Synthesis, biological evaluation and mechanism study of chalcone analogues as novel anti-cancer agents

Article abstract of DOI:10.1039/c5ra14888j

A series of novel chalcone analogues were designed, synthesized and evaluated as anticancer agents. The results of antiproliferative activity tests showed that most of the analogues exhibited moderate to very good antiproliferative activities with GI₅₀ values in the micromol to sub-micromol range. Especially compound 10a gave 0.026 μM to 0.035 μM GI₅₀ for five cancer cell lines. The mechanistic studies including tubulin polymerization inhibition, disruption of microtubule dynamics and cell cycle arrest assay demonstrated that compound 10a could effectively inhibit in vitro cellular tubulin polymerization, interfere with the mitosis, resulting in a prolonged G2/M cell cycle arrest and ultimately lead to cell apoptosis of cancer cells. Taken together, these results suggested that 10a may became a promising lead compound for development of new anticancer drugs.

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Synthesis, Biological Evaluation and Mechanism Study of
Chalcone Analogues as Novel Anti-cancer Agents
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
Jie Chen , Jun Yan , Jinhui Hu , Yanqing Pang , Ling Huang *, Xingshu Li *
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of novel chalcone analogues were designed, synthesized and evaluated as antitcancer agents. The results of
antiproliferative activity test showed most of most analogues exhibited moderated to very good antiproliferative activities
with GI50 values in micromol to sub-micromol range. Especially, compounds 10a, gave 0.026 μM to 0.035 μM of GI50 value
for five cancer cell line. The mechanism studies including tubulin polymerization inhibition, disruption of microtubule
dynamics and cell cycle arrest assay demonstrated that compound 10a could effectively inhibit in vitro cellular tubulin
polymerization, interfere with the mitosis, resulting in a prolonged G2/M cell cycle arrest and ultimately lead to cell
apoptosis of cancer cells. Taken together, these results suggested that 10a may became a promising lead compound for
development of new anticancer drugs.
microtubule polymerization inhibitor. For example, Ducki
incorporated the aryl substitution pattern of CA4 into
Introduction
Tubulin-microtubule system is an important target for the
development of anticancer drugs over the past decades
chalcones and found that some of these compounds exhibited
1
5
good antimitotic properties. Bu reported the synthesis and
evaluation of a series of cytotoxic ortho-aryl chalcones as new
1
because of its crucial involvement in mitosis. Some typical
-3
4
5, 6
1
6
drugs such as taxanes and vinca
central role in the treatment of diverse human cancers, are
alkaloids that played a
scaffold targeting tubulin and mitosis. With millepachine, a
chalcone analogue isolated from the Millettia pachycarpa as
the model, Chen synthesized and evaluated twenty one
dirivatives for their antiproliferative activity and the optimal
7
, 8
well known microtubule-targeting agents.
Combretastatin A-4 (CA-4), a natural cis-stilbene derivative
isolated from the bark of the African willow tree Combretum
caff rum by Pettit and co-workers in 1982, is a typical tubulin
polymerization inhibitor which binds to the colchicine binding
1
7
compound was considered as promising anticancer agent.
Indanones and their derivatives are important bioactive
molecules that have been studied to determine their biological
activities for the treatment of disease including Alzheimer’ s
9
, 10
site.
promising results in human clinical anti-tumor trials as a
CA-4P, the disodium phosphate salt of CA-4, exhibited
1
8
19
disease and cancer. In our previous work, we reported the
evaluation of a series of ortho-(3,4,5-trimethoxybenzoyl)-
1
1
potent vascular disrupting agent. As the cis double bond in
CA-4 is prone to isomerize to the less bioactive trans isomers,
to stabilize the conformation, many CA-4 analogues have been
2
0
acetanilides as tubulin polymerization inhibitors. Considering
both indoles and indanones exhibited varied pharmacological
activities, herein, we designed and evaluated and chalcone
analogues derived from indole aldehydes and indanones as
inhibitors of tubulin polymerization.
1
2
developed in recent years. Among them, the replacement of
benzene ring by benzoheterocyclic structures has received
1
3
much attention.
Chalcones, the precursors of flavonoids and isoflavonoids, are
abundant in edible plants, and have also been shown to
Results and discussion
1
4
display a diverse array of pharmacological activities. Some
chalcon derivatives also exhibited potent activity as
Chemistry. The synthetic routes for the target compounds are
listed in Schemes 1-2. First, the reaction of 2,3,4-
trimethoxybenzaldehyde or 3,4,5-trimethoxybenzaldehyde
2
1
with malonic acid gave propenoic acid derivative
was hydrogenated in the presence of Pd/C to provide
cyclization of catalyzed by 1:10 phosphorus pentoxide-
methanesulfonic acid afforded the substituted indanones 4 in
2,
which
2
0
3
. The
4
2
2
good yield.
On the other hand, 5- (or 6- and 7-)
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Scheme 2. The synthesis of target compounds 10 and 11
.
methoxyindole reacted with DMF in the presence of POCl
2
3
to
3
afford indole-3-aldehyde derivatives
6.
Then, reacted with
2
4
Biological evaluation
CH I to provide the N-methyl products
3
7.
Finally, target
2
compounds 8 were obtained by the aldol reaction of
5
6
or
7
In vitro human cancer cell lines growth inhibition. The
antiproliferative activities evaluation of synthesized
compounds were performed with five human cancer cell lines,
namely, A549 (non-small-cell-lung cancer cell line), HeLa
with indanone 4a in good yields (Schemes 1).
(human epithelial cervical cancer cell line), Bel-7402 (human
liver carcinoma cell line), PC-3 (human prostate cancer cell
line), and K562 (human gastric cancer cell line) using MTT (3-
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
assay with CA-4 as reference. As the results summerized in
table 1, most tested compounds exhibited moderated to very
good antiproliferative activities with GI50 values in micromol to
sub-micromol range. Among them, compound 10a, the aldol
condensation product of 4,5,6-trimethoxy indanone and
indole-4-carbaldehyde, gave the best antiproliferative
activities in all the five cancer cell lines (GI50 = 0.026
A549, 0.027 M for Hela, 0.035 M for Bel-7402, 0.031
PC-3, and 0.031 M for K562). A simple structure–activity
µ
M for
µ
µ
µM for
µ
relationship analysis showed that the antiproliferative activity
was closely related to both the methoxy group position in
indanone moiety and the indole carbaldehyde used.
Compounds 8a-8f, the condensation products of 4,5,6-
trimethoxy indanone with 4-, 5-, or 6-methoxy-3-indole
carbaldehydes (and their N-methyl derivative), gave
moderated activities in most cases, besides 8e and 8f
(
products of 6-methoxy-3-indole carbaldehyde and its N-
methyl analogue) provided good activities with the GI50 range
,DMF,rt. from 0.120 to 1.08
M. It was interesting that most
Scheme 1. The synthesis of target compounds
malonic acid, piperidine, pyridine. (b) Pd/C,
phosphorus pentoxide-methanesulfonic acid,50
e)CH I,NaH,THF,rt. (f)NaOH,EtOH,rt.
8
. Reagents and conditions: (a)
MeOH, 40 (c) 1:10
.(d)POCl
H
2
,
℃.
℃
3
µ
(
3
compounds obtained from 4,5,6-trimethoxy indanone and
indole-4-, or indole-5-carbaldehyde exhibited potent
antiproliferative activities (10a: GI50 range from 0.026 to 0.035
Target compounds 10 and 11 were synthesized with indanones
, indole-(4 or 5) -carbaldehyde and their N-methyl derivatives
by the same aldol condensation procedure.
4
µ
M; 10b: 0.034 to 0.072
.071 to 0.125 M). However, condensation products of 5,6,7-
trimethoxy indanone with indole-4-, or indole-5-carbaldehyde
gave very poor activities (10d 11c and 11d: more than 10
of GI50 for all five human cancer cell lines; 10c: GI50 range from
.03 to 2.13 M).
µM; 11a: 0.034 to 0.072 µM; 11b:
0
µ
,
µM
1
µ
2
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a
Table1．Antiproliferative activity of compounds against five human cancer cell lines
b
GI50 (μΜ)
Compounds
A549
Hela
Bel-7402
PC-3
8.63±0.05
8.24±0.03
8.63±0.02
12.4±0.02
0.539±0.01
1.08±0.03
0.031±0.05
0.062±0.01
1.11±0.28
>10
K562
6.06±0.15
2.88±0.35
1.44±0.02
8.93±0.25
0.615±0.23
0.120±0.03
0.031±0.12
0.039±0.12
1.58±0.03
>10
8
a
2.46±0.02
8.79±0.04
2.37±0.16
7.93±0.19
0.365±0.03
0.910±0.02
0.026±0.03
0.065±0.02
1.03±0.15
>10
2.72±0.56
7.90±0.68
7.33±0.27
11.9±0.20
0.612±0.12
0.160±0.21
0.027±0.01
0.072±0.02
2.13±0.25
>10
5.62±0.05
8.12±0.05
8.09±0.02
12.9±0.37
0.668±0.03
0.250±0.12
0.035±0.04
0.034±0.02
1.59±0.26
>10
8
b
8
c
8
d
e
8
8
f
1
0a
0b
0c
0d
1a
1b
1c
1d
CA-4
1
1
1
1
0.099±0.02
0.071±0.02
>10
0.049±0.03
0.087±0.03
>10
0.056±0.13
0.094±0.02
>10
0.066±0.03
0.125±0.02
>10
0.057±0.04
0.089±0.15
>10
1
1
1
>10
>10
>10
>10
>10
0.018±0.05
0.0028±0.06
0.012±0.001
0.014±0.004
0.011±0.003
a
b
Cell lines were treated with different concentrations of the compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental Section.
GI50 values are indicated as the mean±SD (standard error) of three independent experiments.
In vitro Inhibition of Tubulin Polymerization. Among the In vitro cytotoxicity against human normal cell lines. It is well-
synthesized compounds, we chosed 10a, 10b, 11a and 11b for
known that a main drawback of clinically used anticancer drugs
is their same cytotoxicity against human normal cells.
Therefore, we determined the selectivity of compound 10a
toward three kinds of human normal cells. The HLF (Human
embryonic lung fibroblast), BJ (human dermal fibroblasts) and
MCF-10A (human normal mammary epithelial cell line) cells
were used. The results showed that compound 10a is
associated with a relatively low toxicity towards the non-
analyzing their ability to block the microtuble assembly
because of their good antitumor activity in different cancer cell
lines. With the method originally described by Bonne, D. et al
2
6, 27
with some modification,
and CA-4 as the reference
compound, the results were obtained as listed in table 2. It
could be seen that the tubulin polymerization inhibition (TPI)
activities of these compounds were basiclly consisted with
their antiproliferative activity. Compound 10a, which gave best
antiproliferativ activity in vitro, also displayed most potent TPI cancerous cell line (Table 3). The selectivity ratio were 217.4 -
activity, with 1.99 μM of the IC50. Comparing with 10a 11a 328.5-fold, respectivity. These results demonstrate that 10a
,
gave relatively lower in vitro antiproliferativ activity, gave exhibits lower cytotoxicity towards human normal cells.
weaker TPI activity accordingly. Similarly, their N-methyl
Table 3. Antiproliferative activity of compound 10a against human non-cancerous cells
derivatives, 10b and 11b, also displayed the decrease of their
TPI activities. The correlation between the antiproliferative
line.
activity and TPI activity indicated that the target compounds
indeed target tubulin-microtubule system.
a
b
GI50 (μM)
Selectivity ratio
A549
BJ
0.027±0.01
5.87±0.21
8.87±0.33
6.54±0.29
a
217.4
328.5
242.2
Table 2. Effects of the selected compounds on tubulin polymerization inhibition
HLF
Compd
0a
0b
1a
1b
CA-4
IC50(μM)b
1.99 ± 0.03
5.86±0.05
3.71 ± 0.24
MCF-10A
1
a
Data are presented as the mean ± SE from the dose-response cures of at least
1
b
three independent experiments. Selectivity ratio = (IC50 human normal cells) /
IC50 Hela).
1
(
1
4.36±0.15
To determine the mechanism (stabilizing agents such as
1.25 ± 0.04
2
8
taxanes, or destabilizing agents like such as colchicine) of the
target compounds to tubulin, we carried out the microtubule
dynamics assays with 1.0, 1.25, 2.50, 5.0, 7.5 and 10 μM of
compound 10a, respectively (Figure 1). The representative raw
aInhibition of tubulin polymerization. Tubulin was at 20 μM. b IC50 values are
indicated as the mean±SD (standard error) of three independent experiments.
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data for the polymerization assay of compound 10a showed
that it inhibited tubulin polymerization in a concentration
dependent manner, indicating that it belongs to the
mircotubule destabilizing agents.
Figure1. Effect of compound 10a on mircotubule dynamics.
Disruption of Microtubule Dynamics.
To identify the cellular microtubule morphology that may be Figure 2. Effects of compound 10a on mircrotubule dynamics by
immunoflurescence. Hela cells were treated with compound 10a at different
relevant to the antiproliferative activity of target compounds, concentration (0.01, 0.05, and 0.1μM) or DMSO (0.01%) for 48 h. Nucleuses were
stained with Hochest 33342 (blue), and microtubules were stained with mouse
we performed the disruption of microtubule dynamics assay anti-α-tubulin conjugated with Alex flour 488 (green). Images were taken using
LSM 570 laser confocal microscope.
with 10a by tubulin immunofluorescence. As shown in Figure
2, confocal microscopy studies clearly indicated the disruption
of microtubule system of Hela cells after treated with different Cell cycle arrest at G2/M phase.
concentration of compound 10a. Cells in control group was
Studies has indicated that tubulin-destabilizing agents can block
the cell cycle at the G2/M phase due to microtubule
disruption. To confirm cell cycle arrest effect of 10a, Hela
wrapped by uncondensed chromosomes and the regular slim,
fibrous microtubules. After the concentration of compound
2
9
10a increased, the microtubule network became disorganized
cells were treated with 10a at different concentrations (0.01,
and drastically disrupted at the concentration of 100 nM. The
microtubule spindle strongly shrink around the center of the
cells, and the irregularity of cytoplasm and chromosomes were
also obvious. These abnormal morphology changes of the
microtubule suggested that compound 10a could effectively
disrupted the microtubule dynamics.
0
.05, and 0.1µM) for 48 h with DMSO (0.01%) as the control
group, and then the harvested cells were analysed by flow
cytometry. The results exhibited that 10a led to a significant
cell-cycle arrest at G2/M phase, and displayed a dose-
dependent manner (Figure 3). As shown in Figure 3, in control
group, 10.24% of the cells at G2/M phase, while the percentage
of cells arrest at G2/M phase increased to 25.6%, 51.4% and
5
4.8% when the concentration of compound 10a at 0.01, 0.05,
and 0.1 µM, respectively. Therefore, we draw the conclusion
that compound 10a could arrest cell-cycle progression at the
mitotic phase, which is in agreement with most of the
antimitotic agents.
4
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Figure4. Effect of compound 10a on HeLa cell apoptosis. Cells were treated with
compound 10a (0.01, 0.05, and 0.1 μM) or DMSO (0.01%) for 48 h. The
percentages of cells in each stage of cell apoptosis were quantitated by flow
cytometry. (upper left quadrant) necrotic 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 were shown.
Conclusions
In conclusion, a novel series of new chalcone analogues were
synthesized by the aldol condensation of indanones and indole
carbaldehydes and evaluated as anticancer agents. The SAR
studies showed that the target compounds obtained from
Figure 3. Cell cycle distribution of Hela with or without treatment of compound
1
0a at different concentrations(0.01, 0.05, and 0.1 μM) or DMSO (0.01%) for 48
h. 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 were shown.
4
,5,6-trimethoxy indanone with different indole carbaldehyde
usually gave more potent antiproliferative activities than the
,6,7-trimethoxy indanone derivatives. Most of most
Cell apoptosis
5
Considering the obvious sub-G1 peak was observed in Figure 3,
we hypothesized compound 10a may induce cell apoptosis,
and then, the flow cytometry was performed to validated our
hypothesis. After the Hela cells were treated with compound
analogues exhibited moderated to very good antiproliferative
activities with GI50 values in micromol to sub-micromol range.
Further mechanism studies showed that the synthesized
compounds could effectively inhibit in vitro cellular tubulin
polymerization, interfere with the mitosis, resulting in a
10a at the indicated concentration for 48 h, the harvested cells
were stained with Annexin V-FITC and PI to differentiate the
early and late apoptosis cells. As shown in Figure 4, the total
percentages of early and late apoptosis cells were 18.5%,
prolonged G /M cell cycle arrest and ultimately lead to cell
2
apotosis of cancer cells. Among them, compound 10a gave the
best results in vivo antitumor evaluation and the further in vivo
study of compound 10a is in progress.
57.4%, and 60.9% at the 0.01, 0.05, and 0.1 μM, respectively,
which was significantly increased comparing to the control
group. These results further proved the compound 10a could
inhibit tubulin polymerization, arrest cell cycle at G2/M phase,
and eventally induce cell apoptosis.
Experimental
1
13
H NMR and C NMR spectra were acquired on a Bruker
AvanceIII spectrometer with TMS as the internal standard.
High resolution mass spectra (HR-MS) were recorded on an
Agilent LC–MS 6120 instrument with an ESI mass selective
detector in positive ion mode. Melting points were determined
on an SRS-OptiMelt automated melting point instrument. The
purity of the synthesized compounds was determined by high-
performance liquid chromatography (HPLC) with a TC-C18
column (4.6 × 250 mm, 5 μm), an acetonitrile/water or
methanol/water mobile phase, and a flow rate of 1.00 mL/min.
General procedure for the preparation of 2
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Compounds
2
were prepared in excellent yield (95-100%) by Substituted benzaldehydes
6
and
7
were synthesized according
2
3, 24
Knoevenagel condensation of an aromatic aldehyde with literature method.
To a solution of NaOH (180mg,
OH (10 mL), (200.0mg, 0.9mmol) was
added. After the mixture was stirred for 15 minutes, a
substituted benzaldehyde or (1.02 mmol) in C OH (2
2
1
malonic acid by following a previously reported procedure.
4.5mmol) in C
2
H
5
4
General procedure for the preparation of 3
6
,
7
9
2 5
H
To a stirred suspension of
2 (9.39g, 39.45mmol) in methanol mL) was added dropwise to the solution. The reaction mixture
(
20ml), Pd/C (0.50g) was added and the mixture was was stirred overnight and filtered to yield the crude product.
o
hydrogenated in 1.2 Mpa of hydrogen pressure for 4h at 50 C. The crude product was chromatographed on silica gel and
After cooled to room temperature, the mixture was filtered to eluted with petroleum/ethyl acetate (3:1) as an eluent to
get the crude product. The crude product was afford the proposed compound.
chromatographed on silica gel and eluted with
(
2
E)-4,5,6-trimethoxy-2-((4-methoxy-1H-indol-3-yl)methylene)-
,3-dihydro-1H-inden-1-one (8a)
f
Yellow solid, 299.3mg, yield 87.7%. mp:229.3 =
petroleum/ethyl acetate (8:1) as an eluent to afford the
proposed compound.
℃
-231.7℃. R
3
-(2,3,4-trimethoxyphenyl)propanoic acid(3a)
White solid ,9.1g, yield 96%. mp:78.1 -79.2
petroleum/ethyl acetate=3:1). H-NMR(400 MHz, CDCl
-1
0
.57 (petroleum/ethyl acetate=1:1). IR(KBr,cm ) 3151, 3116,
1
℃
℃
. R
f
= 0.52 3045, 3010, 2937, 1662, 1598, 1317, 727. H NMR (400 MHz,
) δ DMSO) δ 8.35 (s, 1H), 7.91 (s, 1H), 7.11 (dd, J = 16.2, 7.7 Hz,
2.03 (s, 1H), 6.53 (s, 1H), 6.86 (s, 1H), 3.72 (s, 3H), 3.77 (s, 3H), 6.70 (d, J = 7.0 Hz, 1H), 4.00 (s, 3H), 3.97 (s, 3H), 3.88 (s,
1
(
3
1
3
1
3
H), 3.65 (s, 3H), 2.77 - 2.81 (t, 2H), 2.49 - 2.54 (t, 2H) .LC- 3H), 3.86 (s, 3H), 3.83 (s, 2H). C NMR (101 MHz, DMSO) δ
-
MS(ESI): 239.2 for
［M+H］
.
191.84, 154.97, 154.33, 150.15, 146.80, 138.00, 134.74,
1
1
34.56, 129.31, 128.83, 128.36, 123.89, 116.75, 112.63,
3
-(3,4,5-trimethoxyphenyl)propanoic acid(3b)
White solid ,8.9g, yield 94%. mp:98.9 -100.5
petroleum/ethyl acetate=3:1). H-NMR (400 MHz, CDCl
05.91, 101.91, 101.44, 61.13, 60.87, 56.53, 55.76, 30.05.
+
℃
℃
. R
f
= 0.42 HRMS (ESI) (m/z) [M+H] calcd for C22
H21NO
5
, 379.1420; found,
1
(
3
) δ 380.1480. Purity: 95.6% (by HPLC).
1
2.05 (s, 1H), 6.42 (s, 1H), 6.39 (s, 1H), 3.70 (s, 3H), 3.72 (s,
(
E)-4,5,6-trimethoxy-2-((4-methoxy-1-methyl-1H-indol-3-
3H), 3.75(s, 3H), 2.69 – 2.76 (t, 2H), 2.48 - 2.54 (t, 2H). LC-
-
yl)methylene)-2,3-dihydro-1H-inden-1-one(8b)
MS(ESI): 239.1 for
［M+H］ .
Yellow solid, 274.6mg, yield 77.6%.mp:145.1
f
℃-146.9℃. R =
General procedure for the preparation of 4
Compound
0g of rapidly stirred 1:10 phosphorus pentoxide- 8.58 (s, 1H), 7.46 (s, 1H), 7.21 (d, J = 10.7 Hz, 2H), 6.97 (d, J =
methanesulfonic acid. The homogeneous reaction mixture was 8.2 Hz, 1H), 6.66 (d, J = 7.8 Hz, 1H), 4.04 (d, J = 3.0 Hz, 6H), 3.98
-1
0
.55 (petroleum/ethyl acetate=1:1).IR(KBr,cm ) 3070, 3037,
1
3
3
(2g, 8.33mmol) was added in small portions to a 2920, 1679, 1610, 1523, 1342, 710. H NMR (400 MHz, CDCl ) δ
3
1
3
stirred at 50
℃
for 1 h.The violet solution was added dropwise (s, 3H), 3.94 (s, 3H), 3.88 (s, 3H), 3.72 (s, 2H). C NMR (101
) δ 192.78, 155.40, 154.18, 149.70, 146.70, 138.43,
to 90 m1 of water and the mixture was stirred rapidly for 5-10 MHz, CDCl
3
min to ensure the hydrolysis of methanesulfonic anhydride. 134.95, 134.54, 130.10, 129.47, 128.76, 123.77, 117.48,
The mixtue was extracted with ethyl acetate and the organic 113.02, 102.79, 101.47, 101.42, 61.11, 60.82, 56.28, 55.36,
+
phase was washed (with solution of sodium bicarbonate and 33.66, 29.90. HRMS (ESI) (m/z) [M+H] calcd for C23
water, succesively), dried (sodium sulfate) and concentrated to 393.1576; found, 394.1629. Purity: 98.1% (by HPLC).
give the crude product, which was purified by column
H23NO
5
,
(
2
E)-4,5,6-trimethoxy-2-((5-methoxy-1H-indol-3-yl)methylene)-
,3-dihydro-1H-inden-1-one(8c)
Yellow solid, 272.3mg, yield 79.8%. mp:216.5 -218.7℃
chromatography using silica gel and petroleum/ethyl acetate
10:1) as an eluent to afford the proposed compound.
(
℃
.
4,5,6-trimethoxy-2,3-dihydro-1H-inden-1-one(4a)
-1
f
R =0.61 (petroleum/ethyl acetate=1:1).IR (KBr,cm )3131,
1
White acicular crystal, 1.44g, yield 78%. mp:87.9
℃
-88.6
= 0.48 (petroleum/ethyl acetate=4:1). H-NMR (400 MHz, MHz, CDCl
CDCl ) δ 7.03 (s, 1H), 3.97 (s, 3H), 3.96 (s, 3H), 3.89 (s, 3H), 3.08 (m, 2H), 7.21 (s, 1H), 6.94 (d, J = 8.7 Hz, 1H), 4.04 (s, 3H), 3.98
3.02 (t, 2H), 2.70 – 2.65 (t, 2H). LC-MS(ESI): 223.1 for
℃.
3047, 3012, 2939, 1670, 1600, 1503, 1315, 810. H NMR (400
1
R
f
3
) δ 8.70 (s, 1H), 8.03 (s, 1H), 7.70 (s, 1H), 7.38 – 7.31
3
1
3
–
［
M+H (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.79 (s, 2H). C NMR (101
+
.
］
MHz, DMSO) δ 191.82, 155.14, 154.33, 150.12, 146.82, 134.73,
1
1
34.50, 131.30, 129.62, 129.01, 128.68, 125.46, 113.42,
5
,6,7-trimethoxy-2,3-dihydro-1H-inden-1-one (4b)
13.32, 111.95, 101.48, 100.18, 61.11, 60.85, 56.49, 55.82,
+
White squamous crystal, 1.37g, yield 74%.mp:113.9
℃
-114.7
℃.
30.02. HRMS (ESI) (m/z) [M+H] calcd for C22
H21NO
5
, 379.1420;
1
R
f
= 0.46 (petroleum/ethyl acetate=4:1). H-NMR (400 MHz, found,380.1480 . Purity: 98.2% (by HPLC).
CDCl ) δ 6.68 (s, 1H), 4.05 (s, 3H), 3.94 (s, 3H), 3.85 (s, 3H), 3.05
3.00 (t, 2H), 2.69 – 2.64 (t, 2H). LC-MS(ESI): 223.1 for M+H
3
(
E)-4,5,6-trimethoxy-2-((5-methoxy-1-methyl-1H-indol-3-
yl)methylene)-2,3-dihydro-1H-inden-1-one(8d)
Yellow solid, 296.2mg, yield 83.7%. mp:197.5 - 199.2℃.
–
［
+
.
］
℃
General procedure for the preparation of 8, 10 and 11
6
| J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
-1
f
R =0.53(petroleum/ethyl acetate=1:1). IR(KBr,cm ) 3037, (E)-2-((1H-indol-4-yl)methylene)-5,6,7-trimethoxy-2,3-dihydro-
1
2
970, 2892, 1670, 1610, 1525, 1314, 1147, 731. H NMR (400 1H-inden-1-one(10c)
MHz, CDCl ) δ 8.00 (s, 1H), 7.52 (s, 1H), 7.38 (d, J = 2.3 Hz, 1H),
.25 (d, J = 9.0 Hz, 1H), 7.21 (s, 1H), 6.96 (dd, J = 8.8, 2.4 Hz,
H), 4.05 (s, 3H), 3.99 (s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.89 (s,
3
Yellow solid, 281.9mg, yield 89.7%.mp:139.3
℃
-1
-142.5
℃
. R
f
=
7
1
3
1
1
1
2
0
3
.43 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3274, 3118,
1
029, 2996, 1675, 1602, 1571, 1108, 697. H NMR (400 MHz,
) δ 8.41 (s, 1H), 8.13 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.47
d, J = 8.1 Hz, 1H), 7.36 – 7.34 (m, 1H), 7.31 (d, J = 7.8 Hz, 1H),
1
3
3
H), 3.75 (d, J = 1.7 Hz, 2H). C NMR (101 MHz, CDCl ) δ
CDCl
3
92.86, 155.42, 154.29, 149.72, 146.90, 134.83, 134.54,
31.90, 131.14, 129.21, 129.04, 125.03, 113.71, 111.88,
(
6
.93 (d, J = 2.2 Hz, 1H), 6.81 (s, 1H), 4.17 (s, 3H), 4.04 (d, J = 1.5
10.65, 101.44, 100.27, 61.13, 60.83, 56.29, 55.91, 33.67,
+
9.80. HRMS (ESI) (m/z) [M+H] calcd for C23
13
Hz, 2H), 3.99 (s, 3H), 3.92 (s, 3H). C NMR (101 MHz, CDCl
3
) δ
H
23NO
5
, 393.1576;
1
1
1
91.28, 159.56, 152.44, 148.02, 141.06, 136.15, 135.18,
found,394.1635. Purity: 95.1% (by HPLC).
29.57, 129.50, 127.33, 125.44, 124.38, 121.85, 120.19,
12.66, 103.63, 101.09, 62.25, 61.55, 56.36, 32.86. HRMS (ESI)
+
(
2
E)-4,5,6-trimethoxy-2-((6-methoxy-1H-indol-3-yl)methylene)-
,3-dihydro-1H-inden-1-one(8e)
f
Yellow solid, 277.1mg, yield 81.2%. mp:209.1 -211.7℃. R =
(m/z) [M+H] calcd for C21
H19NO
4
, 349.1314; found, 350.1367.
Purity: 97.7% (by HPLC).
℃
-1
0
.51 (petroleum/ethyl acetate=1:1). IR (KBr,cm ) 3160, 3058, (E)-4,5,6-trimethoxy-2-((1-methyl-1H-indol-4-yl)methylene)-2,3-
1
3
014, 2988, 1672, 1600, 1498, 1340, 1127, 723. H NMR (400 dihydro-1H-inden-1-one(10b)
MHz, CDCl
3
) δ 8.57 (s, 1H), 8.00 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H),
.61 (d, J = 2.4 Hz, 1H), 7.20 (s, 1H), 6.93 (dd, J = 10.6, 2.0 Hz,
H), 4.03 (s, 3H), 3.98 (s, 3H), 3.93 (s, 3H), 3.86 (s, 3H), 3.77 (d,
Yellow solid, 282.7mg, yield 86.5%. mp:168.0
℃
-170.5
f
℃. R =
7
2
0
2
8
1
6
.39 (petroleum/ethyl acetate=2:1). IR(KBr,cm-1) 3091, 3066,
1
3
978, 1689, 1621, 1510, 1120, 709. H NMR (400 MHz, CDCl ) δ
1
3
J = 1.7 Hz, 2H). C NMR (101 MHz, DMSO) δ 191.82, 156.85,
.17 (d, J= 1.8 Hz, 1H), 7.60 (d, J= 7.2 Hz, 1H),7.39 (d, J= 8.1 Hz,
H), 7.35 (d, J= 7.5 Hz, 1H), 7.23 (s, 1H), 7.17 (d, J= 3.1 Hz,1H),
1
1
6
54.35, 150.14, 146.88, 137.27, 134.66, 134.52, 129.62,
28.24, 125.22, 122.00, 119.37, 112.01, 111.12, 101.51, 95.52,
+
1.13, 60.87, 56.52, 55.68, 30.00. HRMS (ESI) (m/z) [M+H]
.87 – 6.84 (m, 1H), 4.02 (s, 3H), 3.99 (s, 3H), 3.94 (s, 3H), 3.84
13
3
(s, 3H), 3.72(d, J= 1.9 Hz, 2H) C NMR (101 MHz, CDCl ) δ
5
calcd for C22H21NO , 379.1420; found,380.1482. Purity: 99.3%
1
1
1
93.41, 154.37, 149.82, 147.46, 136.95, 136.18, 134.46,
33.89, 130.70, 130.25, 129.88, 127.25, 121.62, 120.20,
(by HPLC).
10.91, 101.59, 99.65, 61.16, 60.79, 56.31, 33.00, 29.57. HRMS
+
(
E)-4,5,6-trimethoxy-2-((6-methoxy-1-methyl-1H-indol-3-
(ESI) (m/z) [M+H] calcd for C22
H21NO
4
, 363.1471; found,
yl)methylene)-2,3-dihydro-1H-inden-1-one(8f)
3
64.1527. Purity: 98.9% (by HPLC).
Yellow solid, 319.2mg, yield 90.2%.mp:165.5
f
℃-167.6℃. R =
0
1
7
6
3
.57 (petroleum/ethyl acetate=1:1). IR(KBr,cm-1) 3074, 2940, (E)-5,6,7-trimethoxy-2-((1-methyl-1H-indol-4-yl)methylene)-2,3-
662, 1604, 1527, 1345, 1090, 730. H NMR(400 MHz, CDCl ) δ dihydro-1H-inden-1-one(10d)
3
1
.93 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.37 (s, 1H), 7.17 (s, 1H),
.91 (d, J = 8.6 Hz, 1H), 6.74 (s, 1H), 4.04 (s, 3H), 3.98 (s, 3H),
Yellow solid, 289.9mg, yield 88.7%. mp:169.5
℃
-1
-171.3
℃
. R
f
=
0
1
.42 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3097, 2930,
1
3
.93 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.65 (s, 2H). C NMR
) δ 192.88, 157.20, 154.02, 149.54, 146.86,
1
683, 1621, 1602, 1471, 1320, 1114, 706. H NMR (400 MHz,
) δ 8.09 (t, J = 2.0 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.37 (d,
J = 8.1 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.16 (d, J = 3.2 Hz, 1H),
(101 MHz, CDCl
3
CDCl
3
1
1
2
3
37.41, 134.52, 129.97, 124.92, 122.61, 119.63, 112.06,
10.65, 101.27, 93.16, 61.10, 60.79, 58.52, 56.26, 55.63, 33.85,
6
.83 (dd, J = 3.1, 0.6 Hz, 1H), 6.78 (s, 1H), 4.14 (s, 3H), 4.01 (d, J
13
1.6 Hz, 2H), 3.97 (s, 3H), 3.90 (s, 3H), 3.84 (s, 3H). C NMR
9.56, 18.90. HRMS (ESI) (m/z) [M+H]
+
calcd for C23
H23NO
5
,
=
93.1576; found,394.1628. Purity: 95.9% (by HPLC).
(
3
101 MHz, CDCl ) δ 191.06, 159.47, 152.49, 147.91, 141.07,
1
1
3
3
36.94, 135.28, 130.08, 129.78, 129.25, 127.57, 124.43,
(
1
E)-2-((1H-indol-4-yl)methylene)-4,5,6-trimethoxy-2,3-dihydro-
H-inden-1-one(10a)
f
Yellow solid, 260.2mg, yield 82.8%.mp:179.8 =
21.50, 119.81, 110.54, 103.56, 99.69, 62.27, 61.55, 56.34,
+
3.03, 32.86. HRMS (ESI) (m/z) [M+H] calcd for C22
63.1471; found, 364.1524. Purity: 98.1% (by HPLC).
4
H21NO ,
℃
-181.9℃. R
-
1
0
2
.39 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3288, 3120,
1
998, 1662, 1602, 1509, 1127, 710. H NMR (400 MHz, CDCl
3
)
(E)-2-((1H-indol-5-yl)methylene)-4,5,6-trimethoxy-2,3-dihydro-
δ 8.43 (s, 1H), 8.18 (s, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 1H-inden-1-one(11a)
8
1
3
1
1
6
.0 Hz, 1H), 7.32 (dd, J = 12.4, 5.0 Hz, 2H), 7.23 (s, 1H), 6.93 (s,
Yellow solid, 249.5mg, yield 79.4%. mp:211.4
℃
-1
-213.3
℃
. R
f
=
H), 4.03 (s, 3H)4.01 (d, J = 1.8 Hz,2H), 3.99 (s, 3H), 3.94 (s,
0
2
8
7
4
.38 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3237, 3035,
1
1
H). C NMR (101 MHz, CDCl
3
3
) δ 193.57, 154.40, 149.84,
3
940, 1677, 1618, 1591, 1481, 710. H NMR (400 MHz, CDCl ) δ
47.53, 136.26, 136.11, 134.50, 133.88, 130.89, 129.72,
27.20, 125.36, 122.06, 120.62, 112.85, 101.65, 101.29, 61.17,
+
0.81, 56.31, 29.58. HRMS (ESI) (m/z) [M+H] calcd for
.42 (s, 1H), 8.03 (s, 1H), 7.81 (s, 1H), 7.58(d, J= 8.4 Hz, 1H),
.48 (d, J= 8.2 Hz, 1H), 7.26 (s, 1H), 7.22 (s, 1H), 6.67(s, 1H),
13
.05 (s, 3H), 4.02 (s, 2H), 3.99 (s, 3H), 3.93 (s, 3H). C NMR
21 4
C H19NO , 349.1314; found, 372.1192. Purity: 99.6% (by
(
3
101 MHz, CDCl ) δ 193.69, 154.38, 149.84, 147.33, 136.58,
HPLC).
1
1
36.03, 135.81, 133.98, 131.82, 128.44, 127.41, 125.51,
25.19, 124.48, 111.63, 103.63, 101.60, 61.16, 60.85, 56.30,
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+
9.43. HRMS (ESI) (m/z) [M+H] calcd for C21
2
4
H19NO , 349.1314; MTT assay. The antiproliferative activity of the target
found, 372.1190. Purity: 95.8% (by HPLC).
compounds towards the human cancer cell lines were
examined by MTT assay. Briefly, cells were harvested in the
(
1
E)-2-((1H-indol-5-yl)methylene)-5,6,7-trimethoxy-2,3-dihydro-
H-inden-1-one(11c)
f
Yellow solid, 284.1mg, yield 90.4%. mp:129.1 -131.3℃. R =
3
logarithmic phase, and plated into the 96-well plates (5×10
cells per well) for 24 h, and then, different concentrations of
the test compounds were added for 48 h in three replicates.
℃
-
1
0
.44 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3345, 3266, Afterward, 20 μL of 5 mg/mL MTT (Sigma) was added into each
1
010, 2980, 1675, 1606, 1592, 1471, 1128, 690. H NMR (400 well and incubated for another 4 h. Finally, the suspension was
3
MHz, CDCl
3
) δ 8.45 (s, 1H), 7.99 (s, 1H), 7.75 (s, 1H), 7.54 (dd, J discarded and 150 μL of DMSO was added to dissolve the dark
8.5, 1.4 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.31 – 7.29 (m, 1H), blue crystals (formazan). The absorbance at 570nm was
.83 (s, 1H), 6.67 – 6.62 (m, 1H), 4.15 (s, 3H), 4.05 (d, J = 1.2 measured using multifunction mircoplate reader
) δ (Moleculardevices, Flex Station 3). All experiments were
91.38, 159.34, 152.38, 147.81, 141.06, 136.39, 134.22, repeated at least three times. The GI50 values and SD values
=
6
a
1
3
Hz, 2H), 4.00 (s, 3H), 3.92 (s, 3H). C NMR (101 MHz, CDCl
3
1
1
1
32.50, 128.39, 127.56, 125.55, 125.05, 124.44, 123.97, were calculated using Grap Pad Prism version 5.0 and Excel
11.60, 103.61, 103.40, 62.24, 61.54, 56.33, 32.80. HRMS (ESI) software, respectively.
+
(
m/z) [M+H] calcd for C21
H19NO
4
, 349.1314; found, 350.1375.
Tublin polymerization assay in vitro. Tublin polymerization
assay was monitored by an increase in fluorescence intensity.
Purified brain tubulin polymerization kit was purchased from
Cytoskeleton (BK110P, Denver, CO). All of the experiments
were performed under the guidance of the manufacturer's
instruction. The final buffer concentration for tubulin
polymerization contained 80.0 mM piperazine-N, N′-bis(2-
ethanesulfonic acid) sequisodium salt (pH 6.9), 2.0 mM MgCl2,
Purity: 98.5% (by HPLC).
(
E)-4,5,6-trimethoxy-2-((1-methyl-1H-indol-5-yl)methylene)-2,3-
dihydro-1H-inden-1-one(11b)
f
Yellow solid, 265.7mg, yield 81.3%. mp:138.1 =
℃
-140.6℃. R
-
1
0
1
.37 (petroleum/ethyl acetate=2:1). IR(KBr,cm ) 3089, 3026,
1
677, 1619, 1591, 1481, 1340, 1120, 708. H NMR (400 MHz,
) δ 8.00 (s, 1H), 7.80 (s, 1H), 7.60 (dd, J = 8.6, 1.5 Hz, 1H),
.39 (d, J = 8.6 Hz, 1H), 7.22 (s, 1H), 7.11 (d, J = 3.1 Hz, 1H),
.60 (dd, J = 3.1, 0.6 Hz, 1H), 4.05 (s, 3H), 4.01 (d, J = 1.8 Hz,
CDCl
3
0.5 mM EGTA, 1 mM GTP, and 10.2% glycerol. Firstly, 5 μL of
7
6
2
tested compounds were added in 96 well, and then warmed to
37 °C for 1 min. The reaction was initiated by the addition of
1
3
H), 3.99 (s, 3H), 3.94 (s, 3H), 3.83 (s, 3H). C NMR (101 MHz,
) δ 193.49, 154.31, 149.80, 147.21, 137.33, 135.92,
35.74, 133.99, 131.60, 130.15, 128.92, 126.84, 124.75,
24.60, 109.71, 102.19, 101.50, 61.14, 60.82, 56.27, 32.95,
5
5 μL of the tubulin reaction mix. Then the fluorescence
CDCl
3
intensity enhancement were recorded over a 60 minute period
at 37 °C (emission wavelength is 410 nm and excitation
wavelength is 340 nm) using a multifunction mircoplate reader
1
1
2
+
4
9.42. HRMS (ESI) (m/z) [M+H] calcd for C22H21NO , 363.1471;
(Moleculardevices, Flex Station 3). All experiments were
found, 364.1530. Purity: 95.9% (by HPLC).
repeated at least three times. The IC50 values and SD values
were calculated using Grap Pad Prism version 5.0 and Excel
software, respectively.
(
E)-5,6,7-trimethoxy-2-((1-methyl-1H-indol-5-yl)methylene)-2,3-
dihydro-1H-inden-1-one(11d)
Yellow solid, 305.3mg, yield 93.4%. mp:156.1
℃
-158.8
℃
. R
f
=
Immunofluorescence Microscopy. Hela cells were plated in a
.56 (petroleum/ethyl acetate=1:1). IR(KBr,cm ) 3039, 3008, confocal culture-dish at 3 × 10 cells/dish, cultured for 24 h
-
1
4
0
1
920, 1683, 1591, 1484, 1160, 714. H NMR (400 MHz, CDCl
2
3
)
and then were incubated with indicated concentrations of
δ 8.00 (s, 1H), 7.80 (s, 1H), 7.60 (dd, J = 8.6, 1.5 Hz, 1H), 7.39 compound 10a for another 48 h. The cells were briefly washed
d, J = 8.6 Hz, 1H), 7.22 (s, 1H), 7.11 (d, J = 3.1 Hz, 1H), 6.60 (dd, with PBS, fixed with pre-warmed (37 °C)
J = 3.1, 0.6 Hz, 1H), 4.05 (s, 3H), 4.01 (d, J = 1.8 Hz, 2H), 3.99 (s, paraformaldehyde for 15 min, and then were permeabilized
(
4
%
1
3
3
1
1
1
3
H), 3.94 (s, 3H), 3.83 (s, 3H). C NMR (101 MHz, CDCl ) δ with 0.5% Triton X-100 for another 15 min and finally blocked
91.23, 159.24, 152.35, 147.74, 141.01, 137.12, 134.12, with 10% goat serum for 30 min. The bolcked cells was
32.37, 130.11, 128.88, 127.06, 124.59, 124.43, 124.16, incubated with FITC-conjugated mouse anti-tubulin antibody
09.64, 103.58, 102.01, 62.26, 61.53, 56.32, 32.98, 32.77. (Sigma) at 4 °C overnight. The following day, cells were washed
+
HRMS (ESI) (m/z) [M+H] calcd for C22
H21NO
4
, 363.1471; found, with PBS for three times and incubated with goat anti-mouse
IgG/Alexa-Fluor 488 (Invitrogen, USA) for 1 h. The nuclei of
cells were labeled with Hochest 33342 (Sigma). After washing,
1 mL PBS was added and the samples were immediately
visualized on a Zeiss LSM 570 laser scanning confocal
3
64.1525. Purity: 97.3% (by HPLC).
Biology
Cell lines and culture. The human cancer cell lines (A549,
Hela, Bel-7402, PC-3, K562) used in this study were grown in
DMEM containing 10% (v/v) heat-inactivated FBS, 100 Cell Cycle analysis. For flow cytometric analysis of DNA
microscope.
5
units/mL penicillin, and 100 μg/mL streptomycin. The cells content, Hela cells were plated in 6-well plates (2×10
were cultivated at 37 °C under a 5% CO
humidity (RH) atmosphere.
2
and 90% relative cells/well) and incubated in the presence or absence of
compound 10a at indicated concentrations for 48 h. Then, the
cells were harvested by centrifugation and fixed with ice-cold
8
| J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
70% ethanol overnight. After removing the ethanol by 8. C. Ballatore, K. R. Brunden, D. M. Huryn, J. Q. Trojanowski, V. M.
centrifugation, the cells were washed with cold 10% PBS, and Lee and A. B. Smith, 3rd, J. Med. Chem., 2012, 55, 8979-8996.
then treated with RNAse A (100 μg/mL, Beyotime) at 37 °C for 9. C. M. Lin, H. H. Ho, G. R. Pettit and E. Hamel, Biochemistry, 1989,
30 min and DNA staining solution PI (Sigma) at 4 °C for 15 min. 28, 6984-6991.
The DNA contents of 10,000 events were harvested by flow 10. G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts and D.
cytometer (Beckman Coulter, Epics XL) at 488 nm. The Garcia-Kendall, Experientia, 1989, 45, 209-211.
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