Synthesis and Biological Evaluation of 1-Methyl-1H-indole–Pyrazoline Hybrids as Potential Tubulin Polymerization Inhibitors

Article abstract of DOI:10.1002/cmdc.201600137

A series of 1-methyl-1H-indole–pyrazoline hybrids were designed, synthesized, and biologically evaluated as potential tubulin polymerization inhibitors. Among them, compound e19 [5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide] showed the most potent inhibitory effect on tubulin assembly (IC₅₀=2.12 μm) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC₅₀values of 0.21–0.31 μm). Further studies confirmed that compound e19 can induce HeLa cell apoptosis, cause cell-cycle arrest in G₂/M phase, and disrupt the cellular microtubule network. These studies, along with molecular docking and 3D-QSAR modeling, provide an important basis for further optimization of compound e19 as a potential anticancer agent.

Full text of DOI:10.1002/cmdc.201600137

DOI: 10.1002/cmdc.201600137
Full Papers
Synthesis and Biological Evaluation of 1-Methyl-1H-indole–
Pyrazoline Hybrids as Potential Tubulin Polymerization
Inhibitors
Ya-Liang Zhang+, Ya-Juan Qin+, Dan-Jie Tang, Meng-Ru Yang, Bo-Yan Li, Yan-Ting Wang,
Hong-Yu Cai, Bao-Zhong Wang,* and Hai-Liang Zhu*[a]
A series of 1-methyl-1H-indole–pyrazoline hybrids were de-
signed, synthesized, and biologically evaluated as potential tu-
bulin polymerization inhibitors. Among them, compound e19
[5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole-1-carboxamide] showed the most
potent inhibitory effect on tubulin assembly (IC50 =2.12 mm)
and in vitro growth inhibitory activity against a panel of four
human cancer cell lines (IC50 values of 0.21–0.31 mm). Further
studies confirmed that compound e19 can induce HeLa cell
apoptosis, cause cell-cycle arrest in G2/M phase, and disrupt
the cellular microtubule network. These studies, along with
molecular docking and 3D-QSAR modeling, provide an impor-
tant basis for further optimization of compound e19 as a po-
tential anticancer agent.
Introduction
Microtubules (MTs) are a broadly exploited target for the devel-
opment of new effective anticancer agents.[1–3] MTs are highly
dynamic polymers mainly composed of a,b-tubulin heterodim-
ers which turn over continuously. The polymerization–depoly-
merization dynamics play a crucial role in various fundamental
cellular processes including mitotic spindle formation, organi-
zation of intracellular structure and intracellular transport, as
well as cell signaling and secretion.[4] Hence, disorder of MTs
function would block cell division to cause cell damage, which
is an established strategy to inhibit the proliferation of cancer
cells.[2,3,5]
In the last two decades, an ever increasing number of syn-
thetic compounds involving indoles have been reported to in-
hibit tubulin polymerization, such as compound 3 (ATIs),[11]
4
(BPROL075)[12] and 5[13] (Figure 1). The indole structure is a cru-
cial structural motif for tubulin inhibition. The indole nucleus
also has been reported to be one of the core nucleuses for
potent inhibitors of tubulin polymerization.[14,15] Introduction of
a methyl group to the N atom of indole contributes to better
inhibition of tubulin polymerization and cytotoxicity.[16,17]
Pyrazolines have gained prominence due to exhibiting
a broad spectrum of biological activities, as well as producing
useful intermediates in several organic preparations. Pyrazoline
derivatives have been reported to have anticancer, antimicrobi-
al and anti-inflammatory activities.[18,19] In addition, the alterna-
tive actions of pyrazolines connected with angiogenesis and
apoptotic mechanisms seem to be promising in anticancer
therapy.[20,21] Some pyrazoline derivatives can also inhibit tubu-
lin polymerization by binding to the colchicine site, such as
compound 6 (Figure 1).
A large number of agents could bind to tubulin and inter-
fere with microtubule assembly. Colchicine (compound 1,
Figure 1), one of the earliest tubulin-targeting drugs, could ef-
fectively obstruct microtubule polymerization. Its binding site
on tubulin has been characterized already.[6,7] However, the
clinical development of colchicine for cancer therapy was not
successful due to its highly adverse side effects.[8] Another
well-known anti-tubulin agent is CA-4 (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 multiple
human cancer cell lines.[9] But CA-4 easily undergoes cis–trans
isomerization under natural light, causing a sharp decrease in
anti-tubulin activity, which limits its further applications.[10] De-
spite a long history of characterization, colchicine binding site
inhibitors have not yet reached the commercial phase as anti-
cancer drugs. Therefore, further studies are required.
So far, only a few studies have been dedicated to the design
and synthesis of anticancer compounds containing both 1-
methyl-1H-indole and pyrazoline. We attempted to integrate
these two groups to screen new basic scaffolds for the design
of a series of novel tubulin assembling inhibitors (compounds
e1–e28, Figure 1) based on the previous researches and mo-
lecular modeling studies. These coupling structures might pos-
sess enhanced anticancer activity. A series of novel 1-methylin-
dol derivatives were recently reported as potent anticancer
agents targeting tubulin by our research group.[22] Here we
search more novel agents to extend our research and gain fur-
ther insight into the ensuing structure–activity relationships
(SARs). Our strategy was to perform modifications on the two
aromatic rings (A and B ring), while the new combined scaffold
[a] Y.-L. Zhang,+ Y.-J. Qin,+ D.-J. Tang, M.-R. Yang, B.-Y. Li, Y.-T. Wang, H.-Y. Cai,
Prof. B.-Z. Wang, Prof. Dr. H.-L. Zhu
State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210023 (P.R. China)
E-mail: zhuhl@nju.edu.cn
[+] These authors contributed equally to this work.
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Figure 1. Structures of known and potential inhibitors of tubulin polymerization.
was identified as the minimum structural requirement for activ-
ity in this new series of compounds.
In this study, we describe the synthetic method and the bio-
activities of this series of compounds. Docking simulations
were also performed to explore the binding modes of these
compounds at the active site of tubulin.
Results and Discussion
Chemistry
Compounds e1–e28 (Table 1) were synthesized by a four-step
procedure outlined in Scheme 1. First of all, three substituted
1H-indoles (a1–a3) in DMF were treated with phosphorus oxy-
Scheme 1. General synthesis of compounds e1–e28. Reagents and condi-
tions: a) DMF, POCl3, 08C, 2 h; b) CH3I, NaH, THF, 08C!RT, 24 h; c) substitut-
ed acetophenone, EtOH, KOH, RT, 24 h; d) aminourea hydrochloride, EtOH,
NaOH, reflux.
Table 1. Structures of compounds e1–e28.
chloride in DMF to generate compounds b1–b3. Introduction
of a methyl group to the N atom of indole was obtained by
substituted indole-3-carbaldehydes (b1–b3) in THF reacting
with sodium hydride and iodomethane. Subsequently, the ob-
tained compounds (c1–c3) were treated with the correspond-
ing acetophenone in the presence of potassium hydroxide in
ethanol to give d1–d28 in yields of 34.2–93.1%. Finally, the
mixture of compounds (d1–d28) and aminourea hydrochloride
in ethanol were held at reflux under stirring and treated with
aqueous sodium hydroxide to get compounds e1–e28. The re-
lated crude compounds were purified by recrystallization. All
synthesized compounds e1–e28 are reported for the first time
and were characterized by 1H NMR spectroscopy, melting
point, ESI-MS and elemental analysis, in accordance with their
depicted structures.
Compd
R1
R2
Compd
R1
R2
e1
e2
e3
e4
e5
e6
e7
e8
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
Br
H
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
4-OCH3
3-OCH3
2-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
4-F
3-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
4-Cl
4-Br
3-Cl
4-Cl
4-Br
H
e9
3-OCH3
3,4-(OCH3)2
3,4,5-(OCH3)3
4-F
4-Cl
H
4-CH3
e10
e11
e12
e13
e14
4-CF3
3-Cl
3-CF3
Crystal structures of compounds e1 and e8
The crystal structures of compounds e1 and e8 were deter-
mined by X-ray diffraction analysis. The crystal data presented
in Table 2 and Figure 2 gave perspective views of compounds
3-F,4-OCH3
2-F,4-OCH3
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Table 2. Crystallographic and experimental data for compounds e1 and
e8.
Table 3. In vitro cell growth inhibitory effects of compounds e1–e28
against human cancer cell lines.
Parameter
e1
e8
Compd
IC50 [mm][a]
MCF-7
HeLa
>10
A549
>10
HepG2
>10
Formula
Formula weight [Da]
C19H18N4O
318.37
C20H20N4O2
348.4
e1
>10
Crystal system
Orthorhombic
Pbca
16.0750(14)
10.1661(9)
20.0996(18)
90
90
90
3284.7(5)
8
1.288
0.083
1344
2.53–25.10
2909
2249
Monoclinic
P21/c
11.5014(7)
16.8971(10)
9.9298(6)
90
113.895(2)
90
1764.36(18)
4
1.312
0.087
736
2.41–25.32
3203
e2
e3
e4
e5
e6
e7
e8
e9
2.13ꢁ0.03
3.30ꢁ0.07
1.43ꢁ0.02
4.06ꢁ0.11
2.45ꢁ0.02
2.99ꢁ0.01
4.97ꢁ0.03
1.56ꢁ0.06
4.24ꢁ0.02
2.68ꢁ0.02
5.63ꢁ0.06
0.49ꢁ0.01
0.46ꢁ0.05
Space group
a [ꢁ]
b [ꢁ]
c [ꢁ]
4.29ꢁ0.05
1.68ꢁ0.03
3.74ꢁ0.05
>10
>10
0.52ꢁ0.09
>10
>10
>10
a [o]
0.38ꢁ0.09
>10
0.63ꢁ0.13
>10
0.39ꢁ0.07
>10
b [o]
g [o]
3.12ꢁ0.04
4.36ꢁ0.02
1.83ꢁ0.03
3.19ꢁ0.03
3.94ꢁ0.26
1.92ꢁ0.09
2.93ꢁ0.07
4.78ꢁ0.01
2.09ꢁ0.02
3.45ꢁ0.09
3.03ꢁ0.03
2.41ꢁ0.02
V [ꢁ3]
Z
e10
e11
e12
e13
e14
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
colchicine[b]
Dc [gcmꢀ3
]
>10
>10
2.55ꢁ0.13
8.43ꢁ0.02 >10
>10
>10
3.24ꢁ0.02
>10
>10
2.80ꢁ0.04
>10
>10
3.57ꢁ0.06
m [mmꢀ1
F [000]
q [o]
]
9.46ꢁ0.07
0.28ꢁ0.03
1.73ꢁ0.02
0.92ꢁ0.02
0.26ꢁ0.03
0.43ꢁ0.09
0.73ꢁ0.05
2.49ꢁ0.03
6.47ꢁ0.08
0.36ꢁ0.07
0.29ꢁ0.03
0.32ꢁ0.03
1.44ꢁ0.23
0.41ꢁ0.04
0.27ꢁ0.04
9.81ꢁ0.08
0.31ꢁ0.09
1.23ꢁ0.03
0.43ꢁ0.01
0.31ꢁ0.02
0.51ꢁ0.02
0.63ꢁ0.08
2.79ꢁ0.01
7.45ꢁ0.27
0.45ꢁ0.01
0.41ꢁ0.01
0.43ꢁ0.03
1.53ꢁ0.17
1.05ꢁ0.07
0.37ꢁ0.15
Reflections collected
Reflections unique
Goodness-of-fit on F2
R1, wR2 [I>2s(I)]
R1, wR2 [all data]
Max, min D1 [eꢁꢀ3
0.25ꢁ0.04
1.14ꢁ0.11
0.77ꢁ0.01
0.21ꢁ0.05
0.52ꢁ0.04
0.58ꢁ0.07
2.56ꢁ0.02
7.83ꢁ0.12
0.31ꢁ0.03
0.28ꢁ0.02
0.33ꢁ0.04
1.05ꢁ0.05
0.47ꢁ0.02
0.34ꢁ0.12
0.32ꢁ0.03
2444
1.045
0.0464, 0.1117
0.0665, 0.1243
0.189, ꢀ0.180
1.52ꢁ0.08
0.59ꢁ0.05
0.29ꢁ0.04
0.68ꢁ0.12
0.82ꢁ0.01
3.01ꢁ0.05
7.22ꢁ0.05
0.64ꢁ0.04
0.51ꢁ0.05
0.57ꢁ0.06
1.42ꢁ0.14
0.90ꢁ0.05
0.42ꢁ0.08
1.03
0.0404, 0.0957
0.0584, 0.1066
0.152, ꢀ0.141
]
e1 and e8 with the atomic labeling system. CCDC 1416450
(e1) and 1416449 (e8) contain the supplementary crystallo-
graphic data for this paper. These data are provided free of
charge by The Cambridge Crystallographic Data Centre.
[a] Values are the average ꢁSD of at least three separate determinations.
[b] Used as positive control.
Biological activities
In vitro antiproliferative activity
The synthesized compounds were evaluated by MTT method
for antiproliferative activity against four different human
cancer cell lines including HeLa (human cervical carcinoma),
MCF-7 (breast adenocarcinoma), A549 (human non-small-cell
lung carcinoma) and HepG2 (human hepatocellular carcinoma).
The results are summarized in Table 3. Some of these com-
pounds displayed excellent in vitro growth inhibitory effects
especially for HeLa cells. Among them, compound e19 could
inhibit HeLa, MCF-7, A549 and HepG2 cells with IC50 values of
0.21, 0.29, 0.26, and 0.31 mm, respectively, similar to the posi-
tive control compound colchicine (Figure 3). The following
summarizes the SARs of these derivatives:
When there was the same substituent on the B ring, com-
pounds with different substituent groups on the A ring were
compared. It was clear that the activity of the tested com-
pounds with different substituents on the A ring increased in
the following order: Br>H>OCH3, which could be observed
from the comparisons: e19>e4>e11, e16>e2>e9, e18>
e3>e10.
Figure 2. Crystal structure diagrams of compounds e1 and e8. H atoms are shown as small spheres of arbitrary radii.
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Table 4. In vitro inhibition of tubulin polymerization by compounds e1–
e28.
Compd
IC50 [mm][a]
Compd
IC50 [mm][a]
e1
e2
e3
e4
e5
e6
e7
e8
39.20ꢁ0.22
17.03ꢁ1.09
11.49ꢁ0.13
4.51ꢁ0.14
8.64ꢁ0.90
23.85ꢁ0.54
11.94ꢁ0.31
40.02ꢁ0.15
29.32ꢁ0.26
22.87ꢁ1.02
6.45ꢁ0.31
37.82ꢁ0.37
41.34ꢁ0.73
8.29ꢁ0.06
2.78ꢁ0.43
e15
e16
e17
e18
e19
e20
e21
e22
e23
e24
e25
e26
e27
e28
35.32ꢁ0.10
2.44ꢁ0.21
9.62ꢁ0.42
6.34ꢁ0.27
2.12ꢁ0.17
4.21ꢁ0.83
4.73ꢁ0.17
9.29ꢁ1.57
33.34ꢁ0.16
3.08ꢁ0.40
2.53ꢁ0.23
2.65ꢁ0.31
10.13ꢁ0.25
3.19ꢁ0.90
e9
e10
e11
e12
e13
e14
colchicine[b]
[a] Values are the average ꢁSD of at least three separate determinations.
Figure 3. Dose–response curves for cytotoxicity in HeLa, MCF-7, A549 and
HepG2 cells treated with compound e19 for 48 h. Results shown are the
mean ꢁSD of at least three separate determinations.
[b] Used as positive control.
Compounds with different substituent groups on the B ring
were compared in a similar way. The inhibitory activities of
these compounds with different substituents on the B ring in-
creased in the following order: e16>e17>e15, e25>e21,
e26>e24. Therefore, a conclusion could be drawn that com-
pounds with a meta-substituted group showed more potent
activities than those of ortho-substituted, while para-substitut-
ed compounds had the lowest activities. With respect to meta-
position, it could be found that electron-donating groups were
better than electron-withdrawing groups, and this could be
observed from the comparisons: e16>e25>e26, e18>e27.
However, things were not the same when it turned to para-po-
sition, if we draw attention to the comparisons: e24>e20>
e21>e22, e14>e23>e15. It seemed that electron-withdraw-
ing groups on this position were better. Moreover, among all
the substituents on the B ring, 3,4,5-(OCH3)3 had the dominant
position.
Figure 4. Tubulin assembly time-course plots following changes in OD350 of
reaction mixtures in the presence of increasing concentrations of compound
e19 (data are from a single representative experiment).
Inhibition of tubulin polymerization
The ability of compounds e1–e28 to inhibit tubulin polymeri-
zation was evaluated in vitro to investigate whether the anti-
proliferative activity of these agents resulted from an interac-
tion with tubulin (Table 4). The IC50 values of these compounds
displayed a similar tendency with their related IC50 values in
the antiproliferative assay. Compound e19 showed the most
potent activity of anti-tubulin polymerization with IC50 value of
2.12 mm. The tubulin assembly time-course plots in OD350 of re-
action mixtures in the presence of compound e19 are shown
in Figure 4. Therefore, it could be concluded that most of the
synthesized compounds could inhibit tubulin assembly and
the antiproliferative effects might be produced partly by an in-
teraction with tubulin.
Compound e19 induces HeLa cell apoptosis in a dose- and
time-dependent manner
The mode of cell death induced by compound e19 was inves-
tigated by flow cytometry of cells double stained with Annex-
in V–FITC and PI.[23] HeLa cells were treated with e19 (0.05, 0.1,
0.2 or 0.3 mm) for 24 h. The percentage of apoptotic cells in-
creased with the concentration of e19 (Figure 5A). Meanwhile,
HeLa cells were treated with e19 (0.2 mm) for 0, 12, 24 or 36 h.
It was found that e19 induced cell apoptosis in HeLa cells in
a time-dependent manner (Figure 5B). Thus, it could be con-
cluded that compound e19 could induce cell apoptosis like
other tubulin binding agents.
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Figure 5. Representative scatter plot of HeLa cells treated with e19 and analyzed by flow cytometry after double staining of the cells with Annexin V–FITC
and PI (at least three separate determinations). A) HeLa cells treated with e19 (0, 0.05, 0.1, 0.2 or 0.3 mm) for 24 h were collected and analyzed; B) HeLa cells
treated with e19 (0.2 mm) for various times (0, 12, 24 or 36 h) were collected and analyzed. The early apoptotic cells are located in the lower right quadrant
(Annexin V–FITC+/PIꢀ cells), and the late apoptotic cells are in the upper right quadrant (Annexin V–FITC+/PI+ cells); *p<0.05, **p<0.01.
Compound e19 induces HeLa cell-cycle arrest in a dose- and
time-dependent manner
36 h or 48 h exposure. Thus, it was established that compound
e19 could induce cell-cycle arrest in mitosis.
The effect of compound e19 on cell cycle in HeLa cells was
tested by flow cytometry. HeLa cells were treated with e19 at
various doses (0.05, 0.1, 0.2, or 0.3 mm) for 24 h, or with e19
(0.3 mm) for various times (0, 6, 12, 24, 36, or 48 h) (Figure 6).
The results demonstrate that compound e19 induced a gradual
accumulation of HeLa cells in G2/M phase in a dose-dependent
manner (Figure 6A). The percent of G2/M cells increased when
treated at a dose as low as 0.05 mm, while at the highest con-
centration (0.3 mm), more than 50% of the cells were arrested
in G2/M. The cell cycle arrested in G2/M phase, accompanied by
a reduction of cells in G1 phase. Figure 6B shows that com-
pound e19 treatment resulted in accumulation of cells in G2/M
phase starting with a 6 h exposure, with peak accumulation
observed by 24 h. However, there was substantial cell apopto-
sis or death after 24 h treatment, leading to a reduction of
cells in G2/M phase and an increase of cells in S phase after
Compound e19 inhibits tubulin polymerization and disrupts
the microtubule system
HeLa cells were treated with e19 (0.2 mm) for 24 h and micro-
tubules were analyzed by immunocytochemistry staining. As
shown in Figure 7, the microtubule network of control cells
displayed normal arrangement and organization, which was
slim and fibrous. After a 24 h treatment, microtubules became
short and wrapped around the nucleus. There was also a signif-
icant reduction in the density of cellular microtubules and the
radial organization of microtubules was perturbed. This phe-
nomenon could also be found in colchicine (0.5 mm)-treated
cells, which were used as the positive control in this assay. In
contrast, paclitaxel (0.5 mm) dramatically enhanced microtubule
polymerization, with an increase in the density of cellular mi-
crotubules and the formation of long thick microtubule bun-
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Figure 6. Effect of compound e19 on cell-cycle progression of HeLa cells was determined by flow cytometry analysis (at least three separate determinations).
A) HeLa cells treated with e19 (0, 0.05, 0.1, 0.2 or 0.3 mm) for 24 h; B) HeLa cells treated with e19 (0.3 mm) for various times (0, 6, 12, 24, 36 or 48 h). (G1 phase,
green; S phase, yellow and G2/M phase, blue); *p<0.05, **p<0.01.
dles. These results further confirmed that the effects were due
to an interaction with tubulin, resulting in a prolonged mitotic
arrest that ultimately leads to cell death.
ded into the colchicine binding pocket via hydrophobic inter-
actions and the binding was stabilized by two hydrogen
bonds. The enzyme surface model (Figure 8A) revealed that
the A ring of e19 was well embedded in the active pocket via
hydrophobic interactions. The oxygen atom of the acid amides
of compound e19 formed one hydrogen bond with the hydro-
gen atom of the hydroxy group of Ser A: 178 (angle OꢀH···O=
134.68, distance=2.49 ꢁ). The oxygen atom of one of the me-
thoxy groups on the A ring of e19 formed another hydrogen
bond with the amino hydrogen of Asn A: 178 (angle NꢀH···O=
142.78, distance=2.46 ꢁ) (Figure 8B, C).
Computational studies
Molecular docking
The interaction of compounds e1–e28 with tubulin crystal
structure (PDB ID: 1SA0) was examined by a docking study
using the DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). This
study was conducted by inserting the compounds into the col-
chicine binding site of tubulin. The results of the CDOCKER_IN-
TERACTION_ENERGY of the synthesized compounds ranged
from ꢀ53.73 to ꢀ40.85 kcalmolꢀ1. Among them, compound
e19 showed the lowest interaction energy, at ꢀ53.73 kcal
molꢀ1. The binding modes between compound e19 and tubu-
lin are depicted in Figure 8.
3D-QSAR model
A 3D-QSAR model was built to obtain a systematic SAR profile
on 1-methyl-1H-indole–pyrazoline hybrids as antitumor agents
and explore more potent and selective tubulin inhibitors. This
study was performed in the built-in QSAR software of DS 3.5
by using the corresponding pIC50 values converted from the
obtained IC50 values (mm) of tubulin inhibition. The training
The amino acid residues interacting with tubulin were la-
beled. In the binding mode, compound e19 was well embed-
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Table 5. The experimental and predicted inhibitory activity of com-
pounds e1–e28 by 3D-QSAR models based on active conformation in
molecular docking.
Compd
Tubulin assembly pIC50
Actual Predicted
Residual error
e1
e2
e3
e4
e5
e6
e7
4.41
4.77
4.94
5.35
5.06
4.62
4.92
4.40
4.53
4.64
5.19
4.42
4.38
5.08
4.45
5.61
5.02
5.20
5.67
5.38
5.33
5.03
4.48
5.51
5.60
5.58
4.99
5.50
4.44
5.04
4.95
5.36
5.05
4.83
4.95
4.53
4.70
4.63
5.20
4.40
4.37
5.00
4.63
5.35
5.05
5.19
5.86
5.36
5.45
5.01
4.68
5.50
5.59
5.56
4.97
5.51
ꢀ0.03
ꢀ0.27
ꢀ0.01
ꢀ0.01
0.01
ꢀ0.21
ꢀ0.03
ꢀ0.13
ꢀ0.17
0.01
ꢀ0.01
0.02
0.01
0.08
ꢀ0.18
0.26
ꢀ0.03
0.01
ꢀ0.19
0.02
ꢀ0.12
0.02
ꢀ0.20
0.01
0.01
0.02
0.02
ꢀ0.01
e8[a]
e9[a]
e10
e11
e12
e13
e14[a]
e15[a]
e16[a]
e17
e18
e19
e20
e21
e22
e23[a]
e24
e25
e26
e27
e28
Figure 7. Effects of e19 (0, 0.2 mm), colchicine (0.5 mm) or paclitaxel (0.5 mm)
on cellular architecture and microtubule network of HeLa cells. Microtubules
tagged with Cy3 (red) and nuclei tagged with DAPI (blue) were observed
with a confocal microscope.
[a] Compounds in the test set; the remainders were used for training.
and test sets were divided by the random diverse molecules
method. The training set accounted for 79% of all the mole-
cules, whereas the test was set at 21%, with 22 and 6 agents,
respectively. The observed and predicted values, correspond-
ing residual values for the training set and test set molecules
in the 3D-QSAR model are presented in Table 5. The graphical
relationship of observed and predicted values is shown in
Figure 9, in which the plot of the observed IC50 versus the pre-
dicted values showed that this model could be used to predict
the activity of 1-methyl-1H-indole–pyrazoline hybrids.
Conclusions
In this study, a series of 1-methyl-1H-indole–pyrazoline hybrids
(e1–e28) were designed, synthesized and evaluated as poten-
tial inhibitors of tubulin polymerization. Most of them showed
potent bioactivity, and compound e19 displayed the most
potent activity against tubulin assembly (IC50 =2.12 mm) and in
vitro growth inhibitory activity against HeLa, MCF-7, A549, and
HepG2 cells with IC50 values of 0.21, 0.29, 0.26, and 0.31 mm, re-
spectively. The mechanism of action studies proved that com-
pound e19 was a potent inducer of apoptosis in HeLa cells
and caused accumulation of cells in G2/M phase of the cell
cycle, as well as severe disruption of the microtubule system.
In addition, molecular docking and 3D-QSAR studies were per-
formed to learn the inhibitor-tubulin interactions. According to
the analysis of the binding modes between compound e19
and tubulin, several interactions were observed with the pro-
tein residues in the colchicine binding site, which may contrib-
ute to its anti-tubulin polymerization and antiproliferative ac-
tivity. The research of these well designed compounds might
be greatly helpful for the development of tubulin polymeri-
zation inhibitors with better activities in the future.
The molecules aligned with the isosurfaces of the 3D-QSAR
model coefficients on electrostatic potential grids (Figure 10A)
and van der Waals grids (Figure 10B) are also listed. The elec-
trostatic map indicates red contours around regions where
high electron density (negative charge) was expected to in-
crease activity, while blue contours represent areas where low
electron density (partial positive charge) was expected to in-
crease activity. Similarly, the steric map indicates areas where
steric bulk was predicted to increase (green) or decrease
(yellow) activity. It is widely accepted that a better inhibitor
based on the 3D-QSAR model should have strong van der
Waals attraction in the green areas and a polar group in the
blue electrostatic potential areas (which are 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 future studies.
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Figure 8. The binding modes between the active conformation of compound e19 and the target protein tubulin (PDB ID: 1SA0) provided by the CDOCKER
protocol (Discovery Studio 3.5). A) The receptor surface model with compound e19. B) 3D diagram of the interaction between compound e19 and the colchi-
cine binding site. Only interacting residues are displayed. The hydrogen bond (green) is displayed as dotted arrows. C) 2D diagram of the interaction between
compound e19 and the colchicine binding site. The hydrogen bond (blue) is displayed as dotted arrows.
Experimental Section
Chemistry
General: All the chemical solvents and reagents used in this study
were analytically pure without further purification. Melting points
were determined on a XT4MP apparatus (Taike Corp, Beijing,
1
China). H NMR spectra were recorded on a Bruker DPX300 model
Spectrometer using tetramethylsilane (TMS) as internal standard
and DMSO or CDCl3 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). ESI-MS
spectra were recorded on a Mariner System 5304 Mass spectrome-
ter. Elemental analyses were performed on a CHN-O-Rapid instru-
ment and were within ꢁ0.4% of the theoretical values.
General procedure for the synthesis of 1H-indole-3-carbalde-
hyde (b1): Compound a1 (35 mmol) in DMF (40 mL) was added to
a stirred and completely mixed solution of POCl3 (20 mL) in DMF
(40 mL) at 08C. After 2 h reaction, the mixture was poured into
300 mL ice water and mixed completely. Then NaOH pellets were
added to adjust the pH to 9.0. The obtained mixture was filtered,
Figure 9. Comparing the predicted pIC50 values of tubulin inhibitory effects
with those determined experimentally by linear fitting.
&
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(E)-3-(5-bromo-1-methyl-1H-indol-3-yl)-1-(3-(trifluoromethyl)phe-
nyl)prop-2-en-1-one (d26): Yellow powder, yield 67.9%; mp: 165–
1
1678C; H NMR (400 MHz, DMSO): d=8.48 (d, J=7.8 Hz, 1H), 8.34
(s, 1H), 8.30 (d, J=1.7 Hz, 1H), 8.25 (s, 1H), 8.04 (t, J=12.8 Hz, 2H),
7.83 (t, J=7.8 Hz, 1H), 7.68 (d, J=15.5 Hz, 1H), 7.57 (d, J=8.7 Hz,
1H), 7.48–7.42 (m, 1H), 3.87: (s, 3H); MS(ESI): 409.0 [M+H]+; Anal.
Calcd for C19H13BrF3NO: C 55.90, H 3.21, N 3.43, found: C 55.91, H
3.20, N 3.42.
(E)-3-(5-bromo-1-methyl-1H-indol-3-yl)-1-(3-fluoro-4-methoxy-
phenyl)prop-2-en-1-one (d27): Yellow powder, yield 57.8%; mp:
1
191–1928C; H NMR (400 MHz, DMSO): d=8.28 (d, J=1.6 Hz, 1H),
8.18 (s, 1H), 8.04 (d, J=8.7 Hz, 1H), 8.01–7.91 (m, 2H), 7.63 (d, J=
15.5 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.46–7.40 (m, 1H), 7.34 (t, J=
8.6 Hz, 1H), 3.96 (s, 3H), 3.86 ppm (s, 3H); MS(ESI): 389.0 [M+H]+;
Anal. Calcd for C19H15BrFNO2: C 58.78, H 3.89, N 3.61, found: C
58.79, H 3.88, N 3.60.
Figure 10. A) Isosurface of the 3D-QSAR model coefficients on electrostatic
potential grids. Blue represents positive coefficients; red represents negative
coefficients. B) Isosurface of the 3D-QSAR model coefficients on van der
Waals grids. Green represents positive coefficients; yellow represents nega-
tive coefficients.
(E)-3-(5-bromo-1-methyl-1H-indol-3-yl)-1-(2-fluoro-4-methoxy-
phenyl)prop-2-en-1-one (d28): Yellow powder, yield 64.6%; mp:
1
and washed with ethyl acetate. Then the filtrate was extracted by
ethyl acetate. Finally, the organic layer was dried over Na2SO4 and
concentrated to give a brown solid, yield 98.1%. The crude b1 was
used in the next step without any further purification. Following
this way, crude compounds b1–b3 were obtained with yields of
95.3–98.1%.
193–1958C; H NMR (400 MHz, DMSO): d=8.28 (d, J=1.6 Hz, 1H),
8.18 (s, 1H), 8.04 (d, J=8.7 Hz, 1H), 8.01–7.91 (m, 2H), 7.63 (d, J=
15.5 Hz, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.46–7.40 (m, 1H), 7.34 (t, J=
8.6 Hz, 1H), 3.96 (s, 3H), 3.86 ppm (s, 3H); MS(ESI): 389.0 [M+H]+;
Anal. Calcd for C19H15BrFNO2: C 58.78, H 3.89, N 3.61, found: C
58.79, H 3.90, N 3.60.
General procedure for the synthesis of 5-(1-methyl-1H-indol-3-
yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (e1): A mix-
ture of d1 (1 mmol) and aminourea hydrochloride (3 mmol) was
held at reflux in ethanol (10 mL) under vigorous stirring. After the
reactants were completely dissolved, a solution of 0.1 g NaOH
(2.5 mmol) in water (2 mL) was added slowly. After the reaction
was complete, the reaction was brought to room temperature and
the solution was removed. It was diluted with water and extracted
with ethyl acetate. Then the organic layer was washed with water
(three times), dried (Na2SO4), evaporated and recrystallized from
ethanol and dichloromethane to give e1, a white powder, yield
21.9%. Using this method, pure compounds e1–e28 were ob-
tained with yields of 10.1–73.0%.
General procedure for the synthesis of 1-methyl-1H-indole-3-
carbaldehyde (c1): NaH (62.5 mmol) in THF (15 mL) was added
slowly to a stirred solution of b1 (25 mmol) in THF (15 mL) at 08C,
then after 15 min stirring, CH3I (33 mmol) was added. The reaction
mixture was moved to room temperature and allowed to continue
for 24 h, after which it was deemed essentially complete by TLC.
Then the solution was removed and the mixture was extracted by
ethyl acetate. The organic layer was washed with brine, dried over
Na2SO4 and concentrated to give crude residue, c1, yield 97.5%.
Similarly, crude compounds c1–c3 were obtained with yields of
93.4–97.5%.
General procedure for the synthesis of (E)-3-(1-methyl-1H-indol-
3-yl)-1-phenylprop-2-en-1-one (d1): c1 (2 mmol) and acetophe-
none (2 mmol) were dissolved in ethanol (20 mL) and KOH
(6 mmol, 336 mg) was added. After the reaction was complete
(24 h), the reaction mixture was filtered and washed with water
and cool ethanol. The crude product was purified by recrystalliza-
tion from ethanol and dichloromethane to give d1, a yellow crys-
tal, yield 93.1%. In this way, pure compounds d1–d28 were ob-
tained with yields of 34.2–93.1%.
5-(1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboxamide (e1): White powder, yield 56.5%; mp: 232–2348C;
1H NMR (400 MHz, DMSO): d=7.90–7.79 (m, 2H), 7.51–7.30 (m,
5H), 7.20 (s, 1H), 7.12 (t, J=7.5 Hz, 1H), 6.95 (t, J=7.4 Hz, 1H), 6.39
(s, 2H), 5.72–5.63 (m, 1H), 3.87–3.67 (m, 4H), 3.26–3.13 ppm (m,
1H); MS(ESI): 319.2 [M+H]+; Anal. Calcd for C19H18N4O: C 71.68, H
5.70, N 17.60, found: C 71.69, H 5.71, N 17.59.
3-(3-methoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (e2): White powder, yield 21.9%; mp:
191–1928C; H NMR (400 MHz, DMSO): d=7.46 (s, 1H), 7.39 (d, J=
8.2 Hz, 1H), 7.37–7.31 (m, 3H), 7.20 (s, 1H), 7.13 (t, J=7.3 Hz, 1H),
7.04–6.91 (m, 2H), 6.47 (s, 2H), 5.70–5.63 (m, 1H), 3.81 (s, 3H),
3.79–3.69 (m, 4H), 3.23–3.14 ppm (m, 1H); MS(ESI): 349.2 [M+H]+;
Anal. Calcd for C20H20N4O2: C 68.95, H 5.79, N 16.08, found: C 68.96,
H 5.78, N 16.07.
(E)-3-(5-bromo-1-methyl-1H-indol-3-yl)-1-(4-(trifluoromethyl)phe-
nyl)prop-2-en-1-one (d24): Yellow powder, yield 46.3%; mp: 188–
1908C; H NMR (400 MHz, DMSO): d=8.30 (d, J=8.4 Hz, 3H), 8.22
(s, 1H), 8.03 (d, J=15.5 Hz, 1H), 7.94 (d, J=8.3 Hz, 2H), 7.65–7.54
(m, 2H), 7.48–7.41 (m, 1H), 3.87 ppm (s, 3H); MS(ESI): 409.0 [M+
H]+; Anal. Calcd for C19H13BrF3NO: C 55.90, H 3.21, N 3.43, found: C
55.91, H 3.22, N 3.42.
1
1
(E)-3-(5-bromo-1-methyl-1H-indol-3-yl)-1-(3-chlorophenyl)prop-2-
en-1-one (d25): Yellow powder, yield 78.5%; mp: 161–1638C;
1H NMR (400 MHz, DMSO): d=8.28 (s, 1H), 8.23 (d, J=6.3 Hz, 2H),
8.15 (d, J=7.8 Hz, 1H), 8.01 (d, J=15.5 Hz, 1H), 7.84 (d, J=7.6 Hz,
1H), 7.62 (d, J=15.5 Hz, 1H), 7.54 (t, J=8.6 Hz, 2H), 7.47–7.38 (m,
1H), 3.87 ppm (s, 3H); MS(ESI): 373.0 [M+H]+; Anal. Calcd for
C18H13BrClNO: C 57.70, H 3.50, N 3.74, found: C 57.71, H 3.49, N
3.73.
3-(3,4-dimethoxyphenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-
1H-pyrazole-1-carboxamide (e3): White powder, yield 72.9%; mp:
239–2418C; 1H NMR (400 MHz, DMSO) d=7.57 (d, J=1.8 Hz, 1H),
7.42–6.32 (m, 2H), 7.25–7.07 (m, 3H), 7.01–6.90 (m, 2H), 6.42 (s,
2H), 5.68–5.59 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.76–3.68 (m, 4H),
3.22–3.13 ppm (m, 1H); MS(ESI): 379.2 [M+H]+; Anal. Calcd for
C21H22N4O3: C 66.65, H 5.86, N 14.81, found: C 66.66, H 5.87, N
14.80.
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5-(1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihy-
dro-1H-pyrazole-1-carboxamide (e4): White powder, yield 66.8%;
mp: 235–2378C; H NMR (400 MHz, DMSO): d=7.78 (d, J=8.6 Hz,
2H), 7.64 (d, J=8.6 Hz, 2H), 7.39 (d, J=8.2 Hz, 1H), 7.33 (d, J=
7.9 Hz, 1H), 7.19 (s, 1H), 7.16–7.09 (m, 1H), 6.95 (t, J=7.2 Hz, 1H),
6.42 (s, 2H), 5.64–5.72 (m, 1H), 3.84–3.68 (m, 4H), 3.22–3.13 ppm
(m, 1H); MS(ESI): 409.2 [M+H]+; Anal. Calcd for C22H24N4O4: C
64.69, H 5.92, N 13.72, found: C 64.68, H 5.93, N 13.71.
5-(5-methoxy-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphen-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (e11): White powder,
yield 24.3%; mp: 196–1988C; 1H NMR (400 MHz, DMSO): d=7.28
(d, J=8.9 Hz, 1H), 7.28 (d, J=8.9 Hz, 1H), 7.15 (s, 2H), 7.15 (s, 2H),
7.12 (s, 1H), 7.12 (s, 1H), 6.87 (d, J=2.3 Hz, 1H), 6.81–6.74 (m, 1H),
6.47 (s, 2H), 5.68–5.62 (m, 1H), 3.83 (s, 6H), 3.78–3.66 (m, 7H), 3.60
(s, 3H), 3.28–3.19 ppm (m, 1H); MS(ESI): 439.2 [M+H]+; Anal. Calcd
for C23H26N4O5: C 63.00, H 5.98, N 12.78, found: C 63.01, H 5.97, N
12.77.
1
3-(4-chlorophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (e5): White powder, yield 16.3%; mp:
233–2348C; H NMR (400 MHz, DMSO): d=7.85 (d, J=8.5 Hz, 2H),
7.52 (d, J=8.5 Hz, 2H), 7.39 (d, J=8.2 Hz, 1H), 7.32 (d, J=7.9 Hz,
1H), 7.20 (s, 1H), 7.13 (t, J=7.5 Hz, 1H), 6.95 (t, J=7.4 Hz, 1H), 6.46
(s, 2H), 5.64–5.73 (m, 1H), 3.85–3.68 (m, 4H), 3.23–3.13 ppm (m,
1H); MS(ESI): 354.1 [M+H]+; Anal. Calcd for C19H17ClN4O: C 64.68,
H 4.86, N 15.88, found: C 64.69, H 4.87, N 15.87.
3-(4-fluorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e12): White powder, yield
51.9%; mp: 194–1968C; 1H NMR (400 MHz, DMSO): d=7.95–7.87
(m, 2H), 7.30 (t, J=9.0 Hz, 3H), 7.15 (s, 1H), 6.87–6.69 (m, 2H), 6.41
(s, 2H), 5.68–5.61 (m, 1H), 3.84–3.63 (m, 4H), 3.56 (s, 3H), 3.23–
3.14 ppm (m, 1H); MS(ESI): 367.2 [M+H]+; Anal. Calcd for
C20H19FN4O2: C 65.56, H 5.23, N 15.29, found: C 65.57, H 5.22, N
15.29.
1
3-(4-bromophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (e6): White powder, yield 73.0%; mp:
231–2338C; H NMR (400 MHz, DMSO): d=7.78 (d, J=8.6 Hz, 2H),
7.64 (d, J=8.6 Hz, 2H), 7.39 (d, J=8.2 Hz, 1H), 7.33 (d, J=7.9 Hz,
1H), 7.19 (s, 1H), 7.16–7.09 (m, 1H), 6.95 (t, J=7.2 Hz, 1H), 6.42 (s,
2H), 5.64–5.72 (m, 1H), 3.84–3.68 (m, 4H), 3.22–3.13 ppm (m, 1H);
MS(ESI): 398.1 [M+H]+; Anal. Calcd for C19H17BrN4O: C 57.44, H
4.31, N 14.10, found: C 57.45, H 4.32, N 14.11.
3-(4-chlorophenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e13): White powder, yield
24.1%; mp: 238–2408C; H NMR (400 MHz, DMSO): d=7.87 (d, J=
8.6 Hz, 2H), 7.51 (d, J=8.6 Hz, 2H), 7.28 (d, J=8.8 Hz, 1H), 7.14 (s,
1H), 6.84–6.71 (m, 2H), 6.43 (s, 2H), 5.69–5.62 (m, 1H), 3.83–3.63
(m, 4H), 3.57 (s, 3H), 3.23–3.12 ppm (m, 1H); MS(ESI): 384.1 [M+
H]+; Anal. Calcd for C20H19ClN4O2: C 62.74, H 5.00, N 14.63, found:
C 62.75, H 5.01, N 14.62.
1
1
3-(3-chlorophenyl)-5-(1-methyl-1H-indol-3-yl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (e7): White powder, yield 57.0%; mp:
190–1918C; H NMR (400 MHz, DMSO): d=8.15 (s, 1H), 7.75 (d, J=
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-
pyrazole-1-carboxamide (e14): White powder, yield 32.7%; mp:
236–2388C; H NMR (400 MHz, DMSO): d=7.92–7.77 (m, 2H), 7.58
1
1
7.8 Hz, 1H), 7.65–7.54 (m, 1H), 7.44–7.36 (m, 2H), 7.33 (d, J=
7.9 Hz, 1H), 7.21 (s, 1H), 7.13 (t, J=7.6 Hz, 1H), 6.96 (t, J=7.4 Hz,
1H), 6.52 (s, 2H), 5.73–5.64 (m, 1H), 3.83–3.68 (m, 4H), 3.25–
3.14 ppm (m, 1H); MS(ESI): 354.1 [M+H]+; Anal. Calcd for
C19H17ClN4O: C 64.68, H 4.86, N 15.88, found: C 64.69, H 4.85, N
15.87.
(d, J=1.6 Hz, 1H), 7.50–7.34 (m, 4H), 7.29–7.20 (m, 2H), 6.45 (s,
2H), 5.71–5.61 (m, 1H), 3.86–3.75 (m, 1H), 3.73 (s, 3H), 3.24–
3.12 ppm (m, 1H); MS(ESI): 398.1 [M+H]+; Anal. Calcd for
C19H17BrN4O: C 57.44, H 4.31, N 14.10, found: C 57.44, H 4.32, N
14.11.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-methoxyphenyl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e15): White powder, yield
5-(5-methoxy-1-methyl-1H-indol-3-yl)-3-phenyl-4,5-dihydro-1H-
pyrazole-1-carboxamide (e8): White crystals, yield 68.9%; mp:
1
10.1%; mp: 236–2388C; H NMR (400 MHz, DMSO): d=7.78 (d, J=
1
8.8 Hz, 2H), 7.57 (d, J=1.7 Hz, 1H), 7.39 (d, J=8.7 Hz, 1H), 7.27–
7.22 (m, 2H), 7.01 (d, J=8.9 Hz, 2H), 6.36 (s, 2H), 5.65–5.59 (m,
1H), 3.84–3.69 (m, 7H), 3.19–3.10 ppm (m, 1H); MS(ESI): 428.1 [M+
H]+; Anal. Calcd for C20H19BrN4O2: C 56.22, H 4.48, N 13.11, found:
C 56.23, H 4.47, N 13.10.
210–2128C; H NMR (400 MHz, DMSO): d=7.94–7.77 (m, 2H), 7.51–
7.37 (m, 3H), 7.28 (d, J=8.8 Hz, 1H), 7.15 (s, 1H), 6.88–6.70 (m,
2H), 6.39 (s, 2H), 5.69–5.62 (m, 1H), 3.82–3.72 (m, 1H), 3.68 (s, 3H),
3.54 (s, 3H), 3.23–3.14 ppm (m, 1H); MS(ESI): 349.2 [M+H]+; Anal.
Calcd for C20H20N4O2: C 68.95, H 5.79, N 16.08, found: C 68.94, H
5.78, N 16.09.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e16): White powder, yield
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(2-methoxyphenyl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e9): White powder, yield
1
52.7%; mp: 169–1718C; H NMR (400 MHz, DMSO): d=7.57 (d, J=
1
1.7 Hz, 1H), 7.46 (d, J=1.7 Hz, 1H), 7.43–7.31 (m, 3H), 7.30–7.19
(m, 2H), 7.06–6.85 (m, 1H), 6.50 (s, 2H), 5.69–5.62 (m, 1H), 3.85–
3.69 (m, 7H), 3.85–3.69 (m, 7H), 3.22–3.12 ppm (m, 1H); MS(ESI):
428.1 [M+H]+; Anal. Calcd for C20H19BrN4O2: C 56.22, H 4.48, N
13.11, found: C 56.21, H 4.47, N 13.12.
20.1%; mp: 210–2128C; H NMR (400 MHz, DMSO): d=7.47 (s, 1H),
7.40–7.32 (m, 2H), 7.28 (d, J=8.8 Hz, 1H), 7.14 (s, 1H), 7.01–6.97
(m, 1H), 6.83 (d, J=2.3 Hz, 1H), 6.79–6.75 (m, 1H), 6.42 (s, 2H),
5.68–5.62 (m, 1H), 3.84–3.71 (m, 4H), 3.68 (s, 3H), 3.56 (s, 3H),
3.22–3.14 ppm (m, 1H); MS(ESI): 379.42 [M+H]+; Anal. Calcd for
C21H22N4O3: C 66.65, H 5.86, N 14.81, found: C 66.66, H 5.85, N
14.82.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(2-methoxyphenyl)-4,5-di-
hydro-1H-pyrazole-1-carboxamide (e17): White powder, yield
63.4%; mp: 210–2128C; 1H NMR (400 MHz, DMSO): d=8.03–7.92
(m, 1H), 7.61 (d, J=1.8 Hz, 1H), 7.49–7.35 (m, 2H), 7.32–7.21 (m,
2H), 7.12 (d, J=8.2 Hz, 1H), 7.08–6.98 (m, 1H), 6.37 (s, 2H), 5.67–
5.55 (m, 1H), 3.90–3.77 (m, 4H), 3.74 (s, 3H), 3.29–3.18 ppm (m,
1H). MS(ESI): 428.1 [M+H]+; Anal. Calcd for C20H19BrN4O2: C 56.22,
H 4.48, N 13.11, found: C 56.22, H 4.47, N 13.12.
3-(3,4-dimethoxyphenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-
4,5-dihydro-1H-pyrazole-1-carboxamide (e10): White powder,
yield 38.2%; mp: 233–2358C; 1H NMR (400 MHz, DMSO): d=7.58
(d, J=1.4 Hz, 1H), 7.32–7.19 (m, 2H), 7.13 (s, 1H), 6.98 (d, J=
8.4 Hz, 1H), 6.85 (d, J=2.1 Hz, 1H), 6.81–6.73 (m, 1H), 6.41 (s, 2H),
5.65–5.58 (m, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.77–3.65 (m, 4H), 3.57
(s, 3H), 3.21–3.12 ppm (m, 1H); MS(ESI): 409.2 [M+H]+; Anal. Calcd
for C22H24N4O4: C 64.69, H 5.92, N 13.72, found: C 64.68, H 5.93, N
13.73.
3-(3,4-dimethoxyphenyl)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-
4,5-dihydro-1H-pyrazole-1-carboxamide (e18): White powder,
1
yield 38.2%; mp: 233–2358C; H NMR (400 MHz, DMSO): d=7.63–
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7.52 (m, 2H), 7.39 (d, J=8.7 Hz, 1H), 7.30–7.15 (m, 3H), 6.98 (d, J=
8.4 Hz, 1H), 6.47 (s, 2H), 5.68–5.58 (m, 1H), 3.83 (s, 3H), 3.79 (s,
3H), 3.77–3.69 (m, 4H), 3.20–3.10 ppm (m, 1H); MS(ESI): 458.1 [M+
H]+; Anal. Calcd for C21H21BrN4O3: C 55.15, H 4.63, N 12.25, found:
C 55.16, H 4.62, N 12.25.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-(trifluoromethyl)phen-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (e26): White crystals,
yield 34.3%; mp: 197–1998C; 1H NMR (400 MHz, DMSO): d=8.28
(s, 1H), 8.06 (d, J=7.8 Hz, 1H), 7.78 (d, J=7.8 Hz, 1H), 7.69 (t, J=
7.8 Hz, 1H), 7.60 (d, J=1.7 Hz, 1H), 7.40 (d, J=8.7 Hz, 1H), 7.32–
7.20 (m, 2H), 6.63 (s, 2H), 5.75–5.67 (m, 1H), 3.88–3.78 (m, 1H),
3.73 (s, 3H), 3.32–3.22 ppm (m, 1H); MS(ESI): 466.0 [M+H]+; Anal.
Calcd for C20H16BrF3N4O: C 51.63, H 3.47, N 12.04, found: C 51.64, H
3.48, N 12.05.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3,4,5-trimethoxyphenyl)-
4,5-dihydro-1H-pyrazole-1-carboxamide (e19): White crystals,
yield 24.6%; mp: 235–2378C; 1H NMR (400 MHz, DMSO): d=7.62
(d, J=1.8 Hz, 1H), 7.39 (d, J=8.7 Hz, 1H), 7.29–7.18 (m, 2H), 7.13
(s, 2H), 6.50 (s, 2H), 5.70–5.62 (m, 1H), 3.83 (s, 6H), 3.81–3.68 (m,
7H), 3.27–3.18 ppm (m, 1H); MS(ESI): 488.1 [M+H]+; Anal. Calcd
for C22H23BrN4O4: C 54.22, H 4.76, N 11.50, found: C 54.22, H 4.75, N
11.51.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-fluoro-4-methoxyphen-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (e27): White powder,
1
yield 16.2%; mp: 228–2308C; H NMR (400 MHz, DMSO): d=7.86–
7.78 (m, 1H), 7.61–7.48 (m, 2H), 7.39 (d, J=8.7 Hz, 1H), 7.31–7.17
(m, 3H), 6.46 (s, 2H), 5.70–5.58 (m, 1H), 3.89 (s, 3H), 3.82–3.66 (m,
4H), 3.21–3.11 ppm (m, 1H); MS(ESI): 446.1 [M+H]+; Anal. Calcd
for C20H16BrF3N4O: C 53.95, H 4.07, N 12.58, found: C 53.94, H 4.08,
N 12.59.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-fluorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carboxamide (e20): White crystals, yield 63.7%;
1
mp: 245–2478C; H NMR (400 MHz, DMSO): d=7.97–7.83 (m, 2H),
7.56 (d, J=1.7 Hz, 1H), 7.39 (d, J=8.7 Hz, 1H), 7.36–7.19 (m, 4H),
6.46 (s, 2H), 5.72–5.58 (m, 1H), 3.85–3.67 (m, 4H), 3.25–3.13 ppm
(m, 1H); MS(ESI): 416.0 [M+H]+; Anal. Calcd for C19H16BrFN4O: C
54.95, H 3.88, N 13.49, found: C 54.94, H 3.88, N 13.48.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(2-fluoro-4-methoxyphen-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (e28): White powder,
1
yield 72.9%; mp: 227–2298C; H NMR (400 MHz, DMSO): d=7.99 (t,
J=8.8 Hz, 1H), 7.59 (d, J=1.7 Hz, 1H), 7.40 (d, J=8.7 Hz, 1H),
7.30–7.18 (m, 2H), 7.00–6.82 (m, 2H), 6.39 (s, 2H), 5.66–5.57 (m,
1H), 3.94–3.79 (m, 4H), 3.73 (s, 3H), 3.19–3.08 ppm (m, 1H);
MS(ESI): 446.1 [M+H]+; Anal. Calcd for C20H16BrF3N4O: C 53.95, H
4.07, N 12.58, found: C 53.96, H 4.06, N 12.59.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-chlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carboxamide (e21): White powder, yield
44.9%; mp: 240–2428C; 1H NMR (400 MHz, DMSO): d=7.91–7.78
(m, 2H), 7.60–7.48 (m, 3H), 7.39 (d, J=8.7 Hz, 1H), 7.29–7.20 (m,
2H), 6.50 (s, 2H), 5.70–5.63 (m, 1H), 3.87–3.66 (m, 4H), 3.22–
3.13 ppm (m, 1H); MS(ESI): 430.0 [M+H]+; Anal. Calcd for
C19H16BrClN4O: C 52.86, H 3.74, N 12.98, found: C 52.87, H 3.73, N
12.97.
Crystal structure determination
Crystal structure determination of compounds e1 and e8 was car-
ried out on a Nonius CAD4 diffractometer equipped with graphite-
mono chromated MoKa (k=0.71073 ꢁ) radiation. The structure
was solved by direct methods and refined on F2 by full-matrix
least-squares methods using SHELX-97.[24] All non-hydrogen atoms
of compounds e1 and e8 were refined with anisotropic thermal
parameters. All hydrogen atoms were placed in geometrically ide-
alized positions and constrained to ride on their parent atoms.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-bromophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carboxamide (e22): White crystals, yield 46.2%;
mp: 245–2478C; H NMR (400 MHz, DMSO): d=7.79 (d, J=8.6 Hz,
2H), 7.65 (d, J=8.6 Hz, 2H), 7.56 (d, J=1.8 Hz, 1H), 7.39 (d, J=
8.7 Hz, 1H), 7.30–7.19 (m, 2H), 6.48 (s, 2H), 5.71–5.63 (m, 1H),
3.85–3.66 (m, 4H), 3.23–3.12 ppm (m, 1H); MS(ESI): 474.0 [M+H]+;
Anal. Calcd for C19H16Br2N4O: C 47.93, H 3.39, N 11.77, found: C
47.94, H 3.38, N 11.76.
1
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-
pyrazole-1-carboxamide (e23): White powder, yield 42.3%; mp:
256–2588C; H NMR (400 MHz, DMSO): d=7.73 (d, J=8.1 Hz, 2H),
Biological assays
1
Antiproliferation activity:[25] Human cervix carcinoma (HeLa), breast
adenocarcinoma (MCF-7), human non-small-cell lung carcinoma
(A549), and human liver hepatocellular carcinoma (HepG2) cell
lines were cultured in DMEM supplemented with fetal bovine
serum (10%),[12] penicillin G (115 UmLꢀ1), and streptomycin
(115 mgmLꢀ1), in CO2 (5%) cell incubator at 378C. Then cell suspen-
sions (100 mL, 1000–5000 cells) were added into each well of a 96-
well plates depending on growth rate and then allowed to adhere
in the incubator. Samples at varying concentrations (ranging from
0.01 to 50 mm) were added to the wells with colchicine as a positive
reference. After 48 h incubation, PBS (10 mL) containing MTT
(5 mgmLꢀ1) was added to each well. The plates were incubated for
a further 4 h and then were centrifuged at 1500 rpm for 10 min to
remove supernatant. After DMSO (150 mL) was added to each well,
the plates were shaken vigorously for 10 min to ensure complete
solubilization of formazan. The absorbance was measured and re-
corded on an ELISA reader (ELx800, BioTek, USA) at a test wave-
length of 490 nm. Three replicate wells were used for each drug
concentration. Each assay was carried out at least three times.
7.56 (d, J=1.7 Hz, 1H), 7.39 (d, J=8.7 Hz, 1H), 7.31–7.20 (m, 2H),
6.40 (s, 2H), 5.70–5.58 (m, 1H), 3.86–3.69 (m, 4H), 3.21–3.10 (m,
1H), 2.35 ppm (s, 3H); MS(ESI): 412.1 [M+H]+; Anal. Calcd for
C20H19BrN4O: C 58.40, H 4.66, N 13.62, found: C 58.39, H 4.65, N
13.63.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(4-(trifluoromethyl)phen-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide (e24): White powder,
yield 30.0%; mp: 230–2328C; 1H NMR (400 MHz, DMSO): d=8.05
(d, J=8.1 Hz, 2H), 7.82 (d, J=8.4 Hz, 2H), 7.57 (d, J=1.5 Hz, 1H),
7.40 (d, J=8.7 Hz, 1H), 7.31–7.20 (m, 2H), 6.58 (s, 2H), 5.75–5.67
(m, 1H), 3.88–3.78 (m, 1H), 3.73 (s, 3H), 3.28–3.18 ppm (m, 1H);
MS(ESI): 466.0 [M+H]+; Anal. Calcd for C20H16BrF3N4O: C 51.63, H
3.47, N 12.04, found: C 51.64, H 3.46, N 12.05.
5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-chlorophenyl)-4,5-dihy-
dro-1H-pyrazole-1-carboxamide (e25): White crystals, yield 46.3%;
mp: 208–2108C; 1H NMR (400 MHz, DMSO): d=8.14 (s, 1H), 7.77
(d, J=7.8 Hz, 1H), 7.65–7.54 (m, 2H), 7.40 (t, J=8.4 Hz, 2H), 7.30–
7.21 (m, 2H), 6.56 (s, 2H), 5.71–5.63 (m, 1H), 3.83–3.66 (m, 4H),
3.26–3.14 ppm (m, 1H); MS(ESI): 430.0 [M+H]+; Anal. Calcd for
C19H16BrClN4O: C 52.86, H 3.74, N 12.98, found: C 52.87, H 3.75, N
12.97.
Tubulin polymerization assay: Tubulin was purified from bovine
brain as described previously.[26,27] To characterize the effect of the
compounds on tubulin assembly in vitro, compounds (ranging
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from 0.1 to 50 mm) were incubated with bovine brain tubulin
(10 mm) in glutamate (0.8m, pH 6.6 with HCl) at 308C and then
cooled to 08C.[28,29] After GTP (0.4 mm) was added, the mixtures
were transferred to cuvettes at 08C in a recording spectrophotom-
eter and warmed up to 308C. Finally, the assembly of tubulin was
observed turbidimetrically at 350 nm. The IC50 was defined as the
compound concentration which inhibited the extent of assembly
by 50% after 20 min incubation.
subset for validating the reliability of the QSAR model by the Di-
verse Molecules protocol in DS 3.5. The selected test compounds
were: e8, e9, e14, e15, e16 and e23. The inhibitory activities of
the compounds (IC50 [mm]) were initially changed into the corre-
sponding pIC50 (ꢀlogIC50) and then used for subsequent QSAR
analysis as the response variable.[30] In Discovery Studio, the
CHARMm force field is used and the electrostatic potential togeth-
er with the van der Waals potential are treated as separate terms.
A +1e point charge is used as the electrostatic potential probe,
while distance-dependent dielectric constant is used to mimic the
solvation effect. As for the van der Waals potential, a carbon atom
with a radius of 1.73 ꢁ is used as probe. A Partial Least-Squares
(PLS) model is built using energy grids as descriptors.[31] The pre-
dictive ability of 3D-QSAR modeling can be evaluated based on
the cross-validated correlation coefficient, which qualifies the pre-
dictive ability of the models. Scrambled test (Y scrambling) was
performed to investigate the risk of chance correlations. The inhibi-
tory potencies of compounds were randomly reordered 30 times
and subject to leave-one-out validation test.[22] The models were
also validated by test sets, in which the compounds were not in-
cluded in the training sets.
Annexin V assay: HeLa cells were plated into a 12-well plate (2.0ꢂ
105 cells per well) and were allowed to adhere for 12 h. After that,
the cells were treated with e19 at final concentrations of 0, 0.05,
0.1, 0.2, or 0.3 mm for 24 h or treated with e19 (0.2 mm) for 0, 12,
24, or 36 h. Then cells were collected and washed twice with pre-
cooled PBS and strained with Annexin V–FITC (10 mL) and PI (5 mL,
5 mgmLꢀ1) in binding buffer (1ꢂ) for 30 min in the dark at room
temperature. Apoptotic cells were monitored on BD Accuri C6
Flow Cytometer (BD, USA). Statistical analysis was done using
Flowjo 7.6.1 software.
Cell-cycle analysis: HeLa cells were seeded in 6-well plates (5.0ꢂ105
cells per well) and incubated at 378C for 24 h. Then the cells were
incubated with e19 at 0, 0.05, 0.1, 0.2, or 0.3 mm for 24 h or treated
with e19 (0.3 mm) for 0, 6, 12, 24, 36, or 48 h. Treated cells were
washed with PBS and fixed with ice-cold ethanol (70%) overnight.
Fixed cells were then resuspended in PBS containing RNase A
(0.1 mgmLꢀ1) and propidium iodide (PI, 5 mgmLꢀ1) and stained at
378C 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.
Acknowledgements
This work was supported by the National Technology Support
Program (No. 2015BAD16B07), by Major Projects on Control and
Rectification of Water Body Pollution (No. 2011ZX07204-001-2214
004), and by a grant (No. J1103512) from the National Natural
Science Foundation of China and Projects (Nos. CXY1409 and
CG1305) from the Science & Technology Bureau of Lianyungang
City of Jiangsu Province.
Confocal microscopy: HeLa cells were plate on glass coverslips and
incubated with colchicine (0.5 mm), paclitaxel (0.5 mm) or e19
(0.2 mm) for 24 h, respectively. Cells were then rinsed with PBS
three times, fixed with paraformaldehyde (4%) for 20 min and per-
meabilized with cold formaldehyde for 10 min. Fixed cells were
washed with PBS and blocked with Albumin from bovine serum
(BSA, 5%) for at least 1 h. After washing with PBS once, the cells
were incubated with anti-tubulin antibody (1:500) in BSA (3%)
overnight at 48C, followed by Cy3-labeled goat anti-mouse IgG
(H+L) (1: 500) in BSA (3%) and incubated for 1 h. Subsequently,
the cells were incubated with DAPI (5 ngmLꢀ1). Images were cap-
tured with Olympus confocal microscope and data was analyzed
using FV-10-ASW.
Keywords: apoptosis
·
indole–pyrazolines
·
inhibitors
·
molecular docking · tubulin
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Revised: April 3, 2016
Published online on && &&, 0000
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
13
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
Y.-L. Zhang, Y.-J. Qin, D.-J. Tang,
M.-R. Yang, B.-Y. Li, Y.-T. Wang, H.-Y. Cai,
B.-Z. Wang,* H.-L. Zhu*
Network disruption! A series of 1-
methyl-1H-indole–pyrazoline hybrids
were designed, synthesized, and biolog-
ically evaluated as potential tubulin
polymerization inhibitors. Among them,
compound e19 showed the most
potent inhibitory effect on tubulin as-
sembly and in vitro growth inhibitory
activity against four human cancer cell
lines. Further studies confirmed that
e19 induces HeLa cell apoptosis and
causes G2/M-phase cell-cycle arrest by
disrupting the cell’s microtubule net-
work.
&& – &&
Synthesis and Biological Evaluation of
1-Methyl-1H-indole–Pyrazoline
Hybrids as Potential Tubulin
Polymerization Inhibitors
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
14
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!