Synthesis, biological evaluation and molecular docking studies of novel 1-(4,5-dihydro-1Hpyrazol-1-yl)ethanone-containing 1-methylindol derivatives as potential tubulin assembling inhibitors

Article abstract of DOI:10.1039/c5ra28141e

A series of novel compounds (6a-6v) containing 1-methylindol and 1-(4,5-dihydro-1H-pyrazol-1-yl) ethanone skeletons were designed, synthesized and biologically evaluated as potential tubulin polymerization inhibitors and anticancer agents. Among them, compound 6q showed the most potent tubulin polymerization inhibitory activity (IC50 = 1.98 mM) and in vitro growth inhibitory activity against A549, MCF-7 and HepG2 cell lines, with IC50 values of 0.15 mM, 0.17 mM, and 0.25 mM respectively, comparable to the positive control. Furthermore, compound 6q was a potent inducer of apoptosis in A549 cells and it had typical cellular effects for microtubule interacting agents, causing arrest of the cell cycle in the G2/M phase. Confocal microscopy assay and molecular docking results further demonstrated that 6q could bind tightly to the colchicine site of tubulin and act as an anti-tubulin agent. These studies, along with 3D-QSAR modeling provided an important basis for further optimization of compound 6q as a potential anticancer agent.

Full text of DOI:10.1039/c5ra28141e

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Page 1 of 41
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Synthesis, biological evaluation and molecular docking studies of novel
-(4,5-dihydro-1H-pyrazol-1-yl)ethanone-containing 1-methylindol derivatives as
potential tubulin assembling inhibitors
1
†
a
†a
a
a
a
MengꢀRu Yang , YaꢀJuan Qin , Chen Chen , YaꢀLiang Zhang , BoꢀYan Li , TianꢀBao
a
b
a
a
Liu , HaiꢀBin Gong* , BaoꢀZhong Wang* , HaiꢀLiang Zhu*
Abstract: A series of novel compounds (6a-6v) containing 1ꢀmethylindol and
1ꢀ(4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ethanone skeleton were designed, synthesized and
3
biologically evaluated as potential tubulin polymerization inhibitors and anticancer
agents. Among them, compound 6q showed the most potent tubulin polymerization
inhibitory activity (IC50 = 1.98
A549, MCFꢀ7 and HepG2 cell lines, with IC50 values of 0.15
M respectively, being comparable to the positive control. Furthermore, compound 6q
ꢀ
M) and in vitro growth inhibitory activity against
ꢀ
M, 0.17 M, and 0.25
ꢀ
ꢀ
was a potent inducer of apoptosis in A549 cells and it had typical cellular effects for
microtubule interacting agents, causing arrest of the cell cycle in G2/M phase.
Confocal microscopy assay and molecular docking results further demonstrated that
6
q could bind tightly to the colchicineꢀsite of tubulin and act as an antiꢀtubulin agent.
These studies, along with 3DꢀQSAR modeling provided an important basis for further
optimization of compound 6q as a potential anticancer agent.
Keywords: tubulin; inhibitors; 1ꢀmethylindol; pyrazoline; molecular docking
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, People’s Republic of China
b
Xuzhou Central Hospital, Xuzhou 221009, People’s Republic of China
*
Corresponding author. Tel. & fax: +86ꢀ25ꢀ89682572. Eꢀmail address:
zhuhl@nju.edu.cn
†
Both authors contributed equally to this work.
1

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1
. Introduction
Microtubules (MTs) are a broadly exploited target for the development of new
1
ꢀ3
effective anticancer agents. MTs are highly dynamic polymers mainly composed of
α, βꢀtubulin heterodimers which turn over continuously. Their
polymerizationꢀdepolymerization dynamics play a crucial role in various fundamental
cellular processes including mitotic spindle formation, organization of intracellular
4
structure, and intracellular transport, as well as cell signaling and secretion. Hence,
interfering with MTs function can block cell division and cause cell damage, which is
2
ꢀ3, 5
an established strategy to inhibit the proliferation of cancer cells.
There are many agents that bind to tubulin and interfere with microtubule
assembly. Colchicine (compound 1, Figure 1), one of the earliest tubulinꢀtargeting
drugs, can effectively interfere with microtubule polymerization and its binding site
6
, 7
on tubulin has been characterized already.
However, due to its highly adverse
8
effects, the clinical development of colchicine for cancer therapy was not successful.
Another wellꢀknown potent antiꢀtubulin agent is CAꢀ4 (compound 2, Figure 1),
which was isolated from the bark of the South African tree Combretum caffrum. It can
potently bind to the colchicine binding site, leading to severe cytotoxicity against
9
multiple human cancer cell lines. But unfortunately, it easily undergoes cisꢁtrans
isomerization under natural light, causing a sharp reduction in the antiꢀtubulin activity,
10
which limits its further applications. Despite a long history of characterization,
colchicine binding site inhibitors have not yet reached the commercial phase as
anticancer drugs to date. Thus, further studies are required.
In the last two decades, an ever increasing number of synthetic indoles have been
reported, which are able to inhibit tubulin polymerization, such as compound 3 (ATIs),
1
1
12
13
4
(BPROL075) and 5 (Figure 1). It could be found that the indole structure is a
crucial structural motif for tubulin inhibition. The indole nucleus also has been
reported to be one of the core nucleuses for potent inhibitors of tubulin
1
4, 15
polymerization.
Furthermore, the addition of methyl to the N atom of indole
1
6, 17
would be better for the inhibition of tubulin polymerization and cytotoxicity.
2

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Also, pyrazolines have gained prominence due to its broad spectrum of
18, 19
biological activities including anticancer, antimicrobial and antiꢀinflammatory,
as
well as producing useful intermediates in several organic preparations. It was also
reported some pyrazoline derivatives can inhibit tubulin polymerization by binding to
2
0
the colchicineꢀsite, such as compound 6. Besides, the presence of acetyl on the N
atom of the fiveꢀmembered ring enhances the inhibition of cell proliferation and the
21
binding of the colchicineꢀsite. For example, compound 7 (Aꢀ204197)
and
22
compound 8 (Figure 1) showed potent antiꢀproliferative activities against multiple
cancer cell lines and tightly bound to the colchicineꢀsite of tubulin.
Therefore, due to these previous researches and molecule modeling studies, we
attempt to integrate 1ꢀmethylindol with 1ꢀ(4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ethanone to
screen novel compounds (6a-6v) as potential antiꢀtubulin agents (Figure 1). These
coupling structures might possess enhanced anticancer activities. A series of novel
1
ꢀmethylindol derivatives targeting tubulin were recently reported as potent anticancer
23
agents in our group. To extend our research and gain further insight in the ensuing
structure–activity relationships (SARs), here we search more novel agents. Our
strategy for further development of active antiꢀtubulin agents was to perform
modifications on the two aromatic rings (A, B ring). Once the new combined scaffold
was identified as the minimum structural requirement for activity in this new series of
compounds, then some substituents were introduced onto the aromatic rings, and the
modifications have been performed to screen a series of active anticancer agents.
So far, few reports have been dedicated to design and synthesize any anticancer
compound containing 1ꢀmethylindol and 1ꢀ(4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ethanone.
Herein, we discussed the synthetic method and structureꢀactivity relationship (SAR)
of this series of compounds, evaluated their anticancer and antiꢀtubulin activities, and
learned the influence on cell cycle and cell apoptosis. In addition, confocal
microscopy assay provided a more directly view of the effect on microtubule system.
Docking simulations are performed using the Xꢀray crystallographic structure of the
tubulin in complex with an inhibitor to explore the binding modes of these
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compounds at the active site.
2
2
. Results and discussion
.1. Chemistry
The synthetic route of compounds 6a-6v (Table 1) was outlined in Scheme 1.
First of all, the substituted 1Hꢀindole (1a-1c) dissolved in DMF was added to a
mixture of POCl and DMF at 0 °C to generate compounds 2a-2c after twoꢀhour
stirring. Then the substituted indoleꢀ3ꢀcarbaldehyde (2a-2c) was dissolved in THF and
reacted with NaH and CH I to introduce the methyl group to the N atom of indole and
3
3
obtain compounds 3a-3c. Subsequently, the obtained compounds (3a-3c) and the
corresponding acetophenone were dissolved in ethanol in the presence of KOH to
give products 4a-4v. After that, the obtained chalcone (4a-4v) was stirred with
hydrazine hydrate in ethanol and refluxed for 2h, and then crude compounds 5a-5v
were gained. Finally, to a stirred solution of acetic acid, EDC·HCl and HOBt in
acetonitrile or dichloromethane, obtained compounds (5a-5v) were added with the
protection of N
2
to get the target compounds 6a-6v. All synthesized compounds 6a-6v
1
are reported for the first time and determined by H NMR, melting test, ESIꢀMS and
elemental analysis, which were consistent with the assigned structures.
2
.2. Crystal structure of compound 6o
The crystal structure of compound 6o was determined by Xꢀray diffraction
analysis. The crystal data presented in Table 2 and Figure 2 gave perspective views
of compound 6o with the atomic labeling system.
2
2
.3. Biological activity
.3.1. In vitro anti-proliferative activity
We prepared three different human cancer cell lines, including A549 (human
nonsmall cell lung carcinoma), MCFꢀ7 (human breast adenocarcinoma) and HepG2
(human liver hepatocellular carcinoma), and one normal cell line (293T) to evaluate
4

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the antiꢀproliferative activities of all the synthesized compounds (6a-6v). The results
were summarized in Table 3. Among all the 22 tested compounds, 6b, 6i, 6q, 6t, 6u
and 6v possessed the highest overall potency with IC50 values ranging from 0.15 to
0
.34
ꢀ
M against all the three typical cancer cell lines. Compound 6q was found to be
M, 0.17
M, and 0.25 ꢀM against A549, MCFꢀ7 and HepG2 cell lines respectively, superior to
the most active among all compounds with the level of IC values of 0.15
ꢀ
50
ꢀ
the positive compound colchicine. Besides, the results of 293T cell line indicated that
these compounds possessed high safety.
Based on the data of Table 3, we found that the substituent group on the Aꢀring
was of great importance to the activity of the tested compounds. Obviously, the
antiꢀproliferative activities of compounds with different substituents in the Aꢀring
increased in the following order: Br > H > OCH , which could be observed from the
3
comparisons: 6n > 6a > 6h, 6q > 6i > 6b and 6v > 6f > 6m.
The inhibitory activity of compounds with ꢀOCH
3
on different positions of the
Bꢀring increased in the following order: 4ꢀOCH (6q)> 3,4,5ꢀ(OCH ) (6s)>
3
3 3
3
3 2 3 3
,4ꢀ(OCH ) (6r)> 2ꢀOCH (6o)> 3ꢀOCH (6p). Thus, we could draw a conclusion that
compounds with pꢁsubstituted group showed the most potent activity, following by
those of oꢁsubstituted, while mꢁsubstituted compounds had the lowest activity. Among
all pꢁsubstituted (Bꢀring) compounds, 6n, 6q, 6t, 6u and 6v (IC50 = 1.08
.21 M, 0.24 M, 0.18 M) exhibited significant antiꢀproliferative activities in the
order of ꢀOCH > ꢀBr > ꢀF > ꢀCl > ꢀH in A549 cell line, while 6a, 6b, 6e and 6f, 6h, 6i,
k, 6l and 6m showed the similar order.
ꢀM, 0.15ꢀM,
0
ꢀ
ꢀ
ꢀ
3
6
2
.3.2. Inhibition of tubulin polymerization
To investigate whether the antiꢀproliferative activities of compounds 6a-6v were
resulted from their interaction with tubulin, we evaluated the ability of these agents to
inhibit tubulin polymerization in vitro (Table 3). These agents inhibited tubulin
assembling with IC50 values ranging from 1.98 to 34.96 ꢀM. Among them, compound
6
q showed the most potent activity of antiꢀtubulin (IC50 = 1.98 ꢀM), better than the
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positive control colchicine (2.35 ꢀM). We also found that the IC50 values of tubulin
polymerization of these compounds displayed a similar tendency with their relevant
IC50 values of the antiꢀproliferative assay. Figure 3 presented the relationship between
the antiꢀproliferative activity against A549 cell line and the tubulin polymerization
inhibitory activity of the top 10 compounds (6b, 6f, 6i, 6o, 6q, 6r, 6s, 6t, 6u and 6v),
2
which indicated that there was a moderate correlation between them with an R value
of 0.90. Therefore, we could conclude that most of the synthesized compounds could
inhibit the tubulin assembly and the antiꢀproliferative effect was produced partly by
their interaction with tubulin.
2
.3.3. Compound 6q induced cell cycle arrest and cell apoptosis
To explore whether compound 6q could arrest the process of mitosis, A549 cells
were treated with 6q of increasing concentrations (0.02
M, 0.2 M) for 24 h and then incubated with propidium iodide (PI) to analyze their
genomic DNA content in flow cytometry assays (Figure 4). We found that, compound
q induced a gradual accumulation of cells in G2/M phase of the cell cycle in a
doseꢀdependent manner with a rise in G2/M cells occurring at a concentration as low
as 0.02 M. When the concentration increased to 0.20 M, up to 90% of the cells were
ꢀM, 0.05 ꢀM, 0.1 ꢀM, 0.15
ꢀ
ꢀ
6
ꢀ
ꢀ
arrested in the G2/M phase. In contrast, there was a continuing decrease in the
proportion of cells in other phase (G1 and S) of the cell cycle.
To explore the correlation between cell cycle arrest and cell death induced by
compound 6q, a biparametric cytofluorimetric analysis using PI and annexinꢀV (stain
DNA and phosphatidylserine residues, respectively) was performed. Dual staining of
annexinꢀV and PI permits discrimination between early apoptotic cells
+
ꢀ
+
+
ꢀ
ꢀ
(
annexinꢀV /PI ), late apoptotic cells (annexinꢀV /PI ), live cells (annexinꢀV /PI ) and
ꢀ
+
necrotic cells (annexinꢀV /PI ). A549 cells were treated with 0, 0.05, 0.1 and 0.15 ꢀM
of 6q for 24 h. As shown in Figure 5, the percentage of both early and late apoptotic
cells increased as the concentration of 6q became higher, rising from 2.34% and 1.76%
at 0 ꢀM initially to 9.0% and 16.2% at 0.15 ꢀM eventually. Thus, we could draw a
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conclusion that compound 6q could induce cell apoptosis in a doseꢀdependent
manner.
2
.3.4. Compound 6q inhibited tubulin polymerization and disrupted the
microtubule system
Our results suggested that 6q can target microtubules in A549 cells. To test this
directly, we treated cells with 0.2 ꢀM of 6q for 24 h and analyzed microtubules by
immunocytochemistry staining. As shown in Figure 6, the microtubule network of
control cells displayed normal arrangement and organization, which was slim and
fibrous. After treatment with 0.2 ꢀM of 6q, microtubules became short and wrapped
around the nucleus. We also noted a significant reduction in the density of cellular
microtubules and the radial organization of microtubules was perturbed, which could
also be found in colchicine (0.5
in this assay. In contrast, 0.5
ꢀ
M)ꢀtreated cells which were used as positive control
ꢀM of paclitaxel dramatically enhanced microtubule
polymerization, with an increase in the density of cellular microtubules and formation
of long thick microtubule bundles. These results further confirmed that the observed
effects were due to interaction with tubulin, resulting in a prolonged mitotic arrest that
ultimately leads to cell death.
2
2
.4. Computational
.4.1. Molecular docking
To gain a better understanding on the binding mode of the synthesized
compounds, we proceeded to examine the interaction of compounds 6a-6v with
tubulin crystal structure (PDB code: 1SA0) using the DS 3.5 (Discovery Studio 3.5,
Accelrys, Co. Ltd). The molecular docking was performed by inserting the
compounds into the colchicine binding site of tubulin. Among them, compound 6q
showed the lowest interaction energy, at ꢀ54.27 kcal /mol. The binding models of
compound 6q and tubulin were depicted in Figure 7. The enzyme surface model
(Figure 7A) revealed that the compound 6q was well embedded in the active pocket
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via hydrophobic interaction. The amino acid residues interacting with tubulin were
labeled. The oxygen atom of acetyl contributes to the hydrogen bonding interaction
with the hydrogen atom on the main chain of Ala B: 250 (angle NꢀH…O = 127.1°,
distance = 2.47 Å). The 2D diagram of the interactions between the colchicine
binding site and colchicine or compound 6q were shown in Figure 7C and D,
respectively. Significant similarities could be found in their tubulinꢀbinding models.
For example, residues Ala B: 250, Met B: 259, Cys B: 241, Leu B: 255, Leu B: 248
and Lys B: 352 were predicted to make contacts with colchicine or compound 6q.
Besides, Figure 7B presented a superimposition of these two compounds. This
molecular docking results along with the biological assay data suggested that
compound 6q was a potential inhibitor of tubulin.
2
.4.2. 3D-QSAR model
To obtain a systematic SAR profile on the synthesized compounds as antitumor agents
and explore more potent and selective tubulin inhibitors, 3DꢀQSAR model was built
by using the corresponding pIC50 values which were converted from the obtained IC50
(
ꢀM) values of tubulin inhibition (the way of this transformation was derived from an
online calculator developed by an Indian Medicinal Chemistry Lab
http://www.sanjeevslab.org/toolsꢀIC .html)) and performed by builtꢀin QSAR
(
5
0
software of DS 3.5. The training and test set were divided by the random diverse
molecules method, where the training set accounted for 80% of all the molecules
while the test was set to 20%, with 18 agents and 4 agents respectively. The observed
and predicted values, corresponding residual values for the training set and test set
molecules in 3DꢀQSAR model, were presented in Table 4. The graphical relationship
of observed and predicted values was showed in Figure 8, in which the plot of the
observed IC50 versus the predicted values showed that this model could be used in
prediction of activity for 1ꢀmethylindolꢀ and pyrazolineꢀcontaining derivatives. Also
the molecules aligned with the isoꢀsurfaces of the 3DꢀQSAR model coefficients on
electrostatic potential grids (Figure 9A) and van der Waals grids (Figure 9B) were
8

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listed. Electrostatic map indicated red contours around regions where high electron
density (negative charge) was expected to increase activity, while blue contours
represent areas where low electron density (partial positive charge) was expected to
increase activity. This model is consistent with the docking results. For example, the
red region near the B ring corresponds to the Lys B: 352 residue. Compounds 6b, 6i
and 6q could fit the red subregion, and this is part of the reason why these small
molecules show potent tubulin inhibitory activity. Similarly, steric map indicated
areas where steric bulk was predicted to increase (green) or decrease (yellow) activity.
It was widely acceptable that a better inhibitor based on the 3DꢀQSAR model should
have strong van der Waals attraction in the green areas and a polar group in the blue
electrostatic potential areas (which were dominant close to the skeleton). This
promising model would provide a guideline to design and optimize more effective
tubulin inhibitors and pave the way for us in the further study.
3
. Conclusions
In this study, a series of novel compounds (6a-6v) containing 1ꢀmethylindol and
ꢀ(4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ethanone skeleton were designed, synthesized and
1
evaluated as potential inhibitors of tubulin polymerization. Most of them showed
potent antiꢀproliferative activity, and compound 6q displayed the most potent activity
against tubulin assembling, A549, MCFꢀ7 and HepG2 cell lines with IC50 values of
1
.98 ꢀM, 0.15 ꢀM, 0.17 ꢀM, and 0.25 ꢀM respectively, which was comparable to the
positive compound colchicine. Furthermore, the mechanism of action studies proved
that compound 6q could cause accumulation of cells in the G2/M phase of the cell
cycle and potently induce apoptosis in A549 cells, as well as severe disruption of the
microtubule system. Besides, molecular docking and 3DꢀQSAR were performed to
learn the inhibitorꢀtubulin interactions. According to the analysis of the binding model
of compound 6q with tubulin, several interactions were observed with the protein
residues in the colchicine binding site, which may contribute to its antiꢀtubulin
polymerization and antiꢀproliferative activities. The research of these well designed
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compounds might be greatly helpful for the development of tubulin polymerization
inhibitors with stronger activities in the future.
4
4
. Experiments
.1. Chemistry general
All reagents and solvents were of analysis or synthesis grade. Melting points
1
were determined on a XT4MP apparatus (Taike Corp, Beijing, China). H NMR
spectra were recorded on a Bruker DPX300 model Spectrometer using
tetramethylsilane (TMS) as internal standard and DMSO or CDCl
3
as solvents
chemical shifts in ppm (d)). TLC was performed on the glass backed silica gel sheets
Silica Gel 60 GF254) and visualized in UV light (254 nm and 365 nm). ESIꢀMS
(
(
spectra were recorded on a Mariner System 5304 Mass spectrometer. Elemental
analyses were performed on a CHNꢀOꢀRapid instrument and were within ± 0.4% of
the theoretical values.
4
.1.1. General procedure for the synthesis of 1H-indole-3-carbaldehyde (2a)
To a stirred and completely mixed solution of POCl (20 mL) in DMF (40 mL),
a (35 mmol) in DMF (40 mL) was added at 0 °C. After a further 2 h reaction, the
3
1
mixture was poured into 300 mL ice water and mixed completely. Then NaOH pellets
were added and adjusted the pH to 9.0. The reaction mixture was filtered, and washed
with ethyl acetate. Then the filtrate was extracted by ethyl acetate. The organic layer
was dried over Na SO and concentrated to give a brown solid, yield 98.1%. The
2
4
crude 2a was used in the next step without any further purification. Following this
way, we got crude compounds 2a-2c with yields of 95.3ꢀ98.1%.
4
.1.2. General procedure for the synthesis of 1-methyl-1H-indole-3-carbaldehyde
(3a)
To a stirred solution of 2a (25 mmol) in THF (15 mL) at 0 °C was added NaH
62.5 mmol) in THF (15 mL) slowly and stirred for 15 min. Then CH I (33 mmol)
(
3
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was added and the reaction mixture was moved to room temperature to react for
further 24 h. The reaction was stopped after the consumption of the starting material
monitored by TLC. Then the solution was removed and the mixture was extracted by
2 4
ethyl acetate. The organic layer was washed with brine, dried over Na SO and
concentrated to give crude residue, 3a, yield 97.5%. Following this way, we got crude
compounds 3a-3c with yields of 93.4ꢀ97.5%.
4
.1.3.
E)-3-(1-methyl-1H-indol-3-yl)-1-phenylprop-2-en-1-one (4a)
To a stirred solution of 3a (2 mmol) and acetophenone (2 mmol) in ethanol (20
General
procedure
for
the
synthesis
of
(
mL), KOH (6 mmol, 336 mg) was added at room temperature. After the reaction was
completed (24 h), the reaction mixture was filtered and washed with water and cool
ethanol. The crude product was purified by recrystallization from ethanol and
dichloromethane to give 4a, a yellow crystal, yield 93.1%. Following this way, we got
pure compounds 4a-4v with yields of 38.5ꢀ95.7%.
4
.1.4.
-methyl-3-(3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1H-indole (5a)
A mixture of 4a (1 mmol) and hydrazine hydrate (2 mmol) was dissolved in
ethanol (5 mL) and refluxed for 2h. The reaction mixture was cooled and kept at
20 °C overnight. Then the solid obtained was filtered off and washed with petroleum
General
procedure
for
the
synthesis
of
1
ꢀ
ether and taken for the next step immediately. Following this way, we got crude
compounds 5a-5v.
4
1
.1.5.
General
procedure
for
the
synthesis
of
-(5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone
(6a)
To a stirred solution of acetic acid (2 mmol), EDC·HCl (3 mmol) and HOBt (1.2
mmol) in acetonitrile or dichloromethane (5 mL), 5a (1 mmol) was added at room
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2
temperature with the protection of N . After 24 h, the reaction mixture was filtered
and washed with cool ethanol and water. The crude product was purified by
recrystallization from ethanol and dichloromethane to give 6a, a white powder, yield
3
0.2%. Following this way, we got pure compounds 6a-6v.
1
White powder. Yield: 30.2%. m. p. 171 ~ 172 °C. H NMR (DMSOꢀd , 400 MHz)
6
δ: 7.93 – 7.76 (m, 2H), 7.57 – 7.43 (m, 3H), 7.40 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 7.9
Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.97 (t, J = 7.3 Hz, 1H), 5.81 (dd, J =
11.8, 4.4 Hz, 1H), 3.84 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0,
+
4
.4 Hz, 1H), 2.26 (s, 3H). MS (ESI) m/z: 318.15 (C20
19 3
H N O, [M+H] ). Anal. Calcd
19 3
for C20H N
O: C, 75.69; H, 6.03; N, 13.24. Found: C, 75.81; H, 6.04; N, 13.22.
4
1
.1.6.
-(3-(4-methoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)
ethan-1-one (6b)
1
Brown crystal. Yield: 34.5%. m. p. 156 ~ 158 °C. H NMR (DMSOꢀd , 400 MHz)
6
δ: 7.77 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.23 (s,
1
H), 7.13 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.96 (t, J = 7.4 Hz, 1H), 5.77
(dd, J = 11.8, 4.2 Hz, 1H), 3.87 – 3.75 (m, 4H), 3.72 (s, 3H), 3.22 (dd, J = 17.9, 4.3
+
Hz, 1H), 2.23 (s, 3H). MS (ESI) m/z: 348.16 (C21
H
21
N
3
O
2
, [M+H] ). Anal. Calcd for
21 21 3 2
C H N O : C, 72.60; H, 6.09; N, 12.10. Found: C, 72.54; H, 6.09; N, 12.11.
4
1
.1.7.
-(3-(3,4-dimethoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1
-yl)ethan-1-one (6c)
1
Brown crystal. Yield: 14.3%. m. p. 169 ~ 171 °C. H NMR (DMSOꢀd , 400 MHz)
6
δ: 7.39 (d, J = 8.4 Hz, 2H), 7.36 – 7.29 (m, 2H), 7.23 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H),
7
.02 (d, J = 8.4 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 5.78 (dd, J = 11.7, 4.2 Hz, 1H), 3.88
–
3.75 (m, 7H), 3.72 (s, 3H), 3.25 (dd, J = 17.8, 4.3 Hz, 1H), 2.25 (s, 3H). MS (ESI)
+
m/z: 378.17 (C22
H
23
N
3
O
3
, [M+H] ). Anal. Calcd for C22
H
23
N
3
O
3
: C, 70.01; H, 6.14; N,
12

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DOI: 10.1039/C5RA28141E
11.13. Found: C, 69.90; H, 6.15; N, 11.15.
4
1
.1.8.
-(5-(1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazo
l-1-yl)ethan-1-one (6d)
White powder. Yield: 43.1%. m. p. 224 ~ 226 °C. H NMR (DMSOꢀd
1
6
, 400 MHz)
δ: 7.38 (t, J = 8.7 Hz, 2H), 7.22 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.09 (s, 2H), 6.99 (t,
J = 7.4 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.87 – 3.76 (m, 7H), 3.72 (d, J = 5.5
Hz, 6H), 3.32 (dd, J = 17.9, 4.4 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 408.18
+
(C
23
H
25
N
3
O
4
, [M+H] ). Anal. Calcd for C23
25
H N
3
O
4
: C, 67.80; H, 6.18; N, 10.31.
Found: C, 67.95; H, 6.17; N, 10.33.
4
1
.1.9.
-(3-(4-chlorophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)et
han-1-one (6e)
White powder. Yield: 11.1%. m. p. 182 ~ 183 °C. H NMR (DMSOꢀd
1
6
, 400 MHz)
δ: 7.84 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.32 (d,
J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 5.81
(dd, J = 11.8, 4.4 Hz, 1H), 3.83 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J =
+
1
8.0, 4.5 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 352.11 (C20
Calcd for C20
1.96.
H18ClN
3
O, [M+H] ). Anal.
H
3
18ClN O: C, 68.28; H, 5.16; N, 11.94. Found: C, 68.21; H, 5.15; N,
1
4
1
.1.10.
-(3-(4-bromophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)et
han-1-one (6f)
White powder. Yield: 17.7%. m. p. 183 ~ 185 °C. H NMR (DMSOꢀd
1
6
, 400 MHz)
δ: 7.77 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.32 (d,
J = 7.9 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 5.81
13

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(dd, J = 11.9, 4.4 Hz, 1H), 3.83 (dd, J = 18.0, 11.9 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J =
+
1
8.0, 4.5 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 396.06(C H BrN O, [M+H] ). Anal.
2
0
18
3
3
Calcd for C20H18BrN O: C, 60.62; H, 4.58; N, 10.60. Found: C, 60.67; H, 4.59; N,
1
0.58.
4
1
.1.11.
-(5-(1-methyl-1H-indol-3-yl)-3-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyra
zol-1-yl)ethan-1-one (6g)
White crystal. Yield: 61.6%. m. p. 218 ~ 220 °C. H NMR (DMSOꢀd
1
6
, 400 MHz)
δ: 8.04 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.33 (d,
J = 7.9 Hz, 1H), 7.28 (s, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 5.85
(dd, J = 11.9, 4.5 Hz, 1H), 3.89 (dd, J = 18.1, 12.0 Hz, 1H), 3.73 (s, 3H), 3.33 – 3.22
+
(
m, 1H), 2.28 (s, 3H). MS (ESI) m/z: 386.14 (C21
H
18
F
3
N
3
O, [M+H] ). Anal. Calcd for
21 18 3 3
C H F N O: C, 65.45; H, 4.71; N, 10.90. Found: C, 65.31; H, 4.70; N, 10.87.
4
1
.1.12.
-(5-(5-methoxy-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)e
than-1-one (6h)
White crystal. Yield: 45.0%. m. p. 175 ~ 176 °C. H NMR (DMSOꢀd
δ: 7.93 – 7.79 (m, 2H), 7.56 – 7.42 (m, 3H), 7.29 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H),
1
6
, 400 MHz)
6
1
3
7
.80 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 5.78 (dd, J = 11.7, 3.8 Hz, 1H), 3.82 (dd, J =
7.9, 11.7 Hz, 1H), 3.68 (s, 3H), 3.54 (s, 3H), 3.25 (dd, J = 17.9, 3.9 Hz, 1H), 2.26 (s,
+
H). MS (ESI) m/z: 348.16 (C21
H
21
N
3
O
2
, [M+H] ). Anal. Calcd for C21
H
21
N
3
O
2
: C,
2.60; H, 6.09; N, 12.10. Found: C, 72.71; H, 6.10; N, 12.12.
4
1
.1.13.
-(5-(5-methoxy-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-p
yrazol-1-yl)ethan-1-one (6i)
Light yellow powder. Yield: 46.7%. m. p. 158 ~ 160 °C. H NMR (DMSOꢀd
1
6
,
14

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4
00 MHz) δ: 7.78 (d, J = 8.8 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 7.03 (d, J
8.9 Hz, 2H), 6.81 (s, 1H), 6.77 (d, J = 8.8 Hz, 1H), 5.75 (dd, J = 11.6, 3.8 Hz, 1H),
=
3
3
7
.84 – 3.69 (m, 4H), 3.68 (s, 3H), 3.56 (s, 3H), 3.21 (dd, J = 17.8, 3.9 Hz, 1H), 2.23 (s,
+
H). MS (ESI) m/z: 378.17 (C22
H
23
N
3
O
3
, [M+H] ). Anal. Calcd for C22
H
23
N
3
O
3
: C,
0.01; H, 6.14; N, 11.13. Found: C, 69.91; H, 6.16; N, 11.13.
4
1
.1.14.
-(3-(3,4-dimethoxyphenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1
H-pyrazol-1-yl)ethan-1-one (6j)
Grey solid. Yield: 10.2%. m. p. 186 ~ 187 °C. H NMR (DMSOꢀd
.42 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.17 (s, 1H), 7.03 (d, J
8.4 Hz, 1H), 6.83 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 5.76 (dd, J = 11.5, 3.4 Hz, 1H),
1
6
, 400 MHz) δ:
7
=
3
3
6
.86 – 3.72 (m, 7H), 3.68 (s, 3H), 3.58 (s, 3H), 3.24 (dd, J = 17.7, 3.6 Hz, 1H), 2.25 (s,
+
H). MS (ESI) m/z: 408.18 (C23
H
25
N
3
O
4
, [M+H] ). Anal. Calcd for C23
H
25
N
3
O
4
: C,
7.80; H, 6.18; N, 10.31. Found: C, 67.93; H, 6.17; N, 10.32.
4
1
.1.15.
-(3-(4-fluorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyr
azol-1-yl)ethan-1-one (6k)
Gray powder. Yield: 29.8%. m. p. 178 ~ 180 °C. H NMR (DMSOꢀd
δ: 7.90 (dd, J = 8.7, 5.5 Hz, 2H), 7.31 (dd, J = 18.0, 8.9 Hz, 3H), 7.19 (s, 1H), 6.85 –
1
6
, 400 MHz)
6
3
.73 (m, 2H), 5.78 (dd, J = 11.7, 3.8 Hz, 1H), 3.82 (dd, J = 17.9, 11.8 Hz, 1H), 3.68 (s,
H), 3.57 (s, 3H), 3.25 (dd, J = 17.9, 3.9 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z: 366.15
+
(C
21
H20FN
3
O
2
, [M+H] ). Anal. Calcd for C21
H
20FN
3
O
2
: C, 69.03; H, 5.52; N, 11.50.
Found: C, 69.31; H, 5.51; N, 11.48.
4
1
.1.16.
-(3-(4-chlorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyr
azol-1-yl)ethan-1-one (6l)
15

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1
White crystal. Yield: 36.7%. m. p. 169 ~ 170 °C. H NMR (DMSOꢀd
δ: 7.86 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.6 Hz, 1H), 7.20 (s,
H), 6.78 (d, J = 8.7 Hz, 2H), 5.79 (dd, J = 11.7, 3.9 Hz, 1H), 3.81 (dd, J = 18.0, 11.8
6
, 400 MHz)
1
Hz, 1H), 3.68 (s, 3H), 3.57 (s, 3H), 3.24 (dd, J = 17.9, 4.0 Hz, 1H), 2.25 (s, 3H). MS
+
(
ESI) m/z: 382.12 (C H ClN O , [M+H] ). Anal. Calcd for C H ClN O : C, 66.05;
21
20
3
2
21 20
3
2
H, 5.28; N, 11.00. Found: C, 65.98; H, 5.27; N, 10.98.
4
1
.1.17.
-(3-(4-bromophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-pyr
azol-1-yl)ethan-1-one (6m)
White crystal. Yield: 38.6%. m. p. 172 ~ 173 °C. H NMR (DMSOꢀd
δ: 7.78 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.5 Hz, 1H), 7.19 (s,
H), 6.78 (d, J = 8.6 Hz, 2H), 5.79 (dd, J = 11.7, 3.9 Hz, 1H), 3.81 (dd, J = 18.0, 11.9
1
6
, 400 MHz)
1
Hz, 1H), 3.68 (s, 3H), 3.58 (s, 3H), 3.24 (dd, J = 18.0, 4.0 Hz, 1H), 2.25 (s, 3H). MS
+
(
ESI) m/z: 426.07 (C H BrN O , [M+H] ). Anal. Calcd for C H BrN O : C, 59.17;
21
20
3
2
21 20
3
2
H, 4.73; N, 9.86. Found: C, 59.33; H, 4.71; N, 9.85.
4
1
.1.18.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)eth
an-1-one (6n)
White powder. Yield: 59.0%. m. p. 173 ~ 174 °C. H NMR (DMSOꢀd
δ: 7.94 – 7.74 (m, 2H), 7.61 (s, 1H), 7.57 – 7.41 (m, 3H), 7.39 (d, J = 8.7 Hz, 1H),
1
6
, 400 MHz)
7
1
.29 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.84 (dd, J =
8.0, 11.8 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS
+
(
ESI) m/z: 396.06 (C H BrN O, [M+H] ). Anal. Calcd for C H BrN O: C, 60.62;
2
0
18
3
20 18
3
H, 4.58; N, 10.60. Found: C, 60.79; H, 4.57; N, 10.63.
4
1
.1.19.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyr
16

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azol-1-yl)ethan-1-one (6o)
1
Light yellow crystal. Yield: 58.5%. m. p. 180 ~ 181 °C. H NMR (DMSOꢀd ,
6
4
00 MHz) δ: 7.85 (d, J = 6.1 Hz, 1H), 7.63 (s, 1H), 7.46 (t, J = 7.0 Hz, 1H), 7.39 (d, J
=
8.7 Hz, 1H), 7.30 (s, 1H), 7.25 (d, J = 6.9 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H), 7.06 (t,
J = 7.9 Hz, 1H), 5.74 (dd, J = 11.8, 4.2 Hz, 1H), 3.88 (dd, J = 18.5, 11.8 Hz, 1H), 3.81
(s, 3H), 3.73 (s, 3H), 3.28 (dd, J = 18.5, 4.2 Hz, 1H), 2.23 (s, 3H). MS (ESI) m/z:
+
4
26.07 (C21
H20BrN
3
O
2
, [M+H] ). Anal. Calcd for C21
H20BrN
3
O
2
: C, 59.17; H, 4.73;
N, 9.86. Found: C, 59.31; H, 4.74; N, 9.84.
4
1
.1.20.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyr
azol-1-yl)ethan-1-one (6p)
White crystal. Yield: 59.9%. m. p. 184 ~ 185 °C. H NMR (DMSOꢀd
δ: 7.60 (s, 1H), 7.44 – 7.36 (m, 3H), 7.34 (s, 1H), 7.29 (s, 1H), 7.26 (d, J = 6.9 Hz,
1
6
, 400 MHz)
1
3
H), 7.11 – 7.02 (m, 1H), 5.80 (dd, J = 11.7, 4.3 Hz, 1H), 3.89 – 3.78 (m, 4H), 3.72 (s,
H), 3.26 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 426.07
+
(C
21
H20BrN
3
O
2
, [M+H] ). Anal. Calcd for C21
H
20BrN
3
O
2
: C, 59.17; H, 4.73; N, 9.86.
Found: C, 59.24; H, 4.72; N, 9.87.
4
1
.1.21.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyr
azol-1-yl)ethan-1-one (6q)
White crystal. Yield: 75.8%. m. p. 208 ~ 209 °C. H NMR (DMSOꢀd
1
6
, 400 MHz)
δ: 7.77 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.28 (s, 1H), 7.25 (d,
J = 7.0 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 5.77 (dd, J = 11.6, 4.1 Hz, 1H), 3.91 – 3.75
(m, 4H), 3.72 (s, 3H), 3.21 (dd, J = 17.9, 4.2 Hz, 1H), 2.25 (s, 3H). MS (ESI) m/z:
+
4
26.07 (C21
H20BrN
3
O
2
, [M+H] ). Anal. Calcd for C21
H20BrN
3
O
2
: C, 59.17; H, 4.73;
N, 9.86. Found: C, 59.19; H, 4.72; N, 9.85.
17

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4
1
.1.22.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)ethan-1-one (6r)
White powder. Yield: 64.8%. m. p. 229 ~ 231 °C. H NMR (DMSOꢀd
δ: 7.61 (s, 1H), 7.43 – 7.36 (m, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 9.1 Hz, 2H),
1
6
, 400 MHz)
7
3
.03 (d, J = 8.4 Hz, 1H), 5.78 (dd, J = 11.6, 4.0 Hz, 1H), 3.85 – 3.75 (m, 7H), 3.72 (s,
H), 3.24 (dd, J = 17.9, 4.1 Hz, 1H), 2.27 (s, 3H). MS (ESI) m/z: 456.08
+
(C
22
H22BrN
3
O
3
, [M+H] ). Anal. Calcd for C22
H
22BrN
3
O
3
: C, 57.90; H, 4.86; N, 9.21.
Found: C, 57.98; H, 4.87; N, 9.22.
4
1
.1.23.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1
H-pyrazol-1-yl)ethan-1-one (6s)
Pink powder. Yield: 40.6%. m. p. 228 ~ 229 °C. H NMR (DMSOꢀd
δ: 7.65 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.26 (t, J = 5.1 Hz, 2H), 7.09 (s, 2H), 5.82
1
6
, 400 MHz)
(dd, J = 11.6, 4.1 Hz, 1H), 3.87 – 3.76 (m, 7H), 3.72 (s, 6H), 3.30 – 3.28 (m, 1H),
+
2
.29 (s, 3H). MS (ESI) m/z: 486.10 (C23
H
24BrN
3
O
4
, [M+H] ). Anal. Calcd for
23 3 4
C H24BrN O : C, 56.80; H, 4.97; N, 8.64. Found: C, 56.81; H, 4.97; N, 8.62.
4
1
.1.24.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyraz
ol-1-yl)ethan-1-one (6t)
White solid. Yield: 38.6%. m. p. 199 ~ 200 °C. H NMR (DMSOꢀd
.89 (dd, J = 8.7, 5.5 Hz, 2H), 7.60 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.33 (t, J = 8.9
Hz, 2H), 7.30 (s, 1H), 7.25 (d, J = 6.9 Hz, 1H), 5.81 (dd, J = 11.7, 4.2 Hz, 1H), 3.83
1
6
, 400 MHz) δ:
7
(dd, J = 18.0, 11.7 Hz, 1H), 3.72 (s, 3H), 3.26 (dd, J = 18.0, 4.3 Hz, 1H), 2.26 (s, 3H).
+
MS (ESI) m/z: 414.05 (C20
H
17BrFN
3
O, [M+H] ). Anal. Calcd for C20
H17BrFN
3
O: C,
5
7.99; H, 4.14; N, 10.14. Found: C, 57.88; H, 4.14; N, 10.16.
18

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4
1
.1.25.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyraz
ol-1-yl)ethan-1-one (6u)
White powder. Yield: 48.5%. m. p. 200 ~ 201 °C. H NMR (DMSOꢀd
δ: 7.84 (d, J = 8.5 Hz, 2H), 7.60 (s, 1H), 7.54 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.7 Hz,
H), 7.29 (s, 1H), 7.25 (d, J = 8.7 Hz, 1H), 5.81 (dd, J = 11.7, 4.1 Hz, 1H), 3.82 (dd, J
1
6
, 400 MHz)
1
=
18.0, 11.8 Hz, 1H), 3.71 (s, 3H), 3.25 (dd, J = 18.0, 4.3 Hz, 1H), 2.27 (s, 3H). MS
+
(
ESI) m/z: 430.02 (C20
H
17BrClN
3
O, [M+H] ). Anal. Calcd for C20
H
17BrClN
3
O: C,
5
5.77; H, 3.98; N, 9.76. Found: C, 55.91; H, 3.97; N, 9.78.
4
1
.1.26.
-(5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyraz
ol-1-yl)ethan-1-one (6v)
White powder. Yield: 37.0%. m. p. 206 ~ 207 °C. H NMR (DMSOꢀd
δ: 7.77 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.6 Hz, 2H), 7.59 (s, 1H), 7.39 (d, J = 8.7 Hz,
H), 7.30 (s, 1H), 7.25 (d, J = 7.0 Hz, 1H), 5.81 (dd, J = 11.7, 4.3 Hz, 1H), 3.82 (dd, J
1
6
, 400 MHz)
1
=
18.0, 11.8 Hz, 1H), 3.72 (s, 3H), 3.25 (dd, J = 18.0, 4.4 Hz, 1H), 2.27 (s, 3H). MS
+
(ESI) m/z: 473.97 (C20
H17Br
2
N
3
O, [M+H] ). Anal. Calcd for C20
H17Br
2
N
3
O: C, 50.55;
H, 3.61; N, 8.84. Found: C, 50.42; H, 3.61; N, 8.86.
4
.2. Crystal structure determination
Crystal structure determination of compound 6o was carried out on a Nonius
CAD4 diffractometer equipped with graphiteꢀmono chromated MoKa (k = 0.71073 Å)
2
radiation. The structure was solved by direct methods and refined on F by fullꢀ
24
matrix leastꢀsquares methods using SHELXꢀ97.
All nonꢀhydrogen atoms of
compound 6o were refined anisotropically. All hydrogen atoms were placed in
geometrically idealized positions and constrained to ride on their respective parent
atoms.
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4
4
.3. Biological assays
.3.1. Anti-proliferation activity
Human nonsmall cell lung carcinoma (A549), human breast adenocarcinoma
MCFꢀ7), human liver hepatocellular carcinoma (HepG2) and human renal epithelial
cells (293T) were cultured in DMEM supplemented with 10% fetal bovine serum, 115
units/mL of penicillin G, and 115 g/mL of streptomycin, in 5% CO cell incubator at
7 °C. Then 100 L cell suspensions (1000 to 5000 cells) were added into each well
(
ꢀ
2
3
ꢀ
of 96ꢀwell plates depending on growth rate and allowed to adhere in the incubator.
After that, tested samples at preꢀset concentrations were added to 96 wells with
colchicine as positive reference. After 48h incubation, 10 ꢀL of PBS containing 5
mg/mL of MTT was added to each well. The plates were incubated for further 4 h and
then were centrifuged at 1500 rpm for 10 min to remove supernatant. After 150 mL of
DMSO was added to each well, the plates were shaken vigorously for 10 min to
ensure complete solubilization. The absorbance was measured and recorded on an
ELISA reader (ELx800, BioTek, USA) at a test wavelength of 490 nm. In all the
experiments three replicate wells were used for each drug concentration. Each assay
was carried out at least three times.
4
.3.2. Tubulin polymerization assay
2
5, 26
Tubulin was purified from bovine brain as described previously.
To
characterize the effect of the compounds on tubulin assembly in vitro, different
concentrations of compounds were incubated with 10 M bovine brain tubulin in
ꢀ
glutamate buffer at 30 °C and then cooled to 0 °C. After 0.4 mM GTP added, the
mixtures were transferred to cuvettes at 0 °C in a recording spectrophotometer and
warmed up to 30 °C. Finally, the assembly of tubulin was observed turbidimetrically
at 350 nm. The IC50 was defined as the compound concentration inhibited the extent
of assembly by 50% after 20 min incubation.
4
.3.3. Cell Cycle Analysis
20

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5
A549 cells were plated in a 6ꢀwell plate (5.0 × 10 cells/well) and incubated at
3
7 °C for 24 h. Then the cells were treated with 6q at 0, 0.02, 0.05, 0.1, 0.15 and 0.2
ꢀ
M for 24 h. Treated cells were washed with PBS and fixed with 70% iceꢀcold ethanol
overnight. Then, fixed cells were resuspended in PBS containing 0.1 mg/mL RNase A
and 5 g/mL propidium iodide (PI) and stained at 37 °C for 30 min. Cell cycle
ꢀ
distribution was analyzed by BD Accuri C6 Flow Cytometer (BD, USA). The
percentage of cells in the G1, S and G2/M phases of the cell cycle were determined
using the Flowjo 7.6.1 software after cell debris exclusion.
4
.3.4. Annexin-V Assay
A549 cells were plated into a 12ꢀwell plate (2.0 × 10 cells/well) and were
allowed to adhere for 12 h. After that, the cells were treated with 6q at final
concentrations of 0, 0.05, 0.1 and 0.15 M for 24 h. Then cells were collected and
washed twice with precooled PBS and strained with 10 L of Annexin VꢀFITC and 5
L of PI (5 g/mL) in 1 × binding buffer for 30 min in the dark. Apoptotic cells were
5
ꢀ
ꢀ
ꢀ
ꢀ
monitored on BD Accuri C6 Flow Cytometer (BD, USA). Statistics were analyzed by
Flowjo 7.6.1 software.
4
.3.5. Confocal microscopy assay
A549 cells were seeded on glass coverslips and incubated with 0.5 ꢀM paclitaxel,
0
.5 ꢀM colchicine and 0.2 ꢀM 6q, for 24 h respectively. Cells were then rinsed with
PBS for three times, fixed with 4% paraformaldehyde for 20 min and permeabilized
with cold formaldehyde for 10 min. After that, fixed cells were washed with PBS and
blocked with 5% Albumin from bovine serum (BSA) for at least 1 h. After washed
with PBS once, the cells were incubated with antiꢀtubulin antibody (1:500) in 3%
BSA overnight at 4 °C, followed by Cy3ꢀlabeled goat antiꢀmouse IgG (H + L) (1: 500)
in 3% BSA and incubated for 1 h. Subsequently, the cells were incubated with DAPI
(5 ng/mL). Images were captured with Olympus confocal microscope and data was
analyzed using FVꢀ10ꢀASW.
21

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4
.4. Docking simulations
Molecular docking of compound 6q into the three dimensional Xꢀray structure of
tubulin (PDB code: 1SA0) was carried out using the DS 3.5 (Discovery Studio 3.5) as
implemented through the graphical user interface DSꢀCDOCKER protocol. All bound
water and ligands were eliminated from the protein and the polar hydrogen was added.
The whole tubulin complex was defined as a receptor and the site sphere was selected
based on the ligand binding location of colchicine (CN2700), then the CN2700
molecule was removed and replaced by 6q during the molecular docking procedure.
Types of interactions of the docked protein with ligand were analyzed after the end of
molecular docking.
4
.5. 3D-QSAR
Ligandꢀbased 3DꢀQSAR approach was performed by QSAR software of DS 3.5.
The training sets consisted of inhibitors with the corresponding pIC50 values which
were converted from the obtained IC50 M), and test sets comprised compounds of
(ꢀ
data sets. All the definition of the descriptors could be seen in the “Help” of DS 3.5
software and they were calculated by QSAR protocol of DS 3.5. The alignment
conformation of each molecule was the one with lowest interaction energy in the
docked results of CDOCKER. The predictive ability of 3DꢀQSAR modeling can be
evaluated based on the crossꢀvalidated correlation coefficient, which qualifies the
predictive ability of the models. Scrambled test (Y scrambling) was performed to
investigate the risk of chance correlations. The inhibitory potencies of compounds
were randomly reordered for 30 times and subject to leaveꢀoneꢀout validation test
respectively. The models were also validated by test sets, in which the compounds
were not included in the training sets.
Acknowledgements
The work was financed by the Projects (Nos. CXY1409 & CG1305) from the
22

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Science & Technology Bureau of Lianyungang City of Jiangsu Province.
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Figure captions:
Table 1. Structures of compounds 6a-6v
Table 2. Crystallographic and experimental data for compound 6o
Table 3. In vitro inhibitory effects of compounds 6a-6v against human tumor cell
lines, normal cell line and tubulin polymerization
Table 4. The experimental and predicted inhibitory activity of compounds (6a-6v) by
3
DꢀQSAR models based upon active conformation achieved by molecular docking
Figure 1. Chemical structures of inhibitors and potential inhibitors of tubulin
polymerization
Figure 2. Crystal structure diagram of compound 6o. H atoms are shown as small
spheres of arbitrary radii.
Figure 3. Correlation between the antiꢀproliferative activity against A549 cell line and
the tubulin polymerization inhibitory activity of the top 10 compounds
Figure 4. Effect of compound 6q on cell cycle progression of A549 cells was
determined by flow cytometry analysis. (G1 phase, blue; S phase, yellow and G2/M
phase, green)
Figure 5. Representative Scatter Plot of A549 cells treated with 6q at 0, 0.05, 0.1 and
0
.15 ꢀM and analyzed by flow cytometry after double staining of the cells with
AnnexinꢀVꢀFITC and PI.
Figure 6. Effects of paclitaxel (0.5 ꢀM), colchicine (0.5 ꢀM) and 6q (0.2 ꢀM) on
cellular architecture and microtubule network of A549 cells. Microtubules tagged
with Cy3 (red) and nuclei tagged with DAPI (blue) were observed with a confocal
microscope.
Figure 7. The binding mode between the active conformation of compound 6q and
the target protein tubulin (PDB code: 1SA0) provided by the CDOCKER protocol
(
Discovery Studio 3.5, Accelrys, Co. Ltd). (A). The receptor surface model with
compound 6q; (B). 3D diagram of the interactions between colchicine (burlywood) &
q (cyan) and the colchicine binding site. Only interacting residues are displayed. The
Hꢀbond (green arrow) is displayed as dotted arrows; (C). 2D diagram of the
6
25

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interactions between colchicine and the colchicine binding site; (D). 2D diagram of
the interactions between compound 6q and the colchicine binding site. The Hꢀbond
(green arrow) is displayed as dotted arrows.
Figure 8. Comparing the predicted pIC50 value of tubulin inhibitory activities with
that of the experiment by linear fitting curve
Figure 9. (A). Isosurface of the 3DꢀQSAR model coefficients on electrostatic
potential grids. Blue represents positive coefficients; red represents negative
coefficients. (B). Isosurface of the 3DꢀQSAR model coefficients on Van der Waals
grids. Green represents positive coefficients; yellow represents negative coefficients.
Scheme 1. General synthesis of compounds 6a-6v
26

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Table 1. Structures of compounds 6a-6v
Compounds
R
1
R
2
R
3
R
4
R
5
6
6
6
a
H
H
H
H
H
H
H
H
H
H
H
H
OCH
OCH
H
H
OCH
OCH
OCH
Cl
H
H
H
OCH
H
b
c
3
3
3
3
3
6d
3
6
e
6f
H
H
H
Br
H
6
6
6
g
h
i
j
H
H
H
H
H
H
H
H
H
OCH
H
H
H
H
H
H
H
OCH
H
H
H
H
H
OCH
H
OCH
OCH
H
CF
H
OCH
OCH
F
Cl
Br
H
H
3
H
H
H
H
H
H
H
H
H
H
H
H
OCH
H
OCH
OCH
OCH
OCH
OCH
OCH
Br
Br
Br
Br
Br
3
3
3
3
3
3
3
3
6
3
6k
6
l
6m
6
6
6
6
n
o
p
q
3
3
H
OCH
OCH
OCH
F
3
3
3
6
6
6
r
s
t
3
3
Br
Br
3
H
6
u
v
Br
H
H
Cl
H
6
Br
H
H
Br
H
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Table 2. Crystallographic and experimental data for compound 6o
Compound
Formula
6o
21 3 2
C H20BrN O
Formula weight
Crystal system
Space group
a (Å)
426.31
Monoclinic
P 21/c
16.5763(14)
8.0259(7)
15.6946(13)
90
109.896(2)
90
1963.4(3)
4
1.442
b (Å)
c (Å)
o
α ( )
o
β
( )
o
γ ( )
3
V (Å )
Z
ꢀ3
D (g cm )
c
F (000)
872
θ rang (º)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mm )
Goodnessꢀofꢀfit on F
2.62ꢀ25.06
16844/3457
3457/0/247
2.114
ꢀ1
2
1.050
R
R
1
, wR
, wR
2
[I
>
2
σ
(I)]
0.0494, 0.1100
0.0821, 0.1265
0.578, ꢀ0.653
1
2
[all data]
ꢀ
3
Max, min ꢀρ (e Å )
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Table 3. In vitro inhibitory effects of compounds 6a-6v against human tumor cell
lines, normal cell line and tubulin polymerization
IC50±SD (ꢀM)
Compounds
a
a
a
b
c
A549
MCFꢀ7
HepG2
293T
105.41 ± 4.04 32.53 ± 1.97
129.37 ± 3.93 4.33 ± 0.24
Tubulin
6
6
a
5.18 ± 1.03
0.19 ± 0.02
4.52 ± 0.12
2.36 ± 0.23
1.68 ± 0.39
0.87 ± 0.11
1.24 ± 0.18
11.75 ± 1.29
0.22 ± 0.02
8.31 ± 0.41
4.92 ± 0.73
6.43 ± 0.14
1.42 ± 0.29
1.08 ± 0.27
0.71 ± 0.34
1.24 ± 0.26
0.15 ± 0.03
0.51 ± 0.01
0.31 ± 0.04
0.21 ±0.10
0.24 ±0.07
0.18 ± 0.01
0.21 ± 0.06
6.35 ± 0.33
0.22 ± 0.01
5.27 ± 0.68
3.03 ± 0.23
1.43 ± 0.05
1.46 ± 0.36
2.40 ± 0.31
13.21 ± 0.13
0.31 ± 0.11
10.09 ± 0.52
5.78 ± 0.07
8.12 ± 0.36
1.60 ± 0.27
1.51 ± 0.47
0.85 ± 0.13
0.97 ± 0.25
0.17 ± 0.05
0.55 ± 0.02
0.38 ± 0.09
0.19 ± 0.04
0.27 ± 0.12
0.20 ± 0.04
0.22 ± 0.12
9.22 ± 1.98
0.26 ± 0.08
5.46 ± 0.29
3.12 ± 0.20
2.24 ± 0.08
1.71 ± 0.11
1.92 ± 0.56
b
6
c
234.10 ± 4.50 23.19 ± 1.46
269.48 ± 3.84 11.45 ± 0.53
6
d
6
e
48.68 ± 6.05
>300
22.02 ± 1.19
14.32 ± 0.41
6
f
6
6
g
139.34 ± 0.82 26.14 ± 0.47
h
14.09 ± 0.15 298.46 ± 3.19 34.96 ± 1.34
6
i
0.34 ± 0.09
9.76 ± 1.78
6.63 ± 0.17
7.93 ± 0.16
1.88 ± 0.24
1.20 ± 0 03
0.92 ± 0.13
1.14 ± 0.21
0.25 ± 0.05
0.54 ± 0.09
0.42 ± 0.06
0.29 ± 0.01
0.34 ± 0.04
0.26 ± 0.05
0.32 ± 0.09
243.59 ± 6.11
5.75 ± 0.17
6
j
187.09 ± 1.41 19.62 ± 0.55
168.46 ± 5.85 13.05 ± 0.20
6
k
6
l
>300
19.76 ± 1.95
6
m
134.51 ± 1.89 10.39 ± 0.18
6
n
o
p
q
32.79 ± 0.68
>300
20.54 ± 0.40
15.92 ± 0.81
6
6
6
205.86 ± 2.67 29.64 ± 2.68
245.92 ± 3.44
89.18 ± 2.52
>300
1.98 ± 0.25
10.03 ± 0.50
7.81 ± 0.39
4.22 ± 0.13
5.85 ± 0.37
3.16 ± 0.42
2.35 ± 0.34
6
r
6
6
s
t
100.23 ± 4.67
79.68 ± 2.88
46.46 ± 1.34
172 ± 5.52
6
u
v
6
d
Colchicine
All experiments were independently performed at least three times.
a
Inhibition of the growth of tumor cell lines
b
Inhibition of the growth of nomal cell line
c
Inhibition of tubulin polymerization
d
Used as positive control
29