The convenient aqueous synthesis and biological evaluation of ortho-(3,4,5-trimethoxybenzoyl)-acetanilides as novel anti-cancer agents

Article abstract of DOI:10.1039/c4ra07998a

A series of new ortho-(3,4,5-trimethoxybenzoyl)-acetanilides were synthesised by the cross-coupling reaction catalyzed with Pd catalyst in aqueous medium, with polyethylene glycol as additive under very mild conditions. The evaluation of these compounds as tubulin polymerization inhibitors indicated that most of these compounds were potential anti-cancer agents. Among them, compound 13, 2-hydroxy-N-(5-methoxy-2-(3,4,5-trimethoxy benzoyl)phenyl) acetamide exhibited excellent antiproliferative activity against various human cancer cell lines (GI₅₀= 71 nM for human HeLa cell line), and good activity for inhibiting tubulin polymerization (IC₅₀= 2.94 μM). The mechanism study indicated that compound 13 could arrest cell-cycle progression at the mitosis phase, block the formation of cdc2/cyclin B1 complex, down-regulate the p-Cdc 25C and the anti-apoptotic proteins Bcl-2 and Bcl-XL, and up-regulate the pro-apoptotic proteins Bax and Bad. This journal is

Full text of DOI:10.1039/c4ra07998a

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PAPER
The convenient aqueous synthesis and biological
evaluation of ortho-(3,4,5-trimethoxybenzoyl)-
acetanilides as novel anti-cancer agents†
Cite this: RSC Adv., 2014, 4, 41510
Jianfei Sheng, Fei Mao, Jun Yan, Ling Huang* and Xingshu Li*
A series of new ortho-(3,4,5-trimethoxybenzoyl)-acetanilides were synthesised by the cross-coupling
reaction catalyzed with Pd catalyst in aqueous medium, with polyethylene glycol as additive under very
mild conditions. The evaluation of these compounds as tubulin polymerization inhibitors indicated that
most of these compounds were potential anti-cancer agents. Among them, compound 13, 2-hydroxy-
N-(5-methoxy-2-(3,4,5-trimethoxy benzoyl)phenyl) acetamide exhibited excellent antiproliferative
activity against various human cancer cell lines (GI50 ¼ 71 nM for human HeLa cell line), and good
activity for inhibiting tubulin polymerization (IC50 ¼ 2.94 mM). The mechanism study indicated that
compound 13 could arrest cell-cycle progression at the mitosis phase, block the formation of cdc2/
cyclin B1 complex, down-regulate the p-Cdc 25C and the anti-apoptotic proteins Bcl-2 and Bcl-XL, and
up-regulate the pro-apoptotic proteins Bax and Bad.
Received 2nd August 2014
Accepted 27th August 2014
DOI: 10.1039/c4ra07998a
www.rsc.org/advances
both academically and practical standpoints, it would be very
interested to develop a highly effective strategy for the prepa-
Introduction
Microtubules are cytoskeletal laments consisting of a, ration of the new effective antimitotic agents by simple, espe-
b-tubulin heterodimers and are essential in a wide range of cially one-step procedure.
cellular processes including cell shape maintenance, intracel-
In view of direct synthesis being able to minimize waste/side
lular transport, cell signaling, separation of duplicated chro- product formation, transition-metal-catalyzed direct C–H bond
mosomes during cell division, and are required for cell life functionalization/cross-coupling as a valuable tool for the
1–3
cycle. The crucial involvement of microtubules in mitosis modular and facile synthesis of structurally similar, yet diver-
4
,5
makes them an attractive target for anticancer drugs.
There are many chemically diverse compounds targeting the A lot of papers have been published on transition metals (e.g.,
sied organic molecules have attracted increasing attention.
6,7
tubulin-microtubule system. Among them, combretastatin Pd, Ir, Rh, Ru, Cu, Fe, etc.) facilitating the cross-coupling reac-
A-4 (CA-4, 1), a natural cis-stilbene derivative rst isolated from tion in recent years. Recently, we and other groups reported
the bark of the African willow tree Combretum caffrum in 1982,
is a typical example which strongly inhibits tubulin polymeri- between anilides and aromatic aldehydes in toluene.
14
8
the palladium-catalyzed cross-dehydrogenative coupling
15–17
zation (with about 1.9 mM of the IC50) by binding to the Subsequently, Nov ´a k described that the reaction could be per-
colchicine binding site, and then, the disruption of tubulin formed mildly in aqueous with the addition of detergent,
1
8,19
assembly results in mitotic arrest, and subsequent cancer cell accompanying the reaction rate acceleration.
death. However, the cis double bond in CA-4 is prone to
9
Although a lot of progress have been made in transition-
isomerize to the more thermodynamic stable, but less bioactive metal-catalyzed carbon–carbon coupling reactions, there were
trans isomers (with the IC50 above 50 mM against tubulin poly- very limited reports on the application of this methodology to
10
merization). For overcoming this disadvantage, numerous the preparation of complex molecules and useful medical
modications to stabilize the conformation by replacement of compounds. Herein, we disclose the synthesis of a series of new
the olenic bridge with a ring or carbonyl have been developed ortho-(3,4,5-trimethoxybenzoyl)-acetanilides catalyzed with Pd
11–13
in recent years.
Generally, several reaction steps were catalyst in aqueous with polyethyleneglycol as additive, espe-
needed for the synthesis of these compounds, therefore, from cially the evaluation of them as tubulin polymerization inhibi-
tors, and the study of their anticancer mechanism of these
compounds.
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006,
The design strategy of these target compounds were showed
in Fig. 1. As 3,4,5-trimethoxy groups on the A-ring and the cis-
orientation of the two aryl rings on olenic bond were respon-
sible for efficient binding to tubulin,
China. E-mail: huangl72@mail.sysu.edu.cn; lixsh@mail.sysu.edu.cn; Fax: +86-20-
3
994-3051; +86-20-3994-3050; Tel: +86-20-3994-3051; +86-20-3994-3050
†
Electronic supplementary information (ESI) available: NMR spectrums of target
20,21
we focused on the
compounds. See DOI: 10.1039/c4ra07998a
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also attempted to reduce the amount of Pd(OAc)
2
, however, low
yields were obtained (Table 1, entry 8–9).
With this optimal reaction conditions, compounds 1–12
were prepared by the reaction of substituted anilides and 3,4,5-
trimethoxybenzaldehyde (Scheme 1).
Compounds 13–17 were prepared with 12 as the interme-
diate as the route of Scheme 2. The reaction of 12 with CH
3
OH
2
8
in presence of CH ONa gave compound 13 in good yield.
3
Fig. 1 Structures of CA-4 and the ortho-acylacetanilide analogues.
Similarly, 12 reacted with methylamine, ethylamine and N,N-
dimethylamine in the presence of potassium carbohydrate to
provide compounds 14, 15 and 16 in 86–90% yields, respec-
modication of B-ring and the bridging double bond, using the
simple coupling reaction of commercially available 3,4,5-tri-
methoxybenzaldehyde and different anilides to obtaine these
bioactive ortho-(3,4,5-trimethoxybenzoyl)-acetanilides.
29
tively. Finally, compound 17 was obtained by the reaction of 16
30
3 2 2
with CH I in CH Cl .
In vitro cell growth inhibition
To evaluate the antiproliferative activities of all the synthesized
compounds, human cancer cell lines HeLa (human epithelial
cervical cancer cell line), A549 (non-small cell lung carcinoma),
MCF-7 (human breast cancer cell line), and HCT-8 (human
Results and discussion
Chemistry
The study of one-step synthesis of the new ortho-acylacetani- colon cancer cell line) were tested using MTT (3-(4,5-dime-
lides 1–12 is shown in Table 1 and Scheme 1. Inspired by thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with
Nov ´a k's method with the addition of detergent to accelerate the CA-4 as the reference compound.
coupling reaction, and considering polyethylene glycol (PEG),
The results shown in Table 2 demonstrated that most of the
which is soluble in both water and organic solvents, has been synthesized compounds displayed good anti-proliferative
successfully used as reaction medium and additive in a variety activity with GI50 values in micromol to sub-micromol range.
22–27
of organic reactions,
methoxyacetanilide and 3,4,5-trimethoxybenzaldehyde as the with 71 nM of GI50 value for HeLa cell line; 1, GI50 ¼ 113 nM for
prototypical substrates, 5% Pd(OAc) as the catalyst and poly- HeLa). A simple structure–activity relationship analysis showed
we studied the reaction with 3- Among them, compounds 13 and 1 gave the best results (13,
2
ethylene glycol as the additive. A screening of optimal condi- that the anti-proliferative activity closely related to the
tions indicated that the use of PEG was rather signicant (Table substituted groups in B ring, and especially, the methoxy group
1, entries 1–4: 5% mol of PEG-2000, 63% yield; 10% mol of PEG, in R
1 3
(C-3) position was necessary. Compounds 2–7, with H, CH
71% yield; 20% mol of PEG, 91% yield; 50% mol of PEG, trace and Cl in this position exhibited relatively poor activities with
product). The temperature effect was also important for the GI50 values from 0.90 to 36.8 mM respectively for the four cell
ꢀ
ꢀ
reaction; it could proceed smoothly at 40 C or 60 C, but only lines. On the other hand, compounds 9, 10, 11, 13, 14, and 15,
gave 38% yield at room temperature (Table 1, entries 3, 6–7). We which possess OCH
3
in R
1
position, showed moderate to very
a
Table 1 Optimization of reaction conditions
ꢀ
b
Entry
Pd(OAc)
5%
5%
5%
5%
5%
5%
5%
1.5%
3%
2
(equiv.)
PEG-2000 (equiv.)
5%
H
1
1
1
1
0
1
1
1
1
2
O (mL)
T ( C)
40
40
40
40
40
rt
60
40
TFA (equiv.)
26%
26%
26%
26%
26%
26%
26%
26%
TBHP (equiv.)
Yield (%)
63
71
1
2
3
4
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
10%
20%
50%
100%
20%
20%
20%
20%
91
Trace
Trace
38
c
5
6
7
8
9
89
Trace
47
40
26%
a
2
Reactions was carried out using amide (1.0 mmol), 3,4,5-trimethoxybenzaldehyde (1.0 mmol), Pd(OAc) , PEG-2000, tert-butylhydroperoxide TBHP
c
b
(4.0 mmol), triuoroacetic acid TFA (0.26 mmol, 26% mol), water (1.0 mL) under air for 12 h. Isolated yields. PEG-2000 was also as solvent.
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Scheme 1 Reaction conditions: corresponding amide (1.0 mmol), 3,4,5-trimethoxybenzaldehyde (1.0 mmol), Pd(OAc)
2
(0.05 mmol, 5 mol%),
PEG-2000 (0.2 mmol, 20 mol%), tert-butylhydroperoxide TBHP (4.0 mmol), trifluoroacetic acid TFA (0.26 mmol, 26 mol%), water (1.0 mL) at
ꢀ
4
0 C under air for 12 h. Isolated yields (1–12) were reported (reaction times for each substrate were not optimized).
good activities (e.g., with 692 nM to 71 nM of GI50 values for compounds 1, 8, 13, and 15, which showed relatively better
HeLa, and 750 nM to 71 nM of GI50 values for A549). When activity, were choosed to study for their ability to block the
moving the methoxy substitution from C-3 to the C-4 position of assembly of tubulin (Table 3) by the method originally
31,32
the B ring (compound 1 vs. 2), almost 10-fold decrease in anti- described by Bonne, D. et al. with moderate modication,
proliferative activity was observed (from 113 nM to 1420 nM). and CA-4 was examined as reference compound. We rstly use
Using the naphthalene ring to replace the benzene ring 10 mM of the tested compounds for the assays. At this concen-
(
2
compound 3 vs. 4), the antiproliferative activity (GI50 from tration, compound 13 was found to inhibit tubulin polymeri-
0.1 mM to 4.88 mM) was signicantly increased. zation the most (97.8%), while compounds 1, 8, 15 gave 59.3%,
2.9%, and 31.9% inhibitions, respectively. Then, the complete
3
dose-response curves were obtained by assaying these
compounds with inhibition rates and the results were
Inhibition of tubulin polymerization
To investigate whether the antiproliferative activities of these summarized in Table 3. The representative raw data for the
compounds were related to their interaction with tubulin, polymerization assay of compound 13 is shown in Fig. 2. It can
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ꢀ
ꢀ
ꢀ
Scheme 2 Conditions: (a) CH
3
ONa, CH
3
OH, reflux; (b) NH
2
CH
3
, K
2
CO
3
, 50 C; (c) NH
2
CH
2
CH
3
, K
2
CO
3
, 50 C; (d) NH(CH
3
)
2
, K
2
CO
3
, 50 C; (e)
3
CH I, rt.
be seen that compounds 1 and 13 were effective tubulin poly- morphological alterations such as shrinkage, membrane bleb-
merization inhibitors with IC50 values of 7.99 and 2.94 mM, bing and chromatin condensation that are normally associated
respectively. Compounds, 8 and 15, which had relatively poor with the occurrence of apoptotic cell death.
anti-proliferative activities, exhibited weaker ability to inhibit
tubulin polymerization (with IC50 values more than 10 mM) than
that of 1 and 13, which suggest that the anti-proliferative activity
Analysis of cell cycle arrest
It is well-known that CA-4 blocks the cell cycle in the G /M phase
2
of these compounds was correlated with their tubulin poly-
due to microtubule depolymerization and cytoskeleton disrup-
merization inhibition. Moreover, the chemical stability of the
tion. The cell growth inhibitory potency of compound 13
target compound 13 and CA-4 were evaluated by HPLC (see
prompted us to evaluate its effects on the cell cycle (Fig. 3). Aer
ESI†). The results show that there is no degradation of
HeLa cells were treated with DMSO (0.01%) or compound 13 at
different concentrations (0.01, 0.05, 0.1, and 0.5 mM) for 24 h
and 48 h, the analysis (Fig. 4) of harvested cells by ow cytom-
etry indicated that 13 resulted in signicant cell-cycle arrest at
compound 13 up to 5 day at room temperature. While three
impurities (should be the isomerize or decomposed products)
of CA-4 in acetonitrile appeared in 24 h, which is the same as
33
those reported in literature. These results indicated that
replacement of the bridging double bond of CA-4 with carbonyl
group could enhance the congurational stability.
G /M phase, showing a time and dose-dependent manner. The
2
lowest concentration of 13 induced no signicant distribution
of HeLa cells through the cell cycle (22.0% of cells at G /M phase
2
for 24 h, and 23.0% for 48 h). Whereas, when the concentration
of 13 was increased to 0.05, 0.1, and 0.5 mM for 24 h, the
The morphological alterations of HeLa cells caused by 13
2
Base on the assay of antiproliferative activity and tubulin poly- percentage of cells at G /M phase correspondingly increased to
merization inhibition, we choose compound 13 for further 39.51%, 52.75%, and 56.06%, respectively (Fig. 4A), and the
biological study. Optical microscope was applied to identify the percentage of cells at G /M phase were 58.70%, 60.07%, and
2
morphological alterations of HeLa cells aer treated with 13. As 63.86% (Fig. 4B) aer treatment of compound 13 at the same
shown in Fig. 3, when exposed to serial concentrations of 13 concentration for 48 h. Futhermore, when the cells were
(0.01, 0.05, 0.1, and 0.5 mM) for 24 h and 48 h, the cells exhibited exposed to the high concentration, the obvious typical
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a
Table 2 Antiproliferative activity of compounds 1–11, 13–17 and CA-4 against four human cancer cell lines
b
GI50 (mM)
Compd
R
1
R
2
R
3
HeLa
A549
MCF-7
HCT-8
1
2
3
4
5
6
7
8
9
OCH
H
—
3
H
OCH
—
H
H
CH
H
H
H
H
H
H
H
H
H
H
—
CH
CH
—
3
0.113 ꢁ 0.11
1.40 ꢁ 0.014
4.88 ꢁ 0.31
20.1 ꢁ 2.15
23.5 ꢁ 1.16
2.15 ꢁ 0.15
36.8 ꢁ 4.13
0.54 ꢁ 0.03
0.32 ꢁ 0.01
0.23 ꢁ 0.02
0.38 ꢁ 0.05
0.071 ꢁ 0.013
0.692 ꢁ 0.158
0.47 ꢁ 0.11
2.1 ꢁ 0.006
>10
0.18 ꢁ 0.01
1.29 ꢁ 0.19
2.65 ꢁ 0.03
10.4 ꢁ 0.16
13.4 ꢁ 0.62
2.00 ꢁ 0.35
7.60 ꢁ 1.93
0.50 ꢁ 0.035
0.36 ꢁ 0.064
0.50 ꢁ 0.03
0.50 ꢁ 0.102
0.071 ꢁ 0.004
0.613 ꢁ 0.059
0.590 ꢁ 0.004
0.75 ꢁ 0.03
21.4 ꢁ 3.39
0.018 ꢁ 0.05
0.88 ꢁ 0.02
0.90 ꢁ 0.03
1.71 ꢁ 0.02
14.2 ꢁ 1.05
16.1 ꢁ 3.17
3.56 ꢁ 0.22
12.5 ꢁ 1.2
0.34 ꢁ 0.06
1.59 ꢁ 0.02
0.676 ꢁ 0.05
16.8 ꢁ 1.10
20.5 ꢁ 0.56
2.59 ꢁ 0.69
21.5 ꢁ 1.57
0.89 ꢁ 0.05
1.68 ꢁ 0.86
0.84 ꢁ 0.06
1.23 ꢁ 0.75
0.12 ꢁ 0.02
0.78 ꢁ 0.01
0.79 ꢁ 0.01
1.56 ꢁ 0.39
35.6 ꢁ 0.45
0.025 ꢁ 0.02
3
3
H
CH
CH
CH
CH
Ph
3
3
3
3
CH
H
3
3
Cl
H
4.34 ꢁ 1.28
2.34 ꢁ 0.03
2.60 ꢁ 0.4
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
—
3
3
3
3
3
3
3
3
CH
2
CH
3
10
11
13
14
15
16
17
n-Propyl
Isopropyl
0.91 ꢁ 0.01
0.23 ꢁ 0.01
0.55 ꢁ 0.13
0.79 ꢁ 0.02
1.05 ꢁ 0.05
31.5 ꢁ 2.59
0.009 ꢁ 0.01
CH
CH
CH
CH
CH
—
2
2
2
2
2
OH
NHCH
NCH CH
2 3
N(CH
N(CH
3
3
)
)
2
3
3
CA-4
0.0028 ꢁ 0.06
a
Cell lines were treated with different concentrations of the compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental section. GI50 values are indicated as the mean ꢁ SD (standard error) of three independent experiments.
Table 3 Effects of the selected compounds on tublin polymerization
a
inhibition
Tyr15 and phosphorylation of cdc25c phosphatase on Ser216
negatively regulate the activation of the cdc2/cyclin B1 complex.
Thus, dephosphorylation of these proteins is necessary to
activate the cdc2/cyclin B1 complex, and is mediated by the
phosphatase cdc25c, which dephosphorylates cdc2 and auto-
b
Compd
IC50 (mM)
1
8
7.99 ꢁ 0.05
>10
1
1
CA-4
3
5
2.94 ꢁ 0.02 dephosphorylates itself. Phosphorylation of cdc25c directly
>10
stimulates both its phosphatase and autophosphatase activi-
1.2 ꢁ 0.2
ties, a condition required to trigger cdc2/cyclin B1 induction of
a
b
Inhibition of tubulin polymerization. Tublin was at 20 mM. IC50
values are indicated as the mean ꢁ SD (standard error) of three
independent experiments.
apoptosis sub-peak was observed in DNA histogram. These
results revealed that compound 13 could arrest cell-cycle
progression at the mitotic phase, which is in agreement with the
reported properties for most of the antimitotic agents.
Effect of compound 13 on cell-cycle-related-protein expression
2
In view of the signicant ability of compound 13 to induce a G /
M arrest, we further studied the correlation between 13-induced
G /M arrest and alterations of proteins expression that regulate
2
cell division. The formation of a cdc2/cyclin B1 complex is an
important event for cell mitosis. Phosphorylation of cdc2 on Fig. 2 Effects of compound 13 on mircrotubule dynamics.
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Fig. 3 The micrographs of HeLa cells exposed to compound 13 (0.01, 0.05, 0.1, and 0.5 mM) or 0.01% DMSO (control) for 24 h (A) and 48 h (B),
magnification ꢃ200).
(
Fig. 4 Effect of compound 13 on cell-cycle arrest of HeLa cells. Cells were treated with compound 13 (0.01, 0.05, 0.1, and 0.5 mM) or DMSO
0.01%) for 24 h (A) and 48 h (B). The DNA contents of each cell phase were analyzed by flow cytometry. The experiments were performed for
three times, and the results of representative experiments are shown.
(
34,35
entry of cells into mitosis.
Thus, the protein level of cyclin B1 expose on their surface the phospholipid phosphatidylserine.
and the status of both p-cdc2 and cdc25c were examined by Thus, this analysis is able to distinguish between live cells
ꢂ
ꢂ
+
ꢂ
Western blot analysis. As shown in Fig. 5, treatment of HeLa (annexin-V /PI ), early apoptotic cells (annexin-V /PI ), late
+
+
ꢂ
cells with 13 at the lowest concentration (0.01 mM) caused no apoptotic cells (annexin-V /PI ), and necrotic cells (annexin-V /
signicantly variations in cyclin B1, p-Cdc2, and Cdc25C. PI ). As shown in Fig. 6A, when HeLa cells were treated with 13
+
However, with the concentration increasing to 0.05, 0.1, and 0.5 at different concentrations (0.01, 0.05, 0.1, and 0.5 mM) or
mM, a dramatic decrease in the cyclin B1 expression, the phos- DMSO (0.01%) for 24 h, comparing with the control cells, an
phorylated form of Cdc2 (p-Cdc2) and Cdc25C, was observed. accumulation of annexin-V positive cells was observed, which
These results, which were perfectly consistent with the cell cycle meaning the apoptotic cells were increased. And this tendency
arrest analysis, further illustrated the mechanism of the cell- was also obvious aer the cells treated with 13 for 48 h. These
cycle arrest effect of compound 13.
data showed 13 induce cell apoptosis in a concentration-and
time-dependent manner.
Apoptosis assay
Effect of 13 on apoptosis-related-protein expression
To evaluate the mode of cell death induced by 13, a ow
cytometry was performed using propidium iodide (PI), which It is well-known that tubulin polymerization inhibitors affect
only stains DNA and enters only dead cells, and the protein signaling pathways that involve the regulation of the Bcl-2
3
6
annexin-V, which selectively binds to the apoptotic cells that family of proteins. Several pro-apoptotic family proteins
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Fig. 7 Effects of 13 on apoptosis-related proteins. HeLa cells were
treated with 13 for 24 h at the concentrations of 0.01, 0.05, 0.1, and 0.5
Fig. 5 Effects of 13 on G
2
/M regulatory proteins. HeLa cells were mM. The cells were harvested and lysed for the detection of Bad, Bax,
treated with 13 for 24 h at the concentrations of 0.01, 0.05, 0.1, and Bcl-xl, Bcl-2 expression by Western blot analysis, and b-actin was used
.5 mM. The cells were harvested and lysed for the detection of as a control. The experiments were performed for three times, and the
0
cyclin B1, p-Cdc2, Cdc25C expression by Western blot analysis, and results of representative experiments are shown.
b-actin was used as a control. The experiments were performed for
three times, and the results of representative experiments are
shown.
Conclusion
In conclusion, we have developed a new series of ortho-(3,4,5-
trimethoxybenzoyl)-acetanilides by the simple cross-coupling
reaction in aqueous solution with polyethylene glycol as addi-
tive at a very mild conditions. The evaluation of these
compounds as anti-cancer agents exhibited excellent anti-
proliferative activity against various human cancer cell lines
and good activity for inhibiting tubulin polymerization. The
study of the relation of the structure and activity indicated that
the methoxyl group on C-3 position of B ring is crucial for the
activity, and at the same time the acetyl group was helpful to
improve their activity. It is worth noting that compound 13,
which bears a hydroxyl in acetyl group, showed the best anti-
proliferative activity (GI50 ¼ 71 nM, for human HeLa cell line)
and inhibited tubulin polymerization with IC50 value of
(
e.g., Bax, Bid, Bim, and Bad) promote the release of cyto-
chrome C and further induce cell apoptosis, whereas anti-
apoptotic members (Bcl-2, Bcl-XL) are capable of antago-
nizing the pro-apoptotic proteins and preventing the cells
3
7–39
from apoptosis.
To evaluate the effects of 13 on apoptosis-
related proteins, HeLa cells were treated with 13 at different
concertrations (0.01, 0.05, 0.1, and 0.5 mM) for 24 h. As shown
in Fig. 7, the expression of the pro-apoptotic protein Bax and
Bad was strongly up-regulated aer the treatment, whereas
the expression of the anti-apoptotic protein Bcl-2 and Bcl-XL
was obviously down-regulated. These results illustrated
that the anti-proliferative activity of 13 was due to the regu-
lation of apoptosis-related protein and eventually led to cell
death.
2.94 mM. The mechanism study indicated that compound 13
Fig. 6 Effect of compound 13 on HeLa cell apoptosis. Cells were treated with compound 13 (0.01, 0.05, 0.1, and 0.5 mM) or DMSO (0.01%) for
4 h (A) and 48 h (B). The percentages of cells in each stage of cell apoptosis were quantitated by flow cytometry. (upper left quadrant) necrotic
2
cells; (upper right quadrant) late apoptotic cells; (bottom left quadrant) live cells; and (bottom right quadrant) early apoptotic cells. The
experiments were performed three times, and representative experiments are shown.
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+
could arrest cell-cycle progression at the mitosis phase, block (ESI) (M + H) 360.1440 C19
6
H22NO , requires 360.1442. Purity:
the formation of cdc2/cyclin B1 complex and down-regulated 98.3% (by HPLC).
the p-Cdc 25C. The western blot analysis revealed the pro-
apoptotic protein Bax and Bad was strongly up-regulated and White solid; 71% yield; mp: 144.4–145.6 C; H NMR (400 MHz,
the anti-apoptotic protein Bcl-2 and Bcl-XL was obviously down- CDCl
) d 9.98 (s, 1H), 8.39 (d, J ¼ 9.1 Hz, 1H), 7.13 (dd, J ¼ 9.1, 2.9
N-(4-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)acetamide (2).
ꢀ
1
3
regulated. The further study of compound 13 as anti-cancer Hz, 1H), 7.07 (d, J ¼ 2.9 Hz, 1H), 7.01 (s, 2H), 3.95 (s, 3H), 3.88 (s,
1
3
agent in vitro and in vivo is in progress.
6H), 3.77 (s, 3H), 2.17 (s, 3H). C NMR (101 MHz, CDCl
3
) d 197.48,
68.72, 154.33, 152.86, 142.44, 132.93, 132.78, 125.99, 123.95,
19.02, 117.31, 107.79, 60.96, 56.32, 55.67, 24.81. HRMS found
1
1
(
Experimental
Materials and methods
+
ESI) (M + H) 360.1442 C H NO , requires 360.1442. Purity:
1
9
22
6
98.1% (by HPLC).
All reagents used in the synthesis were obtained commercially
N-(2-(3,4,5-Trimethoxybenzoyl)naphthalen-1-yl)acetamide (3).
and used without further purication, unless otherwise speci- White solid; 53% yield; mp: 209.7–211.1 C; H NMR (400 MHz,
ꢀ
1
1
13

ed. The H NMR and C NMR spectra were recorded using CDCl
3
) d 8.79 (s, 1H), 7.98 (d, J ¼ 7.4 Hz, 1H), 7.86 (d, J ¼ 6.0 Hz,
TMS as the internal standard on a Bruker BioSpin GmbH spec- 1H), 7.72 (d, J ¼ 8.4 Hz, 1H), 7.59 (d, J ¼ 3.5 Hz, 2H), 7.44 (d, J ¼
1
3
trometer at 400 and 101 MHz, respectively, and the coupling 8.5 Hz, 1H), 7.13 (s, 2H), 3.95 (s, 3H), 3.85 (s, 6H), 2.23 (s, 3H).
C
constants are reported in hertz. The reactions were monitored by NMR (101 MHz, CDCl ) d 197.14, 169.68, 152.86, 142.73, 134.78,
3
thinlayer chromatography (TLC) on glass-packed precoated 132.44, 132.11, 130.84, 129.00, 128.00, 127.63, 126.74, 125.55,
silica gel plates and visualized in an iodine chamber or with a UV 125.21, 124.51, 108.06, 60.91, 56.25, 23.64. HRMS found (ESI)
+
lamp. Flash column chromatography was performed using silica (M + H) 380.1495 C22
H22NO , requires 380.1492. Purity: 99.3%
5
gel (200ꢂ300 mesh) purchased from Qingdao Haiyang Chemical (by HPLC).
Co. Ltd. The high-resolution mass spectra were obtained using a
N-(2-(3,4,5-Trimethoxybenzoyl)phenyl)acetamide (4). White
Shimadzu LCMS-ITTOF mass spectrometer. The term “dried” solid; 66% yield; mp: 171.5–172.8 C; H NMR (400 MHz, CDCl )
3
ꢀ
1
refers to the use of anhydrous sodium sulfate. The purity ($95%) d 10.49 (s, 1H), 8.56 (d, J ¼ 8.7 Hz, 1H), 7.57 (t, J ¼ 6.5 Hz, 2H),
of the samples was determined by high-performance liquid 7.11 (t, J ¼ 7.6 Hz, 1H), 6.97 (s, 2H), 3.95 (s, 3H), 3.87 (s, 6H), 2.22
1
3
chromatography (HPLC), conducted on a Shimadzu LC-20AT (s, 3H). C NMR (101 MHz, CDCl ) d 198.31, 169.03, 152.86,
3
series system, a TC-C18 column (4.6 mm ꢃ 250 mm, 5 mm), 142.28, 139.98, 133.91, 133.39, 132.82, 124.01, 122.08, 121.92,
+
eluted with a 40 : 60 acetonitrile–1% Ammonium acetate, at a 107.75, 60.96, 56.31, 25.10. HRMS found (ESI) (M + H) 330.1339
ꢂ1
ow rate of 1 mL min
.
C
18
H20NO
5
, requires 330.1336. Purity: 99.5% (by HPLC).
N-(5-Methyl-2-(3,4,5-trimethoxybenzoyl)phenyl)acetamide (5).
ꢀ
1
4
0–42
White solid; 73% yield; mp: 148.5–150.2 C; H NMR (400 MHz,
CDCl
Preparation of substituted acetanilide substrates
3
) d 10.71 (s, 1H), 8.44 (s, 1H), 7.49 (d, J ¼ 7.9 Hz, 1H), 6.96 (s,
All the substituted anilides were prepared from their corre-
sponding precursors with their corresponding acyl chlorides
2
2
1
1
H), 6.92 (d, J ¼ 7.7 Hz, 1H), 3.95 (s, 3H), 3.79 (d, J ¼ 73.4 Hz, 6H),
1
3
.45 (s, 3H), 2.23 (s, 3H). C NMR (101 MHz, CDCl ) d 198.27,
3
in CH
2
Cl
2
according to the literature method without
69.08, 152.82, 145.42, 141.98, 140.32, 133.79, 133.22, 122.92,
22.06, 121.16, 107.57, 60.94, 56.28, 25.20, 22.07. HRMS found
modications.
+
(ESI) (M + H) 344.1494 C19
H
22NO
5
, requires 344.1492. Purity:
1
5,18,19
General procedures for the coupling reactions
98.7% (by HPLC).
Substituted anilide (1.0 mmol), 3,4,5-trimethoxybenzaldehyde
N-(4-Methyl-2-(3,4,5-trimethoxybenzoyl)phenyl)acetamide (6).
ꢀ
1
(
1.0 mmol), Pd(OAc)
2
(12 mg, 0.05 mmol) and PEG-2000 White solid; 67% yield; mp: 128.7–131.0 C; H NMR (400 MHz,
) d 10.34 (s, 1H), 8.44 (d, J ¼ 8.3 Hz, 1H), 7.40 (d, J ¼ 10.6 Hz,
(400 mg, 0.2 mmol) were loaded into a Schlenk tube, and then CDCl
3
water (1.0 mL) was added. Aer the mixture was stirred for 2 2H), 7.00 (s, 2H), 3.98 (s, 3H), 3.90 (s, 6H), 2.34 (s, 3H), 2.21 (s,
1
3
minutes, TBHP (4.0 mmol) and triuoroacetic acid (0.26 mmol) 3H). C NMR (101 MHz, CDCl
were added at the room temperature. The mixture was stirred at 137.46, 134.53, 133.45, 132.94, 131.76, 124.22, 122.02, 107.76,
) d 198.30, 168.93, 152.86, 142.25,
3
ꢀ
+
40
C for 12 hours and then cooled to room temperature. 60.99, 56.34, 25.05, 20.71. HRMS found (ESI) (M + H) 344.1490
Saturated solution of K CO was added to the mixture and , requires 344.1492. Purity: 96.1% (by HPLC).
N-(5-Chloro-2-(3,4,5-trimethoxybenzoyl)phenyl)acetamide (7).
2
3
19 5
C H22NO
diluted with ethyl acetate. The organic layer was separated,
washed with brine and concentrated under reduced pressure. White solid; 48% yield; mp: 158.6–160.1 C; H NMR (400 MHz,
The crude products were puried by ash column chromatog- CDCl
) d 10.66 (s, 1H), 8.71 (s, 1H), 7.54 (d, J ¼ 8.4 Hz, 1H), 7.09
raphy on silica gel (200–300 mesh) to afford the desired product. (d, J ¼ 8.4 Hz, 1H), 6.94 (s, 2H), 3.96 (s, 3H), 3.89 (s, 6H), 2.24 (s,
ꢀ
1
3
1
3
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)acetamide (1). 3H). C NMR (101 MHz, CDCl ) d 197.68, 169.13, 152.97, 142.43,
3
ꢀ
1
White solid; 91% yield; mp: 156.7–157.9 C; H NMR (400 MHz, 141.27, 140.45, 133.99, 133.20, 122.22, 121.70, 121.62, 107.59,
+
CDCl ) d 11.30 (s, 1H), 8.35 (d, J ¼ 2.4 Hz, 1H), 7.56 (d, J ¼ 8.9 Hz, 61.01, 56.35, 25.19. HRMS found (ESI) (M + H) 364.0946
3
1
3
1
1
H), 6.89 (s, 2H), 6.59 (d, J ¼ 8.9 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H),
18 5
C H19ClNO , requires 364.0946. Purity: 99.8% (by HPLC).
13
.87 (s, 6H), 2.24 (s, 3H). C NMR (101 MHz, CDCl ) d 197.82,
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)benzamide
3
ꢀ
1
69.41, 164.45, 152.83, 143.47, 141.55, 135.65, 134.40, 115.82, (8). White solid; 86% yield; mp: 156.4–158.2 C; H NMR (400
08.81, 107.22, 105.12, 60.90, 56.28, 55.60, 25.37. HRMS found MHz, CDCl
3
) d 12.43 (s, 1H), 8.62 (d, J ¼ 2.6 Hz, 1H), 8.17–8.03
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 41510–41520 | 41517

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Paper
+
(
m, 2H), 7.64 (d, J ¼ 8.9 Hz, 1H), 7.60–7.49 (m, 3H), 6.92 (s, 2H), 55.66. HRMS found (ESI) (M + H) 376.1393 C19
7
H22NO ,
6
6
1
1
.64 (dd, J ¼ 8.9, 2.6 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.89 (s, requires 376.1391. Purity: 99.8% (by HPLC).
1
3
H). C NMR (101 MHz, CDCl ) d 198.48, 166.19, 164.77,
3
General procedure for the synthesis of 14–16 (ref. 29)
52.89, 144.12, 141.42, 136.13, 134.63, 134.53, 132.13, 128.87,
27.44, 115.98, 109.38, 107.09, 104.91, 60.98, 56.32, 55.74. To a stirred solution of 2-bromo-N-(5-methoxy-2-(3,4,5-tri-
+
HRMS found (ESI) (M + H) 422.1600 C24
22.1598. Purity: 97.4% (by HPLC).
H
24NO
6
, requires methoxybenzoyl)phenyl) acetamide (12, 50 mg) in acetone,
K CO (30 mg) and the corresponding amine were added. Aer
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)propionamide stirred at 50 C for 5 h, the mixture was extracted with ethyl
4
2
3
ꢀ
ꢀ
1
(
9). White solid; 81% yield; mp: 141.7–143.1 C; H NMR (400 acetate (3 ꢃ 10 mL), washed with brine (twice), dried over
MHz, CDCl ) d 11.37 (s, 1H), 8.41 (d, J ¼ 2.6 Hz, 1H), 7.57 (d, J ¼ 8.9 Na SO and concentrated in vacue to give the crude product
3
2
4
Hz, 1H), 6.89 (s, 2H), 6.59 (dd, J ¼ 8.9, 2.6 Hz, 1H), 3.92 (d, J ¼ 7.0 which was puried by column chromatography (petroleum
Hz, 6H), 3.87 (s, 6H), 2.50 (q, J ¼ 7.6 Hz, 2H), 1.29 (t, J ¼ 7.6 Hz, ether–ethyl acetate, 5 : 1) to give a yellow solid.
13
3
1
6
3
H). C NMR (101 MHz, CDCl
3
) d 197.93, 173.35, 164.52, 152.83,
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)-2-(methyl-
43.69, 141.41, 135.74, 134.52, 115.72, 109.00, 107.12, 104.85, amino)acetamide (14). Yellow solid; 90% yield; mp:
+
ꢀ
1
0.94, 56.27, 55.62, 31.59, 9.45. HRMS found (ESI) (M + H)
74.1599 C20 requires 374.1598. Purity: 99.9% (by HPLC).
123.6–125.4 C; H NMR (400 MHz, CDCl ) d 12.04 (s, 1H), 8.42
3
H
24NO
6
(d, J ¼ 2.5 Hz, 1H), 7.55 (d, J ¼ 8.8 Hz, 1H), 6.93 (s, 2H), 6.61 (dd,
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl) butyramide J ¼ 8.8, 2.6 Hz, 1H), 3.92 (d, J ¼ 3.9 Hz, 6H), 3.87 (s, 6H), 3.43 (s,
ꢀ
1
13
(
10). White solid; 76% yield; mp: 90.7–92.1 C; H NMR 2H), 2.55 (s, 3H). C NMR (101 MHz, CDCl ) d 196.95, 171.76,
3
(
400 MHz, CDCl ) d 11.37 (s, 1H), 8.41 (d, J ¼ 2.6 Hz, 1H), 7.57 (d, 164.09, 152.77, 142.40, 141.48, 135.31, 134.48, 117.04, 108.91,
3
J ¼ 8.9 Hz, 1H), 6.89 (s, 2H), 6.59 (dd, J ¼ 8.9, 2.6 Hz, 1H), 3.92 107.34, 105.45, 60.91, 56.27, 55.84, 55.59, 36.84. HRMS found
+
(
(
1
1
1
3
d, J ¼ 7.3 Hz, 6H), 3.88 (s, 6H), 2.44 (t, J ¼ 7.5 Hz, 2H), 1.88–1.74 (ESI) (M + H) 389.1710 C H N O , requires 389.1707. Purity:
2
0
25 2 6
13
m, 2H), 1.03 (t, J ¼ 7.4 Hz, 3H). C NMR (101 MHz, CDCl
3
) d 99.5% (by HPLC).
97.95, 172.63, 164.55, 152.85, 143.70, 141.48, 135.76, 134.52, 2-(Ethylamino)-N-(5-methoxy-2-(3,4,5-trimethoxybenzoyl)
15.70, 109.01, 107.18, 104.86, 60.95, 56.29, 55.64, 40.56, 18.89, phenyl)acetamide (15). Yellow solid; 86% yield; mp: 99.1–100.3
+
ꢀ
1
3.73. HRMS found (ESI) (M + H) 388.1754 C21
H
26NO
6
, requires
C; H NMR (400 MHz, CDCl
3
) d 12.04 (s, 1H), 8.41 (d, J ¼ 2.5 Hz,
88.1755. Purity: 99.8% (by HPLC).
N-(5-Methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl)isobutyramide Hz, 1H), 3.92 (d, J ¼ 5.9 Hz, 6H), 3.87 (s, 6H), 3.46 (s, 2H), 2.75
1H), 7.53 (d, J ¼ 8.8 Hz, 1H), 6.93 (s, 2H), 6.61 (dd, J ¼ 8.8, 2.6
ꢀ
1
13
(
11). White solid; 69% yield; mp: 117.1–118.8 C; H NMR (400 (q, J ¼ 7.1 Hz, 2H), 1.20 (t, J ¼ 7.1 Hz, 3H). CNMR (101 MHz,
MHz, CDCl
3
) d 11.48 (s, 1H), 8.44 (d, J ¼ 2.6 Hz, 1H), 7.58 (d, J ¼ 8.9 CDCl ) d 196.70, 172.20, 163.97, 152.76, 142.29, 141.45, 135.15,
3
Hz, 1H), 6.86 (d, J ¼ 19.9 Hz, 2H), 6.59 (dd, J ¼ 8.9, 2.6 Hz, 1H), 3.93 134.49, 117.20, 108.86, 107.33, 105.44, 60.89, 56.26, 55.56,
+
(s, 3H), 3.92 (s, 3H), 3.88 (s, 6H), 2.73–2.54 (m, 1H), 1.32 (s, 3H), 53.54, 44.53, 15.19. HRMS found (ESI) (M + H) 403.1866
13
1
1
1
.30 (s, 3H). C NMR (101 MHz, CDCl
52.84, 143.95, 141.38, 135.83, 134.58, 115.68, 109.14, 107.11,
04.68, 60.94, 56.28, 55.63, 37.46, 19.46. HRMS found (ESI) (M + phenyl) acetamide (16). Yellow solid; 88%yield; mp: 138.4–
3
) d 198.00, 176.72, 164.58,
C
21
H
27
N
2
O
6
, requires 403.1864. Purity: 98.8% (by HPLC).
2-(Dimethylamino)-N-(5-methoxy-2-(3,4,5-trimethoxy benzoyl)-
+
ꢀ
1
H) 388.1755 C H NO , requires 388.1755. Purity: 99.0% (by 140.3 C; H NMR (400 MHz, CDCl ) d 11.94 (s, 1H), 8.39 (d, J ¼ 2.5
2
26
6
3
HPLC).
Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 1H), 6.92 (s, 2H), 6.61 (dd, J ¼ 8.8, 2.6
2
-Bromo-N-(5-methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl) Hz, 1H), 3.92 (d, J ¼ 5.2 Hz, 7H), 3.87 (s, 6H), 3.14 (s, 2H), 2.42
ꢀ
1 13
acetamide (12). White solid; 76% yield; mp: 127.1–128.8 C; H (s, 6H). C NMR (101 MHz, CDCl ) d 196.80, 170.90, 164.00,
3
NMR (400 MHz, CDCl
3
) d 11.93 (s, 1H), 8.30 (d, J ¼ 2.5 Hz, 1H), 152.80, 142.28, 141.33, 135.30, 134.64, 117.21, 108.93, 107.17,
7
1
.61 (d, J ¼ 8.9 Hz, 1H), 6.91 (s, 2H), 6.66 (dd, J ¼ 8.9, 2.6 Hz, 105.52, 64.34, 60.90, 56.28, 55.58, 46.07. HRMS found (ESI) (M +
1
3
+
27 2 6
H), 4.03 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.88 (s, 6H). C NMR H) 403.1866 C21H N O , requires 403.1864. Purity: 97.6%
(101 MHz, CDCl ) d 197.56, 165.35, 164.30, 152.86, 142.45, (by HPLC).
3
1
5
41.64, 135.73, 134.14, 116.64, 109.49, 107.30, 105.57, 60.97,
6.32, 55.70, 29.50. LCMS found (ESI) 437.0 (M + H) .
Synthesis of 2-(5-methoxy-2-(3,4,5-trimethoxybenzoyl) phe-
+
30
nylamino)-N,N,N-trimethyl-2-oxoethanaminium (17).
To a
Synthesis of 2-hydroxy-N-(5-methoxy-2-(3,4,5-trimethoxy stirred solution of 2-(dimethylamino)-N-(5-methoxy-2-(3,4,5-tri-
18
benzoyl)phenyl) acetamide (13). To a stirred solution of 2- methoxybenzoyl)phenyl)acetamide (50 mg) in CH Cl , CH I (85
2
2
3
bromo-N-(5-methoxy-2-(3,4,5-trimethoxybenzoyl)phenyl) acet- mg) and triethylamine (35 mg) were added and then the mixture
amide (12, 60 mg) in CH OH (dry) at room temperature, was stirred at room temperature overnight. The solid was
CH ONa (30 mg) was added and then reuxed for 6 h. The ltered and dried to give a white solid. 91% yield; mp: 127.6–
3
3
ꢀ
1
mixture was evaporated under reduced pressure and the residue 128.9 C; H NMR (400 MHz, CDCl ) d 11.48 (s, 1H), 7.86 (d, J ¼
3
was puried by column chromatography (petroleum ether–ethyl 2.5 Hz, 1H), 7.48 (d, J ¼ 8.8 Hz, 1H), 6.84 (s, 2H), 6.67 (dd, J ¼
acetate, 5 : 1) to give the product as a white solid. 85% yield; mp: 8.8, 2.5 Hz, 1H), 5.10 (s, 2H), 3.92 (s, 3H), 3.89 (s, 3H), 3.83 (s,
ꢀ
1
13
1
46.8–148.7 C; H NMR (400 MHz, CDCl ) d 11.84 (s, 1H), 8.37 6H), 3.68 (s, 9H). C NMR (101 MHz, CDCl ) d 196.73, 163.76,
3
3
(
1
s, 1H), 7.60 (s, 1H), 6.91 (s, 2H), 6.64 (d, J ¼ 9.0 Hz, 1H), 5.36 (s, 161.69, 152.74, 141.63, 140.00, 134.97, 133.55, 118.64, 110.11,
1
3
H), 4.31 (s, 2H), 4.19–3.68 (m, 12H). C NMR (101 MHz, 107.35, 65.20, 60.94, 56.38, 55.76, 55.10. HRMS found (ESI)
+
CDCl
3 29 2 6
) d 197.54, 171.38, 164.33, 152.80, 142.29, 141.60, 135.62, (M ) 417.2018 C22H N O , requires 417.2020. Purity: 99.1%
134.22, 116.56, 109.17, 107.31, 105.53, 62.97, 60.97, 56.29, (by HPLC).
41518 | RSC Adv., 2014, 4, 41510–41520
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Paper
RSC Advances
Cell culture and maintenance
binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM
CaCl at Ph 7.4) at room temperature for 15 min. PI was then
2
Four cancer cell lines, HeLa (human epithelial cervical cancer),
A549 (non-small cell lung carcinoma), MCF-7 (human breast
cancer cell line), and HCT-8 (human colon cancer cell line) were
obtained from the laboratory animal center of Sun Yat-sen
University, which were cultivated in the Dulbecco's modied
Eagle medium (DMEM, GIBCO), containing 10% (v/v) heat
dropwise added to the medium at cold temperature, and the
mixture was incubated for 10 min. Almost 10 000 events were
collected for each sample and analyzed by ow cytometer
(Beckman Coulter, Epics XL). The percentage of cells under-
going apoptosis was calculated using EXPO32 ADC Analysis
ꢂ1 soware.
inactivated fetal bovine serum (FBS, GIBCO), 100 IU mL
ꢂ1
penicillin and 100 mg mL streptomycin (GIBCO) in culture
ꢀ
Tublin polymerization assay in vitro
dish in a humidied atmosphere of 5% CO at 37 C.
2
Tublin polymerization is followed by uorescence enhance-
ment due to the incorporation of a uorescent reporter into
microtubules as polymerization occurs. The tublin polymeri-
zation assay was monitored by an increase in uorescence
Antiproliferative activity assays in vitro
The antiproliferative activity of the target compounds were
examined in four human cancer cell lines (HeLa, HCT-8, A549
and MCF-7) by MTT assay. When the cells grew in the loga-
ꢀ
emission at 410 nm over a 60 minute period at 37 C (excitation
3
wavelength is 340 nm) using a modication of methods
described by Bonne, D. et al. Puried brain tubulin polymeri-
zation kit was purchased from Cytoskeleton (BK110P, Denver,
CO). The nal buffer concentrations for tubulin polymerization
rithmic phase, 5 ꢃ 10 cells per well cells were harvested and
plated into the 96-well plates for 24 h, and then the cells were
exposed to different concentrations of the test compounds for
ꢂ1
48 h in three replicates. Aerward, 20 mL of MTT (5 mg mL
,
0
contained 80.0 mM piperazine-N,N -bis(2-ethanesulfonic acid)
Sigma) was added and incubated for another 4 h. Then, the
suspension was discarded and 150 mL of DMSO was added to
each well. Aer shooked the plateds to dissolve the dark blue
crystals (formazan) for 10 min, the absorbance at 570 nm was
measured using a multifunction mircoplate reader (Molec-
ulardevices, Flex Station 3). All experiments were repeated at
least three times. The data were calculated using Grap Pad
Prism version 5.0. The IC50 values were tted using a nonlinear
regression model with a sigmodial dose response.
sequisodium salt (pH 6.9), 2.0 mM MgCl
2
, 0.5 mM ethylene
glycol bis(b-aminoethyl ether)-N,N,N ,N -tetraacetic acid (EGTA),
mM GTP, and 10.2% glycerol. Firstly, 5 mL of test compounds
0
0
1
were added in different concentrations, and then were warmed
ꢀ
to 37 C for 1 min. The reaction was initiated by the addition of
55 mL of the tubulin reaction mix.
Western blot analysis
5
HeLa cells (8.0 ꢃ 10 cells per dish) were incubated with or
without at various concentrations (0.01, 0.05, 0.1, and 0.5 mM)
for 24 h. Aer incubation, the cells were collected by centrifu-
gation and washed twice with phosphate-buffered saline chilled
Flow cytometric analysis of cell-cycle
For ow cytometric analysis of DNA content, HeLa cells were
5
plated in 6-well plates (3 ꢃ 10 cells per well) and incubated in
ꢀ
ꢀ
ꢀ
to 0 C. The cells were then suspended in a lysis buffer at 0 C
the presence or absence of compound 13 at various concentra-
tions (0.01, 0.05, 0.1 and 0.5 mM) for 24 or 48 h. The cells were
harvested with 0.25% trypsin and xed with ice-cold 70%
ethanol overnight. Ethanol was removed by centrifugation, and
for 30 min, and then centrifuged at 15 000 g for 30 min at 4 C.
Total protein content was determined with the BCA protein kit
(Pierce, Italy), and Bovine Serum Albumin (BSA) as a standard.
The protein extracts were reconstituted in sample loading
the cells were washed with cold 10% phosphate buffer solution
buffer (62 mM Tris–HCl, 2% SDS, 10% glycerol, and 5%
ꢂ1
(
3
PBS), then, treated with RNAse A (100 mg mL , Beyotime) at
ꢀ
b-mercaptoethanol), and the mixture was boiled at 100 C for 5
ꢀ ꢀ
7 C for 30 min and DNA staining solution (PI, Beyotime) at 4 C
min. Equal amounts (50 mg) of denatured protein samples were
loaded into each lane, separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) on 8ꢂ12%
polyacrylamide gradient, and then transferred onto a poly-
vinylidene diuoride (PVDF) membrane and then were blocked
with 5% non-fat dried milk in PBS containing 1% Tween-20 for
for 15 min. The DNA contents of 10 000 events were measured by
ow cytometer (Beckman Coulter, Epics XL) at 488 nm. The data

regarding the number of cells in different phases of the cell cycle
were analyzed by EXPO32 ADC Analysis soware.
Flow cytometric analysis of apoptotic cells
2
h at room temperature followed by incubation with primary
5
HeLa cells were plated in 6-well plates (3 ꢃ 10 cells per well) antibodies overnight and either horseradish peroxidase-conju-
and incubated in the presence or absence of compound 13 at gated goat anti-rabbit or anti-mouse antibodies for 2 h. The
various concentrations (0.01, 0.05, 0.1, and 0.5 mM) for 24 h or blots were detected by ImageQuant LAS 4000 mini (Fujilm)
48 h to induce cell apoptosis; 0.1% DMSO was used as a vehicle with the Super-Signal West Pico chemiluminescence substrate
control. The percentages of apoptotic cells were estimated by (Thermo Scientic, IL). Western blot analyses were performed
staining with Annexin-V-FITC and PI (Beyotime Annexin-V-FITC using antibodies against cyclin B1 (Cell Signaling, Boston,
Apoptosis Detection Kit) according to the manufacturer's U.S.A.), Cdc2 (Cell Signaling, Boston, U.S.A.). Cdc25C (Cell
instructions with moderate modication. Aer the cells were Signaling, Boston, U.S.A.), Bcl-2 (Cell Signaling, Boston, U.S.A.),
induced for 24 h or 48 h, both treated and untreated cells were Bax (Cell Signaling, Boston, U.S.A.), Bad (Santa Cruz, Texas,
harvested and incubated with 5 mL of Annexin-V-FITC in U.S.A.), Bcl-xl (Santa Cruz, Texas, U.S.A.). Densitometry analyses
This journal is © The Royal Society of Chemistry 2014
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were performed using quantitative soware (AlphaEase, 19 F. Szab ´o , D. Simk ´o and Z. Nov ´a k, RSC Adv., 2014, 4, 3883.
Genetic Technology Inc. Miami, FL) with the control repre- 20 R. Romagnoli, P. G. Baraldi, C. Lopez-Cara, D. Preti,
senting 1-fold as shown just below the data.
M. Aghazadeh Tabrizi, J. Balzarini, M. Bassetto,
A. Brancale, X.-H. Fu, Y. Gao, J. Li, S.-Z. Zhang, E. Hamel,
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Acknowledgements
56, 9296.
We thank the Natural Science Foundation of China (no. 21 J.-P. Liou, C.-W. Chang, J.-S. Song, Y.-N. Yang, C.-F. Yeh,
21302235, 20972198), the Ph.D. Programs Foundation of the
H.-Y. Tseng, Y.-K. Lo, Y.-L. Chang, C.-M. Chang and
Ministry of Education of China (20120171120045) and the
H.-P. Hsieh, J. Med. Chem., 2002, 45, 2556.
Opening Project of Guangdong Provincial Key Laboratory of 22 A. Haimov and R. Neumann, Chem. Commun., 2002, 876.
New Drug Design and Evaluation (2011A060901014) for nan- 23 S. Chandrasekhar, C. Narsihmulu, S. S. Sultana and
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41520 | RSC Adv., 2014, 4, 41510–41520
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