6j on cell cycle distribution was assessed on SMMC-7721 cells, exhibiting a cell cycle arrest at the G2/M phase. Moreover, tubulin polymerization assays and immunofluorescence assays elucidated that compound 6j could significantly inhibit tubulin polymerization and disrupt the intracellular microtubule network. A molecular docking study provided insight into the binding mode of compound 6j in the colchicine site of tubulin. In addition, compound 6j was found to induce apoptosis of SMMC-7721 cells, an increase of intracellular ROS level and a loss of mitochondrial membrane potential in a dose-dependent manner. These findings provided new molecular scaffolds for the further development of novel antitumor agents targeting tubulin polymerization.

Full text of DOI:10.1039/c8ra02078g

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Synthesis and biological evaluation of 2-aryl-
benzimidazole derivatives of dehydroabietic acid as
Cite this: RSC Adv., 2018, 8, 17511
novel tubulin polymerization inhibitors†
Ting-Ting Miao,‡ Xu-Bing Tao,‡ Dong-Dong Li, Hao Chen, Xiao-Yan Jin, Yi Geng,
Shi-Fa Wang
and Wen Gu
*
A series of novel 2-aryl-benzimidazole derivatives of dehydroabietic acid were synthesized and
1
13
characterized by IR, H NMR, C NMR, MS and elemental analyses. All the target compounds were
evaluated for their in vitro cytotoxic activity against SMMC-7721, MDA-MB-231, HeLa and CT-26 cancer
cell lines and the normal hepatocyte cell line QSG-7701 through MTT assays. Among them, compound
6
j displayed the most potent cytotoxic activity with IC50 values of 0.08 ꢀ 0.01, 0.19 ꢀ 0.04, 0.23 ꢀ 0.05
and 0.42 ꢀ 0.07 mM, respectively, and substantially reduced cytotoxicity against QSG-7701 cells (5.82 ꢀ
.38 mM). The treatment of SMMC-7721 cells with compound 6j led to considerable inhibition of cell
0
migration ability. The influence of compound 6j on cell cycle distribution was assessed on SMMC-7721
cells, exhibiting a cell cycle arrest at the G2/M phase. Moreover, tubulin polymerization assays and
immunofluorescence assays elucidated that compound 6j could significantly inhibit tubulin
polymerization and disrupt the intracellular microtubule network. A molecular docking study provided
insight into the binding mode of compound 6j in the colchicine site of tubulin. In addition, compound 6j
was found to induce apoptosis of SMMC-7721 cells, an increase of intracellular ROS level and a loss of
Received 8th March 2018
Accepted 30th April 2018
DOI: 10.1039/c8ra02078g
mitochondrial membrane potential in
a dose-dependent manner. These findings provided new
rsc.li/rsc-advances
molecular scaffolds for the further development of novel antitumor agents targeting tubulin polymerization.
In the past several decades, antimitotic agents interacting
with the taxane- or vinca alkaloid-binding sites have acquired
Introduction
Microtubules, as key components of the cytoskeleton, are tremendous success in clinical oncology. However, compounds
dynamic polymers of a, b-tubulin and involved in a series of targeting the colchicine-binding site have drawn increasing
important cellular processes including regulation of motility, interest from medicinal chemists in recent years for their high
cell signaling, maintenance of cell shape, cell proliferation and anticancer potency, selective toxicity toward tumor vasculature
1
–4
intracellular transport.
microtubules in the cell cycle, tubulin has become an attractive pump mediated multidrug resistance.
target for the development of novel antimitotic agents for bretastatin A-4 (CA-4) (Fig. 1) is a natural (Z)-stilbene isolated
Due to the multiple functions of and promising ability to overcome P-glycoprotein (P-gp) efflux
9–11
For example, com-
5,6
cancer therapy. Antimitotic agents are generally divided into from the South African bush willow tree Combretum caffrum by
12
two types: microtubule stabilizing agents (e.g., paclitaxel, epo- Pettit and co-workers in 1989. Through binding to the
thilones) and tubulin polymerization inhibitors (e.g., vinca colchicine site, CA-4 can signicantly inhibit tubulin polymer-
alkaloids, colchicine). Three major binding sites on microtu- ization and exhibit a potent cytotoxicity at nanomolar level
bules have been identied as the taxane-, vinca alkaloid- and against a wide range of human cancer cells. Moreover, CA-4
7
colchicine-binding sites. Disruption of microtubule dynamics displayed a selective toxicity towards the vascular network of
by these compounds will lead to cell cycle arrest at G2/M phase tumors, inducing an irreversible shutdown of blood ow to
8
13
and induction of apoptosis.
neoplastic cells. Some water soluble prodrugs of CA-4 were
14
subjected to clinical trials, among which the phosphate diso-
dium prodrug (CA-4P, fosbretabulin, Zybrestat™) received the
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass, orphan drug status (USA and Europe) in 2016 to treat neuro-
Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, College of Chemical
endocrine tumors (NETs) and glioblastoma multiform
Engineering, Nanjing Forestry University, Nanjing 210037, P. R. China. E-mail:
njguwen@163.com
15,16
(GBM).
Therefore, antimitotic agents targeting the
colchicine-binding site have proved to be a promising source for
the development of novel and potent anticancer drugs.
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra02078g
These two authors contributed equally to this study.
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Fig. 1 Structures of some reported antimitotic agents targeting colchicine-binding site and the newly synthesized 2-aryl-benzimidazole
derivatives of dehydroabietic acid (6a–k and 7a–k).
Scheme 1 Reagents and conditions: (a) (i) SOCl
2
, benzene, reflux, 3 h, (ii) MeOH, reflux, 5 h; (b) NBS, MeCN, rt, 24 h; (c) fuming HNO
3
, H
2
SO
4
,
ꢁ
ꢁ
0
C, 40 min; (d) Fe, EtOH, H
2
O, HCl, reflux, 4 h; (e) substituted arylaldehyde, p-toluenesulfonic acid, EtOH, 85 C, 24 h; (f) 3,4,5-trimethoxy-
ꢁ
phenylboronic acid, Pd(PPh
3
) , K
4 2
3 2
CO , THF, H O, 85 C, 24 h.
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Table 1 The in vitro cytotoxic activities of the tested compounds (1–5, 6a–k and 7a–k) against four cancer cell lines (SMMC-7721, MDA-MB-231,
HeLa and CT-26) and one normal hepatocyte cell line (QSG-7701)
IC50 (mM)
Compound
SMMC-7721
MDA-MB-231
HeLa
CT-26
QSG-7701
a
1
2
3
4
5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
43.43 ꢀ 2.81
16.21 ꢀ 1.73
>50
8.01 ꢀ 0.76
17.73 ꢀ 3.02
>50
>50
>50
>50
>50
0.23 ꢀ 0.05
0.98 ꢀ 0.11
13.12 ꢀ 1.70
>50
7.63 ꢀ 1.03
12.89 ꢀ 1.26
18.34 ꢀ 2.98
17.21 ꢀ 2.32
>50
>50
>50
>50
35.13 ꢀ 3.87
38.52 ꢀ 3.79
6.34 ꢀ 0.83
25.26 ꢀ 4.96
2.82 ꢀ 0.97
3.39 ꢀ 0.33
>50
NT
NT
NT
NT
NT
>50
NT
>50
>50
NT
NT
NT
NT
31.80 ꢀ 3.22
38.76 ꢀ 2.15
>50
>50
6
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
12.47 ꢀ 0.45
>50
41.72 ꢀ 2.06
>50
18.21 ꢀ 1.20
11.97 ꢀ 1.31
>50
33.66 ꢀ 4.70
19.21 ꢀ 0.93
35.26 ꢀ 1.77
>50
0.19 ꢀ 0.04
0.56 ꢀ 0.13
>50
1.52 ꢀ 0.77
5.27 ꢀ 0.27
12.28 ꢀ 2.46
16.49 ꢀ 1.87
40.81 ꢀ 3.32
16.58 ꢀ 1.40
>50
0.08 ꢀ 0.01
1.47 ꢀ 0.08
9.38 ꢀ 1.02
>50
>50
>50
>50
21.23 ꢀ 2.23
0.42 ꢀ 0.07
0.67 ꢀ 0.03
8.25 ꢀ 0.50
15.60 ꢀ 1.57
8.95 ꢀ 0.17
2.70 ꢀ 0.12
10.41 ꢀ 1.91
3.44 ꢀ 0.75
13.43 ꢀ 2.35
2.76 ꢀ 0.26
23.52 ꢀ 2.38
0.59 ꢀ 0.08
0.45 ꢀ 0.03
1.37 ꢀ 0.11
NT
j
5.82 ꢀ 0.38
8.43 ꢀ 1.06
>50
k
a
b
c
d
e
f
g
h
i
>50
NT
3.29 ꢀ 0.13
0.69 ꢀ 0.21
6.42 ꢀ 0.04
18.20 ꢀ 1.32
10.14 ꢀ 1.38
15.74 ꢀ 0.87
8.84 ꢀ 1.08
>50
0.83 ꢀ 0.20
0.21 ꢀ 0.02
0.76 ꢀ 0.06
36.07 ꢀ 2.32
>50
>50
>50
NT
>50
NT
>50
11.00 ꢀ 2.68
>50
>50
>50
2.35 ꢀ 1.09
1.95 ꢀ 0.56
0.92 ꢀ 0.07
29.88 ꢀ 3.07
>50
NT
j
k
3.75 ꢀ 0.78
5.86 ꢀ 0.63
0.87 ꢀ 0.05
12.83 ꢀ 1.27
11.78 ꢀ 2.12
13.75 ꢀ 1.08
Nocodazole
a
NT: Not tested.
Nitrogen-containing heterocycles play a vital role in the eld These ndings suggested that benzimidazole and TMP moieties
of medicinal chemistry because they can easily interact with could be useful pharmacophores for the design of novel tubulin
17
biomolecules of living systems. Among them, benzimidazole polymerization inhibitors.
has been considered as a prominent heterocyclic scaffold found
Natural products provide a rich source for new medicinal
in many natural and synthetic drugs which exhibit a wide range leads and play a major role in drug discovery, especially in the
18
31
of therapeutic properties. As antitumor agents, benzimidazole area of cancer therapy. Dehydroabietic acid (DAA, 1) is
derivatives can exert their antitumor activity by acting on a natural diterpenic resin acid which can be easily isolated from
various targets such as topoisomerase inhibitors, DNA inter- Pinus rosin or commercial disproportionated rosin. DAA and its
calating agents, androgen receptor antagonists, antiangiogenic derivatives has been found to exhibit a broad spectrum of bio-
agents, poly(ADP-ribose) polymerase (PARP) inhibitors, dihy- logical activities, such as antimicrobial, antiprotozoal, antiviral,
3
2–37
drofolate reductase inhibitors, protein tyrosine kinase inhibi- antiulcer, anti-aging and radical scavenging activities.
tors and tubulin polymerization inhibitors.
19–25
For example, Especially, a lot of DAA derivatives have been reported exhibit-
nocodazole (NSC-238189), with benzimidazole as its basic ing prominent anticancer activity through various mechanisms
3
8
39
40
scaffold, is a well-known anticancer agent that signicantly including DNA binding, induction of apoptosis or oncosis
41
inhibits the polymerization of tubulin at nanomolar concen- and inhibition of cancer cell migration. These results suggest
tration. Besides nocodazole, a number of benzimidazole that dehydroabietic acid is a promising lead compound for the
26
27
28
29
derivatives such as denibulin, CXI-benzo-84, NSC:761109/1,
investigation of new anticancer agents, and the incorporation of
etc. have also been reported as potent tubulin polymerization benzimidazole and TMP moieties to the structure of DAA may
inhibitors binding to the colchicine site (Fig. 1). On the other result in novel hybrid molecules with improved antimitotic
hand, some above-mentioned compounds such as colchicine properties. In continuation of our research on novel bioactive
42,43
and CA-4 shared a common 3,4,5-trimethoxyphenyl (TMP) derivatives of DAA,
a series of new 2-aryl-benzimidazole
moiety in their chemical structures, implying this moiety might derivatives of DAA were designed and synthesized, which were
30
be crucial for their anti-tubulin polymerization activities.
assayed for their in vitro antiproliferative activity against several
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cell lines. In addition, the possible anticancer mechanisms of displayed two peaks at m/z 506 and 508, corresponding to the
+
these derivatives as tubulin polymerization inhibitor and quasimolcular ions [M + H] , whcih also suggested the presence
apoptosis inducer were also preliminarily explored.
of a bromine atom. The data of molecular weight in combina-
tion with the elemental analysis conrmed the molecular
formula of compound 6j as C27
H
28BrN
3
O
2
. The IR spectrum of
ꢂ1
Results and discussion
Chemistry
6j exhibited a broad strong peaks at 3325 cm corresponding
to the N–H stretch vibrations and two strong C–H vibration
ꢂ1
bands at 2929 and 2866 cm attributed to the C–H stretch
The target compounds (6a–k and 7a–k) were synthesized according
to the method outlined in Scheme 1. The key intermediate methyl
vibrations. In addition, a very strong absorption band at
ꢂ1
1
703 cm was due to the C]O stretch vibrations of the methyl
12-bromo-13,14-dinitro-deisopropyldehydroabietate
(4)
was
1
ester moiety. The H-NMR spectrum of 6j showed three singlets
at d 1.28, 1.32 and 3.69 ppm, attributed to methyl protons at C-
synthesized from dehydroabietic acid based on the procedure re-
44
ported in a previous literature. Subsequently, two nitro groups of
compound 4 were reduced by Fe powder to afford the diamino
derivatives 5 in 57% yield, which was then reacted with different
1
9, C-20 and 18-ester group, respectively. The peaks in the range
of d 1.50–3.20 ppm could be attributed to the eleven protons of
ve aliphatic methylenes and an aliphatic methine at C-5. The

0
substituted arylaldehyde to get a series of 2 -aryl-12-bromo-
six peaks appeared at d 6.62, 7.28, 7.32, 7.45, 7.90 and 8.29 ppm
could be assigned as the aromatic protons in the benzimidazole
and indole moieties, and the N–H signals appeared as a broad
benzimidazole derivatives (6a–k) in 60–83% yield. Furthermore,
compounds 6a–k were reacted with 3,4,5-trimethoxy-
phenylboronic acid in the presence of Pd(PPh
3
)
4
and K
2
CO
3
to
13
single peak at d 8.44 ppm. In the C-NMR spectrum of 6j, there
0
afford the corresponding 2 -aryl-12-(3,4,5-trimethoxyphenyl)-
benzimidazole derivatives (7a–k) in 50–73% yield. All the synthe-
sized compounds were puried by recrystallization or silica gel
column chromatography.
were 27 peaks appearing in the range from d 16–180 ppm.
Among them, een peaks at d 100–155 ppm, including six
tertiary carbons and nine quaternary carbons, were due to the
aromatic carbons on the benzimidazole and indole moieties. In
addition, The peaks at d 52.2 and 179.5 ppm could be attributed
to the signals of methyl carbon and carbonyl carbon on C-18
The structures of compounds 6a–k and 7a–k were charac-
1
13
terized on the basis of MS, IR, H NMR, C NMR and elemental
analyses. In a typical example, the ESIMS of compound 6j
Fig. 2 Effects of compound 6j on cell cycle progression of SMMC-7721 cells. The analysis of cell cycle distribution was performed by using
propidium iodide staining method. Cells were treated with 0.1, 0.2 and 0.5 mM of 6j for 24 h.
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ring. These signals clearly indicated the presence of 3,4,5-tri-
methoxyphenyl moiety in compound 7j.
Biological evaluation
In vitro antiproliferative activity. The in vitro antiproliferative
activity of DAA derivatives (1–5, 6a–k, 7a–k) were evaluated by
MTT assay against human hepatocarcinoma cell line (SMMC-
7
721), human breast cancer cell line (MDA-MB-231), human
cervical carcinoma cell line (HeLa), mouse colon cancer cell line
CT-26) and human normal hepatocyte cell line (QSG-7701).
(
Nocodazole was co-assayed as the positive control and DMEM
culture medium containing 0.1% DMSO was used as negative
Fig. 3 Effect of compound 6j on tubulin polymerization. Colchicine
(
10 mM) and taxol (10 mM) was used as the positive control (purple) and
control. The results of the tested compounds expressed as IC50
negative control (red), respectively, and G-PEM buffer was used as the
blank control (blue). Tubulin was also treated with compound 6j at 5 values (concentration required to inhibit tumor cell prolifera-
mM (yellow) and 10 mM (green) concentrations. The reaction was tion by 50%) were presented in Table 1.
ꢁ
monitored at OD 340 nm at 37 C for 1 h.
From the results, the target compounds exhibited varying
degrees of cytotoxic activity against four cancer cell lines.
ester group, respectively. The assignments of the signals in the
H and C NMR spectra of compound 6j were in good accor-
dance with its structure. As for compound 7j, its ESIMS spec-
Specically, compounds 6c, 6d, 6j, 6k, 7c, 7j and 7k showed
signicant anticancer activity against SMMC-7721 cells with
IC50 values below 5 mM, compounds 6j, 6k, 7c, 7j and 7k dis-
1
13
trum and elemental analysis data also conrmed its molecular played strong cytotoxicity against MDA-MB-231 cells,
1
formula C36
H
39
N
2
O
5
. The H NMR spectrum of 7j exhibited two compounds 6j, 6k, 7j and 7k showed strong inhibition against
) and 3.96 ppm (OCH
singlets at d 3.93 (OCH
3
3
ꢃ 2) and a 2H HeLa cells, and compounds 6c, 6d, 6j, 6k, 7d, 7f, 7h, 7j and 7k
0
0
00
13
singlet at d 6.82 ppm (H-2 and H-6 ). The C NMR spectra of 7j
also demonstrated six additional peaks compared with
compound 6j. Among them, the peaks at d 56.5 and 61.1 ppm
were attributed to the three methoxyl groups in the benzene
exhibited pronounced activity against CT-26 cells (IC50 < 5 mM).
In general, compounds 6a–k exhibited stronger activity against
SMMC-7721 cells than the other three cancer cell lines, while
compounds 7a–k were most cytotoxic to CT-26 cells. Among
these derivatives, compound 6j exhibited the most potent
anticancer activity against all the four cancer cell lines, with IC50
values of 0.08 ꢀ 0.01, 0.19 ꢀ 0.04, 0.23 ꢀ 0.05 and 0.42 ꢀ 0.07
mM, respectively, stronger than the positive control nocodazole.
In addition, compounds 6k, 7j and 7k also exhibited potent
cytotoxic activity at low mM range. It is worth noting that
compounds 6j, 6k and 7j, 7k were signicantly less cytotoxic to
human normal hepatocyte cell line QSG-7701, which indicated
that these compounds possessed potent and selective anti-
proliferative activity on the tested cancer cell lines.
Based on the above observation, some interesting structure–
activity relationships (SAR) could be concluded. As shown in
Table 1, the cytotoxic activities of most target derivatives (6a–k,
7a–k) with benzimidazole moieties were superior to DAA (1) and
intermediates 2–5, which indicated that the introduction of
benzimidazole moiety could signicantly improve the cytotox-
icity. However, compounds 7a–k did not exhibit much stronger
cytotoxic activities than compounds 6a–k, suggesting that the
displacement of 12-Br with 12-(3,4,5-trimethoxyphenyl) moiety
were not obviously advantageous to anticancer activity as ex-
0
pected. On the other hand, the 2 -aryl groups on benzimidazole
rings also signicantly affected the cytotoxic potencies of the
target compounds. For series 6a–k, compounds 6c, 6f, 6h with p-

uoro, p-chloro and p-bromo substituents on the benzene rings
exhibited substantially stronger cytotoxic activity than
compounds 6b and 6i with p-methyl and p-methoxy groups. In
addition, compound 6c with p-uoro group was more cytotoxic
than 6d with m-uoro group which was also more cytotoxic than
Fig. 4 Effect of compound 6j on the microtubule network of SMMC-
7721 cells. SMMC-7721 cells were untreated (control) and treated with
compound 6j at 0.1, 0.2 and 0.5 mM for 24 h. Microtubules and
unassembled tubulin were shown in green and the nuclei were in blue. 6e with o-uoro group. The similar results could also be
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Fig. 5 Effect of compound 6j on in vitro migration potential of SMMC-7721 cells. Scratches were created with sterile 200 mL pipette and images
were captured using phase contrast microscopy at 0, 24 and 48 h after treatment with 0, 0.1, 0.2 and 0.5 mM of compound 6j.
Fig. 6 Annexin V-FITC/propidium iodide (PI) dual staining assay. SMMC-7721 cells were treated with compound 6j at 0, 0.1, 0.2, 0.5, 1.0 and 2.0
mM for 24 h, stained with Annexin V-FITC/PI and analyzed for apoptosis through flow cytometry. The percentage of cells positive for AV and/or PI
is reported in the quadrants. Cells in the lower left quadrant (AVꢂ/PIꢂ): live cells; lower right quadrant (AV+/PIꢂ): early apoptotic cells; upper right
quadrant (AV+/PI+): late apoptotic cells; upper left quadrant (AVꢂ/PI+): necrotic cells.
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Fig. 7 Effect of compound 6j on production of reactive oxygen species (ROS) in SMMC-7721 cells. SMMC-7721 cells were stained with DCF-DA
after treatment with different concentrations of compound 6j (0, 0.2, 0.5, 1.0 and 2.0 mM) for 24 h. Fluorescent microscopic images of SMMC-
7721 cells were acquired using fluorescence microscopy.
observed in series 7a–k. These results indicated that the obviously increased the speed of tubulin polymerization and
electron-withdrawing groups (–F, ꢂCl and ꢂBr) on the benzene the nal absorbance (OD340) compared with the blank control,
rings will be more benecial to the cytotoxic activity than indicating that it could enhance the tubulin polymerization. In
electron-donating ones (ꢂMe and ꢂOMe), and the uoro or contrast, compound 6j substantially decreased the polymeriza-
chloro groups on p-position will also be more favorable to the tion speed and the nal absorbance in a dose-dependent
cytotoxicity than those on m- or o-positions. Notably, manner, and the inhibition potency of 6j was stronger than
compounds 6j, 6k and 7j, 7k exhibited much more potent colchicine at 10 mM concentration. These results clearly indicate
anticancer activities than other derivatives, indicating that the that compound 6j can signicantly inhibit tubulin polymeri-
presence of 1H-indol-5-yl and 1H-indol-3-yl moieties, especially zation in vitro.
1
H-indol-5-yl moiety, would be more propitious to the interac-
Immunouorescence staining. Since the tubulin polymeri-
zation inhibitory effect of compound 6j has been veried, we
tion between these derivatives and their potential target.
Cell cycle analysis. Based on the results of the cytotoxic subsequently performed the immunouorescent staining assay
evaluation, compound 6j with the most potent cytotoxicity was to observe its effect on the intracellular microtubule system of
chosen for the in-depth mechanism study. Flow cytometry was SMMC-7721 cells. As shown in Fig. 4, the nucleus and micro-
used to analyze the effects of the DAA derivative on cell growth tubules in the untreated group appeared as the normal state
and division. SMMC-7721 cells were treated with different characterized by the slim and brous microtubules, well-
concentrations of compound 6j (0, 0.1, 0.2 and 0.5 mM) for 24 h. organized structure (green) surrounding the uncondensed
Cell cycle distribution was investigated by ow cytometric nuclei (blue). However, with increasing concentrations of
analysis aer staining the DNA of the treated cells with propi- compound 6j (0.1–0.5 mM), the meshy microtubule networks
dium iodide (PI). As shown in Fig. 2, the percentage of cells in shrank around the nuclei in a dose-dependent manner. In 0.5
G2/M phase gradually increased from 8.86% (0 mM) to 13.65% mM group, most microtubule networks disappeared, and most
(0.1 mM), 18.37% (0.2 mM) and 26.42% (0.5 mM), while the G0/G1 cell nuclei became irregularly shaped and highly condensed,
phase cells decreased from 65.93% to 49.30%. These results which is a typical characteristic of cell apoptosis. These results
suggested that compound 6j could arrest the cell cycle of indicated that compound 6j could disrupt microtubule organi-
SMMC-7721 cells at G2/M phase.
zations and therefore interfered with the mitosis of SMMC-7721
Tubulin polymerization inhibition. The signicant cell cells.
growth inhibitory activity of compound 6j in combination with
Wound healing assay. Metastasis is a multistep process
its G2/M phase arresting property enlightened us to investigate involving cancer cell motility and invasion which represents
its possible inhibition on tubulin polymerization. The in vitro a key difficulty for cancer therapy. Thus, the inhibition of
tubulin polymerization inhibition assay was carried out by metastasis is vital for efficient cancer treatment. Microtubule
using the Tubulin Polymerization Assay Kit (Cytoskeleton, Inc., plays an important role in cell motility and migration. As
USA) according to the protocol in the manual. Porcine brain compound 6j inhibits tubulin polymerization, the capacity of
tubulin was incubated with compound 6j at concentrations of 5 migration inhibition was also investigated through wound
45
and 10 mM. The positive control colchicine and negative control healing assay. In this assay, standard scratches (wounds) were
taxol were co-assayed at the concentration of 10 mM, and G-PEM made in conuent monolayers of SMMC-7721 cells and incu-
buffer was used as the blank control. As shown in Fig. 3, taxol bated with different concentrations (0, 0.1, 0.2 and 0.5 mM) of
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m
Fig. 8 Effect of compound 6j on mitochondrial membrane potential (DJ ). SMMC-7721 cells were treated with different concentrations of
compound 6j (0, 0.1, 0.2, 0.5, 1.0 and 2.0 mM), incubated with JC-1 and analyzed by flow cytometer.
compound 6j. The migration of SMMC-7721 cells was recorded signicantly increased from 10.12% to 80.08% aer treatment
by microscopic observations at 0, 24 and 48 h. As shown in with different concentrations of compound 6j (0, 0.1, 0.2, 0.5,
Fig. 5, there was almost complete healing of wound in control 1.0 and 2.0 mM) for 24 h. The results suggested that compound
group aer 48 h, which indicated the considerable migration of 6j could induce the apoptosis of SMMC-7721 cells in a dose-
cells. However, healing was strongly suppressed in 0.2 and 0.5 dependent manner.
mM groups, suggesting that compound 6j could inhibit the
ROS generation assay. Reactive oxygen species (ROS) are
migration of SMMC-7721 cells.
highly harmful elements to cells as they exert oxidative stress
Cell apoptosis analysis. In order to investigate whether and nally cause cellular damage. Excessive ROS generation
39
compound 6j could induce cell apoptosis, SMMC-7721 cells renders cells vulnerable to apoptosis. To determine whether
treated with different concentrations of compound 6j were compound 6j triggers ROS generation in SMMC-7721 cells to
subjected to Annexin V-FITC/propidium iodide (PI) dual stain- induce apoptosis, the cells were treated with different concen-
ing followed by ow cytometry assay. As shown in Fig. 6, the trations (0, 0.2, 0.5, 1 and 2 mM) of compound 6j for 24 h, and
percentage of early and late apoptotic cells (lower right quad- the ROS generation was evaluated using the uorescent probe
rant, AV+/PIꢂ and upper right quadrant, AV+/PI+, respectively) 2,7-dichlorouorescein diacetate (DCF-DA) by uorescence
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Fig. 9 The binding modes between the active conformations of compound 6j, colchicine and tubulin. (A) The 3D diagram of interactions
between the two compounds and the colchicine bing site. (B) The 2D diagram of interactions between the two compounds and the colchicine
binding site. The H-bonds were displayed as dashed arrows, and the s–p interactions were shown as orange lines.
microscopy. As shown in Fig. 7, SMMC-7721 cells treated with high red uorescence. However, in apoptotic cells whose
compound 6j appeared strong green uorescence in a dose- mitochondrial potential has collapsed, JC-1 exists in cytosol as
dependent manner, indicating that compound 6j signicantly a monomer which emits green uorescence. SMMC-7721 cells
induced ROS generation. This phenomenon implied that the treated with different concentrations (0, 0.1, 0.2, 0.5, 1.0 and 2.0
increment of ROS might play an important role as an early mM) of compound 6j for 24 h were stained with JC-1 and then
mediator in compound 6j-induced apoptosis.
subjected to ow cytometry. The results were shown in Fig. 8.
As presented in Fig. 8, the percentage of cells with low DJ
JC-1 mitochondrial membrane potential assay. As the most
m
important organelle, mitochondria play key roles not only on (lower right quadrant) increased from 6.47% (control) to 11.35%
supplying metabolic energy in the form of ATP, but also on (0.1 mM), 15.66% (0.2 mM), 20.16% (0.5 mM), 31.30% (1.0 mM) and
regulating the signal transmission during the apoptosis of 53.12% (2.0 mM), which implied that compound 6j could cause the
46
cancer cells. Besides, the mitochondrial dysfunction can be decrease of mitochondrial membrane potential in a dose depen-
aroused under high ROS exposure, leading to the collapse of dent manner, and probably induced the apoptosis of SMMC-7721
4
8
mitochondrial membrane potential (DJ ), which is a charac- cells through the intrinsic mitochondrial-mediated pathway.
teristic phenomenon of early apoptosis. In order to further
m
47
Molecular docking. In order to investigate the binding
investigate the apoptosis-inducing effect of target compound 6j, modes of dehydroabietic acid derivatives with the tubulin
mitochondrial membrane potential changes were detected protein, the top active compound 6j and the reference ligand
using the uorescent probe JC-1. As a lipophilic cationic dye, JC- colchicine were selected and docked into the colchicine-binding
49
1
can easily pass through the plasma membrane into cells and site between a- and b-tubulin protein. (PDB ID: 1SA0).
accumulates in actively respiring mitochondria. In control cells, Molecular docking studies were performed by using GLIDE
50
JC-1 can form aggregates in mitochondrial matrix and present docking module of Schr ¨o dinger suite 2015-1.
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As shown in Fig. 9(A), it was observed that the obtained binding measured on a Nexus 870 FT-IR spectrometer, and the absorp-
ꢂ1
pose of compound 6j could be well accommodated inside the tion bands were expressed in cm . The ESI-MS spectra were
colchicine-binding site of tubulin. The position of the imidazole recorded on a Mariner System 5304 mass spectrometer.
moiety and dehydroabietic acid as a core scaffold of the derivative Elemental analyses were carried out by Elementar Vario El cube
1
13
seemed substantially consistent in the active pocket, which was elemental analyzer. H and C NMR spectra were accomplished
surmised that this series of the DAA derivatives would adopt in CDCl or DMSO-d on Bruker AV-300 and AV-500 NMR
3
6
similar kind of binding mode interacting with the tubulin protein. spectrometers using TMS as internal standard. Reactions and
Fig. 9(B) illustrate the detailed protein-inhibitor interaction of the resulted products were monitored by TLC which was carried
compound 6j with key residues of the colchicine binding site. It out on TLC silica gel 60 F254 aluminium sheets from Merck
could been observed that the N–H on the indole moiety of KGaA, Darmstadt, Germany and visualized in UV light (254 nm).
compound 6j established a hydrogen bond with the important Silica gel (300–400 mesh) for column chromatography was
active site residue Val B: 238, while the ester group of compound 6j purchased from Qingdao Marine Chemical Factory, China. The
also formed a hydrogen bond with Lys B: 254, which implied that reagents (chemicals), all being of A.R. grade, were purchased
the indole and methyl ester moieties played an important role in from Shanghai Chemical Reagent Company (Shanghai, China)
its anti-tubulin polymerization activity. In addition, the docking and Energy Chemical (Shanghai, China). Disproportionated
results showed that compound 6j displayed potent interaction rosin was provided by Zhongbang Chemicals Co., Ltd. (Zhaoq-
ꢂ1
energy (ꢂ8.888 kcal mol ), superior to the docking score of ing, China), from which dehydroabietic acid (1, 97%) was iso-
ꢂ1
51
colchicine (ꢂ6.557 kcal mol ), which can further proved the lated according to the published method.
effectiveness of this series of DAA derivatives targeting tubulin
polymerization.
General procedure for the synthesis of compounds 6a–k
The key intermediate (4) was synthesized from dehydroabietic
acid (1) according to the procedure previously reported, which
Conclusion
44
In this work, a series of new benzimidazole derivatives of dehy- was further treated as follows to afford compounds 6a–k and
droabietic acid were synthesized and evaluated for their in vitro 7a–k (Scheme 1). To a solution of compound 4 (0.44 g, 1.0
anticancer potential against four cancer cell lines (SMMC-7721, mmol) in 20 mL of EtOH was added reduced iron powder (0.6 g,
2
MDA-MB-231, HeLa and CT-26). In MTT assay, compound 6j, 6k, 10.6 mmol), H O (2 mL) and 10 drops of concentrated HCl. The
7j and 7k displayed signicant growth inhibitory activities against mixture was stirred under reux for 4 h. Aer cooling, the
several cancer cell lines comparable or superior to the positive mixture was ltered to remove iron powder. The solution was
ꢂ1
control nocodazole. Among them, compound 6j exhibited the neutralized with aqueous NaOH (2 mol L ) and concentrated
most potent anticancer activity against all test cancer cell lines and in vacuo. The residue was puried by column chromatography
it was found to be comparatively safe towards human normal on silica gel, eluting with CH Cl –MeOH (20 : 1, v/v) to give 5 as
2 2
hepatocyte cell line QSG-7701. The treatment of SMMC-7721 cells a yellow resin (0.22 g, yield 57%). The spectral data of the
with compound 6j led to inhibition of cell migration ability. product were in accordance with those in the previous
44
Subsequently, the cell cycle analysis indicated that compound 6j literature.
could arrest SMMC-7721 cells at G2/M phase in a dose-dependent
To a solution of compound 5 (0.66 g, 1.5 mmol) in 20 mL of
manner, which implied that compound 6j might interact with the anhydrous EtOH was added p-toluenesulfonic acid (25.8 mg,
microtubule systems of cancer cells. Tubulin polymerization 0.15 mmol) and 2.2 mmol of different substituted arylaldehyde.
ꢁ
inhibition assay and immunouorescence staining further The reaction mixture was stirred at 85 C for 24 h under
conrmed that compound 6j could considerably inhibit the poly- nitrogen atmosphere and monitored by TLC. At the end of
merization of tubulin and thus disrupt the microtubule network of reaction, the mixture was poured into 200 mL of ice-cold water,
cancer cells. The molecular docking study provided insight into which was extracted with EtOAc for three times (3 ꢃ 200 mL).
the binding mode of compound 6j in the colchicine-binding site of The organic phase was combined, washed with water, saturated
tubulin. On the other hand, Annexin V-FITC/PI dual staining assay NaHCO solution and brine, dried over anhydrous Na SO and
3
2
4
suggested that compound 6j could induce the apoptosis of SMMC- then concentrated in vacuo to give the crude product, which was
721 cells. The treatment with compound 6j also caused the puried by silica gel chromatography (petroleum ether–acetone
7
collapse of mitochondrial membrane potential and enhanced 100 : 1–10 : 1, v/v) to afford compounds 6a–k.
0
intracellular ROS levels in SMMC-7721 cells. Therefore, compound
Methyl
2 -phenyl-12-bromo-13,14-imidazolyl-deisopropyl-
ꢁ
6
j represented a novel lead for potent tubulin polymerization dehydroabietate (6a). White powder solid; mp 123–125 C;
1
inhibitors that paved the way for the discovery of new promising yield: 73.8%; H NMR (300 MHz, CDCl
3
) d: 1.28 (s, 3H), 1.31 (s,
anticancer agents.
3H), 1.50–1.81 (m, 7H), 1.92 (m, 1H), 2.30–2.37 (m, 2H), 3.09 (m,
1
8
1
1
H), 3.17 (brs, 1H), 3.68 (s, 3H), 7.35 (s, 1H), 7.45–7.48 (m, 3H),
1
3
.04 (dd, J ¼ 7.9, 1.8 Hz, 2H); C NMR (75 MHz, CDCl
3
) d: 16.6,
Experimental
Chemistry
8.7, 21.1, 25.3, 25.4, 36.8, 37.7, 38.6, 45.3, 47.9, 52.2, 104.7,
21.2, 122.5, 126.9, 129.0, 129.8, 130.3, 138.3, 139.8, 145.7,
ꢂ1
Melting points were measured on an XT-4 apparatus (Taike 151.5, 179.2; IR (KBr, cm ): 3303, 2930, 2864, 1726, 1619, 1579,
Corp., Beijing, China) and were uncorrected. IR spectra were 1470, 1454, 1385, 1254, 1193, 1129, 1035, 857, 737; MS (ESI): m/z
17520 | RSC Adv., 2018, 8, 17511–17526
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+
4
5
67.1 [M + H] ; anal. calcd for C25
H
27BrN
2
O
2
: C 64.24; H 5.82; N 3H), 1.32 (s, 3H), 1.50–2.10 (m, 7H), 1.93 (m, 1H), 2.30–2.37 (m,
.99; found: C 64.31; H 5.78, N 6.03.
2H), 3.05–3.16 (m, 2H), 3.68 (s, 3H), 7.36 (s, 1H), 7.46 (d, J ¼
0
13
Methyl 2 -(p-tolyl)-12-bromo-13,14-imidazolyl-deisopropyl- 8.1 Hz, 2H), 7.98 (d, J ¼ 8.3 Hz, 2H); C NMR (75 MHz, CDCl ) d:
3
ꢁ
dehydroabietate (6b). White powder solid; mp 143–145 C; 16.6, 18.7, 21.1, 25.3, 25.4, 36.8, 37.7, 38.5, 45.2, 47.9, 52.2,
1
yield: 71.6%; H NMR (500 MHz, CDCl
3
) d: 1.27 (s, 3H), 1.31 (s, 105.1, 122.8, 128.1, 128.3, 129.3, 136.4, 139.7, 141.3, 146.0,
ꢂ1
3
3
7
H), 1.50–1.81 (m, 7H), 1.92 (m, 1H), 2.30–2.36 (m, 2H), 2.41 (s, 150.3, 179.3; IR (KBr, cm ): 3355, 2924, 2852, 1724, 1666, 1577,
H), 3.07 (brs, 1H), 3.17 (brs, 1H), 3.68 (s, 3H), 7.28 (d, J ¼ 1494, 1454, 1244, 1126, 1046, 1015, 764, 732. MS (ESI): m/z 501.1
1
3
+
.9 Hz, 2H), 7.33 (s, 1H), 7.93 (d, J ¼ 8.0 Hz, 2H); C NMR (75 [M + H] ; anal. calcd (%) for C25
2 2
H26BrClN O : C, 59.83; H,
MHz, CDCl
3
) d: 16.6, 18.7, 21.1, 21.6, 25.4, 36.8, 37.6, 38.6, 45.2, 5.22; N, 5.58; found (%): C, 59.76; H, 5.27; N, 5.62.
0
2 -(2-chlorophenyl)-12-bromo-13,14-imidazolyl-
4
1
1
7.9, 52.2, 104.6, 122.4, 126.8, 126.9, 129.8, 134.1, 139.2, 140.7,
45.6, 151.7, 179.2; IR (KBr, cm ): 3308, 2929, 2867, 1725, 1623, deisopropyl-dehydroabietate (6g). Yellow powder solid; mp
579, 1476, 1429, 1380, 1253, 1190, 1128, 1037, 824, 726; MS 142–143 C; yield: 75.8%; H NMR (300 MHz, CDCl ) d: 1.29 (s,
ESI): m/z 481.1 [M + H] ; anal. calcd for C26
Methyl
ꢂ1
ꢁ
1
3
+
(
H
29BrN
2
O
2
: C 64.87; 3H), 1.32 (s, 3H), 1.50–2.10 (m, 7H), 1.94 (m, 1H), 2.30–2.42 (m,
H 6.07; N 5.82; found: C 64.81; H 6.13, N 5.76.
2H), 3.02–3.30 (m, 2H), 3.69 (s, 3H), 7.39 (s, 1H), 7.41–7.45 (m,
0
13
Methyl
2 -(4-uorophenyl)-12-bromo-13,14-imidazolyl- 2H), 7.49 (dd, J ¼ 7.2, 1.8 Hz, 1H), 8.43 (d, J ¼ 7.2 Hz, 1H);
C
3
deisopropyl-dehydroabietate (6c). White powder solid; mp NMR (75 MHz, CDCl ) d: 16.6, 18.7, 21.0, 25.34, 25.36, 36.7, 37.7,
ꢁ
1
1
3
2
2
49–151 C; yield: 82.6%; H NMR (500 MHz, CDCl
3
) d: 1.28 (s, 38.6, 45.2, 47.8, 52.1, 105.8, 122.8, 127.6, 128.4, 129.0, 130.6,
ꢂ1
H), 1.32 (s, 3H), 1.50–1.81 (m, 7H), 1.92 (m, 1H), 2.30–2.36 (m, 131.1, 132.5, 138.7, 141.1, 145.8, 150.4, 179.1; IR (KBr, cm ):
H), 3.06 (brs, 1H), 3.17 (brs, 1H), 3.68 (s, 3H), 7.16 (t, J ¼ 8.5 Hz, 3301, 2929, 2866, 1725, 1617, 1578, 1537, 1451, 1404, 1250,
1
3
H), 7.35 (s, 1H), 8.02 (dd, J ¼ 8.5, 5.2 Hz, 2H); C NMR (75 1192, 1129, 1052, 1035, 962, 764, 735. MS (ESI): m/z 501.1 [M +
+
MHz, CDCl
3
) d: 16.6, 18.7, 21.1, 25.3, 25.4, 36.8, 37.7, 38.6, 45.2, H] ; anal. calcd (%) for C25
H26BrClN
2
O
2
: C, 59.83; H, 5.22; N,
4
8
7.9, 52.2, 103.0, 116.2 (d, J ¼ 21.9 Hz), 122.6, 126.1, 128.9 (d, J ¼ 5.58; found (%): C, 59.88; H, 5.29; N, 5.51.
0
2 -(4-bromophenyl)-12-bromo-13,14-imidazolyl-
.5 Hz), 139.2, 140.6, 145.9, 150.6, 164.1 (d, J ¼ 249.1 Hz), 179.3;
Methyl
ꢂ1
IR (KBr, cm ): 3313, 2931, 2863, 1729, 1620, 1607, 1493, 1471, deisopropyl-dehydroabietate (6h). Yellow powder solid; mp
ꢁ
1
1431, 1381, 1250, 1160, 1128, 841, 736; MS (ESI): m/z 485.1 [M + 141–142 C; yield: 74.2%; H NMR (300 MHz, CDCl
3
) d: 1.28 (s,
+
H] ; anal. calcd for C H BrFN O : C 61.86; H 5.40; N 5.77; 3H), 1.32 (s, 3H), 1.50–2.10 (m, 8H), 2.30–2.37 (m, 2H), 3.05–3.17
2
5
26
2 2
found: C 61.84; H 5.42, N 5.78.
(m, 2H), 3.68 (s, 3H), 7.36 (s, 1H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.92
13
0
Methyl
2 -(3-uorophenyl)-12-bromo-13,14-imidazolyl- (d, J ¼ 8.3 Hz, 2H); C NMR (75 MHz, CDCl ) d: 16.6, 18.7, 21.1,
3
deisopropyl-dehydroabietate (6d). Yellow powder solid; mp 25.4, 25.5, 36.8, 37.7, 38.5, 45.2, 47.9, 52.2, 105.2, 122.8, 124.9,
ꢁ
1
1
3
2
1
42–144 C; yield: 74.2%; H NMR (300 MHz, CDCl
3
) d: 1.28 (s, 128.4, 129.0, 131.1, 132.2, 136.9, 139.2, 145.2, 150.5, 179.3; IR
ꢂ1
H), 1.32 (s, 3H), 1.50–1.82 (m, 7H), 1.93 (m, 1H), 2.30–2.36 (m, (KBr, cm ): 3317, 2923, 2852, 1725, 1468, 1428, 1382, 1254,
H), 3.07 (m, 1H), 3.16 (m, 1H), 3.69 (s, 3H), 7.16 (t, J ¼ 8.4 Hz, 1191, 1129, 1072, 1038, 1010, 834, 727. MS (ESI): m/z 545.0 [M +
1
3
+
H), 7.36 (s, 1H), 7.46 (m, 1H), 7.79 (d, J ¼ 8.1 Hz, 2H); C NMR H] ; anal. calcd (%) for C25
2 2 2
H26Br N O : C, 54.96; H, 4.80; N,
(
75 MHz, CDCl ) d: 16.6, 18.7, 21.1, 25.3, 25.4, 36.8, 37.7, 38.6, 5.13; found (%): C, 55.03; H, 4.84; N, 5.08.
3
0
4
5.3, 47.9, 52.2, 105.9, 114.1 (d, J ¼ 23.4 Hz), 117.3 (d, J ¼ 21.2
Methyl
2 -(4-methoxyphenyl)-12-bromo-13,14-imidazolyl-
Hz), 122.4 (d, J ¼ 2.8 Hz), 122.9, 127.3 (d, J ¼ 7.1 Hz), 130.8 (d, J deisopropyl-dehydroabietate (6i). White powder solid; mp
ꢁ
1
¼
8.2 Hz), 136.1, 140.2, 145.2, 147.9, 164.1 (d, J ¼ 249.1 Hz), 140–142 C; yield: 61.3%; H NMR (500 MHz, CDCl
3
) d: 1.27 (s,
ꢂ1
1
79.3; IR (KBr, cm ): 3285, 2926, 2854, 1724, 1665, 1586, 1461, 3H), 1.31 (s, 3H), 1.50–1.81 (m, 7H), 1.91 (m, 1H), 2.33 (m, 2H),
+
1
255, 1198, 1129, 861, 790; MS (ESI): m/z 485.1 [M + H] ; anal. 3.03 (m, 1H), 3.17 (brs, 1H), 3.67 (s, 3H), 3.86 (s, 3H), 6.98 (d, J ¼
1
3
calcd for C25
H
26BrFN
2
O
2
: C 61.86; H 5.40; N 5.77; found: C 8.5 Hz, 2H), 7.31 (s, 1H), 7.98 (d, J ¼ 8.4 Hz, 2H); C NMR (75
61.93; H 5.36, N 5.71.
3
MHz, CDCl ) d: 16.6, 18.7, 21.1, 25.3, 25.4, 36.8, 37.6, 38.5, 45.2,
0
Methyl
2 -(2-uorophenyl)-12-bromo-13,14-imidazolyl- 47.8, 52.2, 55.5, 105.9, 114.4, 122.2, 122.4, 128.5, 136.2, 138.7,
ꢂ1
deisopropyl-dehydroabietate (6e). Yellow powder solid; mp 145.4, 151.9, 161.3, 179.3; IR (KBr, cm ): 3331, 2934, 2863,
ꢁ
1
1
3
2
2
2
2
38–139 C; yield: 72.7%; H NMR (300 MHz, CDCl ) d: 1.28 (s, 1724, 1613, 1581, 1496, 1474, 1435, 1385, 1254, 1178, 1129,
3
+
H), 1.32 (s, 3H), 1.50–1.82 (m, 7H), 1.95 (m, 1H), 2.31–2.37 (m, 1033, 838, 737; MS (ESI): m/z [M + H] : 497.1; anal. calcd for
H), 3.07–3.16 (m, 2H), 3.69 (s, 3H), 7.20 (m, 1H), 7.29–7.34 (m,
H), 7.40–7.48 (m, 2H); C NMR (75 MHz, CDCl
C
26
H29BrN
2
O
3
: C 62.78; H 5.88; N 5.63; found: C 62.70; H 5.93, N
1
3
3
) d: 16.6, 18.7, 5.67.
Methyl
0
1.1, 25.3, 36.7, 37.7, 38.6, 45.3, 47.8, 52.2, 106.4, 116.2 (d, J ¼
2 -(1H-indol-5-yl)-12-bromo-13,14-imidazolyl-
3.0 Hz), 122.8, 125.3 (d, J ¼ 2.9 Hz), 130.8 (d, J ¼ 23.8 Hz), 131.8 deisopropyl-dehydroabietate (6j). White powder solid; mp
ꢁ
1
(
1
d, J ¼ 9.1 Hz), 137.8, 139.8, 145.9, 151.8, 160.4 (d, J ¼ 245.6 Hz), 179–181 C; yield: 74.4%; H NMR (500 MHz, CDCl ) d: 1.28 (s,
3
ꢂ1
79.1; IR (KBr, cm ): 3379, 2921, 2780, 2360, 1712, 1455, 1245, 3H), 1.32 (s, 3H), 1.50–1.82 (m, 7H), 1.94 (m, 1H), 2.31–2.37 (m,
+
1128, 1045, 880, 758, 692. MS (ESI): m/z 486.4 [M + H] ; anal. 2H), 3.09 (m, 1H), 3.21 (brs, 1H), 3.69 (s, 3H), 6.62 (s, 1H), 7.28
calcd (%) for C25
H
26BrFN
2
O
2
: C, 61.86; H, 5.40; N, 5.77; found (s, 1H), 7.32 (s, 1H), 7.45 (d, J ¼ 8.1 Hz, 1H), 7.90 (d, J ¼ 8.2 Hz,
13
(%): C, 61.92; H, 5.37; N, 5.82.
1H), 8.29 (s, 1H), 8.44 (brs, 1H); C NMR (75 MHz, CDCl
3
) d:
0
Methyl
2 -(4-chlorophenyl)-12-bromo-13,14-imidazolyl- 16.6, 18.7, 21.1, 25.3, 25.4, 36.8, 37.5, 38.5, 45.2, 47.8, 52.2,
deisopropyl-dehydroabietate (6f). Yellow powder solid; mp 103.1, 107.6, 111.8, 116.3, 119.8, 121.1, 121.2, 121.9, 125.8,
ꢁ
1
ꢂ1
1
39–141 C; yield: 81.8%; H NMR (300 MHz, CDCl
3
) d: 1.28 (s, 128.0, 136.2, 137.0, 139.8, 145.2, 153.9, 179.5; IR (KBr, cm ):
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3
1
325, 2929, 2866, 1703, 1620, 1581, 1542, 1452, 1397, 1250, 1382, 1245, 1186, 1127, 1021, 826, 731; MS (ESI): m/z 569.3 [M +
+
+
130, 1035, 811, 735; MS (ESI): m/z 506.1 [M + H] ; anal. calcd for H] ; anal. calcd for C H N O : C 73.92; H 7.09; N 4.93; found:
3
5
40 2 5
C H BrN O : C 64.03; H 5.57; N 8.30; found: C 64.09; H 5.53, N C 73.87; H 7.13, N 4.99.
2
7
28
3 2
0
8
.37.
Methyl
deisopropyl-dehydroabietate (6k). White powder solid; mp powder solid; mp 139–141 C; yield: 50.1%; H NMR (500 MHz,
Methyl
2 -(4-uorophenyl)-12-(3,4,5-trimethoxyphenyl)-
0
2 -(1H-indol-3-yl)-12-bromo-13,14-imidazolyl- 13,14-imidazolyl-deisopropyl-dehydroabietate
(7c). White
ꢁ
1
ꢁ
1
139–140 C; yield: 60.2%; H NMR (300 MHz, DMSO-d
6 3
) d: 1.23 CDCl ) d: 1.26 (s, 3H), 1.34 (s, 3H), 1.60–1.85 (m, 7H), 2.01 (m,
(
s, 3H), 1.25 (s, 3H), 1.36–1.51 (m, 2H), 1.63–2.00 (m, 5H), 2.15– 1H), 2.41 (d, J ¼ 12.3 Hz, 1H), 2.46 (d, J ¼ 13.7 Hz, 1H), 3.17 (brs,
2
3
1
2
1
1
1
.39 (m, 2H), 2.37 (m, 1H), 2.90 (m, 1H), 3.06 (m, 1H), 3.64 (s, 1H), 3.26 (brs, 1H), 3.70 (s, 3H), 3.92 (s, 3H), 3.95 (s, 6H), 6.84 (s,
1
3
H), 7.23 (m, 3H), 7.48 (d, J ¼ 5.2 Hz, 1H), 8.25 (s, 1H), 8.56 (s, 2H), 7.16 (t, J ¼ 8.3 Hz, 2H), 7.36 (s, 1H), 8.03 (brs, 2H); C NMR
1
3
H), 11.60 (s, 1H); C NMR (75 MHz, CDCl ) d: 16.6, 18.7, 21.1, (75 MHz, CDCl ) d: 16.7, 18.8, 21.3, 25.5, 25.6, 36.9, 37.6, 38.7,
3
3
5.4, 36.8, 37.6, 38.6, 45.2, 47.8, 52.2, 106.0, 106.9, 112.2, 120.1, 45.5, 48.0, 52.1, 56.5, 61.1, 106.3, 116.2 (d, J ¼ 21.9 Hz), 119.2,
21.4, 121.8, 123.1, 125.1, 126.1, 129.7, 135.7, 136.5, 138.5, 119.3, 123.6, 124.1, 124.5, 124.6, 128.6 (d, J ¼ 8.5 Hz), 137.7,
ꢂ1
45.3, 149.4, 179.3; IR (KBr, cm ): 3393, 3327, 2929, 2870, 1724, 140.1, 147.3, 153.6, 154.4, 164.0 (d, J ¼ 249.8 Hz), 179.3; IR
ꢂ1
705, 1627, 1587, 1542, 1456, 1390, 1247, 1129, 1023, 821, 743; (KBr, cm ): 3308, 2959, 2925, 2855, 1726, 1598, 1579, 1493,
+
MS (ESI): m/z 506.1 [M + H] ; anal. calcd for C27
6
H28BrN
3
O
2
: C 1433, 1387, 1245, 1158, 1128, 1021, 843, 735; MS (ESI): m/z 573.3
+
4.03; H 5.57; N 8.30; found: C 63.95; H 5.64, N 8.26.
2 5
[M + H] ; anal. calcd for C34H37FN O : C 71.31; H 6.51; N 4.89;
found: C 71.38; H 6.48, N 4.83.
0
Methyl
2 -(3-uorophenyl)-12-(3,4,5-trimethoxyphenyl)-
General procedure for the synthesis of compounds 7a–k
To a solution of compound 6 (0.15 mmol) in 22 mL of THF/H
1
3,14-imidazolyl-deisopropyl-dehydroabietate (7d). Yellow
ꢁ
1
2
O
powder solid; mp 156–158 C; yield: 72.9%; H NMR (300 MHz,
10 : 1) was added 3,4,5-trimethoxyphenylboronic acid (63 mg, CDCl ) d: 1.26 (s, 3H), 1.35 (s, 3H), 1.50–1.90 (m, 7H), 2.00 (m,
(
3
0
0
.3 mmol), K CO3 (415 mg, 3 mmol) and Pd(PPh ) (17 mg, 1H), 2.42 (d, J ¼ 12.5 Hz, 1H), 2.49 (m, 1H), 3.10 (m, 1H), 3.22
2
3 4
ꢁ
.015 mmol). The reaction mixture was stirred at 85 C for 24 h (m, 1H), 3.70 (s, 3H), 3.92 (s, 3H), 3.95 (s, 6H), 6.82 (s, 2H), 7.14
under nitrogen atmosphere and monitored by TLC. Subse- (t, J ¼ 7.6 Hz, 1H), 7.35 (s, 1H), 7.45 (m, 1H), 7.77 (d, J ¼ 8.3 Hz,
1
3
quently, the mixture was poured into ice-cold water (200 mL) 2H); C NMR (125 MHz, CDCl
and extracted EtOAc for three times (3 ꢃ 200 mL). The organic 36.9, 37.7, 38.6, 45.5, 48.0, 52.2, 56.5, 61.1, 106.3, 113.8 (d, J ¼
layer was combined, washed with water, saturated NaHCO
) d: 16.7, 18.8, 21.3, 25.4, 25.6,
3
23.5 Hz), 116.9 (d, J ¼ 20.9 Hz), 119.2, 119.5, 122.2, 124.6, 130.7
3
solution and brine, dried over anhydrous Na
2
SO
4
and then (d, J ¼ 8.2 Hz), 132.5 (d, J ¼ 8.4 Hz), 134.7, 136.7, 137.9, 139.6,
ꢂ1
concentrated in vacuo to give the crude product, which was 144.9, 149.9, 153.6, 163.2 (d, J ¼ 245.2 Hz), 179.4; IR (KBr, cm ):
puried by silica gel chromatography (petroleum ether-acetone 3305, 2928, 2854, 1724, 1659, 1582, 1492, 1462, 1243, 1126,
+
1
00 : 1–10 : 1, v/v) to afford compounds 7a–k.
1006, 790, 760; MS (ESI): m/z 573.3 [M + H] ; anal. calcd for
0
Methyl
2 -phenyl-12-(3,4,5-trimethoxyphenyl)-13,14-
C
34
H37FN
2
O
5
: C 71.31; H 6.51; N 4.89; found: C 71.22; H 6.57, N
imidazolyl-deisopropyl-dehydroabietate (7a). White powder 4.92.
ꢁ
1
0
solid; mp 153–155 C; yield: 62.5%; H NMR (500 MHz, CDCl
3
)
Methyl
2 -(2-uorophenyl)-12-(3,4,5-trimethoxyphenyl)-
d: 1.35 (s, 6H), 1.50–1.82 (m, 7H), 2.00 (m, 1H), 2.42 (d, J ¼ 13,14-imidazolyl-deisopropyl-dehydroabietate (7e). Yellow
ꢁ
1
1
3
(
2.4 Hz, 1H), 2.47 (m, 1H), 3.07 (brs, 1H), 3.14 (m, 1H), 3.70 (s, powder solid; mp 155–157 C; yield: 53.1%; H NMR (300 MHz,
H), 3.93 (s, 6H), 3.96 (s, 3H), 6.80 (s, 2H), 7.36 (s, 1H), 7.40–7.49 CDCl ) d: 1.35 (s, 6H), 1.51–1.90 (m, 7H), 2.01 (m, 1H), 2.42 (d, J
3
1
3
m, 3H), 8.02 (brs, 2H); C NMR (75 MHz, CDCl ) d: 16.7, 18.8, ¼ 11.7 Hz, 1H), 2.48 (m, 1H), 3.07 (brs, 1H), 3.30 (m, 1H), 3.69 (s,
3
2
1
1
3
1
1.3, 25.5, 25.6, 36.8, 37.6, 38.7, 45.5, 48.0, 52.1, 56.5, 61.1, 3H), 3.95 (s, 6H), 3.99 (s, 3H), 6.84 (s, 2H), 7.20 (m, 1H), 7.31–
1
3
06.2, 118.9, 119.8, 126.2, 126.6, 128.8, 129.1, 130.1, 134.9, 7.43 (m, 4H); C NMR (75 MHz, CDCl
3
) d: 16.7, 18.8, 21.3, 25.4,
ꢂ1
37.6, 140.3, 140.7, 147.6, 151.1, 153.7, 179.2; IR (KBr, cm ): 25.5, 36.8, 37.7, 38.7, 45.5, 48.0, 52.1, 56.5, 61.1, 107.5, 116.2 (d, J
318, 2935, 2870, 1724, 1581, 1496, 1454, 1378, 1246, 1180, ¼ 22.8 Hz), 117.8 (d, J ¼ 9.9 Hz), 118.6, 119.5, 123.4 (d, J ¼ 24.9
+
128, 1023, 833, 735; MS (ESI): m/z 555.3 [M + H] ; anal. calcd for Hz), 125.3, 130.4, 131.4 (d, J ¼ 6.8 Hz), 134.7, 137.8, 136.6, 140.0,
ꢂ1
C
34
H
38
N
2
O
5
: C 73.62; H 6.91; N 5.05; found: C 73.69; H 6.88, N 146.6, 152.8, 153.9, 160.4 (d, J ¼ 245.0 Hz), 179.2; IR (KBr, cm ):
5
.10.
Methyl
imidazolyl-deisopropyl-dehydroabietate (7b). White powder
3353, 2928, 2867, 2280, 1723, 1581, 1494, 1464, 1245, 1178,
0
+
2 -(p-tolyl)-12-(3,4,5-trimethoxyphenyl)-13,14- 1065, 1015, 824, 761. MS (ESI): m/z 573.3 [M + H] ; anal. calcd for
C
34
H
37FN
2
O
5
: C 71.31; H 6.51; N 4.89; found: C 71.36; H 6.59, N
ꢁ
1
solid; mp 133–135 C; yield: 56.3%; H NMR (500 MHz, CDCl
d: 1.26 (s, 3H), 1.34 (s, 3H), 1.50–1.86 (m, 7H), 2.01 (m, 1H), 2.41
3
)
4.81.
Methyl
0
2 -(4-chlorophenyl)-12-(3,4,5-trimethoxyphenyl)-
(
s, 3H), 2.45 (m, 2H), 3.19 (brs, 1H), 3.28 (brs, 1H), 3.69 (s, 3H), 13,14-imidazolyl-deisopropyl-dehydroabietate
(7f).
Yellow
ꢁ
1
3
7
1
5
1
.93 (s, 3H), 3.95 (s, 6H), 6.82 (s, 2H), 7.29 (d, J ¼ 7.9 Hz, 2H), powder solid; mp 168–170 C; yield: 59.9%; H NMR (300 MHz,
1
3
.37 (s, 1H), 7.92 (d, J ¼ 7.4 Hz, 2H); C NMR (75 MHz, CDCl
3
3
) d: CDCl ) d: 1.26 (s, 3H), 1.35 (s, 3H), 1.50–1.85 (m, 7H), 1.99 (m,
6.7, 18.8, 21.3, 21.6, 25.5, 25.6, 36.8, 37.6, 38.7, 45.4, 47.9, 52.1, 1H), 2.41 (d, J ¼ 12.5 Hz, 1H), 2.48 (m, 1H), 3.16 (brs, 1H), 3,23
6.5, 61.1, 106.2, 119.0, 119.7, 126.8, 127.0, 128.6, 128.8, 129.8, (brs, 1H), 3.69 (s, 3H), 3.92 (s, 3H), 3.95 (s, 6H), 6.82 (s, 2H), 7.35
1
3
32.2, 137.8, 140.0, 140.5, 144.6, 151.3, 153.6, 179.2; IR (s, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.97 (d, J ¼ 7.7 Hz, 2H); C NMR
ꢂ1
(KBr, cm ): 3330, 2925, 2854, 1725, 1666, 1581, 1497, 1459, (75 MHz, CDCl
3
) d: 16.7, 18.8, 21.3, 25.4, 25.6, 36.9, 37.6, 38.6,
17522 | RSC Adv., 2018, 8, 17511–17526
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4
1
1
1
5.5, 48.0, 52.2, 56.5, 61.1, 106.3, 119.0, 119.2, 124.5, 127.9, 103.2, 106.3, 111.8, 118.4, 119.3, 119.7, 121.1, 122.0, 125.8,
28.8, 129.3, 134.8, 136.0, 137.8, 139.0, 140.8, 141.3, 150.1, 128.2, 129.0, 131.0, 135.2, 137.0, 137.7, 140.9, 144.1, 153.2,
53.6, 179.4; IR (KBr, cm ): 3317, 2926, 2853, 1725, 1581, 1495, 153.6, 179.4; IR (KBr, cm ): 3345, 2959, 2924, 2853, 1724, 1620,
ꢂ1
ꢂ1
461, 1428, 1381, 1244, 1127, 1014, 836, 730. MS (ESI): m/z 589.2 1580, 1498, 1456, 1379, 1258, 1126, 836, 737; MS (ESI): m/z 593.3
+
+
[M + H] ; anal. calcd (%) for C34
H37ClN
2 5 39 2 5
O : C, 69.32; H, 6.33; N, [M + H] ; anal. calcd for C36H N O : C 72.83; H 6.62; N 7.08;
4
.76; found (%): C, 69.38; H, 6.25; N, 4.82.
found: C 72.85; H 6.62, N 7.05.
0
0
Methyl
2 -(2-chlorophenyl)-12-(3,4,5-trimethoxyphenyl)-
Methyl 2 -(1H-indol-3-yl)-12-(3,4,5-trimethoxyphenyl)-13,14-
1
3,14-imidazolyl-deisopropyl-dehydroabietate (7g). Yellow imidazolyl-deisopropyl-dehydroabietate (7k). White powder
ꢁ
1
ꢁ
1
3
powder solid; mp 157–159 C; yield: 72.4%; H NMR (300 MHz, solid; mp 199–201 C; yield: 56.2%; H NMR (500 MHz, CDCl )
CDCl ) d: 1.26 (s, 3H), 1.35 (s, 3H), 1.50–2.10 (m, 7H), 2.42 (d, J ¼ d: 1.27 (s, 3H), 1.33 (s, 3H), 1.50–1.80 (m, 7H), 1.98 (m, 1H), 2.38
3
1
1
1.7 Hz, 1H), 2.48 (m, 1H), 3.06 (brs, 1H), 3.31 (m, 1H), 3.50 (m, (d, J ¼ 12.2 Hz, 1H), 2.46 (d, J ¼ 12.0 Hz, 1H), 3.11 (m, 1H), 3.21
H), 3.69 (s, 3H), 3.93 (s, 3H), 3.96 (s, 6H), 6.85 (s, 2H), 7.35–7.43 (m, 1H), 3.66 (s, 3H), 3.89 (s, 3H), 3.93 (s, 6H), 7.06–7.22 (m, 5H),
1
3
13
(
m, 3H), 7.48 (d, J ¼ 7.6 Hz, 1H), 8.44 (brs, 1H); C NMR (75 7.25 (s, 1H), 7.67 (s, 1H), 8.21 (s, 1H), 9.59 (brs, 1H); C NMR (75
MHz, CDCl ) d: 16.7, 18.8, 21.3, 25.4, 25.6, 36.8, 37.7, 38.8, 45.5, MHz, CDCl ) d: 16.7, 18.8, 21.4, 25.5, 25.7, 36.9, 37.6, 38.7, 45.4,
8.0, 52.1, 56.4, 61.1, 106.9, 119.5, 119.7, 126.7, 127.7, 128.8, 48.0, 52.1, 56.4, 61.1, 106.1, 106.2, 112.0, 118.2, 120.3, 120.4,
29.5, 130.0, 130.7, 132.4, 133.2, 137.8, 139.5, 140.3, 146.1, 121.3, 123.1, 125.4, 126.1, 127.0, 131.1, 135.3, 136.6, 137.7,
3
3
4
1
1
1
5
6
ꢂ1
ꢂ1
48.5, 153.4, 179.2; IR (KBr, cm ): 3355, 2924, 2852, 1724, 1666, 139.9, 144.2, 148.9, 153.6, 179.4; IR (KBr, cm ): 3398, 3058,
577, 1494, 1454, 1244, 1126, 1046, 1016, 764, 732. MS (ESI): m/z 2926, 2853, 1725, 1624, 1584, 1498, 1455, 1427, 1377, 1295,
+
+
89.2 [M + H] ; anal. calcd (%) for C H ClN O : C, 69.32; H, 1125, 833, 743; MS (ESI): m/z 593.3 [M + H] ; anal. calcd for
3
4
37
2 5
.33; N, 4.76; found (%): C, 69.26; H, 6.37; N, 4.71.
C H N O : C 72.83; H 6.62; N 7.08; found: C 72.80; H 6.63, N
36 39 2 5
2 -(4-bromophenyl)-12-(3,4,5-trimethoxyphenyl)- 7.09.
0
Methyl
1
3,14-imidazolyl-deisopropyl-dehydroabietate (7h). Yellow
ꢁ
1
powder solid; mp 150–152 C; yield: 54.1%; HNMR (300 MHz, Cell lines and culture
CDCl ) d: 1.26 (s, 3H), 1.35 (s, 3H), 1.50–1.90 (m, 7H), 2.00 (m,
H), 2.42 (d, J ¼ 11.8 Hz, 1H), 2.48 (m, 1H), 3.08 (brs, 1H), 3.28
brs, 1H), 3.69 (s, 3H), 3.92 (s, 3H), 3.95 (s, 6H), 6.82 (s, 2H), 7.36
3
The human hepatocarcinoma cell line (SMMC-7721), human
breast cancer cell line (MDA-MB-231), human cervical carci-
noma cell line (HeLa), mouse colon cancer cell line (CT-26) and
human normal hepatocyte cells (QSG-7701) used in this study
were all obtained from Keygene Biotech., China. These cell lines
were maintained in DMEM medium supplemented with 10%
1
(
(
(
1
3
s, 1H), 7.67 (d, J ¼ 8.2 Hz, 2H), 8.10 (d, J ¼ 9.3 Hz, 2H); C NMR
75 MHz, CDCl ) d: 16.7, 18.8, 21.3, 25.4, 25.6, 36.8, 37.6, 38.6,
3
4
1
1
1
6
5
5.5, 48.0, 52.1, 56.4, 61.1, 104.7, 118.4, 119.7, 127.0, 127.6,
29.0, 129.2, 131.0, 136.2, 137.8, 138.4, 142.8, 144.5, 150.9,
heat-inactivated fetal bovine serum in a humidied atmosphere
ꢂ1
53.8, 179.3; IR (KBr, cm ): 3285, 2928, 2854, 1724, 1584, 1459,
ꢁ
of 5% CO at 37 C.
2
343, 1245, 1188, 1093, 1008, 878, 825, 745. MS (ESI): m/z 632.2,
+
34.2 [M + H] ; anal. calcd (%) for C34
H37BrN
2
O
5
: C, 64.45; H,
Cytotoxic assay
.89; N, 4.42; found (%): C, 64.36; H, 5.92; N, 4.38.
0
The in vitro cytotoxic activities of the benzimidazole derivatives
of dehydroabietic acid were evaluated against tested cell lines
via the MTT colorimetric method. Briey, different tumor cells
were grown in DMEM medium supplemented with 10% fetal
Methyl
2 -(4-methoxyphenyl)-12-(3,4,5-trimethoxyphenyl)-
13,14-imidazolyl-deisopropyl-dehydroabietate
(7i).
White
ꢁ
1
powder solid; mp 153–155 C; yield: 70.7%; H NMR (500 MHz,
CDCl ) d: 1.26 (s, 3H), 1.34 (s, 3H), 1.50–1.84 (m, 7H), 2.01 (m,
H), 2.40 (d, J ¼ 12.2 Hz, 2H), 2.46 (brs, 1H), 3.30 (m, 1H), 3.69
s, 3H), 3.87 (s, 3H), 3.92 (s, 6H), 3.95 (s, 3H), 6.80 (s, 2H), 7.00
3
ꢂ1
bovine serum, penicillin (100 U mL ), and streptomycin (50 mg
1
ꢂ1
mL ). Cells were harvested at log phase of growth and seeded
(
(
(
5
1
3
in 96-well plates (100 mL per well at a density of 2 ꢃ 10 cells
d, J ¼ 8.2 Hz, 2H), 7.35 (s, 1H), 7.96 (d, J ¼ 8.2 Hz, 2H); C NMR
ꢂ1
ꢁ
mL ). Aer 24 h incubation at 37 C and 5% CO to allow cell
2
75 MHz, CDCl ) d: 16.7, 18.8, 21.3, 25.5, 25.6, 36.8, 37.6, 38.7,
3
attachment, cultures were exposed to various concentrations of
the isolated compounds for 48 h. Finally, MTT solution (2.5 mg
4
1
1
1
5
4
5.5, 48.0, 52.1, 55.6, 56.5, 61.1, 107.3, 113.9, 114.2, 114.5, 119.7,
21.5, 126.4, 128.1, 131.0, 136.7, 138.6, 140.6, 151.4, 153.2,
ꢂ1
ꢂ1
mL in PBS) was added (40 mL per well). Plates were further
62.9, 179.3; IR (KBr, cm ): 3330, 2925, 2854, 1725, 1666, 1581,
ꢁ
incubated for 4 h at 37 C, and the formazan crystals formed
497, 1459, 1382, 1245, 1186, 1127, 1021, 826, 731; MS (ESI): m/z
84.3 [M + H] ; anal. calcd for C35
.79; found: C 71.87; H 6.92, N 4.81.
+
were dissolved by adding 150 mL per well of DMSO. Absorption
at 570 nm was measured with an ELISA plate reader. The results
were expressed as IC50 values with standard deviations, which
was dened as the concentration at which 50% survival of cells
was discerned. Nocodazole was co-assayed as positive control.
H
40
N
2
O
6
: C 71.90; H 6.90; N
0
Methyl 2 -(1H-indol-5-yl)-12-(3,4,5-trimethoxyphenyl)-13,14-
imidazolyl-deisopropyl-dehydroabietate (7j). White powder
solid; mp 189–191 C; yield: 61.8%; H NMR (300 MHz, CDCl )
ꢁ
1
3
d: 1.26 (s, 3H), 1.35 (s, 3H), 1.50–1.80 (m, 7H), 2.02 (m, 1H), 2.43
In vitro tubulin polymerization inhibition
(
(
d, J ¼ 12.1 Hz, 1H), 2.47 (d, J ¼ 12.2 Hz, 1H), 3.21 (brs, 2H), 3.70
s, 3H), 3.93 (s, 3H), 3.96 (s, 6H), 6.62 (s, 1H), 6.82 (s, 2H), 7.25 (s, The tubulin polymerization assay was accomplished using
1
8
1
H), 7.31 (s, 1H), 7.46 (d, J ¼ 8.5 Hz, 1H), 7.91 (d, J ¼ 8.3 Hz, 1H), a commercial assay kit (cat. #BK004P) purchased from Cyto-
13
.26 (s, 1H), 8.47 (brs, 1H); C NMR (75 MHz, CDCl
3
) d: 16.7, skeleton, Inc. (Danvers, MA, USA). Tubulin isolated from
8.8, 21.3, 25.5, 25.6, 36.9, 37.6, 38.7, 45.5, 48.0, 52.1, 56.5, 61.1, porcine brain tissue was used in this tubulin polymerization
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assay kit. Tubulin polymerizations are followed by an increase resuspended in ice-cold PBS. Aer this, the cells were treated
ꢂ1
ꢁ
in optical density at 340 nm (OD340) over a 60 min period at with 100 mL of 100 mg mL RNAse A at 37 C for 30 min, fol-
ꢁ
ꢂ1
3
7 C due to the light scattered by microtubule polymer. At rst, lowed by incubation with 400 mL of 1 mg mL DNA staining
tubulin (4 mg) was disovled in 1 mL of G-PEM buffer containing solution propidium iodide (PI) in the dark at 4 C for 30 min.
0 mM piperazine-N,N -bis(2-ethanesulfonic acid) sequisodium The samples were then analyzed for their DNA content by ow
ꢁ
0
8
2
salt (PIPES, pH 6.9), 2.0 mM MgCl , 0.5 mM EGTA and 1 mM cytometry (Becton-Dickinson FACSCalibur, New York, USA).
GTP to prepare tubulin solution. Subsequently, 10 mL of 10 ꢃ
stock solutions of the tested compounds at different concen-
Annexin V-FITC/PI dual staining assay
trations were added to a prewarmed 96-well plate and incubated
ꢁ
at 37 C for 2 min, then 90 mL of tubulin solution was also added Apoptosis was discriminated with the Annexin V-FITC/PI dual
5
into each well to initiate the reaction. The optical intensity staining assay. SMMC-7721 cells were seeded at 1 ꢃ 10 cells per
(OD340) of each well was recorded every 60 s for 60 min (Cytation well in 10% fetal calf serum (FBS)-DMEM into six-well plates
3
Cell Imaging Multi-Mode Reader, BioTek Instruments, Inc., and treated with or without compound 6j for 24 h. The cells
USA). Colchicine and taxol were co-assayed as the positive were detached with 0.25% trypsin, washed twice with ice-cold
control and negative control, respectively. G-PEM buffer was PBS and then resuspended in 500 mL of 1ꢃ binding buffer
used as the blank control.
2
(0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl ). The
solution was transferred into 5 mL culture tubes, and 5 mL of
Annexin V-FITC (BD, Pharmingen) and 5 mL of PI were added to
each tube. The cells were gently vortexed, and incubated for
30 min at 25 C in the dark. The analysis was performed on
a ow cytometry (Becton-Dickinson FACSCalibur, New York,
Immunouorescence
The intracellular microtubule morphology was investigated
using the protocol previously reported. For the immunouo-
ꢁ
52
5
rescence studies, SMMC-7721 cells (3 ꢃ 10 cells per well) were
2
USA).
seeded on 10 mm confocal culture dishes (NEST Biotech-
nology, China) for 24 h and then incubated in the absence or
presence of compound 6j at the indicated concentrations for
another 12 h. Aer being washed with phosphate buffer solu-
tion (PBS) and xed in 4% paraformaldehyde for 15 min, the
cells were permeabilized with 0.5% Triton X-100 for 15 min and
blocked for 30 min with 10% goat serum. Then, the cells were
incubated with mouse anti-tubulin antibody (ZSGB-Bio, China)
ROS generation assay
ROS generation assay was performed by using the reactive
oxygen species assay kit (Beyotime Biotech., China). Intracel-
lular ROS generation was tested through dichlorodihydro uo-
rescein diacetate (DCFH-DA) assay. DCFH-DA is taken up by
SMMC-7721 cells, and then activated by esterase-mediated
cleavage of acetate to form dichlorodihydro uorescein
ꢁ
at 4 C overnight and incubated with goat anti-mouse IgG/Alexa-
Fluor 488 antibody (ZSGB-Bio, China) at room temperature for
(DCFH), which is trapped in the cells. DCFH is converted to
1
h. Aer the nuclei were stained with Hoechst 33258 (Beyotime

uorescein DCF in the presence of ROS. SMMC-7721 cells were
Biotech., China) in the dark at room temperature for 30 min, the
samples were immediately visualized on a Zeiss LSM 570 laser
scanning confocal microscope (Carl Zeiss, Germany).
seeded in six-well plates and incubated with different concen-
trations of compound 6j for 24 h. Aer removing the compound
solution, cells were treated with 10 mM of DCFH-DA at 37 C for
ꢁ
2
0 min. Subsequently, the cells were washed with PBS for three
Wound healing assay
times and then exposed to light. Immediately aer light expo-
sure, cell images were acquired through an inverted uores-
cence microscope (Olympus 1X71 Inverted System Microscope,
Olympus, Japan).
5
SMMC-7721 cells (5 ꢃ 10 cells per well) were grown in a 12-well
plate for 24 h. Scratches were made in conuent monolayers
using 200 mL pipette tip. Then, wounds were washed twice with
PBS to remove non-adherent cell debris. The media containing
different concentrations (0, 0.1, 0.2 and 0.5 mM) of compound 6j
were added to the corresponding wells. Cells which migrated JC-1 mitochondrial membrane potential assay
across the wound area were photographed under the phase
contrast microscope (Olympus 1X71 Inverted System Micro-
scope, Olympus, Japan) aer 24 and 48 h treatment.
The JC-1 mitochondrial membrane potential assay kit (Keygene
Biotech., China) was employed to measure mitochondrial
depolarization in SMMC-7721 cells. Briey, cells cultured in six-
well plates aer indicated treatments were incubated with an
Cell cycle analysis
ꢂ1
ꢁ
equal volume of JC-1 staining solution (5 mg mL ) at 37 C for
For the ow cytometric analysis of the DNA content, SMMC- 20 min and rinsed twice with PBS. Mitochondrial membrane
5
7
721 cells were seeded in 6-well plates (5 ꢃ 10 cells per well) potentials were monitored by determining the relative amounts
and incubated in the absence or presence of compound 6j at of dual emissions from mitochondrial JC-1 monomers or
indicated concentrations for 24 h. Aer treatment, cells were aggregates using ow cytometry (Becton-Dickinson FACSCali-
detached with 0.25% trypsin, harvested by centrifugation, bur, New York, USA). Mitochondrial depolarization is indicated
washed twice with ice-cold PBS and then xed and per- by an increase in the percentage of cells with low DJm (green
ꢁ
meabilized with ice-cold ethanol at 4 C overnight. Ethanol was uorescence, lower right quadrant) compared with cells with
removed by centrifugation, and the cells were washed twice and high DJ
m
(red uorescence, upper right quadrant).
17524 | RSC Adv., 2018, 8, 17511–17526
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Paper
RSC Advances
Molecular modelling
8 P. K. Sorger, M. Dobles, R. Tournebize and A. A. Hyman,
Curr. Opin. Cell Biol., 1997, 9, 807–814.
9 Y. Lu, J. Chen, M. Xiao, W. Li and D. D. Miller, Pharm. Res.,
2012, 29, 2943–2971.
X-ray structures of the tubulin co-crystal was retrieved from the
PDB (accession code: 1SA0) and prepared using the Protein
Preparation Wizard workow from Schr ¨o dinger suite,
53
50
1
0 M.-T. Cui, L. Jiang, M. Goto, P.-L. Hsu, L. Li, Q. Zhang,
L. Wei, S.-J. Yuan, E. Hamel, S. L. Morris-Natschke,
K.-H. Lee and L. Xie, J. Med. Chem., 2017, 60, 5586–5598.
1 G. F. Mangiatordi, D. Trisciuzzi, D. Alberga, N. Denora,
R. M. Iacobazzi, D. Gadaleta, M. Catto and O. Nicolotti,
Eur. J. Med. Chem., 2017, 139, 792–803.
including optimization of the hydrogen bond network and
a short energy minimization with position restraints on heavy
atoms using the OPLS_2005 force eld. Ligands were then freely
docked in the colchicine binding site located between chains A
and B using the standard protocol implemented in Maestro,
version 10.1. van der Waals (vdW) scaling of 0.8 and partial cut-
off of 0.15 were set to soen the potential for non-polar sites,
and no constraints were specied. The best docked pose ranked
by GlideScore value was recorded, and saved for each ligand.
The structures of complexes were analysed for interactions and
the 3D pose of the most active compound 6j was displayed using
Discovery studio 3.5 client.
1
1
1
1
2 G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts and
D. Garcia-Kendall, Experientia, 1989, 45, 209–211.
3 G. G. Dark, S. A. Hill, V. E. Prise, G. M. Tozer, G. R. Pettit and
D. J. Chaplin, Cancer Res., 1997, 57, 1829–1834.
4 G. J. Rustin, S. M. Galbraith, H. Anderson, M. Stratford,
L. K. Folkes, L. Sena, L. Gumbrell and P. M. Price, J. Clin.
Oncol., 2003, 21, 2815–2822.
15 S. L. Young and D. J. Chaplin, Expert Opin. Invest. Drugs,
2
004, 13, 1171–1182.
Conflicts of interest
1
6 I. Kheli, T. Naret, D. Renko, A. Hamze, G. Bernadat,
J. Bignon, C. Lenoir, J. Dubois, J.-D. Brion, O. Provot and
M. Alami, Eur. J. Med. Chem., 2017, 127, 1025–1034.
There are no conicts to declare.
1
7 N. Shrivastava, M. J. Naim, M. J. Alam, F. Nawaz, S. Ahmed
and O. Alam, Arch. Pharm., 2017, 350, e1700040.
8 A. Kamal, B. Nagaseshadri, V. L. Nayak, V. Srinivasulu,
M. Sathish, J. S. Kapure and C. S. Reddy, Bioorg. Chem.,
Acknowledgements
1
This work was supported by the National Natural Science
Foundation of China (31770616), the Natural Science Founda-
tion of Jiangsu Province (BK201516), the Natural Science
Foundation for Colleges and Universities in Jiangsu Province
2015, 63, 72–84.
1
2
9 P. Singla, V. Luxami, R. Singh, V. Tandon and K. Paul, Eur. J.
Med. Chem., 2017, 126, 24–35.
0 P. Purushottamachar, A. M. Godbole, L. K. Gediya,
M. S. Martin, T. S. Vasaitis, A. K. Kwegyir-Afful,
S. Ramalingam, Z. Ates-Alagoz and V. C. O. Njar, J. Med.
Chem., 2013, 56, 4880–4898.
(17KJA220002), Top-notch Academic Programs Project of
Jiangsu Higher Education Institutions (PPZY2015C221) and
a Project Funded by the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions (PAPD). The
authors are grateful to the Advanced Analysis & Testing Center
of Nanjing Forestry University for the measurement of NMR
data.
2
1 A. Temirak, Y. M. Shaker, F. A. F. Ragab, M. M. Ali, H. I. Ali
and H. I. El Diwani, Eur. J. Med. Chem., 2014, 87, 868–880.
2 A. W. White, R. Almassy, A. H. Calvert, N. J. Curtin,
R. J. Griffin, Z. Hostomsky, K. Maegley, D. R. Newell,
S. Srinivasan and B. T. Golding, J. Med. Chem., 2000, 43,
2
Notes and references
4084–4097.
1
2
3
4
J. Seligmann and C. Twelves, Future Med. Chem., 2013, 5, 23 P. Singla, V. Luxami and K. Paul, Bioorg. Med. Chem., 2015,
39–352. 23, 1691–1700.
X. C. Huang, R. Z. Huang, L. X. Li, S. H. Gou and H. S. Wang, 24 W. Yan, X. Y. Wang, Y. Dai, B. Zhao, X. Y. Yang, J. Fan,
3
Eur. J. Med. Chem., 2017, 132, 11–25.
N. G. Vindya, N. Sharma, M. Yadav and K. R. Ethiraj, Curr.
Top. Med. Chem., 2015, 15, 73–82.
Y. L. Gao, F. W. Meng, Y. M. Wang, C. Luo, J. Ai,
M. Y. Geng and W. H. Duan, J. Med. Chem., 2016, 59,
6690–6708.
M. S. Poruchynsky, E. Komlodi-Pasztor, S. Trostel, 25 F. C. Torres, M. E. Garc ´ı a-Rubi n˜ o, C. Lozano-L ´o pez,
J. Wilkerson, M. Regairaz, Y. Pommier, X. Zhang,
T. K. Maity, R. Robey, M. Burotto, D. Sackett, U. Guha and
D. F. Kawano, V. L. Eier-Lima, G. L. von Poser and
J. M. Campos, Curr. Med. Chem., 2015, 22, 1312–1323.
A. T. Fojo, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 1571–1576. 26 R. J. Vasquez, B. Howell, A. M. Yvon, P. Wadsworth and
5
6
M. A. Jordan and L. Wilson, Nat. Rev. Cancer, 2004, 4, 253–
65.
A. E. Prota, K. Bargsten, D. Zurwerra, J. J. Field, J. F. D ´ı az,
L. Cassimeris, Mol. Biol. Cell, 1997, 8, 973–985.
27 W. Shi and D. W. Siemann, Anticancer Res., 2005, 25, 3899–
3904.
2
K.-H. Altmann and M. O. Steinmetz, Science, 2013, 339, 28 A. Rai, T. K. Gupta, S. Kini, A. Kunwar, A. Surolia and
87–590. D. Panda, Biochem. Pharmacol., 2013, 86, 378–391.
Y. Lu, J. Chen, J. Wang, C.-M. Li, S. Ahn, C. M. Barrett, 29 A. Kamal, T. S. Reddy, M. V. P. S. Vishnuvardhan,
5
7
J. T. Dalton, W. Li and D. D. Miller, J. Med. Chem., 2014,
7, 7355–7366.
V. D. Nimbarte, A. V. S. Rao, V. Srinivasulu and
N. Shankaraiah, Bioorg. Med. Chem., 2015, 23, 4608–4623.
5
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17511–17526 | 17525

View Article Online
RSC Advances
Paper
3
3
3
0 C. Alvarez, R. Alvarez, P. Corchete, C. Perez-Melero, R. Pelaez 41 R. Z. Huang, G. B. Liang, X. C. Huang, B. Zhang, M. M. Zhou,
and M. Medarde, Eur. J. Med. Chem., 2010, 45, 588–597.
1 D. J. Newman, G. M. Cragg and K. M. Snader, J. Nat. Prod.,
Z. X. Liao and H. S. Wang, Eur. J. Med. Chem., 2017, 138, 979–
992.
2
003, 66, 1022–1037.
42 W. Gu, T. T. Miao, D. W. Hua, X. Y. Jin, X. B. Tao, C. B. Huang
and S. F. Wang, Bioorg. Med. Chem. Lett., 2017, 27, 1296–
1300.
2 W. M. Zhang, T. Yang, X. Y. Pan, X. L. Liu, H. X. Lin,
Z. B. Gao, C. G. Yang and Y. M. Cui, Eur. J. Med. Chem.,
2
017, 127, 917–927.
43 W. Gu, S. Wang, X. Y. Jin, Y. L. Zhang, D. W. Hua, T. T. Miao,
X. B. Tao and S. F. Wang, Molecules, 2017, 22, 1154.
44 T. Fonseca, B. Gigante, M. M. Marques, T. L. Gilchrist and
E. De Clercq, Bioorg. Med. Chem., 2004, 12, 103–112.
3
3 M. W. Pertino, C. Vega, M. Rol ´o n, C. Coronel, A. R. de Arias
and G. Schmeda-Hirschmann, Molecules, 2017, 22, 369.
4 V. C. Roa-Linares, Y. M. Brand, L. S. Agudelo-Gomez,
3
V. Tangarife-Castano, L. A. Betancur-Galvis, J. C. Gallego- 45 S. D. Guggilapu, L. Guntuku, T. S. Reddy, A. Nagarsenkar,
Gomez and M. A. Gonzalez, Eur. J. Med. Chem., 2016, 108,
9–88.
D. K. Sigalapalli, V. G. M. Naidu, S. K. Bhargava and
N. B. Bathini, Eur. J. Med. Chem., 2017, 138, 83–95.
7
3
3
5 H. Wada, S. Kodato, M. Kawamori, T. Morikawa, H. Nakai, 46 J.-C. Martinou and R. J. Youle, Dev. Cell, 2011, 21, 92–101.
M. Takeda, S. Saito, Y. Onoda and H. Tamaki, Chem. 47 M. L. Circu and T. Y. Aw, Free Radicals Biol. Med., 2010, 48,
Pharm. Bull., 1985, 33, 1472–1487.
6 J. Kim, Y. G. Kang, J. S. Park, J. H. Lee, J. Y. Lee, J. M. Shin, 48 S. Orrenius, B. Zhivotovsky and P. Nicotera, Nat. Rev. Mol.
D. H. Choi, Y. U. Cho, W. G. Kim, D. B. Seo, T. R. Lee, Cell Biol., 2003, 4, 552–565.
Y. Miyamoto and K. T. No, Mol. Cell. Endocrinol., 2015, 49 R. B. Ravelli, B. Gigant, P. A. Curmi, I. Jourdain, S. Lachkar,
12, 216–225. A. Sobel and M. Knossow, Nature, 2004, 428, 198–202.
7 M. A. Esteves, N. Narender, M. J. Marcelo-Curto and 50 Schr ¨o dinger Suite 2015-1 Protein Preparation Wizard, Epik
749–762.
4
3
3
B. Gigante, J. Nat. Prod., 2001, 64, 761–766.
8 B. L. Fei, W. S. Xu, W. L. Gao, J. Zhang, Y. Zhao, J. Y. Long,
C. E. Anson and A. K. Powell, J. Photochem. Photobiol., B,
version 3.2, Schr ¨o dinger, LLC, New York, NY, 2015, Impact
version 6.7, Schr ¨o dinger, LLC, New York, NY, 2015, Prime
version 4.0, Schr ¨o dinger, LLC, New York, NY, 2015.
51 N. J. Halbrook and R. V. Lawrence, J. Org. Chem., 1966, 31,
4246–4247.
52 J. Yan, J. H. Hu, B. J. An, L. Huang and X. S. Li, Eur. J. Med.
Chem., 2017, 125, 663–675.
53 F. C. Bernstein, T. F. Koetzle, G. J. Williams, E. E. Meyer Jr,
M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi
and M. Tasumi, J. Mol. Biol., 1977, 11, 535–542.
2015, 142, 77–85.
3
9 X. C. Huang, R. Z. Huang, Z. X. Liao, Y. M. Pan, S. H. Gou and
H. S. Wang, Eur. J. Med. Chem., 2016, 108, 381–391.
0 D. J. Luo, Q. Ni, A. L. Ji, W. Gu, J. H. Wu and C. P. Jiang,
BioMed Res. Int., 2016, 2016, 2581061.
4
17526 | RSC Adv., 2018, 8, 17511–17526
This journal is © The Royal Society of Chemistry 2018