Synthesis and biological evaluation of (1-aryl-1H-pyrazol-4-yl) (3,4,5-trimethoxyphenyl)methanone derivatives as tubulin inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.05.058

A series of (1-aryl-1H-pyrazol-4-yl) (3,4,5-trimethoxyphenyl)methanones (8a-p, 9a-p) and ketoxime (10c) derivatives were designed and synthesized as antitubulin agents. All of the target compounds were evaluated for the in vitro anti-proliferative activities against three tumor cell lines (A549, HT-1080, SGC-7901). The most promising compounds in this class were (1-(p-tolyl)-1H-pyrazol-4-yl) (3,4,5-trimethoxyphenyl)methanone (9c) and its ketoxime derivative (10c), which significantly inhibited tumor cells growth with IC₅₀ value of 0.054–0.16 μM. Meanwhile, compound 9c exhibited effectively inhibitory activity of tubulin polymerization. Consistent with its antitubulin activity, compound 9c could destructively damage microtubule network and arrest SGC-7901 cell cycle at G2/M phase significantly. The structure-activity relationship (SAR) and conformational analysis indicate that methyl group at C4-position of C-ring is critical for the activities and the amino group at the C5-position of B-ring plays a negative role in maintaining bioactivity. Furthermore, a molecular docking study was performed to elucidate its binding mode at the colchicine site in the tubulin heterodimer.

Full text of DOI:10.1016/j.ejmech.2018.05.058

Accepted Manuscript
Synthesis and biological evaluation of (1-aryl-1H-pyrazol-4-yl) (3,4,5-
trimethoxyphenyl)methanone derivatives as tubulin inhibitors
Min'an Zhai, Long Wang, Shiyuan Liu, Lijing Wang, Peng Yan, Junfang Wang, Jingbo
Zhang, Haifei Guo, Qi Guan, Kai Bao, Yingliang Wu, Weige Zhang
PII:
S0223-5234(18)30489-6
10.1016/j.ejmech.2018.05.058
EJMECH 10469
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 April 2018
Revised Date: 30 May 2018
Accepted Date: 31 May 2018
Please cite this article as: Min'. Zhai, L. Wang, S. Liu, L. Wang, P. Yan, J. Wang, J. Zhang, H. Guo, Q.
Guan, K. Bao, Y. Wu, W. Zhang, Synthesis and biological evaluation of (1-aryl-1H-pyrazol-4-yl) (3,4,5-
trimethoxyphenyl)methanone derivatives as tubulin inhibitors, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.05.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of
(1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone derivatives as
tubulin inhibitors
Min’an Zhaia, Long Wanga, Shiyuan Liua, Lijing Wangb, Peng Yana, Junfang Wangb, Jingbo Zhanga, Haifei Guoa,
Qi Guana, Kai Baoc*, Yingliang Wub* ,Weige Zhanga*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China
b
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, China
c
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District,
Shenyang 110016, China
Corresponding author. Tel.: +86 24 43520220; fax: +86 24 23986393
E-mail: zhangweige2000@sina.com (W. Zhang), yingliang_1016@163.com (Y. Wu), baokai@syphu.edu.cn (K.
Bao).

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Abstract
A series of (1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanones (8a-p, 9a-p) and
ketoxime (10c) derivatives were designed and synthesized as antitubulin agents. All of the target
compounds were evaluated for the in vitro anti-proliferative activities against three tumor cell
lines (A549, HT-1080, SGC-7901). The most promising compounds in this class were
(1-(p-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9c) and its ketoxime derivative
(10c), which significantly inhibited tumor cells growth with IC50 value of 0.054-0.16 µM.
Meanwhile, compound 9c exhibited effectively inhibitory activity of tubulin polymerization.
Consistent with its antitubulin activity, compound 9c could destructively damage microtubule
network and arrest SGC-7901 cell cycle at G2/M phase significantly. The structure-activity
relationship (SAR) and conformational analysis indicate that methyl group at C4-position of
C-ring is critical for the activities and the amino group at the C5-position of B-ring plays a
negative role in maintaining bioactivity. Furthermore, a molecular docking study was performed to
elucidate its binding mode at the colchicine site in the tubulin heterodimer.
Keywords: anti-proliferative, antitubulin, G2/M phase, conformational analysis, molecular
docking.

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1. Introduction
Microtubules are the main constituents of the cytoskeleton in eukaryocyte which are composed of
α,β-tubulin heterodimers.[1,2] The tubular polymers of tubulin are always in a dynamic process of
polymerization and depolymerization.[3] Some microtubule polymerization inhibitors such as
CA-4 (1, Figure 1A) have already been validated to break the balance of (de)polymerization via
binding to colchicine sites, resulting in damaging or killing tumor cells.[4-8] Therefore microtubule
inhibitors have great prospects in cancer chemotherapy.
In recent years, 4-substituted methoxybenzoyl-aryl-thiazole (SMART 2, Figure 1A) was
discovered and synthesized successively as colchicine binding site inhibitor (CBSI), and it
exhibited cytotoxic potency in the low nanomolar range in vitro and ultimately induced cell cycle
arrest in G2/M phase.[9] The chemical structures of SMART and its analogues are constituted of
three conjugated aromatic rings (A-ring: 3,4,5-trimethoxyphenyl, B-ring: five-membered aromatic
heterocycle, and C-ring: substituted phenyl, respectively, Figure 1A) and a ketone linkage between
B- and C-ring. In general, the favorite design strategy of SMART analogues was such that
replacing the thiazole fragment with other five-membered aromatic heterocycles according to
bioisosterism theory and the original antiproliferative activities could often be maintained or
increased to some extent (3, 4, Figure 1A).[10-14] Meanwhile, some small chemical groups on
five-membered aromatic heterocycles, such as amino and methyl group, were well-tolerated (5, 6,
Figure 1A).[15-17] Additionally, several sp2 linkages, such as oxime and cyanoimine, were
introduced into SMART molecule to replace the carbonyl group, which could also maintain or
increase the antiproliferative activities.[17] Those together offered numerous possibilities for
further optimization and design of SMART analogues.
Pyrazole is a five-membered aromatic heterocycle, which contains two adjacent nitrogen
atoms, and its derivatives have been consistently focused on because of its ease of preparation and
numerous potential pharmacological properties including anti-depressant, anti-microbial,
anti-convulsant and anti-tubulin activities.[18-20] For example, a series of 1,5-diaryl substituted
pyrazole compounds were reported as inhibitors of tubulin polymerization in our previous work (7,
Figure 1A).[21]
In our study, pyrazole fragment was introduced into SMART skeleton to replace the central
thiazole moiety. Thus, a series of (1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone

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(8a-p, 9a-p, Figure 1B) and ketoxime (10c, Figure 1B) derivatives were designed and synthesized.
The newly synthesized target compounds were investigated for their bioactivities to explore the
preliminary structure-activity relationships (SAR) and the molecular modeling was carried out to
elucidate its possible binding mode in tubulin.
Figure 1. (A) Chemical structures of CA-4 (1), SMART (2) and their analogues 3-7; (B) target compounds (8a-p,
9a-p, 10c)
2. Results and discussion
2.1. Chemistry
The
general
synthetic
approach
for
the
preparation
of
(1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone and ketoxime derivatives (8-10) was
illustrated in Scheme 1. First of all, one step direct preparation of
3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (12) was achieved by treatment of
3,4,5-trimethoxybenzaldehyde (11), copper chloride and potassium hydroxide under oxygen
atmosphere in N,N-Dimethyl acetamide at room temperature.[22] Subsequently, ketone (12) reacted
with N,N-Diphenylformamidine to form enamine (13) in high yield.[23] Substituted
phenylhydrazine hydrochlorides (15) were synthesized from the corresponding phenylamine
(14).[24] Treatment of enamine (13) with corresponding phenylhydrazin hydrochlorides (15) in the
presence of triethylamine proceeded with high regioselectivity and exclusively cyclized onto the

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nitrile to form 5-amino-1H-pyrazole compounds (8a-p) under microwave irradiation.[25] Then, the
deaminating pyrazole compounds (9a-p) were synthesized via deamination by using isoamyl
nitrite in THF under reflux condition.[26] Finally, 9c was reacted with 10-fold amount of
hydroxylamine hydrochloride and sodium acetate to afford ketoxime 10c consisting of Z and E
isomers.[27]
Scheme 1. Synthetic route of the target compounds. Reagents and conditions: (i) CH3CN, CuCl2, KOH, O2, DMA,
25 oC, 12 h; (ii) N,N-Diphenylformamidine, xylene, reflux, 2 h; (iii) NaNO2, HCl (conc.), 0 oC, 0.5 h;
.
SnCl2 H2O/HCl (conc.), 0 oC, 4 h; (iv) EtOH, Et3N, MW 120 oC, 10 min; (v) isoamyl nitrite, THF, reflux, 1 h; ( )
NH2OH.HCl, CH3CO2Na, EtOH, reflux, 2 h.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity assay and discussion
The antiproliferative effect of the synthesized compounds 8a-p, 9a-p and 10c against three human
cancer cell lines (A549: human lung adenocarcinoma epithelial cell; HT-1080: human
fibrosarcoma cell; SGC-7901: human gastric adenocarcinoma cell) were performed by using the
standard MTT assay in vitro, while SMART was used as the positive control. The results (IC50
values) were summarized in Table 1. According to the experimental data, almost all of compounds
8a-p showed low antiproliferative activity. But the deaminating compounds 9a-p derivated from
compounds 8a-p displayed moderate antiproliferative activities against three different human
tumor cell lines. Compound 9c, which contained a methyl group at the C4-positon of C-ring,

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showed the most potent antiproliferative activity against A549 cell, HT-1080 cell and SGC-7901
cell, with IC50 values of 0.11, 0.16 and 0.054 µM, respectively. To our surprise, a sharply decline
of antiproliferative activity was observed when other substituents were introduced to replace the
methyl group at C4-position of C-ring. Interestingly, compound 10c, as a derivative of compound
9c with oxime linkage maintained potent antiproliferative activity against multiple tumor cell lines.
Compared with compounds 8a-p, the enhanced activities of compounds 9a-p indicated that the
amino of B-ring play a negative effect in maintaining the antiproliferative activities.
Table 1. In vitro antiproliferative activity of the target compounds
(IC50 ± SD, µM)a
Compound
A549
HT-1080
SGC-7901
11.3 ± 0.72
>30
5.0 ± 0.21
>30
24.7 ± 2.6
>30
8a
8b
8c
8d
8e
8f
20.6 ± 2.1
>30
>30
29.6 ± 1.9
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
8g
8h
8i
>30
>30
>30
>30
>30
>30
>30
>30
>30
8j
>30
>30
>30
8k
8l
>30
>30
>30
>30
>30
>30
8m
8n
8o
8p
9a
9b
9c
9d
9e
9f
>30
>30
>30
>30
>30
>30
>30
>30
>30
8.6 ± 0.17
>30
19.4 ± 2.3
>30
>30
>30
0.11 ± 0.013
14.0 ± 1.1
>30
0.16 ± 0.02
2.8 ± 0.19
>30
0.054 ± 0.008
6.5 ± 0.28
>30
20.4 ± 0.7
>30
3.7 ± 0.23
>30
16.7 ± 0.16
>30
9g
9h
9i
>30
>30
>30
>30
>30
>30
18.7 ± 1.4
16.3 ± 0.19
>30
>30
17.9 ± 1.6
7.8 ± 0.42
>30
9j
3.1 ± 0.11
17.0 ± 1.3
>30
9k
9l
>30
>30
9m
9n
2.6 ± 0.3
12.9 ± 2.0
3.7 ± 0.5

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2.3 ± 0.1
>30
1.6 ± 0.09
22.6 ± 3.1
1.9 ± 0.2
>30
9o
9p
10cb
SMART (2)c
0.14 ± 0.02
0.032 ± 0.013
0.090 ± 0.017
0.027 ± 0.009
0.071 ± 0.014
0.023 ± 0.008
a IC50: 50% inhibitive concentration after 72 h of drug exposure, as determined by using the standard MTT assays
and the values were average of three parallel experiments. b Consist of Z/E isomer (10.8:1, measured by HPLC). c
SMART was used as positive control.
To investigate the cytotoxicity of two compounds with potent bioactivity (9c and 10c),
normal fibroblasts L929 cell line was selected to perform the cytotoxicity test and SMART was
used as positive control. As shown in Table 2, compounds 9c, 10c and SMART which inhibited
the growth of three cancer cell lines potently in vitro, displayed weak inhibitory effect on L929
cells with IC50 values of 65.7, 96.2 and 21.0 µM, respectively. The results meant that these
compounds have a relatively low cytotoxicity against L929 cell line.
Table 2. Cytotoxicity test of compounds 9c, 10c and SMART on normal fibroblasts L929 cells
(IC50 ± SD, µM)
Compound
65.7 ± 4.1
96.2 ± 6.6
21.0 ± 1.3
9c
10c
SMART (2)
The tremendous differences of antiproliferative activity between 8c and 9c impelled us
continuously to explore the essential reasons. One possible reason is that they adopted different
conformation when they interacted with colchicine binding site, which is known to be decisive for
producing bioactivity. From the perspective of chemical structure, SMART has two relatively
stable and clearly distinct conformations, “straight” and “bent” conformation, respectively (Figure
2). We believed that SMART adopted a single bioactive conformation to interact with tubulin
dimer, however, due to the lack of co-crystal structure of tubulin-SMART complex, we speculated
that SMART and its analogues may adopt “bent” conformation to interact with colchicine binding
site according to the crystal structure of SMART and the potential binding mode of them in
previous literatures.[10, 14, 17, 28] Herein density functional theory (DFT) calculation was performed
to compare the energy between “straight” and “bent” conformation in this study (see
supplementary data, Table S1).
As shown in the calculation results (Table 2), the energy of “bent” conformation of SMART

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(p-Me) was much lower than “straight” conformation (-13.02 kJ/mol). Meanwhile, the estimated
proportion of “bent” conformation was over 99.5%. Hence, SMART most probably bind to
colchicine site with “bent” conformation as the speculation.
In contrast, the “bent” conformation of 8c was a higher energy situation (+25.47 kJ/mol,
Table 2) and the proportion of “bent” conformation of 8c was less than 0.1%. Too low proportion
of “bent” conformation may be the primary cause of the loss of bioactivity. We speculated that the
role of amino group in maintaining conformation is twofold: (i) hydrogen bond between amino
and carbonyl group is beneficial for stabilizing “straight” conformation (Figure 3A); and (ii) the
steric hindrance of amino with 3,4,5-trimethoxylphenyl group has an adverse effect for stabilizing
“bent” conformation (Figure 3B). This result further showed that “bent” conformation may be
essential for bioactivity.
The “straight” conformation of the most potent compound 9c was dominant. Nevertheless it
only had a small energy difference compared to the “bent” conformation (+2.21 kJ/mol) and the
proportion of “bent” conformation of 9c could reach 29.2%. Therefore we speculated that a certain
ratio of “bent” conformation and the relatively low rotation energy barrier between two
conformations of 9c may be the reasons of its relatively high activity.
Figure 2. Two conformations of SMART (p-Me) and target compounds
Table 2. Energy of different conformations
Compound
Ebent-Estraight (a.u.)a Ebent-Estraight (kJ/mol)b Estimated proportion of “bent” conformationc
SMART (p-Me)d
-0.00496
+0.00970
+0.00084
-13.02
+25.47
+2.21
99.5%
<0.1%
8c
9c
29.2%
c
a 1 a.u. = 2625.50 kJ·mol-1; b negative value represents “bent” conformation is dominant; propotion was obtained
from equilibrium theory; d SMART with methyl group at C4-position of C-ring, it has stronger antitumor activity
than SMART (without substituent groups on C-ring).[9]

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Figure 3. Intramolecular interactions of 8c: (A) hydrogen bond and (B) steric hindrance.
2.2.2. In vitro tubulin polymerization assay
In order to examine whether the compounds interact with tubulin and inhibit tubulin
polymerization, we chose the most active compound 9c to evaluate for its inhibition of tubulin
polymerization in vitro. Microtubule polymerization inhibitor (SMART) and microtubule
stabilizing agent (Taxol) were used as the positive and negative controls, respectively. As the data
shown in Figure 4, compound 9c inhibited tubulin assembly (IC50: 9.17 µM) in a
concentration-dependent manner. In contrast, taxol could raise the proportion of tubulin
polymerization in comparison with the untreated cells. The results suggested that compound 9c
interferes with the microtubule polymerization in a way similar to the positive control SMART. In
other words, tubulin is most likely the target of this series of compounds.
Figure 4. Effects of compound 9c on tubulin polymerization. Tubulin was pre-incubated for 1 min with 9c at 2.5
µM, 5 µM, 10 µM and 20 µM, respectively, SMART (2) at 2.5 µM, Taxol at 5 µM or the vehicle DMSO before
GTP was added to start the tubulin polymerization reaction. The reaction was monitored at 37 oC.
2.2.3. Immunofluorescence staining studies
For further study the effects of compound 9c on microtubule, confocal microscopy was used to

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monitor the alterations of microtubules and cytoskeleton. As shown in Figure 5, untreated cells
(control group) maintain a well-organised and spindle microtubular network, whereas the
morphology of microtubular network was diffuse around the nuclei when exposed SGC-7901 cells
to SMART or compound 9c (2-fold IC50 concentration). The result indicated that compound 9c is
most likely to target tubulin and it can disrupt microtubule formation in accordance with SMART.
Figure 5. Compound 9c interfered microtubule polymerization in SGC-7901 tumor cells. The SGC-7901 tumor
cells were treated with SMART or compound 9c at 2-fold IC50 concentration for 24 h then stained with
anti-α-tubulin-FITC specific antibodies followed by DAPI. Unassembled tubulin and microtubules were colored
green, and DNA was colored blue.
2.2.4. Cell cycle study by flow cytometry
It has been confirmed that inhibitors of tubulin polymerization, such as SMART could induce cell
cycle arrest in G2/M phase. Therefore, SMART was selected as positive control to investigate the
impacts of compound 9c on the cell cycle period of SGC-7901 cells. Treatment of cells for 24 h
with SMART (2-fold IC50) and compound 9c (0.5-fold IC50, IC50 and 2-fold IC50, respectively)
resulted in accumulation of SGC-7901 cells in G2/M phase obviously, as shown in Figure 6A. In
the vehicle control group, about 15% of SGC-7901 cells were distributed in G2/M phase, while
the percentage of cells in G2/M phase could be extended up to around 68% significantly in a
dose-dependent manner by treatment with 9c (Figure 6B). This phenomenon indirectly proved that

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9c might be a new antitubulin agent which was consistent with the conclusion of previous assays.
Figure 6. (A) Cell cycle distribution of SGC-7901 cells treated with SMART (46 nM) and 9c (27 nM, 54 nM, 108
nM) for 24 h; (B) The proportion of SGC-7901 cells in G2/M phase.
2.3. Molecular modeling
To investigate the possible binding of the newly synthesized compounds to the colchicine site of
tubulin, a molecular docking study of compound 9c was performed by using CDOCKER program
of Discovery Studio 3.0 software (PDB: 5LYJ). As the results showed in Figure 7, SMART and
compound 9c were embedded into the colchicine-binding site by “bent” conformation in a similar
way (Figure 7A). The carbonyl linkages of SMART and 9c produced hydrogen bonding
interactions with amino group of β-Asp 251 (SMART: 2.97 Å; 9c: 2.95 Å. Figure 7B and 7C).
Meanwhile, the stronger hydrogen bonds existed between the oxygen atom of C4-position
methoxyl of A-ring and sulfydryl group of β-Cys 241 (SMART: 2.58 Å; 9c: 2.52 Å. Figure 7B and
7C). In addition, the methyl groups at C4-position of C-rings of SMART (p-Me) and 9c produced
alkyl hydrophobic interactions with β-Lys 352 (SMART: 5.22 Å; 9c: 4.96 Å. Figure 7B and 7C)
and α-Val 181 (SMART: 4.04 Å; 9c: 4.03 Å. Figure 7B and 7C). As shown in Figure 7E and 7F,
the methyl group at C4-position of C-ring of 9c could embed into the hydrophobic pocket well,
which might explain the reason that 9c has most potent activity in this series of compounds.
Meanwhile, the result about alkyl hydrophobic interaction was consistent with the fact that
SMART (p-Me) and ABI (p-Me) possessed most potent antiproliferative activities compared to
SMART and ABI with other substituents on C-rings. The docking data indicated that compound 9c
may play its bioactive role through binding to colchicine site.

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Figure 7. (A) Superimposition of SMART (gray) and 9c (green) in colchicine-binding site; (B) Interactions of
SMART with tubulin (green dotted lines represent H-bond); (C) Interactions of 9c with tubulin (green dotted lines
represent H-bond); (D) 2D diagram of 9c-tubulin interactions; (E) The hydrophobic surface on the receptor (9c:
green); (F) Superimposition of 9a (yellow) and 9c (green).
3. Conclusion
In
summary,
a
series
of
novel
tubulin
polymerization
inhibitors,
(1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone and its ketoxime derivatives, were
designed and synthesized as analogues of SMART. All of the targeted compounds were screened
for antiproliferative activity against three human tumor cell lines, and the results indicated that 9c
and 10c showed potent antiproliferative activity with IC50 value of 0.054-0.16 µM in vitro. SAR

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studies suggested that the methyl group at C4-position of C-ring played an important role for the
potent biological activities. The results of DFT calculation and conformational analysis indicated
that a certain ratio of “bent” conformation was probably necessary for exerting the biological
effects. Our speculation was supported by the fact that the introduction of amino group to
C5-position of B-ring of compound 9c could lead to a decrease of “bent” conformation and the
loss of antiproliferative activity. Further biological assays of 9c containing tubulin polymerization
and immunofluorescence staining in vitro demonstrated that 9c inhibited tubulin assembly
effectively and destroyed cellular microtubule strongly. Additionally, molecular modeling results
showed that 9c may be a potential colchicine site binder.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and instruments
All reagents and solvents were obtained from commercial suppliers and used without any further
purification. The progress of reactions was monitored by thin layer chromatography (TLC) with
precoated silica gel polyester GF254 plates (thickness 0.25 mm). The purification of products was
performed using column chromatography (200-300 mesh, Qingdao PUKE) or silica gel plate (0.25
mm, Qingdao PUKE). The melting points were determined with a hot-stage microscope (X-4,
Beijing Taike Ltd.) and were uncorrected. Mass spectra (MS) for the intermediates were obtained
on an Agilent 1100-sl mass spectrometer (Agilent Co. Ltd.) equipped with electrospray ionization
(ESI). High resolution accurate mass determinations (HRMS) for the target compounds were
obtained using an Agilent 6530 Accurate-Mass Q-TOF LCMS (Agilent Co. Ltd.) equipped with
1
electrospray ionisation (ESI). H NMR (400 or 600 MHz) and 13C NMR (100 or 150 MHz)
spectra were recorded on a Bruker Avance spectrometers in DMSO-d6 or CDCl3 containing 0.03%
(v/v) of tetramethylsilane (TMS) as an internal reference at room temperature.
4.1.2. General procedure for synthesis of substituted phenylhydrazine hydrochloride (15)
Concentrated hydrochloric acid (30 mmol, 2.5 mL, 12 M) was added to phenylaniline (10
mmol) and the resulting white suspension was cooled to 0 , then appropriate amount of water
was added to dissolve the white solid. Sodium nitrite (11 mmol) in 10 mL of water, precooled to 0
oC, was slowly added to the solution of phenylaniline hydrochloride. The temperature of the
reaction was maintained at 0 oC for 0.5 h. The diazonium salt solution was treated with a solution

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of stannous chloride hydrate (40 mmol) in 15 mL of concentrate hydrochloric acid (12 M), then
o
stirred for another 4 h at 0 C. A light brown solid which formed was collected by filtration,
washed with little precooled ethyl alcohol and dichloromethane, the crude product was used
without further purification.
4.1.3. Synthesis of 3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (12)
3,4,5-Trimethoxybenzaldehyde (5.0 mmol), potassium hydroxide (15.0 mmol), copper
chloride dehydrate (0.05 mmol), acetonitrile (50.0 mmol) and N,N-dimethylformamide (20 mL)
were added to a 100 mL flask, the mixture was stirred vigorously under O2 circumstance (balloon)
o
for 24 h at 25 C. After the reaction was completed, the redundant acetonitrile was evaporated
under reduced pressure, then added 200 mL 10% hydrochloric acid and stirred for 0.5 h, the solid
1
was filtered and dried to get product, pale yellow solid, 0.90 g, yield 75%. H NMR (600 MHz,
CDCl3): δ 3.93 (6H, s), 3.95 (3H, s), 4.08 (2H, s), 7.15 (2H, s) ppm; MS (ESI): [M+H]+ = 236.1.
4.1.4. Synthesis of 3-phenylamino-2-(3,4,5-trimethoxy- benzoyl)acrylonitrile (13)
A
solution of 3-oxo-3-(3,4,5-trimethoxyphenyl)propanenitrile (2.1 mmol) and
N,N-diphenylformamidine (2.1 mmol) in 10 mL xylenes was heated at reflux for 2 h under
nitrogen. When the reaction was completed, cooled the mixture to room temperature and a large
amount of solid precipitated, filtered and washed with appropriate ethyl acetate,
3-phenylamino-2-(3,4,5-trimethoxybenzoyl)- acrylonitrile was collect as a pale yellow solid, 0.58
g, yield 81%. 1H NMR (600 MHz, CDCl3): δ 3.93 (3H, s), 3.94 (6H, s), 7.22 (2H, d, J = 7.9 Hz),
7.29 (3H, m), 7.45 (2H, t, J = 7.9 Hz), 8.07 (1H, d, J = 12.6 Hz), 12.86 (1H, d, J = 12.6 Hz) ppm;
MS (ESI) : [M+H]+ = 339.1.
4.1.5.
General
procedure
for
preparation
of
(5-amino-1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8a-p)
A mixture of 3-(phenylamino)-2-(3,4,5-trimethoxybenzoyl)acrylonitrile (1 mmol), substituted
phenylhydrazine hydrochloride (1 mmol) and trimethylamine(1 mmol) in ethanol was irradiated in
o
the sealed tube at 120 C for 10 minutes using a CEM Discover microwave synthesizer. After
cooling to room temperature, the mixture was concentrated in vacuo, purification by column
chromatography on the silica gel to get the pure product.
4.1.5.1. (5-Amino-1-phenyl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8a)
White solid (0.30 g, 86%); mp: 178-180 ; 1H NMR (400 MHz, CDCl3): δ 3.93 (9H, s), 6.09 (2H,

ACCEPTED MANUSCRIPT
s), 7.09 (2H, s), 7.44 (1H, m), 7.55 (4H, m), 7.85 (1H, s) ppm; 13C NMR (100 MHz, CDCl3): δ
188.8, 153.1 (×2), 150.6, 141.8, 141.0, 137.1, 135.0, 130.0 (×2), 128.5, 124.0 (×2), 105.6 (×2),
104.6, 60.9, 58.3(×2) ppm; HRMS (ESI): calcd. for C19H20N3O4 [M+H]+ 354.1448, found
354.1448; C19H19N3NaO4 [M+Na]+ 376.1268, found 376.1270.
4.1.5.2. (5-Amino-1-(o-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8b)
White solid (0.28 g, 76%); mp: 102-105 oC; 1H NMR (400 MHz, CDCl3): δ 2.20 (3H, s), 3.91 (3H,
s), 3.92 (6H, s), 5.78 (2H, s), 7.09 (2H, s), 7.36 (4H, m), 7.84 (1H, s) ppm; 13C NMR (100MHz,
CDCl3): δ 188.5, 153.1 (×2), 151.3, 141.5, 141.0, 136.6, 135.1, 135.0, 131.8, 130.2, 127.6, 127.3,
105.7 (×2), 103.8, 61.0, 56.3 (×2), 17.5 ppm; HRMS (ESI): calcd. for C20H22N3O4 [M+H]+
368.1605, found 368.1606; C20H21N3NaO4 [M+Na]+ 390.1424, found 390.1428.
4.1.5.3. (5-Amino-1-(p-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8c)
White solid (0.29 g, 79%); mp: 171-173 oC; 1H NMR (400 MHz, CDCl3): δ 2.40 (3H, s), 3.90 (6H,
s), 3.91 (3H, s), 6.09 (2H, s), 7.08 (2H, s), 7.31 (2H, d, J = 8.2 Hz), 7.42 (2H, d, J = 8.2 Hz),
7.81(1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 188.7, 153.1 (×2), 150.5, 141.6, 141.0, 138.7,
135.1, 134.5, 130.5 (×2), 123.9 (×2), 105.7 (×2), 104.5, 61.0, 56.3 (×2), 21.2 ppm; HRMS (ESI):
calcd. for C20H22N3O4 [M+H]+ 368.1605, found 368.1613; C20H21N3NaO4 [M+Na]+ 390.1424,
found 390.1435.
4.1.5.4. (5-Amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8d)
White solid (0.26 g, 69%); mp: 123-125 oC; 1H NMR (400 MHz, CDCl3): δ 3.94 (9H, s), 6.08 (2H,
s), 7.09 (2H, s), 7.21 (3H, m), 7.55 (2H, m), 7.85 (1H, s), 8.05 (1H, d) ppm; 13C NMR (100MHz,
CDCl3): δ 188.72, 160.89, 153.15 (×2), 150.67, 141.85, 134.92, 126.17, 126.08, 117.04 (×2),
116.81 (×2), 105.66 (×2), 104.55, 60.97, 56.27 (×2) ppm; HRMS (ESI): calcd. for
C19H18FN3NaO4 [M+Na]+ 394.1174, found 394.1186.
4.1.5.5. (5-Amino-1-(2-chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8e)
White solid (0.26 g, 65%); mp: 136-138 oC; 1H NMR (400 MHz, CDCl3): δ 3.93 (3H, s), 3.94 (6H,
s), 5.98 (2H, s), 7.11 (2H, s), 7.49 (3H, m), 7.63 (1H, m), 7.90 (1H, s) ppm; 13C NMR (100MHz,
CDCl3): δ 188.6, 153.1 (×2), 151.8, 142.1, 141.0, 135.0, 134.2, 132.2, 131.3, 130.9, 129.8, 128.3,
105.7 (×2), 103.8, 61.0, 56.3 (×2) ppm; HRMS (ESI): calcd. for C19H19ClN3O4 [M+H]+ 388.1059,
found 388.1058; C19H18ClN3NaO4 [M+Na]+ 410.0878, found 410.0880.
4.1.5.6. (5-Amino-1-(3-chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8f)

ACCEPTED MANUSCRIPT
White solid (0.34 g, 87%); mp: 139-142 oC; 1H NMR (400 MHz, CDCl3): δ 3.90 (9H, s), 6.19 (2H,
s), 7.04 (2H, s), 7.36 (1H, m), 7.45(2H, m), 7.59 (1H, s), 7.83 (1H, s) ppm; 13C NMR (100MHz,
CDCl3): δ 188.7, 153.1 (×2), 150.6, 142.2, 141.0, 138.3, 135.6, 134.8, 130.9, 128.4, 124.1, 121.6,
105.6 (×2), 104.7, 61.0, 56.3 (×2) ppm; HRMS (ESI): calcd. for C19H18ClN3NaO4 [M+Na]+
410.0878, found 410.0895.
4.1.5.7. (5-Amino-1-(4-chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8g)
White solid (0.32 g, 82%); mp: 200-203 oC; 1H NMR (400 MHz, CDCl3): δ 3.93 (6H, s), 3.93 (3H,
s), 6.11 (2H, s), 7.08 (2H, s), 7.53 (4H, m), 7.86 (1H, s)ppm; 13C NMR (100MHz, CDCl3): δ 188.8,
153.2 (×2), 150.6, 142.1, 141.1, 135.7, 134.9, 134.3, 130.1 (×2), 125.1 (×2), 105.7 (×2), 104.7,
61.0, 56.3 (×2) ppm; HRMS (ESI): calcd. for C19H19ClN3O4 [M+H]+ 388.1059, found 388.1073;
C19H18ClN3NaO4 [M+Na]+ 410.0878, found 410.0891.
4.1.5.8. (5-Amino-1-(2-bromophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8h)
o
White solid (0.31 g, 71%); mp: 123-126 C; 1H NMR (400 MHz, CDCl3): δ 3.91 (3H, s), 3.92
(6H, s),5.93 (2H, s), 7.06 (2H, s), 7.34 (1H, m), 7.44 (1H, m), 7.73 (1H, d, J = 8.2 Hz), 7.83 (1H, s)
ppm; 13C NMR (100MHz, CDCl3): δ 188.5, 153.1 (×2), 151.7, 141.9, 141.0, 135.8, 135.0, 134.1,
131.6, 130.0, 128.9, 122.1, 105.7 (×2) , 103.8, 60.9, 56.3 (×2) ppm; HRMS (ESI): calcd. for
C19H19BrN3O4 [M+H]+ 432.0553, found 432.0553; C19H18BrN3NaO4 [M+Na]+ 454.0373, found
454.0377.
4.1.5.9. (5-Amino-1-(3-bromophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8i)
o
White solid (0.35 g, 81%); mp: 83-86 C; 1H NMR (400 MHz, CDCl3): δ 3.91 (9H, s), 6.15 (2H,
s), 7.06 (2H, s), 7.40 (1H, dd, J = 7.8 Hz, J = 8.3 Hz), 7.52 (1H, d, J= 8.3 Hz), 7.55 (1H, d, J = 7.8
Hz), 7.77 (1H, s), 7.83 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 188.7, 153.1 (×2), 150.6,
142.2, 141.1, 138.4, 134.8, 131.4, 131.1, 127.0, 123.4, 122.1, 105.7 (×2), 104.7, 61.0, 56.3 (×2)
ppm; HRMS (ESI): calcd. for C19H18BrN3NaO4 [M+Na]+ 454.0373, found 454.0393.
4.1.5.10.
(5-Amino-1-(4-methoxyphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone
(8j)
White solid (0.31 g, 80%); mp: 184-187 oC; 1H NMR (400 MHz, CDCl3): δ 3.83 (3H, s), 3.92 (9H,
s), 6.04 (2H, s), 7.00 (2H, d, J = 7.9 Hz), 7.08 (2H, s)，7.43 (2H, d, J = 7.9 Hz), 7.79 (1H, s) ppm;
13C NMR (100MHz, CDCl3): δ 188.6, 159.6, 153.1 (×2), 150.6, 141.5, 140.9, 135.1, 129.8, 125.8
(×2), 115.0 (×2), 105.6 (×2), 104.4, 60.9, 56.2 (×2), 55.6 ppm; HRMS (ESI): calcd. for

ACCEPTED MANUSCRIPT
C20H22N3O5 [M+H]+ 384.1554, found 384.1566; C20H21N3NaO5 [M+Na]+ 406.1373, found
406.1388.
4.1.5.11. (5-Amino-1-(4-ethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (8k)
o
1
White solid (0.32 g, 85%); mp: 161-163 C; H NMR (400 MHz, CDCl3): δ 1.28 (3H, t, J = 7.6
Hz), 2.72 (2H, q, J = 7.6 Hz), 3.93 (9H, s), 6.07 (2H, s), 7.09 (2H, s), 7.36 (2H, d, J = 8.2 Hz),
7.46 (2H, d, J = 8.2 Hz), 7.84 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 188.7, 153.1 (×2),
150.5, 145.0, 141.6, 141.0, 135.1, 134.7, 129.3 (×2), 124.0 (×2), 105.6 (×2), 104.5, 61.0, 56.3 (×2),
28.6, 15.5 ppm; HRMS (ESI): calcd. for C21H24N3O4 [M+H]+ 382.1761, found 382.1765;
C21H23N3NaO4 [M+Na]+ 404.1581, found 404.1584.
4.1.5.12. (5-Amino-1-(4-isopropylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone
(8l)
o
1
White solid (0.31 g, 79%); mp: 165-166 C; H NMR (400 MHz, CDCl3): δ 1.30 (6H, d, J = 6.9
Hz), 2.99 (1H, m, J = 6.9 Hz), 3.93 (9H, s), 6.04 (2H, s), 7.10 (2H, s), 7.40 (2H, d, J = 8.3 Hz),
7.47 (2H, d, J = 8.3 Hz), 7.84 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 188.7, 153.1 (×2),
150.5, 149.6, 141.6, 141.0, 135.1, 134.7, 127.9 (×2), 124.0 (×2), 105.6 (×2), 104.5, 61.0, 56.3 (×2),
33.9, 23.9 (×2) ppm; HRMS (ESI): calcd. for C22H26N3O4 [M+H]+ 396.1918, found 396.1915;
C22H25N3NaO4 [M+Na]+ 418.1737, found 418.1738.
4.1.5.13.
(5-Amino-1-(3-chloro-2-methylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)
methanone (8m)
White solid (0.34 g, 86%); mp: 153-155 oC; 1H NMR (400 MHz, CDCl3): δ 2.24 (3H, s), 3.94 (3H,
s), 3.95 (6H, s), 7.12 (2H, s), 7.33 (2H, m), 7.56 (1H, m), 7.89 (1H,s) ppm; 13C NMR (100MHz,
CDCl3): δ 188.5, 153.2 (×2), 151.4, 141.6, 141.1, 136.4, 136.2, 135.4, 134.9, 131.1, 127.7, 126.3,
105.9 (×2), 103.8, 61.0, 56.3 (×2), 15.2 ppm; HRMS (ESI): calcd. for C20H21ClN3O4 [M+H]+
402.1215, found 402.1213; C20H20ClN3NaO4 [M+Na]+ 424.1035, found 424.1034.
4.1.5.14.
(5-Amino-1-(2,5-dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxy-
phenyl)methanone (8n)
White solid (0.29 g, 76%); mp: 167-169 oC; 1H NMR (400 MHz, CDCl3): δ 2.14 (3H, s), 2.36 (3H,
s), 3.93 (9H, s), 5.84 (2H, s), 7.08 (2H, s), 7.14 (1H, s), 7.19 (1H, d, J = 7.7 Hz), 7.24 (1H, d, J =
7.7 Hz), 7.82 (1H, s) ppm; 13C NMR (100 MHz, CDCl3): δ 188.4, 153.0 (×2), 151.2, 141.3, 140.9,
137.2, 135.1, 134.8, 133.0, 131.4, 130.8, 128.0, 105.6 (×2), 103.7, 60.8, 56.2 (×2), 20.6, 16.9 ppm;

ACCEPTED MANUSCRIPT
HRMS (ESI): calcd. for C21H24N3O4 [M+H]+ 382.1761, found 382.1777.
4.1.5.15. (5-Amino-1-(3,5-dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone
(8o)
White solid (0.28 g, 74%); mp: 102-104 oC; 1H NMR (400 MHz, CDCl3): δ 2.38 (6H, s), 3.92 (9H,
s), 6.12 (2H, s), 7.05 (1H, s), 7.08 (2H, s), 7.17 (2H, s), 7.82 (1H, s) ppm; 13C NMR (100MHz,
CDCl3): δ 188.7, 153.1 (×2), 150.5, 141.6, 140.9, 139.9 (×2), 136.9, 135.1, 130.1, 121.6 (×2),
105.7 (×2), 104.4, 60.9, 56.2 (×2), 21.2 (×2) ppm; HRMS (ESI): calcd. for C21H24N3O4 [M+H]+
382.1761, found 382.1776.
4.1.5.16.
(5-Amino-1-(2,4-dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxy-
phenyl)methanone (8p)
White solid (0.25 g, 66%); mp: 149-151 oC; 1H NMR (400 MHz, CDCl3): δ 2.16 (3H, s), 2.40 (3H,
s), 3.93 (3H, s), 3.94 (6H, s), 7.10 (2H, s), 7.15 (1H, d, J = 7.9 Hz), 7.22(2H, d, J = 7.9 Hz), 7.85
(1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 188.5, 153.1 (×2), 151.3, 141.2, 141.0, 140.4, 136.2,
135.1, 132.3, 132.2, 127.9, 127.4, 105.7 (×2), 103.7, 60.9, 56.3 (×2), 21.2, 17.4 ppm; HRMS (ESI):
calcd. for C21H24N3O4 [M+H]+ 382.1761, found 382.1779.
4.1.6.
General
procedure
for
preparation
of
(1-aryl-1H-pyrazol-4-yl)(3,4,5-tri-
methoxyphenyl)methanone (9a-p)
A mixture of (5-amino-1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (0.5 mmol)
and isoamyl nitrite (2 mmol) in dry tetrahydrofuran was refluxed for 3 h and then cooled to room
temperature, the mixture was concentrated and evaporated in vacuo, purification by column
chromatography on the silica gel to get the pure product.
4.1.6.1. (1-Phenyl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9a)
Pale yellow solid (0.12 g, 70%); mp: 119-121 oC; 1H NMR (400 MHz ,CDCl3) δ 3.92 (6H, s), 3.94
(3H, s), 7.17 (2H, s), 7.38 (1H, m), 7.50 (2H, m), 7.75 (2H, m), 8.15 (1H, s), 8.50 (1H, s) ppm; 13C
NMR (100MHz, CDCl3): δ 188.7, 153.1 (×2), 142.6, 142.0, 139.2, 133.9, 130.7, 129.7 (×2), 127.8,
124.1, 119.6 (×2), 106.5 (×2), 60.9, 56.3 (×2) ppm; HRMS (ESI): calcd. for C19H19N2O4 [M+H]+
339.1339, found 339.1338; C19H18N2NaO4 [M+Na]+ 361.1159, found 361.1162.
4.1.6.2. (1-(o-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9b)
Pale yellow solid (0.14 g, 78%); mp: 98-100 oC; 1H NMR (400 MHz, CDCl3): δ 2.29 (3H, s), 3.91
(3H, s), 3.93 (6H, s), 7.17 (2H, s), 7.34 (4H, m), 8.15 (1H, s), 8.16 (1H, s) ppm; 13C NMR

ACCEPTED MANUSCRIPT
(100MHz, CDCl3): δ 187.9, 153.1 (×2), 142.0, 139.0, 134.7, 134.1, 133.5, 131.5, 129.3, 126.9,
126.0, 123.2, 121.0, 106.6(×2), 61.0, 56.4 (×2), 18.0 ppm; HRMS (ESI): calcd. for C20H21N2O4
[M+H]+ 353.1496, found 353.1495; C20H20N2NaO4 [M+Na]+ 375.1315, found 375.1314.
4.1.6.3. (1-(p-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9c)
Pale yellow solid (0.12 g, 68%); purity: 99.05%; mp: 102-104 oC; 1H NMR (400 MHz, CDCl3): δ
2.39 (3H, s), 3.90 (6H, s), 3.92 (3H, s), 7.15 (2H, s), 7.27 (2H, d, J = 8.2 Hz), 7.60 (2H, d, J = 8.2
Hz), 8.11 (1H, s), 8.44 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.7, 153.1 (×2), 142.4,
142.0, 137.7, 137.0, 134.0, 130.5, 130.2 (×2), 123.9, 119.6 (×2), 106.5(×2), 60.9, 56.3 (×2), 21.0
ppm; HRMS (ESI): calcd. for C20H20N2NaO4 [M+Na]+ 375.1315, found 375.1307.
4.1.6.4. (1-(4-Fluorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9d)
o
1
Pale yellow solid (0.10 g, 57%); mp: 108-111 C; H NMR (400 MHz, CDCl3): δ 3.98 (9H, s),
7.22 (4H, m), 7.76 (2H, d), 8.17 (1H, s), 8.56 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.73,
163.08, 160.62, 153.19 (×2), 142.66, 142.14, 135.63, 133.86, 121.61, 121.52, 116.71 (×2), 116.48
(×2), 106.53 (×2), 61.00, 56.38 (×2) ppm; HRMS (ESI): calcd. for C19H18FN2O4 [M+H]+
357.1245, found 357.1248; C19H17FN2NaO4 [M+Na]+ 379.1065, found 379.1067.
4.16.5. (1-(2-Chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9e)
Pale yellow solid (0.12 g, 62%); mp: 96-98 oC; 1H NMR (400 MHz, CDCl3): δ 3.93 (6H, s), 3.95
(3H, s), 7.20 (2H, s), 7.43 (1H, m), 7.57 (1H, m), 7.66 (1H, m), 8.22 (1H, s), 8.42 (1H, s) ppm; 13C
NMR (100MHz, CDCl3): δ 187.6, 153.2 (×2), 142.5, 142.0, 137.2, 135.4, 134.0, 130.8, 130.0,
128.1, 128.0, 127.7, 123.4, 106.5 (×2), 61.0, 56.3(×2) ppm; HRMS (ESI): calcd. for
C19H17ClN2NaO4 [M+Na]+ 395.0769, found 395.0771.
4.1.6.6. (1-(3-Chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9f)
o
1
Pale yellow solid (0.13 g, 68%); mp: 134-136 C; H NMR (400 MHz, CDCl3): δ 3.93 (6H, s),
3.96 (3H, s), 7.16(2H, s), 7.36(1H, d, J = 8.2 Hz), 7.44 (1H, dd, J = 8.0 Hz, J = 8.2 Hz), 7.65 (1H,
d, J = 8.0 Hz), 7.83 (1H, s), 8.17 (1H, s), 8.51 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 186.6,
152.2 (×2), 141.8, 141.2, 139.1, 134.6, 132.7, 129.7, 129.6, 126.8, 123.4, 119.0, 116.4, 105.5 (×2),
60.0, 55.4 (×2) ppm; HRMS (ESI): calcd. for C19H17ClN2NaO4 [M+Na]+ 395.0769, found
395.0791.
4.1.6.7. (1-(4-Chlorophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9g)

ACCEPTED MANUSCRIPT
o
1
Pale yellow solid (0.16 g, 85%); mp: 158-160 C; H NMR (400 MHz, CDCl3): δ 3.91 (6H, s),
3.94 (3H, s), 7.14 (2H, s), 7.47 (2H, d, J = 8.9 Hz), 7.69 (2H, d, J = 8.9 Hz), 8.13 (1H, s), 8.46 (1H,
s) ppm; 13C NMR (100MHz, CDCl3): δ 187.6, 153.1 (×2), 150.4, 142.7, 137.7, 133.7, 133.4, 130.5,
129.8 (×2), 124.3, 120.8 (×2), 106.5 (×2), 61.0, 56.3 (×2) ppm; HRMS (ESI): calcd. for
C19H17ClN2NaO4 [M+Na]+ 395.0769, found 395.0785.
4.1.6.8. (1-(2-Bromophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone(9h)
o
1
Pale yellow solid (0.16 g, 79%); mp: 184-186 C; H NMR (400 MHz, CDCl3): δ 3.93 (6H,
s),3.94 (3H, s), 7.20 (2H, s)7.35 (1H, m), 7.48 (1H, m), 7.60 (1H, m), 7.75 (1H, m), 8.23 (1H, s),
8.37 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.66, 153.13 (×2), 142.44, 138.84, 135.42,
133.90, 130.56, 128.57, 128.22, 123.21, 119.87 (×2), 118.27, 114.10, 106.49 (×2), 60.96, 56.34
(×2) ppm; HRMS (ESI): calcd. for C19H18BrN2O4 [M+H]+ 417.0444, found 417.0442.
4.1.6.9. (1-(3-Bromophenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9i)
Pale yellow solid (0.15 g, 74%); mp: 128-131 oC; 1H NMR (400 MHz, CDCl3): δ 3.94 (6H, s),3.96
(3H, s), 7.18 (2H, s), 7.39 (1H, dd, J = 7.8 Hz, J = 8.3 Hz), 7.52 (1H, d, J = 7.8 Hz), 7.69 (1H, d, J
= 8.3 Hz), 7.99 (1H, s), 8.16 (1H, s), 8.50 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.6,
153.2 (×2), 142.8, 142.2, 140.2, 133.7, 131.0, 130.7, 130.7, 124.5, 123.4, 123.0, 118.0, 106.6 (×2),
61.0, 56.4 (×2) ppm; HRMS (ESI): calcd. for C19H18BrN2O4 [M+H]+ 417.0444, found 417.0454.
4.1.6.10. (1-(4-Methoxyphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9j)
Pale yellow solid (0.14 g, 76%); mp: 99-101 oC; 1H NMR (400 MHz, CDCl3): δ 3.86 (3H, s), 3.92
(6H, s),3.95 (3H, s), 7.00 (2H, d, J = 8.9 Hz), 7.17 (2H, s), 7.64 (2H, d, J = 8.9 Hz), 8.12 (1H, s),
8.41 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.9, 159.2, 153.1 (×2), 142.3, 134.0, 132.8,
130.7, 123.8, 121.3 (×2), 114.7 (×2), 106.5 (×2), 61.0, 56.3 (×2), 55.6 ppm; HRMS (ESI): calcd.
for C20H21N2O5 [M+H]+ 369.1445, found 369.1466.
4.1.6.11. (1-(4-Ethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9k)
Pale yellow solid (0.09 g, 51%); mp: 132-134 oC; 1H NMR (400 MHz, CDCl3): δ 1.28 (3H, t, J =
7.6 Hz), 2.71 (2H, q, J = 7.6 Hz), 3.93 (6H, s), 3.96 (3H, s), 7.18 (2H, s), 7.33 (2H, d, J = 8.2 Hz),
7.65 (2H, d, J = 8.2 Hz), 8.14 (1H, s), 8.46 (1H, s)ppm; 13C NMR (100MHz, CDCl3): δ 187.9,
153.2 (×2), 144.2, 142.4, 142.0, 137.2, 134.0, 130.6, 129.1 (×2), 123.9, 119.7 (×2), 106.5 (×2),
61.0, 56.4 (×2), 28.4, 15.5 ppm; HRMS (ESI): calcd. for C21H23N2O4 [M+H]+ 367.1652, found
367.1655; C21H22N2NaO4 [M+Na]+ 389.1472, found 389.1478.

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4.1.6.12. (1-(4-Isopropylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9l)
Pale yellow solid (0.11 g, 59%); mp: 120-122 oC; 1H NMR (400 MHz, CDCl3): δ 1.29 (6H, d, J =
6.9 Hz), 2.99 (1H, m, J = 6.9 Hz), 3.93 (6H, s), 3.96 (3H, s), 7.17 (2H, s), 7.36 (2H, d, J = 8.4 Hz),
7.65 (2H, d, J = 8.4 Hz), 8.15 (1H, s), 8.46 (1H, s) ppm; 13C NMR (100MHz, CDCl3): δ 187.9,
153.2 (×2), 148.8, 142.4, 142.0, 137.2, 134.0, 130.6, 127.6 (×2), 123.9, 119.7 (×2), 106.5 (×2),
61.0, 56.4 (×2), 33.8, 23.9 (×2) ppm; HRMS (ESI): calcd. for C22H24N2NaO4 [M+Na]+ 403.1628,
found 403.1632.
4.1.6.13.
(1-(3-Chloro-2-methylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone
(9m)
o
Pale yellow solid (0.14 g, 72%); mp: 139-141 C; 1H NMR (400 MHz, CDCl3): δ 2.29 (3H, s),
3.93 (6H, s), 3.94 (3H, s), 7.17 (2H, s), 7.28 (2H, m), 7.51 (1H, m), 8.15(1H, s), 8.17(1H, s) ppm;
13C NMR (100MHz, CDCl3): δ 187.7, 153.2 (×2), 142.3, 142.2, 140.2, 136.1, 134.9, 133.9, 132.8,
130.4, 127.2, 124.9, 123.4, 106.6 (×2), 61.0, 56.4 (×2), 15.6 ppm; HRMS (ESI): calcd. for
C20H19ClN2NaO4 [M+Na]+ 409.0926, found 409.0928.
4.1.6.14. (1-(2,5-Dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9n)
o
1
Pale yellow solid (0.13 g, 70%); mp: 138-140 C; H NMR (400 MHz, CDCl3): δ 2.24 (3H, s),
2.34 (3H, s), 3.90 (6H, s), 3.91 (3H, s), 7.18 (5H, m), 8.14 (1H, s), 8.15 (1H, s) ppm; 13C NMR
(100MHz, CDCl3): δ 187.8, 153.1 (×2), 142.0, 141.9, 138.7, 136.8, 134.6, 134.1, 131.3, 130.0,
129.9, 126.5, 123.1, 106.5(×2), 61.0, 56.3(×2), 20.7, 17.6 ppm; HRMS (ESI): calcd. for
C21H23N2O4 [M+H]+ 367.1652, found 367.1666.
4.1.6.15. (1-(3,5-Dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9o)
o
Pale yellow solid (0.15 g, 80%); mp: 144-146 C; 1H NMR (400 MHz, CDCl3): δ 2.39 (6H, s),
3.91 (6H, s), 3.95 (3H, s), 7.01 (1H, s), 7.16 (2H, s), 7.36 (2H, s), 8.13(1H, s), 8.45 (1H, s) ppm;
13C NMR (100MHz, CDCl3): δ 187.8, 153.2 (×2), 142.4, 142.1, 139.6 (×2), 139.2, 134.0, 130.7,
129.5, 123.9, 117.5 (×2), 106.5 (×2), 61.0, 56.4 (×2), 21.3 (×2) ppm; HRMS (ESI): calcd. for
C21H23N2O4 [M+H]+ 367.1652, found 367.1667.
4.1.6.16. (1-(2,4-Dimethylphenyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (9p)
o
Pale yellow solid (0.13 g, 69%); mp: 119-121 C; 1H NMR (400 MHz, CDCl3): δ 2.25 (3H, s),
2.39 (3H, s), 3.93 (6H, s), 3.94 (3H, s), 7.14 (5H, m), 8.17 (2H, s) ppm; 13C NMR (100MHz,
CDCl3): δ 187.9, 153.1 (×2), 142.0, 139.3, 136.6, 134.8, 134.1, 133.2, 132.1, 127.4, 125.8, 123.2,

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106.5 (×2), 61.0, 56.3 (×2), 21.1, 17.9 ppm; HRMS (ESI): calcd. for C21H23N2O4 [M+H]+
367.1652, found 367.1669.
4.1.7. General procedure for preparation of (1-(p-tolyl)-1H-pyrazol-4-yl)(3,4,5-tri-
methoxyphenyl)methanone oxime (10c)
A suspension of (1-(p-tolyl)-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (0.1 mmol),
hydroxylamine hydrochloride (1 mmol) and sodium acetate (1 mmol) in ethyl alcohol was
refluxed for 2 h. After completion of the reaction, the mixture was concentrated and evaporated in
vacuo, then added 10 mL water, the mixture was extracted by dichloromethane (10 mL × 3), the
organic layer was combined and washed with 10 mL water, dried over magnesium sulfate and
concentrated. The residue was subjected to column chromatography on the silica gel to get the
pure product. White solid (0.03 g, 93%, Z/E 10.8:1); purity: 98.57%; mp: 130-132 oC; (Z isomer):
1H NMR (400 MHz , DMSO-d6) δ 2.35 (3H, s), 3.71 (3H, s), 3.79 (6H, s), 6.81 (2H, s), 7.30 (2H,
d, J = 8.3 Hz), 7.77 (2H, d, J = 8.3 Hz), 8.03 (1H, s), 8.80 (1H, s), 11.67 (1H, s) ppm; 13C NMR
(100MHz, DMSO-d6): δ 153.1, 148.1, 142.5, 138.4, 137.4, 136.5, 132.4, 130.3 (×2), 130.0, 119.2
(×2), 115.3, 106.0 (×2), 60.4, 56.2 (×2), 20.8 ppm; (E isomer): 1H NMR (400 MHz , DMSO-d6) δ
2.32 (3H, s), 3.73 (3H, s), 3.77 (6H, s), 6.70 (2H, s), 7.26 (2H, d, J = 8.5 Hz), 7.73 (2H, d, J = 8.5
Hz), 7.83 (1H, s), 8.30 (1H, s), 11.02 (1H, s) ppm; HRMS (ESI): calcd. for C20H21N3NaO4
[M+Na]+ 390.1424, found 390.1415.
4.2. Biological evaluation
4.2.1. MTT assay
The in vitro antiproliferative activities of SMART and all the target compounds (8a-p, 9a-p and
10c) were tested by MTT assay according to the report in a previous literature.[29]
4.2.2. Inhibition of tubulin polymerization
The tubulin polymerization assay was conducted by using the commercial tubulin polymerization
assay kit (Cytoskeleton-Cat. #BK011P) according to the manufacturer’s protocol.
4.2.3. Immunofluorescence assay
Immunofluorescence staining studies of SMART and the investigated compound 9c were
performed using the reported method.[30]
4.2.4. Cell cycle analysis by flow cytometry
Cell cycle analysis assay was followed the procedures, as previous report.[31]

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4.3. Computational assay
4.3.1. DFT computation
Structural optimization of SMART (p-Me), target compound 8c and 9c were performed at
B3LYP/6-31G(d) level of Gaussian 09 software.[32] The results of optimization showed the energy
of two conformations for any molecule, and the unit a.u. of original energy value was converted
into standard unit kJ·mol-1 (1 a.u. = 2625.50 kJ·mol-1). Thermodynamic equilibrium was calculated
as followed formulas:
∆G ≈ Ebent - Estraight = -RTlnK (R = 8.314 J·mol-1·K-1, T = 300 K)
Thus, constant K could be obtained, and
K = [bent]/[straight] = nbent/nstraight
[bent] and [straight] are the concentration of each conformation, nbent and nstraight are amount of
each conformation, thus,
Estimated proportion of “bent” conformation = nbent/(nstraight+nbent) = K/(K+1)
The results were given in Table S1 (Supplementary data) and Table 2.
4.3.2. Molecular modeling studies
The molecular modeling studies were investigated using Accelrys Discovery Studio 3.0. The
X-ray crystal structures of tubulin in complex with CA-4 (PDB: 5LYJ) was downloaded from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb). The ligand 9c was energy-minimised with
the CHARMm force-field and docked into colchicine binding site using CDOCKER protocol. The
pictures in Figure 6 were edited with Discovery Studio 4.5 Visualizer.
Acknowledgements
We gratefully acknowledge the National Natural Science Foundation of China (81673293,
81502932 and 81602969), Fund for Fostering Talents in Basic Science of the National Natural
Science Foundation of China (J1103606), Doctoral Research Funding of Liaoning Province
(201601144), Education Fund Item of Liaoning Province (201610163L12), and Science and
Technology Project of Shenyang City (17-231-1-41) and Liaoning Province Undergraduate
Training Program for Innovation (201610163019, 201710163000202 and 201710163000094) for
the generous financial support. This work was also supported by Program for Innovative Research
Team of the Ministry of Education and Program for Liaoning Innovative Research Team in
University.

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Highlights
‹
A set of (1-aryl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methanone derivatives were
synthesized.
‹
Compounds 9c and 10c showed potent antiproliferative activities with IC50 values of
0.054-0.17 µM.
‹
‹
Compound 9c inhibited tubulin polymerization and caused cells arrest in G2/M phase.
Conformational analysis indicated that a certain ratio of “bent” conformation was probably
necessary for exerting the biological effects.
‹
The binding mode of 9c was determined by docking studies.