Design, synthesis and biological evaluation of novel pyrazoline-containing derivatives as potential tubulin assembling inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.058

Abstract A series of novel pyrazoline-containing derivatives (15-47) has been designed, synthesized and evaluated for their biological activities. Among them, compound 18 displayed the most potent antiproliferative activity against A549, MCF-7 and HepG-2 cells line (IC₅₀ Combining double low line 0.07 μM, 0.05 μM, 0.03 μM, respectively) and the tubulin polymerization inhibitory activity (IC₅₀ Combining double low line 1.88 μM), being comparable to CA-4. Furthermore, we also tested that compound 18 was a potent inducer of apoptosis in HepG-2 cells and it had cellular effects typical for microtubule interacting agents, causing accumulation of cells in the G2/M phase of the cell cycle. These studies, along with molecular docking, provided a new molecular scaffold for the further development of antitumor agents that target tubulin.

Full text of DOI:10.1016/j.ejmech.2015.02.058

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Novel Pyrazoline-Containing
Derivatives as Potential Tubulin Assembling Inhibitors
Ya-Juan Qin, Yu-jing Li, Ai-Qin Jiang, Meng-Ru Yang, Qi-Zhang Zhu, Hong Dong,
Hai-Liang Zhu
PII:
S0223-5234(15)00154-3
10.1016/j.ejmech.2015.02.058
EJMECH 7740
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 August 2014
Revised Date: 5 November 2014
Accepted Date: 28 February 2015
Please cite this article as: Y.-J. Qin, Y.-j. Li, A.-Q. Jiang, M.-R. Yang, Q.-Z. Zhu, H. Dong, H.-L.
Zhu, Design, Synthesis and Biological Evaluation of Novel Pyrazoline-Containing Derivatives as
Potential Tubulin Assembling Inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.02.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Pyrazoline-Containing
Derivatives as Potential Tubulin Assembling Inhibitors
Ya-Juan Qina†, Yu-jing Lia†, Ai-Qin Jiangb*, Meng-Ru Yanga, Qi-Zhang Zhua,
Hong-Donga*, Hai-Liang Zhua*
a State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science,
Nanjing University, Nanjing 210093, P. R. China
b School of Medicine, Nanjing University, Nanjing 210093, P. R. China
† Both authors contributed equally to this work.
Abstract
A series of novel pyrazoline-containing derivatives (15-47) has been designed,
synthesized and evaluated for their biological activities. Among them, compound 18
displayed the most potent antiproliferative activity against A549, MCF-7 and HepG-2
cells line (IC50 = 0.07 μM, 0.05 μM, 0.03 μM, respectively) and the tubulin
polymerization inhibitory activity (IC50 = 1.88 μM), being comparable to CA-4.
Furthermore, we also tested that compound 18 was a potent inducer of apoptosis in
HepG-2 cells and it had cellular effects typical for microtubule interacting agents,
causing accumulation of cells in the G2/M phase of the cell cycle. These studies,
along with molecular docking, provided a new molecular scaffold for the further
development of antitumor agents that target tubulin.
Keywords: tubulin polymerization inhibitors; pyrazoline; molecular docking
Abbreviations: CA-4, combretastatin A-4; SAR: structure-activity relationship; IC50,
half maximal inhibitory concentration; MTT, 3-(4, 5-Dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide
*Corresponding authors. Tel: +86-25-8359 2572; Fax: +86-25-8359 2672.
E-mail address: zhuhl@nju.edu.cn
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Introduction
Microtubules are cytoskeletal structures [1] and critical elements in various
fundamental cellular processes such as cell division, formation, and maintenance of
cell shape, motility, cell signaling, secretion, and intracellular transport. [2] This is
one of the reasons why microtubules are an attractive target for anticancer agents. So
far, microtubule-targeting agents could be simply classified into microtubule stabilizer
and microtubule distabilizer according to the mechanism by interfering with
microtubule dynamics. Further divided, there are four major binding sites provided by
the microtubule: the taxane site and the laulimalide/peloruside A site for microtubule
stabilizer, and the vinca site and the colchicine site for microtubule destabilizer. [3, 4]
In the last decade years, there has been a continuous interest in the exploitation of
novel small molecules which are able to inhibit tubulin polymerization. Compound 1
(Combretastatin A-4) [5, 6] (CA-4, Figure 1), isolated from the bark of the South
African tree Combretum caffrum, [7] is one of the prominent tubulin-binding
molecules by affecting microtubule dynamics. CA-4 strongly inhibits the tubulin
polymerization by binding to the colchicine site of the tubulin. [8] Other natural
products such as compound 2 (Colchicine) [9] (Figure 1), compound 3
(Podophyllotoxin) [9, 10] (Figure 1) and compound 4 (Steganacin) [9] (Figure 1)
exhibit potent tubulin polymerization inhibitory activity by binding to the colchicine
binding site and their distinctive structural features play a fundamental role in the
design of tubulin polymerization inhibitors. Analysis of these microtubule inhibitors
indicates that the 3, 4, 5-trimethoxyphenyl groups seem to play an important role in
their bioactivity. Cis-olefin configuration at the bridge of CA-4 has been reported as
prerequisites for the activity of potent anti-tubulin polymerization. [11] Cis
configuration of CA-4 is prone to isomerize to transform during storage and
administration, which produces a dramatic reduction in the anti-tubulin activity. [12]
Therefore, to retain the cis-olefin configuration of CA-4, it has been reported that the
isomerization from cis- to trans-olefin can be avoided by incorporating the double
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bond in five-member aromatic heterocyclic rings [12], such as pyrazole,[13]
imidazole,[14] thiazole [13] and so on. For example, some synthesized compounds
such as compound 5 (CA-NH2) [15], 6 (A-105972) [8], 7 (A-204197) [16], 8
(SMART) [17], 9 (Pyrazole) [18] and 10 (RABI) [19] (Figure 1) also strongly inhibit
the tubulin polymerization by binding to the colchicine site of the tubulin. In 2001,
compound 6 (A-105972), reported by Abbott, a novel colchicine-site binding agent,
was selected for biological profiling from a high throughput screening of more than
60,000 compounds, which was based on inhibition of cell proliferation. [8]
Subsequently, Abbott extended their initial observation and reported that compound 7
(A-204197) showed more significant tubulin polymerization inhibitory activity and
also showed bioactivity against several MDR positive cell lines. [16] Moreover, we
have performed docking simulations using the X-ray crystallographic structure of the
tubulin in complex with a tubulin inhibitor (PDB code: 1SA0) to explore the binding
modes of these compounds at the active site.
These previous researches and docking simulations encouraged us to design and
synthesize compounds 15-47 (Table 1) which were pyrazoline-containing derivatives.
This combined substructures—the pyrazoline ring along with methylphenyl
group—might possess synergistic tubulin inhibitory effect. Some novel potent
potential tubulin inhibitors were recently reported as potent anticancer agents
targeting tubulin in our group and some had demonstrated potent tubulin inhibitory
activity. [11, 20] In order to extend our research, here we describe the synthesis of
these target compounds and the ensuing structure-activity relationship (SAR) studies.
Biological evaluation indicated that some of the synthesized compounds were potent
inhibitors of tubulin polymerization. Molecular Docking simulations were performed
using the X-ray crystallographic structure of the tubulin in complex with an inhibitor
(PDB code: 1SA0) to explore the binding modes of these compounds at the active
site.
2. Results and discussion
2.1. Chemistry
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The synthetic route of compounds 15-47 is outlined in Scheme 1. Firstly, a solution
of 3, 4-dihydroxybenzaldehyde in DMF was added drop wise to a suspension of
CH2Cl2 and K2CO3 in DMF and then we obtained compound 12. Secondly,
compounds 13a-13m were prepared according to the procedure reported by our group
with some modifications. [21] Compounds 13a-13m were synthesized from
compound 12 reacting with the corresponding acetophenone at room temperature. The
mixture of corresponding compounds 3a-3m and hydrazine hydrate in EtOH was
refluxed under stirring for 4 h and the precipitate formed was filtered off, washed with
cool ethanol to get compounds 14a-14m, respectively. Finally, compounds 14a-14m
reacted with the corresponding benzoic acid, together with EDCI and HOBt,
respectively. The mixture was refluxed under stirring for 24 h. The corresponding
crude product were crystallized with ethanol to give target compounds 15-47.
1
Compounds 15-47 were fully characterized by H NMR, ESI-MS and elemental
analysis.
2.2. Crystal structure of compound 23
Among these compounds, the crystal structure of compound 23 was determined by
X-ray diffraction analysis. The crystal data presented in Figure 2 and Table 2 gave
perspective views of compound 23 with the atomic labeling system.
2.3. Biological activity
2.3.1. In Vitro Antiproliferative Activities.
To test the antiproliferative activities of the synthesized compounds 15-47, we
evaluated the inhibition activities of them against A549 (human lung cancer cell line),
MCF-7 (human breast cancer cell line) and HepG-2 (human liver hepatocellular
carcinoma cell line). Cells were treated with increasing concentrations of the
compounds 15-47, and viabilities were measured by the MTT assay. The results were
summarized in Table 3. Among them, Compound 18 displayed the most potent
inhibitory activity against three cell lines. Meanwhile, compound 18 could inhibit
A549, MCF-7 and HepG-2 at the level of IC50 value less than 0.1 µM. (IC50 = 0.07
μM, 0.05 μM, 0.03 μM, respectively), comparable to the positive compound CA-4.
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Based on the data of Table 3 obtained, we drew that when R2=3, 4, 5-trimethyl
group, the activity of the tested compounds may be correlated to the variation and
modifications of the structure. Obviously, the antiproliferative activities of
compounds 15-19 were more potent than compounds 21-27. In comparison to these
target compounds, the inhibitory activity of these compounds with different
substituents on the B ring increased in the following order: 4-OCH3 > 4-OCH2CH3 >
4-F>4-CH3 > 4-H > 4-Br > 4-Cl; 4-OCH3 > 3, 4-diOCH3 > 3-OCH3 > 2-OCH3; 4-Cl >
3-Cl; 4-CH3 > 3-CH3. Then, we drew three conclusions as follows: the para-position
substituent is more potent than the meta-position and ortho-position on the B ring; the
electron-donating group on the B ring may have improved the antiproliferative
activity of the agents; when the substitutions on the B ring are 3, 4, 5-trimethyl, the
inhibitory activity is lower. We guessed 3, 4, 5-trimethyl increased the steric.
It was also found that when the substituent on the C ring changed to 4-Br, 4-Cl, the
antiproliferative activities of most compounds were lower or totally lost. For example,
the antiproliferative activities of compounds 32, 33, 41, 47 are all moderate. Even the
IC50 values of compounds 28-30, 34, 35, 37-39 are more than 40 μM. We also drew
that when the substituent is 4-OCH3 on the B ring, the electron-donating group on the
C ring may have improved antiproliferative activity of the agents. What was
surprising was that compounds 23, 40 and 46 displayed a potent inhibitory activity
against A459 MCF-7 and HepG-2. We guessed that the 4-F on the B ring or C ring
played a very important role.
2.3.2. Inhibition of tubulin polymerization
To investigate whether the antiproliferative activities of compounds 15-47 derived
from an interaction with tubulin or not, these agents were evaluated for their
inhibition of tubulin polymerization (Table 3). As expected, compound 18 displayed
the most potent activity of anti-tubulin polymerization (IC50 = 1.88 μM) and it
suggested that the IC50 values of these compounds showed a similar tendency with
their relevant IC50 values of the antiproliferative assay. However, when comparing the
inhibition of tubulin polymerization versus the growth inhibitory effects, we found a
positive correlation for most, but not all, of the active compounds.
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The next molecular docking of all the synthesized compounds will help us to
analyze these results.
2.3.3. Compound 18 induced apoptosis.
To characterize the mode of cell death induced by compound 18, a biparametric
cytofluorimetric analysis was performed using propidium iodide (PI), which stains
DNA and enters only dead cells, and fluorescent immunolabeling of the protein
annexin-V, which binds to phosphatidyl serine (PS) in a highly selective manner. [22]
HepG-2 cells were treated with compound 18 at 0.01 μM, 0.03 μM and 0.05 μM for
24 h, respectively and DMSO served as the control. The results showed that the
percentage of cell apoptosis was 3.89 % at 0 μM for compound 18, and cell apoptosis
was increased to 5.12%, 8.72%, and 13.4% at 0.01 μM, 0.03 μM, and 0.05 μM,
respectively (Figure 3). Thus, compound 18 could cause cell apoptosis like other
tubulin-binding agents.
2.3.4. Analysis of Cell Cycle.
The effect of compound 18 on cell cycle progression was examined by flow
cytometry in HepG-2 cells (Figure 4). At higher concentration of 0.10 ~ 0.05 μM up
to 52.20 % of the cells were arrested in G2/M. There was a concomitant decrease of
cells in the other phases of the cell cycle (G1 and S) as shown in Figure 4. These
findings confirmed a continuing impairment of cell division and proved that
compound 8 was a potent antitubulin agent.
2.4. Docking simulations
To gain better understanding on the potency of the synthesized compounds 15-47
and guide further SAR studies, we proceeded to examine the interaction of
compounds 15-47 with tubulin crystal structure (PDB code: 1SA0) using the
Discovery
Studio
(version
3.5).
We
described
the
CDOCKER_INTERACTION_ENERGY of molecular docking with Figure 5. It is
clear that compound 18, shows lowest interaction energy of all the synthesized
compounds and the interaction energy reached up to -60.69 kcal/mol.
To take more directly insight into the binding mode of compound 18 and tubulin,
we presented the docking results with two pictures (Figure 6A, Figure 6B) Visual
6

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inspection of the pose of compound 18 into tubulin binding site revealed that
compound 18 was tightly embedded into the ATP binding pocket (Figure 7). In the
binding model, compound 18 is nicely bound to tubulin protein in the ATP-binding
site via four hydrogen bonds. The oxygen atom of methoxy on the B ring contributes
to the hydrogen bonding interaction (O…H-N: 2.5 Å) with the hydrogen atom on the
main chain of LYS 254. The oxygen atom of methoxy on the C ring contributes to the
hydrogen bonding interactions (O…H-N: 2.1 Å, O…H-N: 1.9 Å) with the hydrogen
atom on the main chain of CYS241. The oxygen atom of the benzo [d] [1, 3] dioxole
forms the hydrogen bonding interactions with LYS 352(O…H-N: 2.5 Å).
3. Conclusions
In this study, a series of novel pyrazoline-containing derivatives (compounds 15-47)
have been synthesized and evaluated for their biological activities. Among these small
molecules, most of the compounds showed potent in vitro inhibitory activity in the
tubulin polymerization and cellular assays. Among these small molecules, compound
18 displayed the most potent activity against tubulin assembling, A549, MCF-7 and
HepG-2 cell lines (IC50 = 1.88 μM, 0.07μM, 0.05μM, 0.03μM, respectively), being
comparable to the positive compound CA-4. Furthermore, we also showed that
compound 18 was a potent inducer of apoptosis in HepG-2 cells and it had cellular
effects typical for microtubule interacting agents, causing accumulation of cells in the
G2/M phase of the cell cycle. These results, along with molecular docking
observations, could provide an important basis for further development of compound
18 as a potent tubulin polymerization inhibitors. Compound 23 and 46 also have
attract our attentions. Further studies to improve tubulin polymerization inhibitors and
kinases selectivity are now in progress.
4. Experimental.
4.1. Chemistry general
All chemicals and reagents of analytical grade used were purchased from Aldrich
(USA). Melting points (uncorrected) were determined on a XT4MP apparatus (Taike
1
Corp, Beijing, China). All the H NMR spectra were recorded on a Bruker DPX 300
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model Spectrometer at 25 ℃ with TMS and solvent signals allotted as internal
standards, and chemical shifts were reported in ppm (d). ESI-MS spectra were
recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument and were within 0.4% of the theoretical
values. TLC was performed on the glass-backed silica gel sheets (Silica Gel 60
GF254) and visualized in UV light (254 nm). Column chromatography was performed
using silica gel (200-300 mesh) eluting with ethyl acetate and petroleum ether.
4.1.1. Synthesis of 1, 3-benzodioxole-5-carbaldehyde (12) [22]
A solution of 3, 4-dihydroxybenzaldehyde (10 g, 0.073 mol, 11) in DMF (150 mL)
was added dropwise to a suspension of CH2Cl2 (7 mL, 0.108 mol) and K2CO3 (20 g,
0.144 mol) in DMF (300 mL). The mixture was stirred and heated to reflux for 4 h
then cooled and filtered. The filtrate was concentrated, diluted with water and
extracted with diethyl ether (3100 mL). The filtered cake was washed with diethyl
ether (100 mL). The ethereal extracts were combined, washed with 10% NaOH (100
mL), water (100 mL), dried (Na2SO4) and evaporated to give 1,
3-benzodioxole-5-carbaldehyde (12) a light brown solid. The crude 12 was used in the
next step without any further purification.
4.1.2. (E)-3-(benzo[d] [1,3]dioxol-5-yl)-1-(4-ethoxyphenyl)prop-2-en-1-one (13a)
[21]
To a stirred solution of 1-(3-ethoxyphenyl) ethanone (1 mmol) and 1,
3-benzodioxole-5-carbaldehyde (12, 1 mmol) in ethanol (30 mL), 6 mol KOH (4 mL)
was added and the reaction mixture was stirred until the solids fully formed. The
products were filtrated and washed carefully with ice water and cool ethanol, and
purified
by crystallization
from
ethanol
in
refrigerator
to
give
(E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(3-ethoxyphenyl)prop-2-en-1-one (13a) a yellow
powder, yield 60%. The corrsesponding acetophenone following this way, we get pure
compound 13b-13m with a yield of 60 - 87%.
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4.1.3. 5-(benzo[d] [1,3]dioxol-5-yl)-3-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazole)
(14a) [21]
To a solution of compound 13a (1 mmol) in isopropanol (5 mL) hydrazine hydrate
(0.2 mL, 4 mmol) was added. The mixture was refluxed under stirring for 4 h, stored
at 4 - 5 ℃ for 1 h, and the precipitate formed was filtered off, washed with cool
isopropanol (10 ml ), next washed with petroleum ether(10 ml) for three times. We
can get white powder, yield 40%. The crude 14a was used in the next step without
any further purification. The compound 13a-13m following this way, we get crude
compound 14b-14m with a yield of 30-55%.
4.1.4.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,
4, 5-trimethoxyphenyl) methanone (15)
White powders, yield 47.7%. mp: 102 ~ 103 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.71 (s, 1H), 7.68 (s, 1H), 7.32 (s, 2H), 7.03 (s, 1H), 7.01 (s, 1H), 6.87 (t, J = 6.00 Hz,
2H), 6.77 (d, J = 5.88 Hz, 1H), 5.99(s, 2H), 5.65 (dd, J = 3.48 and 8.61 Hz, 1H), 4.08
(q, J = 5.19 Hz, 2H), 3.84 (s, 7H), 3.75 (s, 3H), 3.14 (dd, J = 3.63 and 13.50 Hz, 1H),
1.34 (t, J = 5.22 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz) δ: 164.36, 160.79, 155.58,
125.46, 147.94, 146.81, 140.23, 136.92, 129.85, 128.84, 123.93, 119.35, 115.23,
108.78, 108.07, 106.70, 101.46, 63.79, 61.21, 60.60, 56.43, 55.39, 41.73, 15.00. MS
(ESI): 505.53 [M+H] +. Anal. calc. for C28H28N2O7: C, 66.66; H, 5.59; N, 5.55. Found:
C, 66.67; H, 5.58; N, 5.54.
4.1.5.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
3, 4, 5-trimethoxyphenyl) methanone (16)
White powders, yield 46.3%. mp: 178 ~ 179 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.79 ~ 7.81 (m, 1H), 7.42 ~ 7.47 (m, 1H), 7.33 (s, 2H), 7.13 (d, J = 6.21 Hz, 1H), 7.03
(t, J = 5.76 Hz, 1H), 6.87 (t, J = 6.00 Hz, 2H), 6.78 (d, J = 5.94 Hz, 1H), 5.99 (s, 2H),
5.62 (dd, J = 3.51 and 8.67 Hz, 1H), 3.92 (dd, J = 9.39 and 13.95 Hz, 1H), 3.82 (s,
13
6H), 3.81 (s, 3H), 3.74 (s, 3H), 3.18 (dd, J = 3.63 and 13.98 Hz, 1H). C NMR
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(DMSO-d6, 100 MHz) δ: 164.57, 158.62, 154.98, 152.46, 147.96, 146.81, 140.32,
137.04, 132.34, 129.84,129.04, 129.04, 121.23, 120.39, 119.28, 113.02, 108.80,
108.20, 106.62, 101.46, 61.14, 60.59, 56.46, 56.20, 45.01. MS (ESI): 491.50 [M+H] +.
Anal. calc. for C27H26N2O7: C, 66.11; H, 5.34; N, 5.71. Found: C, 66.12; H, 5.33; N,
5.70.
4.1.6
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
3, 4, 5-trimethoxyphenyl) methanone (17)
White powders, yield 40.6%. mp: 156 ~ 157 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.36 ~ 7.42 (m, 4H), 7.29 (s, 1H), 7.06 (d, J = 5.97 Hz, 1H), 6.87 (d, J = 6.00 Hz, 2H),
6.78 (d, J = 5.91 Hz, 1H), 5.99 (s, 2H), 5.68 (dd, J = 3.60 and 8.73 Hz, 1H), 3.84 (s,
7H), 3.79 (s, 3H), 3.75 (s, 3H), 3.19 (dd, J = 3.72 and 13.62 Hz, 1H). MS (ESI):
+
491.50 [M+H] . Anal. calc. for C27H26N2O7: C, 66.11; H, 5.34; N, 5.71. Found: C,
66.12; H, 5.33; N, 5.70.
4.1.7
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
3, 4, 5-trimethoxyphenyl) methanone (18)
White powders, yield 50.6%. mp: 107 ~ 108 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.72 (s, 1H), 7.70 (s, 1H), 7.33 (s, 2H), 7.05 (s, 1H), 7.02 (s, 1H), 6.86 (t, J = 5.97 Hz,
2H), 6.77 (d, J = 5.88 Hz, 1H), 5.99 (s, 2H), 5.65 (dd, J = 3.45 and 8.61 Hz, 1H), 3.83
(s, 7H), 3.80 (s, 3H), 3.75 (s, 3H), 3.14 (dd, J = 3.60 and 13.50 Hz, 1H). MS (ESI):
+
491.50 [M+H] . Anal. calc. for C27H26N2O7: C, 66.11; H, 5.34; N, 5.71. Found: C,
66.10; H, 5.33; N, 5.70.
4.1.8
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)(3, 4, 5-trimethoxyphenyl) methanone (19)
White powders, yield 51.5%. mp: 115 ~ 116 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.40 (s, 2H), 7.34 (d, J = 1.32 Hz, 1H), 7.29 (dd, J = 1.38 and 6.24 Hz, 1H), 7.04 (d, J
= 6.36 Hz, 1H), 6.88 (d, J = 6.00 Hz, 1H), 6.83 (s, 1H), 6.76 (d, J = 5.91 Hz, 1H),
5.99 (s, 2H), 5.67 (dd, J = 3.33 and 8.61 Hz, 1H), 3.85 (s, 7H), 3.80 (s, 3H) , 3.78 (s,
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13
3H), 3.75 (s, 3H), 3.17 (dd, J = 3.48 and 13.50 Hz, 1H). C NMR (DMSO-d6, 100
MHz) δ: 164.00, 155.91, 152.48, 151.43, 149.27, 147,95, 147.95, 146.81, 140.35,
138.99, 129.57, 124.13, 121.13, 119.28, 111.99, 109.36, 108.78, 108.25, 106.63,
101.46, 61.32, 60.61, 56.40, 56.09, 56.09, 55.79, 55.38, 41.66. MS (ESI): 521.53
[M+H] +. Anal. calc. for C28H28N2O8: C, 64.61; H, 5.42; N, 5.38. Found: C, 64.60; H,
5.43; N, 5.37.
4.1.9.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(3, 4, 5-trimethoxyphenyl) methanone (20)
White powders, yield 51.2%. mp: 206 ~ 207 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.42 (s, 2H), 7.06 (s, 2H), 6.89 (d, J = 5.97 Hz, 1H), 6.84 (s, 1H), 6.77 (d, J = 5.97 Hz,
1H), 6.00 (s, 2H), 5.72 (dd, J = 3.36 and 8.67 Hz, 1H), 3.85 (s, 7H), 3.82 (s, 6H), 3.75
(s, 3H), 3.71 (s, 3H), 3.26 (dd, J = 3.48 and 13.62 Hz, 1H). 13C NMR (DMSO-d6, 100
MHz) δ: 164.00, 156.01, 153.52, 152.50, 147.97, 146.83, 140.46, 140.03, 136.93,
129.37, 127.02, 119.24, 108.80, 108.29, 106.58, 104.60, 101.48, 61.51, 60.63, 56.41,
+
56.35, 41.67. MS (ESI): 551.56 [M+H] . Anal. calc. for C29H30N2O9: C, 63.27; H,
5.49; N, 5.09. Found: C, 63.26; H, 5.48; N, 5.08.
4.1.10.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,
4, 5-trimethoxyphenyl) methanone (21)
White powders, yield 49.5%. mp: 107 ~ 108 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.70 (s, 4H), 7.29 (s, 2H), 6.88 (d, J = 5.88 Hz, 2H), 6.79 (d, J = 6.03 Hz, 1H), 6.00(s,
2H), 5.69 (dd, J = 3.78 and 8.73 Hz, 1H), 3.84 (s, 7H), 3.75 (s, 3H), 3.18 (dd, J = 3.87
+
and 13.62 Hz, 1H). MS (ESI): 540.37 [M+H] . Anal. calc. for C26H23BrN2O6: C,
57.90; H, 4.30; N, 5.19. Found: C, 57.91; H, 4.31; N, 5.18.
4.1.11
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,
4, 5-trimethoxyphenyl) methanone (22)
11

ACCEPTED MANUSCRIPT
White powders, yield 53.5%. mp: 151 ~ 152 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.78 (s, 1H), 7.76 (s, 1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.29 (s, 2H), 6.89 (s, 1H), 6.87 (s,
1H), 6.80 (d, J = 6.03 Hz, 1H), 6.00(s, 2H), 5.69 (dd, J = 3.78 and 8.76 Hz, 1H), 3.84
(s, 7H), 3.76 (s, 3H), 3.18 (dd, J = 3.84 and 13.59 Hz, 1H). MS (ESI): 495.92 [M+H]+.
Anal. calc. for C26H23ClN2O6: C, 63.10; H, 4.68; N, 5.66. Found: C, 63.11; H, 4.69; N,
5.65.
4.1.12.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,
4, 5-trimethoxyphenyl) methanone (23)
White powders, yield 45.9%. mp: 168 ~ 169 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.82 (q, J = 4.14 Hz, 2H), 7.35 (s, 1H), 7.33 (s, 1H), 7.30 (d, J = 4.50 Hz, 2H), 6.88 (d,
J = 6.00 Hz, 2H), 6.79 (d, J = 5.97 Hz, 1H), 5.99 (s, 2H), 5.68 (dd, J = 3.69 and 8.73
Hz, 1H), 3.83 (s, 7H), 3.75 (s, 3H), 3.18 (dd, J = 3.78 and 17.34 Hz, 1H). MS (ESI):
+
479.47 [M+H] . Anal. calc. for C26H23FN2O6: C, 65.27; H, 4.85; N, 5.85. Found: C,
65.26; H, 4.84; N, 5.86.
4.1.13.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,
4, 5-trimethoxyphenyl) methanone (24)
White powders, yield 42.6%. mp: 169 ~ 170 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.95 (s, 1H), 7.75 (d, J = 5.85 Hz, 1H), 7.68 (d, J = 6.54 Hz, 1H), 7.45 (t, J = 5.94 Hz,
1H), 7.33 (s, 2H), 6.88 (t, J = 2.25 Hz, 2H), 6.79 (d, J = 6.06 Hz, 1H), 6.00 (s, 2H),
5.69 (dd, J = 3.66 and 8.76 Hz, 1H), 3.85 (s, 7H), 3.75 (s, 3H), 3.20 (dd, J = 3.81 and
13.71 Hz, 1H). MS (ESI): 495.92 [M+H] +. Anal. calc. for C26H23ClN2O6: C, 63.10; H,
4.68; N, 5.66. Found: C, 63.09; H, 4.67; N, 5.67.
4.1.14.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,4,5-trime
thoxyphenyl) methanone (25)
White powders, yield 42.6%. mp: 145 ~ 146 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.67 (s, 1H), 7.65 (s, 1H), 7.33 (s, 2H), 7.29 (s, 1H), 7.27 (s, 1H), 6.86 (t, J = 6.00 Hz,
2H), 6.78 (d, J = 5.94 Hz, 1H), 5.99 (s, 2H), 5.66 (dd, J = 3.57 and 8.64 Hz, 1H), 3.83
12

ACCEPTED MANUSCRIPT
(s, 7H), 3.75 (s, 3H), 3.15 (dd, J = 3.69 and 13.56 Hz, 1H), 2.34 (s, 3H). MS (ESI):
+
475.51 [M+H] . Anal. calc. for C27H26N2O6: C, 68.34; H, 5.52; N, 5.90. Found: C,
68.33; H, 5.51; N, 5.91.
4.1.15.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(3,4,5-trime
thoxyphenyl) methanone (26)
White powders, yield 49.6%. mp: 159 ~ 160 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.61 (s, 1H), 7.56 (d, J = 5.76 Hz, 1H), 7.35 ~ 7.38 (m, 3H), 7.29 (d, J = 5.67 Hz, 1H),
6.87 (t, J = 6.00 Hz, 2H), 6.77 (d, J = 5.88 Hz, 1H), 5.99 (s, 2H), 5.68 (dd, J = 3.48
and 8.67 Hz, 1H), 3.85 (s, 7H), 3.75 (s, 3H), 3.16 (dd, J = 3.60 and 13.56 Hz, 1H),
+
2.34 (s, 3H). MS (ESI): 475.51 [M+H] . Anal. calc. for C27H26N2O6: C, 68.34; H,
5.52; N, 5.90. Found: C, 68.35; H, 5.51; N, 5.91.
4.1.16.
(5-(benzo[d][1,3]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(3,4,5-trimet
hoxyphenyl) methanone (27)
White powders, yield 37.6%. mp: 149 ~ 150 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.76 ~ 7.78 (m, 2H), 7.48 (d, J = 4.80 Hz, 3H), 7.32 (s, 2H), 6.88 (d, J = 6.06 Hz, 2H),
6.79 (d, J = 6.03 Hz, 1H), 5.99 (s, 2H), 5.68 (dd, J = 3.63 and 8.67 Hz, 1H), 3.84 (s,
+
7H), 3.75 (s, 3H), 3.18 (dd, J = 3.75 and 13.59 Hz, 1H). MS (ESI): 461.48 [M+H] .
Anal. calc. for C26H24N2O6: C, 67.82; H, 5.25; N, 6.08. Found: C, 67.81; H, 5.24; N,
6.07.
4.1.17.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-
bromophenyl) methanone (28)
White powders, yield 57.3%. mp: 147 ~ 148 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.84 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.00 (s,
1H), 6.98 (s, 1H), 6.87 (t, J = 6.00 Hz, 2H), 6.78 (d, J = 5.97 Hz, 1H), 6.00 (s, 2H),
5.66 (dd, J = 3.54 and 8.64 Hz, 1H), 4.06 (q, J = 5.19 Hz, 2H), 3.83 (dd, J = 8.76 and
13.53 Hz, 1H) , 3.15 (dd, J = 3.66 and 13.53 Hz, 1H), 1.33 (t, J = 5.19 Hz, 3H). MS
+
(ESI): 494.35 [M+H] . Anal. calc. for C25H21BrN2O4: C, 60.86; H, 4.29; N, 5.68.
13

ACCEPTED MANUSCRIPT
Found: C, 60.89; H, 4.28; N, 5.67.
4.1.18.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-bromophenyl) methanone (29)
White powders, yield 43.2%. mp: 148 ~ 149 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.88 (s, 1H), 7.85 (s, 1H), 7.67 ~ 7.72 (m, 3H), 7.42 ~ 7.46 (m, 1H), 7.11 (d, J = 6.18
Hz, 1H), 7.00 (t, J = 5.76 Hz, 1H), 6.86 (t, J = 5.97 Hz, 2H), 6.76 (d, J = 5.97 Hz, 1H),
5.99 (s, 2H), 5.63 (dd, J = 3.51 and 8.67 Hz, 1H), 3.91 (dd, J = 8.79 and 13.95 Hz,
13
1H), 3.82 (s, 3H), 3.19 (dd, J = 3.60 and 13.95 Hz, 1H). C NMR (DMSO-d6, 100
MHz) δ: 164.60, 158.59, 155.38, 148.02, 146.89, 136.76, 134.18, 132.39, 132.15,
131.25, 129.41, 124.94, 121.13, 120.18, 119.28, 112.85, 108.83, 106.54, 101.49,
+
60.66, 56.23, 45.17. MS (ESI): 480.32 [M+H] . Anal. calc. for C24H19BrN2O4: C,
60.14; H, 4.00; N, 5.84. Found: C, 60.15; H, 4.01; N, 5.85.
4.1.19.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-bromophenyl) methanone (30)
White powders, yield 56.3%. mp: 159 ~ 160 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.86 (s, 1H), 7.84 (s, 1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.32 ~ 7.41 (m, 2H), 7.24 (s, 1H),
7.06 (d, J = 6.00 Hz, 1H), 6.90 (s, 1H), 6.87 (d, J = 2.70 Hz, 1H), 6.80 (d, J = 5.91 Hz,
1H), 6.00 (s, 2H), 5.70 (dd, J = 3.63 and 8.70 Hz, 1H), 3.88 (dd, J = 8.85 and 13.65
Hz, 1H), 3.80 (s, 3H), 3.22 (dd, J = 3.75 and 13.65 Hz, 1H). MS (ESI): 480.32 [M+H]
+. Anal. calc. for C24H19BrN2O4: C, 60.14; H, 4.00; N, 5.84. Found: C, 60.15; H, 5.83;
N, 5.85.
4.1.20.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-bromophenyl) methanone (31)
White powders, yield 40.2%. mp: 175 ~ 176 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.85 (s, 1H), 7.83 (s, 1H), 7.69 (t, J = 6.42 Hz, 4H), 7.02 (s, 1H), 7.00 (s, 1H), 6.87 (t,
J = 5.97 Hz, 2H), 6.79 (d, J = 5.91 Hz, 1H), 6.00 (s, 2H), 5.66 (dd, J = 3.54 and 8.64
Hz, 1H), 3.80 (s, 4H), 3.16 (dd, J = 3.66 and 13.53 Hz, 1H). MS (ESI): 480.32 [M+H]
14

ACCEPTED MANUSCRIPT
+. Anal. calc. for C24H19BrN2O4: C, 60.14; H, 4.00; N, 5.84. Found: C, 60.13; H, 4.01;
N, 5.85.
4.1.21.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)(4-bromophenyl) methanone (32)
White powders, yield 39.5%. mp: 181 ~ 182 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.88 (d, J = 6.21 Hz, 2H), 7.72 (s, 1H), 7.70 (s, 1H), 7.30 (s, 1H), 7.28 (s, 1H), 7.03 (d,
J = 6.57 Hz, 1H), 6.88 (t, J = 6.00 Hz, 2H), 6.78 (d, J = 5.97 Hz, 1H), 6.00 (s, 2H),
5.68 (dd, J = 3.42 and 8.61 Hz, 1H), 3.81 (s, 4H), 3.79 (s, 3H), 3.20 (dd, J = 3.54 and
13.53 Hz, 1H). MS (ESI): 510.35 [M+H] +. Anal. calc. for C25H21BrN2O5: C, 58.95; H,
4.16; N, 5.50. Found: C, 58.94; H, 4.15; N, 5.51.
4.1.22.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(4-bromophenyl) methanone (33)
White powders, yield 51.6%. mp: 190 ~ 191 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.90 (d, J = 6.18 Hz, 2H), 7.72 (s, 1H), 7.70 (s, 1H), 7.03 (s, 2H), 6.88 (t, J = 6.00 Hz,
2H), 6.78 (d, J = 5.88 Hz, 1H), 6.00 (s, 2H), 5.71 (dd, J = 3.39 and 8.64 Hz, 1H), 3.82
(s, 7H), 3.71 (s, 3H), 3.28 (dd, J = 3.54 and 13.62 Hz, 1H). 13C NMR (DMSO-d6, 100
MHz) δ: 164.36, 155.31, 153.47, 148.05, 146.93, 140.13, 136.60, 133.95, 132.29,
131.26, 126.82, 125.12, 119.28, 108.81, 106.54, 104. 83, 101.51, 61.14, 60.62, 56.45,
42.07. MS (ESI): 540.37 [M+H]+. Anal. calc. for C26H23BrN2O6: C, 57.90; H, 4.30; N,
5.19. Found: C, 57.91; H, 4.31; N, 5.18.
4.1.23.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-
bromophenyl) methanone (34)
White powders, yield 45.7%. mp: 186 ~ 187 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.82 (s, 1H), 7.80 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.65 (s, 4H), 6.87 (t, J = 6.00 Hz,
2H), 6.79 (d, J = 6.12 Hz, 1H), 6.00 (s, 2H), 5.68 (dd, J = 3.78 and 8.73 Hz, 1H), 3.86
13
(dd, J = 8.88 and 13.65 Hz, 1H), 3.18 (dd, J = 3.90 and 13.68 Hz, 1H). C NMR
(DMSO-d6, 100 MHz) δ: 164.97, 155.29, 148.05, 146.98, 136.40, 134.10, 132.26,
15

ACCEPTED MANUSCRIPT
132.01, 131.34, 130.64, 129.24, 125.01, 124.38, 119.54, 108.79, 106.71, 101.51,
61.12, 41.89. MS (ESI): 529.19 [M+H] +. Anal. calc. for C23H16Br2N2O3: C, 52.30; H,
3.05; N, 5.30. Found: C, 52.31; H, 3.04; N, 5.31.
4.1.24.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-
bromophenyl) methanone (35)
White powders, yield 50.5%. mp: 173 ~ 174 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.82 (d, J = 5.79 Hz, 2H), 7.71 (dd, J = 10.17 Hz, 4H), 7.52 (d, J = 6.06 Hz, 2H), 6.84
(d, J = 18.21 Hz, 3H), 5.99 (s, 2H), 5.69 (dd, J = 3.30 and 8.43 Hz, 1H), 3.87 (dd, J =
9.03 and 13.53 Hz, 1H), 3.19 (dd, J = 3.93 and 13.59 Hz, 1H). MS (ESI): 484.74
+
[M+H] . Anal. calc. for C23H16BrClN2O3: C, 57.11; H, 3.33; N, 5.79. Found: C,
57.10; H, 3.34; N, 5.78.
4.1.25.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-b
romophenyl) methanone (36)
White powders, yield 52.5%. mp: 153 ~ 154 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.76 ~ 7.84 (m, 4H), 7.71 (s, 1H), 7.69 (s, 1H), 7.30 (t, J = 6.63 Hz, 2H), 6.88 (d, J =
5.85 Hz, 2H), 6.80 (d, J = 6.03 Hz, 1H), 5.99 (s, 2H), 5.68 (dd, J = 3.69 and 8.70 Hz,
1H), 3.89 (dd, J = 8.82 and 13.62 Hz, 1H), 3.20 (dd, J = 3.81 and 13.65 Hz, 1H). MS
+
(ESI): 468.29 [M+H] . Anal. calc. for C23H16BrFN2O3: C, 59.12; H, 3.45; N, 5.99.
Found: C, 59.11; H, 3.46; N, 6.00.
4.1.26.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-bromoph
enyl) methanone (37)
White powders, yield 55.7%. mp: 159 ~ 160 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.84 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7.69 (s, 1H), 7.63 (s, 1H), 7.61 (s, 1H), 7.27 (s,
1H), 7.25 (s, 1H), 6.87 (t, J = 6.00 Hz, 2H), 6.78 (d, J = 5.94 Hz, 1H), 5.99 (s, 2H),
5.67 (dd, J = 3.60 and 8.64 Hz, 1H), 3.85 (dd, J = 8.82 and 13.59 Hz, 1H), 3.16 (dd, J
+
= 3.75 and 13.59 Hz, 1H), 2.34 (s, 3H). MS (ESI): 464.32 [M+H] . Anal. calc. for
C24H19BrN2O3: C, 62.22; H, 4.13; N, 6.05. Found: C, 62.21; H, 4.14; N, 6.06.
16

ACCEPTED MANUSCRIPT
4.1.27.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)(4-bromop
henyl) methanone (38)
White powders, yield 47.7%. mp: 155 ~ 156 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.85 (s, 1H), 7.82 (s, 1H), 7.71 (s, 1H), 7.69 (s, 1H), 7.54 (s, 2H), 7.34 (t, J = 5.76 Hz,
1H), 7.28 (d, J = 5.70 Hz, 1H), 6.87 (t, J = 6.00 Hz, 2H), 6.79 (d, J = 5.94 Hz, 1H),
5.99 (s, 2H), 5.68 (dd, J = 3.57 and 8.67 Hz, 1H), 3.86 (dd, J = 8.82 and 13.59 Hz,
+
1H), 3.18 (dd, J = 3.66 and 13.59 Hz, 1H), 2.33 (s, 3H). MS (ESI): 464.32 [M+H] .
Anal. calc. for C24H19BrN2O3: C, 62.22; H, 4.13; N, 6.05. Found: C, 62.21; H, 4.14; N,
6.04.
4.1.28.
(5-(benzo[d][1,3]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)(4-bromophe
nyl) methanone (39)
White powders, yield 48.1%. mp: 144 ~ 145 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.85 (s, 1H), 7.83 (s, 1H), 7.72 (t, J = 6.27 Hz, 4H), 7.46 (d, J = 5.22 Hz, 3H), 6.88 (t,
J = 6.00 Hz, 2H), 6.80 (d, J = 6.03 Hz, 1H), 6.00 (s, 2H), 5.69 (dd, J = 3.69 and 8.73
Hz, 1H), 3.88 (dd, J = 8.82 and 13.62 Hz, 1H), 3.20 (dd, J = 3.75 and 13.59 Hz, 1H).
MS (ESI): 450.30 [M+H] +. Anal. calc. for C23H17BrN2O3: C, 61.48; H, 3.81; N, 6.23.
Found: C, 61.47; H, 3.82; N, 6.24.
4.1.29.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-fluorophenyl) methanone (40)
White powders, yield 42.6%. mp: 154 ~ 155 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.99 (t, J = 5.85 Hz, 2H), 7.69 (s, 1H), 7.67 (s, 1H), 7.32 (t, J = 6.63 Hz, 2H), 7.03 (s,
1H), 7.01 (s, 1H), 6.87 (t, J = 5.97 Hz, 2H), 6.79 (d, J = 5.94 Hz, 1H), 5.99 (s, 2H),
5.67 (dd, J = 3.54 and 8.64 Hz, 1H), 3.84 (dd, J = 8.79 and 13.53 Hz, 1H), 3.80 (s,
3H), 3.15 (dd, J = 3.66 and 13.53 Hz, 1H). MS (ESI): 419.42 [M+H] +. Anal. calc. for
C24H19FN2O4: C, 68.89; H, 4.58; N, 6.70. Found: C, 68.88; H, 4.59; N, 6.69.
4.1.30.
17

ACCEPTED MANUSCRIPT
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-chlorophenyl) methanone (41)
White powders, yield 48.6%. mp: 140 ~ 141 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.93 (s, 1H), 7.91 (s, 1H), 7.69 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.55 (s, 1H), 7.02 (s,
1H), 7.00 (s, 1H), 7.87 (t, J = 6.00 Hz, 2H), 6.79 (d, J = 5.91 Hz, 1H), 6.00 (s, 2H),
5.67 (dd, J = J = 3.56 and 8.68 Hz, 1H), 3.84 (dd, J = 8.75 and 13.50 Hz, 1H), 3.80 (s,
3H), 3.16 (dd, J = 3.66 and 13.59 Hz, 1H). MS (ESI): 435.87 [M+H] +. Anal. calc. for
C24H19ClN2O4: C, 66.29; H, 4.40; N, 6.44. Found: C, 66.28; H, 4.41; N, 6.43.
4.1.31.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
3-methoxyphenyl) methanone (42)
White powders, yield 35.9%. mp: 166 ~ 167 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.85 (s, 1H), 7.83 (s, 1H), 7.65 (s, 1H), 7.62 (s, 1H), 7.47 ~ 7.54 (m, 2H), 7.03 (s, 1H),
7.00 (s, 1H), 6.88 (t, J = 6.00 Hz, 2H), 6.79 (d, J = 5.97 Hz, 1H), 6.00 (s, 2H), 5.68
(dd, J = 3.50 and 8.66 Hz, 1H), 3.84 (s, 4H), 3.80 (s, 3H), 3.19 (dd, J = 3.66 and 13.59
Hz, 1H). MS (ESI): 431.45 [M+H] +. Anal. calc. for C25H22N2O5: C, 69.76; H, 5.15; N,
6.51. Found: C, 69.77; H, 5.14; N, 6.50.
4.1.32.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
4-methoxyphenyl) methanone (43)
White powders, yield 31.2%. mp: 146 ~ 147 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.97 (t, J = 5.81 Hz, 2H), 7.66 (s, 1H), 7.64 (s, 1H), 7.32 (t, J = 6.60 Hz, 2H), 7.03 (s,
1H), 7.00 (s, 1H), 6.87 (t, J = 5.97 Hz, 2H), 6.79 (d, J = 5.94 Hz, 1H), 5.99 (s, 2H),
5.68 (dd, J = 3.50 and 8.66 Hz, 1H), 3.84 (s, 4H), 3.80 (s, 3H), 3.19 (dd, J = 3.66 and
+
13.59 Hz, 1H). MS (ESI): 431.45 [M+H] . Anal. calc. for C25H22N2O5: C, 69.76; H,
5.15; N, 6.51. Found: C, 69.78; H, 5.14; N, 6.50.
4.1.33.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
3,4-dimethoxyphenyl) methanone (44)
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White powders, yield 44.6%. mp: 150 ~ 151 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.80 (s, 1H), 7.78 (s, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.47 ~ 7.54 (m, 1H), 7.01 (s, 1H),
7.00 (s, 1H), 6.87 (t, J = 6.00 Hz, 2H), 6.78 (d, J = 5.97 Hz, 1H), 6.00 (s, 2H), 5.68
(dd, J = 3.50 and 8.66 Hz, 1H), 3.84 (s, 4H), 3.82 (s, 3H), 3.80 (s, 3H), 3.18 (dd, J =
+
3.66 and 13.59 Hz, 1H). MS (ESI): 461.48 [M+H] . Anal. calc. for C26H24N2O6: C,
67.82; H, 5.25; N, 6.08. Found: C, 67.81; H, 5.24; N, 6.09.
4.1.34.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)(
phenyl) methanone (45)
White powders, yield 41.9%. mp: 152 ~ 153 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.89 (s, 1H), 7.87 (s, 1H), 7.67 (s, 1H), 7.65 (s, 1H), 7.47 ~ 7.54 (m, 3H), 7.03 (s, 1H),
7.00 (s, 1H), 6.88 (t, J = 6.00 Hz, 2H), 6.79 (d, J = 5.97 Hz, 1H), 6.00 (s, 2H), 5.68
(dd, J = 3.51 and 8.67 Hz, 1H), 3.84 (dd, J = 8.76 and 13.50 Hz, 1H), 3.80 (s, 3H),
+
3.19 (dd, J = 3.63 and 13.50 Hz, 1H). MS (ESI): 401.43 [M+H] . Anal. calc. for
C24H20N2O4: C, 71.99; H, 5.03; N, 7.00. Found: C, 71.98; H, 5.04; N, 7.01.
4.1.35.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(4-fluorophenyl)methanone(46)
White powders, yield 35.9%. mp: 121 ~ 122 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
8.02 (s, 2H), 7.33 (t, J = 6.42 Hz, 2H), 7.03 (s, 2H), 6.88 (t, J = 5.91 Hz, 2H), 6.78 (d,
J = 5.37 Hz, 1H), 6.00 (s, 2H), 5.71 (dd, J = 3.50 and 8.61 Hz, 1H), 3.82 (s, 7H), 3.71
(s, 3H), 3.27 (dd, J = 3.06 and 13.59 Hz, 1H). MS (ESI): 479.47 [M+H] +. Anal. calc.
for C26H23FN2O6: C, 65.27; H, 4.85; N, 5.85. Found: C, 65.26; H, 4.86; N, 5.84.
4.1.36.
(5-(benzo[d][1,3]dioxol-5-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-
1-yl)(4-chlorophenyl)methanone(47)
White powders, yield 34.6%. mp: 128 ~ 129 ℃. 1H NMR (DMSO-d6, 300 MHz) δ:
7.97 (d, J = 5.61 Hz, 2H), 7.57 (d, J = 6.12 Hz, 2H), 7.03 (s, 2H), 6.88 (t, J = 5.91 Hz,
2H), 6.79 (d, J = 5.55 Hz, 1H), 6.00 (s, 2H), 5.71 (dd, J = 2.97 and 8.43 Hz, 1H), 3.82
(s, 7H), 3.71 (s, 3H), 3.27 (dd, J = 3.03 and 13.62 Hz, 1H). MS (ESI): 495.92 [M+H]
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+. Anal. calc. for C26H23ClN2O6: C, 53.62; H, 3.97; N, 5.66. Found: C, 53.61; H, 3.96;
N, 5.67.
4.2. Biological Assays
4.2.1. Antiproliferation assay [24-26]
A549 human lung cancer cells, MCF-7 human breast cancer cells and HepG-2
human liver cancer cells cultured in DMEM/10% (V/V) fetal bovine serum, in 5%
CO2 water saturated atmosphere at 37 ℃ . A549, MCF-7 Cell and HepG-2
suspensions (10000/mL) were prepared and 100 μL/well dispensed into 96-well plates
(Costar) giving 1000 cells/well respectively. The plates were returned to the incubator
for 18 h to allow the cells to reattach. These compounds were initially prepared at 20
mM in DMSO. Aliquots (200 μL) were diluted into 20 mL culture medium giving 200
μM, and 10 serial dilutions of 3×prepared. Aliquots (100 μL) of each dilution were
added to the wells, giving doses ranging from 100 μM to 0.005 μM. After a further
incubated at 37 ℃ for 24 h in a humidified atmosphere with 5% CO2, the cell
viability was assessed by the fresh MTT (Sigma, 4 mg/mL in PBS) reduction assay
and carried out strictly according to the manufacturer instructions. Each assay was
carried out for at least three times. The results were summarized in Table 3.
4.2.2. Tubulin polymerization assay
Bovine brain tubulin was purified as described previously. [27] To evaluate the
effect of the compounds on tubulin assembly in vitro, varying concentrations of
compounds were preincubated with 10 μM bovine brain tubulin in glutamate buffer at
30 ℃ and then cooled to 0 ℃. [28, 29] After the addition of 0.4 mM GTP, the
mixtures were transferred to 0 ℃ cuvettes in a recording spectrophotometer and
warmed up to 30 ℃. Then, the assembly of tubulin was observed turbidimetrically at
350 nm. The IC50 was defined as the compound concentration that inhibited the extent
of assembly by 50% after 20 min incubation..
4.2.3. Annexin-V Assay
Approximately 105 cells HepG-2 cells /well were plated in a 12 well plate and
allowed to adhere. After 12 h, the medium was replaced with fresh culture medium
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containing compound 18 at final concentrations of 0, 0.01, 0.03 and 0.05 μM. Then
cells were harvested after 24 h.
They were trypsinized, washed in PBS and centrifuged at 2000 rpm for 5 min. The
pellet was then resuspended in 500 μL of staining solution (containing 5 μL Annexin
V-PE and 5 μL PI in Binding Buffer), mixed gently and incubated for 15 min at room
temperature (15 - 25℃) in dark. The samples were then read in a FACScalibur flow
cytometer (USA) at 488 nm excitation. Analyses were performed by the software
supplied in the instrument.
4.2.4. Flow Cytometric Analysis of Cell Cycle Distribution.
For flow cytometric analysis of DNA content, 5×105 HepG-2 cells in exponential
growth were treated with different concentrations of compound 18 for 48 h. After the
incubation, the cells were collected, centrifuged, and fixed with ice-cold ethanol
(70%). The cells were treated with lysis buffer containing RNase A and 0.1% Triton
X-100 and stained with PI. Samples were analyzed on a Cytomic FC500 flow
cytometer (Beckman Coulter). DNA histograms were analyzed using Mod Fit for
Windows.
4.3. Molecular docking
Molecular docking of compounds 15-47 into the three dimensional X-ray structure
of tubulin (PDB code: 1SA0) was carried out using the Discovery Studio (version 3.5)
as implemented through the graphical user interface DS- CDOCKER protocol. [30]
The three-dimensional structures of the aforementioned compounds were
constructed using Chem. 3D ultra 12.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2010)], then they were energetically
minimized by using MMFF94 with 5000 iterations and minimum RMS gradient of
0.10. The crystal structures of two TS proteins complex were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters
and ligands were eliminated from the protein and the polar hydrogen was added to the
proteins.
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Acknowledgments
The work was financed by a grant (No. J1103512) from National Natural Science
Foundation of China and Major Projects on Control and Rectification of Water Body
Pollution (No. 2011ZX07204-001-004).
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Figure captions:
Figure 1 Inhibitors and Potential Inhibitors of Tubulin Polymerization
Figure 2 Crystal structure diagram of compound 23. H atoms are shown as small
spheres of arbitrary radii.
Figure 3 Representative flow cytometric histograms of apoptotic HepG-2 cells after
24 h treatment with compound 18. The cells were harvested and labeled with
Annexin-V-FITC and PI and then analyzed by flow cytometry.
Figure 4 Effects of compound 18 on cell cycle progression of HepG-2 cells were
determined by flow cytometry analysis. HepG-2 cells were treated with different
concentrations of compound 18 for 48 h. The percentage of cells in each cycle phase
was indicated.
Figure 5 The CDOCKER_INTERACTION_ENERGY (kcal/mol) obtained from the
docking study of compounds 15-47 by the CDOCKER protocol (Discovery Studio 3.5,
Accelrys, Co. Ltd). It is clear that compound 18, showed lowest interaction energy of
all the synthesized compounds and the interaction energy reached up to -60.69
kcal/mol.
Figure 6 The binding mode between the active conformation of compound 18 and the
target protein tubulin (PDB code: 1SA0) provided by the CDOCKER protocol
(Discovery Studio 3.5, Accelrys, Co. Ltd). We employed 2D diagram (Figure 6A),
3D interaction map (Figure 6B) to display the interaction between 18 and the targeted
protein. In the binding model, compound 18 is nicely bound to tubulin via four
hydrogen bonds.
Figure 7 The surface model structure to display the interaction between compound 18
and the targeted protein tubulin.
Scheme 1 General synthesis of compounds 15 - 37.
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Figure 1. Inhibitors and Potential Inhibitors of Tubulin Polymerization.
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Figure 2. Crystal structure diagram of compound 23. H atoms are shown as small
spheres of arbitrary radii.
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Figure 3. Representative flow cytometric histograms of apoptotic HepG-2 cells after
24 h treatment with compound 18. The cells were harvested and labeled with
Annexin-V-FITC and PI and then analyzed by flow cytometry.
28